Functional genomic dissection of human brain development, degeneration, and dysfunction in pediatric neurogenetic disorders

Abstract

Human disease genetics has implicated hundreds of genes in brain development, maintenance, and function. Experimentally tractable animal models and culture systems have contributed greatly to understanding the function of these genes, but have limitations, beginning with their inability to capture human-specific biology.

The Introduction chapter provides background on recent experimental approaches for studying and modeling human neurogenetic diseases. The combination of modern technological developments in functional genomics applied to human brain tissue and stem-cell based models has offered novel ways to molecularly dissect human neurological disease.

Chapter 1 focuses on applying this modern human-based genomic approach to probe the molecular mechanisms underlying a human neurogenetic disorder in which the genetic cause has been known for over 25 years. Ataxia-Telangiectasia (A-T) is an autosomal recessive multi-system disorder caused by mutations in the gene ATM that presents with progressive cerebellar neurodegeneration. Understanding disease pathogenesis has been hampered by the inability to generate animal models that recapitulate hallmark human features of disease. We address this gap by analyzing single-nucleus RNA-sequencing (snRNAseq) of postmortem brain tissue from individuals with A-T, generating mechanistic hypotheses that can then be tested using patient-derived induced pluripotent stem cells (iPSCs). We identified transcriptomic signatures of microglial inflammation in our snRNAseq analyses of postmortem A-T brains. To experimentally corroborate this finding, we studied microglia and neurons generated from A-T patient versus control iPSCs. Transcriptomic profiling of A-T iPSC-derived microglia confirmed cell-intrinsic microglial activation of cytokine production and innate immune response pathways compared to controls. Furthermore, adding A-T microglia to co-cultures with either control or A-T iPSC-derived neurons was sufficient to induce cytotoxicity. Taken together, these studies reveal that cell-intrinsic microglial activation may play a critical role in the development and progression of neurodegeneration in Ataxia Telangiectasia.

Chapter 2 describes the discovery of a novel genetic cause of a neurodevelopmental disorder (NDD) that presents with Autism Spectrum Disorder (ASD), attention-deficit hyperactivity disorder (ADHD), and behavioral dysregulation. We assembled a cohort of 38 individuals with de novo loss-of-function (LoF) variants in the ciliogenic RFX family of transcription factors (RFX3, RFX4, and RFX7) and demonstrate that pathogenic variants in these genes are associated with an overlapping neurobehavioral phenotype. We show that RFX3, RFX4, and RFX7 have enriched expression in the developing and adult human brain and their X-box binding motif is enriched in cis-regulatory regions of known ASD risk genes. These results implicate deleterious variation in RFX transcription factors in cases of monogenic NDD and highlight them as potential critical transcriptional regulators of neurobiological processes underlying NDD pathogenesis.

In Chapter 3 we dissect the molecular roles of RFX3, using human iPSC-derived neurons and forebrain organoids to understand how haploinsufficiency of RFX3 alters brain development and neuronal function. Transcriptomic analyses of RFX3 dosage demonstrated disruption of ciliary gene expression in RFX3-/- neurons (expected based on prior studies of RFX3 in mice and worms) while analyses of RFX3+/- neurons revealed a previously unanticipated role for RFX3 in synaptic gene expression. RFX3 deficiency led to decreased synchronization of neural network activity and impaired induction of CREB targets in response to neuronal depolarization. Our results highlight a novel role of the ciliogenic transcription factor RFX3 in shaping activity dependent responses and synaptic plasticity in human neurons that is disrupted by haploinsufficiency.

Chapter 4 highlights the potential for gene therapy development for neurogenetic conditions including RFX3 haploinsufficiency. Antisense oligonucleotides (ASO) are short, synthetic, sequences that can modulate gene expression levels in a titratable manner through several different mechanisms. ASOs have the potential to restore functional protein levels in neurogenetic disease by targeting mRNA processing inefficiencies. We found that the wild-type allele of RFX3 has a naturally occurring skipped exon that leads to nonsense-mediated decay (NMD). ASOs can promote inclusion of the exon, thereby rescuing the transcript from NMD and increasing RFX3 levels in haploinsufficient human neurons. Our results suggest that the neurodevelopmental disorder caused by RFX3 haploinsufficiency may be amenable to ASO-based therapy.

The Conclusion chapter synthesizes the insights from the studies presented and delineates potential future directions. Taken together, this thesis highlights how functional genomics applied to human brain tissue and iPSC-derived models of pediatric neurogenetic disorders can enable mechanistic and interventional insights.