RESUMO
Fused in sarcoma (FUS) is a DNA/RNA-binding protein that is involved in DNA repair and RNA processing. FUS is associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the molecular mechanisms underlying FUS-mediated neurodegeneration are largely unknown. Here, using a Drosophila model, we showed that the overexpression of glutathione transferase omega 2 (GstO2) reduces cytoplasmic FUS aggregates and prevents neurodegenerative phenotypes, including neurotoxicity and mitochondrial dysfunction. We found a FUS glutathionylation site at the 447th cysteine residue in the RanBP2-type ZnF domain. The glutathionylation of FUS induces FUS aggregation by promoting phase separation. GstO2 reduced cytoplasmic FUS aggregation by deglutathionylation in Drosophila brains. Moreover, we demonstrated that the overexpression of human GSTO1, the homolog of Drosophila GstO2, attenuates FUS-induced neurotoxicity and cytoplasmic FUS accumulation in mouse neuronal cells. Thus, the modulation of FUS glutathionylation might be a promising therapeutic strategy for FUS-associated neurodegenerative diseases.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Drosophila/metabolismo , Camundongos , Mutação/genética , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismoRESUMO
BACKGROUND: Cytoplasmic inclusions of transactive response DNA binding protein of 43 kDa (TDP-43) in neurons and astrocytes are a feature of some neurodegenerative diseases, such as frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). However, the role of TDP-43 in astrocyte pathology remains largely unknown. METHODS: To investigate whether TDP-43 overexpression in primary astrocytes could induce inflammation, we transfected primary astrocytes with plasmids encoding Gfp or TDP-43-Gfp. The inflammatory response and upregulation of PTP1B in transfected cells were examined using quantitative RT-PCR and immunoblot analysis. Neurotoxicity was analysed in a transwell coculture system of primary cortical neurons with astrocytes and cultured neurons treated with astrocyte-conditioned medium (ACM). We also examined the lifespan, performed climbing assays and analysed immunohistochemical data in pan-glial TDP-43-expressing flies in the presence or absence of a Ptp61f RNAi transgene. RESULTS: PTP1B inhibition suppressed TDP-43-induced secretion of inflammatory cytokines (interleukin 1 beta (IL-1ß), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α)) in primary astrocytes. Using a neuron-astrocyte coculture system and astrocyte-conditioned media treatment, we demonstrated that PTP1B inhibition attenuated neuronal death and mitochondrial dysfunction caused by overexpression of TDP-43 in astrocytes. In addition, neuromuscular junction (NMJ) defects, a shortened lifespan, inflammation and climbing defects caused by pan-glial overexpression of TDP-43 were significantly rescued by downregulation of ptp61f (the Drosophila homologue of PTP1B) in flies. CONCLUSIONS: These results indicate that PTP1B inhibition mitigates the neuronal toxicity caused by TDP-43-induced inflammation in mammalian astrocytes and Drosophila glial cells.
Assuntos
Astrócitos/metabolismo , Proteínas de Ligação a DNA/biossíntese , Mediadores da Inflamação/metabolismo , Degeneração Neural/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/biossíntese , Animais , Animais Geneticamente Modificados , Astrócitos/patologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Drosophila , Expressão Gênica , Mediadores da Inflamação/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/genética , Degeneração Neural/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/genéticaRESUMO
Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified â¼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.
Assuntos
Movimento Celular , Glioma/patologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais , Fuso Acromático/genética , Quinases Ativadas por p21/genética , Animais , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Modelos Animais de Doenças , Epistasia Genética , Feminino , Glioma/genética , Glioma/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitose , Inibidores de Proteínas Quinases/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Quinases Ativadas por p21/metabolismoRESUMO
Kinases are critical intracellular signaling proteins. To better understand kinase-mediated signal transduction, a large-scale human-yeast genetic interaction screen was performed. Among 597 human kinase genes tested, 28 displayed strong toxicity in yeast when overexpressed. En masse transformation of these toxic kinase genes into 4653 homozygous diploid yeast deletion mutants followed by barcode sequencing identified yeast toxicity modifiers and thus their human orthologs. Subsequent network analyses and functional grouping revealed that the 28 kinases and their 676 interaction partners (corresponding to a total of 969 genetic interactions) are enriched in cell death and survival (34%), small-molecule biochemistry (18%) and molecular transport (11%), among others. In the subnetwork analyses, a few kinases were commonly associated with glioma, cell migration and cell death/survival. Our analysis enabled the creation of a first draft of the kinase genetic interactome network and identified multiple drug targets for inflammatory diseases and cancer, in which deregulated kinase signaling plays a pathogenic role.
Assuntos
Epistasia Genética , Proteínas Serina-Treonina Quinases/genética , Proteoma/genética , Saccharomyces cerevisiae/genética , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/metabolismoRESUMO
Optineurin (OPTN) is an autophagy receptor protein that has been implicated in glaucoma and amyotrophic lateral sclerosis. OPTN-mediated autophagy is a complex process involving many autophagy-regulating proteins. Autophagy plays a critical role in removing damaged organelles, intracellular pathogens, and protein aggregates to maintain cellular homeostasis. We identified Ypt1 as a novel interaction partner of OPTN by performing a large-scale yeast-human two-hybrid assay. Coimmunoprecipitation assay showed that OPTN interacted with Rab1, the mammalian homolog of yeast Ypt1, in N2a mouse neuroblastoma cell line. We confirmed this interaction by confocal microscopy showing intracellular colocalization of the two proteins. We observed that a zinc finger domain of OPTN is important for Rab1a binding. Rab1a activity is also required for the binding with OPTN. The role of the OPTN-Rab1a complex in neuronal autophagy was determined by measuring the translocation of microtubule-associated protein light chain 3-EGFP to autophagosomes. In N2a cells, OPTN-induced autophagosome formation was inhibited by Rab1a knockdown, indicating the important role of OPTN-Rab1a interaction in neuronal autophagy processes. Similarly, in N2a cells overexpressing Rab1a, serum starvation-induced formation of autophagosome was enhanced, while OPTN knockdown reduced the Rab1a-induced autophagy. These results show that the OPTN-Rab1a complex modulates autophagosome formation in neuroblastoma cells.