LRK-1/LRRK2 and AP-3 regulate trafficking of synaptic vesicle precursors through active zone protein SYD-2/Liprin-α.

Synaptic vesicle proteins (SVps) are transported by the motor UNC-104/KIF1A. We show that SVps travel in heterogeneous carriers in C. elegans neuronal processes, with some SVp carriers co-transporting lysosomal proteins (SV-lysosomes). LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 play a critical role in the sorting of SVps and lysosomal proteins away from each other at the SV-lysosomal intermediate trafficking compartment. Both SVp carriers lacking lysosomal proteins and SV-lysosomes are dependent on the motor UNC-104/KIF1A for their transport. In lrk-1 mutants, both SVp carriers and SV-lysosomes can travel in axons in the absence of UNC-104, suggesting that LRK-1 plays an important role to enable UNC-104 dependent transport of synaptic vesicle proteins. Additionally, LRK-1 acts upstream of the AP-3 complex and regulates its membrane localization. In the absence of the AP-3 complex, the SV-lysosomes become more dependent on the UNC-104-SYD-2/Liprin-α complex for their transport. Therefore, SYD-2 acts to link upstream trafficking events with the transport of SVps likely through its interaction with the motor UNC-104. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. SYD-2 acts in concert with AP complexes to ensure polarized trafficking & transport of SVps.

A Conserved Requirement for RME-8/DNAJC13 in Neuronal Autolysosome Reformation.

Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autolysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain proteostasis and cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. Cell biological studies have linked the DNA-J domain Hsc70 co-chaperone RME-8/DNAJC13 to endosomal coat protein regulation, while human genetics studies have linked RME-8/DNAJC13 to neurological disease, including Parkinsonism and Essential Tremor. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing on C. elegans mechanosensory neurons in the intact animal, and in primary mouse cortical neurons in culture. We find that 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 bec-1/beclin and vps-15/PIK3R4/p150 that regulate type-III PI3-kinase 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 conserved but previously unrecognized role in autolysosome reformation, likely affecting ALR tubule initiation and/or severing. Additionally, in both systems, we found that loss of RME-8/DNAJC13 appeared to reduce 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.

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.

Active zone protein SYD-2/Liprin-α acts downstream of LRK-1/LRRK2 to regulate polarized trafficking of synaptic vesicle precursors through clathrin adaptor protein complexes.

Synaptic vesicle proteins (SVps) are thought to travel in heterogeneous carriers dependent on the motor UNC-104/KIF1A. In C. elegans neurons, we found that some SVps are transported along with lysosomal proteins by the motor UNC-104/KIF1A. LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 are critical for the separation of lysosomal proteins from SVp transport carriers. In lrk-1 mutants, both SVp carriers and SVp carriers containing lysosomal proteins are independent of UNC-104, suggesting that LRK-1 plays a key role in ensuring UNC-104-dependent transport of SVps. Additionally, LRK-1 likely acts upstream of the AP-3 complex and regulates the membrane localization of AP-3. The action of AP-3 is necessary for the active zone protein SYD-2/Liprin-α to facilitate the transport of SVp carriers. In the absence of the AP-3 complex, SYD-2/Liprin-α acts with UNC-104 to instead facilitate the transport of SVp carriers containing lysosomal proteins. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. We propose that SYD-2 acts in concert with both the AP-1 and AP-3 complexes to ensure polarized trafficking of SVps.

Selective disruption of Drp1-independent mitophagy and mitolysosome trafficking by an Alzheimer's disease relevant tau modification in a novel Caenorhabditis elegans model.

Accumulation of inappropriately phosphorylated tau into neurofibrillary tangles is a defining feature of Alzheimer's disease, with Tau pT231 being an early harbinger of tau pathology. Previously, we demonstrated that expressing a single genomic copy of human phosphomimetic mutant tau (T231E) in Caenorhabditis elegans drove age-dependent neurodegeneration. A critical finding was that T231E, unlike wild-type tau, completely and selectively suppressed oxidative stress-induced mitophagy. Here, we used dynamic imaging approaches to analyze T231E-associated changes in mitochondria and mitolysosome morphology, abundance, trafficking, and stress-induced mitophagy as a function of mitochondrial fission mediator dynamin-related protein 1, which has been demonstrated to interact with hyper phosphorylated tau and contribute to Alzheimer's disease pathogenesis, as well as Pink1, a well-recognized mediator of mitochondrial quality control that works together with Parkin to support stress-induced mitophagy. T231E impacted both mitophagy and mitolysosome neurite trafficking with exquisite selectivity, sparing macroautophagy as well as lysosome and autolysosome trafficking. Both oxidative-stress-induced mitophagy and the ability of T231E to suppress it were independent of drp-1, but at least partially dependent on pink-1. Organelle trafficking was more complicated, with drp-1 and pink-1 mutants exerting independent effects, but generally supported the idea that the mitophagy phenotype is of greater physiologic impact in T231E. Collectively, our results refine the mechanistic pathway through which T231E causes neurodegeneration, demonstrating pathologic selectivity for mutations that mimic tauopathy-associated post-translational modifications, physiologic selectivity for organelles that contain damaged mitochondria, and molecular selectivity for dynamin-related protein 1-independent, Pink1-dependent, perhaps adaptive, and mitophagy.