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Inhibition of mitochondrial protein import by mutant huntingtin

Inhibition of mitochondrial protein import by mutant huntingtin

Research has shown that mitochondrial dysfunction is associated with neuronal loss in Huntington’s disease (HD). However, it is unclear how mutant huntingtin (Htt) may cause such dysfunction. Researchers at University of Pittsburg and Washington University have discovered evidence of a direct relationship between mutant Htt and the mitochondrial protein import machinery1. This study has many implications for HD research including the development of mitochondrial protein import-based therapies.

Background^

Mitochondria are specialized subunits within a cell often referred to as the ‘energy powerhouse’ as they are responsible for conversion of nutrients into energy for the cell. Mitochondria can be found in every cell of the human body (with the exception of red blood cells) and they are also responsible for calcium homeostasis and the regulation of apoptosis (programmed cell death) 2.

Unfortunately, mitochondria may not function properly due to the obstruction of important pathways, which can compromise the energy production of the cell. When mitochondria in the brain malfunction, less energy is generated within the neuronal cells, which can lead to cell injury, and eventually cell death. Therefore, neuronal cells need healthy mitochondria to survive. There is evidence that mitochondrial dysfunction may be a critical driver of HD pathophysiology1.

The mitochondrial protein import pathway is crucial for healthy mitochondrial function since proteins are often created in the cytoplasm before traveling through several complexes to make their way to the mitochondria. To stress the importance of this pathway, mitochondria contain approximately 1,500 different proteins, yet 99% of which are encoded by the nuclear genome.

The Study^

Initially, the scientists observed that mutant Htt protein, unlike normal Htt protein, was present within brain mitochondria of HD patients. They hypothesized the existence of an interaction between mHtt and some mitochondrial proteins. The researchers conducted a series of experiments using mice models and cell lines to determine what that interaction is exactly and if that interaction negatively affects the mitochondria function.

In order to further understand how this deficiency in mitochondrial protein import is occurring, the scientists further investigated the role of Htt protein using an in vitro protein import assay with a radiolabeled precursor matrix protein (pOTC) which allows the measurement of the import activity for many mitochondrial proteins. They performed this assay in normal mitochondria in the presence of either normal (23Q) or mutant (97Q) recombinant Htt fusion proteins (shorter N-terminal Htt fragments that have been tagged to allow for purification and increased expression3) and observed that, if the Htt fragment carries a polyQ expansion, it will inhibit the uptake of preproteins into the mitochondria through direct association with the TIM23 mitochondrial protein import complex1. (The TIM23 complex assists in the movements of certain proteins across the inner mitochondrial membrane into the mitochondrial matrix.) The mutant Htt does, in fact, cause mitochondrial dysfunction by interfering with the mitochondria’s protein import function.

To understand how to prevent such dysfunction, the scientists overexpressed the TIM23 complex of the mitochondria. This resulted in more pre-proteins being able to travel through the membrane and into the mitochondria, and consequently, successfully limited the problematic cell death caused by the mutant Htt. This observation confirms that the purposeful overexpression of the TIM23 complex may actually be a key therapeutic target as cell death could be avoided despite a presence of mutant Htt.

It is important to note that there is a brain-specific reduction in the activity of protein import in neuronal mitochondria, rather than an increased sensitivity to import dysfunction, as previously hypothesized. Because of the energetic demands of synaptic transmission, synaptosomal mitochondria might be more sensitive to changes in protein import.

Finally, since HD is an age-dependent progressive neurodegenerative disease, the researchers decide to investigate how age-related insults like oxidative stress (link to definition) might compound the progression of the disease. For this experiment, mitochondrial protein import activity was observed in the presence of a sub lethal dose of hydrogen peroxide. This dosage did not impact the wild type neurons. However, it did significantly decrease import activity in the 195CAG HD neurons.

Conclusion^

These results suggest that mutant Htt inhibits mitochondrial protein import via a direct interaction with the import machinery and have important implications in respect to the development of future HD therapies. Scientists can now apply these findings to target mechanisms that prevent the mutant Htt from blocking mitochondrial protein import in an attempt to prevent or delay neuronal cell death due to mitochondrial dysfunction. Such therapeutic approach could have the potential to slow down the progression of the disease and reduce the need to focus on the more difficult task of neurogenesis and network repair.

For Further Reading^

1.Yano, Hiroko, et al. “Inhibition of mitochondrial protein import by mutant huntingtin.” Nature neuroscience (2014).

2.Wiedemann, Nils, Ann E. Frazier, and Nikolaus Pfanner. “The protein import machinery of mitochondria.” Journal of Biological Chemistry 279.15 (2004): 14473-14476.

3. “GST-tagged Proteins – Production and Purification.” GST-tagged Proteins. N.p., n.d. Web. 02 July 2014.

4. . Johri, A. & Beal, M.F. Antioxidants in Huntington’s disease. Biochem. Biophys. Acta 1822, 664–674 (2012).

K. Powers 2014

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Trojan Therapy

The blood-brain barrier (BBB) is a layer of cells that block most molecules from entering the brain in order to protect this sensitive organ from external invaders. It does its job so well that delivering treatments to the brain is extremely difficult and poses a great challenge to drug development.

BBB

 

BBB2

Researchers from the University of Nebraska Medical Center, the University of North Carolina, and Lomonosov Moscow State University in Russia have found a potential solution. In early 2013, these collaborators announced that they were successful in delivering an enzyme to the site of cell death within the brain of a mouse experiencing poor motor function characteristic of Parkinson’s disease.  The researchers accomplished targeted delivery through a method called Trojan therapy. This article will explain the concept of Trojan therapy, as well as its potential application for Huntington’s disease.

What is Trojan Therapy?^

The method for drug delivery is named for the Trojan War tale, during which one army snuck past the defenses of their enemy by disguising themselves within large wooden horses. The horse was presented as a gift and when it was brought behind the city’s defenses the army used the element of surprise to defeat the enemy forces.

Trojan therapy works in a similar fashion. The blood-brain barrier serves as a fortress wall for the brain. In this case, researchers disguised potential treatments within macrophages, immune cells that are allowed entry past the blood-brain barrier, making it possible to deliver the targeted therapy straight to the cells that need it.

The scientists utilized an antioxidant enzyme (proteins that combat oxidative free radicals within the body) known as catalase, to prevent cell death within the mouse brain affected by Parkinson’s disease. They engineered macrophages to carry the genetic material for catalase past the blood-brain barrier and into the brain. Once inside the brain, the macrophages attached to dying neurons, stimulating signals that called for backup to repair these neurons.

The Science behind Trojan Therapy^

Oxidative stress can lead to cell damage by producing free radicals, highly reactive molecules that can indiscriminately react with and damage cellular components. Reducing excess free radicals with antioxidants is an attractive possible method to reducing inflammation caused by cell damage in Parkinson’s brains. In the past, introducing antioxidant therapies has proven unsuccessful because the treatments were not able to overcome the blood-brain barrier.

In order for this type of therapy to function properly, blood-borne macrophages must be able to carry antioxidant proteins across the blood-brain barrier to affected areas of the brain. In this study, the scientists loaded the macrophages with nanozymes, a type of enzyme that was used to clear out free radicals from the brain.

BBB3

Once the macrophage finds its way into the brain, there are three options for the nanozyme to reach its specific target in the brain:

1) Transient fusion of cellular membranes

2) Formation of macrophage bridges (physical structures that repair cellular gaps) and cellular movement mechanisms and,

3) Release of exosomes – large vesicles or storage pockets in the cell – containing the nanozyme. 1

Exosomes appear to be most promising for targeting cell death as research has shown they are excellent long-distance cellular transporters. They effectively transport many biological molecules involved in making proteins from the information contained in genetic material, including proteins, mRNA, and microRNA (Zomer et al, 2010). Researchers engineered macrophages to release exosomes containing DNA, mRNA, transcription factors (proteins that attach to DNA and control transcription of genetic information to messenger RNA) and an encoded catalase protein to assist in reducing inflammation of the brain. In Parkinson’s disease mouse models, the researchers discovered that sustained production of catalase resulted in inflammatory and neuroprotective outcomes, meaning cell death and brain atrophy was not as likely in those mouse models treated by the macrophage Trojan therapy.

The expression of the encoded catalase included within the macrophage increased over time, while DNA levels remained constant, which has implications for the therapeutic efficacy. An antioxidant with greater positive outcomes might be able to be transferred to needy neurons through this mechanism of delivery.

Conclusion of Findings^

The control group for this experiment utilized mouse models without inflammation.  The experimental group included mice exhibiting motor signs of PD. After injecting the PD mice with the macrophages, noticeable changes occurred. The fluorescent markers used to tag the progress of the materials packaged within the macrophage showed thorough coverage of the brain, especially to regions of inflammation where cell repair support was needed. These mice were able to perform just as well as the control mice on balance tests after receiving the Trojan therapy.

Oxidative stress can lead to cell damage. Reducing excess free radicals is an attractive method of reducing inflammation in PD brains. In the past, these methods have proven unsuccessful due a lack of mechanisms to deliver therapeutic drugs across the blood-brain barrier. The research conducted by this collaboration of scientists is the first to find a potential route for overcoming these obstacles. In fact, this method might be more effective than traditional viral vector gene therapy used for these purposes.

The research by the University of North Carolina and Lomonosov Moscow State University highlights various issues faced by clinical researchers. As outlined in this article, ability to cross the blood-brain barrier plays a major role in therapy design. Additionally, as the brain is a very sensitive organ, it is difficult to gauge the toxicity thresholds for various treatments and medications. Exosomes, unfortunately, are not a silver bullet either. The efficiency of loading drugs into exosomes is a challenge, as well as the difficulty in targeting the exosome to a particular cell type or organ.

Scientist Andrew Feigin of Hofstra North Shore-LIJ School of Medicine is currently leading a human-based clinical trial in which he is trying to use virus-delivered gene therapy for PD. This method has more sustained impacts than macrophages, but is much harder to get through the brain as the immune system fights these foreign viruses trying to pass the blood-brain barrier.

Researcher Elena Batrakova of University of North Carolina aims to deliver growth factors into the brain utilizing macrophages, in order to stimulate the growth of neurons. She believes that macrophages are the solution to reversing neuronal death in patients with PD. To-date this research has only been conducted in mouse models. In order to start human trials, scientists need to prove the safety of this therapy, as well as assure this therapy can be repeated successfully. This process could take several more years before human trials begin.

What does this research means for HD?^

While this study focuses on Parkinson’s disease, overcoming the challenge of effectively delivering medicine across the blood-brain barrier could be useful in preventing damage or repairing brain cells in any of the neurodegenerative disorders, including Huntington’s disease. If HD scientists develop drugs or treatments that could slow down or reverse neuronal death in Huntington’s disease patients, macrophage-based delivery, as pioneered in this research, could be a method for getting that medication across the blood brain barrier and into regions of the brain where the medication is needed. If a method of drug delivery, such as Trojan therapy, could be proven safe, effective, and broadly applicable, it would allow scientists to concentrate on drug development.

For Further Reading

1. Haney, M. J., Zhao, Y., Harrison, E. B., Mahajan, V., Ahmed, S., He, Z., … & Batrakova, E. V. (2013). Specific Transfection of Inflamed Brain by Macrophages: A New Therapeutic Strategy for Neurodegenerative Diseases. PloS one, 8(4), e61852.
2. Ahmed, Abdul-Kareem. “A Trojan Treatment for Parkinson’s Disease.” MIT Technology Review. MIT, 14 Oct. 2013. Web. 04 Nov. 2013.
3. Zomer A, Vendrig T, Hopmans ES, van Eijndhoven M, Middeldorp JM, et al. (2010) Exosomes: Fit to deliver small RNA. Commun Integr Biol 3: 447–450.


KP 12/6/13 More

Weight Loss: Demystifying a Medical Mystery

While Huntington’s disease is traditionally thought of as a disease of the brain, its effects are much more widespread: many people with HD lose a dangerous amount of weight, complicating a disease that is already complicated enough. Although weight loss is one of the most serious non-neurological problems of HD, scientists don’t fully understand why it occurs. This medical mystery has driven scientists deep into the biology underlying weight loss in HD. Researchers have recently turned up a few potential explanations, and our increased understanding of this symptom is leading scientists to look at possible new ways of treating the disease.

Weight Loss in HD^

People with HD tend to weigh less than those without the disease. A group of researchers from the Huntington Study Group followed 927 people with early-stage HD. For a description of the stages of HD, please click here. The investigators found that people with early-stage HD weighed an average of 10 kilograms (22 pounds) less than age-matched controls, which are people of the same age who don’t have the disease. Another study found that people with HD lose an average of 0.9 pounds per year, which stands in stark contrast to the average American, who gains 0.4-2 pounds yearly.

Unfortunately, while 0.9 pounds doesn’t seem like much, that’s just an average; some people with HD lose so much weight that their health is impacted. Weight loss worsens other aspects of the disease as underweight patients become malnourished and weak. Underweight patients are more susceptible to infection, and take longer to recover from illness, operations, and wounds. Weight loss also increases the likelihood of developing pressure ulcers, commonly known as bedsores, as bedridden patients have less fat tissue to cushion them from pressure. Patients who lose the most weight report a lower quality of life, and are more likely to feel apathetic and depressed. In the late stages of the disease, some patients lose so much weight that they need a feeding tube to stay healthy, as described here. On the other hand, people who start out heavier fare better; people who have a high body-mass index (BMI) when symptoms begin progress more slowly through the disease. Visit this website for an explanation of BMI and a for BMI calculator.

A Medical Mystery^

While weight loss is one of the most serious non-neurological problems associated with HD, doctors don’t understand why it happens. Many suggestions have been put forth, but most of them have been disproved, forcing researchers to dig deeper to understand this phenomenon.

Chorea^

Doctors once believed that weight loss was due to chorea, the uncontrolled movements characteristic of HD. Doctors thought that people with HD lost weight because they burned extra energy as a result of the involuntary movements of chorea. However, three experiments indicate that chorea can’t be fully responsible for weight loss.

The first piece of evidence comes from looking at the early stages of the disease. People who have just been diagnosed with HD – and therefore have very mild symptoms – already weigh less than people without the disease. As mentioned earlier, people in early-stage HD weigh an average of 10 kg less than those who are not affected by the disease. Another group of researchers arrived at similar results; a study of 361 people with early-stage HD found that they have BMIs an average of 2 points lower than those without the disease, even if the patients had just been diagnosed with HD within that year and hadn’t yet begun to experience choreic movements. Researchers concluded that chorea alone could not explain why people with HD have lower BMIs, and that other factors are at play.

Other studies suggest that chorea may not have as much of an impact as doctors once thought. Pratley et al. measured how much movement chorea caused, in an attempt to quantify how much weight patients lose due to choreic movement. After measuring the movements of 17 people with mild to moderate HD for a week, they found that chorea caused people with HD to move more than people without the disease when sedentary: people with HD moved 14% more than people without HD while sitting or lying down. However, people with HD do less voluntary activity. Study participants with HD walked around and exercised less than people without the disease. In the end, Pratley et al. were surprised to discover that sedentary over-activity balanced out voluntary under-activity: people in the early and middle stages of HD don’t actually move more than people without the disease.

A similar study by the European Huntington’s Disease Initiative Study Group (EHDI) measured weight loss in 517 people with HD, and found no correlation between the amount of weight people lost and the severity of their motor symptoms; people with good scores on tests measuring motor symptoms (such as the UHDRS) were just as likely to lose weight as those with bad motor scores. For more information on diagnostic tests like the UHDRS, click here.

The final strike against the chorea theory comes from observations of people with late-stage HD. Weight loss is most drastic in the final stages of HD, despite the fact that chorea has usually ceased and patients are largely bedridden. So while chorea contributes to weight loss in HD, it cannot stand as the sole explanation.

Reduced Food Intake^

Others suggest that people with HD lose weight because they have trouble eating; as the disease progresses, it becomes increasingly difficult to perform the complicated series of movements needed to eat, chew, and swallow.

However, this theory is also not enough to fully explain the weight loss. Studies have shown that people with HD actually tend to eat more than people without the disease; a study of 25 people with HD found that they ate an average of almost 400 calories more each day than people without the disease. Others report that they’ve had patients who eat up to 5000 calories a day – over twice the average daily caloric intake – just to maintain their weight.

So two popular explanations for weight loss in HD – chorea and insufficient diet – cannot entirely explain why people with HD lose so much weight.

Possible Biological Causes^

Though the reasons for the mysterious weight loss are unclear, scientists are currently testing a few ideas.

Abnormalities in Energy Metabolism^

One leading idea has to do with metabolism, the way the body burns calories to produce energy. HD researchers have long suspected that the disease-causing form of huntingtin (hereafter described as mutant huntingtin) interferes with energy metabolism, as described here. Results from a recent study suggest that this interference might contribute to weight loss.

After discovering that weight loss is not correlated with motor symptoms, scientists from the EHDI Study Group looked for other factors that might be to blame. They found that weight loss could be partially predicted by the number of CAG repeats on a patient’s copy of the mutant huntingtin gene; for every additional CAG repeat a patient had, they lost on average an extra 0.136 BMI points (0.8 pounds) over the course of the three year period that the study was conducted. For an explanation of CAG repeats, please click here.

The same holds true in mouse models of HD. The EHDI Study Group found that the more CAG repeats an HD mouse had, the more it tended to eat. Yet paradoxically, the mice with the most CAG repeats lost the most weight. So people and mice with more CAG repeats lose more weight.

The EHDI investigators suspect that this is due to the long tail of the mutant huntingtin protein. People with more CAG repeats produce mutant huntingtin with a longer tail, as described here. The EHDI investigators suggest that the mutant huntingtin protein interferes with the way cells make energy, and that longer-tailed proteins cause more problems. Mutant huntingtin has been shown to disrupt proteins that are needed to make energy and can damage mitochondria, the “energy factory” of our cells, as described here. In support of the theory that proteins with longer tails are more problematic, scientists at the MacDonald lab in Boston studied cells engineered to express mutant huntingtin. They found that cells with more CAG repeats made less ATP, the energy currency of the cell. So it seems possible that the more CAG repeats individuals have, the less efficient their cells are at converting calories to energy.

Hormonal Shifts^

A second school of thought suggests that weight loss is due to hormonal disturbances in people with HD. Hormones are the body’s chemical messengers, and are important for regulating physiological processes, like hunger. The hypothalamus secretes many hormones, so when HD causes cells in the hypothalamus to malfunction and die, hormone production is disturbed.

Some of the hormonal signals that the hypothalamus sends out go to the gut and fat tissue, and direct processes like eating and burning energy – processes that are very important in maintaining a healthy weight. Therefore, some scientists think that cell death in the hypothalamus causes hormonal changes that might contribute to weight loss and other problems such as sleep disturbances, as described here.

Dysfunctional Digestion^

Further insights have come from studying the way mutant huntingtin interacts with the digestive system. Certain symptoms of HD have hinted that the disease might affect the gut; apart from weight loss, people with HD often experience nutritional deficiencies, cramps, and wasting of skeletal muscles. People with HD are also prone to gastritis, a disease where the stomach lining becomes irritated or swollen.

Despite these symptoms, many HD researchers have traditionally thought that mutant huntingtin only affected the brain – a belief that struck some as strange because the protein is made and found throughout the body. However, results from a recent study suggest that mutant huntingtin in the gut might interfere with important digestive processes, thus contributing to weight loss.

In the study, van der Burg and colleagues looked at R6/2 mice, which are mouse models of HD described in greater detail here. They noticed several physiological changes that could all impact digestion. First, they noticed that the small intestines of HD mice were 10-15% shorter than those of normal mice, and that they had smaller villi, the tiny finger-like projections in the gut that take up nutrients. On top of that, scientists noticed that the mucus lining of the gut of the HD mice was 20-30% thinner. Since all of these structures are needed for nutrient absorption, these findings suggest that HD mice can’t take up nutrients as efficiently as normal mice.

Furthermore, the group found that the HD mice were missing a few key hormones that control the speed at which food passes through the body. This caused an increase in ‘transit time’: the food passed more slowly through the gut. Longer transit time might foster bacterial growth; if food takes longer to pass through the gut, harmful bacterial have more time and a better opportunity to flourish. This could make the small intestine irritated and inflamed, which could cause malabsorption of nutrients, chronic diarrhea, nausea, bloating, flatus, and weight loss. Those bacteria might also use up nutrients that the body would have otherwise taken up.

To see whether these physiological differences actually have an impact on digestion, researchers then compared the feces of HD mice to those of normal mice. They found that HD mice excreted more of what they ate, suggesting that they absorbed fewer calories and nutrients from their food. Notably, the mice that were the worst at absorbing nutrients from their food lost the most weight.

Van der Burg et al. had a few ideas as to what mutant huntingtin might be doing to interfere with digestion. Since the protein is present in gut cells, it could interfere with cell function and nutrient absorption. They also thought that mutant huntingtin might affect transcription, the process by which DNA is converted into protein as described here. If mutant huntingtin affects transcription in gut cells, it could cause a decrease in levels of important proteins needed for cells to survive and function properly.

While findings in HD mice don’t always translate to humans, these results indicate that scientists might benefit from studying the way HD affects digestion in people. Van der Burg et al. suggest that such research might help doctors improve their understanding of nutritional supplements for HD, and might even change the way we think about how people with HD metabolize and react to medicine.

Solutions^

Weight loss in HD has long puzzled doctors, patients, and caretakers alike. Two popular explanations of the phenomenon – chorea and reduced food intake – have been debunked as major contributors to weight loss. However, scientists have made new in-roads in recent years. By discovering that mutant huntingtin might disrupt energy metabolism, digestion, and hormones in HD mice, scientists have enhanced our understanding of HD, which may pave the way to new treatments and therapies. For example, the hypothesis that weight loss is linked to abnormalities in energy metabolism suggests that energy-boosting drugs – namely creatine and Coenzyme Q10 – are strong candidates to fight HD, as described in these articles here. Each further discovery about HD leads to a greater understanding of the disease, and brings hope for patients and families.

Sources^

1.     Aziz NA, van der Burg JM, Landwehrmeyer GB, Brundin P, Stijnen T; EHDI Study Group, Roos RA. Weight loss in Huntington disease increases with higher CAG repeat number. Neurology. 2008 Nov 4;71(19):1506-13.

This medium-difficulty study describes how people with more CAG repeats lose more weight – and provides some theories as to why that might be the case.

2.     Djoussé L, Knowlton B, Cupples LA, Marder K, Shoulson I, Myers RH. Weight loss in early stage of Huntington’s disease. Neurology. 2002 Nov 12;59(9):1325-30.

This medium-difficulty article describes weight loss in people with early-stage HD

3.     Hamilton JM, Wolfson T, Peavy GM, Jacobson MW, Corey-Bloom J; Huntington Study Group. Rate and correlates of weight change in Huntington’s disease. J Neurol Neurosurg Psychiatry. 2004 Feb;75(2):209-12.

This medium-difficulty article describes weight loss in people with early-stage HD

4.     Kremer HP, Roos RA. Weight loss in Huntington’s disease. Arch Neurol. 1992 Apr;49(4):349.

This short, medium-difficulty column suggests that cell death in the hypothalamus could contribute to weight loss

5.     Petersén A, Björkqvist M. Hypothalamic-endocrine aspects in Huntington’s disease. Eur J Neurosci. 2006 Aug;24(4):961-7. Epub 2006 Aug 21. Review

This technical article describes how hormonal changes in people with HD might lead to weight loss

6.     Pollard J, Best R, Imbrigilo S, Klasner E, Rublin A, Sanders G, Simpson W. A Caregiver’s Guide for Advanced-Stage Huntington’s Disease. Huntington’s Disease Society of America, 1999.

This easy-to-read handbook is a very helpful resource for caregivers taking care of people in late-stage HD

7.     Pratley RE, Salbe AD, Ravussin E, Caviness JN. Higher sedentary energy expenditure in patients with Huntington’s disease. Ann Neurol. 2000 Jan;47(1):64-70

This study measured movements of people with HD, and found that their total energy expenditure was the same as that of people without the disease, and is somewhat technical

8.     Seong IS, Ivanova E, Lee JM, Choo YS, Fossale E, Anderson M, Gusella JF, Laramie JM, Myers RH, Lesort M, MacDonald ME. HD CAG repeat implicates a dominant property of huntingtin in mitochondrial energy metabolism. Hum Mol Genet. 2005 Oct 1;14(19):2871-80. Epub 2005 Aug 22.

This technical article describes how huntingtin interferes with energy metabolism in a CAG-dependent fashion

9.     Trejo A, Tarrats RM, Alonso ME, Boll MC, Ochoa A, Velásquez L. Assessment of the nutrition status of patients with Huntington’s disease. Nutrition. 2004 Feb;20(2):192-6.

This medium-difficulty paper discusses the result of the study on 25 HD patients that ate an average of 400 calories more than controls each day.

10. van der Burg JM, Winqvist A, Aziz NA, Maat-Schieman ML, Roos RA, Bates GP, Brundin P, Björkqvist M, Wierup N. Gastrointestinal dysfunction contributes to weight loss in Huntington’s disease mice. Neurobiol Dis. 2011 Oct;44(1):1-8. Epub 2011 May 23.

This technical article describes the impact of huntingtin on digestion in HD mice

M. Hedlin 11.16.11

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University of California at San Francisco 2010 HD Research Symposium

HOPES summary of the talks from scientists and clinicians

Note: This article includes references to Dimebon, which is no longer being considered as a potential treatment for HD after the HORIZON clinical trial showed that Dimebon was not better than a placebo. For more information, click here

Lisa Ellerby, PhD, Buck Institute for Age Research^

Target Validation in Huntington’s Disease

Dr. Ellerby’s talk focused on the question: “What are possible biological targets for drugs designed to treat HD?” Therapeutic drugs work by targeting specific processes in the human body that have gone awry in disease. For example, the common symptom chorea in HD is thought to be a result of the increased activity of the neurotransmitter dopamine. Tetrabenazine is used to treat chorea because the drug reduces the amount of dopamine in the brain.

At present, the pharmaceutical industry is focused on developing therapeutics for approximately 200 to 300 different targets in the human body that may be related to HD. However, those few hundred targets represent a very small subset of all possible biological targets and it is possible that drugs that have been created for other diseases could have therapeutic benefits for individuals with HD. One example of this is Dimebon, a drug that was originally used as an anti-histamine and is now being studied in clinical trials for HD because of its neuroprotective effects.

Dr. Ellerby is interested in identifying targets that play specific roles in the death of neurons in HD. While it is well known that the mutated Huntingtin protein (Htt) results in the neurodegeneration characteristic of HD, Dr. Ellerby’s research is important because it can provide insight into how this neuronal death occurs. Her lab used small interfering RNA (siRNA) to block the production of different proteins and then assessed the effects of these knockdowns on neuronal death. If shutting off a particular target results in decreased neuronal death, it is possible that this target plays a role in neurodegeneration due to mutant Htt. These experiments found that blocking the activity of proteases, enzymes that break down other proteins, reduces the death of neurons in a cellular model of HD. Specifically, Dr. Ellerby mentioned that decreasing the level of a family of proteases known as matrix metalloproteases (MMP) reduces the toxicity of mutant Htt. By using siRNA to identify targets that play a role in HD, Dr. Ellerby’s research is laying a foundation for the discovery and development of drugs that can prevent, treat, and reverse the devastating effects of HD.


Jill Larimore, BSc, 4th year graduate student in neurobiology at UCSF^

Immune System Dysfunction in Huntington’s Disease

Ms. Larimore, as a member of Dr. Muchowski’s lab, researches the effect of HD on immune system function. Using yeast and animal models, her lab explores the HD on the molecular level in order to find new therapeutic targets. Ms. Larimore began her talk by explaining the immune system of the brain and the role of microglia cells. These specialized cells differ from those found in the peripheral immune system (i.e. the immune system which operates in all parts of the body besides the brain and spinal cord). Although the blood-brain barrier normally keeps these immune systems apart, Larimore’s research interestingly showed that there was peripheral immune system activation in HD patients. This indicates that the neurological symptoms of HD are either paralleled in the peripheral immune system or communication between cells traverses the blood-brain barrier to connect the two immune systems.

The Muchowski lab’s research also showed that HD increased inflammatory response in both the neural and peripheral immune system, even before manifestations of HD symptoms. Inflammation is an acute immune response that counters tissue injury by releasing chemical signals in the area of injury. Physical inflammation acts as a barrier against the spread of infection while immune cells repair the damaged tissue. Although normally beneficial, inflammation can be harmful when it becomes chronic and remains after healing. In HD patients, a key immune protein, interleukin-6 (IL-6) was found at higher concentrations both in the brain and body. IL-6 helps activate and regulate inflammatory response in the immune system, and indicates immune activity when found in heightened concentrations. In the brain, microglia activation increased as well, indicating microglia were responding to tissue damage in the brain. While immune activation could potentially be a natural healing response, Muchowski’s lab hypothesized that it was chronic inflammation that contributes to HD progression.

Similar inflammatory symptoms found in other neurodegenerative diseases have been extensively researched. Treatments have been found to regulate the heightened inflammatory response that occurs when certain immune proteins are activated. In mouse models of Alzheimer’s disease, the cannabinoid type 2 receptor (CB2) in the brain was found to decrease IL-6 and other proteins involved in the immune response in both the peripheral and neural immune systems. Inhibition of the CB2 receptor in a mouse model of HD worsened symptoms, as shown in behavioral assays (testing the mouse for motor functions and balance). Activating the CB2 receptor resulted in improved coordination and motor function, and slowed the onset of HD symptoms. Because CB2 therapeutics are already in clinical trials for autoimmune diseases, if CB2 is found to be beneficial in HD models by decreasing inflammation in the brain and the peripheral immune system these drugs could potentially be clinically tested as a therapy for HD.


Jan Nolta, PhD, Stem Cell Program, UC Davis^

Working toward mesenchymal stem cell-based therapies for HD

Dr. Jan Nolta, director of stem cell research at UC Davis, presented on recent developments in her work on therapies for HD using mesenchymal stem cells (MSCs).  MSCs have been found to deliver protein products throughout tissue for 18 months at a time. MSCs can potentially be engineered to deliver proteins that help prevent neurodegeneration to the brain. MSCs themselves exhibit neuroprotective activities. They restore synaptic connections, decrease inflammation, decrease neuron death and increase vascularization. Using vessels in the brain as train tracks, they are able to travel throughout the brain to assist other cells. Videos taken under a microscope show that MSCs are “social cells,” meaning they are constantly communicating with other cells around them. By interacting with another cell, an MSC can sense the needs of that particular cell and initiate a flow of appropriate nutrients directly into the other cell. In this way, MSCs act as cellular “paramedics” of the body.

One possibility for an HD therapy involves injecting MSCs into the brain where the cells could help reduce neurodegeneration by saving damaged neurons. Scientists at UC Davis conducted tests on non-human primates to ensure that injecting MSCs into the brain is safe for humans. Safety testing was recently completed with MSCs being injected through the skull into the brains of fetal non-human primates. Fortunately, results showed that after 5 months, the MSCs were still present. This means MSCs will be able to stay in the brain for a good length of time, theoretically assisting neurons and preventing additional cell death. Also, no tumors or tissue abnormalities were detected, indicating that MSC injection is largely safe. More studies about the intracranial transplantation and long-term MSC safety are needed.

Research on MSCs in Dr. Nolta’s lab currently involves three main goals: test bone marrow-derived MSCs to see if they restore neurons in non-human primates, test MSCs for the ability to secrete factors like brain-derived neurotrophic factor (BDNF) that help brain cell function, and to investigate MSC production of siRNA. Dr. Nolta was happy to announce that the FDA recently approved injection of MSCs into the central nervous system of individuals with another disease called amyotrophic lateral sclerosis. This sets an important precedent that will increase the likelihood that Dr. Nolta’s eventual therapy will work in other diseases.


Michael Geschwind, MD PhD, UC San Francisco Memory and Aging Center^

Update on Clinical Studies and Trials in Huntington’s Disease

Dr. Michael Geschwind, a neurologist at the UCSF Memory and Aging Center, provided updates about clinical trials in HD. First, he reviewed the two types of clinical research: observational and clinical trials. An observational study is a type of study in which individuals are observed and certain outcomes are measured (such as motor control or mental function) but no attempt is made to affect the outcome in the form of treatment or therapy. In contrast, a randomized double-blind clinical trial is a study in which each individual is assigned randomly to a treatment group (experimental therapy) or a control group (placebo or standard therapy) and the outcomes are compared. Currently, there is important and promising HD-related clinical research being conducted both within and outside of the United States. The following paragraphs summarize the significant points regarding recent or ongoing studies in the HD field.

The PREDICT-HD study is an observational study that began in 2001, was expanded in 2008, and is still underway. The ultimate goal of the PREDICT-HD study is to define the earliest biological and clinical features of HD before at-risk individuals have diagnosable symptoms of the disease. While the current approach is to treat HD at the beginning of the onset of symptoms, this study aims to help design future studies of experimental drugs aimed at slowing or postponing the onset of HD in the at-risk population prior to observable symptoms. The PREDICT-HD study has identified markers that were shown to appear long before an individual would expect to be diagnosed. One marker is CAG repeat length: CAG stands for the nucleotides (DNA building blocks), cytosine, adenine, and guanine. The HD mutation consists of multiple repeats of CAG in the DNA.  This study validated the CAG repeat length-age formula, in which the CAG repeat length for an individual could estimate the average age of HD onset.  In general, fewer than 30 repeats is considered normal, whereas more than 39 repeats means the person will likely develop HD in a normal lifespan. To read more about CAG repeat lengths click here. Other markers such as the size of ventricles in the brain and the volume of other specific brain areas (i.e. striatum) were also found.  In short, the PREDICT study has validated models for predicting motor onset of HD, which will ultimately increase the likelihood of treating HD before patients become symptomatic.

The DIMOND-HD study was a phase II clinical trial investigating the efficacy of the drug Dimebon, which is a small molecule that inhibits nerve cell death. This drug has been shown to decrease cognitive impairment in Alzheimer’s patients, and has been shown to improve cognition and memory in rats. Dimebon is often referred to as latrepirdine. The DIMOND-HD study evaluated the safety of administering Dimebon for 3 months and the efficacy of Dimebon in improving cognitive, motor, and overall function in subjects with Huntington’s Disease. The study was completed in the summer of 2008, and showed that Dimebon is a well-tolerated drug that generally improves cognition in HD. The researchers concluded that the drug should be tested in phase III clinical trials, which has resulted in the HORIZON trial described below. To read more about Dimebon click here.

The HORIZON study is a randomized, double-blind, placebo-controlled study that is ongoing at 37 sites, spanning 7 countries. The study is in phase III of clinical trials, and also aims to expand upon the results of the DIMOND-HD study and determine if Dimebon (latrepiridine) safely improves cognition in patients with Huntington’s disease.

The HART study is also a randomized, double-blind, placebo controlled study that is ongoing in both Europe and North America. The purpose of the study is to determine if ACR-16, also known as pridopidine and Huntexil, is effective and safe in the symptomatic treatment of HD. ACR-16 is a dopamine stabilizer, which means that it works to help regulate the many functions of dopamine in the striatum and other areas of the brain. ACR-16 has passed phase II of the clinical trials in Europe, and has been allowed to be tested in stage III in North America (US and Canada). Initial results of the clinical trials are promising, and have shown that ACR-16 can improve motor control and may translate to 0.5-1.5 years of disease improvement in voluntary and involuntary movements. To read more about ACR-16, click

The following table outlines the types of characteristics researchers are looking for in each of the ongoing HD Clinical Trials described above. For more information, click on the links provided.

Inclusion Criteria for HD Clinical Trials

PREDICT-HD StudyFor more information on the PREDICT-HD study click here Gene negative and gene positive individuals: specifically, men and women at risk for HD, who have been tested for the HD gene mutation, and who have not been diagnosed with symptoms of HD (CAG > or equal to 36 for CAG-expanded group or CAG < 36 for CAG-norm group).
• 18 years of age or older
• Able to commit to a minimum of 5 yearly evaluations
• Commitment of a companion to attend visits or complete surveys via mail
• Able to undergo a MRI
HORIZON StudyFor more information on the HORIZON study click here • Have clinical features of HD and a CAG polyglutamine repeat expansion ≥ 36• Have cognitive impairment as noted by the following:

1. A screening MMSE and a baseline (pre-dose) MMSE score between 10 and 26 (inclusive); and

2. A subjective assessment of cognitive impairment with decline from pre-HD levels by the Investigator after interviewing the subject and caregiver;

•  Are willing and able to give informed consent

•  Aged 30 years or older

• Have a caregiver who assists/spends time with the subject at least five days per week for at least three hours per day and has intimate knowledge of the subject’s cognitive, functional, and emotional states, and of the subject’s personal care.

HART StudyFor more information on the HART study click here • Able to provide written Informed Consent prior to any study related procedure, including consent to genotyping of the CYP2D6 gene.• Clinical features of HD, and a positive family history and/or the presence of ≥ 36 CAG repeats in the Huntington gene.

• Male or female age ≥ 30 years.

• Willing and able to take oral medication and to comply with the study specific procedures.

• Ambulatory, being able to travel to the assessment center, and judged by the Investigator as likely to be able to continue to travel for the duration of the study.

• Availability of a caregiver or family member to accompany the subject to two visits.

• A sum of ≥ 10 points on the mMS at the screening visit.

• For subjects taking allowed antidepressants or other psychotropic medication, the dosing of medication must have been kept constant for at least 6 weeks before enrollment.

Works Cited

http://www.huntington-study-group.org/Portals/0/PREDICTArticle.pdf

http://www.huntington-study-group.org/ClinicalResearch/ClinicalTrialsObservationalStudiesinProgress/PREDICTHD/tabid/85/Default.aspx

http://hddrugworks.org/index.php?Itemid=24&id=189&option=com_content&task=view

http://www.molecularneurodegeneration.com/content/3/1/15

http://www.hdsa.org/research/clinical-trials/ongoing-clinical-trials/acr16.html

F. Clum, C. Garnett, T. Wang and A, Lanctot, 2010

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Induced Pluripotent Stem Cells: The Future of Tissue Generation

As viable human brain tissue is not available for use in studying disease development and creating therapies for neurological disorders like Huntington’s disease (HD), researchers desperately needed an alternative cell source for this purpose.  Embryonic stem cells fit this role but have many disadvantages, especially for treatments, including immune rejection by the recipient. Some of these drawbacks have been overcome by a recent discovery that revolutionized the face of stem cell biology.  In 2006, Shinya Yamanaka’s research group at Kyoto University made a groundbreaking announcement: they had discovered that adult cells could be genetically engineered to revert back to apluripotent, stem cell-like state.  As iPSC (induced pluripotent stem cell) production rapidly improved, the cells were soon able to compete with traditional fetal, embryonic, and adult stem cells. The primary advantages of iPSCs compared to other stem cells are: a) iPSCs can be created from the tissue of the same patient that will receive the transplantation, thus avoiding immune rejection, and b) the lack of ethical implications because cells are harvested from a willing adult without harming them.  These patient-specific cells can be used to study diseases in vitro, to test drugs on a human model without endangering anyone, and to hopefully act as tissue replacement for diseased and damaged cells.

Like other stem cells, iPSCs have the ability to proliferate indefinitely in vitro, creating a theoretically unlimited source of cells. Like embryonic stem cells,  iPSCs can also differentiate into any cell of the body, regardless of the original tissue from which they are created.  Scientists have found how to direct the differentiation of pluripotent stem cells into many types of target tissue, including neural tissue.  iPSCs demonstrate that by the introduction of just four genes into somatic cells that normally cannot differentiate at all, cells can be created that can differentiate into every cell type in the body.  The early results of iPSC differentiation studies look promising. For example, human fibroblasts have been successfully turned into iPSCs that then are differentiated into insulin-producing cells, a result that holds much potential for the treatment of diabetes.  Mouse iPSCs have been differentiated into cardiovascular (heart muscle) cells, that actually show the contractile beating expected of heart tissue.

Although there are many problems that still must be addressed for iPS technology, such as the tendency for tumors to evolve after iPSC transplantation and the low efficiency of the technology, iPSCs could completely change how diseases are approached in biomedical research.  For HD and other neurological disorders, iPSCs could create perfect models for the cells of the central nervous system that are harmed in the diseases.

Stem cell biology is a very hot topic in modern medicine, yet much is still unknown about the mechanisms underlying pluripotency and differentiation. In order for safe, controllable, and efficient cellular reprogramming to be achieved, there must be more knowledge on the regulation of stem cell states and transitions. iPSCs show that specialized cells and tissue can be transformed into other types of cells, proving cells are much more flexible than previously thought.  As the study of HD will greatly benefit from this new, unlimited source of neural cells for research and cell therapy, iPSCs may be able to provide new and innovative treatments for HD.

The Discovery of iPSCs^

The creation of pluripotent cells has been widely studied for decades. In 1976, the first method of fusion of an adult somatic cell with embryonic cells to create pluripotent stem cells was reported. However, fusion with embryonic cells created unstable cells that were rejected by immune systems after transplantation. If the genes that induced pluripotency could be isolated from their parent embryos and injected into somatic cells, these problems could be avoided.

Yamanaka’s research team studied twenty-four genes expressed by embryonic stem cells in an effort to track down these essential genes that induce pluripotency. To detect pluripotency, they looked for cells expressing genes that were traditionally expressed only in embryos.  They discovered that the addition of four genes induced a cell into a pluripotent state capable of then becoming many different cell types.

The Four Factors^

Subsequent studies showed that other gene combinations were also successful in reengineering cells into iPSCs, but none were as efficient as the first four. Adding other genes that are expressed in early development was shown to increase reprogramming efficiency, and the specific genes needed varied depending on the cell type that was being forced back to its pluripotent state.  As the four factors and their alternatives were largely discovered by trial and error, it is not known how the genes induce pluripotency. Discovering how genes work may point to ways of improving the efficiency of the process and assessing the quality of iPSCs.

The specific genes that induce iPSCs tell scientists a lot about the characteristics of the cells themselves. Pluripotent stem cells are very closely related to tumor cells. Both can survive and proliferate indefinitely, and a test of pluripotency is whether a cell can create a tumor.  It is therefore no surprise that two tumor-related genes, c-Myc and Klf4, are needed to create iPSCs.  Another requirement of pluripotent stem cells is open and active chromatin structures (for more information on chromosomes, click here and DNA transcription click here). The c-Myc gene codes for proteins that loosen the chromatic structure, stimulating differentiation.  Klf4 impedes proliferation.  c-Myc and Klf4 in this way  regulate the balance between proliferation and differentiation.  If only c-Myc and Klf4 are used in the engineering of iPSCs, tumor cells will arise—instead of pluripotent stem cells. Oct3/4 and Sox2 are required to direct cell fate towards a more embryonic stem cells (ESC)-like phenotype.  Oct3/4 directs specific differentiation, such as neural and cardiac differentiation, while Sox2 maintains pluripotency.  Oct3/4 and Sox2 together ensure that iPSCs are indeed pluripotent stem cells and not tumor cells.

Amounts and Timing of Reprogramming Factors^

The programming of iPSCs depends both on the original cell type being transformed and the levels of each reprogramming factor that is expressed.  Expressing Oct3/4 more than the other genes increases efficiency.  Increasing the expression of any of the other three genes decreases the efficiency.  There is clearly a correlation between gene expression ratio and reprogramming efficiency, but the optimal ratio is likely to vary depending on the cell type being reprogrammed. For instance, when neural progrenitor cells are reprogrammed, they do not require Sox2 as they express this gene sufficiently already.  The level of expression of other important genes for maintaining pluripotency also can affect the reprogramming process and the quality of the resulting cells.

The effect gene expression ratio has on reprogramming may explain why efficiency is typically so low (less than 1% of cells are reprogrammed successfully).  Reprogramming is a slow process, and so the timing of various events may also exert a great influence over thecell’s success. The minimum time for the full reprogramming of a mouse somatic cell into an iPSC is between eight and twelve days. The timing of the mechanism for cellular reprogramming may also be a reason for low efficiency, as the cells can only proceed if the right molecular events happen in the correct order.

Creation of a Germline Competent Model^

In the first studies of iPSCs, the cells were shown to be similar to ESCs in morphology and proliferation.  But the cells were not germline-competent, in other words they were unable to differentiate into cells that expressed genes of the parent cells, and so they could not give rise to adult chimeras when transplanted into blastocysts.  As chimeras play key roles in biomedical research, scientists identified iPSCs through a stricter gene marker that only identified iPSCS that were germline competent. It was found that cells that expressed Nanog, a gene closely tied to pluripotency, were germline competent. These cells also were virtually indistinguishable from ESCs in gene expression, and were more stable.  The transgenes were better silenced in the Nanog identified cells although 20% of the iPSCs still developed tumors due to the reactivation of c-Myc.  Unfortunately this stricter criterion also decreased efficiency to only 0.001-0.03%. While subsequent studies improved this efficiency by varying methods, the fact remains that iPSCs are generated with incredibly low efficiency.

Characteristics of iPSCs^

iPSCs exhibit many characteristics that are related to their pluripotency. They lose proteins that are common to somatic cells and gain proteins common to embryonic cells. They also lose the G1 checkpoint in their cell cycle control mechanism, which embryonic stem cells lack as well. During the reprogramming of somatic cells in the iPS mechanism, the cell cycle structure of stem cells must be reestablished. Another distinguishing characteristic of pluripotent stem cells is their open chromatin structure, as this is needed to maintain pluripotency and to access genes rapidly for differentiation. iPSCs have the open chromatin structure associated with ESCs and other pluripotent cells.  Finally, female iPSCs show reactivation of the somatically silenced X chromosome.  A very early step of stem cell differentiation is the inactivation of one of the two X chromosomes in female mammals, a random process. By the reactivation of this X chromosome, iPSCs show that they are truly pluripotent and identical to ESCs.

Non-retroviral Methods of iPSC Production^

A huge barrier to the eventual use of iPSC-derived treatments is the use of retroviruses to force the expression of the four key genes, discussed above, and activating their transcription factors. Retroviruses can carry target DNA that is inserted into a host cell’s genome upon injection, making them ideal for incorporating the four genes into target cells. However, this DNA and the rest of the viruses’ genomes remain in the host genome, which can lead to transcription of unwanted genes and greatly increases the risk of tumors. The expression of the four transgenes must be silenced after reprogramming to avoid harmful gene expression.  c-Myc, a tumor-promoting gene, especially must be silenced after cellular reprogramming or the risk of tumor development becomes too great for clinical use. These retroviral methods in which the transgenes are still present in the pluripotent cells pose a danger to safety, and also are less closely related to ESCs in gene expression than their non-retroviral alternatives.  Methods of reprogramming iPSCs without transgene expression in the reprogrammed cell is therefore essential not only for potential therapies and clinical applications, but also for reliable and accurate in vitro models of diseases.  Yet, the low efficiency of alternatives remains a worry. Whether these methods will be viable for human clinical use remains to be seen.

Excisions^

The excision strategy (transient transfection) of iPSC generation allows the transgenes to briefly integrate into the genome but then removes them once reprogramming is achieved. An example of this site/enzyme combination is the loxP site and the Cre enzyme.  In a study of Parkinson’s disease (PD), specific iPSCs, this loxP/Cre combination was used to generate the iPSCs. Neural differentiation was then induced on the iPSCs to test whether they could differentiate into dopaminergic neurons, the cells harmed in PD.  The differentiation was successful, indicating the transgenes had been excised. However, a loxP site remains in iPSC genome as does some residual viral DNA, so there is still a small potential for insertional mutagenesis. The piggyBac site/enzyme system on the other hand is capable of excising itself completely, not leaving any remnants of external DNA in the iPSC genome.  The piggyBac system also was much more efficient than other non-retroviral methods, with comparable efficiency to retroviral methods, but with the added benefits of safety and ease of application.

Adenoviral Methods^

Adenoviral methods do not pose the same threats as retroviral methods of generating iPSCs. Adenoviruses work like all viruses by hijacking their hosts’ cellular machinery to replicate their own genome and reproduce, but unlike retroviruses they do not incorporate their genome into the host DNA. Because the transgenes are never even incorporated into the host’s genome they do not have to be excised. Instead, the genes are expressed directly from the virusgenome. iPSCs created by adenoviral methods demonstrated pluripotency, but have extremely low reprogramming efficiency.   Viralgenomic material could not be detected in any of the iPSCs, and no tumor formation was reported. This suggests that the use of non-integrating adenoviral methods substantially lowers the threat of tumorgenesis. The successful creation of iPSCs from adenoviral methods proves definitively that safer, non-retroviral methods can also successfully reengineer cells.

Non-DNA methods of iPSC generation^

Recent studies have implied that perhaps genetic material is not required for iPS cellular reprogramming.  The substitution of transgenes with small molecules that promote iPSC generation would be a safe, clinically appropriate way of creating iPSCs, though it remains to be seen if small molecules will be able to completelyreplace genetic methods of iPSC generation or are just useful as supplementary aids to the process.  Protein transduction is a different method shown to entirely replace gene delivery. In this method fusion proteins are created, which fuse each of the transgenes to a cell-penetrating peptide sequence that allows it to cross the cellular membrane. Reprogramming without DNA intermediates should eliminate the risk of tumorgenesis and distorted gene expression due to the reactivation of the transgenes.

Issues Facing the Use of iPSCs^

With iPSC research being a hotspot for several years now, many of the problems the technology first faced have been studied and resolved.  iPSCs are now germline competent,  can be generated from many different types of human and animal somatic tissue, and can be generated in a  variety of retrovirus-free methods. This lack of retroviruses ended worries about transgene reactivation and subsequent tumorgenesis. The nature of the transgenes in question made the risk of tumor development particularly prevalent, as two of the genes, c-Myc and Klf4, directly inducing tumorgenesis.  Retroviral delivery posed a threat to safety in its increased risk of tumorgenesis and in its tendency to alter gene expression. When other methods were established that did not require retroviruses, these concerns were put to rest, yet these new methods’ efficiencies must be improved and some issues still remain concerning the safety of iPSCs and their abilities to act on par with any other pluripotent cell.

Tumorgenesis^

Even without the use of retrovirsues, tumorgenesis is still a large concern for iPSCs, especially if they are ever to be used as cell replacement therapies. Using retroviral methods, twenty percent of iPSCs developed tumors in one study, and though this number has significantly lowered, it must become negligible for iPSCs to be considered for clinical use.  It is telling that the assay for pluripotency in stem cells is the ability to form teratomas, or tumors. This test of “stemness” illustrates the precariously close link between stem cells and tumor cells.  There are several proposals on how to prevent this tumor formation.  The idea to sort cells before transplantation and after differentiation, so that only well-differentiated neural progenitors will be transplanted, is one such proposal.  Another proposal is to genetically modify iPSCs so that they will have a suicide gene to self-destruct when tumors are created. Finally, some antioxidants, such as Resveratrol, have been shown to have tumor-suppressing qualities, and could potentially aid in any treatment proposed to prevent tumors (for an article about the potential of Resveratrol for the treatment of HD, click here).

Differentiation^

Directed differentiation has been a perennial problem in stem cell biology, and iPSCs bring their own unique characteristics to the dilemma.  As with ESCs, iPSCs sometimes have the tendency to not fully differentiate. Also, as with all stem cell research with neurodegenerative diseases, a more efficient and comprehensive method to differentiate cells into neural progenitors and specific neuronal tissue must be discovered, as current methods are imperfect and slow.

Quality Assessment and Variability^

In iPSC research there is a need to establish methods to evaluate the reprogramming process and the final quality of the cells.  To create human iPSCs suitable for cell replacement therapies, there must be tests to ensure that all pluripotent cells have differentiated, and that the cells have not been genetically altered during reprogramming or during differentiation.  With cells derived from diseased individuals for an autologous treatment, there is naturally the concern that the underlying genetic cause of the disease remains in the iPSCs and will manifest itself in the same way. Some studies have indicated that iPSC lines differ drastically, which makes the reproducibility of any particular phenotype difficult.  Analyzing this variability may help discover which somatic tissue is best for generating iPSCs.

Efficiency^

A problem that has not been significantly improved upon since the beginnings of iPSC research is the technology’s low efficiency. Some hypothesize that the addition of other factors would greatly aid the reprogramming process, and that reprogramming success depends on specific amounts and ratios of the four factors, which are only achieved by chance in a small percentage of the cells. Modifying the culture conditions is another area of study for increasing efficiency and rate of iPSC production. For cellular transplantation therapies, other questions must also be considered, such as the optimal cell dose and source tissue, and the best way to deliver the cells. There are potential solutions to this problem, though. Induction efficiencies have been improved up to a hundred times initial values by use of different somatic starting cells and the aid of small molecules. Although there are barriers to iPSC production, research in this field is still in its infancy and has made impressive gains for the short time it has been going on. As more studies are conducted on iPSCs, these issues may be resolved and iPSCs may enter a state capable of clinical use.

Origin Cells Used^

Another potential way to improve iPSC generation efficiency is to establish the best somatic cells type to reprogram for the cleanest, easiest reprogramming. Many different tissue types have been reprogrammed, including fibroblasts, neural progenitor cell, and stomach epithelial (stomach lining) cells. Certain cell types are much more efficient and rapid than others. There is also the probability that subtly varying iPSCs are generated from different types of starting tissue, some of which may prove to be useful for research or replacement purposes.

An interesting “type” of somatic cell was used in studies of secondary iPSCs.  iPSCS were initially generated and then implanted into blastocysts to create chimeric animals. Somatic cells from these chimeras were then removed and iPSCs were generated from these cells, creating secondary iPSCs.  These secondary iPSCs were generated more efficiently. The differentiation status of thecells to be reprogrammed also affects efficiency, as adult progenitor cells are reprogrammed at three hundred times the efficiency of completely differentiated somatic cells.

Transdifferentiation^

An interesting possibility for the reprogramming methods of iPSCs is the potential for transdifferentiation. It may not always be necessary to reprogram cells all the way back to their most primitive pluripotent stem cell state, and instead reprogram one type of adult somatic tissue directly into a different type, bypassing the lengthy processes of complete reprogramming and subsequent differentiation.  For example, in theory fibroblasts that can be easily and safely obtained from a patient’s skin could be converted into neurons or heart muscle cells without ever passing through a pluripotent stage. This would have advantages not only in the conservation of time and resources but also for safety, as transdifferentiation does not pose the risk of tumorgenesis as the cells never are pluripotent. Unfortunately, the technology for such processes is very difficult. To reprogram cells directly into a different cell type, the qualities and characteristics of the desired cell type must be comprehensively understood. For iPSCs the desired cell type was embryonic stem cells, which were very well researched and characterized, but for many types of cell of interest, including cells of the central nervous system, there are still many unanswered questions about the target cell population. Excitingly, the Wernig lab at Stanford has recently created induced neurons (iN) directly from mouse fibroblasts.

Paramedic functions^

A potential use of iPSCs for cellular therapy that can be applied much more quickly than actual replacement of damaged tissue is the transplant of pluripotent cells as support cells rather than replacement neurons. These cells offer neuroprotection by preventing inflammation and producing neurotrophic factors (for the therapeutic use of neurotrophic factors in HD, click here).  In various studies, the transplantation of iPSCs has significantly improved host neuronal survival and function.  This bystander mechanism of therapy is of huge immediate potential in iPSCs, and Dr. Nolta’s lab recently submitted a request for a clinical study of the same mechanism using mesenchymal stem cells to the FDA. For a detailed study of the use of iPSCs for this purpose click here.

Conclusions^

Stem cell biology has been an area of great interest and intense debate since its inception, and iPSC technology has furthered this research and created hope for potential therapeutic applications. While there are still many barriers to the clinical use of stem cells, iPSCs may help elucidate the nature of both pluripotent stem cells and of many disease pathologies to reach an eventual concrete connection between the two. With their potential for autologous cell replacement and disease modeling in vitro iPSCs are the future of stem cell research, and as such they are key players in the battle against HD.

For Further Reading^

Abeliovich, Asa and Claudia A. Doege. “Reprogramming Therapeutics: iPS Cell Prospects for Neurodegenerative Disease.” Neuron. 12 Feb, 2009, 61 (3): 337-39.

Short, approachable article reviewing two studies deriving iPSCs from patients with neurological disorders.

Cox, Jesse L. and Angie Rizzino. “Induced pluripotent stem cells: what lies beyond the paradigm shift.” Experimental Biology and Medicine. Feb 2010, 235 (2): 148-58.

Very detailed, mostly accessible review of the state of iPS research and the discoveries to date, as well as what iPS cells mean for stem cell biology and modern medical approaches. Perfect thorough introduction to iPS technology.

Crook, Jeremy Micah, and Nao Rei Kobayashi. “Human stem cells for modeling neurological disorders: Accelerating the drug discovery pipeline.” Journal of Cellular Biochemistry. 105 (6): 1361-66.

Accessible, interesting article that argues the greatest potential for iPSCs is to test potential drugs for neurological diseases in vitro and find problems early on in the drug development, saving time and resources.

Gunaseeli, I., et al. “Induced Pluripotent Stem Cells as a Model for Accelerated Patient- and Disease-specific Drug Discovery.” Current Medicinal Chemistry. 2010, 17: 759-766.

Readable review on the future of iPS cells, comparing them with other stem cells and elucidating their pontential drawbacks. Good summary of the landmark discoveries in iPS technology to date.

Haruhisa, Inoue. “Neurodegenerative disease-specific induced pluripotent stem cell research.” Experimental Cell Research. 2010.

General overview of use of iPS cells specific to neurological diseases for modeling diseases in vitro and eventually using as a cellular replacement therapy. Good, non-technical overview of the various potential pathways of iPS technology.

Hung, Chia-Wei, et al.  “Stem Cell-Based Neuroprotective and Neurorestorative Strategies.” International Journal of Molecular Science. 2010, 11(5): 2039–2055.

Overview of various neurological diseases and the potential of stem cell therapeutics, either using adult neural stem cells or iPS stem cells. Experiment descriptions are fairly technical, but the review’s reflections and discussion are accessible and interesting.

Laowtammathron, Chuti, et al. “Monkey hybrid stem cells develop cellular features of Huntington’s disease.” BioMed Center Cell Biology. 2010, 11 (12).

Detailed article on the establishment of pluripotent HD monkey model cell line and its use in the study of Huntington’s.

Marchetto, Maria C.N., et al. “Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases.” Human Molecular Genetics. 2010, 19 (1).

Fairly technical review describing the use of iPSCs for modeling neurological disorders.

Niclis, J.C., et al. “Human embryonic stem cell models of Huntington’s Disease.” Reproductive Biomedicine Online. July 2009, 19 (1): 106-13.

Detailed, technical article on the use of human embryonic stem cell lines for HD.

O’Malley, James. “New strategies to generate induced pluripotent stem cells”. Current Opinions in Biotechnology. Oct. 2009: 20 (5): 516-21.

Longer technical article on the various strategies to generate iPS cells without using potentially dangerous viral vectors.

Okita, Keisuke, et al. “Generation of germline-competent induced pluripotent stem cells.” Nature. 19 Jul, 2007, 448(7151):313-17.

Fairly technical article about an early study in iPS research, where cells were selected for Nanog expression rather than the less pertinent gene Fbx15. This higher caliber of selected cells were germline-competent.

Okita, Keisuke, et al. Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors.” Science. 7 Nov, 2008, 322 (5903): 949-53.

Technical article about the advancements in finding non-viral, clinically applicable methods of creating iPS cells.

Orlacchio, A., et al. “Stem Cells: An Overview of the Current Status of Therapies for Central and Peripheral Nervous System Diseases.” Current Medicinal Chemistry. 2010, 17: 595-608.

Technical review on the various types of stem cells used in the studies of neurological diseases and the progress made to date with these cells.

Park, In-Hyun, et al. “Disease-Specific Induced Pluripotent Stem Cells.” Cell. 2008, 134 (5): 877-86.

Fairly accessible article on the creation of iPS cells with genetic defects, as tools for studying the symptoms and experimenting with treatments of various diseases.

Robbins, Reisha D., et al. “Inducible pluripotent stem cells: not quite ready for prime time?” Current Opinion in Organ Transplantation. 15 (1): 61-57.

Clear review of the barriers facing clinical use of iPSCs, accessible and realistic.

Soldner, Frank, et al.Parkinson’s Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors.” Cell. 6 Mar, 2009, 136 (5): 964-77.

Technical article about first successful derivation of iPS cells from a patient with a neurodegenerative disease without using viral vectors. Relevant to HD research as a protocol that will likely be followed for subsequent creation of neurodegenerative iPSC lines for in vitro study.

Stradtfeld, Matthias, et al. “Induced Pluripotent Stem Cells Generated Without Viral Integration.” Science. 7 Nov, 2008, 322 (5903): 945-49.

Technical article outlining a method for creating iPS cells using excisable adenoviruses, rather. than retroviruses that have the potential to harm the cells.

Takahashi, Kazutoshi, et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors” Cell. 30 Nov, 2007, 131(5): 861-72.

Landmark article in the discovery of induced pluripotent stem cells and the factors that create them. Short, but fairly technical.

Yamanaka, Shinya. “Induction of pluripotent stem cells from mouse fibroblasts by four transcription factors.” Cell Proliferation. Feb, 2008, 41 (Suppl. 1):51-6

Short review, less technical summary of first iPS discovery by Yamanaka. Perfect for quick overview of the basics of iPS cell generation.

Yamanaka, Shinya. “Strategies and New Developments in the Generation of Patient-Specific Pluripotent Stem Cells.” Cell: Stem Cell. 7 June 2007, 1(1): 39-49.

Comprehensive review of various methods for creating pluripotent stem cells with a detailed introduction to iPSC methods. Fairly accessible, and very thorough.

A. Lanctot 2011

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Adipose Derived Stem Cells and HD: Real Applications of iPSCs

While stem cells have always been heralded as the future of cellular replacement therapies, recent stem cell research has explored the potential “bystander” or “paramedic” effects of stem cells, which use stem cells to repair damaged cells rather than replacing them. Bystander therapies do not require the stem cells to become the type of cell that is damaged (to differentiate into this cell type),  but rather can help damaged neurons by changing the host environment. The phenotype of the undifferentiated, stem cell that are still pluripotent, able to differentiate into many different cell types, may provide therapeutic benefits in Huntington’s Disease (HD) by releasing neurotrophic factors that promote neuron growth and survival and arrest the mutant huntingtin protein’s negative influence on key cellular survival and energetic pathways. This paramedic function of stem cells might be harnessed to prevent mechanisms of HD that cause harmful symptoms rather than replacing damaged cells, as an alternative approach to traditional drug therapy for the treatment of neurodegenerative diseases.

Researchers in South Korea have recently found that adipose-derived stem cells (ASCs) can serve the same “paramedic” function in HD as is observed in the mesenchymal stem cells that Dr. Nolta is currently researching at the University of California, Davis. Similar to mesenchymal stem cells, adipose-derived stem cells do not create the ethical debates that embryonic stem cells do, as they are removed from adults during elective surgery, not from embryos in vitro. But in contrast to mesenchymal stem cells, ASCs are not found naturally in the body, but are rather multipotent stem cells created by iPS, or induced pluripotent stem cells (for more information about the new technology of iPS, click here). Derived from fat tissue taken from consenting patients, ASCs have the double advantage of easy access and minimal ethical implications. Like all stem cells, ASCs have the ability to differentiate into different somatic cells, though the mechanisms of differentiation are still unclear and scientists do not know how to direct differentiation of ASCs into certain types of tissue.  This study was not concerned with the differentiation of ASCs, but rather their neuroprotective abilities, such as the release of growth factors that are essential in combating many of the symptoms of HD.

How the Bystander Mechanism Works^

Current research using HD mouse models indicates that the use of fetal striatal tissue to replace the damaged striatal tissue in HD mouse models is not possible at this time, as the replacement tissue does not alter the toxicity of the mutant huntingtin protein.  This is like replacing a wall that has been eaten away by termites without doing anything to remove the termites: the new wall will soon also be harmed by the pests. Even if this barrier was overcome, the use of stem cells to replace damaged tissue is hampered by many other problems, such as a lack of donor tissue and rejection by the immune system, identical problems to organ replacement.  Rather than replacing damaged tissue, a different approach could use stem cells to preemptively prevent HD from harming neuronal tissue.  Scientists have noted that with other diseases, stem cells often act through a bystander mechanism, preventing the symptoms of the disease from manifesting, rather than directly replacing damaged cells. This novel approach to stem cell use does not require the extensive technology of differentiation, transplantation, and incorporation with host tissue that cell replacement needs. Instead, the bystander mechanism approach of stem cells takes advantage of the cells’ ability to release factors in their pluripotent state that combat the symptoms of HD. The researchers in this study wished to test this mechanism using ASCs, knowing that the cells could not only differentiate into cells such as neurons and glial cells that could be useful in combating HD, but could also release growth factors that may slow the symptoms of HD (to learn about growth factors and their role in fighting HD, click here.

HD symptoms may be caused by the alterations the mutant huntingtin protein makes to the transcription of DNA by interfering with transcription factors (for more information about the HD’s affect on transcription, click here) Transcriptional interference and mitochondrial dysfunction are key aspects of HD pathology. Mutant huntingtin protein aggregates impede important transcription factors such as the CREB-binding protein which is essential for transcription of pathways key to cell survival.  In HD, one way neural cell death is induced is by inhibiting this transcription factor.  ASCs could slow neurodegeneration and neural death by releasing neurotrophic factors that help prevent premature cell death. Neurotrophic factors encourage the maintenance, growth, and survival of neurons and so serve as a counterforce to mutant huntingtin’s stimulation of cell death. Another transcription factor, PGC-1, controls the creation of mitochondria, and when it is repressed by mutant huntingtin, reduced numbers of mitochondria causes the cell to receive insufficient energy. This makes the cell susceptible to glutamate, an excitatory neurotransmitter that can harm neurons and may be involved in many of the symptoms of HD. ASCs increase PGC-1 expression by preventing the huntingtin protein from impeding its transcriptional regulation, and so prevent glutamate levels from becoming harmfully high in the neuron.

Effects of ASCs in Three Different Models^

The effect of ASC transplantation was tested in three models, a knockout rat model, a transgenic mouse model and an in vitro cell model (for more information about different types of experimental models, click here). All models showed the positive effects of ASCs. The rat model showed that the ASCs were neuroprotective, reducing neuronal death. More specifically, this was observed by comparing the size of the rats’ ventricles of the brain, which are enlarged by the loss of neural tissue in HD. The ventricles of the ASC-treated rats were smaller than those of the control HD model rats, indicating less tissue loss. Ventricle volume is closely correlated with HD progression, as larger ventricles indicate greater loss of neural tissue. By showing a decreased ventricle volume, ASC rats seem to exhibit less cell death, resulting in less loss of neural tissue. In addition, compared to the HD control mice, the mouse model with ASC transplantation had a delayed decline in motor function, had less mutant huntingtin aggregates, and  lived longer.

Potential Problems with ASCs for Clinical Use^

The promising results of the three models hint at the potential of ASCs to delay the onset of HD symptoms, but there is much that still needs to be researched.  It was found that the transplanted ASCs were not evenly distributed across the striatum as expected, but rather most ASCs remained at the initial site of transplantation. This may have impeded the effectiveness of the ASCs and may even be harmful in long-term models. The proliferation of the ASCs was slow once transplanted, so the documented ability of stem cells to proliferate in actual organisms does not seem to apply to ASC transplantation in HD models.  This study did not test whether the same results could be achieved in model organisms that are longer lived. Whether ASCs would be effective in humans, for extended periods of time, has not yet been determined.  Furthermore, the ASCs must be prevented from differentiating into cells that would be harmful in the brain. Currently, the research for directed differentiation of pluripotent cells is rudimentary, and there is particular risk associated with the spontaneous differentiation of ASCs in vivo. The potential risk of the cells to differentiate into other tissue like heart or skin cells should be tested. Another problem of stem cells is that they may be rejected by the patient’s immune system, though this problem is greatly reduced with ASCs. In this study, human ASCs were successfully injected into rat and mouse models without the aid of immunosuppressants, which is encouraging. To further prevent the risk of rejection, the patient’s own adipose cells could be used to create the ASCs. But ASCs derived from patients with the mutant huntingtin protein have yet to be tested and it is possible that these cells may be damaged or not fully effective.

Conclusions^

Researchers have shown that ASCs may have the potential to protect mechanisms of transcription and rescue degenerating neurons by combating the detrimental actions of huntingtin aggregates through the release of growth factors. This bystander mechanism is a novel approach to using stem cells, as they have been traditionally thought of as replacement cells for damaged tissue. The value of stem cells to replace damaged tissue with healthy, fully-differentiated replacement cells cannot be dismissed, especially with new iPS technology and the ability to engineer replacement cells from the patient who is to receive them, reducing the risk of immune rejection. Unfortunately, much more research must be done before stem cells will be used in clinical therapies for cellular replacement, but a more immediate potential for stem cells is in a paramedic capacity, where differentiation and incorporation with the host tissue is not required. By influencing key events in the pathogenesis of HD, ASCs may delay the onset of harmful symptoms. Current research has shown that ASC transplantation may allow for the expression of transcription pathways that HD suppresses, reduce the number of toxic huntingtin aggregates, and decrease the extent of neuron death in mouse models. Its therapeutic use still requires much more research and exploration, and then must make the leap from animal models to human trials, but ASCs have the potential to rescue degenerating neurons and prevent HD symptoms. This ability of stem cells to not only replace damaged tissue, but also prevent tissue damage, holds promise for the treatment of HD.

For Further Reading

Lee, et al. “Slowed Progression in Models of Huntington Disease by Adipose Stem Cell Transplantation.Annals of Neurology. 2009, 66(5): 671-681.

Technical but well-explained article on a specific study of the use iPS cells derived from adipose cells in three HD models: induced rat, transgenic mouse, and in vitro.

A. Lanctot 2011


While stem cells have always been heralded as the future of cellular replacement therapies, recent stem cell research has explored the potential “bystander” or “paramedic” effects of stem cells, which use stem cells to repair damaged cells rather than replacing them. Bystander therapies do not require the stem cells to become the type of cell that is damaged (to differentiate into this cell type), but rather can help damaged neurons by changing the host environment. The phenotype of the undifferentiated, stem cell that are still pluripotent, able to differentiate into many different cell types, may provide therapeutic benefits in Huntington’s Disease (HD) by releasing neurotrophic factors that promote neuron growth and survival and arrest the mutant huntingtin protein’s negative influence on key cellular survival and energetic pathways. This paramedic function of stem cells might be harnessed to prevent mechanisms of HD that cause harmful symptoms rather than replacing damaged cells, as an alternative approach to traditional drug therapy for the treatment of neurodegenerative diseases.

Researchers in South Korea have recently found that adipose-derived stem cells (ASCs) can serve the same “paramedic” function in HD as is observed in the mesenchymal stem cells that Dr. Nolta is currently researching at the University of California, Davis. Similar to mesenchymal stem cells, adipose-derived stem cells do not create the ethical debates that embryonic stem cells do, as they are removed from adults during elective surgery, not from embryos in vitro. But in contrast to mesenchymal stem cells, ASCs are not found naturally in the body, but are rather multipotent stem cells created by iPS, or induced pluripotent stem cells (for more information about the new technology of iPS, click http://hopes.stanford.edu/n3821/research-and-hd/induced-pluripotent-stem-cells). Derived from fat tissue taken from consenting patients, ASCs have the double advantage of easy access and minimal ethical implications. Like all stem cells, ASCs have the ability to differentiate into different somatic cells, though the mechanisms of differentiation are still unclear and scientists do not know how to direct differentiation of ASCs into certain types of tissue. This study was not concerned with the differentiation of ASCs, but rather their neuroprotective abilities, such as the release of growth factors that are essential in combating many of the symptoms of HD.

How the Bystander Mechanism Works

Current research using HD mouse models indicates that the use of fetal striatal tissue to replace the damaged striatal tissue in HD mouse models is not possible at this time, as the replacement tissue does not alter the toxicity of the mutant huntingtin protein. This is like replacing a wall that has been eaten away by termites without doing anything to remove the termites: the new wall will soon also be harmed by the pests. Even if this barrier was overcome, the use of stem cells to replace damaged tissue is hampered by many other problems, such as a lack of donor tissue and rejection by the immune system, identical problems to organ replacement. Rather than replacing damaged tissue, a different approach could use stem cells to preemptively prevent HD from harming neuronal tissue. Scientists have noted that with other diseases, stem cells often act through a bystander mechanism, preventing the symptoms of the disease from manifesting, rather than directly replacing damaged cells. This novel approach to stem cell use does not require the extensive technology of differentiation, transplantation, and incorporation with host tissue that cell replacement needs. Instead, the bystander mechanism approach of stem cells takes advantage of the cells’ ability to release factors in their pluripotent state that combat the symptoms of HD. The researchers in this study wished to test this mechanism using ASCs, knowing that the cells could not only differentiate into cells such as neurons and glial cells that could be useful in combating HD, but could also release growth factors that may slow the symptoms of HD (to learn about growth factors and their role in fighting HD, click (http://hopes.stanford.edu/treatmts/neurotrophicfactors/nf0.html|here).

HD symptoms may be caused by the alterations the mutant huntingtin protein makes to the transcription of DNA by interfering with transcription factors (for more information about the HD’s affect on transcription, click http://hopes.stanford.edu/causes/huntprot/p5.html |here) Transcriptional interference and mitochondrial dysfunction are key aspects of HD pathology. Mutant huntingtin protein aggregates impede important transcription factors such as the CREB-binding protein which is essential for transcription of pathways key to cell survival. In HD, one way neural cell death is induced is by inhibiting this transcription factor. ASCs could slow neurodegeneration and neural death by releasing neurotrophic factors that help prevent premature cell death. Neurotrophic factors encourage the maintenance, growth, and survival of neurons and so serve as a counterforce to mutant huntingtin’s stimulation of cell death. Another transcription factor, PGC-1http://www3.interscience.wiley.com/giflibrary/12/alpha.gif, controls the creation of mitochondria, and when it is repressed by mutant huntingtin, reduced numbers of mitochondria causes the cell to receive insufficient energy. This makes the cell susceptible to glutamate, an excitatory neurotransmitter that can harm neurons and may be involved in many of the symptoms of HD. ASCs increase PGC-1http://www3.interscience.wiley.com/giflibrary/12/alpha.gif expression by preventing the huntingtin protein from impeding its transcriptional regulation, and so prevent glutamate levels from becoming harmfully high in the neuron.

Effects of ASCs in Three Different Models

The effect of ASC transplantation was tested in three models, a knockout rat model, a transgenic mouse model and an in vitro cell model (for more information about different types of experimental models, click http://hopes.stanford.edu/rltdsci/studyhd/am00.html|here). All models showed the positive effects of ASCs. The rat model showed that the ASCs were neuroprotective, reducing neuronal death. More specifically, this was observed by comparing the size of the rats’ ventricles of the brain, which are enlarged by the loss of neural tissue in HD. The ventricles of the ASC-treated rats were smaller than those of the control HD model rats, indicating less tissue loss. Ventricle volume is closely correlated with HD progression, as larger ventricles indicate greater loss of neural tissue. By showing a decreased ventricle volume, ASC rats seem to exhibit less cell death, resulting in less loss of neural tissue. In addition, compared to the HD control mice, the mouse model with ASC transplantation had a delayed decline in motor function, had less mutant huntingtin aggregates, and lived longer.

Potential Problems with ASCs for Clinical Use

The promising results of the three models hint at the potential of ASCs to delay the onset of HD symptoms, but there is much that still needs to be researched. It was found that the transplanted ASCs were not evenly distributed across the striatum as expected, but rather most ASCs remained at the initial site of transplantation. This may have impeded the effectiveness of the ASCs and may even be harmful in long-term models. The proliferation of the ASCs was slow once transplanted, so the documented ability of stem cells to proliferate in actual organisms does not seem to apply to ASC transplantation in HD models. This study did not test whether the same results could be achieved in model organisms that are longer lived. Whether ASCs would be effective in humans, for extended periods of time, has not yet been determined. Furthermore, the ASCs must be prevented from differentiating into cells that would be harmful in the brain. Currently, the research for directed differentiation of pluripotent cells is rudimentary, and there is particular risk associated with the spontaneous differentiation of ASCs in vivo. The potential risk of the cells to differentiate into other tissue like heart or skin cells should be tested. Another problem of stem cells is that they may be rejected by the patient’s immune system, though this problem is greatly reduced with ASCs. In this study, human ASCs were successfully injected into rat and mouse models without the aid of immunosuppressants, which is encouraging. To further prevent the risk of rejection, the patient’s own adipose cells could be used to create the ASCs. But ASCs derived from patients with the mutant huntingtin protein have yet to be tested and it is possible that these cells may be damaged or not fully effective.

Conclusions

Researchers have shown that ASCs may have the potential to protect mechanisms of transcription and rescue degenerating neurons by combating the detrimental actions of huntingtin aggregates through the release of growth factors. This bystander mechanism is a novel approach to using stem cells, as they have been traditionally thought of as replacement cells for damaged tissue. The value of stem cells to replace damaged tissue with healthy, fully-differentiated replacement cells cannot be dismissed, especially with new iPS technology and the ability to engineer replacement cells from the patient who is to receive them, reducing the risk of immune rejection. Unfortunately, much more research must be done before stem cells will be used in clinical therapies for cellular replacement, but a more immediate potential for stem cells is in a paramedic capacity, where differentiation and incorporation with the host tissue is not required. By influencing key events in the pathogenesis of HD, ASCs may delay the onset of harmful symptoms. Current research has shown that ASC transplantation may allow for the expression of transcription pathways that HD suppresses, reduce the number of toxic huntingtin aggregates, and decrease the extent of neuron death in mouse models. Its therapeutic use still requires much more research and exploration, and then must make the leap from animal models to human trials, but ASCs have the potential to rescue degenerating neurons and prevent HD symptoms. This ability of stem cells to not only replace damaged tissue, but also prevent tissue damage, holds promise for the treatment of HD.

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Induced Neurons

Despite predictions of transdifferentiation being a technology of the future, Dr. Marius Wernig’s lab at Stanford has recently discovered a method of reengineering neurons directly from fibroblasts by the the forced expression of transgenes. This is the same method by which induced pluriptent stem cells (iPSCs) are produced, and transdifferentiation, the engineering of cells so they change their type without preceding through an intermediate stage, has been suggested as the logical progression from iPSCs. Its success suggests that, along with iPSC technology, cellular differentiation and cell fate are far more flexible than previously thought.  The new discovery that somatic cells can be turned into completely different cell types by a cocktail of a few genes revolutionizes previous thought about the unchangeable nature of fully differentiated cells, and adds new theories as to how cell fate is determined. The research also importantly shows that reengineering cells with transcription factors can create the complex structures and functions of somatic cells, not simply undifferentiated stem cells.  A combination of three transcription factors that are specifically found in the brain were shown to be able to convert both embryonic and postnatal fibroblasts directly into neurons, in much the same way iPSCs are created, but without preceding through any pluripotent stem cell intermediate. The lab tested nineteen potential genes to see what combination could efficiently convert mouse fibroblasts into neurons in vitro. The gene Asc11 alone could generate immature neurons with undeveloped properties. Another eighteen genes were then tested in combination with Ascl1, and five genes (Brn2, Brn4, Myt1l, Zic1, and Olig2) substantially improved neuron development. While none of these five genes generated induced neuronal (iN) cells when tested individually, it was found that the three factor combination of Ascl1, Brn2, and Myt1l produced the highest quantity of neurons with the most mature action potentials. As a result this combination was concluded to be the ideal grouping of factors for inducing neuron phenotypes.

Induced Neurons: Are they just like normal neurons?^

A difficulty in evaluating whether transdifferentation is successful is that unlike the relatively simple phenotypes of undifferentiated cells, somatic cells have far too many characteristics that must be tested to ensure that complete reengineering occurred. Induced neuronal cells (iNs) express neuron-specific proteins, generate action potentials and form functional synapses but it is still not known how, if at all, the iNs differ from normal neurons. The cells expressed three neuron marker proteins, MAP2, NeuN, and synapsin, and produced spontaneous action potentials, which are the distribution of charged ions across a neuron membrane that causes a signal to be propelled down the neuron’s axon and to the next neuron. Action potentials promote neural communication as they allow a message to cross a cell before neurotransmitters become involved in its movement between two cells. These action potentials were blocked when a sodium ion inhibitor, tetradotoxin, was introduced, as would occur in normal brain tissue. In normal brains, the concentration of sodium across the membrane of the neuron is essential for the cells to generate action potential and relay electrochemical messages through the neuron and onto the connecting cells. The cells’ expression of functional membrane channel proteins that allow sodium ions to flow in and out of the neuron supports the claim that iN cells and normal neurons exhibit identical membrane properties.

Problems in iN Generation^

The iN cells had various phenotypes, but did not exhibit all types of nervous tissue. Most iNs were excitatory neurons, while almost none contained periperin, a protein characteristic of neurons in the peripheral nervous system. The majority of iN cells were excitatory cells that expressed markers indicative of cortical identity, i.e. specific to cells typically found in the cortex. Further research may focus on the generation of iN cells of other specific neuronal subtypes, not to mention the generation of iNs from human cells (currently only mouse iNs have been successfully engineered).  Additional neural transcription factors may aid in creating neurons of more specific phenotypes.

Like induced pluripotent stem cells, iNs go through a gradual process of reengineering. While immature neuron-like cells can been seen as early as three days after infection, it takes five days for branching neuronal cells to form. Physical maturation continues over several weeks.  The efficiency of converting cells into iNs ranged from 1.8–7.7%, which is substantially better than efficiency of iPSC production. However, iNs cannot proliferate like iPSCs, so creating a larger quantity of cells is crucial.

An important question concerning iNs involves their ability to function as neurons by forming functional communication with other cells. This is a crucial requirement if they are to be used as tissue replacement in the future. The researchers tested whether iN cells have the capacity to form functional synapses with other iN cells and whether iN cells were capable of integrating into preexisting neural networks. When iN cells were grown with actual neurons, spontaneous and rhythmic neural activity was observed. The cells could receive synaptic inputs from the normal neurons, demonstrating their ability to integrate into preexisting tissue. It was also shown that iN cells are capable of forming functional synapses with each other.

Transgenes that Engineer Neurons^

Like iPSCs, the gene combination for creating iN cells allows some leniency and variation, but a certain “recipe” seems the most effective. It remains to be seen if different genes or different ratios of the genes that Dr. Wernig’s lab identified will even further improve efficiency of iN production. While Ascl1 alone is sufficient to induce some neuronal traits, such as expression of proteins that generate action potentials, the addition of Brn2 and Myt1l creates more mature cells with increased efficiency up to 19.5%. The highly efficient production of iNs makes it unlikely that they are merely formed from rare stem or precursor cells in the starting cell population, as great care was taken to exclude neural tissue in the isolation of the initial cell population, and no neurons or neural progenitor cells were detected in the culture. Future studies are nonetheless needed to unequivocally demonstrate that cells that have their own unique morphologies can be directly converted into neurons, and that the iNs are not mere derivatives of stem cells.

Why certain neuronal subpopulations (such as the cortical neurons) are more favored than others is another aspect of iN technology that remains to be researched. It may be that high expression of neural cell-fate determining factors directs certain cell types to form, so they are reengineered more often. Different cell types are produced during development by sets of transcription factors that cause cell type specific proteins to be produced. Each cell can be thought of as a person who walks by a lot of doors, but only has one key (specific transcription factors) that allows him to open one door, i.e. the cell can only become the type specified by its transcription factors.

iN cells are a possible alternative to iPSCs for generating patient-specific neurons. The generation of iN cells is fast, efficient, and has the major advantage over iPSCs that it does not go through a stage of pluripotent stem cells that are susceptible to tumor production.  The iN cells could also provide new methods for studying cellular identity and neural development. They have potential uses in neurological disease-modeling, drug discovery, and regenerative medicine. Formerly, transdifferentiation was never thought of as anything besides a futuristic version of cell engineering that would take many years to accomplish. The Wernig lab has shown that cells with more complex morphologies can indeed be generated directly from other cell types using much the same method as iPSCs, and although much remains to be tested, this new technology may revolutionize cell therapies as more cell types are derived.

For Further Reading^

Vierbuchen, et al. “Direct conversion of fibroblasts to functional neurons by defined factors.” Nature. 25 Feb. 2010, 463 (7284):1035-1041.

Well written, fairly accessible article. Some parts a bit technical but very nice section on the next steps for iN.

A. Lanctot 2011.


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Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a type of multipotent stem cell, meaning that they can give rise to many but not all types of cells in the body. MSCs secrete substances, including cytokines and growth factors, that are essential to cell growth and help repair damaged tissue. Researchers are still exploring the functions of human MSCs in the body, but current knowledge about the stem cells suggests that they play an important role in cell repair, acting as a sort of “cellular paramedic.”

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Mesenchymal Stem Cells: The Cellular Paramedic^

MSCs can be thought of as “cellular paramedics,” helping to restore damaged cells and tissue. As mentioned before, MSCs are able to secrete substances like cytokines and growth factors that can promote tissue repair. MSCs have even been shown to transfer products as large as mitochondria to damaged cells that need help. Specifically, MSCs stimulate angiogenesis, the process of new blood vessel formation, which has been linked to neurogenesis, the process by which new nerve cells are produced. The factors secreted by MSCs also reduce the harmful effects of oxidative damage and apoptosis.

When researchers discovered the “paramedic” quality of MSCs, they conducted several experiments to see how MSCs could potentially be used to help treat human diseases. In one experiment, MSCs obtained from humans were injected into mice that had some type of tissue damage and did not have a functional immune system. The MSCs were labeled so that scientists could track where they migrated after being injected into the mice. The researchers observed that the cells migrated throughout the damaged tissues apparently evenly and continued to be present in the tissue for a substantial period of time. The continued presence of MSCs is important to therapeutic development because it indicates that potential positive long-term effects of a treatment might be capable of persisting.

In additional experiments, scientists found that MSCs function differently in chronic disease models than in more temporary conditions like injury and trauma. In mouse models of acute injury, injected MSCs responded by helping to repair the tissue but were not present in the tissue for a substantial period of time, as they were in mouse models of chronic disease. For more information on mouse models, click here. When the experiment was repeated in injured mice without functional immune systems, the MSCs were again only temporarily present in the tissue. This suggests that temporary presence of MSCs is not a result of the host immune system. It is important to establish what types of environment foster the sustained presence of MSCs in the tissue so that clinicians can increase the effectiveness of future treatments using MSCs.

Research Using Mesenchymal Stem Cells^

The unique ability of MSCs to secrete their own growth factors enables scientists to culture them in a laboratory with relatively little maintenance. Furthermore, MSCs multiply rapidly in cell cultures. As a result, compared to other cells, it is easier to grow many MSCs after obtaining a limited number of the cells from a patient. Cells that are grown in vitro often develop different characteristics or stop multiplying after a period of time. However, MSCs have been found to maintain their characteristics and the ability to multiply even after many cycles of replication. Most other cells require expensive cytokines and growth factors to grow in vitro, so the low maintenance of MSCs increases their appeal as a source of stem cells to investigate potential treatments.

In the body, MSCs are found in the bone marrow, umbilical cord tissue, and fat pads.  MSCs are relatively rare in the bone marrow, comprising only 1 out of every 10,000 cells. On the other hand, umbilical cord blood and fat are rich sources of MSCs. Human MSCs can be harvested with minimal patient discomfort by tapping into an individual’s marrow space or fat pads.

Unlike most other cells, MSCs can be transferred between organisms with little immune rejection, in which the immune system of the organism receiving the transplant attacks the foreign tissue being transplanted. Scientists have found that MSCs suppress the immune system and reduce inflammation, making them good candidates for transplantation or injection into a host because they can avoid rejection by the host’s immune system.

The ease with which MSCs can be obtained, cultured, and transferred into a host without immune rejection is one reason why researchers are hopeful that MSCs may offer a promising way for scientists to develop treatments for neurodegeneration.

MSCs and the Brain^

In the brain, MSCs can help repair neurodegeneration by providing neurotrophic factors, proteins in the nervous system that promote the growth of nerve cells. For example, detailed experiments have shown that human MSCs express the neurotrophic factor BDNF (brain-derived neurotrophic factor) but do not express certain types of neurotrophins. You can read more about BDNF by visiting the HOPES article here. Interestingly, MSCs still exhibited their “cellular paramedic” effects when BDNF activity is blocked by an antibody, suggesting that MSCs secrete factors other than BDNF that help with cellular growth in the brain.  The effects of these factors allow nerve cells to carry out several processes that support survival: axon extension, growth, and cell attachment. In essence, MSCs change the tissue environment to enhance cell growth and regeneration in the brain.

Experiments done on mice with nerve cell injury have shown that MSCs injected into the brain promote recovery by secreting neurotrophic factors that facilitate nerve cell survival and regeneration. More relevant to HD are animal experiments showing that MSCs have the potential to repair striatal degeneration.

Bantubungi et al conducted experiments using rat MSCs to help treat rat models of HD with parts of their striatum removed. The research showed that MSCs proliferated more rapidly in the rat brains with striatal lesions than in healthy rat brains, suggesting that MSCs selectively respond to areas needing repair.  Furthermore, the scientists identified a protein called stem cell factor that encouraged proliferation and directed migration of the MSCs to damaged tissue. Stem cell factor is a naturally occurring protein that plays an important role in communication between cells. The experiment by the scientists suggests that MSCs do in fact play an important role in the brain and have potential to become a cellular therapy for neural repair.

Amin et al. conducted an experiment in which rat models of Huntington’s disease with damage to one side of the brain responded positively to MSC implantation into the brain. Specifically, damage within the striatum, the region of the brain drastically affected in HD, was significantly reduced in rats that received MSC implantations.

It is important to note that although MSCs promote cell growth and repair in the brain, scientists have not yet confirmed that MSCs can become mature nerve cells with the ability to signal, or communicate with, other nerve cells. Other types of stem cells, such as neural stem cells have been found to generate mature nerve cells. MSCs may not be able to become mature nerve cells themselves.

Genetically Engineered MSCs^

In addition to exploiting the natural ability of MSCs to help repair damaged nerve cells, scientists have found ways to genetically engineer MSCs to enhance their reparative properties in the brain. Scientists can introduce genes into MSCs that cause them to produce a greater quantity of factors such as cytokines and neurotrophins. Even after genetically engineered MSCs are allowed to multiply through several generations, they retain these genetic characteristics that boost production of helpful factors.  Furthermore, MSCs have proven to be robust cells: genetic engineering does not hinder the cells’ ability to multiply or grow. It is important however to continue these types of studies to ensure there are no unintended side effects of enhanced neutrophin and cytokine productions in MSCs or other cells and tissues in the body.

An experiment conducted by Dey et al. showed that mouse models of HD responded positively to treatment by MSCs. When the MSCs were genetically engineered to produce greater quantities of BDNF, the delay in disease progression was even more drastic in the mice.

The potential to genetically engineer MSCs to deliver factors such as BDNF is important because directly injecting some of these factors is not effective. Transplanted MSCs, as indicated in the studies mentioned above, have been shown to disperse throughout damaged tissue for a sustained period of time. The characteristics of the compounds themselves often prevent them from having a sustained physiological effect on their own. Therefore, the genetically engineered MSCs serve as a vehicle to enable effective delivery of helpful factors into the brain.

Another exciting possibility is to have MSCs themselves become vehicles for delivering genetic material that can help with diseases like HD. Dr. Jan Nolta’s research group at University of California Davis, for example, hopes to have MSCs deliver molecules for RNA interference, a type of gene therapy, into the cells of HD patients. You can read more about RNAi in the HOPES article here. This area of research is still in its preliminary stages and may take several years to obtain approval from government agencies such as the Food and Drug Administration (FDA). Nevertheless, it holds promise as a potential future treatment for HD.

A Potential Treatment for HD Using MSCs^

Future cellular therapies using MSCs would involve delivering MSCs into the brain, which has been approached in a number of different ways. Scientists have proposed delivering MSCs through an injection directly into the brain, an injection into the space surrounding the spinal cord, or a route through the nose (e.g. a nasal spray).

Although clinical trials using MSCs in humans have not yet been approved in the United States, one human cellular therapy trial has been conducted in France. In the trial, neural stem cells rather than MSCs were used. Five patients with HD received transplants from human fetal neural stem cells. After two years, three out of the five patients demonstrated motor and cognitive improvements. While this experiment provides hopeful evidence that stem cell therapies may provide a treatment for HD, the results should be interpreted with caution. First, two of the patients did not show significant improvements. Second, as noted before, neural stem cells and MSCs have different characteristics. Therefore, the results from this experiment do not indicate whether MSCs would provide an effective treatment. Finally, after four to six years, the patients showed clinical decline once again, suggesting that additional research is required before an effective long-term treatment is developed.

In addition to showing that stem cells are in fact an effective treatment for HD, researchers must also show that implanting MSCs into the brain is a safe procedure before treatments can continue to be developed. One of the main concerns with MSCs is that they could cause abnormal cell growth. Abnormal growth could result in extra bone or tumor formation. In particular, MSCs have been found to migrate to areas in the body that contain tumors. This could be dangerous if the MSCs excrete factors that encourage angiogenesis, cell growth, and cell proliferation within the tumor. For safety reasons, proposed clinical trials for cellular therapies exclude anyone who has had a brain tumor or other cancer within the past 5 years. Before treatment, an MRI will be administered to ensure the absence of any brain tumors.

Extensive biological safety trials have been conducted with MSCs by Dr. Gerhard Bauer and Dr. Jan Nolta at University of California at Davis. They have performed numerous experiments over the past decade on different animal models including mice, rats, and primates, to test if MSCs can be safely injected or grafted without tumorous growths.  Additionally, a successful clinical trial in France with five HD patients suggests that transplantation of stem cells into the brain can be done without negative health consequences. However, more evidence for the biological safety of injecting MSCs into the brain is needed to meet the rigorous safety standards of the FDA in the United States

MSCs have potential to be a safe and effective therapy for HD. While there is promising evidence from animal research that MSCs can slow neurodegeneration, specifically in the striatum, there are still many aspects of the potential therapy that require additional experimentation.

Further reading:^

1.     Aggarwal, S. and M. F. Pittenger (2005). “Human mesenchymal stem cells modulate allogeneic immune cell responses.” Blood 105(4): 1815-1822.

A technical paper that discusses how MSCs interact with the immune system.

2.     Aizman, I., C. C. Tate, et al. (2009). “Extracellular matrix produced by bone marrow stromal cells and by their derivative, SB623 cells, supports neural cell growth.” J Neurosci Res 87(14): 3198-3206.

A technical paper that talks about the various compounds secreted by MSCs

3.     Amin, E. M., B. A. Reza, et al. (2008). “Microanatomical evidences for potential of mesenchymal stem cells in amelioration of striatal degeneration.” Neurol Res 30(10): 1086-1090.

This paper discusses how MSCs might help counter nerve cell damage in the striatum, and is difficult

4.     Bachoud-Levi, A. C., V. Gaura, et al. (2006). “Effect of fetal neural transplants in patients with Huntington’s disease 6 years after surgery: a long-term follow-up study.” Lancet Neurol 5(4): 303-309.

This is the study in which human fetal neural stem cells were transplanted into HD patients

5.     Bantubungi, K., D. Blum, et al. (2008). “Stem cell factor and mesenchymal and neural stem cell transplantation in a rat model of Huntington’s disease.” Mol Cell Neurosci 37(3): 454-470.

A technical paper that discusses transplantation of MSCs in rats

6.     Crigler, L., R. C. Robey, et al. (2006). “Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis.”

This paper discusses some of the compounds that MSCs secrete that play a role in nerve cell health, and is quite difficult

7.     Danielyan, L., R. Schafer, et al. (2009). “Intranasal delivery of cells to the brain.” Eur J Cell Biol 88(6): 315-324.

This paper discusses one of the several ways MSCs might be delivered through the brain.

8.     Dey, N. D., M. C. Bombard, et al. (2010). “Genetically engineered mesenchymal stem cells reduce behavioral deficits in the YAC 128 mouse model of Huntington’s disease.” Behav Brain Res 214(2): 193-200.

This technical paper discusses how MSCs helped behavior problems in a mouse model of HD.

9.     Joyce, N., G. Annett, et al. (2010). “Mesenchymal stem cells for the treatment of neurodegenerative disease.” Regen Med 5(6): 933-946.

This paper discusses potential applications of MSCs in medicine, and is of medium difficulty

10.  Meyerrose, T. E., M. Roberts, et al. (2008). “Lentiviral-transduced human mesenchymal stem cells persistently express therapeutic levels of enzyme in a xenotransplantation model of human disease.” Stem Cells 26(7): 1713-1722.

This technical paper discusses how MSCs migrate through the nervous system when introduced into a mouse’s brain

11.  Spees, J. L., S. D. Olson, et al. (2006). “Mitochondrial transfer between cells can rescue aerobic respiration.” Proc Natl Acad Sci U S A 103(5): 1283-1288.

This technical paper describes how transfer of mitochondria between cells can help the cell that receives the mitochonfrion

12.  Wineman, J., K. Moore, et al. (1996). “Functional heterogeneity of the hematopoietic microenvironment: rare stromal elements maintain long-term repopulating stem cells.” Blood 87(10): 4082-4090.

This technical paper discusses the conditions needed to grow MSCs

T. Wang, 7-25-11

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Dietary Restriction

stress

essential

Judging from common phrases like “breakfast is the most important meal of the day”, “people should eat whenever they are hungry”, and the recently popular “eat five small meals per day instead of three large ones,” public opinion seems to run against going hungry even for short periods of time. However, current research suggests that moderate hunger may actually be healthy. Scientists studying rats and mice on so-called “dietary restriction” have found that these rodents rank significantly better on various measures of health than their counterparts who are fed a more abundant diet.

In addition to studying dietary restriction (DR) in normal rats, scientists have also studied DR in “rodent models” for various neurodegenerative diseases, such as Huntington’s disease (HD), Alzheimer’s disease, and Parkinson’s disease. (In rodent models, researchers either mimic the effects of a given disease via chemicals, or use genetics to actually breed the rodents to manifest an animal form of the disorder.) Here, too, research has demonstrated beneficial effects of dietary restriction: rodent models fed on a dietary restriction system show significant relief from various disease symptoms in comparison to normally fed rodent models. Although more research is needed into the effects of DR on humans, this research suggests the possibility that “cutting back” slightly on traditional daily eating routines could be a benefit to one’s health, especially for people with certain neurodegenerative diseases including HD.

What is meant by dietary restriction? Why is it helpful in rodents? What kinds of things might it do for a person who has HD? This chapter seeks to answer these and other questions, discussing a wide array of topics surrounding the issue of dietary restriction.

The Two Different Types of Dietary Restriction^

The rodents in dietary restriction (DR) studies may be subjected to one of the following dietary restriction schemes: intermittent fasting or daily caloric reduction. (Despite the differences between these two types of dietary restriction, it is important to note from the start that they both maintain a steady intake of vitamins and minerals.) In daily caloric reduction, rodents are fed on a normal, daily schedule, but are fed approximately 50-70% of the calories in the normal lab diet of that species of rodent. In intermittent fasting, rodents are typically placed on an alternating schedule: one day they will fast, then the next day they will be fed. The net number of calories varies, though, for rodents on intermittent fasting. A few rodents will eat a “double meal” on the day they are fed, thus bringing a two-day total (one day fasting, one day eating) to 100% of their normal caloric intake. However, most of the time when intermittent fasting rodents are fed, they will eat roughly what a normal lab rodent eats when fed every day—thus bringing a two-day total to somewhere around 50% of the normal caloric intake. So, the final outcome is similar by either DR scheme: whether through daily caloric reduction or intermittent fasting, mice and rats generally end up with a net reduction in their daily caloric intake.

Interestingly, both forms of DR have been shown to have positive health benefits for rodents. For instance, one study showed that rodents in either DR scheme exhibited “anti-aging” changes (these changes included reduced body temperature and decreased blood glucose and insulin levels). In addition, both types of DR have been shown to have positive effects on the brain (both were shown to combat oxidative stress on proteins and DNA in nerve cells) and to extend rodent life spans. While these results clearly indicate that both types of DR can be quite beneficial, one study suggests that intermittent fasting is more effective at increasing the expression of certain proteins that are very helpful to maintaining the health of nerve cells (these proteins will be described in more detail in the following sections).

How do these two different dietary restriction schemes in rodents correspond to human dietary restriction? Unfortunately, the exact answer to this question has not yet been determined. Some researchers suggest that having a daily intake of 1800-2200 calories (for a moderately active adult) can significantly reduce the risk of age-related neurological disorders like stroke, Alzheimer’s disease, and Parkinson’s disease. (Note: Because HD is passed on in the genes, dietary restriction does not reduce the risk of being genetically predisposed to HD. However, as the rest of this chapter will discuss, research suggests that DR may combat the HD disease process and thus potentially slow down symptom progression.) Other researchers shy away from suggesting an actual number of daily calories, but rather encourage a “low” daily calorie intake (meaning enough calories to fuel all of the functions of the body’s cells, but little or no excess). Still other researchers suggest that foregoing one or two meals per day may be an alternative to reducing the size of each meal. Thus, the lack of a definitive answer to how the rodent research applies to humans should serve as an important disclaimer: one should eat sensibly and not do anything rash like following the rodent studies directly (for instance, researchers warn against the idea of fasting every other day, as was done in some of the rodent trials).

An Evolutionary Explanation for Why Dietary Restriction is Effective^

Despite the fact that it runs counter to some popular beliefs, the concept behind dietary restriction is quite logical. Over the course of human history, access to food was anything but constant, owing to seasonal changes, drought, climate change, migration, etc. Because of a frequent lack of food, the body’s cells evolved mechanisms to cope with the stress that resulted from not having a steady abundance of energy. In fact, over thousands of years, this inconsistent diet actually shaped cellular metabolism, tailoring it to break down food in a way that accounted for the very long time lag between meals. Nowadays, when searching for food is as simple as walking to the grocery store, our frequent and relatively steady intake of food may actually overwhelm our cellular metabolism. The result is that mitochondria, the energy factories of our cells, may begin to produce harmful by-products like free radicals, which can contribute to the degradation and destruction of our cells over time (this can happen in the cells of any person, not simply those with neurological diseases). Thus, one way to think about the link between dietary restriction and healthy cells is that DR allows our cells to metabolize food in manageable amounts, in a manner that is in line with how evolution has programmed the cells’ metabolic machinery to function.

How HD Kills Nerve Cells and How Dietary Restriction Combats It^

Although scientists are uncertain about the exact cause of nerve cell death in HD, they believe that four different harmful phenomena are involved. These phenomena – impaired energy metabolism, oxidative stress, excitotoxicity, and apoptosis – are each believed to be somehow evoked by processes that the mutant huntingtin protein sets in motion. (For background information on the Huntington gene and huntingtin protein, click here.) The exciting news about dietary restriction is that it was shown in rodent studies to be capable of combating all four phenomena, increasing the health and the life span of these nerve cells (and in turn having beneficial effects on disease symptoms, which will be discussed in a later section).

The disease-fighting effects of dietary restriction appear to be the result of its ability to mildly stress the body’s cells. This manipulation encourages cells to produce two special types of proteins that help cells cope with stress: neurotrophic factors and protein chaperones (for more on these proteins, click here and here, respectively). More specifically, brain-derived neurotrophic factor (BDNF) and two protein chaperones known as HSP-70 and GRP-78 appear to play a very large role in opposing the effects of HD on nerve cells. In order to understand how dietary restriction promotes the health of nerve cells, let’s look at each of the four phenomena involved in the degeneration of nerve cells in HD and ask, How do neurotrophic factors and protein chaperones combat their effects?

Impaired Energy Metabolism^

When thinking about cells and metabolism, the first word that comes to mind is mitochondria (singular: mitochondrion), the energy factories of cells. Mitochondria are responsible for using food to make the majority of our cells’ ATP, which is the chemical fuel that our cells’ proteins use to perform their life-sustaining functions. In addition, mitochondria play a key role in maintaining a certain balance of calcium in cells, which is critical to cell survival. Through an unknown mechanism, huntingtin proteins in the nerve cells of people who have HD cause a great deal of problems for mitochondria, interfering with their ability to produce adequate energy and maintain normal calcium levels. (In addition, mitochondria produce dangerous numbers of free radicals, which will be discussed under “Oxidative Stress” below). It is believed that these effects on the cell may play a strong role in the onset of HD, as well as the disease’s progression.

In rodent models of neurodegenerative diseases, the helpful neurotrophic factor BDNF has the ability to increase nerve cells’ resistance to the stress that chemicals put on mitochondria. Two protein chaperones called HSP-70 and GRP-78 are also helpful: they (as well as BDNF) have been shown to stabilize cellular calcium levels. The levels of BDNF, HSP-70, and GRP-78 are all increased in the cells of those rodents in the dietary restriction regimen (in comparison to the normally fed rodents). Thus, these proteins appear to explain why dietary restriction preserves the function of mitochondria. Furthermore, normal mitochondrial function leads to the maintenance of relatively normal levels of ATP production and a safe balance of calcium in cells (as well as reducing the generation of harmful free radicals). This makes cells a lot more resistant to the degeneration that is caused by HD.

Oxidative stress^

In addition to the problems with ATP production and calcium levels that arise when mitochondria are harmed in HD, another major problem arises as well: excessive production of free radicals. It is actually normal for mitochondria to produce some small number of free radicals. However, in HD, the production of free radicals is significantly increased. Over time, this increase of free radicals leads to oxidative stress, which is very harmful to cells and plays a strong role in nerve cell degeneration.

In rodent models of neurodegenerative disorders, where the rodents are given chemicals to elicit oxidative stress in cells, BDNF and HSP-70 are able to increase nerve cell resistance to such stress. It is believed that these proteins induce the production of antioxidant enzymes in order to combat the oxidative stress. Since dietary restriction increases the levels of these proteins, it is not surprising that the number of free radicals in the cells of dietary restriction rats was significantly less than that of normally fed rats. Through the stimulation of BDNF and HSP-70 (and likely other proteins that play a part), dietary restriction suppresses free radical production by a significant amount, thus reducing oxidative stress and making cells much healthier.

Excitotoxicity^

Nerve cells communicate with one another using certain chemicals called neurotransmitters. Typically, the message that is communicated between “sender nerve cell” and “receiver nerve cell” is encoded by two different things: the type of neurotransmitter that is released and how much of this neurotransmitter is released (another aspect of communication – the type of receptors on the receiver neuron – is also involved, but this topic is beyond the scope of this discussion). When a sender nerve cell overstimulates a receiver nerve cell by consistently sending too much neurotransmitter, the receiver nerve cell becomes damaged and may ultimately die. This overstimulation resulting in nerve cell harm is known as excitotoxicity. Scientists believe that excitotoxicity may take place due to certain events: after a stroke, after trauma to the central nervous system (for instance, after a car accident), or during the course of a neurodegenerative disease like HD.

With regard to neurodegenerative diseases, two very important compounds can influence a nerve cell’s vulnerability to excitotoxicity: glutamate and glucose. Glucose is a sugar that is involved in many cellular processes, one of which is to allow nerve cells to adequately send and receive glutamate. Glutamate is a neurotransmitter and is typically the culprit in excitotoxicity. Thus, efficient transport of these two compounds across cell membranes is essential to not only making nerve cell communication effective, but also to protecting nerve cells against excitotoxicity. Indeed, in rodent models of neurodegenerative diseases, chemicals that impaired glucose and glutamate uptake by nerve cells were believed to make the cells more vulnerable to excitotoxicity and degeneration.

Dietary restriction is beneficial in combating this method of cellular degradation because it decreases the impact of the chemicals that impair glucose and glutamate uptake. The result of this action is that levels of glucose and glutamate transport remain closer to normal, resulting in a decreased vulnerability to excitotoxicity. In addition to their other beneficial effects in cells, BDNF and HSP-70 appear again to be the heroes in protecting nerve cells against excitotoxicity (although it is quite possible that other proteins induced by dietary restriction also play a role in protecting against this phenomenon).

Apoptosis^

Apoptosis means “programmed cell death;” it is a mechanism by which a cell can lead to its own destruction. In the development of human brain (and the brains of other animals), apoptosis is frequently used to get rid of nerve cells that are unhealthy or improperly placed and allow for better-suited nerve cells to thrive. In this regard, apoptosis is a very natural event. However, the huntingtin protein in people who have HD can start a cascade of events that leads to apoptosis in otherwise perfectly good cells. Thus, in HD, apoptosis is not only one of the mechanisms by which the disease can degrade nerve cells, but it is often the thing that ultimately kills nerve cells (with impaired energy metabolism, oxidative stress, and excitotoxicity being contributors to a cell’s vulnerability to apoptosis).

As indicated by the fact that they are much more prevalent in nerve cells of mice with HD than mice without the disease, one of the key components in the apoptosis cascade in HD cells appears to be a group of proteins known as caspases. This is where dietary restriction comes into play: in nerve cells from HD mice fed normally, there was a significantly greater amount of caspase-1 (one of the caspase proteins) than in nerve cells from HD mice fed according to the dietary restriction regimen. BDNF, produced in large quantities in rodents on dietary restriction, induces the expression of certain “anti-apoptotic” proteins (specifically, Bcl-2 proteins), which most likely accounts for these effects. Thus, dietary restriction’s ability to interfere with the programmed cell death of nerve cells allows these nerve cells to stay alive and perform their physiological functions for a longer period of time. (For more on caspases, click here.)

How Dietary Restriction Reduces Huntingtin Protein Aggregation^

Although impaired energy metabolism, oxidative stress, excitotoxicity, and apoptosis are the phenomena that actually degrade nerve cells, the more fundamental issue in nerve cells of people with HD is huntingtin protein aggregation. Despite the fact that recent research has dispelled the idea that mutant huntingtin aggregates directly cause nerve cell death, scientists believe that they may have potentially critical indirect effects on the disease process. For instance, researchers have found a correlation between increased aggregation of huntingtin and nerve cell death via apoptosis (click here for more information on correlation and causation). Thus, the neuronal inclusions composed of huntingtin proteins (and other proteins that huntingtin captures) are a truly important aspect of HD. This is why, in addition to its other tremendous benefits, another beneficial effect of dietary restriction is its ability to decrease the number of neuronal inclusions in nerve cells.

The way that dietary restriction reduces neuronal inclusions may be through the initiation of a process called autophagy, which captures the huntingtin protein aggregates and disposes of them. But DR’s initiation of autophagy is somewhat complex. As mentioned previously in the section about excitotoxicity, glucose is a very important compound and its transport into cells of people with HD is somehow hindered. (In fact, because of this difficulty of moving glucose from the blood into cells, many people with HD suffer from a condition called hyperglycemia, which means that their blood sugar is too high.) Dietary restriction (through the induction of BDNF and HSP-70) improves the transport of glucose into cells. The presence of higher amounts of glucose in cells has been shown to decrease the activity of a protein called mTOR, which is a negative regulator of autophagy. Thus, by decreasing mTOR’s activity, the cell’s high glucose concentrations effectively “release the brake” on autophagy, allowing it to do its job of clearing huntingtin aggregates from the cell. This process is summarized in the schematic below:

DR -> BDNF + HSP-70 -> improved glucose transport -> less mTOR activity -> more autophagy -> more huntingtin aggregates cleared from cell

(Interestingly, in addition to this long process of reducing neuronal inclusions, a much shorter mechanism accomplishes the same task: HSP-70, which is produced in large quantities in dietary restriction, has been shown to directly interact with the mutant huntingtin protein and thus reduce the number of aggregates in cells.)

The reduction of aggregates is positively correlated with reducing cell death. Clearly, this is a very positive result for individuals with HD because the more nerve cells that survive, the better.

The Effects of Dietary Restriction on the Onset and Progression of HD^

As explained in the previous two sections, dietary restriction has a wonderful ability to combat nerve cell degeneration in rodent models of HD. Since the disease is characterized by its ability to kill nerve cells (and thus lead to the recognizable symptoms of the disorder), it is quite logical to expect that by combating nerve cell degeneration, dietary restriction would delay the onset of disease symptoms and slow the progression of HD. Indeed, this has been the case in rodent models of the disease. In a study of mice with HD, at age 8 weeks these mice were split into two groups, one that was fed according to a dietary restriction regimen and the other that was fed normally. In comparison to normally fed mice, the onset of behavioral symptoms of HD in the dietary restriction mice was delayed by an average of 12 days. With regard to survival times, by age 21 weeks, all of the normally fed mice had died, while only 40% of the dietary restriction mice had died. On average, the dietary restriction regimen increased survival time by about 2 weeks. Considering the short life of mice (which is approximately 2-3 years for normal mice, and even shorter for mice with HD), 12 days and 2 weeks are relatively long lengths of time, which indicates that dietary restriction has a profound effect on delaying symptom onset and increasing the survival time of those individuals with HD.

The Effects of Dietary Restriction on the Symptoms of HD^

In addition to increasing the survival time of individuals with HD and delaying the symptoms of the disease, dietary restriction also has been shown to significantly reduce the severity of HD symptoms. In order to understand how dietary restriction accomplishes this task, let us discuss the disease symptoms in the context of the parts of the body with which they are associated.

Not every nerve cell in the body is affected by HD; only those nerve cells in the basal ganglia (especially the striatum) and the cerebral cortex are degenerated by the disease. The basal ganglia play a strong role in the brain’s regulation of motion (for more information about the neurobiology behind HD, click here). Or, if you’d like to see a general overview of the brain and its parts, click here). Thus, since nerve cells in the basal ganglia of dietary restriction rodents showed increased resistance to degeneration (due to the processes described in section 3), we can now understand why these rodents showed significant improvements in their motor performance in comparison to normally fed rodents. Indeed, by promoting the health and survival of nerve cells in the basal ganglia, dietary restriction allows these cells to perform their normal duties, which results in relatively strong motor performance despite having HD.

The cerebral cortex also plays a role in regulating the body’s movements, often working with the basal ganglia to perform this task. In addition, the cerebral cortex is involved in many other functions, such as cognitive tasks (like learning and memory) and emotional tasks. In HD mice, brain atrophy (the wasting away of nerve cells after they die) results in the thinning of the cerebral cortex. However, one study showed that the atrophy in dietary restriction rats was much less significant in comparison to normally fed rats, a fact that is explained by dietary restriction’s ability to combat the disease process and keep nerve cells alive. Although the HD researchers performing this study did not make a direct link between the reduced brain atrophy and the cognitive and emotional symptoms of HD, some other research on aging suggests that this reduced atrophy may help HD symptoms as well. These aging studies indicate that dietary restriction rodents perform better than normally fed rodents on learning and memory tasks as they age (the aging process is thought to partially damage nerve cells, albeit to a far less degree than neurodegenerative diseases do). Thus, although learning and memory alterations are not specifically linked to HD, it is possible that other cognitive (and perhaps even emotional) tasks that are related to HD may also be improved by dietary restriction. (However, regardless of whether or not this turns out to be true, surely everyone would welcome improvements in learning and memory!)

Aside from neurological symptoms of HD, one ailment that people with HD often have is hyperglycemia, a dangerous condition involving high blood sugar. By helping to improve the transport of glucose in cells (which is described above in the section on “Excitotoxicity”), dietary restriction helps to normalize blood sugar levels and take away the hyperglycemia.

Finally, the last non-neurological symptom of HD that we will discuss is weight loss. Since we typically associate eating more calories with gaining more weight, one study of HD mice produced a seemingly paradoxical result: mice who practiced dietary restriction actually lost less weight than those who were fed normally! However, if one looks at the typical story of humans who have HD, these results are not so perplexing after all: despite eating more and more calories each day, people with HD often find that they still cannot combat their progressive weight loss. Thus, perhaps the weight loss in HD is not an issue of calories, but instead an issue of tissue “wasting” (that is to say, large amounts of cell death). As illustrated in section 3, dietary restriction is able to combat the disease process, thus allowing cells to be healthy and survive for a far greater amount of time. The more tissue that remains healthy, the less weight will be lost. Thus, one can solve the problem of HD-induced weight loss by actually eating less instead of more.

The Use of Drug Therapy in Place of Dietary Restriction^

In some of the rodent studies mentioned in the sections above, a substance called 2-deoxy-D-glucose (abbreviated “2-DG” or “2DOG”) was shown to produce levels of HSP-70 and GPR-78 that were similar to those produced by dietary restriction. In addition, 2-DG was shown to be even more effective at inducing autophagy and decreasing huntingtin protein aggregation than a high level of glucose in cells. These results indicate that the helpful effects of dietary restriction on cells may also be obtainable through the use of a dietary supplement like 2-DG. However, scientists have not yet determined whether or not long-term use of 2-DG might lead to harmful side effects. More research is necessary to determine whether or not the use of a supplement like 2-DG for an extended period of time is safe.

Important Cautionary Note^

The rodent research on dietary restriction has produced encouraging results for combating Huntington’s disease. However, as mentioned in the first section, exactly how the rodent research translates into dietary restriction recommendations for people is not yet clear. Thus, rather than starting a dietary restriction regimen on one’s own, we at HOPES urge people to first consult their physician to see if dietary restriction might be safe and effective for them.

Seeing as weight loss is a dangerous symptom of HD, dietary restriction is unlikely to be a helpful strategy for patients with the disease.

For further reading^

  1. Duan W, Guo Z, Jiang H, Ware M, Li XJ, Mattson MP. Dietary restriction normalizes glucose metabolism and BDNF levels, slows disease progression, and increases survival in huntingtin mutant mice. Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2911-6. Epub 2003 Feb 14. PMID: 12589027 [PubMed - indexed for MEDLINE]
    A technical paper that illustrates the many beneficial effects of dietary on combatting the symptoms of HD. Covers the topic from many levels, including cell biology, neuroanatomy, and physiology.
  2. Guo Z, Ersoz A, Butterfield DA, Mattson MP. Beneficial effects of dietary restriction on cerebral cortical synaptic terminals: preservation of glucose and glutamate transport and mitochondrial function after exposure to amyloid beta-peptide, iron, and 3-nitropropionic acid. J Neurochem. 2000 Jul;75(1):314-20. PMID: 10854276 [PubMed - indexed for MEDLINE]
    A technical paper about the effects of HSP-70 and GRP-78 on combating neurodegeneration in HD (as well as Parkinson’s disease and Alzheimer’s disease).
  3. Mattson MP, Duan W, Guo Z. Meal size and frequency affect neuronal plasticity and vulnerability to disease: cellular and molecular mechanisms. J Neurochem. 2003 Feb;84(3):417-31. Review. PMID: 12558961 [PubMed - indexed for MEDLINE]
    A technical paper that provides an overview of dietary restriction’s ability to induce the production of proteins like neurotrophic factors and protein chaperones. Also discusses how these proteins combat neurodegeneration.
  4. Ravikumar B, Stewart A, Kita H, Kato K, Duden R, Rubinsztein DC. Raised intracellular glucose concentrations reduce aggregation and cell death caused by mutant huntingtin exon 1 by decreasing mTOR phosphorylation and inducing autophagy. Hum Mol Genet. 2003 May 1;12(9):985-94. PMID: 12700167 [PubMed - indexed for MEDLINE]
    A technical paper that shows how an increase in cellular glucose concentration (or the administration of 2-DG) reduces huntingtin protein aggregation in cells.

-M. Stenerson, 1/6/04

More

Stem Cells

In the last few years, stem cell research has become the latest buzz in the popular media as well as the scientific world. It was the subject of President George W. Bush’s first prime-time television address. It is continuously on the cover of popular news magazines. So what is all the fuss about?

Stem cells hold the potential to treat or even cure many of the diseases that continue to mystify scientists today, such as Parkinson’s Disease, Alzheimer’s Disease, diabetes, and Huntington’s Disease (HD). However, stem cell research is controversial, as most of the stem cell lines available today are derived from embryos or fetuses.

basic ups

The following chapter aims to explain the science behind stem cells and their potential to treat HD.

Stem Cell Basics^

What is a stem cell?^

Most of the cells that make up the organs and tissues of the body are highly specialized for their specific jobs. The red blood cell, for example, is specifically crafted to carry oxygen from the lungs to the tissues. A comparison can be made with today’s society where most workers are trained to perform a specific trade. The days of the generic fix-it man are gone; instead, the electrician, the plumber, and the cable guy fill specific niches.

Likewise, in the human body, most cells are specialized for certain jobs. In fact, most cells lead very standard lives – they grow up, do the same job every day, and then eventually retire and pass away. These cells, such as nerve cells or skin cells, are called specialized cells. They are mature cells that have characteristic shapes and are committed to performing specific functions (See Figure 1). Once these cells have matured, they are usually incapable of reproducing themselves. They essentially remain “childless” for their whole lives.

If mature specialized cells cannot leave “children” behind when they die, how does your body make new cells? For example, when you cut your skin, how do you grow new skin cells? When you get blood drawn, how do you make new blood cells?

It turns out that stem cells solve this unique problem. A stem cell can reproduce itself over and over again (a special trick known as “self-renewal” or “self-replication”). With every replication, the stem cell produces one new stem cell and one new specialized cell. Stem cells can often give rise to a number of different cell types. For example, blood stem cells can produce both red blood cells and white blood cells. In this way, stem cells are not committed to produce a single cell type. Instead, a stem cell remains uncommitted until it receives a specific signal to divide and produce one of the various specialized cells.

In more formal terms, a stem cell is a special kind of cell that has the ability to divide for indefinite periods of time and to give rise to the mature, specialized cells that make up an organism. A stem cell is uncommitted and remains uncommitted until it receives a signal to differentiate (become a specialized cell). (See Figure 2).

What are the different kinds of stem cells?^

There are three main types of stem cells under scientific study today:

  • Embryonic stem (ES) cells: ES cells are taken from the very early stages of embryo development and can give rise to all of the cells of the human body, except the placenta and other supportive tissues in the womb.
  • Embryonic germ (EG) cells: EG cells are taken from the later stages of embryo development and are slightly less “powerful” in their ability to divide.
  • Adult stem cells: Adult stem cells are found in the tissues of a fully developed child or adult and can only produce a limited number of cell types.

These three types of stem cells are easiest to understand in a discussion of human development. Human development begins when a sperm fertilizes an egg and creates a single cell, known as a zygote, which has the potential to form an entire organism. This single cell is said to be totipotent, meaning it has the “total” potential to give rise to all types of cells. About 24 hours after fertilization, the zygote divides into two identical totipotent cells, and is now known as an embryo. About five days after fertilization and after several cycles of cell division, these cells begin to specialize and form a hollow sphere, called a blastocyst. The blastocyst has an outer layer of cells that make up the shape of a sphere and a cluster of cells, known as the inner cell mass, inside the sphere. The outer layer of cells will eventually form the placenta. The inner cell mass will eventually form all the tissues of the human body. The inner cell mass cannot form an organism on its own, however, because it is unable to produce the placenta and the other supporting tissues necessary for development in a woman’s uterus. Therefore, the inner cell mass cells are said to be pluripotent, meaning they have the potential to give rise to most of the tissues required to produce an organism. In other words, they can give rise to all the cells of the human body, excluding the supportive tissues used in the womb. (See Figure 3).

Embryonic stem cells, which are also pluripotent, are isolated directly from the inner cell mass at this blastocyst stage. In 1998, researchers first isolated ES cells from human embryos that were obtained from in vitro fertilization clinics. Although these embryos were originally intended for reproduction, they were in “excess” and were headed for the trash. Instead of being disposed, however, they were donated to research.

Five to 10 weeks after fertilization, the growing embryo, now called a fetus, develops a region known as the gonadal ridge. The gonadal ridge contains the primordial germ cells, which will eventually develop into eggs or sperm.

Embryonic germ cells are isolated from these primordial germ cells of the 5- to 10- week old fetus. Like ES cells, EG cells are also pluripotent.

As the human fetus continues to develop, pluripotent stem cells specialize into stem cells that are geared for specific tissues. For example, they become blood stem cells (which produce blood cells) or skin stem cells (which produce skin cells). These specialized stem cells are said to be multipotent, meaning they can give rise to many, but not all, types of cells.

While all three types of stem cells discussed above (ES cells, EG cells, and multipotent stem cells) are found in the developing human, only multipotent stem cells are found in children and adults. Therefore, multipotent stem cells are often referred to as adult stem cells. Unlike other stem cells, adult stem cells are only found in specialized tissues and can only give rise to the specialized cell types that make up that tissue. Currently, adult stem cells have been found in the bone marrow, blood, blood vessels, skeletal muscle, skin, lining of the digestive track, dental pulp of the tooth, liver, pancreas, cornea and retina of the eye, and brain.

Stem Cell Research^

What kind of research is being conducted?^

Stem cells are being investigated in various areas of scientific research. The most notable research areas are described below:

I. Basic Research
On the most fundamental level, stem cells are used to study the early events of human development. This research may one day explain the cause of birth defects and help devise new approaches to correct or prevent them. Also, research on the genes and chemicals that control human development may help researchers manipulate stem cells to become specialized for transplantation or genetic engineering.

II. Transplantation Research
Stem cells may hold the key to restoring many vital bodily functions by replacing cells lost in various devastating diseases. Many diseases and disorders, such as Huntington’s disease, disrupt specific cellular functions or destroy certain tissues in the body. The goal, therefore, is to coax stem cells to develop into the desired specialized cells, which can then be used as a renewable source of replacement cells or tissues. This process could possibly treat HD and other conditions such as Parkinson’s and Alzheimer’s diseases, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis.

III. Genetic Engineering Research
Stem cells could be used as a vehicle for delivering genes to specific tissues in the body. The goal is to add genes to stem cells that would then coax the stem cell to differentiate into a specific cell type or force the stem cell to produce a desired protein product. Currently, researchers are trying to use specialized cells derived from stem cells to target specific cancer cells and directly deliver treatments that could destroy them.

IV. Drug Testing and Toxin Screening
Currently, animal models are used to test drug safety and efficacy and to screen potential toxins. Animal models, however, cannot always predict the effects that a drug or toxin may have on human cells. Therefore, if human stem cells can be used to generate cells that are important for certain drug or toxin screenings, these cells may offer a safer, more reliable test by mimicking a more realistic human environment.

V. Chromosomal Abnormality Testing
Stem cells might also be used to explore the effects of chromosomal abnormalities in early human development. As a result, we might be able to understand and monitor the development of early childhood tumors, many of which are embryonic in origin.

What are the advantages and disadvantages of using embryonic stem cells, embryonic germ cells, and adult stem cells for research?^

At first glance, embryonic stem cells, embryonic germ cells, and adult stem cells all present similar possibilities for scientific research. They are all stem cells, after all, and therefore share some key characteristics and hold similar potential. For example, they all have the ability to self-replicate for indefinite periods of time in the human body and can give rise to specialized cells. The overall purpose behind research with all types of stemm cells, therefore, is very similar. It has also been shown that all three cell types can be isolated from other cells and kept in a specific laboratory environment that keeps them unspecialized. This is crucial for controlled scientific research. Upon experimentation, it has also been shown that all stem cell types will replicate and specialize when transplanted into an animal with a lowered immune system. The cells then undergo “homing,” a process where the transplanted cells are attracted to and travel to an injured site when transplanted into an animal that has been injured or diseased. Homing provides hope that the transplantation of stem cells will be a clinically useful procedure.

Despite these general similarities, there are some important differences between embryonic stem cells, embryonic germ cells, and adult stem cells. The origins of these three cell types define their differences: ES cells are derived from the inner cell mass of the blastocyst in a developing embryo, EG cells are obtained from the primordial germ cells of a fetus, and adult stem cells are found in developed, specialized tissues. The differences between ES, EG, and adult stem cells result in different advantages and disadvantages for each stem cell type in scientific research and development.

ES and EG cells have some clear advantages over adult stem cells concerning research and clinical usefulness. For example, ES and EG cells are pluripotent, meaning they have the potential to give rise to all types of cells in the body. Adult stem cells are multipotent, meaning they only have the potential to give rise to a limited number of cell types. So far, no adult stem cells have proven to be pluripotent. This means that ES and EG cells could potentially provide a renewable source of replacement cells for any tissue in the human body. Adult stem cells, however, would only be clinically useful for the specific adult tissue that the stem cells came from. ES and EG cells are also relatively abundant in the developing organism, especially compared to adult stem cells, which are scarce in the adult body. As a result, ES and EG cells are much easier to identify, isolate, and purify compared to adult stem cells, which are very difficult to identify, isolate and purify in the lab. This makes research with ES and EG cells all around easier than research with adult stem cells.

On the flip side, adult stem cells have some distinct advantages over ES and EG cells. For example, adult stem cells are around for an organism’s lifetime, while ES and EG cells are only found in the developing organism. This allows a longer time frame for adult stem cells to be studied in an individual. Also, removal of stem cells from an embryo will result in the death of the embryo. Removal of adult stem cells, however, does not involve the death of an embryo, and is therefore less ethically complicated. Furthermore, adult stem cells pose no chance of immune rejection after transplantation because they can be transplanted back into the adult that they came from. ES and EG cells are derived from embryos and fetuses, however, and are transplanted into people with different genetic make-ups. Therefore, rejection is an issue only with the use of ES and EG cells.

Finally, ES cells have a strong advantage and disadvantage over the other stem cell types. First, ES cells are able to replicate in the laboratory far better than either EG or adult stem cells. ES cells can self-renew for up to 2 years, doubling up to 300 times. EG cells can only double a maximum of 70-80 times. Meanwhile, adult stem cells only have a limited ability to replicate in the lab. Replication in the laboratory is critical for research to continue. On the other hand, ES cells are the most likely to develop into tumors. If undifferentiated ES cells are taken from the lab and injected into a mouse, a benign tumor can develop. For this reason, scientists do not plan to use undifferentiated ES cells for transplants or other therapeutic applications. EG cells do not form these tumors, however. At this point, it is not known whether tumors will form with transplanted adult stem cells.

The similarities and differences of ES, EG, and adult stem cells are summarized in the chart below:

Tbl Z-1: ES, EG, & Adult Stem Cells - Similarities

Tbl Z-2: ES, EG, & Adult Stem Cells - Differences I

Tbl Z-3: ES, EG, & Adult Stem Cells - Differences II

Which are more useful – pluripotent stem cells or adult stem cells?^

Based on what scientists currently know, it is unclear whether pluripotent or adult stem cells will be more useful for the development of therapies. As far as scientists can tell at this point, neither one is probably better than the other.

Both pluripotent and adult stem cells have their advantages and disadvantages (see chart below). For example, the main advantage of pluripotent stem cells is their ability to produce any specialized cell in the human body. However, because they are derived from human embryos or fetuses, they are also very controversial.

Adult stem cells, on the other hand, are unlikely to be rejected by a patient’s immune system because they can be isolated from a patient, coaxed to divide and specialize, and then transplanted back into the patient. Because stem cells are isolated from an adult, they are also unlikely to cause ethical concerns. However, adult stem cells have not been isolated for all tissues of the body, which limits the types of tissues they can be used for.

Recently, there has been research on adult stem cell plasticity, the ability of an adult stem cell from one tissue to generate specialized cells of another tissue. Thus far, there have been contradicting results. Time will tell whether or not adult stem cells can actually demonstrate plasticity. For more information on cell plasticity, click here.

Many scientists agree that pluripotent and adult stem cells might be better suited for different treatments

Tbl Z-4: Pluripotent vs. Multipotent Stem Cells

What challenges are researchers facing?^

While stem cell research shows great promise, researchers continue to face many biological, technological, and ethical challenges that must be overcome before innovations can be developed and incorporated into clinical practice.

First, more basic research must be done in order to fully understand the events that lead to cell specialization in humans. Currently, scientists are working to produce reliable, reproducible conditions that will direct stem cells to become the specific types of cells and tissues that are needed for transplantation.

Also, before mature cells derived from ES or EG stem cells can be used for transplantation, scientists must overcome the problem of immune rejection. Because these cells are genetically different from the recipient, their incompatibility must be minimized.

Adult stem cell research has also faced many difficulties, including finding, isolating and identifying the cells, growing the adult stem cells in the laboratory and demonstrating plasticity.

In addition to these technological challenges, researchers must also face the ethical controversy surrounding the use of ES and EG cells. If stem cells are used in clinical practice, researchers, doctors, and society at large must agree on acceptable ethical guidelines.

Stem Cell Research and Huntington’s Disease^

What is the potential for using stem cells to treat HD?^

Huntington’s Disease is a neurodegenerative disorder that is characterized by the death of nerve cells in the striatum. (To learn more about the neurobiology behind HD, click here.) Until recently, it was believed that neurons in the adult human brain and spinal cord could not regenerate. Once dead, the neurons were thought to be gone for good. In the mid-1990s, however, researchers discovered that stem cells in the adult brain could give rise to new neurons and neural support cells. With their ability to regenerate and produce new nerve cells, neural stem cells might be able to replace or repair the cells that are destroyed by HD, thus restoring lost function.

In fact, researchers have already discovered how to coax embryonic and adult mouse stem cells to develop into neurons that produce a neurotransmitter called gamma-aminobutyric acid – the type of neurons that are mainly lost in HD.

More research could potentially lead to the following:

  1. If these stem cells can produce nerve cells in the laboratory, they could be transplanted into the striatum to replace the lost nerve cells, or;
  2. If the adult stem cells already present in the patient’s brain could be stimulated to produce more neurons, they might be able to “self-repair” the striatum.

Either way, further stem cell research could yield new treatments to HD given enough time, research, and luck.

What is fetal neural transplantation? What does this have to do with HD and stem cells?^

Fetal neural transplantation is a surgical technique that involves removing nerve cells from an aborted fetus and transplanting them into a human patient. Clinical trials have attempted to use this technique as a treatment for HD by removing striatal nerve cells (those mainly affected by HD) from a human fetus and grafting them into the brain of an adult patient. The therapeutic value of fetal transplantation has been promising so far. Notable improvements include increases in brain activity and motor and cognitive functions. Although the initial results have been encouraging, the clinical usefulness of fetal neural transplantation for HD treatment remains unclear.

The use of human fetal tissue creates a major roadblock to the development of this technique for two reasons. Technically, fetal tissue is difficult to obtain and prepare. Ethically, the use of fetal tissue raises serious concerns. Therefore, the development of an alternative source of nerve cells for neural grafting will be crucial for the continuation of neural transplantation research. Stem cells currently hold great potential as an alternative source. Theoretically, neural stem cells could be developed in the laboratory and then grafted into the patient’s brain. Ultimately, the future of fetal neural transplantation as a clinically effective HD therapy relies heavily on the future of stem cell research.

Will stem cell research provide the cure for HD?^

Researchers generally do not believe that stem cell research will be the “magic cure” for HD. Rather, it is likely to be part of the fight against the neurodegeneration seen in HD. Ultimately, the medical and scientific community will need to improve early diagnosis, reduce the severity of cell loss, combat inflammation, provide new neurons (which is where stem cells factor in), and utilize progressive rehabilitation techniques to allow complete regeneration. While stem cells may not cure HD, they could serve as a crucial component to effective treatment.

For further reading^

  1. Allison, Wes. “Preliminary success of fetal brain-cell transplantation in Huntington’s Disease.” The Lancet.
    A short, but fairly technical article.
  2. Begley, Sharon. “Cellular Divide.” Newsweek, 9 July 2001: 22-27.
    An easy-to-read explanation of stem cells and an update on progress as of July 2001.
  3. Bjorklund, Anders and Ollie Lindvall. “Cell replacement therapies for central nervous system disorders.” Nature Neuroscience, June 2000, 3 (6): 537-544.
    A technical paper discussing the progress of fetal neural transplantation in treating Parkinson’s and Huntington’s Disease.
  4. Freeman, Thomas, et.al. “Tranplanted fetal striatum in Huntington’s disease: Phenotypic development and lack of pathology.” Proceedings of the National Academy of Sciences of the United States of America, 5 December 2000, 97 (25): 13877-13882.
    A highly technical paper discussing the potential of fetal neural tissue to treat HD.
  5. Gibbs, W. Wayt. “Biological Alchemy.” Scientific American, February 2001: 16-17.
    A less technical article depicting the discovery of neural adult stem cells and discussing the possible plasticity of adult stem cells.
  6. Mitchell, Steve. “Rare stem cells produces many cell types.” United Press International, 21 June 2002.
    A short, easy-to-read article about adult stem cell plasticity.
  7. Stem Cells: A Primer.” National Institutes of Health, May 2000.
    A comprehensive, easy-to-read explanation of stem cells and their potential applications. Great online resource.
  8. Stem Cells: Potential for Good?” The Economist, 18 August 2001: 59-61.
    A thorough explanation of stem cells and the controversy surrounding their development and use.
  9. Stem Cells: Scientific Progress and Future Research Directions.” Department of Health and Human Services, June 2001.
    An extensive, fairly technical summary of everything you would want to know about stem cells.
  10. Weiss, Samuel. “Stem Cells and Huntington Disease.” Horizon, Huntington Society of Canada Newsletter, Summer 2001, No. 101: 1-2.
    An easy-to-read explanation of stem cells and their potential to treat HD.

-J. Czaja, 3-07-03

More

The Scientific Approach

When it comes to scientific research, the public wants results and we want them fast. This is especially true of research on chronic or fatal human diseases such as diabetes, cancer, and Parkinson’s, which affect millions of people in the United States alone. Because the public loves good news, the media is quick to report stories involving major scientific breakthroughs (or what appear to be). On June 16, 2006, for example, the Canadian press released an article entitled “Canadians cure Huntington’s disease in modified mice.”

As I learned firsthand this summer as an intern at Dr. Marcy MacDonald’s Huntington’s disease (HD) laboratory in the Center for Human Genetic Research at Massachusetts General Hospital (MGH) in Boston, the disease is far from cured, even in mice. In fact, the research community is still years, perhaps decades, away from finding drug treatments that target the genetic mutation whose deleterious effects lead to HD, a neurological disorder with symptoms that typically begin in middle age. HD is termed “neurodegenerative” because it involves a progressive loss of nerve cells in the brain. The disease affects men and women alike, occurring at a rate of about one in every 10,000 in most Western countries.

While science journalism is not, for the most part, intentionally fraudulent or misleading, it sometimes gives people the wrong impression about scientific findings by the way it interprets the data from recent articles in science journals like Cell and Human Molecular Genetics. When the Canadian scientists reported that they had inhibited an enzyme that cleaves, or cuts, the mutated HD protein (huntingtin) in mice, thus preventing the degeneration of the nerve cells in the brain, the press trumpeted it as a cure.

When the media takes such leaps or oversimplifies a complex, highly nuanced finding, it presents a skewed picture of the actual process of scientific research and discovery. In reality, the scientific method—that is, the process of empirical investigation into the validity or invalidity of a scientific claim or hypothesis—relies on replication and critical testing of each new finding, which takes a considerable amount of time. Not only does it require patience from both the scientists and the public, but it also requires a great deal of intensive effort that includes collaboration between research teams in different parts of the country and around the world.

Scientists rarely work alone or in isolation. To do so would be highly inefficient, especially since one scientist or group of scientists does not have expertise in every skill necessary to carry out an entire large experiment from start to finish. When teams of scientists work together and share ideas and materials (such as cell lines, which the MacDonald lab frequently sends to other labs), they are able to produce results in less time. However, “less time” does not mean instantaneously; collaborative work, while certainly more efficient than solitary work, still requires many years of sustained effort to find results that translate into good news for disease sufferers.

Although the scientific community values collaboration, it does not necessarily frown upon competition. Competition to test new ideas, to try and “knock them out of the ring,” is built into the scientific method (described later in The Scientific Method) and is, in a manner of speaking, one hallmark of the scientific endeavor.

Blueprints^

One of the greatest rewards of scientific research is the “Eureka!” moment—that sudden gleeful breakthrough that can occur after much effort and many months of work. When a scientist experiences this lightning flash of insight, all the smaller discoveries of years past come together in a meaningful way, like the pieces of a puzzle, forming a much larger discovery. Indeed, HD can be compared to an enormous puzzle, the outlines of which are known, and the rest of which is still a mystery.

The genetic nature of the disease provides a kind of framework for discoveries about the changes that take place in the body on the cellular and molecular levels during the course of the disease. The macro-level changes observed in people with HD also provide guidelines about the micro-level changes occurring within their brains and bodies as the disease progresses.

With a rough outline to use as a guide, scientists can begin finding new pieces of the puzzle and fitting the puzzle pieces together to form recognizable pieces of the bigger picture. By putting the newly discovered pieces in place, scientists can make strides toward finding effective treatments not just for the symptoms of HD but also for the root genetic defect, or mutation, that causes the disease.

In 1993, an international team of researchers, which included Dr. MacDonald and her colleague, Dr. James Gusella, identified the responsible mutation. They found a CAG triplet repeat expansion in a region of human chromosome 4. Found in the nucleus (the information center) of cells, chromosome 4 is, like other chromosomes (we have 23 pairs of them), comprised of the DNA and associated proteins. Lengths of DNA in a chromosome make up genes, which are the functional units of heredity in humans and other organisms.

Each person inherits two copies (called alleles) of each gene, one from mother and one from father (the only exception being genes on sex chromosomes). Because HD is inherited as a genetically dominant character, a person needs only one mutated copy of the gene, called the expanded HD CAG allele, to inherit the disease. (For more information on genes and chromosomes, please click here

Genes are often compared to blueprints for making proteins. If the blueprint is defective, a defective protein will be made. Unlike the non-HD allele, which makes huntingtin protein with fewer than about 37 glutamines (one of the building blocks of the protein), the expanded HD allele makes an abnormal version of huntingtin, with an excess of glutamines (more than 37 or so of them in a row). Due to this mutation, the expanded glutamine version of the huntingtin protein does something—or is a byproduct of another process that does something—that contributes to the slow destruction of nerve cells in the brain. While the onset of symptoms can vary widely, the onset typically occurs between the ages of 30 and 50, after a substantial percentage of the nerve cells have died. There is also a juvenile form of the disease whose symptoms commonly appear before the age of 20. For more information on juvenile HD, please click here.

Physicians typically group the symptoms into three categories: movement, cognitive, and psychiatric. Movement symptoms include uncontrollable movements such as twisting and turning (known as “chorea”), rigidity, falling down, difficulty physically producing speech, and, in the later stages of the disease, difficulty swallowing, which can lead to significant weight loss. Cognitive symptoms include the altered organization and generally slowed processing of information in the brain. The most common psychiatric symptom of HD is depression; other symptoms include personality changes, anxiety, obsession, delirium, and mania. Denial of having HD is also a common symptom of the disease.

Presymptomatic genetic testing is available for those at risk for HD (i.e. people whose mother and/or father were diagnosed with the disease). While there is currently no cure for HD, there are drugs available to treat some of the symptoms, particularly chorea and depression. Some HD researchers, however, are beginning to develop and test drugs that target the presymptomatic effects of the genetic mutation that causes the disease.

Dr. MacDonald is one such researcher who works at the beginning of the disease pathway. She and Dr. Gusella, now director of the Center for Human Genetic Research, believe that the most effective treatments will be those that are specifically designed to reverse the first effects of the genetic mutation. These effects may impart altered physiology that is intrinsic to being born with and living with the HD mutation from birth. Scientists are still a long way from fully understanding the biology of the disease and the underlying mechanisms of nerve cell degeneration.

The Scientific Method^

HD research can also be compared to erecting a building without knowing its dimensions. As of now, researchers only have a vague idea of the shape of the “building,” as specified by the genetic information on chromosome 4. In time, as scientists learn more about the cellular and molecular basis of the disease, they will have a clearer idea of what the “building” actually looks like.

Creating a firm foundation, as the first order of business, is key. Before moving forward with his or her research, a scientist must look to the relevant data from past research and attempt to replicate the key results of other scientists. This step is to make sure that the foundation is sturdy before beginning to build the first wall, or the second. And after completing the second wall, one must make sure that it does not fall when the wind blows, so to speak, in the face of different experiments designed to knock it down.

The idea is to avoid building a flimsy house of cards, but rather to make a solid structure that can be inhabited (and tested) for many years and decades by future generations of scientists. Progress can be thought of either as building a new piece of the foundation that may not initially be connected to the rest, or it can be the addition of new pieces to the growing structure on the original foundation. Progress is accomplished by employing the scientific method as follows:

  • Repeat earlier findings in your specific area of inquiry.
  • Make one or more hypotheses—that is, succinct propositions about what you expect to find if you are right about a process or phenomenon. Be sure your propositions are suitable for empirical testing in laboratory experiments.
  • Conduct an appropriate experiment, controlling for (that is, holding constant) conditions other than those specified in your hypotheses. Carefully observe and note what happens in detail. These details, in combination with the conditions under which they were obtained, are your results.
  • Check your results against the original hypotheses: Do they support one or another of your propositions? Are they what you expected or predicted from one argument or another? Or do they require that you reject all your original hypotheses because you saw something new or unexpected?
  • Explain what you saw and what that tells us. You may need to modify the original hypotheses or, if necessary, you may need to make entirely new ones that are consistent with your findings.
  • Repeat this process until you have eliminated all but one remaining hypothesis. Note that the last hypothesis “left standing” is not what we might call “proven”; it is simply our best bet, given current knowledge. It, too, may be rejected one day when we have better information and understanding.

Although not all research progresses in such a linear fashion, the scientific method can nevertheless be conceptualized as a flowchart:

Fig. 1. “How scientific investigations proceed.” (from Jones et al, 1994.)

The most time-consuming aspect of research—indeed, the heart of any scientific endeavor—is the continual knocking down and building up of the various parts of the knowledge-structure. Scientists can only make progress by first attempting to disprove previous hypotheses, including their own, to ensure the strength of the structure’s foundation. Researchers must also allow time to investigate unexpected results and decide how they fit (or don’t fit) into the emergent structure.

Overall, the scientific method provides scientists with an orderly, systematic way of approaching their research that, in the end, guarantees progress. But it is a multi-step process that cannot be shortened as a result of pressures either from the scientific community or the public without weakening the entire structure. The pace of research can, however, be accelerated by adding more trained scientists, by building and using machines that can allow experiments or observations more quickly and without bias, and by increasing the rate of flow of accurate information about the research, both within the scientific community and from scientists to the public which, directly or indirectly, funds most of these efforts.

Genetic Research at Massachusetts General Hospital (MGH)^

The MacDonald lab is located in the brand-new Richard B. Simches Research Center, just up the street from the main MGH campus. Designed to facilitate communication between the various research groups, the building features wide hallways, open spaces, and meeting rooms equipped with audiovisual equipment for presentations. A spiral staircase, representing the double helix structure of the DNA molecule, connects the fifth and sixth floors, which make up the Center for Human Genetic Research (CHGR), through to the seventh floor, which houses the Molecular Biology Department.

One of five new thematic centers launched at the Hospital, the CHGR strives to facilitate the genetic research cycle, which begins with basic research, driven by scientists’ interest in questions pertaining to the biology behind a genetic disease. In basic research, biologists try to make new discoveries about the disease. For example, by studying animal models relevant to a given disease, scientists can try to observe new phenotypes in animals (that is, observable properties, particularly those associated with gene effects) that can then be looked for in human patients as well. Or researchers may try to use these new phenotypes to develop novel assays (chemical analyses) that can be used to discover drug compounds that may prevent the disease-associated phenotype.

The next stage of the cycle is the applied, or engineering-type, research, which puts the discoveries of basic research into practice. For example, researchers, usually in biotechnology or pharmaceutical companies, may use a variant of the assay discovered in the academic research lab to test a wide variety of drug compounds to see which of them effectively alter the outcome. Then, they may give the effective ones to animals and evaluate the outcomes, modifying the compounds by changing the chemical structure and retesting them, in successive rounds, to make them perform better, with fewer untoward side effects.

At this stage, researchers often look for drug targets, or molecules that can be expected to enhance or inhibit the disease. The best drug targets provide a direct route to what should be changed in a patient on the molecular level. Testing drugs in animal models helps researchers to identify targets and prioritize the best ones for further testing.

The third stage of the research cycle is clinical research, in which physicians and clinical researchers administer drugs to patients in government-approved clinical trials. Observations made at this stage often give rise to hypotheses at the basic research stage, and the cycle begins again, as illustrated in the diagram below.


Fig. 2. The genetic research cycle.
The cycle begins with basic research in academic labs, continues with applied research in biotech or pharmaceutical labs, and ends with clinical research in hospitals. Observations from the clinical phase may be used in basic research, and the cycle begins again.

Genetic research, or research of any kind, is therefore not monolithic; there are various stages of the research effort that operate in different facilities, with different kinds of people, and on different timelines for completing experiments and trials. Because each type of research has different goals, it requires funding from different sources.

The HD researchers at the CHGR are among the many people at MGH working to facilitate the genetic research cycle for HD. The MassGeneral Institute for Neurodegenerative Disease (MIND), directed by Dr. Anne Young, Chief of Neurology, makes discoveries in the basic realm and aims to translate them into prevention and treatments of neurodegenerative diseases like HD and Alzheimer’s. MIND, therefore, serves as a bridge between basic and clinical research. On the clinical side, the Department of Neurology helps HD patients to manage their symptoms through medical treatment, such as drug regimens, some of which may be experimental, in the cadre of clinical trials.

The study of HD at Massachusetts General Hospital via the scientific method can be compared to what scientists call a “fractal,” a geometric pattern that is repeated at ever-smaller scales, as in the diagram below. Whatever the size or scale of the problem—whether a researcher is looking at a molecule, an organism, or an entire population—the process has a regular structure (derived from the scientific method) and resembles the greater whole.


Fig. 3. “Construction of a Fractal Snowflake.” (from MSN Encarta.)

The basic triangle shape is reflected at every stage in the process of forming the larger design, just as the scientific method is reflected at every level of research from the smallest to the biggest detail.

As part of the CHGR, the MacDonald lab performs basic research and takes a molecular genetic approach to understanding HD. The researchers examine the DNA sequences of genes—the HD gene, in particular—to understand how changes in gene expression and protein structure are affected by the HD mutation. Gene expression is the process by which a gene’s DNA sequence is converted into proteins that are involved in cellular processes both structurally and functionally.

Studying the genetic expression of the HD gene (both the HD and non-HD causing alleles) can provide scientists with clues about how the nerve cells stay healthy or get sick. Determining the temporal order of the early steps in the disease pathway will eventually lead to the development of drug compounds that prevent these steps from occurring. As biological models for the disease, the MacDonald lab uses genetically altered mice and cells derived from them. Because the mice have high numbers of glutamine repeats in the huntingtin protein, as a result of the same HD CAG mutations that cause HD, they are likely to reveal the earliest presymptomatic changes to manifest with HD in humans.

From Journalism to Science^

When I arrived at the lab at the beginning of July, I was eager to make a discovery of my own that would, in some small way, help scientists to find a cure for HD. I imagined myself working feverishly under the fume hood, swirling neon-colored chemicals in Erlenmeyer flasks. I envisioned myself peering into a high-powered microscope to observe the elusive structure of the huntingtin protein. As ridiculous as it sounds, I even imagined jumping up from my chair and crying, “Eureka!” as I bounded down the hallway in triumph.

As I settled into the daily routines of the lab, however, I saw the fantasy evaporate before my eyes. My biggest discovery this summer was that, while some discoveries may come in the form of intense flashes of insight, this is a rare event—except in the movies, of course. Getting to this point is much more prosaic.

Working in a lab was a brand-new experience for me. I am, however, familiar with the biology behind HD. For the past three years I have worked for Huntington’s Outreach Project for Education, at Stanford (HOPES), a student-led educational service project working to build a global Web resource on HD. Our site is a “layperson’s guide” to the scientific intricacies of HD and HD-related research. Akin to science journalism, my work has consisted of writing news briefs on the latest research, drugs, and other treatments, as well as interviewing eminent scientists and writing articles about their work on HD. My first interview was with Drs. MacDonald and Gusella during the summer of 2004, published on the website as the first chapter in a section called Research Frontiers. The two researchers discussed at length their approaches to HD and touched upon some of the myths of scientific research, including the ever-popular notion of a sudden cure or “magic bullet.”

Several months ago, just prior to my graduation from Stanford, Dr. MacDonald invited me to do an eight-week internship at her lab in this summer. After earning my BA in English with a minor in Human Biology, I headed east to Boston to begin my work. I was going from writing about science to actually doing science—a big leap.

Dr. MacDonald introduced me to Drs. Gill Gregory and Surya Reis, the two postdoctoral fellows who would be supervising my independent project. In preparation for conducting future research in their area of specialty, postdocs are in the last phase of their training, preparing them to start their own research laboratories, each working on a piece of the research puzzle. Research technologists, on the other hand, are responsible for performing one or more experiments that may either be varied or more routine, requiring long-term concerted expertise. For instance, Lakshmi Mysore, who has been working on HD for twenty years, specializes in genotyping, or determining the genetic makeup of an organism. The lab also depends upon the work of animal technicians, such as Edith Toral Lopez, who oversees the breeding of the animals and provides the genetically altered mice used in experiments.

During my first week I was outfitted with a white lab coat and notebook, given a tour of the lab, briefed on safety procedures and experimental protocols, and taught basic lab skills such as pipetting (using a syringe-like instrument to measure and transfer liquids from one container to another), taking care of tissue cultures, and transferring cells onto cover slips to be mounted on slides for viewing under the microscope.

Tissue cultures are a means of keeping populations of cells alive outside the body in a nutrient-rich liquid called a medium. I was responsible for monitoring the cells’ growth rate from day to day and splitting up the cells onto new dishes with fresh medium when the old dishes became too full because the cells had multiplied. The cells came from the brains—specifically, the striatum, the part of the brain that is first affected in HD—of mutant mouse embryos (those with 109 glutamine repeats in the huntingtin protein) and their normal (“wild-type”) counterparts.

My Summer Project^

My task as an intern would be to complete a small project within the context of Surya’s and Gill’s research. Each of the postdocs takes a slightly different approach to detecting the subtle differences between mutant and wild-type nerve cells. Surya uses immunocytochemistry (IC), a method of staining cells with antibodies so that she can pinpoint the location of the huntingtin protein, for example, in the nuclei. Meanwhile, Gill uses immunohistochemistry (IH), a method of staining tissue slices (from the striatum, in this case) with the same antibodies, also to locate huntingtin in the nerve cells.

Antibodies are proteins made by the body’s immune system as a defense against foreign material, such as bacteria or viruses, which enters the body. These Y-shaped proteins attack and neutralize the substances, called antigens, that triggered the immune response. Each antibody has a specific antigen to which it binds. The IC and IH methods make use of an antibody’s ability to recognize a particular antigen, rather than its ability to attack and neutralize it. Please see below for a diagram of an antibody.


Fig. 4. The structure of an antibody. (from Wikipedia.)

To visualize the location of the huntingtin protein in the nucleus of a mouse nerve cell, researchers use a technique called immunostaining as part of the IC and IH methods. After fixating, or preserving, a cell sample or tissue slice on a cover slip or slide, they add a small amount of a primary antibody. The primary antibody recognizes and binds to a specific place on the huntingtin protein’s surface, called an epitope. Then, a secondary antibody that comes from another animal is used to detect the first. The secondary antibody contains a fluorescent molecule, which allows the researchers to see the position of the huntingtin in the cell under the powerful confocal microscope. Multiple secondary antibodies bind to the primary, thereby amplifying the fluorescent signal. Please see below for a schematic diagram of immunostaining.


Fig. 5. Immunostaining.
The primary antibody recognizes the polyglutamine tract of the huntingtin protein, and the secondary antibody recognizes the primary. The secondary antibody contains a fluorescent molecule to help scientists visualize the huntingtin under a microscope. Multiple secondary antibodies bind to the primary for fluorescent signal amplification.

Using confocal microscopy, scientists can visualize the huntingtin protein molecules to which the primary antibody binds. They can therefore show the localization of huntingtin and make conclusions as to the site-specific function of the protein (both mutant and wild-type) in the cells.

My project fit neatly into this experimental framework and had two objectives. The wet lab component of the project, performed at the lab bench, involved immunostaining with a primary antibody made at two different commercial laboratories. My goal was to determine which of the two versions was better, and under what conditions, for seeing differences between the wild-type and mutant cells with regard to the staining pattern. See below for a picture of me performing an immunostaining procedure.


Fig. 6. Taylor Altman performs an immunostaining procedure.

She applies a primary antibody stain to mutant and wild-type cells on cover slips.

The dry lab component, performed at my desk, entailed building an electronic database of information about huntingtin antibodies. In a Microsoft Excel spreadsheet, I organized the information by such categories as antibody name, epitope, and animal host (the animal from which the antibody is taken). The database will eventually be turned into a website for use by HD researchers all over the world.

Accomplishments^

Before long, I realized that my goals were unrealistic for the two months I’d be spending at the lab. I saw that I couldn’t build an entire database on huntingtin antibodies in one summer because there are hundreds that have not yet been properly described, and information is often scarce. I did manage to gather sufficient data for eight antibodies, which is a good start.

As for the wet lab project, I completed three modest experiments, each with its own purpose and goal, on the path to determining which of the batches of primary antibodies was better suited to seeing differences between the mutant and wild-type striatal cells. Although I was a bit disappointed that I couldn’t see my project out to its end, I did get a good feel for bench work and for the extensive planning that goes into each experiment.

The first of my small experiments was a primary antibody dilutions test. Dilution is the process of making something weaker or less concentrated. To get different dilutions of the antibody, I added the same amount of antibody to increasingly large amounts of the diluting solution. Then, I applied the dilutions to the mutant and wild-type cells to determine the optimal dilution for seeing the greatest differences between the two types of cells under the confocal microscope. As the dilutions increased, I expected the strength of staining to decrease more rapidly in the wild-type than in the mutant. However, I saw the exact opposite. See below for pictures of the wild-type and mutant cells at the highest and lowest dilutions. Notice the difference in strength of staining between the two.

Fig. 7. Wild-type and mutant cells at lowest and highest dilutions.
Pictures taken at 20x magnification on the confocal microscope. As the dilutions increased, the strength of staining decreased more rapidly for the mutant than the wild-type.

Part of science is dealing with unexpected results. When a scientist initially gets results that seem to go against the hypothesis, he or she must repeat the experiment in order to rule out chance or human error. My second experiment, therefore, was a repeat of the first. Again, I got the same results. Clearly, this was no coincidence.

To look for clues that might explain my results, Surya directed my attention to the method I used to fixate the cells. Using a detergent, I had made tiny holes in the cells’ membranes that allowed the primary and secondary antibodies to flow in. Perhaps I’d used too much detergent and had made the membranes too porous, thereby letting in too much antibody or letting out too many important cellular components. Or there may have been other explanations for the results—maybe the cell types had been mixed up, or maybe the antibody no longer worked. The latter seemed most probable.

My third and final experiment, then, was a test of the amount of detergent, in which I kept the antibody dilutions the same while I varied the concentrations of detergent. On the whole, the results were inconclusive, but Surya plans to run another test in the near future.

Conclusions^

Reflecting on my summer at the lab, I am grateful to Gill and Surya, and to their supervisors Marcy McDonald and James Gusella, for the rewarding experience I had at MGH. Not only was I able to learn about the process of scientific discovery firsthand, but I was also able to become a better science writer by enhancing my knowledge of scientific materials, methods, and terminology. By conducting my own experiments, I began to think like a researcher and to understand the scientific method from the point of view of one who puts it into practice on a daily basis.

I saw a side of science that the public rarely, if ever, sees. The actual process of scientific discovery is much lengthier, more complex, and more nuanced than the media’s portrayal of it. Science is about careful observation and planning, good record keeping, and building a strong foundation for future experiments by continually attempting to disprove previous hypotheses.

Above all, science requires great patience and perseverance. Scientists do not leap from their lab benches crying, “Eureka!” every day, nor do they produce a continual stream of results. Moreover, results do not come cheaply. To my surprise, I learned that it cost about $5,000 for the supplies and small equipment, and about $400,000 for the confocal microscope, to perform one of my “simple” immunostaining experiments (not to mention the still-higher cost of paying everyone’s salaries and keeping the lab running on a daily basis).

Although my future plans don’t include working as a researcher, science will always be a part of my life, whether I choose to become a science journalist or simply a science enthusiast. I now have a much greater understanding and appreciation of the arduous work that scientists do, which gives me hope that someday they will solve the mystery of HD and other neurodegenerative disorders.

For further reading^

  • “Construction of a Fractal Snowflake.” MSN Encarta. 28 Aug. 2006
    <http://images.encarta.msn.com/xrefmedia/aencmed/targets/illus/ilt/T046583A.gif>.
  • Jones, Allan, Rob Reed, and Jonathan Weyers. “How scientific investigations proceed.”
    Practical Skills in Biology. Essex: Addison Wesley Longman Limited, 1994.49.
  • “Schematic of antibody binding to antigen.” Wikipedia. 21 Aug. 2006
    <http://en.wikipedia.org/wiki/Antibody>.
  • Ubelacker, Sheryl. “Canadians cure Huntington’s disease modified mice.” The Globeand Mail. 16 June 2006. 15 Aug. 2006
    <http://www.theglobeandmail.com/servlet/story/RTGAM.20060616.whuntingtonS016/BNStory/Science/home>.

T. Altman, 2006

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