E46K mutant α-synuclein is more degradation resistant and exhibits greater toxic effects than wild-type α-synuclein in Drosophila models of Parkinson's disease

Parkinson's disease (PD) is one of the most common neurodegenerative diseases, which is characterized by progressive motor dysfunction as well as non-motor symptoms. Pathological and genetic studies have demonstrated that alpha-synuclein (alpha Syn) plays key roles in the pathogenesis of PD. Although several missense mutations in the alpha Syn gene have been identified as causes of familial PD, the mechanisms underlying the variance in the clinical phenotypes of familial PD caused by different mutations remain elusive. Here, we established novel Drosophila models expressing either wild-type (WT) alpha Syn or one of five alpha Syn mutants (A30P, E46K, H50Q, G51D, and A53T) using site-specific transgenesis, which express transgenes at equivalent levels. Expression of either WT or mutant alpha Syn in the compound eyes by the GMR-GAL4 driver caused mild rough eye phenotypes with no obvious difference among the mutants. Upon pan-neuronal expression by the nSyb-GAL4 driver, these alpha Syn-expressing flies showed a progressive decline in locomotor function. Notably, we found that E46K, H50Q, G51D, and A53T aSyn-expressing flies showed earlier onset of locomotor dysfunction than WT alpha Syn-expressing flies, suggesting their enhanced toxic effects. Whereas mRNA levels of WT and mutant alpha Syn were almost equivalent, we found that protein expression levels of E46K alpha Syn were higher than those of WT alpha Syn. In vivo chase experiments using the drug-inducible GMR-GeneSwitch driver demonstrated that degradation of E46K alpha Syn protein was significantly slower than WT alpha Syn protein, indicating that the E46K alpha Syn mutant gains resistance to degradation in vivo. We therefore conclude that our novel site-specific transgenic fly models expressing either WT or mutant alpha Syn are useful to explore the mechanisms by which different alpha Syn mutants gain toxic functions in vivo.