Ocsyn, a novel syntaxin-interacting protein enriched in the subapical region of inner hair cells.

Sensory (hair) cells of the inner ear contain two specialized areas of membrane delivery. The first, located at the cell base, is the afferent synapse where rapid delivery of synaptic vesicles is required to convey information about auditory signals with exceedingly high temporal precision. The second area is at the apex. To accommodate the continuous movement of stereocilia and facilitate their repair, recycling of membrane components is required. Intense vesicular traffic is restricted to a narrow band of cytoplasm around the cuticular plate, which anchors stereocilia. Our previous analyses showed that SNARE proteins (syntaxin 1A/SNAP25/VAMP1) are concentrated at both poles of hair cells, consistent with their involvement in membrane delivery at both locations. To investigate further the molecules involved in membrane delivery at these two sites, we constructed a two-hybrid library of the organ of Corti and probed it with syntaxin 1A. Here we report the cloning of a novel syntaxin-binding protein that is concentrated in a previously uncharacterized organelle at the apex of inner hair cells.

Postsynaptic density-93 interacts with the delta2 glutamate receptor subunit at parallel fiber synapses.

The glutamate receptor subunit delta2 has a unique distribution at the parallel fiber-Purkinje cell synapse of the cerebellum, which is developmentally regulated such that delta2 occurs at both parallel fiber synapses and climbing fiber synapses early in development but is restricted to parallel fiber synapses in adult animals. To identify proteins that might be involved in the trafficking or docking of delta2 receptors, we screened a yeast two-hybrid library with the cytosolic C terminus of delta2 and isolated a member of the postsynaptic density (PSD)-95 family of proteins, which are known to interact with the extreme C termini of NMDA receptors. We find that delta2 binds specifically to PSD-93, which is enriched in Purkinje cells. In addition, PSD-93 clusters delta2 when they are coexpressed in heterologous cells, and clustering is disrupted by point mutations of delta2 that disrupt the delta2-PSD-93 interaction. Ultrastructural localization of PSD-93 and delta2 shows they are colocalized at parallel fiber synapses; however, PSD-93 also is present at climbing fiber synapses of the adult rat, where delta2 is not found, indicating that the presence of PSD-93 alone is not sufficient for determining the synaptic expression of delta2.

Induction of cytotoxic T cell responses in newborn mice by DNA immunization.

Cytotoxic T cells (CTL) play a critical role in controlling viral infections. Infection of neonatal NFSIN mice with a high dose of Cas-Br-M murine leukemia virus, a neuropathogenic type C retrovirus, results in virus-induced neurologic disease and in their failure to generate a protective CTL response. Cas-Br-M-specific CTL are necessary in the protection of neonatal mice from Cas-Br-M-induced neurologic disease. Here we demonstrate that intramuscular inoculation of newborn mice with naked DNA expressing the full length Cas-Br-M genome induces a virus-specific CTL-mediated response. This CTL response is mediated by CD8+ T cells, is long lasting and, when transferred to susceptible neonatal recipients, protects them from Cas-induced neurologic disease. We also provide evidence that the intramuscular inoculation of neonates with plasmid DNA encoding only env sequences induces a dose-dependent CTL response in the absence of an anti-MuLV antibody response.