AKAP1 Protects from Cerebral Ischemic Stroke by Inhibiting Drp1-Dependent Mitochondrial Fission.

Mitochondrial fission and fusion impact numerous cellular functions and neurons are particularly sensitive to perturbations in mitochondrial dynamics. Here we describe that male mice lacking the mitochondrial A-kinase anchoring protein 1 (AKAP1) exhibit increased sensitivity in the transient middle cerebral artery occlusion model of focal ischemia. At the ultrastructural level, AKAP1-/- mice have smaller mitochondria and increased contacts between mitochondria and the endoplasmic reticulum in the brain. Mechanistically, deletion of AKAP1 dysregulates complex II of the electron transport chain, increases superoxide production, and impairs Ca2+ homeostasis in neurons subjected to excitotoxic glutamate. Ca2+ deregulation in neurons lacking AKAP1 can be attributed to loss of inhibitory phosphorylation of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) at the protein kinase A (PKA) site Ser637. Our results indicate that inhibition of Drp1-dependent mitochondrial fission by the outer mitochondrial AKAP1/PKA complex protects neurons from ischemic stroke by maintaining respiratory chain activity, inhibiting superoxide production, and delaying Ca2+ deregulation. They also provide the first genetic evidence that Drp1 inhibition may be of therapeutic relevance for the treatment of stroke and neurodegeneration.SIGNIFICANCE STATEMENT Previous work suggests that activation of dynamin-related protein 1 (Drp1) and mitochondrial fission contribute to ischemic injury in the brain. However, the specificity and efficacy of the pharmacological Drp1 inhibitor mdivi-1 that was used has now been discredited by several high-profile studies. Our report is timely and highly impactful because it provides the first evidence that genetic disinhibition of Drp1 via knock-out of the mitochondrial protein kinase A (PKA) scaffold AKAP1 exacerbates stroke injury in mice. Mechanistically, we show that electron transport deficiency, increased superoxide production, and Ca2+ overload result from genetic disinhibition of Drp1. In summary, our work settles current controversies regarding the role of mitochondrial fission in neuronal injury, provides mechanisms, and suggests that fission inhibitors hold promise as future therapeutic agents.

The Complement System Component C5a Produces Thermal Hyperalgesia via Macrophage-to-Nociceptor Signaling That Requires NGF and TRPV1.

UNLABELLED: The complement cascade is a principal component of innate immunity. Recent studies have underscored the importance of C5a and other components of the complement system in inflammatory and neuropathic pain, although the underlying mechanisms are largely unknown. In particular, it is unclear how the complement system communicates with nociceptors and which ion channels and receptors are involved. Here we demonstrate that inflammatory thermal and mechanical hyperalgesia induced by complete Freund's adjuvant was accompanied by C5a upregulation and was markedly reduced by C5a receptor (C5aR1) knock-out or treatment with the C5aR1 antagonist PMX53. Direct administration of C5a into the mouse hindpaw produced strong thermal hyperalgesia, an effect that was absent in TRPV1 knock-out mice, and was blocked by the TRPV1 antagonist AMG9810. Immunohistochemistry of mouse plantar skin showed prominent expression of C5aR1 in macrophages. Additionally, C5a evoked strong Ca(2+) mobilization in macrophages. Macrophage depletion in transgenic macrophage Fas-induced apoptosis mice abolished C5a-dependent thermal hyperalgesia. Examination of inflammatory mediators following C5a injection revealed a rapid upregulation of NGF, a mediator known to sensitize TRPV1. Preinjection of an NGF-neutralizing antibody or Trk inhibitor GNF-5837 prevented C5a-induced thermal hyperalgesia. Notably, NGF-induced thermal hyperalgesia was unaffected by macrophage depletion. Collectively, these results suggest that complement fragment C5a induces thermal hyperalgesia by triggering macrophage-dependent signaling that involves mobilization of NGF and NGF-dependent sensitization of TRPV1. Our findings highlight the importance of macrophage-to-neuron signaling in pain processing and identify C5a, NGF, and TRPV1 as key players in this cross-cellular communication. SIGNIFICANCE STATEMENT: This study provides mechanistic insight into how the complement system, a key component of innate immunity, regulates the development of pain hypersensitivity. We demonstrate a crucial role of the C5a receptor, C5aR1, in the development of inflammatory thermal and mechanical sensitization. By focusing on the mechanisms of C5a-induced thermal hyperalgesia, we show that this process requires recruitment of macrophages and initiation of macrophage-to-nociceptor signaling. At the molecular level, we demonstrate that this signaling depends on NGF and is mediated by the heat-sensitive nociceptive channel TRPV1. This deeper understanding of how immune cells and neurons interact to regulate pain processing is expected to facilitate mechanism-based approaches in the development of new analgesics.

Nerve growth factor (NGF) regulates activity of nuclear factor of activated T-cells (NFAT) in neurons via the phosphatidylinositol 3-kinase (PI3K)-Akt-glycogen synthase kinase 3ß (GSK3ß) pathway.

The Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT) plays an important role in regulating many neuronal functions, including excitability, axonal growth, synaptogenesis, and neuronal survival. NFAT can be activated by action potential firing or depolarization that leads to Ca(2+)/calcineurin-dependent dephosphorylation of NFAT and its translocation to the nucleus. Recent data suggest that NFAT and NFAT-dependent functions in neurons can also be potently regulated by NGF and other neurotrophins. However, the mechanisms of NFAT regulation by neurotrophins are not well understood. Here, we show that in dorsal root ganglion sensory neurons, NGF markedly facilitates NFAT-mediated gene expression induced by mild depolarization. The effects of NGF were not associated with changes in [Ca(2+)]i and were independent of phospholipase C activity. Instead, the facilitatory effect of NGF depended on activation of the PI3K/Akt pathway downstream of the TrkA receptor and on inhibition of glycogen synthase kinase 3ß (GSK3ß), a protein kinase known to phosphorylate NFAT and promote its nuclear export. Knockdown or knockout of NFATc3 eliminated this facilitatory effect. Simultaneous monitoring of EGFP-NFATc3 nuclear translocation and [Ca(2+)]i changes in dorsal root ganglion neurons indicated that NGF slowed the rate of NFATc3 nuclear export but did not affect its nuclear import rate. Collectively, our data suggest that NGF facilitates depolarization-induced NFAT activation by stimulating PI3K/Akt signaling, inactivating GSK3ß, and thereby slowing NFATc3 export from the nucleus. We propose that NFAT serves as an integrator of neurotrophin action and depolarization-driven calcium signaling to regulate neuronal gene expression.

Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum.

Ca2+ is a key intermediary in a variety of signalling pathways and undergoes dynamic changes in its cytoplasmic concentration due to release from stores within the endoplasmic reticulum (ER) and influx from the extracellular environment. In addition to regulating cytoplasmic Ca2+ signals, these responses also affect the concentration of Ca2+ within the ER and mitochondria. Single fluorescent protein-based Ca2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concentration of Ca2+ associated with intracellular signalling pathways. Most GCaMP-type indicators have dissociation constants (Kd) for Ca2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca2+], but poorly suited for use in mitochondria and ER where [Ca2+] can reach concentrations of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 µM (LAR-GECO1) and 12 µM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. In addition, we perform two-colour imaging of intracellular Ca2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca2+ indicators enables monitoring of ER and mitochondrial Ca2+ in combination with GFP-based reporters.

Mutated CaV2.1 channels dysregulate CASK/P2X3 signaling in mouse trigeminal sensory neurons of R192Q Cacna1a knock-in mice.

BACKGROUND: ATP-gated P2X3 receptors of sensory ganglion neurons are important transducers of pain as they adapt their expression and function in response to acute and chronic nociceptive signals. The present study investigated the role of calcium/calmodulin-dependent serine protein kinase (CASK) in controlling P2X3 receptor expression and function in trigeminal ganglia from Cacna1a R192Q-mutated knock-in (KI) mice, a genetic model for familial hemiplegic migraine type-1. RESULTS: KI ganglion neurons showed more abundant CASK/P2X3 receptor complex at membrane level, a result that likely originated from gain-of-function effects of R192Q-mutated CaV2.1 channels and downstream enhanced CaMKII activity. The selective CaV2.1 channel blocker ω-Agatoxin IVA and the CaMKII inhibitor KN-93 were sufficient to return CASK/P2X3 co-expression to WT levels. After CASK silencing, P2X3 receptor expression was decreased in both WT and KI ganglia, supporting the role of CASK in P2X3 receptor stabilization. This process was functionally observed as reduced P2X3 receptor currents. CONCLUSIONS: We propose that, in trigeminal sensory neurons, the CASK/P2X3 complex has a dynamic nature depending on intracellular calcium and related signaling, that are enhanced in a transgenic mouse model of genetic hemiplegic migraine.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a new intracellular modulator of P2X3 receptors.

ATP-gated P2X3 receptors of sensory ganglion neurons are important transducers of painful stimuli and are modulated by extracellular algogenic substances, via changes in the receptor phosphorylation state. The present study investigated the role of calcium/calmodulin-dependent serine protein kinase (CASK) in interacting and controlling P2X3 receptor expression and function in mouse trigeminal ganglia. Most ganglion neurons in situ or in culture co-expressed P2X3 and CASK. CASK was immunoprecipitated with P2X3 receptors from trigeminal ganglia and from P2X3/CASK-cotransfected human embryonic kidney (HEK) cells. Recombinant P2X3/CASK expression in HEK cells increased serine phosphorylation of P2X3 receptors, typically associated with receptor upregulation. CASK deletion mutants also enhanced P2X3 subunit expression. After silencing CASK, cell surface P2X3 receptor expression was decreased, which is consistent with depressed P2X3 currents. The reduction in P2X3 expression levels was reversed by the proteasomal inhibitor MG-132. Moreover, neuronal CASK/P2X3 interaction was up-regulated by nerve growth factor (NGF) signaling and down-regulated by P2X3 agonist-induced desensitization. These data suggest a novel interaction between CASK and P2X3 receptors with positive outcome for receptor stability and function. As CASK-mediated control of P2X3 receptors was dependent on the receptor activation state, CASK represents an intracellular gateway to regulate purinergic nociceptive signaling.

Lipid rafts control P2X3 receptor distribution and function in trigeminal sensory neurons of a transgenic migraine mouse model.

BACKGROUND: A genetic knock-in mouse model expressing the R192Q mutation of the α1-subunit of the Ca(V)2.1 channels frequently found in patients with familial hemiplegic migraine shows functional upregulation of ATP-sensitive P2X3 receptors of trigeminal sensory neurons that transduce nociceptive inputs to the brainstem. In an attempt to understand the basic mechanisms linked to the upregulation of P2X3 receptor activity, we investigated the influence of the lipid domain of these trigeminal sensory neurons on receptor compartmentalization and function. RESULTS: Knock-in neurons were strongly enriched with lipid rafts containing a larger fraction of P2X3 receptors at membrane level. Pretreatment with the Ca(V)2.1 channel blocker ω-agatoxin significantly decreased the lipid raft content of KI membranes. After pharmacologically disrupting the cholesterol component of lipid rafts, P2X3 receptors became confined to non-raft compartments and lost their functional potentiation typically observed in KI neurons with whole-cell patch-clamp recording. Following cholesterol depletion, all P2X3 receptor currents decayed more rapidly and showed delayed recovery indicating that alteration of the lipid raft milieu reduced the effectiveness of P2X3 receptor signalling and changed their desensitization process. Kinetic modeling could reproduce the observed data when slower receptor activation was simulated and entry into desensitization was presumed to be faster. CONCLUSIONS: The more abundant lipid raft compartment of knock-in neurons was enriched in P2X3 receptors that exhibited stronger functional responses. These results suggest that the membrane microenvironment of trigeminal sensory neurons is an important factor in determining sensitization of P2X3 receptors and could contribute to a migraine phenotype by enhancing ATP-mediated responses.