Understanding Huntington's Disease pathogenesis using next generation sequencing analyses

Abstract

Huntington's disease is one of nine expanded (CAG) repeat disorders. The expansion in Huntington's disease lies in the first exon of the huntingtin (HTT) gene and is pathogenic when (CAG)>/= 40 . Individuals with Huntington's disease develop motor, cognitive, and psychiatric symptoms in adulthood. These symptoms progress for approximately 15 years at which time they become fatal. The clinical manifestation of HD largely results from the extreme degeneration of neurons in the striatum and cortex. The HTT gene encodes the huntingtin (HTT) protein. Over the years, researchers have developed a rich understanding of the consequences of loss of wildtype HTT function, gain of toxic mutant HTT function, and mutant HTT RNA toxicity. However, the mechanisms through which pathology develops are still largely ambiguous. Given the widespread involvement of HTT in cellular processes, next generation DNA sequencing technologies offer a rich opportunity to explore genome-wide effects of the HD mutation and may help answer mechanistic questions. The application of many next generation DNA sequencing methods is a new luxury for researchers. DNA sequencing methods have undergone a rapid technical evolution which has accelerated the financial feasibility of applying DNA sequencing involved methods on a routine basis. In this thesis, two high throughput analysis techniques, RNA-Seq and ChIP-Seq, were applied to Huntington's disease models to better understand disease mechanisms, and a third high throughput analysis technique, Ribo-Seq, was optimized for future HD studies. RNA-Seq on Huntington's disease model mice and their wildtype littermates demonstrated extensive and progressive dysregulation of the transcriptome in HD striatum and cortex, with most of the affected genes having a lower steady state expression in mutant tissues. ChIP-Seq with an antibody against trimethylated- Histone3-Lysine4 (H3K4Me3) demonstrated both a general reduction of H3K4me3 levels and a unique histone profile at the promoters of HD downregulated genes. Analysis of RNA-Seq results for splicing changes showed that mutant HTT itself is mis-spliced. This mis-splicing product is translated into a small, pathogenic HTT fragment which may have considerable implications for HD therapeutic design. In addition to CNS degeneration, severe muscle dysfunction is an early clinical observation in HD and many CAG repeat expansion disorders. Proper muscle form and function is dependent on an extensive alternative splicing program. Thus RNASeq data on muscle tissue from mouse models of several CAG expansion disorders was examined for genome-wide splicing alterations. Widespread mis-splicing was detected in the muscle of both Spinocerebellar ataxia 7 and Huntington's disease mouse models and minor splicing dysregulation was detected in Spinal-bulbar muscular atrophy. Lastly, methods were developed to examine translational control and mRNA localization in the brain of Huntington's disease mice. Concurrent Ribo-Seq and RNA-Seq in diseased and wildtype animals would answer if there was altered translational control. The Ribo-Seq protocol designed in cell culture was optimized for use on brain tissue and is ready for application in HD mouse models. Analysis of the localization of mRNA transcripts to neuronal projections can be studied by combining fractionation experiments with RNA-Seq. A method to prepare high quality RNA from isolated neuronal projections was developed and is now applicable to RNA-Seq studies.