Synucleinopathies are a group of neurodegenerative disorders sharing the common feature of misfolding and accumulation of the presynaptic protein α-synuclein into insoluble aggregates. Within this diverse group, Dementia with Lewy Bodies (DLB) is characterized by the aberrant accumulation of α-synuclein in cortical, hippocampal, and brainstem neurons, resulting in multiple cellular stressors that particularly impair dopamine and glutamate neurotransmission and related motor and cognitive function. The wide array of cellular stress and symptoms related to over-abundance of α-synuclein makes DLB and other synucleinopathies challenging to treat. Many currently approved therapies fall short, suggesting that effective approaches will require the targeting of multiple mechanisms in order to treat motor, cognitive and other non-motor aspects of these diseases.

The goal of this dissertation is to determine the contributions of neural stem cell (NSC) transplantation to targeting multiple mechanisms related to α-synuclein over-expression in transgenic models of DLB. NSCs are an ideal candidate for transplantation due to their ability to migrate throughout a region of injury, and produce high levels of neurotrophins or growth factors critical to the survival and plasticity of neurons within the brain.

I have determined that mouse allogenic transplantation of NSCs lead to dramatic and sustained behavioral improvement in transgenic models of DLB. Critically, this improvement is dependent on NSC production of the neurotrophic factor, BDNF, which regulates downstream dopamine and glutamate neurotransmitter systems. Weighted Gene Co-Expression Network Analysis verified the involvement of multiple key networks at the transcriptome level, including neurotrophins, dopamine, glutamate and immune-modulating networks. In order to further investigate the therapeutic potential of this approach, I examined the contributions of human NSC (hNSC) xenotransplantation to improving these multiple mechanisms. I demonstrated that clinical-grade hNSCs also rescue motor and cognitive behavioral deficits in an immune-deficient DLB model, and that this improvement is sustained over time. Moreover, transplantation of hNSCs restored dopamine and glutamate systems through a non-BDNF dependent mechanism. Finally, in contrast to mouse NSCs, hNSCs likely achieve these behavioral and neurochemical benefits by altering α-synuclein conformers.

Microglia are the primary cell type comprising the brain’s immune system and, as such, inflammatory responses in these cells have been implicated in essentially all diseases of the central nervous system. Historically, directly studying human microglia has been experimentally challenging, so much of what we understand has been inferred from the study of animal models despite species-specific genetic variation that underlies the inability of rodent microglia to fully recapitulate the human condition. However, recent advancements in the use of human induced pluripotent stem cells (iPSCs) to generate human microglia surrogates has ushered in a new era of microglia research. While the initial in vitro studies utilizing these iPSC-derived microglia (iMG) were revolutionary, subsequent work has demonstrated that these cells are highly sensitive to their environment and exhibit robust transcriptomic deficiencies when kept in isolation from the brain.Therefore, the development of a model that would allow for the examination of these cells within a surrogate brain environment was necessary for the continued development of our understanding of the roles of microglia in health and disease. By transplanting iPSC-derived hematopoietic progenitor cells into the postnatal brain of humanized, immune-deficient mice, transplanted cells were shown to undergo context-dependent differentiation into microglia and other CNS macrophages. Transcriptomic analyses showed that the resulting cells acquired an ex vivo human microglial gene signature and appropriately responded to acute and chronic inflammatory insults. Most notably, transplanted microglia exhibited robust transcriptional responses to Aβ plaques that only partially overlapped with that of murine microglia, revealing multiple human-specific Aβ-response genes. Further expansion of this model in conjunction with CRISPR gene editing technology has allowed for the direct interrogation of the Alzheimer’s disease (AD)-related triggering receptor expressed in myeloid cells 2 (TREM2) R47H genetic polymorphism. This study demonstrated that a key effect of the R47H mutation is an alteration in the distribution of microglia subpopulations and a reduction in plaque associated microglia. Furthermore, cells with the R47H mutation exhibit an inflammatory signature that is not present in WT cells, suggesting that their lack of responsiveness is also paired with inappropriate inflammation. Importantly, these results have highlighted aspects of the R47H mutation that have not previously been observed in mouse models. Finally, as disruption of activation states in response to amyloid pathology appeared to be a primary result of genetic mutation in xMG, further understanding the complex genetic interactions driving these states was a question of critical importance. Analysis of the epigenomic and transcriptomic landscapes of the individual xMG subpopulations that arise in response to amyloid pathology has provided insight into the chromatin structure and gene expression alterations that underlie the microglia response to Aβ plaques. What these data have begun to demonstrate is that the amyloid-responsive population of microglia appear to rely on chromatin modifications that allow transcription factors in the MiTF/TFE family to upregulate the expression of genes related to phagocytosis and lysosomal function. While there are still aspects of these results that have not been fully elucidated, future studies are underway that will determine the nature of these changes and their ability to drive microglial responses in AD. Taken as a whole, the development of the xMG model and application to the study of the early amyloid response has yielded important new insight into Alzheimer’s disease. While these experiments are only the first steps in what will hopefully become a much broader body of human microglia research, my dissertation work has demonstrated that the xMG model is a powerful tool for the future study of microglial homeostasis and disease-associated inflammatory responses.