Abstract
We characterized phenotypes in RBL-2H3 mast cells transfected with human alpha synuclein (a-syn) using stimulated exocytosis of recycling endosomes as a proxy for similar activities of synaptic vesicles in neurons. We found that low expression of a-syn inhibits stimulated exocytosis and that higher expression causes slight enhancement. NMR measurements of membrane interactions correlate with these functional effects: they are eliminated differentially by mutants that perturb helical structure in the helix 1 (A30P) or NAC/helix-2 (V70P) regions of membrane-bound a-syn, but not by other PD-associated mutants or C-terminal truncation. We further found that a-syn (but not A30P or V70P mutants) associates weakly with mitochondria, but this association increases markedly under conditions of cellular stress. These results highlight the importance of specific structural features of a-syn in regulating vesicle release, and point to a potential role for a-syn in perturbing mitochondrial function under pathological conditions.
Highlights
Parkinson’s disease (PD) is the second most common neurodegenerative disorder.[1]
To visualize stimulated recycling endosomes (REs) exocytosis by microscopy, RBL cells are transfected with DNA for the reporter VAMP8-pHluorin, a v-SNARE protein conjugated to a fluorophore that is quenched when localized to the mildly acidic environment of intracellular REs
As shown vividly by total internal reflection fluorescence (TIRF) microscopy for cells co-transfected with an empty vector and stimulated with thapsigargin, exocytotic events appear as increases in VAMP8pHluorin fluorescence at the plasma membrane where REs are exposed to the neutral pH environment of the extracellular medium (Fig. 2a, Supplementary Movie 1a).[23]
Summary
Parkinson’s disease (PD) is the second most common neurodegenerative disorder.[1]. With increased risk of diagnosis after age 60, PD onset strongly correlates with age, affecting ~1–2% of the population over age 65.2 Clinically, motor dysfunction in PD is associated with death of dopaminergic neurons in the substantia nigra, coupled with the presence of abnormal protein aggregates, known as Lewy bodies, in surviving neurons.[2,3] The primary component of these intraneuronal aggregates is the presynaptic protein alpha synuclein (a-syn).[4]. A-syn has been the subject of intense research with the aim of understanding the relationship between a-syn physiological function and PD pathology, but this connection remains poorly understood at the cellular level
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