Self-Association of CaMKII-Delta in Low ATP/Low pH Conditions

poster abstractCalcium-Calmodulin Dependent Protein Kinase II (CaMKII), an enzyme critical for brain function and involved in learning and memory, becomes inactive and aggregates following ischemic insult such as seen with stroke or traumatic brain injury. The Hudmon Lab’s working model is that loss of CaMKII signaling exacerbates glutamate excitotoxicity, in turn inducing astrocytes to release neurotoxic levels of ATP, causing secondary cell death. CaMKII can autophosphorylate resulting in autonomous activity, and research by Hudmon Lab reveals that CaMKII will inactivate and self-associate when activating under low pH and low ATP conditions. CaMKII is coded by four genes, and for this study we focus on the alpha and delta isoforms. Alpha is found primarily in neurons and readily aggregates under conditions mimicking ischemic stress. However, delta is primarily expressed in astrocytes and its response to ischemic stress is uncharted territory. We ask how CaMKII delta self-associates under autophosphorylation conditions mimicking ischemic stress (low pH, low ATP) and how it differs from CaMKII alpha self-association. We use real-time light scattering and sedimentation assay to elucidate the time-course of delta aggregation as well as the kinase’s sensitivity to differing pH and ATP concentrations. Light scattering suggests that alpha and delta have a similar aggregation profile, but also that delta has reduced light scattering over time. Sedimentation analysis suggests delta truly does aggregate under these conditions and that it undergoes a molecular weight shift, indicative of autophosphorylation-induced inactivation. In future studies, we plan to investigate delta’s kinase activity under aggregation conditions, perform the same experiments detailed here on the gamma isoform, and investigate delta and gamma aggregation directly in astrocytes. If we can understand how all the isoforms of CaMKII aggregate, it may prove to be a novel research topic for therapies aimed at neuroprotection in victims of ischemic insults

Doc2b serves as a scaffolding platform for concurrent binding of multiple Munc18 isoforms in pancreatic islet β-cells

Biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells involves soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) protein-regulated exocytosis. SNARE complex assembly further requires the regulatory proteins Munc18c, Munc18-1 and Doc2b. Munc18-1 and Munc18c are required for first- and second-phase GSIS respectively. These distinct Munc18-1 and Munc18c roles are related to their transient high-affinity binding with their cognate target (t-)SNAREs, Syntaxin 1A and Syntaxin 4 respectively. Doc2b is essential for both phases of GSIS, yet the molecular basis for this remains unresolved. Because Doc2b binds to Munc18-1 and Munc18c via its distinct C2A and C2B domains respectively, we hypothesized that Doc2b may provide a plasma membrane-localized scaffold/platform for transient docking of these Munc18 isoforms during GSIS. Towards this, macromolecular complexes composed of Munc18c, Doc2b and Munc18-1 were detected in β-cells. In vitro interaction assays indicated that Doc2b is required to bridge the interaction between Munc18c and Munc18-1 in the macromolecular complex; Munc18c and Munc18-1 failed to associate in the absence of Doc2b. Competition-based GST-Doc2b interaction assays revealed that Doc2b could simultaneously bind both Munc18-1 and Munc18c. Hence these data support a working model wherein Doc2b functions as a docking platform/scaffold for transient interactions with the multiple Munc18 isoforms operative in insulin release, promoting SNARE assembly

Mitochondrial Ca2+ Uniporter and CaMKII in heart

The influx of cytosolic Ca2+ into mitochondria is mediated primarily by the mitochondrial calcium uniporter (MCU), a small-conductance, Ca2+-selective channel-. MCU modulates intracellular Ca2+ transients and regulates ATP production and cell death. Recently, Joiner et al. reported that MCU is regulated by mitochondrial CaMKII, and this regulation determines stress response in heart. They reported a very large current putatively mediated by MCU that was about two orders of magnitude greater than the MCU current (IMCU) that we previously measured in heart mitochondria. Also, the current traces presented by Joiner et al. showed unusually high fluctuations incompatible with the low single-channel conductance of MCU. Here we performed patch-clamp recordings from mouse heart mitochondria under the exact conditions used by Joiner et al. We confirmed that IMCU in cardiomyocytes is very small and showed that it is not directly regulated by CaMKII. Thus the currents presented by Joiner et al. do not correspond to MCU, and there is no direct electrophysiological evidence that CaMKII regulates MCU

Isoform-Specific Inactivation and Aggregation of CaMKII under Ischemic-Like Conditions

poster abstractCalcium-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, I

Cardiac sodium channel palmitoylation regulates channel availability and myocyte excitability with implications for arrhythmia generation

Cardiac voltage-gated sodium channels (Nav1.5) play an essential role in regulating cardiac electric activity by initiating and propagating action potentials in the heart. Altered Nav1.5 function is associated with multiple cardiac diseases including long-QT3 and Brugada syndrome. Here, we show that Nav1.5 is subject to palmitoylation, a reversible post-translational lipid modification. Palmitoylation increases channel availability and late sodium current activity, leading to enhanced cardiac excitability and prolonged action potential duration. In contrast, blocking palmitoylation increases closed-state channel inactivation and reduces myocyte excitability. We identify four cysteines as possible Nav1.5 palmitoylation substrates. A mutation of one of these is associated with cardiac arrhythmia (C981F), induces a significant enhancement of channel closed-state inactivation and ablates sensitivity to depalmitoylation. Our data indicate that alterations in palmitoylation can substantially control Nav1.5 function and cardiac excitability and this form of post-translational modification is likely an important contributor to acquired and congenital arrhythmias

Disruption of nNOS-NOS1AP protein-protein interactions suppresses neuropathic pain in mice

Elevated 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

Long-term exposure to PGE2 causes homologous desensitization of receptor-mediated activation of protein kinase A

BACKGROUND: Acute exposure to prostaglandin E2 (PGE2) activates EP receptors in sensory neurons which triggers the cAMP-dependent protein kinase A (PKA) signaling cascade resulting in enhanced excitability of the neurons. With long-term exposure to PGE2, however, the activation of PKA does not appear to mediate persistent PGE2-induced sensitization. Consequently, we examined whether homologous desensitization of PGE2-mediated PKA activation occurs after long-term exposure of isolated sensory neurons to the eicosanoid. METHODS: Sensory neuronal cultures were harvested from the dorsal root ganglia of adult male Sprague-Dawley rats. The cultures were pretreated with vehicle or PGE2 and used to examine signaling mechanisms mediating acute versus persistent sensitization by exposure to the eicosanoid using enhanced capsaicin-evoked release of immunoreactive calcitonin gene-related peptide (iCGRP) as an endpoint. Neuronal cultures chronically exposed to vehicle or PGE2 also were used to study the ability of the eicosanoid and other agonists to activate PKA and whether long-term exposure to the prostanoid alters expression of EP receptor subtypes. RESULTS: Acute exposure to 1 μM PGE2 augments the capsaicin-evoked release of iCGRP, and this effect is blocked by the PKA inhibitor H-89. After 5 days of exposure to 1 μM PGE2, administration of the eicosanoid still augments evoked release of iCGRP, but the effect is not attenuated by inhibition of PKA or by inhibition of PI3 kinases. The sensitizing actions of PGE2 after acute and long-term exposure were attenuated by EP2, EP3, and EP4 receptor antagonists, but not by an EP1 antagonist. Exposing neuronal cultures to 1 μM PGE2 for 12 h to 5 days blocks the ability of PGE2 to activate PKA. The offset of the desensitization occurs within 24 h of removal of PGE2 from the cultures. Long-term exposure to PGE2 also results in desensitization of the ability of a selective EP4 receptor agonist, L902688 to activate PKA, but does not alter the ability of cholera toxin, forskolin, or a stable analog of prostacyclin to activate PKA. CONCLUSIONS: Long-term exposure to PGE2 results in homologous desensitization of EP4 receptor activation of PKA, but not to neuronal sensitization suggesting that activation of PKA does not mediate PGE2-induced sensitization after chronic exposure to the eicosanoid