Browsing by Subject "SCA1"
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Item Disrupting Nuclear Localization of Expanded ATXN1 Mitigates SCA1 Phenotypes and Transcriptomic Perturbations Across Brain Regions(2022-02) Handler, HillarySpinocerebellar ataxia type 1 (SCA1) is a dominant neurodegenerative disease caused by ATAXIN1 (ATXN1) trinucleotide repeat expansion and characterized by motor dysfunction, cognitive impairment, and premature death. Degeneration of cerebellar Purkinje cells is a frequent and prominent pathological feature of SCA1. Previous research found that transport of ATXN1 to Purkinje cell nuclei is required for pathology, where mutant ATXN1 alters transcription. In addition to cerebellar degeneration, cell loss and atrophy has been observed in the medulla, cerebral cortex, hippocampus, and striatum of patients with SCA1. These brain regions have not historically been a major focus of SCA1 research. To test the hypothesis that nuclear localization of ATXN1 has a critical role in pathogenesis across diverse regions of the central nervous associated with SCA1, CRISPR-Cas9 was used to develop a knock-in SCA1 mouse model with an amino acid alteration (K772T) in the nuclear localization sequence of the expanded ATXN1 protein. Characterization of these Atxn1175QK772T/2Q mice indicates that nuclear localization of expanded ATXN1 contributes to many SCA1-like phenotypes including motor dysfunction, cognitive deficits, and premature lethality. The K772T mutation reduces nuclear localization, improves extractability, and delays nuclear inclusion formation of expanded ATXN1 throughout the brain. RNA sequencing analyses show that transcriptomic aspects of SCA1 pathogenesis differ between the cerebellum, medulla, cerebral cortex, hippocampus, and striatum. The findings from this project establish that nuclear localization of expanded ATXN1 is a key aspect of SCA1 pathogenesis throughout the brain and suggest that the specific molecular mechanisms of disease progression are largely unique to each of the various brain regions affected.Item Regulation and subcellular compartmentalization of ataxin-1 phosphorylation at Serine776.(2011-03) Lai, ShaojuanSpinocerebellar ataxia type 1 (SCA1) is an autosomal dominant cerebellar ataxia caused by the expansion of a CAG repeat encoding an abnormally long polyglutamine tract in Ataxin-1 protein. Although many studies demonstrate that subcellular distribution of Ataxin-1 and protein folding/degradation pathways modulate neurodegeneration, the mechanism of pathogenesis is not completely understood. Phosphorylation of Ataxin-1 at Serine776 (S776) was previously shown to regulate Ataxin-1's functions and SCA1 pathogenicity. In addition, mice expressing human wild type Ataxin-1-[30Q] with a mutation replacing S776 with a phosphomimicking aspartic acid show similar SCA1 pathology as Ataxin-1-[82Q] mice. Here I investigated the mechanism by which phosphorylation of Ataxin-1 at S776 is regulated. I found in the cerebellum a large proportion of Ataxin-1 is phosphorylated at S776 with phosphorylated S776 enriched in the nucleus. While the kinase activity for Ataxin-1 at S776 is localized to the cerebellar cytoplasm, the phosphatase activity is restricted to the nucleus. PP2A was shown to be the phosphatase for phosphorylated S776 Ataxin-1 (Ataxin-1-pS776). 14-3-3, a protein enriched in the cytoplasm, blocks dephosphorylation of Ataxin-1-pS776 by PP2A in the cytoplasm and may affect the shuttling of Ataxin-1 to the nucleus. This work suggests that Ataxin-1 after it is phosphorylated in the cytoplasm, shuttles to the nucleus where it is dephosphorylated by PP2A. The separation of phosphorylation and dephosphorylation of S776-Ataxin-1 into two subcellular compartments may suggest that they regulate different Ataxin-1 functions in different subcellular compartments.Item The Role of Microglia and Astrocyte in Spinocerebellar Ataxia Type 1(2020-11) Ferro, AustinSpinocerebellar ataxia type 1 (SCA1) is a fatal dominantly inherited neurodegenerative disease. Even though there has been illuminating work on the effect of the disease-causing protein, a polyQ expanded ATAXIN-1 (ATXN1) on neurons, the relative contribution to disease of glia has been unknown. Here I present my work on glial-neuron interactions in the context of SCA1; focusing on the neuroinflammatory and activation of microglia as well as the previously undiscovered cell-autonomous effect of polyQ expanded ATXN1 on astrocytes. Using the Lysm-Cre Ikkβflx/tlx line to assess the role of microglial activation in the transgenic ATXN1[82Q] mouse model of SCA1, I show that inhibition of microglial reactivity does not have a large effect on SCA1 disease pathology. Instead, we found that the Lysm-Cre Ikkβflx/tlx line itself had motor performance deficits in the absence of Purkinje cell degeneration. Correlating with this motor performance deficiency, Lysm-Cre Ikkβflx/tlx mice had a prominent deficiency in climbing fiber removal onto Purkinje cells. Due to the low impact of microglia on SCA1 pathology, I then focus on astrocytes and in particular, astrocytic Kir4.1/Kcnj10. Astrocytic Kcnj10 was downregulated throughout the brain of the knock-in Atxn1154Q/2Q mouse line. To investigate the potential role of cell-autonomous effects of Atxn1 on astrocytic transcription, I used the novel conditional humanized ATXN1floxed 146Q/2Q line to delete polyQ expanded ATXN1 from astrocytes. Conditional astrocytic deletion of polyQ ATXN1 did not influence failure to gain weight nor a prominent effect on rotarod pathology. Yet, there was a trending rescue of Kcnj10 expression in the medulla, suggesting a cell-autonomous effect of ATXN1 on astrocytic transcription. In conclusion, my thesis work concerning the role of both microglia and astrocyte in the pathogenesis of SCA1 has revealed the importance of NFᴋB signaling in cerebellar development as well as the potential cell-autonomous effect of polyQ ATXN1 on astrocytes.Item Transcriptional disruptions and functional correlates in a human induced pluripotent stem cell – derived motor neuron model of Spinocerebellar ataxia type 1(2022-08) Sheeler, CarrieIt is hypothesized that degeneration of the motor neurons (MNs) in the brain stem and spinal cord contributes to premature lethality in spinocerebellar ataxia type 1 (SCA1) by affecting the strength of swallowing and respiratory drive. While we can recreate some aspects of MN pathology in mouse models, loss of MNs has only been seen in SCA1 patient populations. This, in addition to other species differences that exist between rodents and humans, accentuates the need for translational human models of disease which can be used to uncover therapeutically relevant cellular and molecular mechanisms underlying dysfunction in vulnerable cell types. To investigate potential mechanisms underlying SCA1 pathology in human MNs, I developed a human induced pluripotent stem cell (iPSC)-derived MN model of SCA1. This is, to our knowledge, the first cell-type specific iPSC-derived model made to study SCA1. Previous work in mouse models has demonstrated both that ATXN1 regulates cellular transcription (Ingram et al 2016, Rousseaux et al 2018, Friedrich et al 2019) and that its entry into the nuclei of cells is important for the longevity of mouse models of SCA1 (Handler et al 2022). Thus, I predicted that the presence of mutant ATXN1 in hiPSC-derived MNs would drive measurable transcriptomic changes in SCA1 samples compared to unaffected controls. Furthermore, transcriptomic disruptions might indicate functional pathways of interest for future studies into the lethality of SCA1. I performed bulk RNA sequencing on MN-enriched cultures to assess underlying transcriptional changes that may be affecting SCA1 human MNs, and subsequently identified disruptions in key cellular processes including extracellular matrix (ECM) regulation, calcium ion binding, and mitotic cell cycle regulation. Proper regulation of ECM is key to many aspects of neuronal development and function, including extension of neurites, proper placement and presentation of receptors and ion channels, cell-cell communication, synapse formation, and intracellular transport. As such, I predicted that broad changes in ECM regulation might result in measurable changes in some of these capabilities in SCA1 MN enriched cultures. Neurite outgrowth was measured in motor neuron progenitors (pMNs) and immature motor neurons (iMNs). I determined that SCA1 samples do not exhibit remarkable disruptions in neurite outgrowth at either timepoint. Assessment of spontaneous calcium activity exhibited a similar degree of physiological maturity between SCA1 and unaffected control MN enriched cultures. Additionally, measurement of miniature excitatory postsynaptic currents (mEPSCs) demonstrated formation of synapses but no functional differences in SCA1 samples compared to controls. However, glutamate evoked calcium activity demonstrated a reduced amplitude of calcium response in SCA1 cultures. This occurred despite any measurable transcriptomic changes in glutamate receptor expression. This indicates potential underlying disruptions in receptor activity and calcium dynamics in SCA1 MN-enriched cultures and provides a potential avenue of interest for future work investigating disruptions in communication in the SCA1 spinal cord.