Judy A. Garner, PhD - US grants

The vertebrate synapse is a specialized form of cell-cell contact critical in nervous system function. The plasma membrane and underlying associated cytoplasmic proteins of both the pre- and postsynaptic sides of the synapse form highly specialized, ultrastructurally unique complexes that are the intracellular locations of many of the biochemically and physiologically regulated synaptic events. The proposed study is designed to examine the molecular organization of the cortical cytoplasm and associated synaptic plasma membrane within central nervous system presynaptic terminals. This will be accomplished by the powerful combination of three, well-documented technologies: a) labelling of presynaptic, subcellular compartments within the terminal by axonal transport, b) the enrichment of radiolabeled presynaptic terminals by careful and rigorous preparation of synaptosomes and c) the subjection of these radiolabled synatosomes to a battery of experiments, similar to those previously used to elucidate the structural organization of the spectrin-enriched cytoskeleton and the associated plasma membrane of the red blood cell. Information we may thus obtain through basic research on normal biochemistry, molecular organization, and functioning of the synapse will aid A) in understanding the way the presynaptic terminal performs its important biological tasks under the stress of disease states, and B) in discerning the origins and source of malfunctions that affect the synapse directly.

Viruses of the family Herpesviridae, including herpes simplex types 1(HSV1) and 2, varicella zoster virus, cytomegalovirus, and Epstein-Barr Virus are complex enveloped viruses containing linear double-stranded DNA. As a group, they are responsible for a large number of serious human ailments. In its natural host, the adult human, the cold virus HSV1 primarily infects oral mucosal epithelium, followed by infection of sensory neurons innervating that mucosa. HSV1, as with all herpesviruses, is capable of achieving latency, and can be reactivated at a later time. Though relatively rare, HSV1 can also be the primary cause of the more serious herpes simplex encephalitis, as well as systemic disseminated infections. Demonstrating increased risk of the more serious sequelae of HSV1 infection is the portion of the population that is relatively immunosuppressed (neonates, transplant patients, or those with immune-suppressive disorders). It has been well established that ocular herpetic keratitis, HSV1 infection of the eye (approximately 300,000 cases diagnosed yearly) is, after trauma, the most common cause of corneal blindness in this country. Clearly, the multiple deleterious effects of HSV1 infection thus motivate us to attempt a better understanding of host cell responses to viral infection. The targeted intracellular transport of HSV-1 is essential for transmission of virus from an infected cell to its neighbor. The synaptic connections of the infected sensory neuron (which has axons that end in the periphery and central nervous system), and the ability of these viruses to be transferred from one neuron to its synaptically linked neighbor, provide a unique route by which virus can be presented the CNS. The mechanisms by which these neurotropic viruses accomplish that intracellular movement are thus of considerable interest not only to those investigating viral pathogenesis in the central nervous system, but also to those interested in the mechanisms of basic cellular targeting and transport of endogenous macromolecular complexes. The goal of this project is to elucidate mechanisms used by HSV-1 to accomplish targetted transport from its assembly site in the neuronal cell nucleus, through the cytoplasm to the plasma membrane. We first would like to establish definitively whether viral transport depends on the presence of the microtubule cytoskeleton and microtuble-dependent motor proteins. We would like to further investigate the biological form assumed by the virus during its targetted anterograde transport. Finally, we plan to investigate host cell macromolecules that may be recruited by the virus as well as viral proteins involved in that movement.