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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2009)
The neuron-restrictive silencer factor (NRSF), also known as repressor element -1 (RE1) silencing transcription factor (REST), is known to act as a transcriptional repressor of neuron-specific genes in nuclei of non- neuronal cells. REST binds a DNA sequence known as neuron- restrictive silencer elements (NRSEs/RE1s) and recruits co -repressors to carry out silencing chromatin modifications. Recent REST ChIP-seq publications have shown that REST binding is not limited to the canonical RE1 sequence, but binds multiple variations of it across the genome. This study focuses on determining REST's capacity to act as a repressor based on the binding site's degree of correspondence to the canonical RE1 motif. I show that the RE1 right half site acts as a REST- recruiting repressive element. I have also begun studying differential recruitment of REST protein and its cofactors between human embryonic stem cells (hESCs) and hESC- derived neural progenitor cells (NPCs) on a genome-wide scale using chromatin immunoprecipitation followed by massive parallel sequencing of DNA tags (ChIP-seq). I also present preliminary data suggesting that REST may have a second function as a post-transcriptional activator. Increased luciferase expression is observed when reporter constructs were cotransfected with high levels of REST in mouse Neuro2A (N2A) cells. I show that this concentration dependent function of REST is due to the cell's limited capacity to transport and/or retain REST in the nucleus, where it acts as a repressor. With increasing concentrations of REST, a large fraction remains in the cytoplasm, where REST may bind and stabilize the luciferase transcript and/or enhance its translation
    Cover page: Nuclear and Cytoplasmic Roles of the Transcription factor REST
    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2023)

    Stress granules (SGs) are highly dynamic cytoplasmic assemblies of ribonucleoprotein complexes. By stalling mRNA translation and sequestering aggregation-prone proteins, these transient membraneless organelles are thought to be a protective response during cellular stress. Defects in SG assembly and clearance are linked to neurodegenerative disease: stable SG-like inclusions in brain are hallmarks of sporadic amyotrophic lateral sclerosis (ALS) and related disorders, and genetic mutations in SG proteins cause familial forms of these diseases. Recent efforts using biochemical fractionation and protein proximity-labeling techniques have identified over 400 components in SGs. However, it has yet to be determined which proteins are critical for the formation, stabilization, or disassembly of these macromolecules, and whether they may be exploited to attenuate the progression of associated diseases. For my dissertation, I developed a screening platform combining high-content imaging on microraft arrays and pooled CRISPR knockout libraries to systematically evaluate over 1,000 RNA-binding proteins (RBPs) for roles in regulating SG abundance. This easy-to-use workflow is inexpensive and can be adapted to study gene function in the regulation of a wide range of subcellular phenotypes. With this platform, we identified and validated previously uncharacterized RBPs that regulate sodium arsenite-induced SGs when depleted. Next, we used previously published research on the composition of neuronal SGs to examine the effect of depleting specific SG components on neurodegenerative symptoms in animal models of ALS. We discovered new functions of SG proteins (DAZAP1, FAM98A, OTUD4, and SND1) in reducing pathogenic TDP-43 aggregates in human cells and improving symptoms in Drosophila models of ALS. Notably, SG protein OTUD4 was found to have a positive impact on reversing key characteristics of ALS across all models studied. Our research using both human and Drosophila models provide a comprehensive understanding of the connection between SGs and functional decline seen in ALS patients.

      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2020)
      Stress granules (SGs) are evolutionarily conserved condensates of ribonucleoproteins that assemble in response to metabolic stresses. Because aberrant SG formation is associated with amyotrophic lateral sclerosis (ALS), understanding the connection between metabolic activity and SG composition can provide therapeutic insights into neurodegeneration. Here, we identify 17 metabolic enzymes recruited to yeast SGs in response to physiological growth stress. Furthermore, the product of one of these enzymes, AdoMet, is a regulator of SG assembly and composition. Decreases in AdoMet levels increase SG formation, while chronic elevation of AdoMet produces SG remnants lacking proteins associated with the 5' end of transcripts. Interestingly, acute elevation of AdoMet blocks SG formation in yeast and motor neurons. Treatment of ALS-derived motor neurons with AdoMet also suppresses the formation of TDP-43-positive SGs, a hallmark of ALS. Together, these results argue that AdoMet is an evolutionarily conserved regulator of SG composition and assembly with therapeutic potential in neurodegeneration.
        Cover page: Conserved metabolite regulation of stress granule assembly via AdoMet
        • Article
        • Peer Reviewed
        UC Santa Cruz Previously Published Works (2019)
        Glial cells regulate multiple aspects of synaptogenesis. In the absence of Schwann cells, a peripheral glial cell, motor neurons initially innervate muscle but then degenerate. Here, using a genetic approach, we show that neural activity-regulated negative factors produced by muscle drive neurodegeneration in Schwann cell-deficient mice. We find that thrombin, the hepatic serine protease central to the hemostatic coagulation cascade, is one such negative factor. Trancriptomic analysis shows that expression of the antithrombins serpin C1 and D1 is significantly reduced in Schwann cell-deficient mice. In the absence of peripheral neuromuscular activity, neurodegeneration is completely blocked, and expression of prothrombin in muscle is markedly reduced. In the absence of muscle-derived prothrombin, neurodegeneration is also markedly reduced. Together, these results suggest that Schwann cells regulate NMJs by opposing the effects of activity-regulated, muscle-derived negative factors and provide the first genetic evidence that thrombin plays a central role outside of the coagulation system.
          Cover page: Glial cells maintain synapses by inhibiting an activity-dependent retrograde protease signal
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2022)
          Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.
            Cover page: Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis
            • Article
            • Peer Reviewed
            UC Santa Cruz Previously Published Works (2016)
            The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.
              Cover page: Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2018)
              Sporadic amyotrophic lateral sclerosis (sALS) is the most common form of ALS, however, the molecular mechanisms underlying cellular damage and motor neuron degeneration remain elusive. To identify molecular signatures of sALS we performed genome-wide expression profiling in laser capture microdissection-enriched surviving motor neurons (MNs) from lumbar spinal cords of sALS patients with rostral onset and caudal progression. After correcting for immunological background, we discover a highly specific gene expression signature for sALS that is associated with phosphorylated TDP-43 (pTDP-43) pathology. Transcriptome-pathology correlation identified casein kinase 1ε (CSNK1E) mRNA as tightly correlated to levels of pTDP-43 in sALS patients. Enhanced crosslinking and immunoprecipitation in human sALS patient- and healthy control-derived frontal cortex, revealed that TDP-43 binds directly to and regulates the expression of CSNK1E mRNA. Additionally, we were able to show that pTDP-43 itself binds RNA. CK1E, the protein product of CSNK1E, in turn interacts with TDP-43 and promotes cytoplasmic accumulation of pTDP-43 in human stem-cell-derived MNs. Pathological TDP-43 phosphorylation is therefore, reciprocally regulated by CK1E activity and TDP-43 RNA binding. Our framework of transcriptome-pathology correlations identifies candidate genes with relevance to novel mechanisms of neurodegeneration.
                Cover page: Transcriptome–pathology correlation identifies interplay between TDP-43 and the expression of its kinase CK1E in sporadic ALS
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2021)
                Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington's disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of patients with HD and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex. Of these targets, SG components were enriched, including the SG-nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of the brains of patients with HD. Intriguingly, we also observed that the SG-associated TAR DNA-binding protein 43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1 granule-positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD.
                  Cover page: Huntington’s disease mice and human brain tissue exhibit increased G3BP1 granules and TDP43 mislocalization
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2019)
                  Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD.
                    Cover page: Small-Molecule Modulation of TDP-43 Recruitment to Stress Granules Prevents Persistent TDP-43 Accumulation in ALS/FTD
                    • Article
                    • Peer Reviewed
                    UC San Diego Previously Published Works (2024)
                    Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) and enhanced crosslinking and immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with SmD1, small nuclear RNAs, and splicing factor mRNAs in motor neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, and inhibiting SmD1/SMN complex increased CHMP7 nuclear localization. Crucially, overexpressing SmD1 in ALS iPSC-MNs restored CHMP7's cytoplasmic localization and corrected STMN2 splicing. Our findings suggest that early ALS pathogenesis is driven by SMN complex dysregulation.
                      Cover page: Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypesCreative Commons 'BY' version 4.0 license
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