Summaries of 2014 HDSA-Funded HD Human Biology Projects

Development of a Novel Fret-Based HTT Aggregation Assay as a Diagnostic Tool for HD

Dr. Alexander Buntru
Fellow / Max Delbrueck Center for Molecular Medicine / Berlin, Germany

alexander buntruHuntington’s disease (HD) is an inherited neurodegenerative disease caused by a mutant huntingtin (HTT)  protein containing an abnormal expansion of the amino acid glutamine. Above a threshold of 40 glutamine  repeats the age of onset of HD is inversely correlated with the length of the glutamine stretch. Similarly to prion and other protein misfolding diseases, the formation of aggregates is strongly associated with cellular toxicity and neuronal decay. The neuronal decay in HD manifests in cognitive, psychiatric and motor impairments. Currently, there are no drug based therapeutic strategies available to combat this devastating disease. Unfortunately, a reliable biomarker to monitor HD progression is still not available. We have developed a novel fluorescence-based assay that allows the amplification and quantification of minute amounts of HTT aggregates in biological samples. First, we will optimize our assay using brain tissue homogenates of HD model mice and healthy wild-type control mice. We aim to examine whether our assay is suitable to detect HTT aggregates in human derived samples including brain tissue and cerebrospinal fluid (CSF). Using our novel fluorescence-based approach, we aim to develop novel diagnostic methods with high sensitivity and specificity. In the longer term our studies should provide the basis for the development of a sensitive, diagnostic biochemical test for HD.


Human Stem Cell-Derived Neuromuscular Co-culture Platform for Assessing Peripheral Manifestation of Huntington’s Disease

Dr. Barbara Calamini
Research Scientist / Duke University / Durham, North Carolina

barbara_calaminiHuntington’s disease (HD) is a genetic neurodegenerative disease caused by a mutation in a protein known  as huntingtin. This protein is expressed in all tissues, and although the disease is classified as a  neurodegenerative disorder affecting the brain, hallmarks of the disease have also been detected in other organs, including skeletal muscles. It is possible that treating these peripheral organs, such as muscles, could have a therapeutic benefit in HD patients.  In addition, since sampling human brain for drug testing is not possible, more accessible tissues, such as blood, skin and muscle might provide alternative substitutes for research and drug discovery. The aim of these studies is thus to investigate the suitability of skeletal muscle for HD research using stem cells coaxed to become muscle cells.  Genea Biocells, an Australian stem cell company that had previously derived HD human stem cells, has recently developed a proprietary method for making human skeletal muscles. Dr. Calamini will collaborate with Genea Biocells to study stem cell-derived muscle cells and compare them with muscle cells biopsied from HD patients. If successful, these human stem cell-derived muscle cells will provide an alternative resource for drug screening and offer a substitute for difficult to obtain patient tissues and organs.


Microvesicles: Biomarker and Vehicle for the Propagation of Mutant Huntingtin Protein

Dr. Giulia Cisbani Post-Doctoral
Fellow / Centre Hospitalier Universitaire de Québec Research Center, Université Laval / Quebec, Canada

giulia_crisbaniTo date, the problem in Huntington’s disease (HD) has been thought to be due to nerve cells producing  their own, genetically coded for, abnormal mutant huntingtin protein (mHtt), which then causes them to dysfunction and die. In recent years, there has been evidence to suggest that other cells, such as those involved in inflammation, may also contribute to the loss of neuronal cells. However, such cells have only been thought of as having indirect effects and not central to the disease process. Recently, though, we have found in HD patients — who were transplanted with fetal tissue designed to replace cells lost to the disease process — that the abnormal mHtt could be seen in the transplant. This mHtt could only have gotten into the transplant from the patient as the grafted tissue was from unaffected donors. This unique observation forms the basis of this application as we now investigate the theory that mHtt can be transferred between cells from circulating immune cells that can get into the brain via leaky blood vessels. If true, this new theory would have wide ranging implications for HD, and similar diseases, and bring with it a totally new therapeutic approach to this currently incurable condition.


Developing a Robust Segmentation Pipeline That Allows for Consistent Trajectory Estimation of Huntington Disease Gene Positive Individuals Across Multiple Longitudinal MRI Sites

Dr. Eun Young Kim
Post-Doctoral Scholar / University of Iowa / Iowa City, Iowa

eun_young_kimThis study aims to improve the brain imaging measures that can be used in future Huntington’s disease  clinical trials. We expect that our improved measures will reduce the number of participants necessary to conduct a clinical trial to determine whether an experimental treatment impacts disease progression. It is well established that accelerated morphological brain changes exist in HD-gene positive individuals. Researchers often characterize disease progression by monitoring these brain changes using MRI scans. Given that HD is a rare disease, studies often require multicenter collaborations in order to have sufficient sample size. Inevitably, longitudinal measures are highly inconsistent due to scanning environment fluctuations over time. These scanner fluctuation effects can be modeled as ‘noise’ presented in MRI measurements. The noise is often influenced by non-biological factors such as difference in MRI manufacturers, scan sequences, and field strength. We will directly address these noises algorithmically. While several approaches have enhanced gross comparison between groups, their high variability within a single participant limits personalized analysis of a participant’s visits over time. We need consistency in MRI-driven measurement to better understand the trajectory in HD gene positive individuals. We have developed a new segmentation approach to simultaneously reduce variability within subjects and between multiple centers. We demonstrate encouraging preliminary results from the software prototype–The method reduces within-subject variability from longitudinal data selected to have a high degree of heterogeneity. The current prototype implementation is so computationally burdensome that it would be impractical for direct application. We will implement an optimized and automated version of the segmentation approach that maintains robustness and will be applicable to large-scale multicenter data sets. Finally, we will validate that the longitudinal brain imaging outcomes are more sensitive to tracking known clinical variables such as motor, cognitive, and behavioral measures. Based on our pilot study, automation of our new protocol will provide a more powerful brain imaging outcome that allows efficient and economical clinical trials.


At-Home Monitoring of Sleep/Wake Cycles of Huntington’s Disease Patients

Dr. Dawn Loh
Research Associate / UCLA / Los Angeles, California

dawn_lohPoor sleep is a common complaint of HD patients. Complaints of poor sleep may stem from an inability to fall sleep, difficulty in staying asleep in bed, and tiredness during the day, with the resultant outcome of feeling unrested and with possible side effects on mood and cognition (e.g. memory and task performance). The daily cycle of sleep and wake is controlled by the circadian system (circa = about,  dian = a day), which we and others have determined is highly disrupted in animal models of HD, suggesting that the genetic cause of HD leads to disrupted circadian rhythms of sleep and wake. A disrupted circadian rhythm not only leads to poor mood and memory, but also has a negative impact on important bodily functions like cardiovascular function and metabolism. We therefore feel it is critical to seek evidence of circadian disruption in HD patients to corroborate our animal model observations. We propose to record daily rhythms in sleep and wake in HD patients and their caregivers using wristwatch-like devices (actiwatch) equipped with sensitive motion detectors that log activity. Patients will be given sleep survey forms and asked to wear the water-resistant actiwatches in their normal daily routine for 2 weeks. What is particularly unique about our proposal is that we will concurrently ask patients to wear commercially available wristband devices with motion detectors that cost one-tenth of the research-quality actiwatches, auto-update to smartphones, and critically, graph their daily activity on smartphone applications. While these devices are not recommended for clinical diagnoses, they will make it considerably easier for patients and clinicians to get a rapid and accurate image of the sleep-wake cycle. What will be critical is to perform such measurements as early as possible in the HD diagnostic process and take steps to prevent sleep-wake and circadian disruption from further affecting the HD disease progression.


A Novel Systems Medicine Approach for HD Biomarker and Therapeutic Target Discovery

Eleni Mina
Ph.D. Candidate / Leiden University Medical Center, Leiden / The Netherlands

eleni_minaHuntington’s disease (HD) is a neurodegenerative disease with the most prominent pathology in the brain. The disease is still lacking a treatment although huge research efforts have been made within the past 20 years that have led to various medication and therapies that can at least help to manage symptoms. This research is mainly based on HD cell and animal models because brain tissue is obviously not easily accessible, cannot be isolated from living patients and does not allow for longitudinal measurements. Peripheral (non-brain) abnormalities, such as weight loss and skeletal-muscle wasting, have been described extensively in HD, both in patients but also in animal models. Studying those changes may give suggestions for the underlying disease mechanisms and for potential biomarkers that can be of use to track disease progression, develop new therapies and measure response to therapy. In this project we follow a novel way of analyzing and integrating diverse kinds of data and reusing existing information to come up with new hypotheses for mechanistic links between the brain and peripheral pathology. In particular, we analyze blood and brain gene expression data from HD patients and controls to identify common signatures for being able to use a highly accessible tissue such as blood for studying the extensive neurodegeneration that occurs in brain. We link significant HD signatures to additional biological and pharmacological knowledge sources to prioritize biomarkers based on their potential as drug targets. Successful employment of samples from living HD patients for the study of disease progression and response to treatment will have a profound impact on the HD research and patient community. Developing a framework for prioritizing hypotheses that will be able to evaluate the potential efficacy of an experiment is of high importance before investing in long and expensive experiments.


Proximal Mutant Huntingtin Protein Interactions That Occur In a Polyglutamine-Length Dependent Manner in Human HD Brains

Dr. Sonia Podvin
Post-Doctoral Fellow / University of California at San Diego / San Diego, California

sonia_podvinHuntington’s disease (HD) is caused by a mutation in the HTT gene, which encodes huntingtin (htt) protein. The mutation expands a part of the htt protein known as the polyglutamine (polyQ) region, and HD patients with longer polyQ regions develop disease symptoms at an earlier age and progress faster to increasingly severe symptoms and death. This project hypothesizes that the increase in polyQ length leads to changes in mutant htt’s ability to bind and function with other proteins, and a change in direct interactions of proteins with mutant htt provides the initial event leading eventually to neuronal cell death/dysfunction and disease onset. This study addresses the unanswered question in the field of what proteins interact with mutant htt to mediate molecular pathways to disease progression Thus, the goal of this project is to identify proteins that preferentially interact with htt depending on polyQ length in human brain tissues from HD patients and normal controls. We will identify both normal protein interactions that are lost as polyQ length increases, as well as abnormal interactions of proteins that prefer to bind only mutant htt. We will determine the effect of increasing or decreasing levels of each candidate protein on neurotoxicity induced by mutant htt in HD cell models; these studies will simulate possible effects of drugs targeting these candidate proteins, helping us determine which proteins merit further study in HD animal models. The ultimate aim is to identify proteins whose interactions with htt influence HD onset and/or progression in patients; such proteins may represent viable targets for future therapies.


Transcriptome Isoform Networks in Huntington’s Disease

Dr. Shihao Shen
Post-Doctoral Fellow / University of California at San Diego / San Diego, California

shihao_shenA variety of normal neuronal cell functions are disrupted by Huntington’s disease (HD). Mutant huntingtin gene, the disease-casual gene of Huntington’s disease, can derail normal cell functions by a domino effect. In which, mutant huntingtin first disrupts the normal functions of a few master regulators; then the disrupting effect is spread to other genes through the master regulators. One type of master regulators are called transcription factors, which regulate the amount of RNA produced from genes (DNA). A second type of master regulators are called splicing factors, which regulate different forms of RNA produced from genes. Since these master regulators control various downstream targets, when mutant huntingtin disrupts one master regulator, it can potentially disrupt the normal functions of the other genes controlled by the master regulator. There is still much to learn about the effect of mutant huntingtin on various master regulators. In this study, we use our expertise in high performance computational analyses to analyze a large RNA deep sequencing dataset of the brain samples of 80 HD patients and 60 controls. We will use the big genomic data to identify the unknown effects of mutant huntingtin on master regulators. Due to the extensive impacts of master regulators, our findings will provide new insights on HD mechanisms and therapeutic targets.


The Role of Complement and Synaptic Loss in Driving Disease Progression in Huntington’s Disease

Dr. Daniel Wilton
Post-Doctoral Fellow / Boston Children’s Hospital / Harvard Medical School / Boston, Massachusetts

daniel_wiltonSeveral studies have shown that the immune system plays an important role in the pathology of Huntington’s disease (HD); however, the underlying mechanisms that cause HD remain unclear. Our preliminary studies using mouse models of HD reveal that complement, a group of immune proteins associated with clearance of dying cells, are elevated in the striatum early in disease progression and localized to synapses. Moreover, microglia, the brain’s resident immune cells, are activated and recruited to vulnerable synapses early in disease progression. During normal development, complement and microglia work together to eliminate unnecessary synapses, which is necessary for correct brain wiring (Stevens et al., 2007; Schaffer et al., 2012). We hypothesize that this immune related elimination pathway is inappropriately reactivated in the HD brain to remove connections between neurons and that this contributes to the cognitive and motor dysfunctions that are hallmarks of this disease. In this proposal we will first establish if complement is elevated in the brains and cerebrospinal fluid (CSF) of HD patients early in disease pathology; and then determine if changes in complement levels can act as a marker of disease progression correlating with synapse loss, brain imaging studies and cognitive/motor tests. Finally, we will test novel complement blocking strategies in mouse models of HD to see if they can slow synapse loss with the hope that they could potentially be developed into therapeutics for human patients.

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