Laura P. Ranum - US grants

One in 17,000 individuals is afflicted with a genetic form of ataxia. Patients with dominant spinocerebellar ataxia (SCA) display adult-onset, motor coordination deficits that usually lead to death. Clinical signs, age of onset, and disease duration vary substantially both among and within families making the clinical classification of this group of diseases extremely difficult. Future classifications of the ataxias will be based on the gene affected. The genetic heterogeneity of the ataxias provides a unique opportunity to understand more about the pathology of neurodegenerative disease through the isolation and characterization of the different genes which can all lead to varying degrees of neuronal loss in the cerebellum, brain stem, and spinal tracts. Toward this goal, I have identified and collected a previously unreported 10 generation ataxia kindred and have shown that the gene involved in not SCA1, SCA2 or Machado Joseph Disease (MJD). The long term goal of this project is to identify and characterize this novel ataxia gene. Understanding the various molecular systems defects that cause ataxia will lead to a better understanding of the interdependence and functioning of the neuronal systems that degenerate during the SCA disease process. The specific aims are: 1. To localize the Lincoln ataxia gene (L-SCA) to a specific chromosome by linkage analysis. 2. To construct a high resolution (1,2-centiMorgan cM) genetic map for the chromosomal region containing the Lincoln ataxia gene. DNA from L- SCA family members as well as the CEPH panel of reference families will be used. 3. Once the L-SCA sub region has been well defined genetically, the following strategies will be used to isolate the defective gene: A) A physical map will be constructed using pulsed-field gel electrophoresis (PFGE) blots probed with DNA markers that are very closely linked to the ataxia gene; B) Overlapping yeast artificial chromosome clones (a YAC contig) will be isolated for the ataxia gene sub region. Candidate genes will be selected from the DNA contained in the YAC contig and examined for their possible role in the disease process. At this stage, screening for the possible involvement of unstable trinucleotide repeats will be a top priority. 4. Initial characterization of the L-SCA gene. Once the L-SCA gene is identified, our efforts will be directed towards understanding its biology. The sequence of the L-SCA gene (genomic and cDNA) will be obtained which may provide important clues as to its function. If the mutation is variable in affected individuals, as is the CAG repeat length within the SCA-1 gene, the mutation in affected individuals will be correlated with age of onset and clinical features.

DESCRIPTION (Adapted from the Investigator's Abstract): The investigator has evaluated a 5 generation family (MN1) with a previously undefined autosomal dominant muscle disease. Although this family has the clinical features associated with myotonic dystrophy (myotonia, neck and facial weakness, frontal balding, cataracts and cardiac arrhythmias), affected individuals do not have the CTG expansion associated with DM, nor is the disease locus linked to the DM region of chromosome 19. The investigator also excluded linkage to the other genetic loci that cause myotonia, i.e., the muscle sodium channel gene (SCN4A) and the chloride channel gene (CLC-1). The identification of a second locus for myotonic dystrophy will facilitate the identification of additional families with the MN1 form of myotonic dystrophy and will be a first step toward identifying the MN1 gene. Because the MN1 disorder causes the same broad range of clinical features as classic myotonic dystrophy, the eventual isolation and characterization of the gene(s) affected will provide insights into the pathology of both DM and the disease afflicting the MN1 family. Our specific aims are: A) To further characterize patients clinically by performing: 1) blood tests to look for muscle fiber disruption, glucose intolerance, and primary gonadal failure in males; 2) EMG studies to better characterize their myotonia; 3) EKG studies to look for conduction block and arrhythmias; 4) muscle biopsies to characterize their myopathy. B) To identify and study additional branches of the MN1 family that are affected by the disease and to look for additional unrelated families with the MN1 form of myotonic dystrophy. C) To localize the MN1 gene to a specific chromosome by linkage analysis. Preliminary results indicate a high likelihood of detecting linkage by performing a genome screen using a marker spacing of 10 cM (360 markers). D) To construct a high resolution (1-2 cM) genetic map for the chromosomal region containing the MN1 gene. In order to genetically narrow the critical region containing the MN1 gene to a 1-2 cM interval it is anticipated that we will need to identify additional branches of the family and or other families with the MN1 form of "myotonic dystrophy". Once the MN1 locus has been well mapped, candidate genes within the region can be examined for their possible involvement in the disease process. If the number of affected individuals is sufficient to allow us to narrow the candidate region to 1-2 cM, then physical mapping and cloning strategies will be used to isolate the defective gene.

? DESCRIPTION (provided by applicant): Research on spinocerebellar ataxia type 8 (SCA8) supported by this application has led to two discoveries that fundamentally change our understanding of how a broad category of microsatellite expansion mutations are expressed. First, we demonstrated that the SCA8 CTG?CAG expansion is bidirectionally transcribed1. This was the first demonstration that a single expansion mutation could lead to the expression and accumulation of toxic CUG and CAG expansion RNAs and a CAG-encoded polyGln expansion protein1. Second, we discovered that the canonical rules of translation do not apply for CTG?CAG repeats and that CAG and CUG expansion transcripts can express homopolymeric expansion proteins in all three frames without an AUG start codon2. We showed this repeat associated non-ATG (RAN) translation is hairpin-dependent, occurs without frameshifting or RNA editing and is observed in cell culture and SCA8 patient tissues2. RAN translation also occurs in myotonic dystrophy type 1 (DM1)2, C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD9)3-5, and fragile X tremor ataxia syndrome (FXTAS)6. The discoveries of bidirectional transcription and RAN translation change our understanding of how genes are expressed and highlight the need for therapies that target both sense and antisense transcripts as well as RAN proteins. Our central hypothesis is that both RNA and RAN gain of function (GOF) contribute to SCA8 and can be mitigated by therapies based on MBNL1 overexpression or RNA knockdown. Our specific aims will test the following hypotheses: 1) RNA gain of function and RAN translation contribute to SCA8; 2) RAN translation can be modulated by MBNL proteins and stress pathways; 3) antisense oligo (ASO) knockdown of ATXN8 and ATXN8OS will block RNA and RAN effects and reverse disease in an SCA8 mouse model.

Myotonic dystrophy (DM) is a multisystem disease and the most common form of muscular dystrophy in adults. In 1992, one form of DM was shown to be caused by an expanded CTG repeat in the 3' untranslated region of the myotonin protein kinase gene (DMPK) on chromosome 19. Although multiple theories attempt to explain how the CTG expansion causes the broad spectrum of clinical features in DM, there is no consensus about how this mutation, which does not alter the protein coding region of a gene, affects cellular function. We have identified a five-generation family (MN1) with a genetically distinct form of myotonic dystrophy. Affected members have the characteristic features of DM (myotonia, proximal and distal limb weakness, frontal balding, cataracts, and cardiac arrhythmias) but do not have the chromosome 19 mutation. We have mapped the disease locus (DM2) for the MN1 family to a small region of chromosome 3 (Nature Genetics 19:196- 198). This proposal outlines a strategy to identify and characterize the DM2 locus. Understanding what is common to chromosome 19 DM (now designated DM1 by the DM consortium) and DM2 at the molecular level should shed light on the mechanisms responsible for the broad constellation of clinical features present in both diseases. Our specific aims are: 1) to develop a high-resolution map of the DM2 region (0.5-1.0 cM) using haplotype and linkage disequilibrium analysis of 29 DM2/PROMM families from Minnesota and Germany; 2) to identify the expressed genes and repeat motifs in the region and prioritize candidates based on homology and expression patterns; 3) to identify the DM2 mutation; 4) to characterize the DM2 gene and investigate whether or not the pathogenic molecular changes found in DM2 are part of a common pathway also affected in DM1; 5) to determine whether molecular changes affecting RNA splicing, CUG binding proteins, and apamin receptors are similar to those found in DM1.

family genetics; myotonic dystrophy; genetic mapping; autosomal dominant trait; gene mutation; endocrine disorder diagnosis; clinical research; human subject; magnetic resonance imaging; diagnostic tests; biopsy; electromyography;

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on chromosome 19 (DM1) or 3 (DM2/PROMM). DM1 is caused by a CTG expansion in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK) and the promoter region of the homeodomain gene SIX5. Although the DM1 mutation was isolated in 1992, for many years it has been difficult to understand how this mutation, which does not alter the protein-coding portion of a gene, causes this dominantly-inherited multisystemic disorder. Suggested mechanisms have included: DMPK haploinsufficiency; reduced expression of regional genes including the homeodomain gene SIX5; and pathogenic effects of the CUG expansion in RNA. Mouse models have suggested that each of these mechanisms contributes to DM1 pathogenesis and that DM1 may be a regional gene disorder. To clarify the pathogenic mechanism of DM, we have studied a second form of myotonic dystrophy that causes a strikingly similar constellation of clinical features in humans including myotonia, myopathy, iridescent cataracts, cardiac arrhythmias, and the specific set of serological changes characteristic of DM. We mapped the DM2 locus to chromosome 3q21 (Nature Genetics 19:196-198, 1998) and recently reported that DM2 is caused by a repeat expansion in intron 1 of the zinc finger protein nine (ZNF9) gene (Science, 293:864-867). Clinical and molecular parallels between DM1 and DM2 indicate that CUG and CCUG expansions expressed at the RNA but not the protein level can themselves be pathogenic and cause the multisystemic features common to both diseases. We propose generating transgenic mice and cell culture models to test the effects of CCUG repeat-containing transcripts and to better understand the molecular mechanisms involved in the disease process. Our specific aims are: (1) To generate a reversible murine model to test the pathogenicity and characterize the downstream effects of transcripts containing the CCUG expansion expressed in skeletal muscle. (2) To generate and characterize a BAC transgenic mouse model that replicates the multisystemic features of myotonic dystrophy type 2. (3) To develop a cell culture model to evaluate the molecular effects of CCUG length on the formation of RNA foci, cellular differentiation and downstream molecular changes in alternative splicing.

DESCRIPTION (provided by applicant): The Fifth International Conference on Unstable Microsatellites and Human Disease will focus on the multi-disciplinary nature of the research field requiring the gathering of clinicians, diagnosticians, pathologists, geneticists and molecular biologists. In addition to focusing upon the most recent scientific advances, the goals of the meeting include: 1) To enhance the open exchange of information related to microsatellite expansion diseases; 2) To stimulate the initiation of collaborative research between investigators worldwide and to improve the understanding of human diseases related to microsatellite instability and 3) To provide junior investigators with an opportunity to present their work and to interact with more established scientists in the field. This meeting has a proven track record of rapidly sharing new information, stimulating scientific exchange, forming collaborations and has served as an excellent venue for training of young scientists (graduate students, post-doctoral fellows and PIs). This is the premier meeting on these mutations and diseases. Submitted papers that have been presented as podium or poster presentations have been published in the best journals, including Science, Nature, Nature Genetics, Cell, Molecular Cell, American Journal of Human Genetics, Human Molecular Genetics, Molecular and Cellular Biology, and Journal of Biological Chemistry. null

DESCRIPTION (PREPARED BY APPLICANT): We have discovered that p-lll spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred and two additional independently reported SCA5 families. The American and a French family have separate in-frame deletions of 39 and 15 bp, respectively, in the third of 17 spectrin repeat motifs. A third mutation, found in a German family, is located in the calponin homology domain, a region known to bind actin, protein 4.1 and Arp1, which could cause generalized dysfunction of the membrane-cytoskeletal complex or deficits in dynein-dynactin mediated transport. Consistent with Purkinje cell degeneration in SCA5, P-lll spectrin is highly expressed in cerebellar Purkinje cells. Dramatic differences in the glutamate transporter EAAT4, a protein known to interact with p-lll spectrin, and the glutamate receptor delta 2 protein (GluR62) were found by Western and cell fractionation analysis of SCA5 autopsy tissue, suggesting the possibility that mutant p-lll spectrin disrupts the distribution or stability of these proteins. TIRF microscopy performed on cell lines transiently transfected with mutant or wildtype spectrin shows that mutant p-lll spectrin fails to stabilize EAAT4 at the plasma membrane. Our preliminary data show that p-lll spectrin mutations are a novel cause of neurodegenerative disease that may affect the stabilization or trafficking of membrane proteins. The goals of this application are to better understand the role of spectrin in ataxia, the normal function of P-lll spectrin, and the molecular consequences of the SCA5 mutations, including effects on EAAT4 and glutamate transport.

2-Naphthacenecarboxamide, 4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-, (4S-(4alpha,4aalpha,5alpha,5aalpha,6alpha,12aalpha))-; 21+ years old; Abscission; Address; Adult; Affect; Alternate Splicing; Alternative Splicing; Animals; Arrhythmia; Articulation; Atrophy, Muscle; Body Tissues; Brain; Cardiac Arrhythmia; Cataract; Cell/Tissue, Immunohistochemistry; Cerebrum; Characteristics; Clinical; Contracture; Data; Development; Disease; Disorder; Disorder of muscle, unspecified; Doxycycline; Dystrophia Myotonica; Encephalon; Encephalons; Excision; Exons; Extirpation; FLR; Failure (biologic function); Future; Gene Products, RNA; Gene Transcription; Genes; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Global Change; Heart Arrhythmias; Heterogeneous Nuclear RNA; Human; Human, Adult; Human, General; IHC; Immunohistochemistry; Immunohistochemistry Staining Method; Injection of therapeutic agent; Injections; Intervening Sequences; Introns; Joints; Lead; Mammalia; Mammals; Mammals, General; Mammals, Mice; Man (Taxonomy); Man, Modern; Mental Retardation; Methylation; Mice; Modeling; Molecular; Monitor; Murine; Mus; Muscle; Muscle Disease; Muscle Disorders; Muscle Tissue; Muscle disease or syndrome; Muscle, Cardiac; Muscle, Heart; Muscle, Skeletal; Muscle, Voluntary; Muscular Atrophy; Muscular Diseases; Mutation; Myocardium; Myopathic Conditions; Myopathic Diseases and Syndromes; Myopathic disease or syndrome; Myopathy; Myopathy, unspecified; Myotonia; Myotonia Atrophica; Myotonia Dystrophica; Myotonic Dystrophy; Nerve Cells; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neurons; Nuclear; Overexpression; Palate; Pathogenesis; Pathology; Patients; Pattern; Pb element; Personal Satisfaction; Phenotype; Pre-mRNA; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Protein Methylation; Protein Overexpression; Proteins; RNA; RNA Expression; RNA Splicing; RNA Splicing, Alternative; RNA, Messenger, Precursors; RNA, Non-Polyadenylated; RNA-Binding Proteins; Removal; Reporting; Ribonucleic Acid; Serologic; Serological; Skeletal Muscle Tissue; Skeletal muscle structure; Skeletal system; Splicing; Steinert Disease; Surgical Removal; System; System, LOINC Axis 4; Testing; Therapeutic; Thinking; Thinking, function; Time; Tissues; Toxic effect; Toxicities; Trans-Acting Factors; Trans-Activators; Transactivators; Transcript; Transcription; Transcription, Genetic; Transgenes; Transgenic Model; Transgenic Organisms; Variant; Variation; Vibramycin; Zinc Finger Domain; Zinc Finger Motifs; Zinc Fingers; adult human (21+); alpha-6-Deoxyoxytetracycline; base; brain atrophy; cardiac muscle; cataractogenesis; cataractous lenses; cerebral atrophy; cortical atrophy; craniofacial; craniofacies; disease/disorder; executive control; executive function; experiment; experimental research; experimental study; failure; frontal cortex; frontal lobe; gain of function; gene product; genome mutation; heart muscle; heavy metal Pb; heavy metal lead; hnRNA; human disease; in vivo; insight; mRNA Precursor; molecular pathology; mouse model; muscular disorder; neuronal; novel; optic imaging; optical imaging; overexpress; premRNA; recombinase; research study; resection; skeletal; substantia alba; trans acting factor (genetic); transgenic; well-being; white matter

Description (provided by applicant): We demonstrated previously that myotonic dystrophy type 2 (DM2) is caused by a CCTG expansion in intron 1 of the ZA/F9gene. Parallels between the DM1 and DM2 expansion mutations and the characterization of RNA binding proteins that interact with expanded CUG and CCUG repeats have uncovered a novel type of mechanism involving RNA gain-of-function effects. In this model, the accumulation of CUG or CCUG expansion transcripts results in the dysregulation of RNA binding proteins normally involved in controlling developmental changes in alternative splicing of a specific set of pre-mRNA transcripts. The failure of this set of normal developmental splicing changes is thought to occur, at least in part, because the accumulation of CUG and CCUG expansion transcripts in DM1 and DM2 leads to sequestration and depletion of nuclear MBNL1. Remarkably, recent studies performed by Dr. Swanson's group now show that AAV injection of constructs overexpressing Mbnll can rescue the muscle phenotype and reverse the splicing alterations in a skeletal muscle poly(CUG) model of DM1. Although significant progress has been made in understanding the pathology and the molecular mechanisms of RNA pathogenesis in skeletal and cardiac muscle, little is known about the molecular changes underlying a clinically important, but poorly understood, set of CNS effects in DM1 and DM2. Patients with adult onset DM1 and DM2 have strikingly similar multisystemic diseases, with preliminary data suggesting similar cerebral and cerebellar white matter abnormalities as well as executive function deficits. Congenially affected DM1 patients have more severe CNS abnormalities including mental retardation. A possible mechanism for congenital DM1 is that methylation at the DM1 locus in congenital cases is associated with increased expression of DMPK, resulting in higher levels of CUG containing transcripts and a more severe congenital phenotype. The focus of this proposal is to use transgenic models to test the hypothesis that CUG and CCUG repeat toxicity is comparable at the cellular and organismal levels, that alterations in temporal and spatial expression of RNA expansion transcripts cause phenotypic similarities and differences in DM1 and DM2, including CNS effects, and that many of the multisystemic features of the disease are reversible. Our specific aims are: 1) Clinical similarities and differences between DM1 and DM2 result from variations in temporal and spatial expression patterns of the CUG/CCUG expansion transcripts and are independent of both the repeat motif and flanking sequence. 2) CNS specific molecular changes found in myotonic dystrophy are caused by RNA gain of function effects and that molecular and phenotypic similarities and differences between DM1 and DM2 result from variations in temporal and spatial expression patterns of the CUG/CCUG expansions in the brain. 3) To evaluate the effects of increased Mbnll expression in skeletal muscle and the CNS by generating an inducible transgenic mouse model of Mbnll over-expression.

Communication; Florida; Minnesota; Phone; Principal Investigator; Programs (PT); Programs [Publication Type]; Research; Review Committee; SCHED; Schedule; Telephone; conference; day; member; programs; symposium

[unreadable] DESCRIPTION (provided by applicant): The Sixth International Conference on Unstable Microsatellites and Human Disease will focus on the multidisciplinary nature of the research field requiring the gathering of clinicians, diagnosticians, pathologists, geneticists and molecular biologists. In addition to focusing upon the most recent scientific advances, the goals of the meeting include: 1) To enhance the open exchange of information related to microsatellite expansion diseases; 2) To stimulate the initiation of collaborative research between investigators worldwide and to improve the understanding of human diseases related to microsatellite instability and 3) To provide junior investigators with an opportunity to present their work and to interact with more established scientists in the field. This meeting has a proven track record of rapidly sharing new information, stimulating scientific exchange, forming collaborations and has served as an excellent venue for training of young scientists (graduate students, post-doctoral fellows and PIs). This is the premier meeting on these mutations and diseases. Submitted papers that have been presented as podium or poster presentations have been published in the best journals, including Science, Nature, Nature Genetics, Cell, Molecular Cell, American Journal of Human Genetics, Human Molecular Genetics, Molecular and Cellular Biology, and Journal of Biological Chemistry. PUBLIC HEALTH RELEVANCE: The Sixth International Conference on Unstable Microsatellites and Human Disease will continue to focus upon the most recent advances in understanding repeat instability of nucleic acids and proteins and their relationship to human diseases, inherited and other. Since 1991, when this type of mutation was first associated with disease there have been at least 36 different hereditary diseases found to be caused by this form of mutation as well as numerous diseases that are associated with genetic instabilities. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application requests funding for the 2011 Gordon Research Conference on CAG Triplet Repeat Disorders and the associated Graduate Research Seminar to be held at the Il Ciocco Resort in Barga, Italy from June 4-10, 2011. This will be the sixth Gordon Research Conference on CAG Triplet Repeat Disorders. The previous five conferences have alternated between American (Mount Holyoke College, 2001, 2005;Watervalley NH, 2009) and European (Il Ciocco, Italy 2003 and Aussois, France 2007) sites. This is the second year that there will be an associated Graduate Research Seminar. The CAG Triplet Repeat Disorders are a group of largely untreatable inherited neurological disorders which result from an expansion in a CAG trinucleotide repeat in the mutant genes. This group of diseases includes Huntington's disease (HD), spinal and bulbar muscular atrophy (SBMA, Kennedy's disease), spinocerebellar ataxias types 1, 2, 3, 6, 7, and 17, and dentatorubropallidoluysian atrophy (DRPLA). In each case, the CAG repeat lies within the coding region of a gene and results in an abnormally long polyglutamine tract within the mutant protein. Marked similarities in the underlying genetics and neuropathology suggest common pathologic mechanisms among these disorders. Differences in the anatomical distribution of selective neuronal degeneration also make it imperative to unravel the distinguishing factors. Since the identification of the genetic defects, significant insights have been gained into the pathogenesis of these diseases. The field has progressed such that the development of therapeutic interventions is now a reality. To increase the pace of basic research discovery and set in place the contacts and clinical resources necessary to move the basic science into the clinic, a multidisciplinary research effort is required. It is essential that collaborative projects between scientists from diverse disciplines ranging from organic chemistry and fruit fly genetics to neurology and human clinical trials be established. The conference on CAG Triplet Repeat Disorders will gather together young investigators and established senior scientists to deliver provoking lectures on the cutting-edge of science. In keeping with the Gordon Research Conference format, there will be generous time allocated for both structured discussions led by peers and for informal discussion and social interactions to facilitate collaboration. Strong emphasis is placed on training and mentoring of young scientists, and time will be devoted to career issues. All participants will be required to present posters. Priority will be given to women, minorities and persons with disabilities when selecting participants. PUBLIC HEALTH RELEVANCE: The 2011 Gordon Research Conference on CAG Triplet Repeat Disorders and its associated Graduate Research Seminar will bring together researchers and clinicians to discuss cutting- edge information on disease mechanisms and therapeutic interventions for these devastating neurological diseases. In addition, the format of the Gordon Research Conference and the funding sought herein will promote and ensure the attendance and enhanced education of junior scientists, including graduate students, postdoctoral fellows, and junior faculty. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of the individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

DESCRIPTION (provided by applicant): This application requests funding for the 7th International Conference on Unstable Microsatellites in Human Disease (UMDH7) will be held at Mt. Ste. Odile, near Strasbourg France from June 9-14, 2012. The previous conferences have alternated between North/Central and European sites. UMHD7 will bring together top scientists from around the world to discuss high-impact, interdisciplinary research on Repeat Disorders, almost 40 have now been identified. The objective of the conference is to promote scientific discussion and facilitate interdisciplinary exchange by bringing leading researchers in the field together with a broad range of experts representing; clinicians, diagnosticians, neurobiologists, pathologists, geneticists and molecular biologists. Focusing on the most recent advances in the field, the specific aims of the conference are: 1) To enhance the open exchange of information related to repeat expansion diseases. This is one of the few opportunities members of different fields have to attend a conference that represents all of the repeat disorders. 2) To foster stimulation and initiation of global collaborative research to advance our understanding of human diseases related to repeat instability. The UMHD series has a proven track record of successful exchange and collaboration and has encouraged breakthrough research. 3) To provide junior investigators with an opportunity to present their work and interact with leading scientists in multiple and diverse fields. We aim to attract the best young minds using a well thought out and comprehensive program so that they have an opportunity to present their own work in both poster and seminar formats. We aim to make an inclusive and comfortable environment where these junior investigators have a chance to openly exchange ideas and interact with the more senior scientists. In summary, we have developed an innovative and cutting edge scientific program that will emphasize emerging themes in pathogenic and mutagenic mechanisms of the repeat disorders and harness these bodies of knowledge for therapeutic benefit. Sessions include speakers who directly work on repeat disorders as well as speakers who can provide perspectives from other fields. The keynote speakers (Stephen Warren and Nahum Sonenberg) reflect our goal to bring in broad expertise to inform studies into pathogenic mechanisms of disease and various treatment strategies in a forum that would not normally be available. PUBLIC HEALTH RELEVANCE: The 7th International Conference on Unstable Microsatellites in Human Disease will bring together researchers and clinicians to discuss cutting-edge information on disease mechanisms and therapeutic interventions for these devastating diseases. In addition, the format of the Conference and the funding sought herein will promote and ensure the attendance and enhanced education of junior scientists, including graduate students, postdoctoral fellows, and junior faculty.

Repeat-Associated Non-ATG Translation in DM1 and DM2 (Project 1)- Ranum Well-established rules of translational initiation have been used as a cornerstone in molecular biology to understand gene expression and to predict the consequences of disease causing mutations. For myotonic dystrophy (DM) and other microsatellite expansion disorders, repeat expansions (e.g., CAG or CTGs) located in predicted coding- and non-coding regions are thought to cause disease by protein gain-, or loss-of- function or RNA gain-of-function mechanisms. In 2001, we showed that myotonic dystrophy type 2 (DM2) is caused by an intronic CCTG*CAGG expansion in CNBP. The apparent non-coding locations of the DM1 and DM2 expansion mutations and the accumulation of RNA foci in both disorders helped to establish that CUGEXP and CCUGEXP RNAs cause dominant RNA effects. While substantial data support RNA gain-of-function contributions to DM, recent discoveries, which fundamentally change our understanding of how disease-causing mutations are expressed, must also now be considered. First, much of the genome is bidirectionally transcribed, including the DM1 CTG'CAG expansion. Therefore, in addition to DM1 CUGEXP transcripts, mutant DM1 CAGEXP transcripts may also play a role in disease. Second, we recently discovered that the canonical rules of translation do not apply for CTG?CAG repeat expansions and that CAG and CUG expansion transcripts can express homopolymeric expansion proteins in all three frames without an AUG start codon. This Repeat-Associated Non-ATG (RAN) translation is hairpin dependent, occurs without frameshifting or RNA editing and is observed in cell culture and DMpatient tissues. We propose to test the overall hypothesis that RAN translation contributes to DM disease pathogenesis. Our specific aims are to test the hypotheses: 1) that novel polymeric expansion proteins expressed by RAN translation accumulate in DM1 and DM2 brain and contribute to CNS pathology; 2) that RAN proteins are toxic independent of RNA gain of function effects; 3) that RAN translation can be blocked in vivo.

Project Summary The expansion of a microsatellite GGGGCC repeat in the C9orf72 gene has been linked to both familial and sporadic forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While the molecular basis of this disease (C9-ALS/FTD) remains largely unknown, proposed disease mechanisms include C9orf72 loss of function due to haploinsufficiency, RNA gain of function (GOF) leading to protein sequestration and repeat-associated non-ATG (RAN) translation resulting in the production of toxic C9-RAN dipeptide repeat proteins. Based on our prior studies on other microsatellite expansion diseases, this proposal is designed to test our sequestration failure hypothesis, which integrates RNA and RAN gain of function mechanisms. According to this hypothesis, bidirectional sense and antisense C9orf72 transcription results in the recruitment of cellular factors to repeat expansion RNAs to produce sense and antisense RNA foci that sequester these toxic RNAs in the nucleus. Somatic repeat expansion and/or age-related cellular stress results in titration of GGGGCC and GGCCCC RNA binding proteins followed by nucleocytoplasmic export of these RNAs and translation of highly toxic C9-RAN proteins in the cytoplasm that lead to neurodegeneration. We have generated a BAC transgenic model of C9-ALS/FTD that will allow us to test this hypothesis. This mouse develops both the molecular (RNA foci, C9-RAN proteins) and pathophysiological (neuronal loss, paralysis, decreased survival) features of C9-ALS/FTD. In this proposal, we will initially test the hypothesis that RNA GOF effects precede RAN protein accumulation by performing RNA-FISH, transcriptome analysis and immunological assays at various developmental periods and in different brain and spinal cord regions on asymptomatic, pre-symptomatic and symptomatic C9-BAC mice. This information will be used in conjunction with histopathological and electrophysiological assays test the hypothesis that C9-RAN protein accumulation triggers neurodegeneration and the acute disease phase. The possibility that stress pathways modulate RAN translation will also be tested. Finally, we will test whether antisense oligonucleotide (ASO) gapmer-mediated knockdowns of sense, antisense or both sense and antisense C9orf72 transcripts blocks the development of RNA and RNA toxicity in our C9-BAC transgenic mice. Overall, the objective of this study is to define pathogenic mechanisms underlying C9-ALS/FTD disease development and progression and provide an accessible and well-characterized mouse model for therapeutic development.

Project Summary Since we discovered repeat associated non-ATG (RAN) RAN translation in 2011, we and others have shown that RAN proteins accumulate in nine different expansion disorders. These proteins, which can be expressed from both sense and antisense expansion transcripts, accumulate in disease-relevant human tissues including spinocerebellar ataxia type 8 (SCA8) and Huntington disease (HD). We now have evidence that polySer and polyLeu RAN proteins accumulate in a group of spinocebellar ataxias (SCA1, 2, 3, 6 and 7) in which the CAG·CTG expansion mutations are located in polyGln open reading frames. Additionally, we have developed AAV and small molecule approaches to inhibit RAN translation. We will use these tools and genetic approaches to test our central hypotheses that RAN protein pathology is a common feature shared across polyglutamine encoding CAG·CTG expansion disorders and that inhibiting the PKR pathway will reduce RAN protein levels and mitigate disease. We will address our central hypothesis in three specific aims (1) To test the hypothesis that RAN proteins contribute to spinocerebellar ataxias (SCAs) caused by polyglutamine encoding CAG·CTG repeat expansion mutations. (2) To test the hypothesis that SCA and HD RAN proteins are toxic and PKR inhibition will decrease RAN protein levels and improve cellular phenotypes in HD and SCA3 iPSC derived cells (3) : To test the hypothesis that RAN proteins contribute to HD and SCA3 phenotypes in mice independent of polyGln effects using genetic and pharmacological approaches. Taken together these specific aims will determine the contribution of RAN proteins to HD,SCA3 and CAG·CTG repeat expansion disorders and characterize PKR inhibition as a potential therapeutic approach for this large class of devastating repeat expansion diseases.