Altered CRABP1 Expression in Fragile-X syndrome Restricted; Files Only

Abstract

Fragile X syndrome (FXS) is the most common heritable form of intellectual disability (ID) and monogenic cause of autism. FXS occurs due to silencing of the FMR1 gene, which encodes the fragile X mental retardation protein (FMRP), an RNA-binding protein that is important for cell signaling and neurogenesis. While the focus has been to define direct mRNA targets of FMR1, non-canonical disease mechanisms remain unexplored. The Bassell lab has identified a cytoplasmic retinoic acid (RA) binding protein, CRABP1, that is significantly downregulated in human FXS patient postmortem brains and FXS patient induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs) and organoids. RA plays crucial roles in cell cycle regulation and neuronal differentiation, and disrupted RA signaling has been implicated in several neurological disorders, including FXS. Given the role of RA signaling in neurogenesis, my thesis aimed to test this dysfunction of RA signaling and explore the functional consequences of CRABP1 deficiency as a direct result of reduced FMRP. RT-qPCR and western blotting reveal that CRABP1 expression trends down in FMR1-deficient human neuroblastoma cells, although this reduction is more robust at the mRNA level than the protein level. CRABP1 has been shown to mediate early-phase ERK 1/2 phosphorylation in response to RA stimulation, which leads to increased p27^KIP levels and a prolonged G0/G1 phase of the cell cycle. To examine the potential loss of this function of CRABP1 and RA signaling in the context of FXS, either FMR1 or CRABP1 was depleted in human neuroblastoma cells that were subsequently stimulated with RA. In contrast to prior findings, FMR1 and CRABP1-deficient cells did not show a loss of rapid ERK 1/2 phosphorylation. However, p27 ^KIP was robustly reduced in FMR1 and CRABP1-deficient cells, which may contribute to the neurogenic defects observed in FXS patient cells and point towards a crucial role of CRABP1 in regulating early neurogenesis. Future studies will address the underlying mechanism for how FMRP regulates CRABP1 expression in FXS-patient derived cells, and how p27^KIP levels are regulated in FMRP and CRABP1-deficient cells to understand potential impacts of CRABP1 and p27 ^KIP on early neurogenesis.

Table of Contents

   Introduction ………………………………..……………………………………………...1

    Methods ………………………………………………………………………………….13

    Results……………………………………………………………………………………15

    Discussion………………………………………………………………………...……...18

    Future Directions…………………………………………………………………....…...23

    Figures………………………………………………………………………...……........26

    References……………………………………………………………………..…...........33

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Neuroscience
Keyword	Neurogenesis, Brain Development, Fragile-X Syndrome, FMRP
Committee Chair / Thesis Advisor	Bassell, Gary, Emory University
Committee Members	Frenzel, Kristen, Emory University Corbett, Anita, Emory University

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