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The active zone protein Clarinet regulates synaptic sorting of ATG-9 and presynaptic autophagy [1]

['Zhao Xuan', 'Program In Cellular Neuroscience', 'Neurodegeneration', 'Repair', 'Departments Of Neuroscience', 'Cell Biology', 'Yale University School Of Medicine', 'New Haven', 'Connecticut', 'United States Of America']

Date: 2023-04

Autophagy is essential for cellular homeostasis and function. In neurons, autophagosome biogenesis is temporally and spatially regulated to occur near presynaptic sites, in part via the trafficking of autophagy transmembrane protein ATG-9. The molecules that regulate autophagy by sorting ATG-9 at synapses remain largely unknown. Here, we conduct forward genetic screens at single synapses of C. elegans neurons and identify a role for the long isoform of the active zone protein Clarinet (CLA-1L) in regulating sorting of autophagy protein ATG-9 at synapses, and presynaptic autophagy. We determine that disrupting CLA-1L results in abnormal accumulation of ATG-9 containing vesicles enriched with clathrin. The ATG-9 phenotype in cla-1(L) mutants is not observed for other synaptic vesicle proteins, suggesting distinct mechanisms that regulate sorting of ATG-9-containing vesicles and synaptic vesicles. Through genetic analyses, we uncover the adaptor protein complexes that genetically interact with CLA-1 in ATG-9 sorting. We also determine that CLA-1L extends from the active zone to the periactive zone and genetically interacts with periactive zone proteins in ATG-9 sorting. Our findings reveal novel roles for active zone proteins in the sorting of ATG-9 and in presynaptic autophagy.

Funding: SY was supported by China Scholarship Council-Yale World Scholars Program (no grant number)( https://medicine.yale.edu/bbs/training/initiatives/csc/ ). ZX, BC, SY, SEH, LM and DACR were supported by NIH R01NS076558, DP1NS111778 ( https://www.nih.gov/ ) and by an HHMI Scholar Award 55108513 ( https://www.hhmi.org/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Xuan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Our findings support a model whereby CLA-1L bridges the exocytic active zone regions with the endocytic periactive zone to regulate presynaptic sorting of ATG-9, likely via endosome-mediated sorting. Our findings also suggest that in vivo, ATG-9 containing vesicles represent a distinct subpopulation of synaptic vesicles. Our study uncovers molecules and synaptic machinery specifically involved in ATG-9 sorting and underscores the importance of active zone proteins in regulating local sorting of autophagy proteins and presynaptic autophagy.

To better understand the in vivo mechanisms that regulate ATG-9 sorting at synapses and their relationship with synaptic vesicle proteins, we generated transgenic Caenorhabditis elegans (C. elegans) strains that allowed us to simultaneously observe, in single neurons, localization of ATG-9 and synaptic vesicle proteins [ 24 ]. We performed unbiased forward genetic screens for mutants in which ATG-9 localization was differentially affected as compared to synaptic vesicle proteins, and identified an allele that affects the long isoform of the active zone protein Clarinet (CLA-1L). Clarinet bears similarity to Drosophila active zone protein Fife and vertebrate active zone proteins RIM, Piccolo, and Bassoon [ 25 – 29 ]. In cla-1(L) mutants ATG-9, but not synaptic vesicle proteins, abnormally accumulate to subsynaptic regions enriched for clathrin. This abnormal ATG-9 phenotype is suppressed by mutants for synaptic vesicle exocytosis, suggesting that the ATG-9 phenotype in cla-1(L) mutants emerges from defects in ATG-9 sorting during exo-endocytosis. Through genetic analyses, we found that mutants of the clathrin-associated adaptor complexes AP-2 and AP180 phenocopy and enhance the ATG-9 phenotypes observed for cla-1(L) mutant, whereas mutants for the AP-1 adaptor complex and the F-BAR protein syndapin 1 suppress the phenotype. We also observed that CLA-1L extends from the exocytic active zone to the endocytic periactive zone and genetically interacts with the periactive zone proteins EHS-1/EPS15 and ITSN-1/intersectin 1 in mediating ATG-9 sorting at presynaptic sites.

ATG-9, a transmembrane protein necessary for autophagy, is actively trafficked in vesicles to promote local autophagosome biogenesis [ 19 – 24 ]. In neurons, ATG-9 is transported to presynaptic sites. Like synaptic vesicles, ATG-9-containing vesicles undergo activity-dependent exo-endocytosis regulated by canonical synaptic molecules such as endophilin A and synaptojanin 1 [ 24 ]. Activity-dependent exo-endocytosis of ATG-9-containing vesicles supports synaptic autophagy [ 24 ]. Yet, beyond the requirement of endophilin A and synaptojanin 1, the mechanisms that sort ATG-9 at presynaptic sites, the relationship of these mechanisms to those that sort canonical synaptic vesicle proteins and the relationship between ATG-9 and the synaptic machinery is not well understood. Knowledge of the molecules required for specific sorting of ATG-9 at synapses is of critical importance to understand the mechanisms that regulate synaptic autophagy.

Macroautophagy (herein called autophagy) is a well-conserved cellular degradative pathway, and its disruption in neurons results in axonal degeneration, accumulation of protein aggregates, and cell death [ 1 – 4 ]. Autophagy is spatially and temporally regulated in neurons, and autophagosome biogenesis occurs near presynaptic sites and in response to increased neuronal activity states [ 5 – 16 ]. Directed transport of autophagy proteins to presynaptic sites contributes to local autophagosome biogenesis at synapses [ 5 , 9 , 16 – 18 ]. How autophagy proteins are transported, sorted, and locally regulated in neurons to control synaptic autophagy is not well understood.

Results

Clarinet long isoform, CLA-1L, acts cell autonomously to selectively regulate ATG-9 sorting at presynaptic sites The cla-1 gene encodes 3 isoforms: CLA-1L (long), CLA-1M (medium), and CLA-1S (short) (S1A Fig). The long isoform, which contains a repetitive region predicted to be disordered (Fig 1K), is necessary for synaptic vesicle clustering, whereas the shorter isoforms are required for active zone assembly [25]. The isolated allele cla-1(ola285) (a missense mutation in the coding region of cla-1L), as well as the examined allele cla-1(ok560) (a deletion of the promoter and part of the coding region of cla-1L), only affect CLA-1L, but not CLA-1M or CLA-1S. A null allele affecting all isoforms, cla-1(wy1048), did not display a more severe ATG-9 phenotype than the alleles affecting only CLA-1L (S1A and S1B Fig). These findings suggest that the long isoform of Clarinet (CLA-1L) is necessary for presynaptic sorting of ATG-9. To better assess the effects of the lesion of allele ola285 in CLA-1 protein product in vivo, we inserted, via CRISPR, a DNA sequence encoding GFP at the 5′-end of the endogenous cla-1 locus. The 5′-end of cla-1 gene corresponds to the N-terminus of CLA-1L (S1A Fig), so the inserted GFP specifically labels CLA-1L (S6B Fig). We observed that CLA-1L expression levels were reduced in ola285 as compared to wild-type animals (Figs 1N–1O and S1C). Based on the loss of function phenotype of ola285 allele, we hypothesize that the missense mutation results in degradation of CLA-1L. To determine the specific requirement of CLA-1L for ATG-9 sorting at presynaptic sites in AIY, we manipulated the expression of CLA-1L in AIY using a cell-specific knockout strategy [25]. We used a strain in which loxP sites were inserted, via CRISPR, to flank the unique 5′-end gene locus specific to CLA-1L (Figs 1K and S1A). Cell-specific expression of Cre recombinase in AIY, which leads to AIY-specific deletion of the CLA-1L isoform (without affecting CLA-1S and CLA-1M), resulted in the ATG-9 phenotype in AIY (Figs 1R and S1D), which was indistinguishable from that seen for the cla-1 (ok560) allele (Figs 1S and S1D, compare to wild type in Figs 1P, 1Q, and S1D). Together, our data indicate that the allele ola285 affects the long isoform of Clarinet (CLA-1L) and that CLA-1L regulates presynaptic sorting of ATG-9 in a cell-autonomous manner.

The clathrin adaptor complexes, AP-2 and AP180, regulate ATG-9 sorting at presynaptic sites We next examined the genetic relationship between clathrin adaptor protein complexes and CLA-1L in ATG-9 localization. The AP-2 complex mediates clathrin-mediated endocytosis (CME) of synaptic vesicle proteins [44–49], and it has been implicated in the sorting of ATG-9 during autophagy induction in mammalian nonneuronal cells [50–52]. To determine if the AP-2, and the associated AP180, adaptor complexes were required in presynaptic sorting of ATG-9, we examined ATG-9 localization in the null alleles dpy-23(e840)/AP2μ and unc-11(e47)/AP180. We observed that dpy-23(e840)/AP2μ and unc-11(e47)/AP180 mutants phenocopied cla-1(ola285) mutants in ATG-9 presynaptic sorting defects (Fig 5J, 5K, 5M, and 5N). In addition, the expressivity of the ATG-9 sorting defects was enhanced in dpy-23(e840)/AP2μ;cla-1(ola285) double mutant worms (Fig 5L–5N). These findings suggest shared mechanisms that similarly result in defective ATG-9 sorting when clathrin-associated adaptor complexes, or the active zone protein CLA-1L, are disrupted.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002030

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