Russell L. Margolis, MD/PhD - US grants

Trinucleotide repeats consist of three bases consecutively repeated (e.g., CAG CAG CAG CAG CAG CAG CAG) within a region of genomic DNA. Increase in the number of triplets contained within a repeat is a new form of human genetic mutation, known as an expansion mutation. Seven diseases, each with neuropsychiatric features and unusual patterns of inheritance, are caused by expansion mutations. One of these expansion mutations occurs in a gene identified in the laboratory of Dr. Ross. A number of neuropsychiatric disorders, including schizophrenia, bipolar affective disorder, and autism have clinical and neuropathological features and patterns of inheritance similar to the other diseases caused by expansion mutations. In addition, genes with TNRs are frequently transcription factors, and such genes often play a prominent role in neurodevelopment. Together these factors lead to the two hypotheses guiding the proposed research plan: 1) Genes with trinucleotide repeats may regulate aspects of neurodevelopment, and 2) trinucleotide repeat expansion mutation may cause some forms of neuropsychiatric disorders. To test these hypotheses, three specific aims, each tied to a specific career development goal, have been identified. Formal course work and the assistance of collaborators will provide a conceptual context for the technical skills to be acquired in pursuit of each specific aim. In specific aim #1, the cDNA fragments of previously unidentified genes which contain CAG or CCG repeats will be cloned. The repeats within the clones will be examined for length polymorphisms and collaborators will map each polymorphic clone to a specific loci and test patient DNA for expansion mutation of the newly identified genes. In specific aims #2 and #3, mRNA and protein expression of selected genes will be examined, with particular emphasis on expression during neurodevelopment. The overall goal of these studies is to identify and characterize genes that are of neurodevelopmental relevance and in which expansion mutations could lead to neuropsychiatric disease.

Understanding the genetic factors underlying HD provides a powerful approach to identifying points of therapeutic intervention. The goals of the Genetics Core are therefore to use molecular techniques, in conjunction with each of the other Cores and projects in the program, to quantify the known genetic factor, CAG repeat length, that influences the onset and course of HD and to identify unknown genetic factors that influence HD and that cause related disorders. These goals will be met through three specific aims. In specific Aim # 1, we propose to provide clinical genetic testing for HD and similar disorders caused by CAG repeat expansions. There results will be used in every clinical study of the program. In addition, we will assist project with genetic mapping, sequence analysis, and other molecular techniques. In Specific Aim # 2, we will determine if genetic factors other than the CAG repeat in the HD gene influence HD onset on progression. Results will be confirmed by biochemical means, mouse models and through genetic studies of subjects from our collaborators. In Specific Aim # 3, we will identify novel causative mutations of familial disorders similar to HD. DNA from affected subjects found to have an HD-like phenotype and determined in Specific Aim # 1 to have none of the known CAG repeat expansions, will be assayed for the presence of a novel CAG repeat expansion. Any such expansion will be cloned. Genotype-phenotype relationships will be studied and the mutation will be studied. Overall, the proposed studies will facilitate every project of the program, and lead to new approaches for determining diagnosis, prognosis, and treatment of HD and related disorders.

Toxicity of RNA transcripts containing long CUG repeats, a novel mechanism of disease pathogenesis, was recently described in myotonic dystrophy type 1 (DM1), the most common form of adult onset muscular dystrophy. The precise mechanism by which CUG expansions lead to cell toxicity is unclear, though misregulation of splicing may be involved. Our group recently identified an autosomal dominant disorder, Huntington's disease-like 2 (HDL2), with clinical and pathological features almost identical to Huntington's disease (HD), a relentlessly progressive adult onset neurodegenerative disorder. Like HD, HDL2 is caused by a CAG/CTG expansion mutation. To our surprise, the pathogenesis of HDL2, like DM1 and unlike HD, appears to arise at least in part from the toxic effect of RNA transcripts containing long CUG repeats. To test the hypothesis that untranslated CUG expansions can lead to neurotoxicity in the mammalian brain, we propose to generate a transgenic mouse specifically expressing an expanded CUG repeat in the brain. In Specific Aim 1, we will generate CUG transgenic mice, using an untranslatable construct that contains a short fragment of JPH3 with either a normal or an expanded CTG repeat under the control of the brain-specific PrP promoter. In Specific Aim 2, the behavioral, motoric, and pathological phenotype of these mice will be examined. In Specific Aim 3, specific characteristics of CUG repeat expansion diseases will be examined in the mice, with an emphasis on RNA foci and protein aggregation. We predict that the expanded CUG repeat will lead to neurotoxicity, evident in motor behavior and neuropathology. If this prediction is correct, the mice generated here will become invaluable tools for dissecting the pathogenic pathways of DM1, HD, and HDL2. Finding the points of pathogenic convergence in these diseases may provide novel leads for the development of rational therapeutics.

DESCRIPTION (provided by applicant): A novel form of disease pathogenesis, in which cell damage is caused by transcripts with expanded CUG repeats, has been described in myotonic dystrophy type 1 (DM1). Our group recently described another disorder, Huntington's disease-like 2 (HDL2), that is also associated with CUG repeat toxicity. Like DM1, HDL2 is a dominant disorder. Unlike DM1, HDL2 is very similar to Huntington's disease (HD), with mid-life onset of a devastating neurodegenerative process selectively affecting the striatum and cerebral cortex and progressing inevitably to death. We found that HDL2 is caused by a CAG/CTG repeat expansion in junctophilin-3 (JPH3) on chromosome 16q24.3, and that JPH3 transcripts with an expanded repeat accumulate and form foci in neuronal nuclei. Preliminary cell experiments suggest that these transcripts are toxic, and may induce toxicity via redistribution of muscleblind-like protein 1 (MBNL1), a protein also implicated in the muscle and brain abnormalities of DM1. Surprisingly, our preliminary experiments indicate that untranslated huntingtin transcripts containing CAG repeat expansions are also toxic. These preliminary findings have led us to hypothesize that the pathogenesis of HDL2, and perhaps HD, may at least partly stem from transcripts with expanded repeats. More specifically, we hypothesize that RNA-induced neurotoxicity will be determined by the type and length of the repeat, the sequence flanking the repeat, and the cell types in which the repeat is expressed. We also hypothesize that the pathogenesis of this neurotoxicity will involve the splice-regulating protein muscleblind like 1 (MBNL1). Here, we propose to develop and explore cell models that will provide clues about RNA neurotoxicity in both CUG and CAG diseases. If successful, our work will open up new lines of investigation into the pathogenesis of HDL2, HD, and potentially other repeat disorders, with the ultimate goal of developing new targets for the therapeutics of neurodegenerative disease.

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a fatal neurodegenerative disorder, affecting about 30,000 people in the United State, and caused by an expansion of a CAG repeat (encoding polyglutamine) in the gene huntingtin located on chromosome 4p. While ample evidence supports a major role for polyglutamine toxicity in the pathogenesis of Huntington's disease (HD), the complete explanation for HD pathogenesis remains elusive. Recent evidence suggests that transcripts antisense to genes are present throughout the genome. In particular, antisense transcripts with potential roles in disease pathogenesis have been detected at the locus of a number of repeat expansion diseases, including fragile X, myotonic dystrophy type 1, spinocerebellar ataxia type 8, and Huntington's disease-like 2. Our preliminary data indicates that 1) an antisense transcript spanning the CAG/CTG repeat region at the HD locus (termed huntingtin antisense, abbreviated HTTAS) exists and is expressed in multiple brain regions;2) HTTAS promoter activity is inversely proportional to the length of the CAG/CTG repeat, 3) HTTAS is decreased in HD brain compared to control brain, and 4) exogenous overexpression of HTTAS decreases huntingtin (HTT) levels, while knockdown of HTTAS increases HTT levels. We have also developed a series of constructs modeling the HD locus, which demonstrates the repeat-length dependent influence of HTTAS on HTT. Based on this initial data, we hypothesize that HTTAS influences HTT expression in a repeat length-dependent manner, with consequent impact on HD pathogenesis. We will test this hypothesis in Aim 1 by mapping the complete HTTAS gene in the human and mouse genome, confirming the repeat-length dependent actiivty of the promoter, and determining the pattern of HTTAS1 expression in normal and HD brain. In Aim 2, we will use cell models to determine the effect of HTTAS on HTT expression both in cis and in trans, and in the setting of normal and expanded repeats. These experiments will enable us to establish the existence and function of HTTAS, and provide us with sufficient preliminary data to compete for long term funding for this project. Ultimately, understanding the relationship between HTT and HTTAS will provide new insights into the mechanism of HD pathogenesis and may lead to the development of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: Antisense transcripts appear to regulate mutant transcripts in a number of diseases, including several repeat expansion diseases. We propose to test the hypothesis that an antisense transcript at the HD locus regulates huntingtin. If confirmed, antisense regulation would provide a new approach to understanding the pathogenesis of HD, and new approaches for developing therapeutic agents for HD.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease, characterized by abnormalities of movement, cognition and emotion, with relentless progression until death ~20 years after disease onset. HD is caused by expanded CAG repeats in exon 1 of the Huntingtin (HTT) gene. Substantial advances have been made into understanding the neurobiology of HD. Nonetheless, as of yet no treatment exists that stops or substantially slows disease progression, and it has proven difficult to prioritize among the >100 proposed pathogenic mechanisms to focus on those most likely to lead to therapeutic advances. HDL2, discovered and genetically defined by the Margolis group, is a rare, autosomal dominant neurodegenerative disorder, clinically and neuropathologically very similar from HD. Like HD, the neuropathology of HDL2 is characterized by cortical and striatal neurodegeneration and the presence of neuronal protein aggregates. HDL2 is caused by a CTG/CAG expansion on chromosome 16q24. Normal alleles contain 6-28 triplets, while pathogenic repeats range from 40-59 triplets, again remarkably similar to HD. In the CTG orientation, the repeat falls in the gene junctophilin-3 (JPH3). We have hypothesized that the HDL2 mutation leads to neurodegeneration via a combination of loss of JPH3 expression, toxicity of the sense strand transcript containing an expanded CUG repeat, and expression of polyglutamine from a cryptic gene on the antisense strand. The relative contribution and interactions of these mechanisms remains unknown, and modeling HDL2 has proven challenging. We now propose (Aim 1) to generate and characterize induced pluripotent cells from fibroblasts of individuals with HDL2. The pluripotency of the cells will be systematically investigated. In aim 2, we will then differentiate the iPS cells into neurons, including a subpopulation with a striatal phenotype. Cells will be characterized with neuronal and striatal-specific markers to determine the differentiation pattern, and we will determine the extent to which these cells recapitulate findings observed in HDL2 brain and model systems. We will determine the survival, electrophysiological profiles, vulnerability to glutamate toxicity, and vulnerability to BDNF withdrawal of these cells compared to controls. The public health implications of developing HDL2 iPS cells as a tool for studying HDL2 are several fold: improved understanding of HDL2 itself, new insights into fundamental pathogenic processes relevant to other repeat expansion diseases, and the opportunity to find pathogenic points of convergence between HD and HDL2 that will lead to a focus on therapeutic targets of most promise for both diseases.

? DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 12 (SCA12) is a progressive, autosomal dominant, neurodegenerative disorder caused by an expansion of a CAG/CTG trinucleotide repeat on chromosome 5q32; both the disease phenotype and the causative mutation were initially described by our group (Holmes et al, 1999). While the disease is one of most common forms of SCA in India, and scattered SCA12 pedigrees have been detected around the world, perhaps the most intriguing aspect of SCA12 is that the repeat falls in a putative promoter of PPP2R2B, a gene encoding ß regulatory subunits of the trimeric enzyme phosphatase 2A (PP2A). Functional PP2A consists of a structural unit, one of two catalytic units, and one of ~30 regulatory subunits, with the N-terminal region of the regulatory subunits serving to target the holoenzyme to specific intracellular sites. Dysregulation of PP2A has been directly linked to tau hyperphosphorylation in Alzheimer's disease, and to multiple other neurodegenerative diseases. We hypothesize, based on preliminary data from cell overexpression models, that the SCA12 repeat expansion leads to increased expression of PPP2R2B isoform Bß1, and that this overexpression leads to dysregulation of PP2A activity and neurotoxicity. However, it has not been possible to confirm these observations, as human SCA12 brain material is not available and PPP2R2B Is not expressed in leukocytes or lymphoblasts. To test our hypothesis, we will use fibroblasts from skin biopsies of patients with SCA12 to generate induced pluripotent stem cells (iPSCs)(Aim 1). We will then determine the effect of the mutation on PPP2R2B expression and other cellular properties in the fibroblasts and in the IPSCs differentiated into forebrain neurons (Aim 2). The potential public health benefits of this project are three fold: 1) a better understanding of how repetitive DNA can influence gene expression, 2) a better understanding of SCA12 pathogenesis, with the potential of detecting targets for therapeutic agents, and 3) new insight into the role of PP2A in the pathogenesis of neurodegenerative disease.

Summary. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by relentless progression to death ~20 years after disease onset. HD is caused by an expanded CAG repeat in exon 1 of the huntingtin (HTT) gene. Disease pathogenesis is largely a result of expression of the mutant transcript and protein, which have neurotoxic properties. Suppressing the expression of the mutant allele is therefore a promising therapeutic approach, thus far pursued with antibody, oligonucleotide and siRNA strategies. As with any knockdown approach, especially in the CNS, problems of delivery, reversibility, and off-target effects using these methods remain problematic. Surprisingly, relatively little is known about the regulation of the HD locus, and in particular on the mechanisms that regulate HTT expression. We have recently discovered a gene on the strand antisense to HTT at the HD locus, which we have termed huntingtin antisense (HTT-AS). We have demonstrated that increasing expression of HTT-AS decreases expression of HTT, and that HTT-AS expression can be manipulated using small molecules. We hypothesize that HTT-AS itself, and the components that regulate its expression and interaction with HTT, will provide novel therapeutic targets for suppression of HTT expression and hence treatment of HD. A corollary to this hypothesis is that a better understanding of the HD locus, in this case the role of HTT-AS, will be critical in the interpretation of any HTT suppression strategy. We will test this hypothesis with a series of experiments organized into three specific aims, each built on compelling preliminary data. In Aim 1, we will determine if additional HTT-AS exons, splice variants, and promoters exist, determine the effect of repeat expansion on HTT-AS expression, and identify protein factors that regulate HTT-AS promoter activity. In Aim 2, we will determine the mechanisms by which HTT-AS suppresses HTT, including the quantitative effect of different HTT-AS transcripts on the suppression of HTT, and the role of chromatin remodeling on HTT expression. In Aim 3, we will collaborate with NCATS and CHDI to perform a large scale high throughput screen to find and characterize compounds that decrease expression of HTT by specifically increasing expression of HTT-AS. Selected compounds will be validated in cell and mouse models.