Advisory Committee Chair
Advisory Committee Members
William J Britt
Date of Award
Degree Name by School
Doctor of Philosophy (PhD) School of Medicine
Human cytomegalovirus (HCMV) is a complex double stranded DNA betaherpesvirus. Its linear DNA in enclosed in an icosahedral capsid, and has a genome size of about 230kb and encodes about 170 genes. Primary infection with HCMV lead to a lifelong persistent infection. HCMV can also be transmitted across the placenta and infect the developing fetus leading to infection of the newborn infant, an infection which is commonly described as congenital HCMV infection. In the United States, the prevalence of congenital HCMV infection is between 0.2-1.2%. Approximately 5-15% of these newborn infants will develop long-term neurological damage resulting, motor disorder, cognition and mental retardation, and sensorineural hearing loss. Infected infants may also present with microcephaly, pachygryia, cerebellar hypoplasia and other structural manifestation of neurological damage from this intrauterine infection. There is 10% death in the most severely affected infants. Although the neurological sequelae associated with congenital HCMV infections are well characterized, little is known about the pathogenesis of this infection of the developing central nervous system (CNS), including its potential impact on synaptic development in the CNS. To study the pathogenesis of congenital HCMV infection, we have developed a mouse model in which newborn mice are infected intraperitoneally (i.p.) with murine CMV (MCMV), a virus that has a similar genetic background and replication program to HCMV. Following inoculation, the virus infects and replicates in peripheral organs such as the spleen and liver. Infected mice then develop iv viremia that results hematogeous dissemination and replication in all regions of the brain of the infected newborn mouse. Maternal health and fetal environment during pregnancy has a major role in shaping the health of the developing fetus. Evidence from human epidemiological studies and studies in experimental animal model support the association between maternal immune activation and altered neurodevelopment of the offspring, with maternal immune activation associated with increased risk of neurodevelopmental disorders (NDD). Previously, we have described the pathology of MCMV infection in our mouse model. Perhaps the most readily apparent structural changes are seen in the cerebellar cortex of infected mice as this region of brain develops in the postnatal period in rodents. The cerebella of infected mice are significantly smaller in size, have reduced foliation, and have decreased numbers of granular neurons in the inner granular layer of the cerebellar cortex. With these background observations, we sought to study the impact neonatal MCMV infection has on synaptic development in the cerebellum as altered synaptic development has been associated with neurodevelopmental and neurobehavioral disorders in rodents and in humans. During neurodevelopment, connections between neurons are removed in a process that has been termed developmental synaptic pruning. Purkinje neurons (PC) are GABAergic inhibitory neurons located in the cerebellum, and are distinguished by their large soma and vast dendritic branching in the molecular layer of the cerebellar cortex that has been described as arborization. Extending from neurons in the inferior olivary nucclus are climbing fiber (CF) that form multiple excitatory synapses on the Purkinje cell body early the postnatal period. During subsequent maturation of the cellular connectivity of the cerebellum, excessive climbing fibers are removed from the soma of the PC and subsequent v migration of CF to the PC dendrites, resulting in one “winning” climbing fiber per Purkinje cell. The elimination multiple CF synapses occurs after birth as does similar synaptic pruning in other regions of the brain. To date our understanding of mechanisms of pruning are limited to only a few regions of the brain. The most well described mechanism of synaptic pruning involves the capacity of microglia, the resident immune phagocytic cells of the brain, to remove redundant synapses that have been targeted by complement molecules during neurodevelopment. Microglia complement-mediated synaptic pruning mechanism has been well described in the dorsal lateral geniculate nucleus. In this pruning process, astrocytes release TGFb which then causes the production of C1q. C1q then initiates the cleavage of C3 into C3b that in turn attaches to presynaptic neurons. Presynaptic neurons tagged with C3b are recognized by CR3 receptor on microglia, and microglia then engulf synapses formed by the presynaptic neuron, in a process called trogocytosis. During our studies we noted that postnatal day 8 MCMV infected mice have more climbing fiber synapses on Purkinje cell soma when compared to uninfected controls and that infected mice have reduced expression of complement proteins C1q and C3b in the cerebellum. Furthermore, we observed a significant increase in synapse proteins synaptophysin and vGlut2 in the cerebellum of infected mice. Lastly, we have shown that microglia in the cerebella of MCMV infected mice have increased expression of microglia activation marker Iba-1 and reduced Siglec-E, arguing that microglia with an activated, proinflammatory phenotypes have replaced microglia with normal phagocytic and trophic functions in the cerebellum of infected mice. Such a change in microglia function could impact the physiological role of microglia in synaptic pruning. Although, several reports have shown that microglia complement-mediated synaptic pruning does not occur in the vi cerebellum nor does microglia eliminate climbing fibers on Purkinje soma via trogocytosis, the expression of complement and complement associated protein are important for neural stem cell proliferation, differentiation and neuronal migration. Thus, reduced complement expression in MCMV infected neonatal mice may be associated with the observed delay in neurodevelopment. Climbing fiber elimination is competitive, and dependent on synaptic strength, with the “winning” climbing fiber having the greatest synaptic signal with the Purkinje neuron. The P/Q voltage-dependent calcium channel (P/Q VDCC) allows for the influx of Ca2+ into the Purkinje and efflux through AMPA receptors, thus signaling to the adjacent climbing fibers. MCMV infected mice have a significantly reduced cerebellar protein expression of P/Q VDCC, AMPA receptor GluR2 and AMPA associated proteins Stargazin and PSD-95, thus providing an alternative mechanism that can account for the altered synaptic pruning in the cerebella of infected mice. Finally, altered synaptic pruning in MCMV infected mice could result in altered neurodevelopment as measured by neurobehavioral testing. This possibility was confirmed in neurobehavioral testing of 5- week-old mice infected as newborns. Infected mice displayed compulsive behavior with a 2.5-fold increase in marble burying when compared to age-matched controls. Interestingly, high synaptic density has been associated with autism spectrum disorder and obsessive compulsive behaviors. MCMV infected mice treated with prednisolone were shown to have increased expression of both complement and P/Q VDCC. Furthermore, prednisolone treatment improved righting reflex and compulsive marble burying behaviors. Together these findings in this animal model have provided potentially important insight into mechanisms of neurological damage that follow HCMV infection during early neurodevelopment and vii ultimately, could serve as a foundation for treatment and intervention to alter the natural history of disordered CNS development in infants infected with HCMV.
Moulden, Jerome II, "Neonatal Murine Cytomegalovirus Infection Disrupts Synapse Remodeling During Development" (2021). All ETDs from UAB. 547.