Avanti Gokhale

Quantitative Proteomic and Genetic Analyses of the Schizophrenia Susceptibility Factor Dysbindin Identify Novel Roles of the Biogenesis of Lysosome-Related Organelles Complex 1

The Biogenesis of Lysosome-Related Organelles Complex 1 (BLOC-1) is a protein complex containing the schizophrenia susceptibility factor dysbindin, which is encoded by the gene DTNBP1. However, mechanisms engaged by dysbindin defining schizophrenia susceptibility pathways have not been quantitatively elucidated. Here, we discovered prevalent and novel cellular roles of the BLOC-1 complex in neuronal cells by performing large-scale Stable Isotopic Labeling of Cells in Culture (SILAC) quantitative proteomics combined with genetic analyses in dysbindin-null mice (Mus musculus) and the genome of schizophrenia patients. We identified 24 proteins that associate with the BLOC-1 complex, many of which were altered in content/distribution in cells or tissues deficient in BLOC-1. New findings include BLOC-1 interactions with the COG complex, a Golgi apparatus tether, and antioxidant enzymes peroxiredoxins 1–2. Importantly, loci encoding eight of the 24 proteins are affected by genomic copy number variation in schizophrenia patients. Thus, our quantitative proteomic studies expand the functional repertoire of the BLOC-1 complex and provide insight into putative molecular pathways of schizophrenia susceptibility.

Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

The newly discovered Ca2+-activated Cl− channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca2+-activated Cl− currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca2+-binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.

The schizophrenia susceptibility factor dysbindin and its associated complex sort cargoes from cell bodies to the synapse

A novel vesicle transport mechanism is described that requires dysbindin-associated complexes for cargo targeting from neuronal cell bodies to neurites and nerve terminals. The results suggest that mistargeting of specific vesicular cargoes may underlie, in part, the molecular pathogenesis of schizophrenia.

Cell Biology of the BLOC-1 Complex Subunit Dysbindin, a Schizophrenia Susceptibility Gene

There is growing interest in the biology of dysbindin and its genetic locus (DTNBP1) due to genetic variants associated with an increased risk of schizophrenia. Reduced levels of dysbindin mRNA and protein in the hippocampal formation of schizophrenia patients further support involvement of this locus in disease risk. Here, we discuss phylogenetically conserved dysbindin molecular interactions that define its contribution to the assembly of the biogenesis of lysosome-related organelles complex-1 (BLOC-1). We explore fundamental cellular processes where dysbindin and the dysbindin-containing BLOC-1 complex are implicated. We propose that cellular, tissue, and system neurological phenotypes from dysbindin deficiencies in model genetic organisms, and likely individuals affected with schizophrenia, emerge from abnormalities in few core cellular mechanisms controlled by BLOC-1-dysbindin-containing complex rather than from defects in dysbindin itself.

The WASH complex, an endosomal Arp2/3 activator, interacts with the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type IIα

The WASH complex, an endosomal activator of the Arp2/3 complex involved in branched actin polymerization, is identified as a new factor in vesicle traffic mediated by the Hermansky–Pudlak syndrome complex BLOC-1.

Regulation of ciliary motility: conserved protein kinases and phosphatases are targeted and anchored in the ciliary axoneme

Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9+2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation/dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.

Regulation of dynein-driven microtubule sliding by the axonemal protein kinase CK1 in Chlamydomonas flagella

CK1 puts the brakes on dynein activity when added to purified axonemes in vitro, presumably to regulate how flagella bend.

Mutations in the BLOC-1 subunits dysbindin and muted generate divergent and dosage-dependent phenotypes.

Background: Genetic defects affecting subunits of protein complexes are presumed to generate identical diseases in mammals. Results: Two mouse mutants in genes belonging to the BLOC-1 complex have divergent brain and pigmentation phenotypes. Conclusion: Genetic defects affecting subunits of a complex manifest by partially overlapping clinical features. Significance: Disease resulting from mutations in protein complexes may generate a wide range of clinically presentations. Post-mortem analysis has revealed reduced levels of the protein dysbindin in the brains of those suffering from the neurodevelopmental disorder schizophrenia. Consequently, mechanisms controlling the cellular levels of dysbindin and its interacting partners may participate in neurodevelopmental processes impaired in that disorder. To address this question, we studied loss of function mutations in the genes encoding dysbindin and its interacting BLOC-1 subunits. We focused on BLOC-1 mutants affecting synapse composition and function in addition to their established systemic pigmentation, hematological, and lung phenotypes. We tested phenotypic homogeneity and gene dosage effects in the mouse null alleles muted (Bloc1s5mu/mu) and dysbindin (Bloc1s8sdy/sdy). Transcripts of NMDA receptor subunits and GABAergic interneuron markers, as well as expression of BLOC-1 subunit gene products, were affected differently in the brains of Bloc1s5mu/mu and Bloc1s8sdy/sdy mice. Unlike Bloc1s8sdy/sdy, elimination of one or two copies of Bloc1s5 generated indistinguishable pallidin transcript phenotypes. We conclude that monogenic mutations abrogating the expression of a protein complex subunit differentially affect the expression of other complex transcripts and polypeptides as well as their downstream effectors. We propose that the genetic disruption of different subunits of protein complexes and combinations thereof diversifies phenotypic presentation of pathway deficiencies, contributing to the wide phenotypic spectrum and complexity of neurodevelopmental disorders.

Neurodevelopmental disorders: mechanisms and boundary definitions from genomes, interactomes and proteomes.

Neurodevelopmental disorders such as intellectual disability, autism spectrum disorder and schizophrenia lack precise boundaries in their clinical definitions, epidemiology, genetics and protein–protein interactomes. This calls into question the appropriateness of current categorical disease concepts. Recently, there has been a rising tide to reformulate neurodevelopmental nosological entities from biology upward. To facilitate this developing trend, we propose that identification of unique proteomic signatures that can be strongly associated with patient’s risk alleles and proteome-interactome-guided exploration of patient genomes could define biological mechanisms necessary to reformulate disorder definitions.

Molecular Basis of Neurodegeneration and Neurodevelopmental Defects in Menkes Disease

ATP7A mutations impair copper metabolism resulting in three distinct genetic disorders in humans. These diseases are characterized by neurological phenotypes ranging from intellectual disability to neurodegeneration. Severe ATP7A loss-of-function alleles trigger Menkes disease, a copper deficiency condition where systemic and neurodegenerative phenotypes dominate clinical outcomes. The pathogenesis of these manifestations has been attributed to the hypoactivity of a limited number of copper-dependent enzymes, a hypothesis that we refer as the oligoenzymatic pathogenic hypothesis. This hypothesis, which has dominated the field for 25 years, only explains some systemic Menkes phenotypes. However, we argue that this hypothesis does not fully account for the Menkes neurodegeneration or neurodevelopmental phenotypes. Here, we propose revisions of the oligoenzymatic hypothesis that could illuminate the pathogenesis of Menkes neurodegeneration and neurodevelopmental defects through unsuspected overlap with other neurological conditions including Parkinsons, intellectual disability, and schizophrenia.