AbstractThe accumulation of α-synuclein (α-syn)-enriched protein aggregates is thought to arise from dysfunction in degradation systems within the brain. Recently, missense mutations ofSYNJ1encoding the SAC1 and 5’-phosphatase domains have been found in families with hereditary early-onset Parkinsonism. Previous studies showed thatSynj1haploinsufficiency (Synj1+/−) leads to accumulation of the autophagy substrate p62 and pathologic α-syn proteins in the midbrain (MB) and striatum of aged mice. In this study, we aim to investigate the neuronal degradation pathway using theSynj1+/− MB culture from mouse pups of mixed sex as a model. Our data show that GFP-LC3 puncta formation and cumulative mKeima puncta formation are unaltered at baseline inSynj1+/− MB neurons. However, GFP-LAMP1 puncta is reduced with a similar decrease in endogenous proteins, including lysosomal-associated membrane protein (LAMP)1, LAMP2, and LAMP2A. The LAMP1 vesicles are hyperacidified with enhanced enzymatic activity inSynj1+/− MB neurons. Using a combination of light and electron microscopy (EM), we show that endolysosomal changes are primarily associated with a lack of SAC1 activity. Consistently, expressing the SYNJ1 R258Q mutant in N2a cells reduces the lysosome number. Interestingly, the endolysosomal defects inSynj1+/− neurons does not impact the clearance of exogenously expressed wild-type (WT) α-syn; however, the clearance of α-syn A53T was impaired in the axons ofSynj1+/− MB neurons. Taken together, our results suggest axonal vulnerability to endolysosomal defects in Synj1-deficient MB neurons.