Huntington's disease (HD) and Parkinson's disease (PD) are devastating neurodegenerative disorders that are characterized pathologically by degeneration of neurons in the brain and clinically by loss of motor function and cognitive decline in mid to late life. The cause of neuronal degeneration in these diseases is unclear, but both are histologically marked by aggregation of specific proteins in specific brain regions. In HD, fragments of a mutant Huntingtin protein aggregate and cause medium spiny interneurons of the striatum to degenerate. In contrast, PD brains exhibit aggregation of toxic fragments of the alpha synuclein protein throughout the central nervous system and trigger degeneration of dopaminergic neurons in the substantia nigra. Considering the commonalities and differences between these diseases, identifying common biological patterns across HD and PD as well as signatures unique to each may provide significant insight into the molecular mechanisms underlying neurodegeneration as a general process. State-of-the-art high-throughput sequencing technology allows for unbiased, whole genome quantification of RNA molecules within a biological sample that can be used to assess the level of activity, or expression, of thousands of genes simultaneously. In this thesis, I present three studies characterizing the RNA expression profiles of post-mortem HD and PD subjects using high-throughput mRNA sequencing data sets. The first study describes an analysis of differential expression between HD individuals and neurologically normal controls that indicates a widespread increase in immune, neuroinflammatory, and developmental gene expression. The second study expands upon the first study by making methodological improvements and extends the differential expression analysis to include PD subjects, with the goal of comparing and contrasting HD and PD gene expression profiles. This study was designed to identify common mechanisms underlying the neurodegenerative phenotype, transcending those of each unique disease, and has revealed specific biological processes, in particular those related to NFkB inflammation, common to HD and PD. The last study describes a novel methodology for combining mRNA and miRNA expression that seeks to identify associations between mRNA-miRNA modules and continuous clinical variables of interest, including CAG repeat length and clinical age of onset in HD.

ABSTRACT Huntington Disease (HD) is a neurodegenerative disorder that is inherited in an autosomal dominant manner, and for which testing is available. The aim of this retrospective file-based study was to analyse the numbers and demographics of individuals who had diagnostic, predictive or prenatal genetic counselling and/or testing for HD between January 1998 and December 2006 through the Division of Human Genetics, National Health Laboratory Service and University of the Witwatersrand, Johannesburg. Files for 287 individuals who had genetic counselling and/or testing for HD were included in this study, with 77% being diagnostic cases, 20% predictive and 3% prenatal. When the results obtained in this study were compared to a study by Kromberg et al. (1999) done previously in the same Division, it was found that there has been an increase in the number of diagnostic and predictive tests done per year during this study, with diagnostic tests making up a greater percentage of the total number of tests performed. One of the objectives of this study was to characterise the individuals who requested HD testing and to compare the characteristics of those in the diagnostic testing group to those in the predictive testing group. The median age of the individuals in the predictive testing group was 30 years, which was significantly different from the median age of 49 years for individuals in the diagnostic testing group (p<0.001). It was found that there were significantly more women than men requesting predictive testing (p=0.02), while the number of males and females in the diagnostic testing group was similar (p=1.00). There was also a greater percentage of employed (76.4%) versus unemployed (23.6%) individuals in the predictive testing group, while the percentages of employed and unemployed individuals in the diagnostic testing group were similar (45.5% and 54.5% respectively). Significantly more individuals in the diagnostic testing group had children (74.5%) compared to those in the predictive testing group, where 44.6% of individuals had one or more children. There was a greater percentage of white individuals in the predictive testing group (91% white; 3.5% black) compared to the diagnostic testing group (48% white, 42% black). The completion rate of the predictive testing process was 66.7%. In the predictive testing group, 39.5% of individuals tested positive for HD, and in the diagnostic testing group 53% of individuals tested positive for HD. Nine prenatal tests were requested by five different couples, and 7 tests were performed. Three of these fetuses tested positive for HD (including a set of twins) and these two pregnancies were terminated. Overall, there seems to be a lack of awareness of and/or access to the genetic services offered for HD through the Division of Human Genetics, National Health Laboratory Service and University of the Witwatersrand, Johannesburg, particularly among black individuals and the professionals treating them. Information generated from this study can be used to understand the individuals seeking genetic counselling and/or testing for HD better, and can direct efforts to improve awareness and access amongst groups noted to be under-represented. It also serves as a starting point for further research.

Huntington's Disease (HD) is an inherited neurodegenerative disorders caused by CAG repeat expansions in exon 1 of the huntingtin gene (htt). Patients with HD experience profound region specific neural degeneration for reasons that remain incompletely understood. Early studies in HD brain suggest that transcriptional misregulation occurs early in disease, before significant tissue loss and degeneration has occurred. However, a comprehensive understanding of the events that contribute to this remain poorly understood. In this study, we investigate a functional role for small RNA or miRNAs in the central nervous system (CNS) of patients with HD. Our work identifies subsets of miRNAs misregulated in HD. A functional role for these miRNAs was investigated by identifying their predicted targets. We identify a subset of differentially detected miRNAs which are inversely correlated with predicted downstream predicted 3'UTR target genes. We also identify targets of these differentially detected miRNAs including transcriptional regulators REST, CoREST and cFOS. The transcription factor REST silences neuronal gene expression in non-neuronal cells. Polyglutamine expansions in Huntingtin, which cause HD, abrogate REST-mediated Huntingtin binding, and as a result REST translocates to the nucleus, occupies RE1 consensus sites and represses the expression of both coding and non-coding RNAs. In this work, we identify miRNAs (miRNAs) with upstream REST consensus sites that are decreased in HD patient primary motor cortices (BA4). One of these miRNAs, miRNA-9/miRNA-9* is capable of regulating the expression of two components of the REST complex: miRNA-9 targets REST and miRNA-9* targets CoREST. These data provide evidence for a double negative feedback loop between the REST silencing complex and the miRNAs it regulates. In addition to these studies, we identify CNS enriched miRNAs which may differentially regulate human versus non-human primate gene expression. We computationally identified a single nucleotide change from G to A in the 3'UTR of human cFOS 3'UTR which is predicted to be regulated by the brain enriched miRNA-7. A regulatory role for the single nucleotide change in humans (G->A) was assessed by mutating the single nucleotide in the human cFOS 3'UTR (from A->G), as well as by introducing the corresponding human mutation (G->A) into the rhesus and chimpanzee cFOS 3'UTRs. The presence of the A nucleotide in the predicted MRE for miRNA-7 was sufficient to partially abrogate miRNA-7 activity in reporter plasmids. Finally, overexpression of artificial precursor miRNAs in human HEK293 and mouse N2A cell lines confirmed differential targeting of cFOS in human versus mouse cell lines. These data provide evidence for the potential contribution of a single nucleotide change in humans as regards changes in cFOS regulated gene expression. Since cFOS is a transcription factor, downstream affects from altered expression could be significant. Together, this work provides new support for the role of brain enriched miRNAs in the CNS and identifies functional support that their misregulation or altered expression can impact expression of protein coding transcripts in disease brain, and may be relevant to primate brain evolution.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2003. / Vita. Includes bibliographical references.

No description available.

Thesis (Ph. D.)--University of California, San Diego, 1998. / Vita. Includes bibliographical references (leaves 165-174).

Thesis (M.A.)--Carleton University, 1993. / Also available in electronic format on the Internet.

Direct (D1) and indirect (D2) pathway medium spiny neurons (MSNs) in the striatum are severely affected in Huntington’s disease. The aim of this study was to compare the dendritic topology and electrophysiological properties of wild type (WT) D1 and D2 MSNs with those in the Q-175 mouse model of Huntington’s disease. By scanning biocytin-filled MSNs using high-resolution confocal imaging, we quantified the dendritic lengths and complexity of WT and Q-175+/- D1 and D2 MSNs. We correlated these dendritic topological parameters with various electrophysiological properties. Q175+/- D1 MSNs had significantly larger total dendritic lengths, more complex dendritic arbors, and a larger mean number of primary dendrites than their WT counterparts. Q175+/- D2 MSNs had similar total dendritic lengths, dendritic complexities, and mean number of primary branches as the WT D2 MSNs. WT D1 and D2 MSNs were similar in terms of their total dendritic length, total number of intersections, and mean number of primary dendrites suggesting a degree of homogeneity in these cell populations. We found no correlations between membrane resistance, rheobase, EPSC frequency, or EPSC amplitude, and total dendritic length or dendritic complexity of MSNs when observed separately (WT and Q175+/-) or combined, with one exception, a positive correlation between rheobase and total intersections. These findings add to the understanding of the morphology of D1 and D2 MSNs in general, as well as how they are differentially affected by the presence of a CAG expansion in the Q-175+/- mouse model of Huntington’s disease.

Nanomedicine is the fusion between drug therapy and nanotechnology. It is an expanding industry which provides a more efficient and effective way to deliver drugs throughout the body. For individuals suffering from a neurodegenerative disorder, like Huntington’s Disease (HD), current treatments cause major side effects in addition to the disease’s detrimental motor, behavioral and psychiatric symptoms. The goal of the experiment is to encapsulate tetrabenazine, the only FDA approved drug for HD, in a nanoparticle called a micelle. If successful, the new drug nanoparticle can undergo animal testing then clinical trials in hopes of improving patients’ lives.

Indiana University-Purdue University Indianapolis (IUPUI) / Huntington’s disease (HD) is an autosomal dominantly inherited, fatal neurodegenerative disorder for which there is no cure. HD is clinically characterized by progressively worsening motor, cognitive, and psychiatric disturbances. Currently available therapeutics for HD only treat symptoms, but do not address underlying disease pathology. HD pathogenesis is linked to a mutation in the huntingtin gene, which encodes a protein called huntingtin (Htt) that is normally involved in a variety of cellular processes. In healthy individuals, the N-terminus of huntingtin possesses a polyglutamine stretch containing less than 35 glutamines, however, the mutated huntingtin protein (mHtt) has an elongated polyglutamine tract that correlates with the development of HD. The mechanism of deleterious action by mHtt is unknown, but a major hypothesis postulates that mHtt may cause mitochondrial dysfunction. However, the data regarding involvement of mitochondrial impairment in HD pathology are contradictory. Some investigators previously reported, for example, that mHtt suppresses mitochondrial respiratory activity and decreases mitochondrial Ca2+ uptake capacity. However, other investigators found increased respiratory activity and augmented mitochondrial Ca2+ uptake capacity. We used transgenic mouse models of HD to investigate the effect of full-length and fragments of mHtt on oxidative metabolism and Ca2+ handling using a combination of isolated mitochondria, primary neurons, and whole-animal metabolic measurements. We evaluated the effect of full-length mHtt on isolated mitochondria and primary neurons from YAC128 mice. We found no alteration in respiratory activity or Ca2+ uptake capacity, indicative of mitochondrial damage, between mitochondria or neurons from YAC128 mice compared to wild-type (WT) mice. Furthermore, we measured whole animal oxidative metabolism and physical activity level and found that YAC128 mice do not display any decline in metabolic and physical activity. Although full-length mHtt expressing YAC128 mice may be a more faithful genetic recapitulation of HD, data suggests mHtt fragments may be more toxic. To assess the effect of mHtt fragments, we used isolated brain mitochondria and primary striatal neurons from the R6/2 mouse model and found no significant impairment in respiration or Ca2+ handling. Thus, our data strongly support the hypothesis that mHtt does not alter mitochondrial functions assessed either with isolated mitochondria, primary neurons, or whole animals.