Human Cytomegalovirus (HCMV) is a leading cause of congenital birth defects. The most common sequela observed in symptomatic infants is sensorineural hearing loss (SNHL). SNHL is also observed, somewhat perplexingly, during early childhood, developing over a period of years in children born hearing competent and asymptomatic for infection at birth. The most abundant peripheral nervous system (PNS) myelin protein, myelin protein zero (MPZ), is largely responsible for compaction of the PNS myelin sheath. Charcot-Marie-Tooth Type 1B and Dejerine-Sottas syndromes patients, with mutations in MPZ, frequently suffer from late-onset SNHL, similar to that observed in HCMV congenitally-infected children. MPZ expression is dramatically decreased in congenital HCMV-infected tissue samples. Schwann cells (SCs) are the only cells in the body that produce MPZ. HCMV infection of SCs, or sole expression of the HCMV tegument protein pp71 in culture, causes large decreases in MPZ mRNA levels. SCs express MPZ protein only when directly contacting the neurons they sheathe, making a tractable co-culture system essential to studying defects in myelination. Substantial literature describes rat SC/neuron co-culture; however, very little work has reported using only human cells, essential for the study of species-restricted Human CMV pathogenesis.

The project: We have developed an all-human three-dimensional (3D) SC/neuron co-culture system, amenable to the study of HCMV interactions. Our preliminary experiments have produced robust neurite outgrowth, SC/neuron interactions, MPZ expression and myelination. Initial experiments with pp71-expressing SCs produced very different interactions between the cell types and no evidence of myelination.

We propose to 1) Rigorously characterize our all-human in vitro SC/neuron 3D co-culture system designed to study HCMV's influence on MPZ production and myelination ; 2) Assess the ramifications of HCMV infection or sole pp71 expression on the baseline parameters established above; 3) Determine whether loss of MPZ function alone (via CRISPR KO in the SC) can replicate the perturbations we observe during infection- or sole pp71 expression and 4) utilize our co-culture system to examine crosstalk between the extracellular matrix (ECM) and myelin production, MPZ in particular, since the literature suggests a critical link between the ECM and SCs? ability to properly myelinate neurons.