자료유형  

 학위논문

Control Number  

0017161356

International Standard Book Number  

9798382777962

Dewey Decimal Classification Number  

575

Main Entry-Personal Name  

Benoit, Courtney.

Publication, Distribution, etc. (Imprint  

[S.l.] : Harvard University., 2024

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2024

Physical Description  

154 p.

General Note  

Source: Dissertations Abstracts International, Volume: 85-12, Section: B.

General Note  

Advisor: Young-Pearse, Tracy.

Dissertation Note  

Thesis (Ph.D.)--Harvard University, 2024.

Summary, Etc.  

요약Proper human neurodevelopment requires an intricate series of gene regulatory interactions, mediated by the activity of neural transcription factors. Disruption of neurodevelopment has been linked to a wide variety of human disorders, including developmental delay, intellectual disability, autism spectrum disorder, and neuropsychiatric disorders. POU3F2 is a neural-specific POU class III transcription factor that has been implicated in neurodevelopmental and neurodegenerative disorders. Multiple studies have described heterozygous loss-of-function and missense mutations in POU3F2 in individuals with intellectual disability and/or autism spectrum disorder. Further, POU3F2 has been identified as a genome-wide or transcriptome-wide risk locus for bipolar disorder, brain activation in schizophrenia, and autism spectrum disorder. While these studies clearly underscore the importance of proper POU3F2 activity in humans, little is known about the mechanisms underlying its connection to human neurological disorders.To elucidate the mechanistic role of POU3F2 during neurodevelopment, we used CRISPR/Cas9 mutagenesis to induce loss-of-function mutations in POU3F2 in human induced pluripotent stem cell (iPSC) lines, which were then differentiated into neural progenitor cells (NPCs). Using unbiased approaches, we found that POU3F2MUT NPCs show dysregulation of the canonical Wnt signaling pathway. Functional assays demonstrated that POU3F2MUT NPCs exhibit decreased baseline canonical Wnt signaling and decreased proliferation when compared to wildtype cells. Single-cell RNA-sequencing showed that POU3F2MUT NPCs exhibit a shift in NPC subtype favoring increased levels of radial glia at the expense of G2/M and neural stem cell-like progenitors. We found that POU3F2 positively associated with baseline canonical Wnt signaling and negatively associated with markers of radial glia in a large cohort of NPCs derived from genetically diverse individuals exhibiting natural variation in POU3F2 levels. Through a series of unbiased analyses, we show that SOX13 is a transcriptional target of POU3F2 that mediates POU3F2's effects on Wnt signaling in human NPCs. Finally, we provide evidence for prioritization of POU3F2 as a high-confidence autism risk gene through (1) the identification of a protein-protein interaction with ADNP, mutations in which result in syndromic autism, (2) enrichment analyses showing that genes dysregulated in autism are enriched in datasets relating to POU3F2 function, and (3) the identification of an additional five individuals with autism spectrum disorder that exhibit loss-of-function mutations in POU3F2. Together, these studies define POU3F2 as an activator of canonical Wnt signaling which regulates the specification and proliferation of human NPCs and demonstrate its relevance for the study of autism.We further investigated the role of POU3F2 in the maturation of post-mitotic neurons. POU3F2WT and POU3F2MUT iPSCs were differentiated into layer II-III cortical excitatory neurons via a well-established Ngn2 induced neuron (iN) protocol. We assessed the transcriptomic and proteomic profiles of POU3F2MUT iNs and found that POU3F2MUT iNs display an upregulation of pathways related to synaptic maturation and a concurrent increase in synapse density, as measured via immunostaining and colocalization of presynaptic and postsynaptic marker puncta. Despite this, POU3F2MUT iNs were found to exhibit reduced neurite outgrowth during early maturation and reduced neuronal activity, as measured by multi-electrode array. Finally, we identified the gene regulatory network of POU3F2 using CUT&RUN technology to further define the role of POU3F2 in neuronal homeostasis and to prioritize downstream targets of POU3F2.While human neural cell models provide an opportunity to define the molecular pathways involved in neurodevelopmental disorders, we also assessed the functional consequences of Pou3f2 loss on an organismal level using mouse models. To do so, we used a published Pou3f2 floxed mouse which we mated to one of three Cre lines: (1) Emx1-Cre to cause forebrain-specific loss of Pou3f2, and (2) CAG-CreER to induce loss of Pou3f2 in adult mice via tamoxifen injection, and (3) CMV-Cre to cause germline loss of Pou3f2. While additional characterization of these mouse models is ongoing, we found that forebrain-specific loss of Pou3f2 results in upregulation of genes involved in axonogenesis and neuronal maturation, providing further relevance for the results we observed in our human induced neuron model.Together, these studies clearly implicate POU3F2 as a key regulator of neurodevelopment and neuronal maturation. For the former, we show evidence that POU3F2 regulates canonical Wnt signaling in human neural progenitor cells to establish the progenitor pool prior to radial glia specification. For the latter, we demonstrate multiple findings, across human and mouse models, that show loss of POU3F2/Pou3f2 results in dysregulation of pathways relating to neuronal homeostasis and maturation. Based on these results, we provide evidence for the prioritization of POU3F2 as a high-confidence risk gene underlying the etiology of neurodevelopmental and neurodegenerative disorders. 

Subject Added Entry-Topical Term  

Genetics.

Subject Added Entry-Topical Term  

Neurosciences.

Subject Added Entry-Topical Term  

Mental health.

Index Term-Uncontrolled  

Pluripotent stem cell

Index Term-Uncontrolled  

Human neurodevelopment

Index Term-Uncontrolled  

Brain activation

Index Term-Uncontrolled  

Human neurological disorders

Index Term-Uncontrolled  

Autism spectrum disorder

Added Entry-Corporate Name  

Harvard University Medical Sciences

Host Item Entry  

Dissertations Abstracts International. 85-12B.

Electronic Location and Access  

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Control Number  

joongbu:658402