Human cytomegalovirus (HCMV) is a highly prevalent viral pathogen that typically presents asymptomatically in healthy individuals despite lifelong latency. However, in 10%–15% of congenital cases, this beta-herpesvirus demonstrates direct effects on the central nervous system, including microcephaly, cognitive and learning delays, and hearing deficits. HCMV has been widely shown to infect neural progenitor cells, but the permissiveness of fully differentiated neurons to HCMV is controversial and chronically understudied, despite potential associations between HCMV infection and neurodegenerative conditions. Using a model system representative of the human forebrain, we demonstrate that induced pluripotent stem cell-derived excitatory glutamatergic and inhibitory GABAergic neurons are fully permissive to HCMV with complete viral replication, competent virion production, and spread within the culture. Interestingly, while cell proliferation was not induced in these post-mitotic neurons, HCMV did increase expression of proliferative markers Ki67 and proliferative cell nuclear antigen suggesting alterations in cell cycle machinery. These findings are consistent with previous HCMV-mediated changes in various cell types and implicate the ability of viral proteins to alter proliferative pathways to promote virion production. Infection also induces significant structural changes in forebrain neurons, such as the formation of syncytia and the retraction of neurites. Finally, we demonstrate that HCMV disrupts calcium signaling and decreases neurotransmission, with action potential generation effectively silenced after 15 days post-infection. Taken together, our data highlight the potential for forebrain neurons to be permissive to HCMV infection in the central nervous system, which has significant implications for overall brain health and function. IMPORTANCE: Human cytomegalovirus (HCMV) is a highly prevalent viral pathogen that can cause serious neurological deficits in infants experiencing an in utero infection. Also, as a life-long infection, HCMV has been associated with several diseases in the adult brain. HCMV is known to infect early neural progenitor cells, but whether it also infects terminally differentiated neurons is still debated. Here, we differentiated human-induced pluripotent stem cells into neurons for 84–120 days to test the ability of HCMV to infect terminally differentiated neurons and assess the downstream functional consequences. We discovered that mature human neurons are highly permissive to HCMV infection, exhibited late replication hallmarks, and produced infectious virus. Moreover, infection in terminally differentiated neurons essentially eliminated neuron function. These results demonstrate that terminally differentiated human neurons are permissive to HCMV infection, which can significantly alter both structural and functional features of this mature neuron population.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motor neuron loss is the primary phenotype, leading to muscle weakness and wasting, respiratory failure, and death. Although a portion of ALS cases are linked to one of over 50 unique genes, the vast majority of cases are sporadic in nature. However, the mechanisms underlying the motor neuron loss in either familial or sporadic ALS are not entirely clear. Here, we used induced pluripotent stem cells derived from a set of identical twin brothers discordant for ALS to assess the role of astrocytes and microglia on the expression and accumulation of neurofilament proteins in motor neurons. We found that motor neurons derived from the affected twin which exhibited increased transcript levels of all three neurofilament isoforms and increased expression of phosphorylated neurofilament puncta. We further found that treatment of the motor neurons with astrocyte-conditioned medium and microglial-conditioned medium significantly impacted neurofilament deposition. Together, these data suggest that glial-secreted factors can alter neurofilament pathology in ALS iPSC-derived motor neurons.

Human cytomegalovirus (HCMV) is a prolific human herpesvirus that infects most individuals by adulthood. While typically asymptomatic in adults, congenital infection can induce serious neurological symptoms, including hearing loss, visual deficits, cognitive impairment, and microcephaly in 10%–15% of cases. HCMV has been shown to infect most neural cells, with our group recently demonstrating this capacity in stem cell-derived forebrain neurons. Infection of neurons induces deleterious effects on calcium dynamics and electrophysiological function paired with gross restructuring of neuronal morphology. Here, we utilize an induced pluripotent stem cell-derived model of the human forebrain to demonstrate how HCMV infection induces syncytia, drives neurite retraction, and remodels microtubule networks to promote viral production and release. We establish that HCMV downregulates microtubule-associated proteins while largely sparing other cytoskeletal elements. Furthermore, we pharmacologically modulate microtubule dynamics using paclitaxel (stabilize) and colchicine (destabilize) to examine the effects on neurite structure, syncytial morphology, and viral release. With paclitaxel, we found improvement of neurite outgrowth, but neither paclitaxel nor colchicine impacted viral titers. Together, these data suggest that HCMV infection-induced disruption of microtubules in human cortical neurons can be partially mitigated with microtubule stabilization, suggesting a potential avenue for future neuroprotective strategies. IMPORTANCE: Infection by human cytomegalovirus (HCMV) continues to cause significant damage to human health. In the absence of a vaccine, vertical transmission from mother to fetus can result in profound neurological damage impacting quality of life. These studies focus on understanding the impact of HCMV infection on forebrain cortical neurons derived from induced pluripotent stem cells (iPSCs). We show that infection results in loss of neurite extension accompanied by cell-to-cell fusion. These pathogenic changes involve HCMV infection-mediated disruption of the microtubule network in iPSCs from different patient backgrounds. The microtubule stabilization agent paclitaxel partially protected neurite length and altered syncytia morphology without impacting viral replication. This work is part of our continued efforts to define putative strategies to limit HCMV-induced neurological damage.