Dynamic Partitioning of Synaptic Vesicle Pools by the SNARE-Binding Protein Tomosyn.

Neural networks engaged in high-frequency activity rely on sustained synaptic vesicle recycling and coordinated recruitment from functionally distinct synaptic vesicle (SV) pools. However, the molecular pathways matching neural activity to SV dynamics and release requirements remain unclear. Here we identify unique roles of SNARE-binding Tomosyn1 (Tomo1) proteins as activity-dependent substrates that regulate dynamics of SV pool partitioning at rat hippocampal synapses. Our analysis is based on monitoring changes in distinct functionally defined SV pools via V-Glut1-pHluorin fluorescence in cultured hippocampal neurons in response to alterations in presynaptic protein expression. Specifically, we find knockdown of Tomo1 facilitates release efficacy from the Readily Releasable Pool (RRP), and regulates SV distribution to the Total Recycling Pool (TRP), which is matched by a decrease in the SV Resting Pool. Notably, these effects were reversed by Tomo1 rescue and overexpression. Further, we identify that these actions of Tomo1 are regulated via activity-dependent phosphorylation by cyclin-dependent kinase 5 (Cdk5). Assessment of molecular interactions that may contribute to these actions identified Tomo1 interaction with the GTP-bound state of Rab3A, an SV GTPase involved in SV targeting and presynaptic membrane tethering. In addition, Tomo1 via Rab3A-GTP was also observed to interact with Synapsin 1a/b cytoskeletal interacting proteins. Finally, our data indicate that Tomo1 regulation of SV pool sizes serves to adapt presynaptic neurotransmitter release to chronic silencing of network activity. Overall, the results establish Tomo1 proteins as central mediators in neural activity-dependent changes in SV distribution among SV pools. SIGNIFICANCE STATEMENT: Although information transfer at central synapses via sustained high-frequency neural activity requires coordinated synaptic vesicle (SV) recycling, the mechanism(s) by which synapses sense and dynamically modify SV pools to match network demands remains poorly defined. To advance understanding, we quantified SV pool sizes and their sensitivity to neural activity while altering Tomo1 expression, a putative regulator of the presynaptic Readily Releasable Pool. Remarkably, we find Tomo1 actions to extend beyond the Readily Releasable Pool to mediate the Total Recycling Pool and SV Resting Pool distribution, and this action is sensitive to neural activity through Cdk5 phosphorylation of Tomo1. Moreover, Tomo1 appears to exert these actions through interaction with Rab3A-GTP and synapsin proteins. Together, our results argue that Tomo1 is a central mediator of SV availability for neurotransmission.

Hypochlorite converts cysteinyl-dopamine into a cytotoxic product: A possible factor in Parkinson's Disease.

The dopamine oxidation product cysteinyl-dopamine has attracted attention as a contributor to the death of dopaminergic neurons in Parkinson's disease. Treatment of cysteinyl-dopamine with hypochlorite yields an even more cytotoxic product. This product has potent redox-cycling activity and initiates production of superoxide in PC12 cells. Taurine, which scavenges hypochlorite, protects PC12 cells from cysteinyl-dopamine but not from the hypochlorite product, suggesting that the product, not cysteinyl-dopamine itself, is toxic. Furthermore, rotenone, which enhances expression of the hypochlorite-producing enzyme myeloperoxidase, increases the cytotoxicity of cysteinyl-dopamine but not of the hypochlorite product. This suggests that dopamine oxidation to cysteinyl-dopamine followed by hypochlorite-dependent conversion to a cytotoxic redox-cycling product leads to the generation of reactive oxygen species and oxidative stress and may contribute to the death of dopaminergic neurons.