Browsing by Author "Haulcomb, Melissa"

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    4335 Role of PSD95 and nNOS interaction in gene regulation following fear conditioning and implications for molecular mechanisms underlying PTSD
    (Cambridge University Press, 2020-07-29) Patel, Jheel; Dustrude, Erik; Haulcomb, Melissa; Li, Liangping; Jiang, Guanglong; Liu, Yunlong; Lai, Yvonne; Molosh, Andrei; Shekhar, Anantha; Medicine, School of Medicine
    OBJECTIVES/GOALS: Normal fear learning produces avoidance behavior that promotes survival, but excessive and persistent fear after trauma can lead to development of phobias and post-traumatic stress disorder (PTSD). Our goal is to understand the mechanism and identify novel genetic targets underlying fear responses. METHODS/STUDY POPULATION: Involvement of the amygdala in fear acquisition is well established and requires activation of N-methyl-D-aspartic acid receptors (NMDARs). At a cellular level, NMDAR activation leads to production of nitric oxide (NO) by a process mediated by interaction between postsynaptic density protein 95 (PSD95) and neuronal nitric oxide synthase (nNOS). To elucidate mechanisms underlying the role of the PSD95-nNOS-NO pathway in conditioned fear, here we use rodent behavioral paradigms, pharmacological treatment with a small molecular PSD95-nNOS inhibitor, co-immunoprecipitation, Western blotting, and RNA-sequencing. RESULTS/ANTICIPATED RESULTS: We show that fear conditioning enhances the PSD95-nNOS interaction and that the small-molecule ZL006 inhibits this interaction. Treatment with ZL006 also attenuates rodent cued-fear consolidation and prevents fear-mediated shifts in glutamatergic receptor and current densities in the basolateral amygdala (BLA). With RNA-sequencing, expression of 516 genes was altered in the BLA following fear expression; of these genes, 83 were restored by systemic ZL006 treatment. Network data and gene ontology enrichment analysis with Ingenuity Pathway Analysis and DAVID software found that cell-cell interaction, cognition-related pathways, and insulin-like growth factor binding were significantly altered. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results reveal novel genetic targets that underlie plasticity of fear-memory circuitry via their contribution of NMDAR-mediated fear consolidation and can inform future strategies for targeting fear related disorders like PTSD. CONFLICT OF INTEREST DESCRIPTION: Anantha Shekhar and Yvonne Lai are co-founders of Anagin, Inc., which is developing some of the related molecules for the treatment of PTSD.
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    77822 PSD95-nNOS interaction alters the basolateral amygdala transcriptome following fear conditioning: implications for molecular mechanisms underlying PTSD
    (Cambridge University Press, 2021) Patel, Jheel; Haulcomb, Melissa; Li, Liangping; Jiang, Guanglong; Dustrude, Erik; Liu, Yunlong; Lai, Yvonne; Molosh, Andrei; Shekhar, Anantha; Medicine, School of Medicine
    ABSTRACT IMPACT: This research takes a transcriptomic approach to parse genes and molecular pathways that underlie the fear memory circuitry and, in doing so, identifies therapeutic targets that can further be developed into treatments for fear disorders, such as post-traumatic stress disorder. OBJECTIVES/GOALS: Normal fear learning produces avoidance behavior that promotes survival, but excessive and persistent fear after trauma can lead to development of phobias and post-traumatic stress disorder (PTSD). Our goal is to understand the mechanism and identify novel genetic targets underlying fear responses. METHODS/STUDY POPULATION: Involvement of the basolateral amygdala (BLA) in fear acquisition is well established and requires activation of N-methyl-D-aspartic acid receptors (NMDARs). At a cellular level, NMDAR activation leads to production of nitric oxide (NO) by a process mediated by interaction between postsynaptic density protein 95 (PSD95) and neuronal nitric oxide synthase (nNOS). To elucidate mechanisms underlying the role of the PSD95-nNOS-NO pathway in conditioned fear, here we use rodent behavioral paradigms, pharmacological treatment with a small molecule PSD95-nNOS inhibitor, RNA-sequencing, and an AAV-mediated knockdown of the nNOS gene in the BLA. RESULTS/ANTICIPATED RESULTS: We show that treatment with ZL006 attenuates rodent cued-fear consolidation. Additionally, with RNA-sequencing, expression of 516 genes was altered in the BLA following fear expression; of these genes, 83 were restored with systemic ZL006 treatment. Network data and gene ontology enrichment analyses further revealed that cGMP effects, insulin-like growth factor binding, and cognition-related pathways were significantly altered. Finally, we show that a BLA-specific knockdown of nNOS attenuates cued fear consolidation, without adverse effects on other memory and motor behaviors. DISCUSSION/SIGNIFICANCE OF FINDINGS: Via a model of NMDAR-mediated fear consolidation, our results reveal novel pathways and genetic targets that underlie plasticity of fear memory circuitry. Importantly, these results will inform future therapeutic strategies for targeting fear related disorders like PTSD.