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Browsing by Author "Chawla, Aarti"
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Item Disruption of nNOS-NOS1AP protein-protein interactions suppresses neuropathic pain in mice(Wolters Kluwer, 2018-05) Lee, Wan-Hung; Li, Li-Li; Chawla, Aarti; Hudmon, Andy; Lai, Yvonne Y.; Courtney, Michael J.; Hohmann, Andrea G.; Biochemistry and Molecular Biology, School of MedicineElevated N-methyl-D-aspartate receptor (NMDAR) activity is linked to central sensitization and chronic pain. However, NMDAR antagonists display limited therapeutic potential because of their adverse side effects. Novel approaches targeting the NR2B-PSD95-nNOS complex to disrupt signaling pathways downstream of NMDARs show efficacy in preclinical pain models. Here, we evaluated the involvement of interactions between neuronal nitric oxide synthase (nNOS) and the nitric oxide synthase 1 adaptor protein (NOS1AP) in pronociceptive signaling and neuropathic pain. TAT-GESV, a peptide inhibitor of the nNOS-NOS1AP complex, disrupted the in vitro binding between nNOS and its downstream protein partner NOS1AP but not its upstream protein partner postsynaptic density 95 kDa (PSD95). Putative inactive peptides (TAT-cp4GESV and TAT-GESVΔ1) failed to do so. Only the active peptide protected primary cortical neurons from glutamate/glycine-induced excitotoxicity. TAT-GESV, administered intrathecally (i.t.), suppressed mechanical and cold allodynia induced by either the chemotherapeutic agent paclitaxel or a traumatic nerve injury induced by partial sciatic nerve ligation. TAT-GESV also blocked the paclitaxel-induced phosphorylation at Ser15 of p53, a substrate of p38 MAPK. Finally, TAT-GESV (i.t.) did not induce NMDAR-mediated motor ataxia in the rotarod test and did not alter basal nociceptive thresholds in the radiant heat tail-flick test. These observations support the hypothesis that antiallodynic efficacy of an nNOS-NOS1AP disruptor may result, at least in part, from blockade of p38 MAPK-mediated downstream effects. Our studies demonstrate, for the first time, that disrupting nNOS-NOS1AP protein-protein interactions attenuates mechanistically distinct forms of neuropathic pain without unwanted motor ataxic effects of NMDAR antagonists.Item Isoform-Specific Inactivation and Aggregation of CaMKII under Ischemic-Like Conditions(Office of the Vice Chancellor for Research, 2016-04-08) Nelson, Ross; Hudmon, Andy; Johnson, Derrick; Ramaswamy, Swarna; Chawla, AartiCalcium-Calmodulin Dependent Protein Kinase II (CaMKII), an enzyme critical for learning and memory, inactivates and self-associates into sedimentable aggregates following ischemic insults such as stroke or traumatic brain injury; the extent of inactivation correlates increased neuronal dysfunction and death. CaMKII α and β—isoforms found primarily in neurons—are well documented in their response to ischemic stress; α aggregates and undergoes catalytic inactivation quickly while β does not. However, γ and δ—primarily found in glial cells—are not well studied under these conditions. Previous research by our lab suggests that loss of CaMKII signaling in astrocytes may contribute to reduced glutamate uptake and neurotoxic ATP release. Therefore, there is a need to elucidate the role of the astrocytic CaMKII isoforms in ischemic stress. This study aims to investigate CaMKII δ and γ’s response to artificial ischemic conditions compared to CaMKII α. Activity assay of cell lysates expressing the four different human genes of CaMKII (α, β, γ, and δ) reveal that, under artificial ischemic conditions, δ undergoes very minimal loss of activity over time while γ experiences robust inactivation. We then used light scattering to compare α, δ, and γ sedimentation in real time and found that δ had an aggregation profile similar to α yet γ’s was radically different. A follow-up time-course sedimentation assay suggests that δ becomes sedimentable and undergoes an upwards molecular weight shift akin to α over time, indicative of autophosphorylation, but that γ begins partially sedimentable before becoming completely soluble upon activation, contrary to our hypothesis. This suggests that each isoform responds differentially to activation under ischemic-like conditions and that aggregation is not necessarily correlative with inactivation. We are currently characterizing endogenous astrocytic CaMKII expression and activity to later determine if these findings persist in a cellular environment under ischemic-like conditions. Mentor: Andy Hudmon, Stark Neurosciences Research Institute, IU School of Medicine, IUPUI, Indianapolis, IN