PD pathogenesis is associated with misfolding of a-synuclein (a-syn) and mitochondrial dysfunction. Notably, a-syn was shown to interact with, and disrupt, TOMM20, a mitochondrial outer membrane protein. This interaction causes a mitochondrial protein import block, reduced respiration and enhanced generation of reactive oxygen species in cell culture and mammalian brain studies. Our lab extended these findings by reporting that UPRmt stress was increased following overexpression of a-syn variants within the intestine. This was examined via ATFS-1-dependent transcriptional upregulation with the established reporter for the UPRmt, Phsp-6::GFP. These a-syn variants were wild-type human a-syn (WT), two disease variants (A53T and A30P), and a deletion disrupting an N-terminal mitochondrial targeting sequence (delta1-32). The disease variants and the N-terminal mutant induced significantly higher UPRmt activity during both larval development and in adults whereas WT a-syn only induced the UPRmt more than controls in larval development. To determine if UPRmt regulation occurs differently for various familial a-syn variants, the transgenic strains were subjected to RNAi of
clpp-1,
haf-1, and
lonp-1 and then re-analyzed for UPRmt stress. Following knockdown of
lonp-1, all the transgenic worm strains displayed significantly enhanced UPRmt activity compared to their EV RNAi controls. Additionally, all variants, except the delta1-32 line displayed enhanced UPRmt stress following knockdown of
haf-1. One striking difference in these data, however, was that WT and A53T a-syn-expressing worm lines displayed increases in UPRmt stress between larval and adult stages, while the other two lines did not display increases in UPRmt stress over time. It is interesting to note that the protein pool for at least a subset of WT and A53T a-syn is likely localized to mitochondria, based on previously published work, while A30P and delta1-32 a-syn will remain in the aqueous cytoplasm. These studies were extended to neurons, where knockdown of
clpp-1,
haf-1, and
lonp-1 was performed exclusively in dopaminergic neurons of animals overexpressing WT a-syn. Enhanced neurodegeneration was observed after depletion of
clpp-1 and
haf-1, whereas knockdown of
lonp-1 was similar to EV control. In total, these data suggest that WT and A53T a-syn enter mitochondria where CLPP-1 mitochondrial protease and HAF-1 matrix peptide exporter are involved in their degradation and removal.