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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 (2016)

The Role of Wnt Signaling Proteins in Neurodevelopment and Psychiatric Susceptibility

Robert Stanley

Abstract

Proper regulation of cell signaling pathways is required for normal neurodevelopment. Dysfunction in signaling can lead to problems in the development of the brain and increased susceptibility to develop psychiatric disorders. Many components are required to act in concert in multiple intersecting pathways, and mutations in these components can lead to aberrant signaling. Modern human genetics is increasingly identifying genes and gene mutations associated with psychiatric risk via genome-wide sequencing of patients with psychiatric disorders. Once specific genes and their mutations are identified as possible contributors to disease, experimental assays are required to determine the functionality of these genes and their variants in neurodevelopment and signaling.

The scaffolding protein Dix-Domain Containing 1 (Dixdc1) is a positive regulator of Wnt-/β-catenin signaling that interacts with the psychiatric risk gene Disrupted in Schizophrenia 1 (Disc1). We have a utilized a Dixdc1 knock-out mouse line to help determine functions of this gene in neurodevelopment. Using cultured hippocampal neurons and tissue sections from intact brains expressing GFP in cortical neurons, we have interrogated what effect deletion of Dixdc1 has on the development of dendrites, dendritic spines, and synapses in pyramidal neurons of the forebrain. In addition, using sequencing data from patient samples of three different psychiatric disorders (ASD, Schizophrenia, and Bipolar Disorder) we have identified a set of rare missense mutations in the Dixdc1 locus that are present in patients with these diseases. These variants were then functionally tested using several neurodevelopmental and canonical Wnt/Beta-catenin signaling assays.

Our results show that Dixdc1 is required for proper development of spines- both in their quantity and maturation- and excitatory synapses in forebrain pyramidal neurons. There appears to be no effect on dendrite complexity (in a mixed genetic background) or on inhibitory synapse numbers. Additionally, mutant versions of Dixdc1 found in ASD patient samples that have aberrant Wnt signaling are unable to rescue neurodevelopmental phenotypes in KO neurons, while some of these variants that are hyperactive for Wnt/β-catenin signaling have dominant negative effects when expressed in wild type neurons.

    Cover page: The Role of Wnt Signaling Proteins in Neurodevelopement and Psychiatry
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2016)
    The transmembrane protein Vangl2, a key regulator of the Wnt/planar cell polarity (PCP) pathway, is involved in dendrite arbor elaboration, dendritic spine formation and glutamatergic synapse formation in mammalian central nervous system neurons. Cultured forebrain neurons from Vangl2 knockout mice have simpler dendrite arbors, fewer total spines, less mature spines and fewer glutamatergic synapse inputs on their dendrites than control neurons. Neurons from mice heterozygous for a semidominant Vangl2 mutation have similar but not identical phenotypes, and these phenotypes are also observed in Golgi-stained brain tissue from adult mutant mice. Given increasing evidence linking psychiatric pathophysiology to these subneuronal sites and structures, our findings underscore the relevance of core PCP proteins including Vangl2 to the underlying biology of major mental illnesses and their treatment.
      Cover page: The Planar Cell Polarity Transmembrane Protein Vangl2 Promotes Dendrite, Spine and Glutamatergic Synapse Formation in the Mammalian Forebrain
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2021)
      Overexpression and aggregation of α-synuclein (ASyn) are linked to the onset and pathology of Parkinson's disease and related synucleinopathies. Elevated levels of the stress-induced chaperone Hsp70 protect against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations, and inhibitors, we confirm that Hsp70 interacts with ASyn via an as yet unidentified, noncanonical interaction site in the C-terminal domain. Finally, we report a biological role for a similar mode of action in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target the Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70's canonical action. Additionally, these results raise the question of whether other misfolded proteins might also engage Hsp70 via the same noncanonical mechanism.
        Cover page: Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2019)
        In figure 4 the first sentence of the caption has been corrected to "Loss of Sestd1 does not affect β-catenin but increases activity of Rac1 and RhoA in the neonatal and mature hippocampus." The new caption text has been corrected online and in print. The author regrets this error.
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2019)
          SEC14 and Spectrin domain-1 (Sestd1) is a synapse protein that exhibits a striking shift from the presynaptic to postsynaptic space as neurons mature postnatally in the mouse hippocampus. Hippocampal pyramidal neurons from mice with global genetic deletion of Sestd1 have reduced dendrite arbors, spines, and excitatory synapses. Electrophysiologically this correlates with cell-autonomous reductions in both AMPA- and NMDA-excitatory postsynaptic currents in individual hippocampal neurons from which Sestd1 has been deleted in vivo. These neurodevelopmental and functional deficits are associated with increased activation of the Rho family GTPases Rac1 and RhoA. Co-immunoprecipitation and mass spectrometry reveal that the Breakpoint Cluster Region protein, a Rho GTPase activating protein (GAP), forms complexes with Sestd1 in brain tissue. This complements earlier findings that Sestd1 can also partner with other Rho family GAPs and guanine nucleotide exchange factors. Our findings demonstrate that Sestd1 is a developmentally dynamic synaptic regulator of Rho GTPases that contributes to dendrite and excitatory synapse formation within differentiating pyramidal neurons of the forebrain.
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2022)
            Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adapter of a Cullin-3 RING E3 ubiquitin ligase complex responsible for the degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations was identified in clonal hematopoietic conditions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 that escape degradation, drives hematopoietic stem cell (HSC) expansion and leukemia in vivo. Although RIT1 stabilization was sufficient to drive hematopoietic transformation, transformation mediated by LZTR1 loss required MRAS. Proteolysis targeting chimeras (PROTAC) against RAS or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data reveal proteolysis of noncanonical RAS proteins as novel regulators of HSC self-renewal, define the function of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation.

            Significance

            Here we identify that impairing proteolysis of the noncanonical RAS GTPases RIT1 and MRAS via LZTR1 downregulation or leukemia-associated mutations stabilizing RIT1 enhances MAP kinase activation and drives leukemogenesis. Reducing the abundance of GTP-bound KRAS and NRAS overcomes the resistance to FLT3 kinase inhibitors associated with LZTR1 downregulation in leukemia. This article is highlighted in the In This Issue feature, p. 2221.
              Cover page: Impaired proteolysis of non-canonical RAS proteins drives clonal hematopoietic transformation
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2021)
              We report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of receptor binding motif replacements such as N439K and Y453F, known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that, although ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of spike proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of the H69 and V70 residues in the Alpha variant B.1.1.7 spike (where ΔH69/V70 occurs naturally) impairs spike incorporation and entry efficiency of the B.1.1.7 spike pseudotyped virus. Alpha variant B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild-type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.
                Cover page: Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the Alpha variant B.1.1.7
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2021)
                The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.
                  Cover page: SARS-CoV-2 evolution during treatment of chronic infection
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