A conserved requirement for RME-8/DNAJC13 in neuronal autophagic lysosome reformation

Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autophagic lysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. The DNA-J domain HSC70 co-chaperone RME-8/DNAJC13 has been linked to endosomal coat protein regulation and to neurological disease. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing on intact <i>C. elegans</i> mechanosensory neurons, and primary mouse cortical neurons in culture. Loss of RME-8/DNAJC13 in both systems results in accumulation of grossly elongated autolysosomal tubules. Further <i>C. elegans</i> analysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including mutants lacking <i>bec-1/BECN1/Beclin1</i> and <i>vps-15/PIK3R4/p150</i> that regulate the class III phosphatidylinositol 3-kinase (PtdIns3K) VPS-34, and <i>dyn-1</i>/<i>dynamin</i> that severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. In <i>C. elegans</i> we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with <i>bec-1</i> and <i>vps-15</i> mutants. We conclude that RME-8/DNAJC13 plays a previously unrecognized role in ALR, likely affecting autolysosome tubule severing. Additionally, in both systems, loss of RME-8/DNAJC13 reduced macroautophagic/autophagic flux, suggesting feedback regulation from ALR to autophagy. Our results connecting RME-8/DNAJC13 to ALR and autophagy provide a potential mechanism by which RME-8/DNAJC13 could influence neuronal health and the progression of neurodegenerative disease. <b>Abbreviation:</b> ALR, autophagic lysosome reformation; ATG-13/EPG-1, AuTophaGy (yeast Atg homolog)-13; ATG-18, AuTophaGy (yeast Atg homolog)-18; AV, autophagic vacuole; CLIC-1, Clathrin Light Chain-1; EPG-3, Ectopic P Granules-3; EPG-6, Ectopic P Granules-6; LGG-1, LC3, GABARAP and GATE-16 family-1; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; PD, Parkinson disease; PtdIns3P, phosphatidylinositol-3-phosphate; PtdIns(4,5)P₂, phosphatidylinositol-4,5-bisphosphate; RME-8, Receptor Mediated Endocytosis-8; SNX-1, Sorting NeXin-1; VPS-34, related to yeast Vacuolar Protein Sorting factor-34