TRPV4 disrupts mitochondrial transport and causes axonal degeneration via a CaMKII-dependent elevation of intracellular Ca2.
ABSTRACT: The cation channel transient receptor potential vanilloid 4 (TRPV4) is one of the few identified ion channels that can directly cause inherited neurodegeneration syndromes, but the molecular mechanisms are unknown. Here, we show that in vivo expression of a neuropathy-causing TRPV4 mutant (TRPV4R269C) causes dose-dependent neuronal dysfunction and axonal degeneration, which are rescued by genetic or pharmacological blockade of TRPV4 channel activity. TRPV4R269C triggers increased intracellular Ca2+ through a Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism, and CaMKII inhibition prevents both increased intracellular Ca2+ and neurotoxicity in Drosophila and cultured primary mouse neurons. Importantly, TRPV4 activity impairs axonal mitochondrial transport, and TRPV4-mediated neurotoxicity is modulated by the Ca2+-binding mitochondrial GTPase Miro. Our data highlight an integral role for CaMKII in neuronal TRPV4-associated Ca2+ responses, the importance of tightly regulated Ca2+ dynamics for mitochondrial axonal transport, and the therapeutic promise of TRPV4 antagonists for patients with TRPV4-related neurodegenerative diseases.
Project description:OBJECTIVE:The main objective of this study is to define the mechanisms by which mitochondria control vascular smooth muscle cell (VSMC) migration and impact neointimal hyperplasia. APPROACH AND RESULTS:The multifunctional CaMKII (Ca2+/calmodulin-dependent kinase II) in the mitochondrial matrix of VSMC drove a feed-forward circuit with the mitochondrial Ca2+ uniporter (MCU) to promote matrix Ca2+ influx. MCU was necessary for the activation of mitochondrial CaMKII (mtCaMKII), whereas mtCaMKII phosphorylated MCU at the regulatory site S92 that promotes Ca2+ entry. mtCaMKII was necessary and sufficient for platelet-derived growth factor-induced mitochondrial Ca2+ uptake. This effect was dependent on MCU. mtCaMKII and MCU inhibition abrogated VSMC migration and mitochondrial translocation to the leading edge. Overexpression of wild-type MCU, but not MCU S92A, mutant in MCU-/- VSMC rescued migration and mitochondrial mobility. Inhibition of microtubule, but not of actin assembly, blocked mitochondrial mobility. The outer mitochondrial membrane GTPase Miro-1 promotes mitochondrial mobility via microtubule transport but arrests it in subcellular domains of high Ca2+ concentrations. In Miro-1-/- VSMC, mitochondrial mobility and VSMC migration were abolished, and overexpression of mtCaMKII or a CaMKII inhibitory peptide in mitochondria (mtCaMKIIN) had no effect. Consistently, inhibition of mtCaMKII increased and prolonged cytosolic Ca2+ transients. mtCaMKII inhibition diminished phosphorylation of focal adhesion kinase and myosin light chain, leading to reduced focal adhesion turnover and cytoskeletal remodeling. In a transgenic model of selective mitochondrial CaMKII inhibition in VSMC, neointimal hyperplasia was significantly reduced after vascular injury. CONCLUSIONS:These findings identify mitochondrial CaMKII as a key regulator of mitochondrial Ca2+ uptake via MCU, thereby controlling mitochondrial translocation and VSMC migration after vascular injury.
Project description:Mutations in TRPV4 have been linked to three distinct axonal neuropathies. However, the pathogenic mechanism underlying these disorders remains unclear. Both gain and loss of calcium channel activity of the mutant TRPV4 have been suggested. Here, we show that the three previously reported TRPV4 mutant channels have a physiological localization and display an increased calcium channel activity, leading to increased cytotoxicity in three different cell types. Patch clamp experiments showed that cells expressing mutant TRPV4 have much larger whole-cell currents than those expressing the wild-type TRPV4 channel. Single channel recordings showed that the mutant channels have higher open probability, due to a modification of gating, and no change in single-channel conductance. These data support the hypothesis that a "gain of function" mechanism, possibly leading to increased intracellular calcium influx, underlies the pathogenesis of the TRPV4-linked axonal neuropathies, and may have immediate implications for designing rational therapies.
Project description:Rotenone, a neurotoxic pesticide, induces loss of dopaminergic neurons related to Parkinson's disease. Previous studies have shown that rotenone induces neuronal apoptosis partly by triggering hydrogen peroxide (H2O2)-dependent suppression of mTOR pathway. However, the underlying mechanism is not fully understood. Here, we show that rotenone elevates intracellular free calcium ion ([Ca2+]i) level, and activates CaMKII, resulting in inhibition of mTOR signaling and induction of neuronal apoptosis. Chelating [Ca2+]i with BAPTA/AM, preventing extracellular Ca2+ influx using EGTA, inhibiting CaMKII with KN93, or silencing CaMKII significantly attenuated rotenone-induced H2O2 production, mTOR inhibition, and cell death. Interestingly, using TTFA, antimycin A, catalase or Mito-TEMPO, we found that rotenone-induced mitochondrial H2O2 also in turn elevated [Ca2+]i level, thereby stimulating CaMKII, leading to inhibition of mTOR pathway and induction of neuronal apoptosis. Expression of wild type mTOR or constitutively active S6K1, or silencing 4E-BP1 strengthened the inhibitory effects of catalase, Mito-TEMPO, BAPTA/AM or EGTA on rotenone-induced [Ca2+]i elevation, CaMKII phosphorylation and neuronal apoptosis. Together, the results indicate that the crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that how to control over-elevation of intracellular Ca2+ and overproduction of mitochondrial H2O2 may be a new approach to deal with the neurotoxicity of rotenone.
Project description:<h4>Objective</h4>To improve understanding of TRPV4-associated axonal Charcot-Marie-Tooth (CMT) neuropathy phenotypes and their debated pathologic mechanism.<h4>Methods</h4>A total of 17 CMT2C phenotypic families with vocal cord and diaphragmatic involvement and 36 clinically undifferentiated CMT2 subjects underwent sequencing analysis of the coding region of TRPV4. Functional studies of mutant proteins were performed using transiently transfected cells for TRPV4 subcellular localization, basal and stimulated Ca(2+) channel analysis, and cell viability assay with or without channel blockade.<h4>Results</h4>Two TRPV4 mutations R232C and R316H from 17 CMT2C families were identified in the ankyrin repeat domains. The R316H is a novel de novo mutation found in a patient with CMT2C phenotype. The family with R232C mutation had individuals with and without vocal cord and diaphragm involvement. Both mutant TRPV4 proteins had normal subcellular localization in HEK293 and HeLa cells. Cells transfected with R232C and R316H displayed increased intracellular Ca(2+) levels and reversible cell death by the TRPV channel antagonist, ruthenium red.<h4>Conclusion</h4>TRPV4 ankyrin domain alterations including a novel de novo mutation cause axonal CMT2. Individuals with the same mutation may have nondistinct CMT2 or have phenotypic CMT2C with vocal cord paresis. Reversible hypercalcemic gain-of-function of mutant TRPV4 instead of loss-of-function appears to be pathologically important. The reversibility of cell death by channel blockade provides an attractive area of investigation in consideration of treatable axonal degeneration.
Project description:Ca2+ entry plays an important role in modulating endothelial cell migration and tube formation. Transient receptor potential cation channel subfamily V member 4 (TRPV4) is a Ca2+-permeable channel that is widely expressed in endothelial cells. It has been reported that TRPV4 is expressed in HRCECs and regulates Ca2+ entry. However, the function of TRPV4 in human retinal capillary endothelial cells (HRCECs) remains unknown.In this study we used western blot and immunostaining assay to verify TRPV4 expression in HRCECs. And then we pretreated HRCECs with HC067047 and transfected with specific shRNA of TRPV4. The functional presence of TrpV4 was determined by using fluorescence, migration and tube formation assay in TrpV4 knockdown cells or control cells.Using western blot and immunostaining, we confirmed TRPV4 expression in HRCECs. Moreover, inhibition of TRPV4 using the specific inhibitor HC067047 and the knockdown of TRPV4 with shRNA significantly suppressed tube formation and migration by HRCECs.TRPV4 is essential for HRCEC migration and tube formation, and maybe a potential therapeutic target for retinal vascular diseases.
Project description:The adaptation of the skeleton to its mechanical environment is orchestrated by mechanosensitive osteocytes, largely by regulating the abundance of sclerostin, a secreted inhibitor of bone formation. We defined a microtubule-dependent mechanotransduction pathway that linked fluid shear stress to reactive oxygen species (ROS) and calcium (Ca2+) signals that led to a reduction in sclerostin abundance in cultured osteocytes. We demonstrated that microtubules stabilized by detyrosination, a reversible posttranslational modification of polymerized ?-tubulin, determined the stiffness of the cytoskeleton, which set the mechanoresponsive range of cultured osteocytes to fluid shear stress. We showed that fluid shear stress through the microtubule network activated NADPH oxidase 2 (NOX2)-generated ROS that target the Ca2+ channel TRPV4 to elicit Ca2+ influx. Furthermore, tuning the abundance of detyrosinated tubulin affected cytoskeletal stiffness to define the mechanoresponsive range of cultured osteocytes to fluid shear stress. Finally, we demonstrated that NOX2-ROS elicited Ca2+ signals that activated the kinase CaMKII to decrease the abundance of sclerostin protein. Together, these discoveries may identify potentially druggable targets for regulating osteocyte mechanotransduction to affect bone quality.
Project description:TRPV4 (transient receptor potential vanilloid 4) channels are Ca2+-permeable channels that play a key role in regulating vascular tone. In arterial myocytes, opening of TRPV4 channels creates local increases in Ca2+ influx, detectable optically as "TRPV4 sparklets." TRPV4 sparklet activity can be enhanced by the action of the vasoconstrictor angiotensin II (AngII). This modulation depends on the activation of subcellular signaling domains that comprise protein kinase C α (PKCα) bound to the anchoring protein AKAP150. Here, we used super-resolution nanoscopy, patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling approaches to test the hypothesis that AKAP150-dependent modulation of TRPV4 channels is critically dependent on the distance between these two proteins in the sarcolemma of arterial myocytes. Our data show that the distance between AKAP150 and TRPV4 channel clusters varies with sex and arterial bed. Consistent with our hypothesis, we further find that basal and AngII-induced TRPV4 channel activity decays exponentially as the distance between TRPV4 and AKAP150 increases. Our data suggest a maximum radius of action of ∼200 nm for local modulation of TRPV4 channels by AKAP150-associated PKCα.
Project description:The transient receptor potential vallinoid type 4 (TRPV4) is a calcium entry channel known to modulate vascular function by mediating endothelium-dependent vasodilation. The present study investigated if isolated cerebral arterial myocytes of the Fawn Hooded hypertensive (FHH) rat, known to display exaggerated KCa channel current activity and impaired myogenic tone, express TRPV4 channels at the transcript and protein level and exhibit TRPV4-like single-channel cationic current activity. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis detected the expression of mRNA transcript and translated protein of TRPV4 channel in FHH rat cerebral arterial myocytes. Patch clamp recording of single-channel current activity identified the presence of a single-channel cationic current with unitary conductance of ~85 pS and ~96 pS at hyperpolarizing and depolarizing potentials, respectively, that was inhibited by the TRPV4 channel antagonist RN 1734 or HC 067074 and activated by the potent TRPV4 channel agonist GSK1016790A. Application of negative pressure via the interior of the patch pipette increased the NPo of the TRPV4-like single-channel cationic current recorded in cell-attached patches at a patch potential of 60 mV that was inhibited by prior application of the TRPV4 channel antagonist RN 1734 or HC 067047. Treatment with the TRPV4 channel agonist GSK1016790A caused concentration-dependent increase in the NPo of KCa single-channel current recorded in cell-attached patches of cerebral arterial myocytes at a patch potential of 40 mV, which was not influenced by pretreatment with the voltage-gated L-type Ca2+ channel blocker nifedipine or the T-type Ca2+ channel blocker Ni2+. These findings demonstrate that FHH rat cerebral arterial myocytes express mRNA transcript and translated protein for TRPV4 channel and display TRPV4-like single-channel cationic current activity that was stretch-sensitive and activation of which increased the open state probability of KCa single-channel current in these arterial myocytes.
Project description:Myogenic response, a phenomenon in which resistance size arteries and arterioles swiftly constrict or dilate in response to an acute elevation or reduction, respectively, in intravascular pressure is a key component of renal autoregulation mechanisms. Although it is well established that the renal system is functionally immature in neonates, mechanisms that regulate neonatal renal blood flow (RBF) remain poorly understood. In this study, we investigated the hypothesis that members of the transient receptor potential vanilloid (TRPV) channels are molecular components of renal myogenic constriction in newborns. We show that unlike TRPV1-3, TRPV4 channels are predominantly expressed in neonatal pig preglomerular vascular smooth muscle cells (SMCs). Intracellular Ca2+ concentration ([Ca2+]i) elevation induced by osmotic cell swelling was attenuated by TRPV4, L-type Ca2+, and stretch-activated Ca2+ channel blockers but not phospholipase A2 inhibitor. Blockade of TRPV4 channels reversed steady-state myogenic tone and inhibited pressure-induced membrane depolarization, [Ca2+]i elevation, and constriction in distal interlobular arteries. A step increase in arterial pressure induced efficient autoregulation of renal cortical perfusion and total RBF in anesthetized and mechanically ventilated neonatal pigs. Moreover, intrarenal arterial infusion of the TRPV4 channel blockers HC 067047 and RN 1734 attenuated renal autoregulation in the pigs. These data suggest that renal myogenic autoregulation is functional in neonates. Our findings also indicate that TRPV4 channels are mechanosensors in neonatal pig preglomerular vascular SMCs and contribute to renal myogenic autoregulation.
Project description:Ion channels are well placed to transduce environmental cues into signals used by cells to generate a wide range of responses, but little is known about their role in the regulation of RNA metabolism. Here we show that the TRPV4 cation channel binds the DEAD-box RNA helicase DDX3X and regulates its function. TRPV4-mediated Ca2+ influx releases DDX3X from the channel and drives DDX3X nuclear translocation, a process that involves calmodulin (CaM) and the CaM-dependent kinase II. Genetic depletion or pharmacological inhibition of TRPV4 diminishes DDX3X-dependent functions, including nuclear viral export and translation. Furthermore, TRPV4 mediates Ca2+ influx and nuclear accumulation of DDX3X in cells exposed to the Zika virus or the purified viral envelope protein. Consequently, targeting of TRPV4 reduces infectivity of dengue, hepatitis C and Zika viruses. Together, our results highlight the role of TRPV4 in the regulation of DDX3X-dependent control of RNA metabolism and viral infectivity.