ATG2A acts as a tether to regulate autophagosome-lysosome fusion in neural cells

The macroautophagy/autophagy proteins ATG2A and ATG2B transfer lipids for phagophore membrane growth. They also form stable complexes with WDR45 and WDR45B. Our previous study demonstrated that WDR45 and WDR45B mediate autophagosome-lysosome fusion in neural cells. Given the defective autophagosome formation in cells lacking both ATG2s, their role in later autophagy stages is hard to explore. Here, we report that in neuroblastoma-derived Neuro-2a (N2a) cells, knocking down (KD) <i>Atg2a</i>, but not <i>Atg2b</i>, results in significant accumulation of SQSTM1/p62 and MAP1LC3-II/LC3-II, indicating impaired autophagy. <i>Atg2a</i> deficiency does not affect autophagosome formation, but reduces colocalization of autophagosomal LC3 with late endosomal/lysosomal RFP-RAB7, suggesting impaired autophagosome-lysosome fusion. ATG2A interacts with the SNARE proteins STX17, SNAP29, and VAMP8, facilitating their assembly. Overexpression of ATG2A partially rescues the autophagosome-lysosome fusion defects in <i>Wdr45-</i> and <i>Wdr45b</i>-deficient cells. ATG2 and another tether protein, EPG5, function partially redundantly in mediating autophagosome-lysosome fusion. Thus, ATG2A plays a key role in neural autophagy by tethering autophagosomes with lysosomes for fusion. <b>Abbreviations</b>: AAV: adeno-associated virus; ATG2A^r: RNAi-resistant ATG2A; Baf: bafilomycin A₁; co-IP: co-immunoprecipitation; CQ: chloroquine; DKD: double knockdown; DKO: double knockout; ER: endoplasmic reticulum; KD: knockdown; KO: knockout; MIL: membrane-impermeable Halo ligand; MPL: membrane-permeable Halo ligand; N2a: Neuro-2a; NC negative control; PG: phagophore; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; TEM: Transmission electron microscopy; TM: transmembrane domain; WT: wild-type.