SETD5, a gene recently linked to intellectual disability (ID) and autism spectrum disorder (ASD) encodes a putative histone methyltransferase. To date, no causal evidence has linked SETD5 haploinsufficiency with ID/ASD, and a potential underlying mechanism of action remains unknown. Here, we characterized the impact of Setd5 on neurodevelopment using molecular, cellular, and behavioral assays in the Setd5+/- mouse. Setd5+/- animals manifest several autism-like behaviors, including hyperactivity, increased anxiety, cognitive deficits, and altered social interactions. Anatomical differences in cortex were observed in Setd5+/- adult brains, accompanied by a deficit of deep-layer cortical neurons in the early postnatal brain. Setd5+/- cortical neurons displayed significantly reduced synaptic density and neuritic outgrowth. Development of network connectivity was impaired in Setd5+/- neurons. At the molecular level, SETD5 methylated H3K4 in a SET domain-dependent manner. Together, our data converge on a picture of abnormal neurodevelopment driven by Setd5 haploinsufficiency, consistent with a highly penetrant risk factor. Thus, the Setd5+/- animal represents a novel model for ASD and yields insight into the epigenetic regulation of neurodevelopment.

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.

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.

Defects in telomerase and telomere length maintenance have been implicated in difficult to treat genetic disorders, premature senescence underlying aging disorders, and chromosomal instability contributing to increased risk of cancer development. It has relatively recently been noted that patients with congenitally short telomeres and telomerase dysfunction diseases present with microcephaly and associated neurological symptoms. However, few studies have explored the role of telomere dysfunction on neurodevelopment. This proposal aims to study the role of shortened telomeres and telomerase dysfunction using human induced pluripotent stem cells (hiPSCs) to model neurodevelopmental defects. In previous studies, hiPSCs with measurably shortened telomere length, as well as hiPSCs that have been CRISPR engineered with defects in the telomerase reverse transcriptase (TERT) component of the telomerase complex that is important for end replication function, have elucidated that bone marrow failure associated with the disease can be attributed to cellular dysfunction that underlies failure of hematopoietic stem cell development. Using hiPSCs that have similar defects in telomere length and in TERT (the enzymatic component of telomerase), this proposal will utilize a directed differentiation approach to study early neurodevelopment and neuronal maturation to measure critical points of cellular development that potentially give rise to the reduced neuronal volume that characterizes microcephaly. Furthermore, this proposal will utilize a previously published three-dimensional cellular protocol for studying the microcephaly phenotype using cortical organoid culture, which will allow for visualization and measurement of neuronal development related to spatial organization. Finally, the cellular basis of associated neurodevelopmental aberrations will be measured via bioinformatics analysis and functional activity assays, to elucidate the underlying contribution of prematurely shortened telomeres to neuronal activity and maturation.