池田婚姻願（宮内大臣宛様式）

The heat shock response (HSR) is a mechanism to cope with proteotoxic stress by inducing the expression of molecular chaperones and other heat shock response genes. The HSR is evolutionarily well conserved and has been widely studied in bacteria, cell lines and lower eukaryotic model organisms. However, mechanistic insights into the HSR in higher eukaryotes, in particular in mammals, are limited. We have developed an in vivo heat shock protocol to analyze the HSR in mice and dissected heat shock factor 1 (HSF1)-dependent and-independent pathways. Whilst the induction of proteostasis-related genes was dependent on HSF1, the regulation of circadian function related genes, indicating that the circadian clock oscillators have been reset, was independent of its presence. Furthermore, we demonstrate that the in vivo HSR is impaired in mouse models of Huntington's disease but we were unable to corroborate the general repression of transcription that follows a heat shock in lower eukaryotes

Huntington’s Disease iPSC-Derived Brain Microvascular Endothelial Cells Reveal WNT-Mediated Angiogenic and Blood-Brain Barrier Deficits

Brain microvascular endothelial cells (BMECs) are an essential component of the blood-brain barrier (BBB) that shields the brain against toxins and immune cells. While BBB dysfunction exists in neurological disorders, including Huntington's disease (HD), it is not known if BMECs themselves are functionally compromised to promote BBB dysfunction. Further, the underlying mechanisms of BBB dysfunction remain elusive given limitations with mouse models and post-mortem tissue to identify primary deficits. We undertook a transcriptome and functional analysis of human induced pluripotent stem cell (iPSC)-derived BMECs (iBMEC) from HD patients or unaffected controls. We demonstrate that HD iBMECs have intrinsic abnormalities in angiogenesis and barrier properties, as well as in signaling pathways governing these processes. Thus, our findings provide an iPSC-derived BBB model for a neurodegenerative disease and demonstrate autonomous neurovascular deficits that may underlie HD pathology with implications for therapeutics and drug delivery.American Heart Association (12PRE10410000)American Heart Association (CIRMTG2-01152)National Institutes of Health (U.S.) (NIHNS089076

Understanding Huntington's Disease pathogenesis using next generation sequencing analyses

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February 2016.Cataloged from PDF version of thesis. "February 2015."Includes bibliographical references (pages 215-240).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.by Theresa Anne Wasylenko.Ph. D

Developmental alterations in Huntington's disease neural cells and pharmacological rescue in cells and mice

Neural cultures derived from Huntington’s disease (HD) patient-derived induced pluripotent stem cells were used for ‘omics’ analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling, axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development, the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks, gene expression and unique peak profiles associated with dysregulated genes, suggesting a coordinated epigenetic program. Treatment with isoxazole-9, which targets key dysregulated pathways, led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time