Defense Date

7-19-2023

Graduation Date

Fall 12-15-2023

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Pharmacology

School

School of Pharmacy

Committee Chair

Lauren A. O'Donnell

Committee Member

Rehana K. Leak

Committee Member

Megan C. Freeman

Committee Member

Kerry M. Empey

Committee Member

Paula A. Witt- Enderby

Keywords

Measles virus, neural stem cells, hippocampus, sub-ventricular zone, myelination, oligodendrocytes, nestin, O4, Olig2, viral infections, virus

Abstract

Viral infections of the central nervous system (CNS) often result in neurological sequelae and are considered a risk factor for the development of neurological disease. Moreover, the outcomes of a viral infection are largely dependent on the age of the host, with younger hosts developing more profound neuropathology. Perhaps the developing brain coupled with an immature immune system contributes to worse outcomes in the very young. Children suffering from brain infections are more prone to develop enduring neurological impairments even after recovery from the infection. Although majority of the brain growth occurs in utero, neurodevelopment continues throughout the juvenile period and into adulthood. Specifically, processes such as myelination, synaptogenesis, pruning, and refinement of neural circuitry occur in the juvenile and pediatric ages. Thus, a viral infection during this time can disrupt ongoing neurodevelopment and result in long-lasting damage in the surviving host.

Neural stem cells (NSCs) are one of the many neural cells that may be affected during neurotropic viral infections. NSCs are multipotent progenitor cells that give rise to neurons, astrocytes, and oligodendrocytes that populate the brain. The NSC pool is highly dynamic pre- and postnatally, but ultimately contracts with age to two neurogenic niches in the brain [hippocampus and sub-ventricular zone (SVZ)]. In these niches, the NSCs are heterogenous in different stages of differentiation, ranging from the most “stem-like” undifferentiated stage to early commitment to neurons or glia. During a viral infection, NSCs are often disrupted, leading to reduced proliferation, and altered differentiation. Most studies on viruses and NSCs have focused on fetal NSC models in vitro and in vivo, with a handful of investigations in adult animal models of infection. Thus, the impact of viruses on NSCs at the juvenile age is largely unknown, leaving us with a poor understanding of how childhood infections affect NSCs in the short-term and neurological function in the long-term. Here, my aim was to determine the impact of the anti-viral immune response on NSCs in a juvenile animal and track their development and survival into adulthood. In doing so, my goal was to determine the cellular basis for the manifestation of long-term neurological sequela resulting from an infection in childhood.

To determine the impact of the anti-viral immune response on juvenile NSCs, we utilized a transgenic mouse model for CNS measles virus (MeV) pathogenesis. In this model, the expression of the human MeV receptor CD46 is limited to mature neurons by the neuron specific enolase (NSE) promoter. Thus, MeV infection is neuronally restricted and non-neuronal cells such as NSCs remain uninfected. Therefore, any changes in the NSC pool are due to anti-viral immunity and not direct infection by MeV. We specifically assessed the changes in the NSC numbers, proliferation, and differentiation in the two neurogenic niches: the hippocampus and the SVZ. Using juvenile CD46+ mice (10 days old), we examined the immediate effects on NSCs during the acute infection and tracked the NSCs of surviving mice into adulthood. We found that ~25% of the juvenile mice succumb to infection with neurological signs, whereas 75% of mice survive the infection with minimal symptoms. However, in the surviving mice, the viral load is not controlled until 5 months post-infection, suggesting that the juvenile CD46+ mice may serve as a model for persistent RNA virus infections. As NSCs are present at different stages of differentiation we also assessed the impact of the immune response on the various NSC stages. With the establishment of this juvenile model of infection, we determined how NSCs experience the antiviral immune response as bystanders in the microenvironment of the infected brain, both early during acute infection and later in the adult survivors.

During early infection (9 days post-infection; 9 dpi), the NSCs exhibited a stage-dependent decrease in numbers with a loss of mid-stage NSCs and NSCs early in neuronal differentiation, but no loss in undifferentiated NSCs. In surviving adults (90 dpi), the NSC pool had resolved to physiological levels. Thus, NSCs are sensitive to the juvenile immune response in a stage-dependent manner, but ultimately recover. We also observed that the decrease in NSCs is attributed to an increased rate of differentiation rather than a block in proliferation or massive cell death. Specifically, NSC produced greater numbers of both immature neurons and oligodendrocytes progenitor cells (OPCs) early during infection. Even though the NSC pool had normalized by adulthood, the OPCs remained high in both the neurogenic niches, indicating long-term expansion of the OPC lineage after infection. Moreover, both OPCs and mature oligodendrocytes increased at 90 dpi in the hippocampus as well as the SVZ. These results led us to investigate whether myelination is disrupted in the brain, as oligodendrocytes are responsible for producing myelin. In every region in the brain that we examined, there was evidence of abnormal myelination during early infection as well as in the survivors at 90 dpi, indicating that juvenile brain infections lead to enduring disruptions in myelination. Synaptic proteins were also dysregulated at 90 dpi, suggesting that disturbances in myelination are associated with neuronal dysfunction. Further investigation at 150 dpi revealed that some of the surviving mice had declined, with impaired gait and seizures along with a decrease in immature neurons and NSCs in the hippocampus. These studies show that neurological damage can emerge later in life even when the virus and inflammation were previously controlled. Thus, collectively, we have shown that juvenile viral infections induce life-long disruptions at the cellular as well as behavioral levels, even when the initial infection may appear benign.

In order to better assess the role of the immune response in juvenile neurotropic infections, we examined the effects of Fin using CD46+/IFNγ-Knockout (CD46+/IFNγ-KO) mice early during acute infection, when IFNγ expression is high in multiple brain regions. We focused on IFNγ because it is needed to control the virus in this model and because we have previously shown that NSCs are exquisitely sensitive to IFNγ signaling in vitro and in vivo. A lack of IFNγ in juvenile CD46+ mice resulted in more profound sickness and a higher viral load, highlighting the importance of this anti-viral cytokine at this age. Additionally, we observed an increase in both neurogenesis and gliogenesis after infection in CD46+/IFNγ-KO mice, suggesting aberrant NSC differentiation after infection. As the role of individual cytokines is difficult to assess in vivo, we also performed in vitro studies to elucidate the role of IFNγ on NSC differentiation. We observed that IFNγ drives embryonic NSC differentiation toward the glial lineage by inhibiting neuronal differentiation. These studies suggest that IFNγ is important for viral control in the brain at multiple ages, and that it either directly or indirectly shapes the responses of the NSCs to the inflammatory microenvironment.

Overall, the studies in this thesis show that CNS viral infections at the juvenile age can have long-term consequences, even if the host survives with limited symptoms during the acute infection. Juvenile NSCs are sensitive to the antiviral immune response, not only early in infection but also long-term, suggesting that aftereffects of an infection can be profound. We speculate that the impacts we observed on NSCs, OPCs, and myelination elucidates a potential mechanism by which brain infections in childhood may result in long-term behavioral deficits. Based on the development of seizures and motor impairments in the adulthood of infected mice, we propose that the increase in OPCs and oligodendrocytes is a compensatory mechanism of the brain perhaps to bring about myelin stabilization, although these efforts to mobilize OPCs may not be successful in the long-term. Our studies highlight that immune-mediated myelin pathology can be triggered by viral infections that do not directly target the oligodendroglial lineage. However, future studies are needed to identify immune factors that specifically result in aberrant myelination and OPC distortion. Such studies would help identify potential therapeutic targets to ameliorate the effects of childhood infections and provide a better quality of life for the surviving host.

Language

English

Additional Citations

  1. Kamte, Y. S., Chandwani, M. N., London, N. M., Potosnak, C. E., Leak, R. K., & O’Donnell, L. A. (2023). Perturbations in neural stem cell function during a neurotropic viral infection in juvenile mice. Journal of Neurochemistry, 00, 1–21. https://doi.org/10.1111/jnc.15914
  2. Kamte, Y.S.; Chandwani, M.N.; Michaels, A.C.; O’Donnell, L.A. Neural Stem Cells: What Happens When They Go Viral? Viruses 2021, 13, 1468. https://doi.org/10.3390/v13081468

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