HEREDITARY DISEASE FOUNDATION

RESEARCH GRANTS

Anne Ast

Mentor: Erich E. Wanker, Ph.D.

Institution: Max-Delbrück-Center

for Molecular Medicine, Germany

Project Title: Manipulating seeding activity and proteotoxicity of amyloidogenic HTT aggregates by targeted amino acid exchanges

Every protein needs a specific architecture in order to execute its duties within the cell. In Huntington’s disease (HD), the disease-causing huntingtin protein has lost its original folding instruction and forms stable protein clumps. These clumps operate as templates and pass on their corrupted folding pattern to other normally folded huntingtin molecules. We suspect that this process could be a driving force in HD development and progression. Therefore, we want to understand the connection between templated protein misfolding and toxicity and find a way to manipulate this process as a therapeutic approach.

Kristina Becanovic, Ph.D.

Institution: Karolinska Institutet, Sweden

Project Title: Towards the discovery of genetic modifiers and targets for future Huntington’s disease treatments

We all have the same genes with only minor differences on individual sites that affect the activity of genes and how much we have of a particular gene product in the body. These small genetic differences make us unique and different from one another and they are also the reason HD manifests itself differently in different people. In this research study, Dr. Becanovic wants to understand why people with the same number of CAG repeats sometimes have very different ages of symptom onset. The goal of this study is to identify the genetic variants that affect disease onset, so called “genetic modifiers”.

If we succeed in identifying these genetic modifiers, we will be able to use this knowledge to develop better drugs. These would, unlike today's medicines, not just treat symptoms but primarily protect the brain and slow down the development of the disease.

Steven Finkbeiner, M.D., Ph.D.

Institution: J. David Gladstone Institute

University of California, San Francisco

Leslie Thompson, Ph.D.

Institution: University of California, Irvine

Project Title: Assessment of WGS-derived genetic modifiers in differentiated HD-derived iPSCs

Dr. Leslie Thompson and Dr. Steven Finkbeiner, members of the HDF’s Scientific Advisory Board, are both established researchers in the Huntington’s disease field. Leslie and Steve are longtime collaborators and were founding members of a consortium established by the National Institutes of Health. The consortium received funding from HD organizations, including the HDF, to develop human neuron models of HD by reprogramming patient cells to be stem cells and then directing those stem cells to become neurons.

The two investigators developed a collaborative project for the Hereditary Disease Foundation to evaluate genes that may modify HD. Several potential modifying genes are being analyzed in human neurons from HD patients and their effects will be studied. Leslie is using sophisticated techniques to look globally at how those genes affect the expression of other genes in cells. She will specifically look at whether modulation of potential HD modifier genes affects the gene expression changes that she previously found were associated with HD neurons. Steve is using special robotic microscopes he invented to test whether modulation of potential HD modifier genes affects degeneration of human HD neurons. These special microscopes let them follow individual cells, much like patients in a clinical trial, and he has found that two potential HD modifier genes have dramatic effects on neurodegeneration in patient HD neurons which are now being followed up for gene and protein changes in Leslie’s lab.

The results from the two approaches will be integrated into a deeper molecular and cellular understanding of how other genes besides the HD gene affect HD. This will provide a better understanding of how HD occurs and the identification of therapeutic targets that could lead to new treatments.

Charlene Geater, Ph.D.

Mentor: Leslie M. Thompson, Ph.D.

Institution: University of California, Irvine

Project Title: PIAS1 network in HD induced pluripotent stem cells – Dressing Huntington

The mistake in the protein that causes Huntington’s disease can be very subtle!

Imagine that the Huntingtin’s disease protein starts out as a person undressed and deciding what clothing to put on. The protein needs to go out and work – what to wear? The protein wants to go out and party – what to wear?

The protein has different dress options! It is the same underneath, but can wear different outfits – called “post-translational modifications” – that fit with where it needs to go and what it needs to do. Depending on how the huntingtin protein is dressed and where it is going, the protein can act as it should or misbehave in the presence of the HD mutation. How one is dressed and where one is taken can make a difference in whether or not one gets sick and how sick one gets.

SUMO is a protein which is used as an “item of clothing” on the huntingtin protein that influences the balancing act of protein stability in cells. One of the “fashion designers,” called PIAS1, will dress proteins with SUMO, changing how proteins behave.

The Thompson lab previously showed in HD mice that decreasing the amount of PIAS1 helps to slow the progression of HD. Now Dr. Geater will evaluate whether the promising results seen in the HD mouse model translate to nerve cells derived from human HD patients. To do this, she uses an amazing technology that allows skin cells from HD patients and healthy individuals to be “reprogrammed” into stem cells, which can form any cell type of the body. They are then made into neurons, the cells that are severely affected in HD. Using these cells, she will see how reducing or increasing the presence of PIAS1 affects many different processes in HD neurons to gain an understanding of how we can use the system to develop new treatments for HD.

Pan Li, Ph.D.

Mentor: Russell Margolis, Ph.D.

Institution: Johns Hopkins University

Project Title: A high throughput screen for small molecules enhancing the expression of HTTAS-a novel therapeutic strategy for HD

Russell Margolis’ group identified a piece of RNA (the recipe for making proteins) that is widely expressed at low levels in the brain. When this RNA is overexpressed, it lowers the amount of the protein that causes HD. They hypothesize that this RNA will provide novel therapeutic targets for suppression of the abnormal HD protein and, therefore, will be an effective treatment of HD.

Funded by the Hereditary Disease Foundation, Pan Li’s research will explore the mechanism and magnitude of the effect this RNA has on the expression of the HD protein. She will use skin cells, stem cells and mouse models of HD to quantify these findings. This strategy provides an alternative approach to suppress the HD-causing protein, which may have considerable therapeutic potential.

Boxun Lu, Ph.D.

Institution: Fudan University, Shanghai, China

Project Title: Validation of a Gpr52 antagonist’s effect on mHTT levels and toxicity

Huntington's disease is a devastating hereditary disorder that causes neuronal death in specific brain regions, motor functional deficits and psychological symptoms, and eventually premature death. HD is caused by mutation in a single gene called huntingtin, which expresses the abnormal huntingtin protein that is toxic to the neurons. This then leads to abnormal neuronal death via complicated and unclear mechanisms. An effective approach to treating HD is lowering the abnormal protein, which may suppress all kinds of its downstream toxicity. While this could be achieved by several large biological molecules targeting the protein, the delivery of these molecules into the brain is highly challenging and expensive, prohibiting their accessibility to most HD patients. Thus, small molecule compounds that may lower the abnormal protein are highly desired.

The ultimate goal of Dr. Lu’s work is to look for safe compounds that can lower the abnormal protein to treat HD. In order to achieve this, he needs to identify potential drug targets to regulate protein levels. Lu’s previous work identified a few such proteins. In this project, Lu plans to validate one of them, Gpr52, which has the highest potential for drug discovery. He will use a virus to deliver reagents that can decrease Gpr52 in HD mice and test whether the abnormal protein is lowered and the pathology is rescued.

As one of the few HD labs in China, Dr. Lu’s lab also is interested in serving as a connection hub for Chinese HD research to the rest of the worldwide HD research community.

Alejandro Mas-Monteys, Ph.D.

Institution: University of Pennsylvania

The Children's Hospital of Philadelphia

Project Title: Novel approaches for knock down control

Sustained levels of RNA interference machinery for chronic gene silencing may result in unintended side effects. My project will test a new method to control RNA interference machinery expression. I have developed a modular system that can respond to a re-purposed drug to turn the RNA interference machinery off or on. Importantly the drug can reach the brain when given orally. I will test the utility of the system in HD cells cultured from human patients as well as in mouse models of HD.

Jose F. Moruno-Manchon, Ph.D.

Mentor: Andrey S. Tsvetkov, Ph.D.

Institution: The University of Texas McGovern

Medical School

Project Title: Sphingosine kinase 2 and DNA damage in neurons – A lipid kinase, DNA damage and Huntington’s disease

Maintaining genome integrity in neurons is critical for normal functioning of the brain. Every day, the genetic code of our neurons is exposed to harmful stimuli. Neurons have the molecular mechanisms that protect and repair their DNA. However, in brains of HD patients, neurons repair DNA less efficiently, leading to accumulation of DNA damage and eventually neuronal death.

Understanding what goes wrong during the DNA repair process in HD patients, and identifying new molecules involved in DNA damage and repair, may potentially lead to designing drugs that mitigate neurodegeneration in HD patients.

Sphingosine kinase 2 (SK2) is a molecule that generates a fat-like compound that participates in a variety of processes critical for cell life. Dr. Moruno discovered that SK2 is toxic for neurons, its activity is intensified in HD, and that neurons with overactive SK2 exhibit more DNA damage. He also found that a drug that inhibits SK2 reduces neurodegeneration and DNA damage in neurons. Using a novel fluorescence microscopy system and neuron models of HD, Dr. Moruno plans to identify DNA repair proteins that are regulated by SK2, and determine how they participate in DNA damage and repair, and survival of HD neurons. If successful, this project will establish SK2 as a potential therapeutic target to develop treatments for HD.

Christopher Ng, Ph.D.

Mentor: David Housman, Ph.D.

Institution: Massachusetts Institute of Technology

Project Title: Characterization of genetic variants that modify age of onset in Huntington's disease

David Housman helped pioneer the discovery of the genetic marker for Huntington’s disease. The Housman lab now studies how the rest of the human genome controls the age at which a patient with the Huntingtin mutation becomes symptomatic for the disease. Currently a postdoctoral fellow in the Housman lab, Christopher Ng plans to identify other genetic markers that modify HD age of onset by using the extensive resources of patient samples and clinical data collected over a 23 year period from the world’s largest HD family in Venezuela. He plans on characterizing dysfunction of the proteins encoded by these genetic variants in HD patient samples and mice models. Further, his goal is to discover the role of modifier variants in the pathology of the disease by genetic manipulation in patient-derived cells and mice models. Understanding how these genetic variants alter the course of the disease will distinguish the molecular pathways that are most capable of modulating Huntington’s onset. By going from genetic to molecular insights, he hopes to target these modifier pathways to develop protective therapies capable of slowing HD pathology.

Ellen Penney, M.D., Ph.D.

Institution: Massachusetts General Hospital

Project Title: R-loops in HD: Somatic Expansion and DNA Repair

Abnormal DNA/RNA structures, R-loops, have recently been described in several neurodegenerative diseases. These structures are predicted to form at the HD gene. R-loops are known to interact with factors that modify HD onset and progression. I believe R-loop formation may be a pathogenic process that occurs early in HD and ultimately leads to cell dysfunction and death. I’m studying whether R-loops occur in HD and working to identify mechanisms that can prevent their formation. I hope this work will lead to the identification of therapeutic targets for HD and other neurodegenerative disorders in which abnormal R-loop formation occurs.

Anna Pluciennik, Ph.D.

Institution: Thomas Jefferson University

Project title: Crosstalk between DNA repair pathways in Huntington's disease

Huntington’s disease is caused by an expansion of an unstable DNA tract composed of CAG repeats located within the huntingtin gene. Genomic analyses of HD patients have revealed that a number of DNA repair genes influence the age of onset of the disease, presumably due to their effect on the CAG repeat length. Nevertheless, how the proteins encoded by these genes conspire to cause CAG repeat expansion is not well understood. Using biochemical approaches, Dr. Pluciennik will isolate and study neuronal DNA repair protein assemblies that recognize and handle DNA structures formed by CAG repeats. Modulation of such assemblies may represent a viable therapeutic strategy for treating HD.

Piere Rodriguez, Ph.D.

Mentor: Judith Frydman, Ph.D.

Institution: Stanford University

Project Title: Dissection of the functional and structural communication within the Huntingtin protein and its

interaction with the human TRiC chaperonin

The risk of developing Huntington’s disease is tightly linked with the length of a specific region within the Huntingtin protein (Htt). Mutations can make this region longer than normal—i.e. more than 36 amino acids—producing a mutant Htt with characteristics that induce neuronal decay. The underlying mechanism behind this length-dependent toxicity remains largely unexplored. This is mainly because current biochemical/structural methods face two major technical barriers for the study of Htt: 1) Htt’s high aggregation propensity, which hinders the ability to perform experiments under biological conditions, and 2) Htt’s remarkable conformational heterogeneity, which has hindered structural studies of Htt with high resolution. To circumvent these technical barriers, I will use a novel single-molecule approach to obtain insights about the molecular mechanism that render Htt toxic, and to obtain information about Htt not accessible through traditional methods. This information is relevant for the identification of novel therapeutic targets and the development of new drugs against mutant Htt toxicity.

Anne Simonsen, Ph.D.

Institution: University of Oslo, Norway

Project Title: Alfy at the Battlefront against Huntington’s Disease

First identified by the work of Dr. Anne Simonsen, Alfy is a very large protein that targets protein clumps, known as aggregates, to a recycling pathway within the cell known as autophagy. Autophagy captures and then recycles many different things in the cell. Alfy works by telling autophagy to specifically capture and recycle aggregates, and therefore can potentially help fight diseases such as HD.

Recent work on Alfy shows that if the amount of Alfy is increased in the cell, autophagy can capture and recycle more aggregates more quickly. Recently, work led by Drs. Nancy Wexler and David Housman identified a series of patients from the Venezuelan cohort who showed a delayed age of onset of HD by up to 23 years. These patients all possessed a subtle change in the Alfy gene.

Dr. Simonsen proposes to use her expertise in understanding whether this subtle change causes the amount of Alfy in the cell to increase. In addition, she also proposes to identify how Alfy levels are increased, so that therapeutics can be designed to bring about similar changes to all HD patients.

Joan Steffan, Ph.D.

Institution: University of California, Irvine

Project Title: Analysis of an ubiquitin-binding domain within Huntingtin – Analysis of how the Huntingtin protein helps clean up cellular trash

Dr. Steffan’s laboratory is studying how the Huntingtin protein, mutated in Huntington’s disease, functions as part of the cell’s garbage collection system. Huntingtin is a large protein that can function in a process called “autophagy” as a scaffold for cellular trash, helping to target it to the cellular garbage dump, called the lysosome, where it is chewed up and recycled to make nutrients for the cell during stressful times. Trash is tagged by the cell to be carried to the lysosome by a small protein called ubiquitin.

The Steffan laboratory is being funded by the Hereditary Disease Foundation to investigate how the Huntingtin protein interacts with the ubiquitin-tagged trash in order to scaffold it to the lysosome for degradation, and to figure out what kinds of trash may be cleaned up by Huntingtin.

Steffan hypothesizes that mutation of Huntingtin may inhibit its autophagic function, allowing cellular trash to build up and be toxic to neuronal cells in the brain, contributing to Huntington’s disease pathogenesis. Dr. Steffan’s work will help define this autophagic role of Huntingtin so that therapies can be created to improve mutant Huntingtin function to keep the cell clean.

Mary Wertz, Ph.D.

Mentor: Myriam Heiman, Ph.D.

Institution: Broad Institute / Massachusetts Institute of Technology

Project Title: In vivo genome-wide genetic screening for modifiers of mutant Huntingtin toxicity

Although the gene linked to Huntington’s disease, Huntingtin, was discovered over twenty years ago, to date there is no cure or fully effective treatment for this terrible disease. Thus there is a pressing need for basic studies to reveal new therapeutic targets.

Towards this goal, Dr. Wertz’s project will perform a ‘genetic screen’ – a way of systematically testing the effects of each gene in the genome – to identify genes that change the toxicity of the mutant Huntingtin protein. Although such screens have been conducted before, they have not been conducted before in the context of nerve cells in the brain.

With Dr. Wertz’s “in brain” genetic screen, and optimizations of it made possible by HDF funding, she has already identified many potential modulators of mutant Huntingtin toxicity. Many of these candidate modulators are novel to HD biology, giving hope that they will be able to soon elucidate a new class of HD therapeutic targets.

Ai Yamamoto, Ph.D.

Institution: Columbia University

Project Title: ALFY-mediated Degradation and HD

Ai Yamamoto was awarded Hereditary Disease Foundation funding to better understand how brain cells are able to remove abnormal huntingtin, the protein that causes damage in HD. Her focus is a protein called Alfy that helps to package up and dispose of clumps of harmful litter in the brain. In HD animal models, clearing away abnormal huntingtin deposits has led to improvements in symptoms, and Alfy plays an important role in cleanup.

This project aims to closely examine the huntingtin removal process by lowering or boosting levels of Alfy in HD mouse models, and testing how it affects their behavior. A better understanding of Alfy’s function could help to identify therapeutic approaches to speed the elimination of damaging debris in HD brain cells.

William Yang, M.D., Ph.D.

Institution: University of California, Los Angeles

Project Title: Novel Fan1 Knockin Mice to Facilitate Huntington's Disease Genetic Modifier Research

Huntington’s disease is a progressive neurodegenerative disorder caused by a genetic mutation consisting of CAG trinucleotide repeat expansion in the Huntingtin gene. The length of the abnormal DNA repeat is inversely correlated with the age when the patients become first affected by motor symptoms of HD, but other genetic and environmental factors can also contribute to the disease onset. Two recent genetic analyses of HD patients, one focusing on patients with European heritage, and another on those from Venezuela, have identified genetic variants that can either accelerate or delay the onset of HD symptoms. Importantly, both studies suggest a genetic locus containing the DNA repair pathway gene Fan1 may be involved in modifying the age of disease onset in HD. Fan1 was originally discovered as a component of a multi-protein complex that is involved in repairing DNA lesions. The role of the Fan1-containing protein complex has been extensively studied in cell types outside the brain (e.g. in the blood or kidney), but its role in normal brain development, function, and in neurodegeneration in HD remains unclear.

In this project, Dr. Yang will use mouse models to probe the role of Fan1 in normal brain function and in the pathogenesis of HD. First, he will examine if a deficiency of Fan1 could alter the molecular and pathological phenotypes in an HD mouse model expressing intact mutant Huntingtin. Second, he will study mouse models carrying Fan1 genetic variants derived from HD patients to assess if such variants can alter the phenotypic course of HD mouse models. Finally, he will create new mice expressing Fan1 with a protein tag, which will facilitate the visualization Fan1 protein in intact brain cells. The latter model may help to pinpoint the precise role of Fan1 in the healthy brain, and determine how Fan1 function may intersect with HD disease processes. Together, this study will provide novel resources and deeper knowledge on the role of Fan1 in HD, and insights from this study should bridge the gap between the identification of human genetic modifiers of HD and mechanistic insights that can inform novel therapeutic development.

Andrew Yoo, Ph.D.

Institution: Washington University School of Medicine

Project Title: Modeling Huntington's Disease with Patient Neurons Generated by Direct Neuronal Reprogramming

Much of our understanding of human diseases of the brain is often gained post mortem – after death. The study of neuropathology has been an extremely powerful means through which we have been able to gain insight into how diseases such as Huntington’s disease give rise to the devastating symptoms for which they are known. Although neuropathology has also given us insight into how the diseases have begun, it has been difficult to test these ideas, given that these studies require the study of live human brain cells.

Recently, scientists, including Dr. Yoo, have shown that human brain cells can be generated by converting human skin cells, a process known as direct neuronal reprogramming. Using this approach, Dr. Yoo has created brain cells from skin cells collected from HD patients. He has found that these brain cells also recapture features that were previously only described post mortem.

Using this powerful approach, Dr. Yoo will determine if the genes identified by Drs. Nancy Wexler and David Housman and others to alter the course of HD alter the disease-related features in his system. This will give scientists unprecedented insight into how the different pathologic features of the disease might influence actual disease outcome.

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