Retrograde Axonal Autophagy and Endocytic Pathways Are Parallel and Separate in Neurons

Autophagy and endocytic trafficking are two key pathways that regulate the composition and integrity of the neuronal pro-teome. Alterations in these pathways are sufficient to cause neurodevelopmental and neurodegenerative disorders. Thus, defining how autophagy and endocytic pathways are organized in neurons remains a key area of investigation. These path-ways share many features and converge on lysosomes for cargo degradation, but what remains unclear is the degree to which the identity of each pathway is preserved in each compartment of the neuron. Here, we elucidate the degree of intersection between autophagic and endocytic pathways in axons of primary mouse cortical neurons of both sexes. Using microfluidic chambers, we labeled newly-generated bulk endosomes and signaling endosomes in the distal axon, and systematically tracked their trajectories, molecular composition, and functional characteristics relative to autophagosomes. We find that newly -formed endosomes and autophagosomes both undergo retrograde transport in the axon, but as distinct organelle populations. Moreover, these pathways differ in their degree of acidification and association with molecular determinants of organelle maturation. These results suggest that the identity of autophagic and newly endocytosed organelles is preserved for the length of the axon. Lastly, we find that expression of a pathogenic form of a-synuclein, a protein enriched in presynaptic terminals, increases merging between autophagic and endocytic pathways. Thus, aberrant merging of these pathways may represent a mechanism contributing to neuronal dysfunction in Parkinson's disease (PD) and related a-synucleinopathies.