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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2013)

Despite years of incremental progress in our understanding of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's Disease (HD), and amyotrophic lateral sclerosis (ALS), there are still no disease-modifying therapeutics. The discrepancy between the number of lead compounds and approved drugs may partially be a result of the methods used to generate the leads and highlights the need for new technology to obtain more detailed and physiologically relevant information on cellular processes in normal and diseased states. We developed a high-throughput automated microscope system and primary neuron model of HD that allows us to monitor dynamic stress responses in intact, fully differentiated neurons. We are able to assay thousands of conditions, including millions of neurons, in a short period of time, which can reveal completely new aspects of biology and identify lead therapeutics in the span of a few months when conventional methods could take years or fail all together. We use this system to understand how neurons, a long-lived, postmitotic cell type, differ in their acute responses to proteotoxic insults including those associated with malformed protein. We show that neurons have a deficient acute stress response to multiple known stimuli, including thermal stress, Hsp90 inhibition, and proteasome inhibition. We find that neurons have low expression of the two major stress-responsive transcriptional activators in mammals, HSF1 and HSF2. We also find that neurons have high constitutive chaperoning capability through relatively high levels of Hsp90 and Hsc70. High Hsp90, however, decreases the acute stress response through negative regulation of HSF1. By increasing HSF1 levels, we were able to restore the response in neurons and protect them from the toxicity associated with malformed protein. We propose a mechanism for the attenuated acute stress response in neurons that implicates a high Hsp90 to HSF1 ratio, which results in an increased ability to cope with chronic stresses, but decreased ability to respond acutely when basal homeostatic responses are overwhelmed. We conclude that targeted therapies to bolster acute stress responses in neurons through increasing HSF1 expression or activation will therefore benefit HD and other neurodegenerative disorders of protein conformation.

    Cover page: An Automated Microscope System to Monitor Dynamic Stress Responses in Neurons
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2017)
    Mutations in leucine-rich repeat kinase 2 (LRRK2) and α-synuclein lead to Parkinson's disease (PD). Disruption of protein homeostasis is an emerging theme in PD pathogenesis, making mechanisms to reduce the accumulation of misfolded proteins an attractive therapeutic strategy. We determined if activating nuclear factor erythroid 2-related factor (Nrf2), a potential therapeutic target for neurodegeneration, could reduce PD-associated neuron toxicity by modulating the protein homeostasis network. Using a longitudinal imaging platform, we visualized the metabolism and location of mutant LRRK2 and α-synuclein in living neurons at the single-cell level. Nrf2 reduced PD-associated protein toxicity by a cell-autonomous mechanism that was time-dependent. Furthermore, Nrf2 activated distinct mechanisms to handle different misfolded proteins. Nrf2 decreased steady-state levels of α-synuclein in part by increasing α-synuclein degradation. In contrast, Nrf2 sequestered misfolded diffuse LRRK2 into more insoluble and homogeneous inclusion bodies. By identifying the stress response strategies activated by Nrf2, we also highlight endogenous coping responses that might be therapeutically bolstered to treat PD.
      Cover page: Nrf2 mitigates LRRK2- and α-synuclein–induced neurodegeneration by modulating proteostasis
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2018)
      Huntington's disease is a progressive neurodegenerative disorder caused by polyglutamine-expanded mutant huntingtin (mHTT). Here, we show that the deubiquitinase Usp12 rescues mHTT-mediated neurodegeneration in Huntington's disease rodent and patient-derived human neurons, and in Drosophila. The neuroprotective role of Usp12 may be specific amongst related deubiquitinases, as the closely related homolog Usp46 does not suppress mHTT-mediated toxicity. Mechanistically, we identify Usp12 as a potent inducer of neuronal autophagy. Usp12 overexpression accelerates autophagic flux and induces an approximately sixfold increase in autophagic structures as determined by ultrastructural analyses, while suppression of endogenous Usp12 slows autophagy. Surprisingly, the catalytic activity of Usp12 is not required to protect against neurodegeneration or induce autophagy. These findings identify the deubiquitinase Usp12 as a regulator of neuronal proteostasis and mHTT-mediated neurodegeneration.
        Cover page: Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington’s diseaseCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2020)
        An amendment to this paper has been published and can be accessed via a link at the top of the paper.
          Cover page: Author Correction: Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington’s diseaseCreative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2018)
          The original version of this Article incorrectly gave a publication date of 8 October 2018; this should have been 28 September 2018. This has now been corrected in the PDF and HTML versions of the Article.
            Cover page: Publisher Correction: Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington’s diseaseCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2020)
            An amendment to this paper has been published and can be accessed via a link at the top of the paper.
              Cover page: Publisher Correction: Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington’s diseaseCreative Commons 'BY' version 4.0 license
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2016)
              Huntington's disease (HD) is a progressive, adult-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminal region of the protein huntingtin (HTT). There are no cures or disease-modifying therapies for HD. HTT has a highly conserved Akt phosphorylation site at serine 421, and prior work in HD models found that phosphorylation at S421 (S421-P) diminishes the toxicity of mutant HTT (mHTT) fragments in neuronal cultures. However, whether S421-P affects the toxicity of mHTT in vivo remains unknown. In this work, we used murine models to investigate the role of S421-P in HTT-induced neurodegeneration. Specifically, we mutated the human mHTT gene within a BAC to express either an aspartic acid or an alanine at position 421, mimicking tonic phosphorylation (mHTT-S421D mice) or preventing phosphorylation (mHTT-S421A mice), respectively. Mimicking HTT phosphorylation strongly ameliorated mHTT-induced behavioral dysfunction and striatal neurodegeneration, whereas neuronal dysfunction persisted when S421 phosphorylation was blocked. We found that S421 phosphorylation mitigates neurodegeneration by increasing proteasome-dependent turnover of mHTT and reducing the presence of a toxic mHTT conformer. These data indicate that S421 is a potent modifier of mHTT toxicity and offer in vivo validation for S421 as a therapeutic target in HD.
                Cover page: Serine 421 regulates mutant huntingtin toxicity and clearance in mice
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2015)
                Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry.
                  Cover page: Sequence-Level Analysis of the Major European Huntington Disease Haplotype
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