Three Prime Repair Exonuclease 1 (TREX1) gene mutations have been associated with Aicardi-Goutières Syndrome (AGS) – a rare, severe pediatric autoimmune disorder that primarily affects the brain and has a poorly understood etiology. Microglia are brain-resident macrophages indispensable for brain development and implicated in multiple neuroinflammatory diseases. However, the role of TREX1 – a DNase that cleaves cytosolic nucleic acids, preventing viral- and autoimmune-related inflammatory responses – in microglia biology remains to be elucidated. Chapter 1 serves as an introduction and a review of the literature providing key background on the following: human cortical brain development, glial contributions to brain development, organoid models to study human brain development, and using brain organoids for modeling neurodevelopmental disorders. Chapter 2 is an original study investigating TREX1 functions in human microglia. We leverage a model of human embryonic stem cell (hESC)-derived engineered microglia-like cells, bulk and single-cell transcriptomics, optical and transmission electron microscopy, and three-month-old assembloids composed of microglia and regionalized neural organoids to interrogate TREX1 functions in human microglia. Our analyses suggest that TREX1 influences cholesterol metabolism, leading to an active microglial morphology with increased phagocytosis in the absence of TREX1. Notably, regulating cholesterol metabolism with an HMG-CoA reductase inhibitor, FDA-approved atorvastatin, rescues these microglial phenotypes. Chapter 3 details our original work elucidating the functions of TREX1-KO microglia on human brain development. We develop the first in vitro human brain assembloid model where microglia are present during neuro- and gliogenesis, including oligodendrocyte development. Using single-cell transcriptomics, we discover that TREX1-KO microglia selectively affect oligodendrocyte maturation and myelination. Together, these results suggest routes for therapeutic intervention in pathologies such as AGS based on microglia-specific molecular and cellular mechanisms.

Cyclin-dependent kinase-like 5 (CDKL5) syndrome is a neurodevelopmental disorder characterized by early-onset intractable seizures, mental retardation, and hand stereotypies. Studies have shown that loss of CDKL5 protein function is responsible for aberrations in neuronal morphogenesis, and decreased synaptogenesis. In order to understand the effects of CDKL5 mutations on human neural disease pathology, we utilized induced pluripotent stem cell (iPSC) technology to generate human iPSC-derived neurons from CDKL5 patients. Furthermore, to circumvent in vitro iPSC disease modeling limitations and to take advantage of an in vivo brain environment, we integrated these two systems by engrafting human iPSC-derived neural progenitor cells (NPCs) into immunodeficient mice. Here, we report defects in neuronal morphology in CDKL5- mutant neurons differentiated in vitro that are retained in human neurons differentiated in a mouse brain. Furthermore, we found that using a histone deacetylase inhibitor, LMK235, specific to histone deacetylase 4 (HDAC4), was able to rescue defects in synaptogenesis and neural electrical activity in CDKL5 iPSC-derived neurons in vitro. Ultimately, we hope our work will provide further insights into CDKL5 disease pathology and accelerate the development of treatments for this complex neurodevelopmental disorder.

Long interspersed nuclear element-1 (LINE-1) are endogenous, self-propagating genomic sequences that comprise ~17% of the human genome. Retrotransposons play a critical role in genomic and cellular instability, particularly in various forms of human disease. In the diseased state, there is mounting evidence that endogenous retroelements play a role in etiopathogenesis of inflammatory diseases. Mutations in methyl-CpG- binding protein 2 (MeCP2), widely regarded as a global methylator and transcriptional repressor, result in increased LINE-1 expression and activity. Patients with mutations in MeCP2 have subclinical inflammatory phenotypes and cytokine dysregulation. Although de novo LINE-1 activity seems to occur frequently in neurons, little is known about the contribution of this element in glial cells. Astrocytes with mutations in MECP2 are abnormal in several key functions, thus we sought to investigate astrocyte-mediated inflammation caused by MeCP2 loss-of-function mutations. We generated iPSC-derived astrocytes from MeCP2-KO and healthy controls to observe whether LINE-1, which we know to be upregulated, contributes to the inflammatory phenotype observed. We chronically treated our cells with reverse transcriptase inhibitors (RTi) to reduce endogenous LINE-1 activity. Interestingly, we found upregulation of neuroinflammatory- related genes, increased levels of the proinflammatory cytokine IL-6 as well as an increase in reactive oxygen species, extracellular glutamate and glutathione levels in MeCP2-mutant astrocyte cultures when compared to control cells. Remarkably, inhibition of LINE-1 with RTis improved most of these pathological phenotypes in mutated cells. We hope our work brings further attention to mobile genetic elements, as they contribute more to disease pathologies than previously thought.

Microglia, the tissue-resident macrophages of the central nervous system (CNS), are the first line of defense against injury and disease. Given their unique ontology and context-specific functions, they might play key roles during neurodevelopment. Rett syndrome (RTT) is a progressive neurodevelopmental disorder characterized by a mutation in the methyl-CpG-binding protein 2 (MECP2) gene, whereupon affected patients can develop a range of behavioral and neurological symptoms. Although MECP2 is expressed in many different cell types, RTT is attributed to cell autonomous effects from a primary neuronal dysfunction. The role of microglia in RTT pathology has been studied with an ongoing controversy in mouse models. Here, we aim to decipher if human microglia have an impact in RTT pathogenesis by using induced-pluripotent stem cells (iPSCs). We utilized high throughput, multiplex experiments in a holistic approach to investigate the inflammatory signature of RTT microglia in gene expression and cytokine release. Consequentially, we observed genes differentially expressed in RTT microglia with no clear inflammatory phenotype. Additionally, we observed a significant decrease in RTT microglia’s phagocytic ability, congruent with our gene expression analysis. Finally, we increased the complexity of our model by establishing a neuron-microglia co-culture system to study the impact of microglia on neuronal synaptogenesis in both healthy and RTT conditions. Altogether, our data suggests that the lack of MECP2 in microglial cells alters its functions, which might be implicated in RTT pathogenesis and that they can potentially be targeted to develop novel therapies for RTT patients for whom there is currently no cure.