Microsatellite repeats are a phenomenon found in DNA where a short sequence, usually 1-6bps, is repeated dozens to hundreds of times. Microsatellite repeats that are able to be transcribed are termed expanded tandem repeat-containing RNA (xtrRNA) [1]. xtrRNA have been associated with many diseases, such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), which are both caused by a repeat in the C9ORF72 gene. Recent research has been focused on trying to provide treatments for patients with these diseases. This study focuses on creating a drug screening process for therapeutics targeting transcription by stopping or slowing the transcription of C9ORF72 repeat expansions. One project has focused on interrupting the interaction of two transcription factors, SUPT5H and SUPT4H1, to slow transcription. Another project has focused on slowing transcription by using transcriptional inhibitors or nucleoside analogs at low concentrations. Our hypothesis is that if transcription rates are slowed enough, pausing or arrest of RNA polymerase will be induced at complex sequences, including GC-rich regions and repeats. This should reduce synthesis of xtrRNA and provide a starting point for therapeutic development.

Microsatellites, or simple tandem repeat sequences, occur naturally in the human genome and have important roles in genome evolution and function. However, the expansion of microsatellites is associated with over two dozen neurological diseases. A common denominator among the majority of these disorders is the expression of expanded tandem repeat-containing RNA, referred to as xtrRNA, which can mediate molecular disease pathology in multiple ways. Frontotemporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) are two fatal neurodegenerative diseases with significant clinical, neurological and genetic overlap thus referred to as C9FTD/ALS. Currently, gaps in the study of the underlying disease mechanisms persist, which can aid in the identification of promising therapeutic approaches. Access to simple models of neurological repeat expansion disease is critical for investigating biochemical mechanisms and for early therapeutic discovery. To better understand the molecular pathology of C9FTD/ALS repeat expansion disorder, we cloned GGGGCC repeats, which are the leading genetic cause of C9FTD/ALS. We employed a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors and validated the cloning to facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level using these vectors. In this study, we also differentiated C9FTD/ALS patient-derived induced pluripotent stem cells (iPSCs) to neural stem cells (NSCs) to be used as model systems. The use of iPSCs and NSCs to reveal important insights into the pathogenic mechanisms and to generate multiple neural cell types presents an excellent opportunity for researchers to model neurodegenerative diseases for cell therapy and drug discovery. We further investigated potential nuclear export mechanisms for C9FTD/ALS xtrRNA. The nuclear export mechanisms of xtrRNA in C9FTD/ALS are not well studied. ASOs and siRNAs were employed to knockdown genes of interest to study their involvement in the nuclear export of xtrRNA. We saw promising results on knockdown of TorsinA involved in nuclear export of xtrRNAs, corroborated by a substantial increase in the average number of xtrRNA foci in the nucleus. Our initial study provides evidence that TOR1A may be involved in the nuclear export of aberrant C9FTD/ALS repeat-containing RNAs. Due to the lack of reliable and robust assays to detect RAN translation products, the effect of the knockdown of TorsinA in these cell lines still remains to be explored. But the current study lays the groundwork for a deeper understanding of the less-studied nuclear export mechanisms in C9FTD/ALS and could reveal new therapeutic approaches to selectively block the nuclear export of xtrRNA through the use of RNAi and ASOs. The insights gained from this study will help us understand future events in the xtrRNA life cycle such as repeat translation mechanisms.

This dissertation examines the history of the creation and development of the concept of anticipation, a pattern of heredity found in several diseases (e.g. Huntington’s disease and myotonic dystrophy), in which an illness manifests itself earlier and often more severely in successive generations. It reconstructs major arguments in twentieth-century debates about anticipation and analyzes the relations between different research communities and schools of thought. Developments in cutting-edge medicine, biology, and genetics are analyzed; many of these developments were centered in Britain, but saw significant contributions by people working in France, Germany, Switzerland, the Netherlands and North America. Chapter one traces precursor notions in psychiatric and hereditarian thought from 1840 to the coining of the term ‘anticipation’ by the ophthalmologist Edward Nettleship in 1905. Key roles in the following chapters are played by several figures. Prior to World War II, these include: the neuropathologist F.W. Mott, whose advocacy during 1911- 1927 led to anticipation being called “Mott’s law”; the biometrician and eugenicist Karl Pearson, who opposed Mott on methodological and political grounds; and two politically and theoretically opposed Germans – Ernst Rüdin, a leading psychiatrist and eugenicist who came to reject anticipation, and Richard Goldschmidt, a geneticist who offered a peculiar Mendelian explanation. The British psychiatrist and human geneticist, Lionel Penrose, makes a first interwar appearance, but becomes crucial to the story after World War II due to his systematic dismissal of anticipation, which discredited the notion on orthodox Mendelian grounds. The final chapters highlight the contributions of Dutch neurologist Christiaan Höweler, whose 1980s work demonstrated a major hole in Penrose’s reasoning, and British geneticist Peter Harper, whose research helped demonstrate that expanding trinucleotide repeats accounted for the transgenerational worsening without contradicting Mendel and resurrected anticipation as scientifically legitimate. Reception of the concept of anticipation is traced across the century through the examination of textbooks used in different fields. This dissertation argues against established positions regarding the history of the concept, including claims that anticipation’s association with eugenics adequately explains the rejection of the notion after 1945. Rejected, in fact, by many eugenicists from 1912, anticipation was used by physicians until the 1960s.