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 C. elegans 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 C. elegans analysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including mutants lacking bec-1/BECN1/Beclin1 and vps-15/PIK3R4/p150 that regulate the class III phosphatidylinositol 3-kinase (PtdIns3K) VPS-34, and dyn-1/dynamin that severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. In C. elegans we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with bec-1 and vps-15 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.
Abbreviation: 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

自噬体（Autophagosome）与溶酶体（Lysosome）融合，形成自噬溶酶体（Autolysosome）以降解所吞噬的胞质底物。为维持溶酶体功能容量，自噬溶酶体重塑（autophagic lysosome reformation, ALR）需通过基于膜小管的过程，从自噬溶酶体中再生出溶酶体。维持溶酶体功能容量是维持细胞健康的必要条件，尤其在神经元中——溶酶体功能异常已被多次证实与神经退行性疾病密切相关。
含有DNA-J结构域的HSC70共伴侣蛋白RME-8/DNAJC13，已被证实参与内体包被蛋白调控，并与神经系统疾病相关。本研究针对神经元中RME-8/DNAJC13蛋白的功能需求展开全新分析，实验模型包括完整的秀丽隐杆线虫（C. elegans）机械感觉神经元，以及体外培养的原代小鼠皮层神经元。在两种实验系统中缺失RME-8/DNAJC13，均会导致显著伸长的自噬溶酶体小管大量堆积。
进一步针对秀丽隐杆线虫的分析显示，在已知调控哺乳动物ALR的突变体中，同样存在自噬溶酶体小管堆积缺陷；这类突变体包括：调控III型磷脂酰肌醇3-激酶（class III phosphatidylinositol 3-kinase, PtdIns3K）VPS-34的bec-1/BECN1/Beclin1缺失突变体、vps-15/PIK3R4/p150缺失突变体，以及剪切ALR小管的dyn-1/发动蛋白（Dynamin）缺失突变体。网格蛋白（Clathrin）亦是重要的ALR调控因子，参与自噬溶酶体小管的形成与释放过程。
在秀丽隐杆线虫中，我们发现RME-8缺失会导致神经元自噬溶酶体上的网格蛋白严重耗竭，该表型与bec-1和vps-15突变体一致。综上，我们认为RME-8/DNAJC13在ALR中发挥了此前未被发现的功能，其作用机制可能与调控自噬溶酶体小管的剪切过程相关。此外，在两种实验系统中，RME-8/DNAJC13的缺失均会降低巨自噬/自噬流（macroautophagic/autophagic flux）水平，提示ALR对自噬存在反馈调控作用。本研究将RME-8/DNAJC13与ALR及自噬通路建立关联，为阐释RME-8/DNAJC13如何影响神经元健康以及神经退行性疾病的进展提供了潜在的分子机制。
缩写说明：
ALR：自噬溶酶体重塑（autophagic lysosome reformation）；
ATG-13/EPG-1：自噬相关（酵母Atg同源物）-13（AuTophaGy (yeast Atg homolog)-13）；
ATG-18：自噬相关（酵母Atg同源物）-18（AuTophaGy (yeast Atg homolog)-18）；
AV：自噬泡（autophagic vacuole）；
CLIC-1：网格蛋白轻链-1（Clathrin Light Chain-1）；
EPG-3：异位P颗粒-3（Ectopic P Granules-3）；
EPG-6：异位P颗粒-6（Ectopic P Granules-6）；
LGG-1：LC3、GABARAP及GATE-16家族蛋白-1（LC3, GABARAP and GATE-16 family-1）；
MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3）；
PD：帕金森病（Parkinson disease）；
PtdIns3P：磷脂酰肌醇-3-磷酸（phosphatidylinositol-3-phosphate）；
PtdIns(4,5)P2：磷脂酰肌醇-4,5-二磷酸（phosphatidylinositol-4,5-bisphosphate）；
RME-8：受体介导内吞-8（Receptor Mediated Endocytosis-8）；
SNX-1：分选连接蛋白-1（Sorting NeXin-1）；
VPS-34：酵母液泡蛋白分选因子-34同源物（related to yeast Vacuolar Protein Sorting factor-34）