The Structural and Molecular Changes associated with Altered Homeostatic Intrinsic Plasticity in the Axon Initial Segment and Dendrites of Fmr1 KO Neurons Public

Abstract

Fragile-X Syndrome (FXS), often associated with autism and autism associated disorders, is characterized by intellectual disability, sensory hypersensitivity and sleep disorders. Several of the symptoms in FXS have been linked to increased neuronal excitability, a phenotype that has also been demonstrated in the FXS mouse model, the Fmr1knockout (KO). Though the cause for this cortical hyperexcitability remains a mystery, recent work from our lab is supporting the idea that one type of neuronal plasticity called Homeostatic Intrinsic Plasticity (HIP) is involved (Bülow et al., 2019, Cell Reports). HIP regulates the excitability of a neuron in response to activity perturbations and is thereby one of the fundamental mechanisms in maintaining normal neural activity levels during brain development. Studies conducted in our lab on cortical neurons from Fmr1 KO mice have highlighted a dysregulation of HIP mechanisms and that these mechanisms were either exaggerated or absent in two subsets of excitatory neurons (Bülow et al., 2019, Cell Reports). To further expand on these results, we are now testing the hypothesis that altered HIP function is mediated by altered regulation of the Axon Initial Segment (AIS). The AIS is a specialized region at the start of the axon and is the site of action potential initiation due to high expression of voltage-gated sodium channels. In this region, both the length and the location of the AIS have previously been reported to be regulated by HIP, and a few recent studies have also demonstrated dysregulated AIS regulation in neurodegenerative and neurodevelopmental diseases. Additionally, loss of FMRP in FXS is also associated with dysregulation of protein synthesis. Preliminary experiments measuring nascent protein synthesis using puromycin labelling and  experiments analyzing translational regulation in the soma by measuring processing bodies(P bodies) in the soma revealed that Fmr1 KO neurons display altered protein translational regulation. Therefore we tested whether changes to P bodies in the dendrites and AIS in response to TTX/APV treatment would mirror the phenotypic changes seen in the WT and KO neurons from the preliminary soma experiments.

Using immunocytochemical approaches, we find that the AIS is located farther away from the soma in the Fmr1KO at baseline when compared to WT counterparts. This is indicative of unique compensatory mechanisms engaged by the Fmr1 KO neurons at baseline. We also found that Fmr1 KO neurons do not exhibit any significant responses to treatment with gabazine. Immunocytochemical experiments conducted on the dendrites and AIS analyzing the P bodies revealed that there was no significant changes in the Fmr1 KO or WT neurons at baseline or in response to treatment. This data did not align with our initial hypothesis and indicates that P bodies are potentially recruited to the soma following activity deprivation.

Table of Contents

Table of Contents

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

Aims……………………………………………………………………………………………10

Methods……………………………………………………………………………………….11

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

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

Future Directions and Conclusion……………………………………………………….23

References………………………………………………………………………………....….25

Figures…………………………………………………………………………………….…...31

   Fig1. Ion Channels involved in HIP that are regulated by FMRP………………….…..…..32

   Fig2. The Characteristics of the Axon Initial Segment..…………………………….…....…33

   Fig3.Findings from Bülow et al., 2019...……………………………………………......……...34

   Fig 4.Findings from Preliminary TTX/APV Experiments….………………………...……...35

   Fig 5. Findings from Preliminary Puromycin Experiments...……….…………….………..36

   Fig 6. Findings from Preliminary soma P-body Experiments…...………………….……...37

   Fig 7. Image Analysis Example………………………………………………………...........……38

   Fig 8. Example of Images used for Processing Body Analysis…………………...………….39

   Fig 9. Results from Gabazine Experiments(Aim 1)…………………………………….....…..40

   Fig 10. Results from AIS Processing Body Experiments(Aim 2)..………………..………...41  

  Fig 11. Results from Dendritic Processing Body Experiments(Aim 3)………………..…..42

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, Cell Biology, Neuroscience
Mot-clé	Homeostatic Plasticity Molecular Fragile-X Syndrome Structural
Committee Chair / Thesis Advisor	Dr. Gary Bassell, Emory University
Committee Members	Dr. Kristen E Frenzel, Emory University Dr. Anita Corbett, Emory University Dr. Victor Faundez, Emory University Dr. Robert Liu, Emory University

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