Venu M. Nemani

Increased Expression of α-Synuclein Reduces Neurotransmitter Release by Inhibiting Synaptic Vesicle Reclustering after Endocytosis

The protein alpha-synuclein accumulates in the brain of patients with sporadic Parkinsons disease (PD), and increased gene dosage causes a severe, dominantly inherited form of PD, but we know little about the effects of synuclein that precede degeneration. alpha-Synuclein localizes to the nerve terminal, but the knockout has little if any effect on synaptic transmission. In contrast, we now find that the modest overexpression of alpha-synuclein, in the range predicted for gene multiplication and in the absence of overt toxicity, markedly inhibits neurotransmitter release. The mechanism, elucidated by direct imaging of the synaptic vesicle cycle, involves a specific reduction in size of the synaptic vesicle recycling pool. Ultrastructural analysis demonstrates reduced synaptic vesicle density at the active zone, and imaging further reveals a defect in the reclustering of synaptic vesicles after endocytosis. Increased levels of alpha-synuclein thus produce a specific, physiological defect in synaptic vesicle recycling that precedes detectable neuropathology.

Direct Membrane Association Drives Mitochondrial Fission by the Parkinson Disease-associated Protein α-Synuclein

The protein α-synuclein has a central role in Parkinson disease, but the mechanism by which it contributes to neural degeneration remains unknown. We now show that the expression of α-synuclein in mammalian cells, including neurons in vitro and in vivo, causes the fragmentation of mitochondria. The effect is specific for synuclein, with more fragmentation by α- than β- or γ-isoforms, and it is not accompanied by changes in the morphology of other organelles or in mitochondrial membrane potential. However, mitochondrial fragmentation is eventually followed by a decline in respiration and neuronal death. The fragmentation does not require the mitochondrial fission protein Drp1 and involves a direct interaction of synuclein with mitochondrial membranes. In vitro, synuclein fragments artificial membranes containing the mitochondrial lipid cardiolipin, and this effect is specific for the small oligomeric forms of synuclein. α-Synuclein thus exerts a primary and direct effect on the morphology of an organelle long implicated in the pathogenesis of Parkinson disease.

α-Synuclein Overexpression in PC12 and Chromaffin Cells Impairs Catecholamine Release by Interfering with a Late Step in Exocytosis

α-Synuclein (α-syn), a protein implicated in Parkinsons disease pathogenesis, is a presynaptic protein suggested to regulate transmitter release. We explored how α-syn overexpression in PC12 and chromaffin cells, which exhibit low endogenous α-syn levels relative to neurons, affects catecholamine release. Overexpression of wild-type or A30P mutant α-syn in PC12 cell lines inhibited evoked catecholamine release without altering calcium threshold or cooperativity of release. Electron micrographs revealed that vesicular pools were not reduced but that, on the contrary, a marked accumulation of morphologically “docked” vesicles was apparent in the α-syn-overexpressing lines. We used amperometric recordings from chromaffin cells derived from mice that overexpress A30P or wild-type (WT) α-syn, as well as chromaffin cells from control and α-syn null mice, to determine whether the filling of vesicles with the transmitter was altered. The quantal size and shape characteristics of amperometric events were identical for all mouse lines, suggesting that overexpression of WT or mutant α-syn did not affect vesicular transmitter accumulation or the kinetics of vesicle fusion. The frequency and number of exocytotic events per stimulus, however, was lower for both WT and A30P α-syn-overexpressing cells. The α-syn-overexpressing cells exhibited reduced depression of evoked release in response to repeated stimuli, consistent with a smaller population of readily releasable vesicles. We conclude that α-syn overexpression inhibits a vesicle “priming” step, after secretory vesicle trafficking to “docking” sites but before calcium-dependent vesicle membrane fusion.

Neural Activity Controls the Synaptic Accumulation of α-Synuclein

The presynaptic protein α-synuclein has a central role in Parkinsons disease (PD). However, the mechanism by which the protein contributes to neurodegeneration and its normal function remain unknown. α-Synuclein localizes to the nerve terminal and interacts with artificial membranes in vitro but binds weakly to native brain membranes. To characterize the membrane association of α-synuclein in living neurons, we used fluorescence recovery after photobleaching. Despite its enrichment at the synapse, α-synuclein is highly mobile, with rapid exchange between adjacent synapses. In addition, we find that α-synuclein disperses from the nerve terminal in response to neural activity. Dispersion depends on exocytosis, but unlike other synaptic vesicle proteins, α-synuclein dissociates from the synaptic vesicle membrane after fusion. Furthermore, the dispersion of α-synuclein is graded with respect to stimulus intensity. Neural activity thus controls the normal function of α-synuclein at the nerve terminal and may influence its role in PD.

Optical Reporters for the Conformation of α-Synuclein Reveal a Specific Interaction with Mitochondria

The aggregation of abnormally folded proteins is a defining feature of neurodegenerative disease, but it has not previously been possible to assess the conformation of these proteins in a physiologically relevant context, before they form morphologically recognizable aggregates. We now describe FRET-based reporters for the conformation of α-synuclein, a protein central to the pathogenesis of Parkinsons disease (PD). Characterization in vitro shows that α-synuclein adopts a relatively “closed” conformation in solution that converts to “open” on membrane binding. In living cells, the closed conformation predominates. In neurons, however, cell bodies contain a much larger proportion of the open conformation than synaptic boutons. To account for these differences, we also used the reporters to characterize the interaction with native membranes. We find that the conformation of α-synuclein responds selectively to mitochondria, indicating a direct link between α-synuclein and an organelle strongly implicated in the pathogenesis of PD.

The behavior of α-synuclein in neurons†

Despite considerable evidence linking α‐synuclein with membranes in vitro, it has proven difficult to demonstrate membrane association of the protein in vivo. α‐Synuclein localizes to the nerve terminal, but biochemical experiments have not revealed a tight association with membranes. To address the dynamics of the protein in live cells, we have used photobleaching and found that α‐synuclein exhibits high mobility, although distinctly less than an entirely soluble protein. Further, neural activity controls the distribution of α‐synuclein, causing its dispersion from the synapse. In addition to the presumed role of α‐synuclein dynamics in synaptic function, changes in its physiological behavior may underlie the pathological changes associated with Parkinsons disease.

Chapter 18 – The Dynamics of α-Synuclein at the Nerve Terminal

Publisher Summary This chapter discusses the underlying dynamics of α-synuclein at the nerve terminal. α-synuclein fibrils are found in abundance in Lewy bodies, a cardinal pathological lesion found in most cases of Parkinsons disease (PD). α-synuclein can adopt multiple structural conformations depending on its environment, some of which are capable of converting into key intermediates in the assembly of Lewy bodies. Controlling the appearance of these pathogenic intermediates may prevent or at least slow down PD. Under normal circumstances, α-synuclein interacts with synaptic vesicle membranes, localizing to the nerve terminal where it has been proposed to regulate neurotransmitter release. Upon membrane binding, α-synuclein undergoes a major conformational change, from relatively unstructured to highly α-helical. Neuronal activity induces the dissociation of the protein from synaptic vesicle membranes and its dispersion from the synapse. The link between membrane association and conformation indicates that the dynamics of α-synuclein probably has an important role in the pathogenesis of PD. This chapter, while explaining the dynamics of α-synuclein, also elaborates in detail the membrane binding in vitro as well as the membrane interactions in vivo.