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Your Dollars At Work:

A Look at NAF Funded Research

The following are lay summaries from research projects that NAF was able to fund because of generous contributions from our donors. All of these research summaries are of grants funded by NAF for fiscal year 2018. Thank you to each of you who made a donation to last year’s Research Drive “Proud Past… Focused Future.”

Unless you are a scientist, these research summaries can seem like “Greek” to you, however, it does demonstrate the complexity of science, particularly neuroscience. These summaries were submitted directly from the researchers. While they may be difficult to read, we at NAF think it is important to keep you up-to-date on the science that your membership and donations support.


Deciphering the pathophysiology of SCA35

Manuela Basso, PhD
University of Trento
Trento, Italy

Spinocerebellar ataxia type 35 (SCA35) is a rare, autosomal dominant neurodegenerative disorder associated with mutations in TGM6 gene that encode the protein transglutaminase 6 (TG6). In the last years several new mutations have been reported as associated to SCA35 but the mechanisms inducing neuronal degeneration are still uncovered. We previously showed that the toxicity is both linked to a loss and a gain of function of the mutant protein but the neuronal role of the TG6 is still not known. To uncover TG6 function, we generated a knockout animal model that shows early mild signs of motor and coordination impairment. The generation of an in vivo model of SCA35 can be of use to better understand the molecular mechanisms induced by TG6 loss of function and find new therapeutic avenues for this disease.

Investigating the connection between DNA damage repair and nervous system maintenance in a Drosophila model of SCAN1 disease

Valeria Cavaliere, PhD
Università di Bologna
Bologna, Italy

The main goal of our project is the development of a powerful genetic model to investigate pathogenesis of spinocerebellar ataxia with axonal neuropathy-1 (SCAN-1) disease. Patients affected by SCAN-1 develop an adult onset devastating pathology characterized by peripheral axonal motor, sensory neuropathy, distal muscular atrophy, pes cavus and steppage gait. The genetic alteration causative of SCAN-1 is a mutation altering the function of a gene called tdp1 (tyrosyl- DNA phosphodiesterase 1). The product of this gene is an enzyme that plays a key role in DNA repair processes whose defective activity causes failure to reseal broken DNA strands causing neurodegeneration. The tiny fruit fly, Drosophila, is an organism extremely useful for studies on human biology, health and a wide range of pathologies including neurodegenerative diseases. This is because Drosophila genes controlling fundamental cellular functions, such as cell growth and death, are quite identical to those found in human cells. We plan to obtain a Drosophila model of SCAN- 1 disease by applying a well-known genetic approach. This focuses on the expression of the human tdp1 mutant gene in brain tissue of flies lacking the endogenous Gkt protein. This approach would create a fruit fly model that mimics the human pathological condition. Genome-wide transcriptome analysis of this SCAN-1 fly model will get insight into the mechanism of the disease. The identification of the steps of the SCAN-1 pathological cascade in turn will help the development of therapies targeting key molecules acting in these steps.

Apoptosis-Related Genes BCL2, BAX And TP53 As Biomarkers Of Machado-Joseph Disease (MJD/SCA3)

Manuela Lima, PhD
University of the Azores

Despite undeniable progresses in the knowledge concerning the molecular pathology of Machado- Joseph disease (MJD)/Spinocerebellar ataxia type 3 (SCA3), therapeutic compounds remain to be discovered and validated. Interventional trials face several obstacles, namely those related with the clinical outcome measures used, which lack sensitivity for slow-progressing diseases such as MJD, and are devoid of utility in the preclinical stage, a time where molecular alterations are known to be already present. Identification of molecular biomarkers, accessible in a peripheral tissue such as the blood, is therefore of particular importance to allow the fine tracking of disease progression, starting at the preclinical stage, thus facilitating the detection of subtle therapeutic benefits during interventional therapeutic trials. Furthermore, once ameliorating drugs are available, molecular biomarkers could help identifying the molecular onset of disease and aid in the therapeutic strategy, since such drugs are expected to be more efficient if administrated to mutation carriers before overt disease. Building on promising preliminary results, the goal of this project is to validate the apoptosis-related genes as peripheral molecular biomarkers of MJD and disease progression. Expression of these genes will also be studied in brains of MJD patients, where we expect a similar expression pattern to that seen in blood samples. Drug discovery relies on the translation from animal model studies to patient trials - confirming the similarity of molecular alterations occurring in animal models of disease with those observed in patients is therefore of high relevance. To ensure translatability of preclinical trials using YACMJD84.2 transgenic mice we will assess the expression of the apoptosisrelated mouse homologue genes in blood and brain tissues from these commonly used mice in such trials. As YACMJD84.2 express the human disease target and replicate the disease we expect to observe similar expression alterations to the ones found in MJD patients. Here, our goal is to hopefully contribute to include the assessment of expression levels of apoptosis-related genes in a set of outcome measures for MJD and to a better definition of the molecular onset of this disease. Furthermore, understanding alterations in the intrinsic apoptotic pathway, particularly in the brain, could be valuable to pinpoint novel therapeutic targets for MJD.

HAX-1 is a molecular biomarker for cardiomyopathies in Friedreich’s Ataxia

Florence Malisan, PhD
University of Rome “Tor Vergata”
Rome, Italy

Frataxin deficiency, responsible for the hereditary disease Friedreich’s Ataxia (FRDA), is crucial for cell survival as it critically affects viability of neurons, pancreatic beta cells and cardiomyocytes. The heart is affected in approximatively two thirds of FRDA patients with typical manifestation of hypertrophic cardiomyopathy, which can progress to heart failure and death. Except for frataxin, very few genes have been correlated with ataxia and cardiac disease in FRDA. To investigate FRDA pathogenesis, we conducted a wide expression analyses on cells derived from a FRDA patient and we observed HAX-1 as the highest over-expressed gene. HAX-1 is highly expressed in the heart and belongs to a family of proteins involved in the protection of cardiomyocytes from cell death. Frataxin and HAX-1 are therefore both involved in cell death regulation, a mechanism underlying the progression of cardiomyopathies. We further analyzed frataxin and HAX-1 expression level in blood cells from FRDA patients and controls and we found a positive correlation, i.e. in FRDA patients, the levels of frataxin and HAX-1 were lower than in controls. This correlation was stronger when we compared frataxin and HAX-1 levels in FRDA patients showing cardiomyopathy. This project is aimed to confirm HAX-1 expression as new molecular biomarkers to enhance a personalized medicine for cardiomyopathy and to render wise decisions to families being evaluated for the presence or absence of this potentially lethal yet treatable cardiac disorder. The role of biomarkers to predict the onset of future cardiomyopathy, to identify its presence when fully developed, to risk stratify affected patients, and possibly to serve as a biological tool to guide therapy for cardiomyopathy is indeed fundamental. Another aim of this project is to analyze the expression level of circulating molecules such as microRNAs in FRDA and to investigate their link to HAX-1 expression and regulation. This information will be useful in terms of prognosis and therapeutic impact.

Disclosure of genetic information within families affected by hereditary ataxia

Weiyi Mu, ScM, CGC
Johns Hopkins University
Baltimore, MD

People with hereditary ataxia face the complex and difficult challenge of communicating their diagnosis and genetic risk information to family members. Communication about an inherited condition implicates unaffected family members as well as future children – information which may have an impact on important life decisions. Previous studies have found that early disclosure and an open family communication style can help in family members’ development of effective coping strategies, adaptation to the genetic condition, and emotional well-being. While studies have found that many people want to disclose genetic risk information to their family members, some people encounter barriers that can cause them to delay or avoid disclosure. While a number of qualitative studies have explored disclosure behavior in families with other neurological conditions, this process has not been described in families with ataxia. The purpose of this study is to explore disclosure behavior and to identify factors that affect the communication of diagnosis and genetic risk information within families affected by hereditary ataxia. This study also aims to identify barriers, if any, to the disclosure process for this population, and to explore people’s attitudes about their disclosure decisions. The results will be critical to understanding the experience of family members about genetic risk, and in predicting how health professional and disease advocacy groups can help facilitate better genetic risk communication for families.

Phenotypic Characterization of Mouse Models of Infant- and Adult-Onset SCA13

Diane Papazian, PhD
Los Angeles, CA

The goal of this project is to characterize the phenotypes of three unique, newly-generated knock-in mouse models of spinocerebellar ataxia type 13 (SCA13). SCA13 is caused by mutations in the KCNC3 gene, which encodes the Kv3.3 voltage-gated potassium channel. Voltage-gated channels such as Kv3.3 control the passage of ions across cell membranes. In the nervous system, voltage-gated channels are responsible for generating action potentials, which are rapidly moving electrical signals that underlie brain function. Kv3.3 is highly expressed in cerebellar neurons, where it regulates action potential firing. Different Kv3.3 mutations cause two clinical forms of SCA13. In one form, SCA13 emerges in infancy or early childhood, characterized by cerebellar atrophy early in life, motor delay, persistent motor deficits, and intellectual disability. In the other form, SCA13 is an adult-onset disease characterized by progressive ataxia and progressive degeneration of the cerebellum. Our lines of knock-in mice carry distinct Kcnc3 point mutations, R421H, R424H, and F449L, which correspond to the human mutations R420H, R423H, and F448L, respectively. In humans, R420H causes late-onset SCA13, whereas R423H and F448L cause the early-onset form of the disease. The new mouse models provide a novel opportunity to investigate mechanisms that determine the age of onset and trigger cerebellar degeneration and ataxia in SCA13. Such studies are important for identifying new therapies for the disease. Our preliminary data, obtained in zebrafish, a lower vertebrate, suggest that earlyand late-onset mutations have differential effects on action potential firing that trigger the agedependent degeneration of cerebellar Purkinje cells. The knock-in mice now make it feasible to test this hypothesis in a mammalian model system. With support from the National Ataxia Foundation, I propose to begin characterizing the phenotypes of the three lines of SCA13 mice by accomplishing the following Specific Aims: 1) to investigate motor performance at different ages using standard behavioral assays; 2) to investigate age-dependent changes in cerebellar anatomy and the viability of Purkinje cells; and 3) to characterize the effects of SCA13 mutations on action potential firing in Purkinje cells.

National Ataxia Database

Susan Perlman, MD
University of California at Los Angeles
Los Angeles, CA

The Natural History Study of and Genetic Modifiers in Spinocerebellar Ataxias ( 18 | Generations Fall 2017 Identifier: NCT01060371), under the direction of Dr. Tetsuo Ashizawa, continues to recruit subjects and monitor changes in their neurologic examinations. The participating 15 sites see and examine subjects and enter data into the National Ataxia Database (housed at UCLA under the direction of Dr. Jeanette Papp in the Department of Genetics). The Database has served as a valuable repository for data collected in this important collaborative project that has enrolled over 500 individuals with Spinocerebellar Ataxia types 1, 2, 3, and 6. It will now be adding individuals with types 7, 8, and 10. There are over 15,000 patient forms entered in the National Ataxia Database.

Nuclear-mitochondrial pathways of neurodegeneration in cerebellar ataxia

Catarina Quinzii MD
Columbia University Medical Center
New York, USA

Autosomal recessive cerebellar ataxias are heterogeneous neurodegenerative diseases, characterized by incoordination of movement and unsteadiness, due to cerebellar dysfunction. Cerebellar ataxia has emerged as the most common clinical presentation of deficiency of Coenzyme Q10 (CoQ10), a vital molecule required for cells to generate energy and to prevent damage from toxic oxygen radicals. The proposed studies will define the causes underlying CoQ10 deficiency in cerebellar ataxia, and its role in neurodegeneration, and may lead to the identification of novel therapeutic targets. The results of our studies may provide important information relevant also to other neurodegenerative diseases.

Characterization of the disease phenotypes of Ataxia Telangiectasia patient Purkinje cells in vitro

Mustafa Sahin, MD, PhD
Boston Children’s Hospital
Boston, MA

Ataxia-Telangiectasia (A-T) is a rare genetic disorder that is caused by mutations in ATM gene. There are currently no treatments for the cerebellar deficits that cause motor dysfunction and balance problems in the A-T patients. Main output neuron type of the cerebellum are the Purkinje cells, which are GABAergic inhibitory neurons that are innervated by cerebellar granule cells and to project to deep cerebellar nuclei that regulate motor coordination and balance. Previously the restricted availability of human brain tissue for medical research has limited the detailed characterization of the causes of cerebellar degeneration and neurological deficits in the A-T patients. To overcome this limitation, researchers have recently developed new cellular model called human induced pluripotent stem cells (hiPSCs), which can be derived from human somatic cells and converted in a dish into different cellular types, like different neuronal cell populations. These hiPSC derived neurons facilitate the studies of patient specific neurons in laboratories, and allow identification of molecular mechanisms behind the development of various neurological diseases, including A-T. Previously the Sahin lab has developed a novel protocol to differentiate the hiPSC into cerebellar precursors and Purkinje cells. In addition, the Lerou lab has discovered genetic abnormalities in control of NRXN1 expression in the human neuronal precursor cells. To discover the molecular causes for the development of cerebellar dysfunction in A-T patients, we propose to study functional properties of the A-T patient derived hiPSC-derived Purkinje cells compared to healthy control Purkinje cells, and study differentially regulated genes in these cells. Our study will provide valuable insights into the molecular mechanism and potential targets that can be utilized in the future for development of new therapies for the neurological deficits of the A-T patients.

Suppression of polyglutamine aggregation by a new class of chaperone

Matthew K. Scaglione, PhD
Medical College of Wisconsin
Milwaukee, WI

We all have proteins that contain repeats of the amino acid glutamine. However, some individuals have an expansion of these glutamines resulting in very long glutamine repeats. These long glutamine repeats cause the polyglutamine diseases including Spinocerebellar ataxia types 1, 3, 6, 7, and 17. ln these diseases the long strings of glutamine cause proteins to clump up and this results in neuronal death. We have recently shown that the ameoba, Dictyostelium discoideum, normally expresses very long glutamine tracts and is naturally resistant to clumping up of proteins with long polyglutamine repeats. More recently we have identified a single protein that is only found in Dictyostelium that is responsible for suppressing the clumping of these polyglutamine proteins. Here we propose to figure out how this protein prevents the clumping of proteins with long polyglutamine repeats, and produce a mouse that expresses this Dictyostelium protein to determine if it can suppress the underlying cause of polyglutamine diseases including SCAI ,3,6,7, and 17.

Modulation of neuronal proteostasis by serotonin: impact on SCA3 pathogenesis

Andreia Teixeira-Castro, PhD
University of Minho
Braga, Portugal

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is a neurological disorder affecting the specific areas of the brain and the spinal cord of patients. The first symptoms usually start around middle age and include difficulty in walking, due to lack of coordination and balance. Other complications, such as problems with vision, speech, and swallowing may also occur. This is a progressive disease and many patients ultimately need a wheelchair or become bedridden, as there is no treatment currently available. We have previously found that chronic treatment with an antidepressant very strongly improves SCA3-like symptoms in model organisms of the disease. Worms (C. elegans) and mice expressing the mutant human gene and protein display abnormal movement and lack of balance and coordination. Antidepressant treatment reverted these phenotypes and decreased the presence of aggregated ataxin-3. Our next step will be to understand what is the drug doing in the brain that protects against disease. We have recently found that antidepressant treated cells show increased expression of protective genes that enhance protein homeostasis capacity and reduce the risk of formation of aggregated mutant protein. Here, we propose to study in which neurons this protective mechanisms are activated and to study this augmentation in protein homeostasis aptitude as a therapy for SCA3. This study will help us to decipher a series of events that occur in the brain of SCA3 animals and are prevented when the animals are treated with the drug. A better understanding of those events may help the future development of clinical trials in patients and define novel therapies. Besides being useful for SCA3 patients, this antidepressant therapy may also be important for patients suffering from other neurodegenerative disorders, hence it is essential to clarify its mode of action, before advancing to clinical trials.

Function of ATM in Neuronal Survival in Response to Growth Factor Signaling

Da-qing Yang, PhD (In partnership with the Bob Allison Ataxia Research Center)
University of Minnesota
Minneapolis, MN

Ataxia-telangiectasia (A-T) is a neuronal degeneration disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasias, and variable immunodeficiencies. The gene mutated in this disease, ATM (A-T, mutated), encodes a protein kinase. The main characteristic of A-T disease is the progressive neuronal degeneration of cerebellar Purkinje and granular cells. Patients with A-T also exhibit various symptoms ranging from insulin resistance, glucose intolerance, to growth retardation. Many of them also develop type 2 diabetes at an early age. However, the reason for progressive neuronal degeneration in A-T patients remains unclear. ATM is traditionally considered a nuclear protein and a common hypothesis is that defective nuclear function of ATM in response to DNA damage is responsible for neuronal degeneration of A-T. However, it is known that ATM is predominantly in the cytoplasm in human Purkinje cells and mouse cerebellum neuronal cells. Our previous findings have shown that cytoplasmic ATM is an insulin responsive protein that stimulates Akt phosphorylation. We also discovered that in response to insulin, ATM protects differentiated human neuron-like SH-SY5Y cells from serum starvation-induced apoptosis. Based on these results, we hypothesize that ATM, through activation of Akt, promotes neuronal survival in response to insulin and other neural growth factors. We have recently compared the functions of ATM in proliferating and differentiated human neuron-like SH-SY5Y cells. Our results clearly show that ATM switches function from a sensor of DNA damage in proliferating cells to a mediator of growth factor signaling in differentiated human neuron-like cells. Our results further demonstrate that ATM mediates Akt signaling and promotes cell survival in differentiated SH-SY5Y neuronlike cells, which suggests that impaired activation of Akt, rather than defective response to DNA damage, is the reason for neuronal degeneration in human A-T. The goal of this proposal is to further examine the functional link between the ATM protein kinase and neuronal survival in response to insulin and other neural growth factors. Findings from this project may provide novel insights into the role of ATM in the progressive cerebellar ataxia observed in patients with A-T. Furthermore, as many of the ataxia or neuronal degeneration disorders are closely related to insulin resistance and type 2 diabetes, our research may provide an overall better understanding of the ataxia and neuronal degeneration process and may lead to the discovery of novel therapeutic strategies and agents not only for A-T but also for other ataxia or neuronal degeneration disorders.


Elucidating the mechanism of novel spinocerebellar ataxia gene TMEM16K in endolysosomal maturation

Maja Petkovic, PhD
University of California San Francisco (UCSF)
San Francisco, CA

Mutations in the evolutionarily conserved endoplasmic reticulum (ER) protein TMEM 16K are causative for autosomal recessive spinocerebellar ataxia (SCAR10), a debilitating progressive neurodegenerative disease. I have discovered that TMEM16K acts as a critical mediator of endolysosomal maturation. Abnormal protein degradation due to endolysosomal dysfunction has been implicated as the primary contributor in multiple neurodegenerative diseases. This proposal seeks to uncover the molecular mechanics of how TMEM16K and its interactome mediate endolysosomal maturation in aging neurons to provide insight into the pathophysiology of neurodegeneration and could open new avenue for therapeutics.

Uncovering pathogenic mechanisms of cerebellar atrophy in SNX14 deficiency

Navara Aquizu Lopez, PhD
University of Pennsylvania
Philadelphia, PA

Cerebellar ataxias are a heterogeneous group of disorders characterized by imbalance and poor coordination due to impaired function or damage to the cerebellum, which regulates balance and movement. The condition is often genetically inherited and affects ~9 per 100,000 individuals. Although more than 100 genes have been implicated so far, disease mechanisms for most of the cerebellar ataxias are still unknown and treatment options unavailable. This highlights the need for further studies that will provide better understanding of disease mechanisms and advance in treatment options. Spinocerebellar ataxia autosomal recessive 20 (SCAR20), is a particular form of cerebellar ataxia diagnosed early after birth and presenting with progressive shrinking of the cerebellum, intellectual disability and dysmorphic facies. Our recent work identified that mutations in the gene Sorting Nexin 14 (SNX14) are the cause of SCAR20. SNX14 is involved in regulating the function of lysosomes, which are the cellular compartments responsible for the degradation of unwanted materials. Lysosome dysfunction has previously been associated with cerebellar degenerative diseases. However, it is not well understood how lysosome dysfunction leads to cerebellar damage. In order to uncover how SNX14 mutations lead to lysosome dysfunction and cerebellar ataxia, we have generated a mouse model that shows impaired movement reminiscent of patients with SCAR20. Our current work aims to characterize the type of cerebellar damage present in these mice and to look for sign of lysosome dysfunction that may be present in their cerebellum. Future work will be focused on seeking for cellular materials that may toxically accumulate in SCAR20 cerebellum due to lysosome dysfunction, and on identifying routes to prevent cerebellar damage. With this study, we expect to uncover disease mechanisms of SCAR20. Moreover, our research will help us better understand the relevance of lysosomes for the cerebellum and may reveal targets for the development of treatment options for a broader spectrum of spinocerebellar ataxias.

Edgotype: a platform to stratify variants of unknown significance ataxias

Claudio Melo de Gusmao, MD
Boston Children’s Hospital
Boston, MA

Since the release of the first reference sequences of the human genome, incredible advances have occurred in the identification of gene mutations that cause ataxia. Next-generation genetic sequencing is now inexpensive enough to bring this diagnostic capability to clinical practice. This is a critical first step toward therapeutics because it allows patients to be stratified, and for treatments to be matched to specific genetic lesions. And, yet, daunting challenges remain. Our ability to sequence genomes is greatly outpacing our ability to usefully interpret the data. Genes code for proteins, the building blocks of our cells. They are involved in chemical reactions, signaling, interactions with DNA and other proteins, as well as a host of other functions. Some gene mutations will lead to a clear loss of function because no active protein is made, or the protein is made with a slight alteration. The alteration may profoundly affect protein function, or not affect it at all, and we are actually very poor at predicting which of these will occur. When these changes are present and there are no previous reports of this genetic change in association with disease, these are called variants of unknown significance (VUS). Here, we propose a new method developed to look at how gene mutations affect the protein interactions within a living cell. We plan to apply our methods to a large number of ataxia patients. We expect that the data generated in this study will have direct impact on clinical care. First, it will shed light on determining if the variants are disease-causing and stratifying patients for clinical trials and potential therapies. Second, it may have the potential to increase knowledge about disturbances in protein-interactions in ataxias, leading to novel therapeutic targets. Finally, it may lay the groundwork for a new test that can be used in many other types of genetic diseases.

Delineating the PUM1 functional network in mice and humans

Vincenzo A. Gennarino, Ph.D.
Baylor College of Medicine
Houston, TX

The molecular genetic revolution of the 1990’s brought us tremendous knowledge of the genetic mutations that cause many neurological diseases, including many ataxias. Further research into the proteins produced by these genes has revealed that there is another way for a protein to cause havoc in the brain besides being mutated: it might be expressed at levels too low or too high. In the case of several neurodegenerative diseases, including Spinocerebellar Ataxia Type 1 (SCA1) and more common diseases such as Alzheimer’s and Parkinson’s, it has been shown that too much of the disease-driving protein can cause the same disease as if the protein were mutated. What if we could find a way to lower the levels of these proteins in the brain? Could we slow disease progression? These questions led me to search for the factors that regulate the levels of ataxin1, the protein that is mutated in SCA1. I discovered that ataxin1 levels are controlled by an RNA-binding protein called Pumilio1 (PUM1). Take away PUM1 in a mouse model of SCA1, and ataxin1 returns to normal levels, and the SCA1 mice no longer have ataxia. We also noticed, however, that mice lacking Pum1 (the mouse version of the protein) developed other symptoms, such as seizures, and they developed ataxia earlier than the SCA1 mice. This led us to suspect that loss of PUM1 function might underlie some childhood ataxia diseases. We reached out to medical geneticists to search for such patients and so far have identified 20 patients with PUM1 deficiencies: those with deletions or severe missense mutations that eradicate protein function suffer from a neurodevelopmental disorder that causes physical, cognitive, and/or psychosocial delay with seizures and ataxia, whereas patients with a hypomorphic mutation develop a late-onset disorder that causes a mild, slowly progressive ataxia. PUM1 is thus a newly identified genetic cause of two ataxia syndromes. This proposal aims to (1) generate mice carrying the PUM1 mutations identified in humans to better understand the molecular pathogenesis of the human disease and (2) characterize the Pum1 protein-protein interaction network in the mouse brain in order to identify potential druggable targets. Not only is this necessary to understand how PUM1 deficiency causes earlyonset ataxia and neurological dysfunction, but understanding the roles PUM1plays in neurons will help us understand whether manipulating PUM1 levels in the brain could help SCA1 patients (or, indeed, patients with other neurodegenerative diseases). As my preliminary data indicate that Pum1 regulates a number of proteins involved in other neurodevelopmental and neurodegenerative conditions, the proposed studies should reap a considerable return on the NAF’s investment.


Identification of novel pathogenic tandem repeat expansions in spinocerebellar ataxia

Alejandro Martin Trujillo, PhD
School of Medicine at Mount Sinai
New York, NY

Many types of spinocerebellar ataxia (SCA) are caused by expansions of tandemly repeated stretches of DNA (or “TRs”). These are segments of DNA where multiple copies of a sequence are serially repeated dozens, hundreds or even thousands of times, e.g. CAG-CAG-CAG-CAGCAG. The large size of many TR expansions in SCA patients makes them difficult to detect by conventional sequencing methods, such as PCR, or standard genome sequencing technologies that only look at short segments of DNA. As a result, the identification of novel expanded TRs that cause SCA is very difficult. To overcome this limitation, in this project, we seek to identify novel pathogenic TR expansions in patients with unexplained SCA using two novel approaches. First, we will study four candidate TRs we have identified in the genome that each show strong signatures of instability using an optimized Repeat-Primed PCR method that can amplify expanded TRs. Secondly, we will perform whole genome sequencing to look for expanded TRs across the entire genome of individuals with inherited SCA using Pacific Biosciences long-read technology. This approach produces long sequencing reads (read length 10,000-15,000 bp) that, unlike short reads, are able to fully span even the very longest expanded TRs found in patients with SCA. In addition to providing an accurate molecular diagnosis to patients with unexplained SCA, we expected that the results arising from this proposal will contribute to expand our understanding about the mechanisms involved in the development of SCAs as well as to develop novel approaches for diagnosis, genetic counseling and follow-up.


Cellular Mechanisms Underlying Spinocerebellar Ataxia Type 13

Fadi Issa, PhD
East Carolina University
Greenville, NC

Spinocerebellar ataxia type 13 (SCA13) is an autosomal dominant genetic disease caused by mutations in the KCNC3 gene. This gene encodes the Kv3.3 voltagegated potassium channel, which plays an essential role in facilitating proper electrical activity in cerebellar Purkinje neurons. There are two identified SCA13 mutations and depending on the mutation, SCA is characterized by ataxia and cerebellar neurodegeneration during aging or persistent motor deficits and cerebellar mal-development starting early in life. We plan to test the hypothesis that SCA13 mutations have adverse effects on the electrical activity of developing Purkinje neurons that lead to their abnormal development. Changes in neuronal function have been reported in several neurodegenerative diseases, including those caused by toxic, misfolded proteins, but whether changes in neuronal electrical activity are involved in pathogenesis is unknown. SCA13 provides a novel opportunity to investigate the relationship between neuronal activity, development and motor control in the absence of other complicating factors such as misfolded proteins. We are using zebrafish to investigate these questions because zebrafish provides numerous technical advantages making it an attractive system to address our research interests. One important advantage is that neuronal function can be assessed using genetically-encoded calcium indicators to monitor changes in electrical activity that are due to the SCA13 mutations, and we can follow the development of the cerebellum in living animals.

Longitudinal Profiling of Spinocerebellar Ataxia Type 3 Mouse Model’s Molecular Signatures and Specific Biomarkers

Hayley S. McLoughlin, PhD
University of Michigan
Ann Arbor, MI

Spinocerebellar ataxia type 3 (SCA3), the most common dominantly inherited ataxia in the world, is a relentlessly progressive and fatal disease for which currently there is no disease-modifying therapy. With the goal of preventive therapy, several ongoing studies seek to reduce levels of the SCA3 disease protein. Leveraging tissue collected from our recently published gene silencing preclinical study in SCA3, I will longitudinally assess the molecular events in disease-relevant brain regions from two complementary SCA3 mouse models. This global gene profiling over time, the first to be completed in these models, will help identify the molecular events underlying disease and may elucidate protective pathways and biomarkers for SCA3.

Regulation of cerebellar development and connectivity by β-spectrins: implications for the pathophysiology of spinocerebellar ataxias

Damaris N. Lorenzo PhD
UNC- Chapel Hill
Chapel Hill, NC

Spinocerebellar ataxia type 5 (SCA5) and spinocerebellar ataxia autosomal recessive 14 (SCAR14) are progressive neurodegenerative disorders characterized by cerebellar atrophy and profound Purkinje cell loss. In addition, patients with SCAR14 also present with cognitive impairment. Genetically, these disorders are caused by mutations in SPTBN2, the gene encoding the protein βIII-spectrin, which is highly present in the cerebellum. Since the discovery of the genetic cause of SCA5 over ten years ago the number of βIIIspectrin mutations found in patients affected with these ataxias has grown, but our understanding of the underlying cellular mechanisms has lagged and promising treatments for the disease continue to evade us. We have new evidence that suggests that βIIIspectrin’s deficits affect the normal function of βII-spectrin, another member of this protein family. βII- spectrin along with βIII-spectrin, actin, and other partnering proteins appear to cooperate to build a periodically organized, structurally robust tubular membrane skeleton in neurons. Simultaneously, these spectrins seem to modulate intracellular transport of multiple synaptic components. In turn, we believe that the combined structural and transport defects caused by βIII-spectrin lead to severe neuronal dysfunction in the cerebellum. In this project, we will characterize mouse models of βII- and βIII-spectrin-deficient cerebella, and conduct cellular studies of βIII-spectrin with selected SCA5 human mutations to understand the functional duality of β-spectrins as scaffolding proteins and transport effectors. We hope to integrate this knowledge with previous findings into a comprehensive mechanism that explains the pathology of SCA5 and SCAR14, and serves as roadmap for judicious design of therapies.

Polyserine protein toxicity in spinocerebellar ataxia type 12

Pan P. Li, Ph.D.
Johns Hopkins University
Baltimore, MD

Spinocerebellar ataxia type 12 (SCA12) is a rare neurodegenerative disease caused by a repetitive CAG sequence in exon 7 of PPP2R2B, a gene encoding regulatory units of the protein phosphatase 2A. Two ATG start codons upstream of the repeat may translate the repeat into a protein containing a polyserine tract, giving rise to short polySer tracts from the normal allele and long polySer tracts from the mutant allele. We therefore hypothesize that proteins containing long polySer tract may contribute to the pathogenesis of SCA12. PolySer neurotoxicity has never been studied in SCA12, or in any other expansion disease, and our hypothesis therefore represents a novel approach to neurodegeneration. To test this hypothesis, we have successfully generated 8 SCA12 induced pluripotent stem cell (iPSC) lines from three different human SCA12 fibroblast lines. We have generated polyclonal antibodies that could recognize the predicted proteins containing the polySer tracts. Our preliminary evidence suggests that long polySer tracts are toxic to cells and its production may be triggered by repeat assisted non-ATG (RAN) translation. Based on these findings, and the tools that we now have available, we have developed two aims to begin systematically testing the polySer hypothesis of SCA12 pathogenesis. In Aim 1, we will examine the expression of the predicted polySer-containing proteins in 8 SCA12 patient iPSC lines and three control lines, both in undifferentiated state and after differentiation into cortical neurons, using western blots and immunocytochemistry. We will also perform immunohistochemistry to detect polySercontaining protein expression in the one formalin fixed SCA12 patient brain that is available for our study, along with control, Huntington’s disease (HD), and Alzheimer’s disease (AD) brains. We will also determine if RAN translation contributes to the expression of long polySer containing proteins, by modifying an SCA12 iPSC line to prevent ATG translation of PPP2R2B exon 7. In Aim 2, we will explore the hypothesis that expression of long polySer containing protein in SCA12 is neurotoxic, and we will test this using SCA12 iPSC models. We will use vulnerability to BDNF-withdrawal as the readout for toxicity. First, we will examine whether SCA12 cortical neurons differentiated from iPSCs are more vulnerable to BDNF-withdrawal induced toxicity, compared with the control lines. Second, we will determine whether interruptions in long polySer region modify toxicity, by comparing the vulnerability of SCA12 cells with that of an isogenic “interrupted” SCA12 line edited to mimic single amino acid replacement in long polySer region. Third, we will ascertain whether long polySer toxicity can be reproduced by polySer encoded by non-repeat RNA, by replacing the hairpin-forming AGC repeat in an SCA12 iPSC line with a TCC/TCT repeat. This project will allow us to test for the first time the novel hypothesis that proteins containing long polySer tract may contribute to SCA12 pathogenesis, with potential relevance for other neurodegenerative diseases caused by repeat expansions.


Exploring the therapeutic capacity of Aripiprazole and related compounds for Machado-Joseph disease

Maria do Carmo Costa, PhD
University of Michigan
Ann Arbor MI

Disease-modifying therapies are lacking for fatal neurodegenerative disorders including Machado-Joseph disease (MJD), also known as Spinocerebellar ataxia type 3 (SCA3). Our longterm goal is to identify small molecules that are effective to reduce the abundance of toxic ATXN3 protein in brains of MJD patients/carriers and hopefully alleviate disease progression. This project aims 1) to evaluate the potential of aripiprazole to be repurposed for MJD by carrying out a chronic pre-clinical trial of this drug in MJD transgenic mice, and 2) to develop novel therapeutic compounds structurally related with aripiprazole for this disease that are effective to decrease levels of mutant ATXN3 in neurons. Because aripiprazole is already commonly used in humans, a positive trial outcome shown by reduced ATXN3 levels in brains of MJD transgenic mice chronically treated with this drug, and/or by decreased progression of motor dysfunction in these mice, would accelerate the path to clinical testing of aripiprazole in MJD patients. To develop novel therapeutic compounds for MJD, we will carry out structure activity relationship studies building on chemical structures of aripiprazole and two related molecules newly identified by us as efficacious to reduce mutant ATXN3 abundance in a MJD cell line. By tackling two different stages of drug discovery for MJD, we expect to identify a fast route for therapy for MJD patients and/or to develop novel compounds of increased therapeutic potential for this disease.

Launching a blood and spinal fluid based biomarker program for SCAs

Puneet Opal, MD, PhD
Northwestern University
Chicago, IL

There is a critical need for establishing objective, reliable and sensitive biomarkers will enhance our understanding of the natural history of spinocerebellar ataxias (SCAs), provide insights into pathophysiology, and most importantly improve our ability to perform well-powered interventional clinical trials using fewer patients and shorter time-lines. Current biomarker strategies in neurodegeneration fall into two broad categories (1) those based on brain imaging and (2) those based on detecting biochemical differences from bodily fluids such as CSF or blood. In this proposal we will take the lead in establishing a comprehensive biochemical biomarker program for the National Ataxia Foundation. This will involve establishing the operational infrastructure that will include regulatory materials (IRB, consent forms and standard operating procedures) and facilities for the collection and storage of blood and CSF. We will align the biomarker program with the NAF funded natural history studies that are being conducted at the CRC-SCA sites. Patient materials will be linked to the existing web-based CRC-SCA database to serve as a resource to the CRC-SCA consortium of investigators. The study is thus designed as operationalizing a biorepository linked to observational, prospective, multi-center natural history study linked to the CRC-SCA. Although, the request is for one year, we are launching this initiative with the intention that it will become an open-ended, prospective study and will be self-supporting in the future by serving as a platform for clinical trials funded by disease foundations and philanthropy (including NAF), government funding agencies (such as the NIH), and pharmaceutical companies.)

Combinatorial Gene Therapy for Spinocerebellar Ataxia Type 1

Beverly Davidson, PhD
Children’s Hospital of Philadelphia
Philadelphia, PA

There are currently no therapies that delay onset or progression of spinocerebellar ataxia. In earlier work, we showed that gene silencing approaches or gene over-expression approaches delivered individually 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 onset. These data are the foundation for future clinical application of gene silencing studies in SCA1 patients. Here, we propose to expand and improve on this work by testing a combinatorial approach. The goal of this newer approach is to reduce the overall dose of material needed to achieve therapeutic benefit. If our tests in mice are successful, we will seek additional funding to move this forward to SCA1 patients.


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