Activation of a TRPC3-dependent cation current through the neurotrophin BDNF

Nonvoltage-gated cation currents, which are activated following stimulation of phospholipase C (PLC), appear to be major modes for Ca2+ and Na+ entry in mammalian cells. The TRPC channels may mediate some of these conductances since their expression in vitro leads to PLC-dependent cation influx. We found that the TRPC3 protein was highly enriched in neurons of the central nervous system (CNS). The temporal and spatial distribution of TRPC3 paralleled that of the neurotrophin receptor TrkB. Activation of TrkB by brain-derived nerve growth factor (BDNF) led to production of a PLC-dependent, nonselective cation conductance in pontine neurons. Evidence is provided that TRPC3 contributes to this current in vivo. Thus, activation of TrkB and PLC leads to a TRPC3-dependent cation influx in CNS neurons.     

Brain‐derived neurotrophic factor attenuates doxorubicin‐induced cardiac dysfunction through activating Akt signalling in rats

The clinical application of doxorubicin (Dox) is limited by its adverse effect of cardiotoxicity. Previous studies have suggested the cardioprotective effect of brain‐derived neurotrophic factor (BDNF). We hypothesize that BDNF could protect against Dox‐induced cardiotoxicity. Sprague Dawley rats were injected with Dox (2.5 mg/kg, 3 times/week, i.p.), in the presence or absence of recombinant BDNF (0.4 μg/kg, i.v.) for 2 weeks. H9c2 cells were treated with Dox (1 μM) and/or BDNF (400 ng/ml) for 24 hrs. Functional roles of BDNF against Dox‐induced cardiac injury were examined both in vivo and in vitro. Protein level of BDNF was reduced in Dox‐treated rat ventricles, whereas BDNF and its receptor tropomyosin‐related kinase B (TrkB) were markedly up‐regulated after BDNF administration. Brain‐derived neurotrophic factor significantly inhibited Dox‐induced cardiomyocyte apoptosis, oxidative stress and cardiac dysfunction in rats. Meanwhile, BDNF increased cell viability, inhibited apoptosis and DNA damage of Dox‐treated H9c2 cells. Investigations of the underlying mechanisms revealed that BDNF activated Akt and preserved phosphorylation of mammalian target of rapamycin and Bad without affecting p38 mitogen‐activated protein kinase and extracellular regulated protein kinase pathways. Furthermore, the beneficial effect of BDNF was abolished by BDNF scavenger TrkB‐Fc or Akt inhibitor. In conclusion, our findings reveal a potent protective role of BDNF against Dox‐induced cardiotoxicity by activating Akt signalling, which may facilitate the safe use of Dox in cancer treatment.     

TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle

Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca^2 + signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca^2 + entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP–BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20 ng/ml, 48 h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10 μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca^2 + (EGTA; 1 mM) or intracellular Ca^2 + (BAPTA; 5 μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca^2 + influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca^2 + and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.     

Paracrine and autocrine functions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brain-derived endothelial cells

Brain-derived neurotrophic factor (BDNF) is expressed by endothelial cells. We investigated the characteristics of BDNF expression by brain-derived endothelial cells and tested the hypothesis that BDNF serves paracrine and autocrine functions affecting the vasculature of the central nervous system. In addition to expressing TrkB and p75NTR and BDNF under normoxic conditions, these cells increased their expression of BDNF under hypoxia. While the expression of TrkB is unaffected by hypoxia, TrkB exhibits a base-line phosphorylation under normoxic conditions and an increased phosphorylation when BDNF is added. TrkB phosphorylation is decreased when endogenous BDNF is sequestered by soluble TrkB. Exogenous BDNF elicits robust angiogenesis and survival in three-dimensional cultures of these endothelial cells, while sequestration of endogenous BDNF caused significant apoptosis. The effects of BDNF engagement of TrkB appears to be mediated via the phosphatidylinositol (PI) 3-kinase-Akt pathway. Modulation of BDNF levels directly correlate with Akt phosphorylation and inhibitors of PI 3-kinase abrogate the BDNF responses. BDNF-mediated effects on endothelial cell survival/apoptosis correlated directly with activation of caspase 3. These endothelial cells also express p75NTR and respond to its preferred ligand, pro-nerve growth factor (pro-NGF), by undergoing apoptosis. These data support a role for neurotrophins signaling in the dynamic maintenance/differentiation of central nervous system endothelia.     

Berberine Ameliorates MCAO Induced Cerebral Ischemia/Reperfusion Injury via Activation of the BDNF–TrkB–PI3K/Akt Signaling Pathway

Cerebral ischemic injury remains associated with high mortality rates and lacks effective therapeutic intervention. Berberine (BBR) possesses anti-oxidant, anti-inflammatory, and anti-tumor activities, as well as potent neuroprotective effects. Although recent studies have examined the neuroprotective effects of berberine, little is known regarding its usefulness in treating cerebral ischemia. Thus, the aim of this study is to investigate the possible effect and the mechanism of berberine against cerebral ischemic injury using the middle cerebral artery occlusion (MCAO) model. Rats were randomly divided into three groups: control group, MCAO group, and MCAO?+?BBR group. Modified neurological severity score tests (mNSS) and infarct volumes were measured to determine the neuroprotective effects of berberine. Neuronal survival in striatum was examined by TUNEL staining and immunohistochemistry. Western blotting measured the expression of BDNF, TrkB, p-Akt and cleaved caspase-3. The results demonstrated that BBR could significantly protect against MCAO. Berberine also increased the expression of BDNF, TrkB, and p-Akt, which were reduced after MCAO. Furthermore, treatment with BBR declined the apoptosis-related proteins induced by MCAO. However, treatment with LY294002 (PI3K inhibitor) reversed the BBR-induced increases in BDNF and p-Akt proteins and decreased cleaved caspase-3 protein expression in focal cerebral ischemic rats. In summary, our results demonstrated that BBR could exert neuroprotective effects through reduction of striatum apoptosis via the BDNF–TrkB–PI3K/Akt signaling pathway.     

BDNF‐mediated migration of cardiac microvascular endothelial cells is impaired during ageing

This study indicates that brain‐derived neurotrophic factor (BDNF) can promote young cardiac microvascular endothelial cells (CMECs) to migrate via the activation of the BDNF‐TrkB‐FL‐PI3K/Akt pathway, which may benefit angiogenesis after myocardial infarction (MI). However, the ageing of CMECs led to changes in the expression of receptor Trk isoforms in that among the three isoforms (TrkB‐FL, TrkB‐T1 and TrkB‐T2), only one of its truncated isoforms, TrkB‐T1, continued to be expressed, which leads to the dysfunction of its ligand, a decrease in the migration of CMECs and increased injury in ageing hearts. This shift in receptor isoforms in aged CMECs, together with changes in the ageing microenvironment, might predispose ageing hearts to decreased angiogenic potential and increased cardiac pathology.     

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca²⁺ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca²⁺ imaging, we found that the depletion of ER/SR Ca²⁺ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca²⁺ ([Ca²⁺]_i), followed by sustained increase depending on extracellular Ca²⁺. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na⁺/Ca²⁺ exchanger inhibitors, inhibited [Ca²⁺]_i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl₃) or by an increased extracellular Ca²⁺([Ca²⁺]_o) increased the concentration of intracelluar Ca²⁺, whereas, the sustained elevation of [Ca²⁺]_i was reduced in the presence of SKF96365. Similarly, the duration of [Ca²⁺]_i increase was also shortened in the absence of extracellular Ca²⁺. Western blot analysis showed that GdCl₃ increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl₃. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca²⁺-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.     

Inhibition of transient receptor potential canonical channels impairs cytokinesis in human malignant gliomas

Abstract.  Objectives : Glial-derived primary brain tumours, gliomas, are among the fastest growing malignancies and present a huge clinical challenge. Research suggests an important, yet poorly understood, role of ion channels in growth control of normal and malignant cells. In this study, we sought to functionally characterize Transient Receptor Potential Canoncial (TRPC) channels in glioma cell proliferation. TRPC channels form non-selective cation channels that have been suggested to represent a Ca²⁺ influx pathway impacting cellular growth. Materials and Methods : Employing a combination of molecular, biochemical and biophysical techniques, we characterized TRPC channels in glioma cells. Results : We showed consistent expression of four channel family members (TRPC-1, -3, -5, -6) in glioma cell lines and acute patient-derived tissues. These channels gave rise to small, non-voltage-dependent cation currents that were blocked by the TRPC inhibitors GdCl₃, 2-APB, or SKF96365. Importantly, TRPC channels contributed to the resting conductance of glioma cells and their acute pharmacological inhibition caused an ~10 mV hyperpolarization of the cells' resting potential. Additionally, chronic application of the TRPC inhibitor SKF96365 caused near complete growth arrest. A detailed analysis, by fluorescence-activated cell sorting and time-lapse microscopy, showed that growth inhibition occurred at the G₂+ M phase of the cell cycle with cytokinesis defects. Cells underwent incomplete cell divisions and became multinucleate, enlarged cells. Conclusions : Nuclear atypia and enlarged cells are histopathological hallmarks for glioblastoma multiforme , the highest grade glioma, suggesting that a defect in TRPC channel function may contribute to cellular abnormalities in these tumours.     

Role of iNOS-derived reactive nitrogen species and resultant nitrative stress in leukocytes-induced cardiomyocyte apoptosis after myocardial ischemia/reperfusion

Polymorphonuclear leukocyte (PMN) accumulation/activation has been implicated as a primary mechanism underlying MI/R injury. Recent studies have demonstrated that PMNs express inducible nitric oxide synthase (iNOS) and produce toxic reactive nitrogen species (RNS). However, the role of iNOS-derived reactive nitrogen species and resultant nitrative stress in PMN-induced cardiomyocyte apoptosis after MI/R remains unclear. Male adult rats were subjected to 30 min of myocardial ischemia followed by 5 h of reperfusion. Animals were randomized to receive one of the following treatments: MI/R+vehicle; MI/R+L-arginine; PMN depletion followed by MI/R+vehicle; PMN depletion followed by MI/R+L-arginine; MI/R+1400 W; MI/R+1400 W+L-arginine and MI/R+ FeTMPyP. Ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis were determined. PMN depletion virtually abolished ischemia/reperfusion- induced PMN accumulation, attenuated ischemic/reperfusion-induced and L-arginine-enhanced nitrative stress, and reduced ischemic/reperfusion-induced and L-arginine-enhanced cardiomyocyte apoptosis (P values all <0.01). Pre-treatment with 1400 W, a highly selective iNOS inhibitor, had no effect on PMN accumulation in the ischemic/reperfused tissue. However, this treatment reduced ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis to an extent that is comparable as that seen in PMN depletion group. Treatment with FeTMPyP, a peroxynitrite decomposition catalyst, had no effect on either PMN accumulation or total NO production. However, treatment with this ONOO⁻ decomposition catalyst also reduced ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis (P values all <0.01). These results demonstrated that ischemic/reperfusion stimulated PMN accumulation may result in cardiomyocyte injury by an iNOS-derived nitric oxide initiated and peroxynitrite-mediated mechanism. Therapeutic interventions that block PMN accumulation, inhibit iNOS activity or scavenge peroxynitrite may reduce nitrative stress and attenuate tissue injury. Xiao-Liang Wang and Hui-Rong Liu contributed equally to this study.     

Assessment of the role of the inositol 1,4,5-trisphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP(3)R) was examined using the permeant InsP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP(3)R-mediated store release and carbachol-induced Sr(2+) entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP(3)Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP(3)R-independent since InsP(3)Rs are dispensable for the light response. We used triple InsP(3)R knockout DT40 chicken B-cells to further assess the role of InsP(3)Rs in SOC activation. (45)Ca(2+) flux analysis revealed that although DT40 wild-type cells retained normal InsP(3)Rs mediating 2-APB-sensitive Ca(2+) release, the DT40InsP(3)R-k/o cells were devoid of functional InsP(3)Rs. Using intact cells, all parameters of Ca(2+) store function and SOC activation were identical in DT40wt and DT40InsP(3)R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC(50)) were identical. The results indicate that (a) the action of 2-APB on Ca(2+) entry is not mediated by the InsP(3)R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.     

Quercetin Attenuates Cell Apoptosis in Focal Cerebral Ischemia Rat Brain Via Activation of BDNF–TrkB–PI3K/Akt Signaling Pathway

Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF?CTrkB?CPI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF?CTrkB?CPI3K/Akt signaling pathway.     

The Ca2+ release-activated Ca2+ current (ICRAC) mediates store-operated Ca2+ entry in rat microglia

Ca²⁺ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca²⁺ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca²⁺ permeable channels, and limited electrophysiological analyses of Ca²⁺ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are ‘transient receptor potential’ (TRP) channels and the recently cloned Orai1, which produces a Ca²⁺-release-activated Ca²⁺ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca²⁺ entry (SOCE) pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca²⁺-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca²⁺ current with the same properties, including high selectivity for Ca²⁺ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca²⁺-dependent current potentiation, and block by SKF-96365, DES and 50 μM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.     

The Cardioprotective Effect of Vitamin E (Alpha-Tocopherol) Is Strongly Related to Age and Gender in Mice

Vitamin E (VitE) only prevented cardiovascular diseases in some patients and the mechanisms remain unknown. VitE levels can be affected by aging and gender. We hypothesize that age and gender can influence VitE’s cardioprotective effect. Mice were divided into 4 groups according to age and gender, and each group of mice were divided into a control group and a VitE group. The mice were administered water or VitE for 21 days; Afterward, the cardiac function and myocardial infarct size and cardiomyocyte apoptosis were measured after myocardial ischemia reperfusion(MI/R). VitE may significantly improved cardiac function in young male mice and aged female mice by enhancing ERK1/2 activity and reducing JNK activity. Enhanced expression of HSP90 and Bcl-2 were also seen in young male mice. No changes in cardiac function and cardiac proteins were detected in aged male mice and VitE was even liked to exert a reverse effect in cardiac function in young mice by enhancing JNK activity and reducing Bcl-2 expression. Those effects were in accordance with the changes of myocardial infarction size and cardiomyocyte apoptosis in each group of mice. VitE may reduce MI/R injury by inhibiting cardiomyocyte apoptosis in young male mice and aged female mice but not in aged male mice. VitE was possibly harmful for young female mice, shown as increased cardiomyocyte apoptosis after MI/R. Thus, we speculated that the efficacy of VitE in cardiac protection was associated with age and gender.     

Endophilin A2-mediated alleviation of endoplasmic reticulum stress-induced cardiac injury involves the suppression of ERO1α/IP3R signaling pathway

Cardiac injury upon myocardial infarction (MI) is the leading cause of heart failure. The present study aims to investigate the role of EndoA2 in ischemia-induced cardiomyocyte apoptosis and cardiac injury. In vivo, we established an MI mouse model by ligating the left anterior descending (LAD) coronary artery, and intramyocardial injection of adenoviral EndoA2 (Ad-EndoA2) was used to overexpress EndoA2. In vitro, we used the siRNA and Ad-EndoA2 transfection strategies. Here, we reported that EndoA2 expression was remarkably elevated in the infarct border zone of MI mouse hearts and neonatal rat cardiomyocytes (NRCMs) stimulated with oxygen and glucose deprivation (OGD) which mimicked ischemia. We showed that intramyocardial injection of Ad-EndoA2 attenuated cardiomyocyte apoptosis and reduced endoplasmic reticulum (ER) stress in response to MI injury. Using siRNA for knockdown and Ad-EndoA2 for overexpression, we validated that knockdown of EndoA2 in NRCMs exacerbated OGD-induced NRCM apoptosis, whereas overexpression of EndoA2 attenuates OGD-induced cardiomyocyte apoptosis. Mechanistically, knockdown of EndoA2 activated ER stress response, which increases ER oxidoreductase 1α (ERO1α) and inositol 1, 4, 5-trisphosphate receptor (IP₃R) activity, thus led to increased intracellular Ca²⁺ accumulation, followed by elevated calcineurin activity and nuclear factor of activated T-cells (NFAT) dephosphorylation. Pretreatment with the IP₃R inhibitor 2-Aminoethoxydiphenylborate (2-APB) attenuated intracellular Ca²⁺ accumulation, and pretreatment with the Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or the calcineurin inhibitor Cyclosporin A (CsA) inhibited EndoA2-knockdown-induced NRCM apoptosis. Overexpression of EndoA2 led to the opposite effects by suppressing ER-stress-mediated ERO1α/IP₃R signaling pathway. This study demonstrated that EndoA2 protected cardiac function in response to MI via attenuating ER-stress-mediated ERO1α/IP₃R signaling pathway. Targeting EndoA2 is a potential therapeutic strategy for the prevention of postinfarction-induced cardiac injury and heart failure.     

Brain-derived Neurotrophic Factor (BDNF)-induced Mitochondrial Motility Arrest and Presynaptic Docking Contribute to BDNF-enhanced Synaptic Transmission

Appropriate mitochondrial transport and distribution are essential for neurons because of the high energy and Ca²⁺ buffering requirements at synapses. Brain-derived neurotrophic factor (BDNF) plays an essential role in regulating synaptic transmission and plasticity. However, whether and how BDNF can regulate mitochondrial transport and distribution are still unclear. Here, we find that in cultured hippocampal neurons, application of BDNF for 15 min decreased the percentage of moving mitochondria in axons, a process dependent on the activation of the TrkB receptor and its downstream PI3K and phospholipase-Cγ signaling pathways. Moreover, the BDNF-induced mitochondrial stopping requires the activation of transient receptor potential canonical 3 and 6 (TRPC3 and TRPC6) channels and elevated intracellular Ca²⁺ levels. The Ca²⁺ sensor Miro1 plays an important role in this process. Finally, the BDNF-induced mitochondrial stopping leads to the accumulation of more mitochondria at presynaptic sites. Mutant Miro1 lacking the ability to bind Ca²⁺ prevents BDNF-induced mitochondrial presynaptic accumulation and synaptic transmission, suggesting that Miro1-mediated mitochondrial motility is involved in BDNF-induced mitochondrial presynaptic docking and neurotransmission. Together, these data suggest that mitochondrial transport and distribution play essential roles in BDNF-mediated synaptic transmission.     

Overexpression of TRPC3 increases apoptosis but not necrosis in response to ischemia-reperfusion in adult mouse cardiomyocytes

An increase in cytosolic Ca2+ via a capacitative calcium entry (CCE)-mediated pathway, attributed to members of the transient receptor potential (TRP) superfamily, TRPC1 and TRPC3, has been reported to play an important role in regulating cardiomyocyte hypertrophy. Increased cytosolic Ca2+ also plays a critical role in mediating cell death in response to ischemia-reperfusion (I/R). Therefore, we tested the hypothesis that overexpression of TRPC3 in cardiomyocytes will increase sensitivity to I/R injury. Adult cardiomyocytes isolated from wild-type (WT) mice and from mice overexpressing TRPC3 in the heart were subjected to 90 min of ischemia and 3 h of reperfusion. After I/R, viability was 51 +/- 1% in WT mice and 42 +/- 5% in transgenic mice (P < 0.05). Apoptosis assessed by annexin V was significantly increased in the TRPC3 group compared with WT (32 +/- 1% vs. 21 +/- 3%; P < 0.05); however, there was no significant difference in necrosis between groups. Treatment of TRPC3 cells with the CCE inhibitor SKF-96365 (0.5 microM) significantly improved cellular viability (54 +/- 4%) and decreased apoptosis (15 +/- 4%); in contrast, the L-type Ca2+ channel inhibitor verapamil (10 microM) had no effect. Calpain-mediated cleavage of alpha-fodrin was increased approximately threefold in the transgenic group following I/R compared with WT (P < 0.05); this was significantly attenuated by SKF-96365. The calpain inhibitor PD-150606 (25 microM) attenuated the increase in both alpha-fodrin cleavage and apoptosis in the TPRC3 group. Increased TRPC3 expression also increased sensitivity to Ca2+ overload stress, but it did not affect the response to TNF-alpha-induced apoptosis. These results suggest that CCE mediated via TRPC may play a role in cardiomyocyte apoptosis following I/R due, at least in part, to increased calpain activation.