Abnormal oligomerization and aggregation of α-synuclein (α-syn/WT-syn) has been shown to be a precipitating factor in the pathophysiology of Parkinson's disease (PD). Earlier observations on the induced-alternative splicing of α-syn by Parkinsonism mimetics as well as identification of region specific abnormalities in the transcript levels of 112-synclein (112-syn) in diseased subjects underscores the role of 112-syn in the pathophysiology of PD. In the present study, we sought to identify the aggregation potential of 112-syn in the presence or absence of WT-syn to predict its plausible role in protein aggregation events. Results demonstrate that unlike WT-syn, lack of 28 aa in the C-terminus results in the loss of chaperone-like activity with a concomitant gain in vulnerability to heat-induced aggregation and time-dependent fibrillation. The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45°C following 10 min of incubation. The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT) staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37°C. Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner. On contrary, WT-syn synergistically enhanced fibrillation of 112-syn. Overall, the present findings highlight a plausible cross-talk between isoforms of α-syn and the relative abundance of these isoforms may dictate the nature and fate of protein aggregates.
The presynaptic protein, α-synuclein (α-syn), has been shown to play a crucial role in multiple neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), and dementia with Lewy bodies (DLB). The three major domains of α-syn protein were shown to govern its membrane interaction, protein fibrillation, and chaperone activity. So far, four different alternatively spliced isoforms of α-syn, which lack either exon 3 (syn-126) or exon 5 (syn-112) or both (syn-98) resulting in altered function of the proteins, have been identified. In the present study, we have identified the smallest isoform of α-syn due to the skipping of exons 3 and 4 generating a 238 bp transcript. Due to the presence of a premature stop codon, the 238 bp transcript generated a 41 aa N-terminal peptide instead of the 78 aa protein, which is secreted into the extracellular medium when overexpressed in cells. The presence of 41-syn was initially noticed in the substantia nigra of PD autopsy tissues, as well as in cells undergoing oxidative stress. In vitro studies inferred that 41-syn neither aggregates nor alters the aggregation propensity of either WT or 112-syn. Overexpression of 41-syn or treatment of cells with 41-syn peptide did not affect cell viability. However, PC-12 cells treated with 41-syn exhibited a time and dose dependent enhancement in the cellular uptake of dopamine. Based on the physiological role of the N-terminal region of α-syn in modulating membrane trafficking events, we believe that the identification of 41-syn may provide novel impetus in unraveling the physiological basis of alternative splicing events in governing PD pathophysiology.
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