Multivalent electrostatic pi-cation interaction between synaptophysin and synapsin is responsible for the coacervation.

We recently showed that synaptophysin (Syph) and synapsin (Syn) can induce liquid-liquid phase separation (LLPS) to cluster small synaptic-like microvesicles in living cells which are highly reminiscent of SV cluster. However, as there is no physical interaction between them, the underlying mechanism for their coacervation remains unknown. Here, we showed that the coacervation between Syph and Syn is primarily governed by multivalent pi-cation electrostatic interactions among tyrosine residues of Syph C-terminal (Ct) and positively charged Syn. We found that Syph Ct is intrinsically disordered and it alone can form liquid droplets by interactions among themselves at high concentration in a crowding environment in vitro or when assisted by additional interactions by tagging with light-sensitive CRY2PHR or subunits of a multimeric protein in living cells. Syph Ct contains 10 repeated sequences, 9 of them start with tyrosine, and mutating 9 tyrosine to serine (9YS) completely abolished the phase separating property of Syph Ct, indicating tyrosine-mediated pi-interactions are critical. We further found that 9YS mutation failed to coacervate with Syn, and since 9YS retains Syph's negative charge, the results indicate that pi-cation interactions rather than simple charge interactions are responsible for their coacervation. In addition to revealing the underlying mechanism of Syph and Syn coacervation, our results also raise the possibility that physiological regulation of pi-cation interactions between Syph and Syn during synaptic activity may contribute to the dynamics of synaptic vesicle clustering.

Loss of neuronal protein expression in mouse hippocampus after irradiation.

The effects of radiation on neurons are incompletely characterized. We evaluated changes in the expression of neuronal nuclear and other proteins in the mouse hippocampus after 17-Gy whole-brain irradiation. Expression of neuronal nuclei (NeuN), neuron-specific enolas, prospero-related homeobox 1 (Prox1), calbindin D28k, and synaptophysin 1 in the CA1, CA3, and dentate gyrus of the hippocampus was determined by immunohistochemistry; neuronal numbers were estimated by design-based stereology. At 7 days after irradiation, there was a marked reduction of NeuN neurons in CA3. Stereologic estimates confirmed a significant reduction in NeuN neurons in CA3 at 7 days, in the dentate gyrus at 7 days, 3 weeks and 2 months, and in CA1 at 2 months compared with controls; neuron-specific enolase and prospero-related homeobox 1-positive neurons in the CA3 subregion were also decreased at 7 days. The numbers of granule and pyramidal cells identified by 4'6-diamidino-2-phenylindole nuclear staining, however, remained unchanged, and there were no changes in calbindin D28k or synaptophysin 1 immunoreactivity after irradiation. We conclude that irradiation may result in a temporary loss of neuronal protein expression in mouse hippocampus. These changes do not necessarily indicate loss of neurons and indicate the need for caution regarding the use of phenotypic markers such as NeuN to estimate changes in neuronal numbers after irradiation.

Abnormality of synaptic vesicular associated proteins in cerebral cortex and hippocampus after microwave exposure.

Studies were performed to determine the effects of microwave on synaptic vesicles and the expression of synaptic vesicular associated proteins including synapsin I, VAMP-2, syntaxin, and synaptophysin. 25 Wistar rats were exposed to microwave which the average power density was 30 mW/cm(2), and whole body average specific absorption rate was 14.1 W/kg for 5 min. Synaptosome preparations in the cerebral cortex and hippocampus were obtained by isotonic Percoll/sucrose discontinuous gradients at 6 h, 1, 3, and 7 days after radiation. The expression of synaptic vesicular associated proteins was measured using Western blots and image analysis. The interaction between VAMP-2 and syntaxin was examined by coimmunoprecipitation analysis. Synapsin I in the cerebral cortex were decreased at 3 days (P < 0.01) after radiation and in the hippocampus increased at 1 day (P < 0.01), decreased at 3 days (P < 0.01), increased again at 7 days (P < 0.01) after exposure, compared with the sham-treated controls. Synaptophysin were increased in 1-7 days (P < 0.01) after exposure in the cerebral cortex and hippocampus. VAMP-2 were decreased at 1 and 3 days (P < 0.01) and syntaxin were decreased in 6 h to 3 days (P < 0.01) after radiation in the cerebral cortex and hippocampus. The interactions between VAMP-2 and syntaxin were decreased at 3-7 days (P < 0.01) after radiation in the cerebral cortex and hippocampus, compared with the sham-treated controls. These results suggest that 30 mW/cm(2) (SAR 14.1 W/kg) microwave radiation can result in the perturbation of the synaptic vesicles associated proteins: synapsin I, synaptophysin, VAMP-2, and syntaxin. The perturbation could induce the deposit of synaptic vesicle, which might be relative to the dysfunction of the synaptic transmission, even the cognition deficit.