Browsing by Author "Lai, Yvonne"

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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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    Disrupting nNOS–PSD95 Interaction Improves Neurological and Cognitive Recoveries after Traumatic Brain Injury
    (Oxford University Press, 2020-06) Qu, Wenrui; Liu, Nai-Kui; Wu, Xiangbing; Wang, Ying; Xia, Yongzhi; Sun, Yan; Lai, Yvonne; Li, Rui; Shekhar, Anantha; Xu, Xiao-Ming; Psychiatry, School of Medicine
    Excessive activation of N-methyl-D-aspartate receptors (NMDARs) and the resulting neuronal nitric oxide synthase (nNOS) activation plays a crucial role in the pathogenesis of traumatic brain injury (TBI). However, directly inhibiting NMDARs or nNOS produces adverse side effects because they play key physiological roles in the normal brain. Since interaction of nNOS–PSD95 is a key step in NMDAR-mediated excitotoxicity, we investigated whether disrupting nNOS–PSD95 interaction with ZL006, an inhibitor of nNOS–PSD95 interaction, attenuates NMDAR-mediated excitotoxicity. In cortical neuronal cultures, ZL006 treatment significantly reduced glutamate-induced neuronal death. In a mouse model of controlled cortical impact (CCI), administration of ZL006 (10 mg/kg, i.p.) at 30 min postinjury significantly inhibited nNOS–PSD95 interaction, reduced TUNEL- and phospho-p38-positive neurons in the motor cortex. ZL006 treatment also significantly reduced CCI-induced cortical expression of apoptotic markers active caspase-3, PARP-1, ratio of Bcl-2/Bax, and phosphorylated p38 MAPK (p-p38). Functionally, ZL006 treatment significantly improved neuroscores and sensorimotor performance, reduced somatosensory and motor deficits, reversed CCI-induced memory deficits, and attenuated cognitive impairment. Histologically, ZL006 treatment significantly reduced the brain lesion volume. These findings collectively suggest that blocking nNOS–PSD95 interaction represents an attractive strategy for ameliorating consequences of TBI and that its action is mediated via inhibiting neuronal apoptosis and p38 MAPK signaling.
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    Spinophilin-Dependent Regulation of the Phosphorylation, Protein Interactions, and Function of the GluN2B Subunit of the NMDAR and its Implications in Neuronal Cell Death
    (2020-12) Beiraghi Salek, Asma; Baucum II, A.J.; Cummins, Ted; Hudmon, Andy; Lai, Yvonne; Berbari, Nicolas; Meyer, Jason
    Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.