Below are lay summaries submitted for research completed in fiscal year 2019. Each study received a grant from NAF to support their research. To check out more information, click the “+” symbol next to the title of the study. Click the “-” symbol to collapse the lay summary for that study.
Research Seed Money Grants
Research Seed Money Grants are available for new and innovative studies that are relevant to the cause, pathogenesis or treatment of the hereditary or sporadic Ataxias. This type of grant is offered primarily as “seed monies” to assist investigators in the early or pilot phase of their studies and as additional support for ongoing investigations on demonstration of need.
François Berthod, PhD
Study on ARSACS
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a condition characterized by symptoms such as spasticity, ataxia and peripheral neuropathy. It is the second most common recessive ataxia. Studying ARSACS pathophysiology directly with sick neural cells of patients is highly desirable but it is challenging because of the difficulty to access the cells without harming the patients. We and others have shown that it is possible to modify peripheral blood mononuclear cells extracted from blood or fibroblasts isolated from skin biopsies and reprogram them into induced pluripotent stem cells (hiPSC). These hiPSC can then be reprogrammed into neural cells. In our proposal to the NAF, we have proposed to apply this cutting-edge technology to recreate neural cells that mimic ARSACS and that can be studied in the laboratory. Our second objective was to determine whether the ARSACS phenotype can be recreated in patients reprogrammed neural cells in vitro. We report that we have been successful in establishing an ARSACS hiPSC cell line and in reprogramming it into neurons and Schwann cells. We are currently comparing these ARSACS neural cells to healthy one in order to describe an in vitro phenotype of the disease. We have been using a sophisticated tissue engineered approach that allows to recreate in vitro a 3D human tissue innervated with the cells in order to recapitulate the ARSACS phenotype at the motor neuron scale in a physiological microenvironment.
Mirella Dottori, PhD
University of Wollongong
Developing therapies for Friedreich Ataxia using nanoparticles
The major aim of this project is to utilize innovative technologies in bioengineering and stem cell biology to develop an optimal delivery system for delivering Frataxin into the nervous system to treat Friedreich Ataxia.
A critical factor in developing therapies for Friedreich’s Ataxia (FRDA), and many other neurodegenerative diseases, is to identify the most optimal approach for delivering therapeutic molecules into the human nervous system. This is particularly pertinent for some types of proteins, such as Frataxin, or DNA that otherwise cannot be easily taken up by cells.
Advances in bioengineering have developed assembled chemical compounds, called ‘nanoparticles’, that have the capability of encapsulating protein or DNA molecules in a non-viral approach and penetrate into cells. Once inside the cell, nanoparticles can be designed to release their contents, thereby essentially serving a specialized carrier system for delivering therapeutic agents. There are many different nanoparticle types that differ in their physical and chemical properties that influences which cells they can or cannot penetrate into.
The major aim of this project was to determine the optimal nanoparticle type that can penetrate human neurons and deliver its contents, in particular DNA encoding for Frataxin protein. Until recently, this has been a challenge because of access to human brain tissue. With stem cell technologies, we now have the capability of generating sensory neurons from Friedreich’s ataxia induced pluripotent stem cells (FRDA iPSCs). Furthermore, sensory neurons can be cultured in 3D aggregates, called organoids, such that they resemble neuronal-like tissue. Using stem cell-derived sensory culture systems, we performed a screen of different nanoparticle types to determine the physical properties needed to be taken up by sensory neurons and deliver Frataxin DNA. Our results found promising nanoparticle candidates that may be utilized as a non-viral gene therapy strategy to treat FRDA.
These findings were published in the bioengineering journal, ‘Biomaterials Science’1. This data was also used to apply for further funding from other philanthropic and government funding agencies to continue this important research.
We thank the National Ataxia Foundation and all the donors for their support towards our research in developing innovative therapies to treat Friedreich ataxia.
1 Czuba-Wojnilowicz E^, Viventi S^, Howden SE, Maksour S, Hulme AE, Cortez-Jugo C*, Dottori M* and Caruso F* (2020) Particle-mediated delivery of frataxin plasmid to a human sensory neuron model of Friedreich’s ataxia. Biomaterials Science doi: 10.1039/c9bm01757g ^Co-first authors * Co-senior authors
Cherie L. Marvel, PhD
Johns Hopkins University-School of Medicine
Cerebellum and basal ganglia interactions in spinocerebellar ataxia
In this research study, we used functional magnetic resonance imaging (MRI) to examine brain function in people with spinocerebellar ataxia (SCA), Parkinson’s disease (PD), and healthy controls. The primary objective of this research was to study the interactions of the cerebellum, which is affected in SCA, and the basal ganglia, which is affected in PD. Both regions are important for modulating motor function. We hypothesized that these brain regions interacted such that as one region loses its function, the other region helps to compensate for that loss. In the MRI, participants performed a motor tapping task at 1, 2, 3, and 4 Hz speeds. Our data analyses have thus far focused on cerebellar function in the SCA and control groups. Preliminary results indicated that cerebellar function in SCA was compensated not by the basal ganglia, as proposed, but by additional motor regions in the cortex, such as the primary motor cortex and premotor cortex. Importantly, SCA participants were more likely than controls to recruit brain regions on both sides of the brain. This may represent a compensatory mechanism that is used to offset functional loss of the cerebellum. We plan to further examine correlates of the cerebellum and basal ganglia within this dataset in order to better understand how the brain responds to functional loss in SCA. This will help to identify early markers of functional decline, monitor disease progression, and develop targeted interventions in SCA.
Vikram Shakkottai, MD, PhD
University of Michigan
Understanding brainstem dysfunction in SCA1
Cerebellar ataxias, a group of disabling and untreatable neurodegenerative disorders affecting up to 150,000 people in the United States, result in uncoordinated movements and falls, frequently leading to wheelchair confinement, and often premature death. Spinocerebellar ataxia type 1 (SCA1), an inherited form of cerebellar ataxia causes cerebellar and brainstem neuron degeneration. Although the molecular events resulting in cerebellar dysfunction are an area of active investigation, the basis for and the impact of brainstem dysfunction, the likely cause of death in SCA1, remains poorly studied. We examined the basis for brainstem neuronal dysfunction in the inferior olivary nucleus in a mouse model of SCA1. We identified changes in neuronal function in this brainstem nucleus associated with degeneration. Although brainstem inferior olive dysfunction may contribute to degeneration, the motor impairment at this stage of disease is driven primarily by cerebellar dysfunction.
Carlo Wilke (PI), Jeannette Hübener-Schmid, Matthis Synofzik (Co-PIs)
Hertie-Institute for Clinical Brain Research
Neurofilaments as blood biomarkers of SCA3 progression in humans and mice
Spinocerebellar ataxia type 3 (SCA3), the most frequent autosomal‐dominant ataxia worldwide, is a prototypic neurodegenerative repeat‐expansion disorder. As targeted molecular treatments for SCA3 (e.g. antisense oligonucleotides) are coming into reach, easily accessible peripheral biomarkers are warranted, both for human and for preclinical trials. Such biomarkers are particularly important at the presymptomatic disease stage, where disease‐modifying therapies might be most effective, and require cross‐validation in animal models, as well as cross‐validation with associated central nervous system changes.
In two independent multicentric human SCA3 cohorts, blood levels of neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) were each increased at the symptomatic disease stage. NfL levels were increased also at the presymptomatic stage. NfL elevations were present already 7.5 years before the individual expected symptom onset, with levels increasing further in proximity to the conversion from the presymptomatic to the symptomatic disease stage. NfL levels reflected both subjects’ clinical disease severity and disease progression. The neurofilament increases in our human cohorts were paralleled by neurofilament increases in a SCA3 knock‐in mouse model, here also starting already at the presymptomatic stage, closely following the onset of ataxin‐3 aggregation and preceding significant Purkinje cell loss in the brain. These results allowed mapping a larger biomarker cascade of SCA3 disease, capturing differential changes in NfL, pNfH, ataxin‐3 and behavioural biomarker trajectories across disease stages.
Blood levels of neurofilaments, particularly NfL, might provide easily accessible peripheral biomarkers of neuronal damage in SCA3, validated both at the presymptomatic and at symptomatic disease stage in humans and mice and associated with brain pathology changes already at the earliest disease stages. NfL levels might serve as progression, proximity‐to‐onset and, potentially, treatment‐response biomarkers for both human and preclinical trials.
Pioneer SCA Translational Research Award
The Pioneer SCA Translational Research Award is offered for a research project that will facilitate the development of treatments for Spinocerebellar Ataxia.
Beverly Davidson, PhD
The Children’s Hospital of Philadelphia
Gene editing for SCA2 therapy
There are currently no therapies that delay onset or progression of spinocerebellar ataxia. In previous work, we showed that gene silencing approaches had a profound positive impact on disease readouts in two animal models of spinocerebellar ataxia type 1 (SCA1). Additionally, our gene silencing therapies in SCA1 mice reversed behavioral deficits and neuropathology, even when delivered after the onset of disease symptoms. We have also shown that CRISPR-Cas9 gene editing resulted in significant allele-specific silencing of mutant human huntingtin in a Huntington’s disease mouse model.
Here, we evaluated the therapeutic efficacy of AAV-delivered CRISPR-Cas9 gene editing to reduce mutant human ATXN2 in a spinocerebellar ataxia type 2 (SCA2) mouse model. Stereotaxic injection of AAV-CRISPR-Cas9 expression vectors into the deep cerebellar nuclei (DCN) of the cerebellum of SCA2 mice resulted in editing, excision of the CAG repeat, and reduced mutant ATXN2 protein levels in a dose-dependent manner. However, we did not prevent motor coordination deficits in a larger preclinical mouse study. Our data suggests AAV delivery coverage throughout the cerebellum was insufficient to reach efficacious editing levels, and perhaps, non-targeted cells may be contributing to the disease. We are now evaluating efficacy in a different SCA2 mouse model to better understand our AAV-CRISPR-Cas9 gene editing strategies, with the hope of one day translating gene editing therapy to humans with SCA2.
Puneet Opal, MD, PhD
Using VEGF-mimetic nanoparticles to treat SCA
Our laboratory studies spinocerebellar ataxia type 1 (SCA1) that is a neurogenerative disorder where patients first present with symptoms in their third or fourth decade of life. Early symptoms include motor incoordination that becomes progressively worse with patients succumbing to the disease about fifteen years after onset when the airway can no longer be cleared. SCA1 is a polyglutamine disorder, where there is a repeat expansion of the amino acid glutamine in the protein ataxin-1 (ATXN1) causes the protein to have abnormal functions. We found that mutant ATXN1 causes repression of vascular endothelial growth factor (VEGF) that is crucial for maintaining the microvasculature in the brain, especially the cerebellum, the brain region most affected in SCA1. Improper vasculature leads to cell death due to the limited blood flow to the region. We have found that restoring VEGF helps ameliorate the disease in SCA1 mice, and using a novel nanoparticle, VEGF-PA, we were able to effectively treat the mice as seen in the improvement of the motor behavior and tissue samples. Results from this exciting study paves the way for a novel treatment for a currently incurable, devastating disease.
Sokol Todi, PhD
Wayne State University
Mechanisms of aggregation and toxicity in SCA3
The most common dominantly inherited Ataxia in the world is Spinocerebellar Ataxia Type 3, also known as Machado-Joseph Disease. The exact reasons for this disease and therapeutic options for it in the clinic remain elusive. Here, we investigated how the protein that causes SCA3/MJD behaves in an animal, with special focus on a protein-protein interaction mechanism that could be targeted in the clinic to inhibit toxicity.
The protein at the root of SCA3/MJD is known as ataxin-3. We published recently that toxicity caused in neurons by SCA3-causing mutations in ataxin-3 is regulated by one of the proteins with which ataxin-3 interacts. The objectives of this proposal were to: 1) understand how ataxin-3 aggregates and becomes toxic, and 2) devise routes to prevent ataxin-3 aggregation and toxicity for future use in the clinic.
We collected extensive evidence that inhibiting the interaction of ataxin-3 with one of its direct binding partners reduced the toxicity of the SCA3/MJD protein in several models of the disease using the fruit fly, Drosophila melanogaster. We found that a decoy protein that prevents the interaction of ataxin-3 with this protein was strongly protective by reducing ataxin-3 aggregation. Importantly, this approach was not protective in other SCAs, demonstrating the specificity of our approach. Based on this exciting body of work, we are not gearing up to assess the neuroprotective power of our approach in mammalian models of this disease.
Young Investigator - SCA Awards
The Young Investigator – SCA Award was created to encourage young clinical and scientific investigators to pursue a career in the field of Spinocerebellar Ataxia research. It is our hope that Ataxia research will be invigorated by the work of young, talented individuals supported by this award.
James Orengo, MD, PhD
Baylor College of Medicine
Investigating muscle wasting in Spinocerebellar Ataxia, type1
SCA1 is a genetic disorder caused by numerous repeats of an amino acid (glutamine) within the protein called Ataxin1. In SCA1 neurodegeneration occurs in a region of the brain called the cerebellum. The cerebellum is critical for coordinating movements and fine-tuning balance; therefore the hallmark feature of SCA1 is progressive and debilitating incoordination. However, later in life individuals with SCA1 will also develop progressive muscle weakness, a symptom not associated with the cerebellum. The muscles especially affected are those that support safe breathing and swallowing. In fact individuals of SCA1 often pass away prematurely due to complications of aspiration pneumonia, which are directly related to the weakening of these muscles.
Progressive weakness of the muscles supporting breathing and swallowing is a common symptom in diseases with degeneration of motor neurons. The most common disease within this class is amyotrophic lateral sclerosis (ALS) or Lou Gehrig’s disease. Motor neurons are responsible for triggering the voluntary contraction of their target muscles. When motor neurons die they leave behind orphan muscles that subsequently, under the lack of direction, shrink and become weak. Given that SCA1 patients ultimately succumb to symptoms common in diseases involving degeneration of motor neurons, I came up with the hypothesis that motor neuron degeneration occurs in SCA1 and drives premature death. Indeed, I found that the major mouse model of SCA1, recapitulates these findings.
The SCA1 mice have a progressive respiratory dysfunction and it is due to weakness of the muscles supporting breathing. The main muscle supporting respiration, is the diaphragm, and I found that this muscle shows features of having lost motor neuron input. The motor neurons that control the diaphragm live in the cervical spinal cord. When I looked at motor neurons in this area, I found these neurons degenerated and neuronal scaring was taking place. Altogether this data supports my hypothesis that the SCA1 mice pass away prematurely from respiratory dysfunction and that this correlates with motor neuron degeneration.
Yalan Zhang, MD, PhD
Investigating the molecular mechanism of neurodegeneration in spinocerebellar
Spinocerebellar Ataxia type 13 (SCA13) is a human autosomal dominant disease that results in degeneration of the cerebellum. It is caused by mutations in the gene encoding Kv3.3, a protein required for the normal electrical excitability of neurons in the cerebellum. An unusual property of Kv3.3 is that it binds another protein, Hax-1, a so-called “survival” protein that is absolutely required for the survival of the cerebellum. However, our previous data has shown that the mutant G592R Kv3.3 channels have an abnormal interaction with the Hax-1. To examine further how this abnormal interaction leads to neuronal degeneration, we generated a strain of mice that bear the G592R Kv3.3 mutation, using Crispr Cas9 gene editing technology. We then analyzed these mice using proteomic approaches to discover which molecular signaling pathways are altered in the brains of the mutant animals. We found that the activity of one specific enzyme termed Tank-Binding protein kinase (TBK1) is much higher in the cerebellum of the mutant G592R Kv3.3 knock-in mice. This effects is specific to the cerebellum because there was no change in the frontal cortex. Moreover, using a technique termed Western blotting, we determined that, while the level of the active (phosphorylated) form of TBK is increased in cell lines and in cerebellar neurons expressing the mutant G592R Kv3.3 protein, total levels of the TBK1 enzyme do not change. The aim of this project is to investigate the mechanism of how overactivation of TBK1 by the mutant G592R Kv3.3 channel leads to neurodegeneration. This work will provide new insights into the development of potential new drugs that target the Kv3.3/TBK1 pathway.
Young Investigator Awards
The Young Investigator Award was created to encourage young clinical and scientific investigators to pursue a career in the field of Ataxia research. It is our hope that Ataxia research will be invigorated by the work of young, talented individuals supported by this award.
Susana Maria D. A. Garcia, PhD
University of Helsinki
Study on RNAs Contribution to SCA Pathogenesis
Spinocerebellar ataxias (SCAs) comprise more than 40 autosomal dominant neurodegenerative diseases that predominantly affect the cerebellum and brainstem. Among these, the SCAs caused by CAG repeat expansions located in coding regions of specific genes are the most common and comprise SCAs 1-3, 6, 7, 17 and dentatorubral-pallidoluysian atrophy (DRPLA).
The CAG repeats in SCAs encode for a polyglutamine (polyQ) tract and expansions result in the formation of an abnormally long polyQ chain whose altered conformation leads to protein aggregation and plays a key role in disease pathogenesis. However, these are complex disorders and different toxic mechanisms have been identified as contributing to disease pathogenesis. In addition to protein aggregates, expression of these ‘expanded’ CAG repeat sequences with production of transcripts (RNAs) bearing long CAG repeats is known to cause a number of cellular disruptions, yet the mechanism of dysfunction or how these RNAs contribute to SCA toxicity is not well understood.
We used the nematode Caenorhabditis elegans to generate a set of stable model strains that recapitulate SCA RNA toxicity phenotypes separately from the polyQ protein phenotypes. These strains exhibit toxicity, by loss of motility, associated with the expression of RNAs bearing expanded repeats. An assay was optimized for the identification and adaptation to semi-high throughput genetic screening of factors responsible for the regulating RNA toxicity in SCA neuronal dysfunction. This method will be used in our laboratory to dissect how toxic RNAs contribute to SCA pathogenesis. Finally, preliminary data obtained using these model systems suggested new mechanisms contributing to SCA toxicity.
Minggang Fang, PhD
University of Massachusetts Medical School
Identification of druggable FXN repressing factors and small molecules to treat Friedreich Ataxia
Friedreich ataxia (FA) is caused by a peculiar defect in the FXN gene in which a short tract of DNA is repeated hundreds of times, the result of which is to effectively turn down (or “repress”) the FXN gene to a very low level. The FXN gene normally encodes a protein called frataxin that is present in a specific compartment inside cells called the mitochondria, the so-called “energy powerhouse” of the cell. In individuals with FA, repression of the FXN gene leads to reduced levels of frataxin, and the resulting damage to the mitochondria causes injury to the nervous and cardiac systems. Importantly, even though the defective FXN gene leads to reduced frataxin levels, the frataxin protein that is produced is completely normal. Therefore, one potential strategy for treating FA is to increase (or upregulate) expression of the defective FXN gene to normal levels. As an initial step toward developing drugs that can upregulate the defective FXN gene, we have identified proteins that are involved in repressing the defective FXN gene (called FXN–Repressing Factors or FXN-RFs). We then identify drugs that can inhibit the function of the FXN-RFs, enabling the defective FXN gene to be upregulated. Through our work, we have identified a very promising new class of FXN-RFs for which approved drugs that block the function of the protein already exist and have been found to be safe and well tolerated in clinical trials. Over the past year, we have provided evidence that these drugs can upregulate the defective FXN gene in FA neurons cultured in the laboratory and can restore normal mitochondrial function in FA neurons and cardiomyocytes. In addition, we have obtained exciting preliminary evidence that these drugs can upregulate the defective FXN gene and restore mitochondrial function in the heart and brain of mice that have FA. It is our hope that these drugs can be rapidly moved forward in clinical trials for FA.
Parthiv Haldipur, PhD
Seattle Children’s Hospital
Characterization of early human cerebellar development
Spinocerebellar ataxia (SCA) is a group of hereditary incurable ataxias that primarily affect a part of the brain called the cerebellum. The cerebellum is located at the back of the head and responsible for control of movement and motor coordination. Recent studies have shown that a neuronal subtype within the cerebellum called Purkinje cells, is especially vulnerable to ataxias. These cells are among the largest neurons in the human brain and are conspicuous by their characteristic branching of dendrites. They form an integral part of the brain circuitry that controls motor coordination. It is hence not surprising that degeneration of this important cell type results in ataxia.
Our understanding of the development of Purkinje cells and ataxias is largely based on studies carried out in animal models, including the mouse. It is assumed that a significant portion of brain development is conserved between the mouse and human. In other words, development patterns are likely similar between the two species. We carried out a thorough histological characterization of the developing human cerebellum and found striking differences in developmental patterns between the mouse and human, including in a region called the ventricular zone, where Purkinje cells are born. Additionally, we also observe differences in migratory patterns of Purkinje cells also differ between the mouse and human. We also see the presence of a new stem cell niche not found in the mouse.
A common problem with mouse models of disease is that they do not recapitulate the human condition. This is true even of mouse models of ataxia. We believe the reason for this could very well be due to the existence of differences in cerebellar and Purkinje cell developmental patterns between the mouse and human.
We have performed a thorough characterization of early human cerebellar development that resulted in a high impact publication in the journal Science. This paper was well received by the scientific community and has been shared extensively on social media. The study was also featured on Forbes. We have since studied the timing of birth of Purkinje cells in the developing human cerebellum and their subsequent migration to their final location within the cerebellum. A manuscript from this study is currently being drafted. We have also performed single cell RNA sequencing at multiple stages of human development to identify pathways critical to the development of Purkinje cells. This work will be submitted for peer review shortly. Our study has enabled us to identify human-specific features in human cerebellar and Purkinje cell developmental programs. Our data will also represent an important resource to benchmark developmental stage-, cell type-, and molecular-specificity of existing ataxia model systems.
Jarmon Lees, PhD
St Vincent’s Institute of Medical Research
Investigating vascular dysfunction in Friedreich’s ataxia using human induced pluripotent stem cells
Friedreich’s ataxia (FRDA) is a common inherited neuromuscular disorder, caused by a GAA repeat mutation in the Frataxin gene resulting in degeneration of neurons and heart disease. Heart disease is the leading cause of death in FRDA. Interestingly, the heart biopsies of FRDA patients often show involvement of diseased blood vessels in the heart, yet this has never been examined in detail. Clinical reports have documented abnormalities in the blood vessels of the heart in FRDA patients, in which the cells that make up the blood vessels (endothelial cells and smooth muscle cells) proliferate and migrate uncontrollably until they block the blood vessels, reducing blood flow and nutrient supply, and likely contributing to heart disease. The overall objective of this study was to examine the mechanisms underlying the diseased blood vessels in FRDA. To do this, induced pluripotent stem cells (iPSCs) were generated from FRDA patients (FRDA-iPSCs), and control-iPSCs were created using the gene-editing tool CRISPR-cas9 to remove the GAA repeat mutation. FRDA-iPSCs and control-iPSCs were transformed into endothelial and smooth muscle cells. Compared to controls, FRDA endothelial cells have reduced ability to form vessels and higher levels of reactive oxygen species. Similarly, FRDA smooth muscle cells also have higher levels of reactive oxygen species, with reduced ability to proliferate and a higher degree of migration. These results suggested that mutation of the Frataxin gene can cause dysfunction in vascular cells and may contribute to heart disease in FRDA. These pre-clinical human vascular cell models are a promising platform to develop novel treatments for FRDA-associated vascular disease.
Martine Tetreault, PhD
Centre Hospitalier de l’Université de Montréal
Defining the genetic etiology of late-onset episodic ataxias
The identification of the genetic causes and mechanisms associated with rare Mendelian diseases has always been an important challenge for the medical field. The identification of mutations and/or genes associated with a disease has forever been driven by technological progress. High-throughput DNA-sequencing technology has led to the identification of a large number of disease-causing variants or genes. Although, this technology succeeded in improving the diagnosis of rare diseases, there is a significant number of patients that remain without a molecular diagnosis. For most patients, a diagnostic test will be inconclusive since many candidate variants with no clear functional impact are identified. Clinical tests are usually targeting a single gene or a panel of genes and thus are not able to identify variants in potential novel-disease genes. Moreover, our current knowledge and tools available limit our capacity to interpret these variants. For these reasons, the diagnostic yield is reported to be 25-30% in several large studies. The integration of functional genomic data, such as RNA-sequencing, is gaining in popularity to improve variants interpretation and increase our knowledge of molecular mechanisms associated with diseases. In addition to the technical limitations, neurological diseases complexity is increased by the high variability in clinical presentation and genes involved. This variability is not only observed between unrelated patients but also within a single family. The study of late-onset diseases is even more challenging since the familial history is often unclear preventing the identification of additional affected cases that could confirm the genetic nature of the disease as well as the mode of transmission. Episodic ataxias are a group of neurodegenerative diseases characterized by recurrent attacks of ataxia in which high variability is observed. To date, eight forms have been described but there are still patients who remain without a known genetic cause. In this project, we proposed to combine genomic and transcriptomic data to identify the genetic cause in a cohort of unresolved late-onset episodic ataxia patients. We performed Whole Genome Sequencing (WGS) and RNA-sequencing on eight patients from seven families. Using a bioinformatic pipeline, we obtained sequence variants, repeat expansions, alternative splicing events and differential expression data. We first focussed our analysis on know ataxia genes to identify variants that could have been missed with previous diagnostic approaches, such as gene panels. On the remaining cases, we will expand our analysis to novel candidate genes. Using this approach, we have been able to detect a large spectrum of possible aberrations and find variants in know ataxia genes that were not identified using diagnostic gene panels, either because the phenotypes are atypical or because of limitation of the approaches. Our results will have a direct impact on patients by increasing the diagnostic yield of episodic ataxia as well as contributing to our understanding of the biological mechanisms leading to the disease. The identification of genetic causes as well as deregulated pathways will ultimately lead to the development of novel therapeutic strategies.
Post Doc Fellowship
Post-Doctoral Fellowship Awards are to serve as a bridge from post-doctoral positions to junior faculty positions. Recipients have shown a commitment to research in the field of Ataxia.
Geena Skariah, PhD
University of Michigan
Endogenous tagging of RAN peptides in Fragile X-associated Tremor/Ataxia Syndrome
Fragile X associated Tremor Ataxia Syndrome (FXTAS) is an inherited neurodegenerative disorder seen primarily in older males. FXTAS patients develop ataxia, action tremor and dementia and the condition has no effective therapies. FXTAS is caused by the expansion of CGG repeats at the start of the FMR1 gene. These repeats undergo an unusual process called Repeat-Associated Non-AUG (RAN) translation giving rise to toxic proteins. While these proteins can be seen as part of large inclusions in patient neurons; we cannot readily track the endogenous protein from the FMR1 locus precluding accurate quantitation of RAN translation. This study aims to address this issue by using CRISPR-Cas9 technology, which allows precise editing at the genomic locus. Using this approach, we will add small, highly sensitive and quantitative tags at the endogenous FMR1 locus in FXTAS patient cells. Preliminary data suggests that these approaches are viable in human cells. We were able to show proper insertion of two different tags at the FMR1 locus in FXTAS patient stem cells. Future studies will address issues regarding repeat protein expression and detection. Once these tags are incorporated into patient neurons with detectable protein expression, we will use this tool to validate specific chemical and genetic modifiers of RAN translation with the goal of identifying targetable drug-development mechanisms.
Qiong Song, PhD
University of California San Diego
Unveiling molecular machinery of primary cilia in a congenital cerebellar ataxia (Joubert Syndrome)
Joubert Syndrome is one of the most common recessive congenital ataxia caused by genetic mutation. The genes mutated in this disease encode proteins related to a tiny antenna-like structure on the cell surface. These antenna-like structures are called primary cilia. Primary cilia play critical roles in development as a signal hub for communication between cells. Our previous finding indicates many Joubert Syndrome casual genes code for ciliary proteins located at the boundary between cilia and the cell membrane. This region acts as a gate, controlling signaling proteins moving in and out of cells. However, the structure of this gate and how it functions is poorly understood.
State-of-art electron microscope tomography was used to visualize the ciliary gate. We chose a cluster of human neural cells as a study model, as it mimics gastrulation in human development. Our data revealed two novel 9-fold symmetric structures at the ciliary gate. Those two structures, together with known ciliary transition fibers and microtubules, offer a sieve-like gate for proteins moving in and out. We also obtained 3D structural information of the ciliary gate in ultra-high resolution (1.2 nanometer) during several stages of the cell cycle. These rich temporal spatial data significantly improved our understanding of the structure and molecular machinery of the ciliary gate. We hope this knowledge will shed light into the understanding of pathology of Joubert Syndrome, and lead to potential treatment.