Multiple sclerosis (MS) is an autoimmune demyelinating disease characterized by increased susceptibility in women. However, when men get the disease, they demonstrate more rapid disease progression, thus presenting a clinical enigma of the pathogenic events in MS susceptibility and progression. Of the sex-related factors that may contribute to MS susceptibility, much of the literature on the MS model, experimental autoimmune encephalitomyelits (EAE), has focused on the role of sex hormones in immune responses. While sex hormones have been demonstrated to affect autoimmune responses and neurodegeneration in EAE, it does not preclude sex chromosome effects. Indeed, previous work from our lab has demonstrated that the sex chromosome complement of immune cells affected autoimmune responses, with the XX sex chromosome complement, as compared to XY, leading to greater encephalitogenicity during auto-antigen stimulation. These results are consistent with human clinical observations of increased autoimmunity in women vs. men. This dissertation builds upon this finding by exploring the nature of sex chromosome effects in the female bias in autoimmunity through the use of transgenic sex chromosome mouse models that include the following genotypes: XX, XY-, XmY*x and XpY*x. Through a series of experiments, I demonstrate that parental imprinting of the X chromosome gene, forkhead box P3 (Foxp3), a master-regulator of T regulatory (Treg) cell development, results in increased FoxP3+ Tregs in XY- mice vs. XX mice.

While sex chromosome effects have been identified in the immune system, it remained unclear if there were separate sex chromosome effects in the target organ, the CNS, response to an immune attack during EAE. This question is addressed in another portion of my dissertation. I created a bone marrow chimera model with mice bearing a common immune system, but varying sex chromosome complement in the CNS using the aforementioned transgenic sex chromosome mouse models, to examine the effects of sex chromosomes in the CNS during neurodegeneration. Through a series of experiments, I demonstrated that mice with CNS cells expressing XY sex chromosome complement, as compared to XX, led to greater neurodegeneration. Further, increased neurodegenerative response in the XY CNS was due to the maternal X allele. Together, these results demonstrated for the first time an affect of parental imprinting resulting in female bias on the autoimmune responses, and male bias in neurodegeneration during EAE. These findings may bear relevance to the clinical observations of female bias in susceptibility, but increased disability progression in men with MS.

Multiple Sclerosis (MS) is a heterogeneous disease that impacts the brain, spinal cord, and optic nerve. The etiology of MS is not completely understood, but the nature of these autoimmune inflammatory and neurodegenerative lesions has been under investigation for decades. Biological aging has been reported to be more than a significant factor associated with neurodegenerative diseases. Thalamic grey matter atrophy in MS has increasingly been demonstrated with normal aging, and a sexual dimorphism over time has been reported even at the earliest forms of the disease. The thalamus is a critical relay center, and the association of the thalamus in MS has been linked to various clinical symptoms ranging from vision loss, motor deficits, cognitive impairment, numbness, and fatigue. Using the preclinical model of MS, experimental autoimmune encephalomyelitis (EAE), we assessed the neuropathology of thalamic grey matter and spinal cord white matter in sex-matched and age-matched wild-type mice to better understand the motor disability seen in MS and EAE. The experiment with healthy controls and EAE middle age and young mice revealed significant widespread microglia activation, astrocyte activation, and synaptic loss in aged mice. This neuropathology in the MS model during aging suggests that similar neuropathology may contribute to thalamic grey matter atrophy in progressive MS. The role of biological aging witnessed in thalamic network injury in EAE and MS related symptoms is key in highlighting the manifestation of variable lesions that are influenced by biological sex and aging.

Currently, available drugs for multiple sclerosis (MS) are predominantly immune-modulatory. They are effective in reducing relapses, but not in slowing down or stopping progressive disease course. Therefore, there is a need to develop treatment strategies that will provide neuroprotection and halt disability progression. Pregnancy is neuroprotective in patients with MS. A pregnancy hormone and a moderate estrogen receptor beta (ERβ)-ligand, estriol, has been reported to have beneficial effects on reducing relapses and improving cognition when treated in MS patients. Although, the mechanisms of ERβ-ligand mediated treatment effects on neuroprotection remains poorly understood. Here I hypothesized that ERβ signaling on CD11c+ immune cells and Olig1+ oligodendrocytes plays an essential role in providing neuroprotection and enhancing remyelination. To test the hypothesis, I created conditional knockout mice (CKO) with ERβ deleted from each cell type using the Cre-LoxP recombinase system and investigated neuroprotective effects of ERβ-ligand treatment in experimental autoimmune encephalomyelitis (EAE) and cuprizone diet-induced demyelinating disease models. Also, I used RiboTag mice to determine oligodendrocyte specific gene expression to understand the molecular mechanisms of remyelination. Subsequently, I studied the mechanisms of ERβ-ligand treatment effects in oligodendrocytes during remyelination.

The results revealed that ERβ-ligand treatment reduced pro-inflammatory responses through ERβ on CD11c+ myeloid DC/MΦ and increased oligodendrocyte maturation through ERβ on Olig1+ oligodendrocytes during EAE. The use of CKO mice proved that both were necessary as one without the other was not sufficient. Mechanistic insights on remyelination using the RiboTag mice revealed that oligodendrocytes upregulate de novo cholesterol biosynthesis during remyelination after chronic demyelination. Furthermore, when compared to vehicle treated naturally remyelinating mice, ERβ-ligand treated mice showed enhanced remyelination by increasing oligodendrocyte maturation, increasing cholesterol synthesis pathway genes, increasing oligodendrocyte progenitor cells (OPCs), and increasing Sox2 expression in OPCs during remyelination. I extended our investigation of ERβ-ligand treatment in the cuprizone model to the EAE model, to discover that ERβ-ligand treated mice showed neuroprotection by increasing oligodendrocyte maturation, and increasing cholesterol synthesis pathway genes, but not by increasing OPC Sox2 expression. This comprehensive approach using two different demyelinating disease models of MS and two transgenic mouse models provided critical insights regarding treatment effects of ERβ-ligand on neuroprotection and remyelination in vivo.

Estrogen has well documented neuroprotective effects in a variety of clinical and experimental disorders of the central nervous system (CNS). The beneficial effects of estrogens in CNS disorders include mitigation of clinical symptoms as well as attenuation of histopathological signs of neurodegeneration and inflammation. The cellular mechanisms that underlie these CNS effects of estrogens are uncertain, because a number of different cell types express estrogen receptors in the peripheral immune system and CNS. Here, we investigated the potential roles of two endogenous CNS cell types in estrogen-mediated neuroprotection. We selectively deleted estrogen receptor alpha or estrogen receptor beta from either neurons or astrocytes using well-characterized Cre-loxP systems for conditional gene knockout in mice and studied the effects of these conditional gene deletions in a well-characterized model of adoptive experimental autoimmune encephalomyelitis (EAE). We found that the pronounced and significant neuroprotective effects of systemic treatment with ERbeta ligand on clinical function, CNS inflammation, and axonal loss during EAE were not completely prevented by conditional deletion of ERbeta from astrocytes or neurons. Interestingly, we found that the pronounced and significant neuroprotective effects of systemic treatment with ERalpha ligand on clinical function, CNS inflammation, and axonal loss during EAE were completely prevented by conditional deletion of ERalpha from astrocytes, whereas conditional deletion of ERalpha from neurons had no significant effect. Given the differential neuroprotective effects of ERalpha ligand treatment versus ERbeta ligand treatment on astrocytes, as well on T-cell and macrophage inflammation, we looked for molecules within astrocytes that were affected by signaling through ERalpha, but not ERbeta. We found that ERalpha ligand treatment, but not ERbeta ligand treatment, decreased expression of the chemokines CCL2 and CCL7 by astrocytes in EAE. Together our data show that neuroprotection in EAE mediated via ERbeta signaling does not require ERbeta on astrocytes or neurons, whereas neuroprotection in EAE mediated via ERalpha signaling requires ERalpha on astrocytes, and not neurons, and reduces astrocyte expression of chemokines that contribute to CNS inflammation. These findings reveal important cellular differences in the neuroprotective mechanisms of estrogen signaling through ERalpha and ERbeta in EAE.

Like most autoimmune diseases, multiple sclerosis (MS), is more prevalent in women than men, thought to be due to more robust immune responses in women. The effects of sex hormones on the immune response have been extensively studied by our lab and others, but effects of sex chromosomes are widely unknown. Sex chromosomes can elicit gene expression differences between males and females via expression of Y genes, X gene dosage effects, or parent-of-origin differences in DNA methylation of X genes, also called parental imprinting. We previously showed that sex chromosomes modulate immune responses in the animal model of MS experimental autoimmune encephalomyelitis (EAE), however, the mechanisms remained unknown. Chapter 2 of this dissertation identified parent-of-origin differences in DNA methylation of X chromosome genes as a mechanism by which sex chromosomes alter gene expression in immune cells. Through a series of RNA sequencing experiments using the “four core genotypes” model, we discovered a cluster of X genes with higher expression in XY than XX genotypes in CD4+ T lymphocytes. Additionally, DNA methylation studies in X monosomic mice revealed parent-of-origin differences in DNA methylation in which the paternal X is hypermethylated compared to the maternal X and autosomes, which is consistent with higher expression of X genes in XY compared to XX. This work demonstrated how parental imprinting of X chromosome genes can lead to sex differences in gene expression during immune responses. While women are more susceptible to MS, disability progression tends to be worse in men, thought to be due to a more neurodegenerative response to immune attack. This identifies males as a superior target for understanding mechanisms of neurodegeneration. In Chapter 3 of this dissertation, I take a cell-specific and region-specific gene expression approach to investigate the neurodegenerative response in the hippocampus during injury. Through transcriptomics studies in hippocampal astrocytes, I found upregulated expression of genes involved in MHC class I (MHC I) signaling. Since MHC I has an important role in synaptic plasticity, I hypothesize that astrocyte MHC I signals to microglia and neurons to induce aberrant synaptic pruning in adult mice with disease (EAE). I will test this hypothesis with time- and cell-specific conditional knock out of B2m to reduce MHC I cell surface expression in astrocytes during EAE. Additional studies utilizing gonadectomy will determine the effects of sex hormones on MHC I gene expression. Together, these projects seek to fundamentally understand complex mechanisms in MS susceptibility and progression, and how sex affects these aspects of disease. Further understanding of these mechanisms may lead to identifying natural disease modifiers and new translatable targets for neuroinflammatory therapeutics.