UCSF

A unifying feature of major neurodegenerative diseases is the aggregation and deposition of misfolded proteins. However, our understanding of the normal function of the proteins implicated in the pathology of these disorders is markedly narrow. Specifically, whether the abnormal accumulation of these proteins reflects a cause or a consequence of pathology remains controversial.

The presynaptic peripheral membrane protein α-synuclein has been strongly implicated in both familial and sporadic forms of Parkinson’s disease (PD). Point mutations in α-synuclein cause inherited PD, as well as gene duplication and triplication lead to disease, implying a role for the wild-type protein. Recently, it has been demonstrated that overexpression of α-synuclein inhibits exocytosis in primary hippocampal neurons as well as in neuroendocrine cells, implying a role for synuclein in the regulation of transmitter release. However, genetic ablation of α-synuclein and its two family members beta- and gamma-synuclein has yielded very modest changes in exocytosis, raising the question of whether synuclein’s effect on exocytosis reflects a normal function of the protein or a toxic gain-of-function.

To understand the role of synuclein in the regulation of exocytosis, we have utilized total internal reflection microscopy (TIRFM) of primary mouse adrenal chromaffin cells to study the dynamics of individual release events. Using BDNF-pHluorin, a fluorescent reporter of dense core vesicle release, we demonstrate that overexpression of α-synuclein accelerates and loss of endogenous synuclein prolongs the kinetics of exocytosis. Experiments with the integral membrane protein VMAT2-pHluorin confirm that this effect is indicative of altered fusion pore dynamics. Further, we demonstrate a similar effect on dense core vesicle exocytosis in primary hippocampal neurons. Using high-resolution structured illumination microscopy, we observe α-synuclein bound directly to secretory granules. Finally, we characterize the behavior of two familial PD-linked synuclein point mutations, A30P and A53T. These mutants exhibit normal binding to secretory granules and inhibition of exocytosis, but fail to accelerate the kinetics of release, suggesting a normal role for the protein in the dilation of the fusion pore that may be dysregulated in disease.