Dataset Information

Regulators of autophagosome formation in Drosophila muscles.

ABSTRACT: Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.

SUBMITTER: Zirin J

PROVIDER: S-EPMC4334200 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Regulators of autophagosome formation in Drosophila muscles.

Zirin Jonathan J Nieuwenhuis Joppe J Samsonova Anastasia A Tao Rong R Perrimon Norbert N

PLoS genetics 20150218 2

Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammal ...[more]

PMID: 25692684

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Project description:Macroautophagy dysregulation is implicated in multiple neurological disorders, such as Parkinson's disease. While autophagy pathways are heavily researched in heterologous cells and neurons, regulation of autophagy in the astrocyte, the most abundant cell type in the mammalian brain, is less well understood. Missense mutations in the Synj1 gene encoding Synaptojanin1 (Synj1), a neuron-enriched lipid phosphatase, have been linked to Parkinsonism with seizures. Our previous study showed that the Synj1 haploinsufficient (Synj1+/-) mouse exhibits age-dependent autophagy impairment in multiple brain regions. Here, we used cultured astrocytes from Synj1-deficient mice to investigate its role in astrocyte autophagy. We report that Synj1 is expressed in low levels in astrocytes and represses basal autophagosome formation. We demonstrate using cellular imaging that Synj1-deficient astrocytes exhibit hyperactive autophagosome formation, represented by an increase in the size and number of GFP-microtubule-associated protein 1A/1B-light chain 3 structures. Interestingly, Synj1 deficiency is also associated with an impairment in stress-induced autophagy clearance. We show, for the first time, that the Parkinsonism-associated R839C mutation impacts autophagy in astrocytes. The impact of this mutation on the phosphatase function of Synj1 resulted in elevated basal autophagosome formation that mimics Synj1 deletion. We found that the membrane expression of the astrocyte-specific glucose transporter GluT-1 was reduced in Synj1-deficient astrocytes. Consistently, AMP-activated protein kinase activity was elevated, suggesting altered glucose sensing in Synj1-deficient astrocytes. Expressing exogenous GluT-1 in Synj1-deficient astrocytes reversed the autophagy impairment, supporting a role for Synj1 in regulating astrocyte autophagy via disrupting glucose-sensing pathways. Thus, our work suggests a novel mechanism for Synj1-related Parkinsonism involving astrocyte dysfunction.

Dataset Information

Regulators of autophagosome formation in Drosophila muscles.

Publications

Regulators of autophagosome formation in Drosophila muscles.

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