Acidosis,Acid-Sensing Ion Channels,and Neuronal Cell Death

Acidosis is a common feature of many neuronal diseases and often accompanied with adverse consequences such as pain and neuronal injury. Before the discovery of acid-sensing ion channels (ASICs), protons were usually considered as a modulator of other ion channels, such as voltage-gated calcium channels, N-methyl-d-aspartate, and γ-amino butyric acid(A) receptor channels. Accordingly, the functional effects of acidosis were considered as consequences of modulations of these channels. Since the first cloning of ASICs in 1997, the conventional view on acidosis-mediated pain and cell injury has been dramatically changed. To date, ASICs, which are directly activated by extracellular protons, are shown to mediate most of the acidosis-associated physiological and pathological functions. For example, ASIC1a channels are reported to mediate acidosis-induced ischemic neuronal death. In this article, we will review the possible mechanisms that underlie ASIC1a channel-mediated neuronal death and discuss ASIC1a channel modulators involved in this process.     

Role of STIM1 in Regulation of Store-Operated Ca<Superscript>2+</Superscript> Influx in Pheochromocytoma Cells

Changes in the local environment such as pH (acidosis/alkalosis), temperature (hypothermia/hyperthermia), and agonist (glutamate) can adversely affect neuronal function, and are important factors in clinical situations such as anesthesia and intensive care. Regulation of intracellular Ca²⁺ ([Ca²⁺]_i) is key to neuronal function. Stromal interaction molecule (STIM1) has been recently recognized to trigger store-operated Ca²⁺ entry (SOCE), an important component of [Ca²⁺]_i regulation. Using differentiated, fura-2 loaded rat pheochromocytoma (PC12) cells transfected with small interference RNA for STIM1 (or vehicle), we examined the role of STIM1 in SOCE sensitivity to temperature, pH, and glutamate. SOCE was triggered following endoplasmic reticulum depletion. Cells were washed and exposed to altered pH (6.0–8.0), altered temperature (34–40°C), or to glutamate. In non-transfected cells, SOCE was inhibited by acidosis or hypothermia, but increased with alkalosis and hyperthermia. Increasing glutamate concentrations progressively stimulated SOCE. STIM1 siRNA decreased SOCE at normal temperature and pH, and substantially decreased sensitivity to acidosis and hypothermia, eliminating the concentration-dependence to glutamate. Sensitivity of SOCE to these environmental parameters was less altered by decreased extracellular Ca²⁺ alone (with STIM1 intact). We conclude that STIM1 mediates exquisite susceptibility of SOCE to pH, temperature, and glutamate: factors that can adversely affect neuronal function under pathological conditions.     

Acidosis Potentiates Oxidative Neuronal Death by Multiple Mechanisms

Both acidosis and oxidative stress contribute to ischemic brain injury. The present study examines interactions between acidosis and oxidative stress in murine cortical cultures. Acidosis (pH 6.2) was found to potentiate markedly neuronal death induced by H2O2 exposure. To determine if this effect was mediated by decreased antioxidant capacity at low pH, the activities of several antioxidant enzymes were measured. Acidosis was found to reduce the activities of glutathione peroxidase and glutathione S-transferase by 50-60% (p < 0.001) and the activity of glutathione reductase by 20% (p < 0.01) in lysates of the cortical cultures. Like acidosis, direct inhibition of glutathione peroxidase with mercaptosuccinate also potentiated H2O2 toxicity. Because acidosis may accelerate hydroxyl radical production by the Fenton reaction, the effect of iron chelators was also examined. Both desferrioxamine and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, two structurally different iron chelators, significantly reduced H2O2-induced neuronal death under both pH 7.2 and pH 6.2 conditions. These results suggest that the increased cell death produced by severe acidosis during cerebral ischemia may result in part from exacerbation of oxidative injury. This exacerbation may result from both impaired antioxidant enzyme functions and increased intracellular free iron levels.     

Endogenous arginine-phenylalanine-amide-related peptides alter steady-state desensitization of ASIC1a

The acid-sensing ion channels (ASICs) are proton-gated, voltage-insensitive cation channels expressed throughout the nervous system. ASIC1a plays a role in learning, pain, and fear-related behaviors. In addition, activation of ASIC1a during prolonged acidosis following cerebral ischemia induces neuronal death. ASICs undergo steady-state desensitization, a characteristic that limits ASIC1a activity and may play a prominent role in the prevention of ASIC1a-evoked neuronal death. In this study, we found exogenous and endogenous arginine-phenylalanine-amide (RF-amide)-related peptides decreased the pH sensitivity of ASIC1a steady-state desensitization. During conditions that normally induced steady-state desensitization, these peptides profoundly enhanced ASIC1a activity. We also determined that human ASIC1a required more acidic pH to undergo steady-state desensitization compared with mouse ASIC1a. Surprisingly, steady-state desensitization of human ASIC1a was also affected by a greater number of peptides compared with mouse ASIC1a. Mutation of five amino acids in a region of the extracellular domain changed the characteristics of human ASIC1a to those of mouse ASIC1a, suggesting that this region plays a pivotal role in neuropeptide and pH sensitivity of steady-state desensitization. Overall, these experiments lend vital insight into steady-state desensitization of ASIC1a and expand our understanding of the structural determinants of RF-amide-related peptide modulation. Furthermore, our finding that endogenous peptides shift steady-state desensitization suggests that RF-amides could impact the role of ASIC1a in both pain and neuronal damage following stroke and ischemia.     

Role of transporters and ion channels in neuronal injury under hypoxia

The aims of the current study were to 1) examine the effects of hypoxia and acidosis on cultured cortical neurons and 2) explore the role of transporters and ion channels in hypoxic injury. Cell injury was measured in cultured neurons or hippocampal slices following hypoxia (1% O(2)) or acidosis (medium pH 6.8) treatment. Inhibitors of transporters and ion channels were employed to investigate their roles in hypoxic injury. Our results showed that 1) neuronal damage was apparent at 5-7 days of hypoxia exposure, i.e., 36-41% of total lactate dehydrogenase was released to medium and 2) acidosis alone did not lead to significant injury compared with nonacidic, normoxic controls. Pharmacological studies revealed 1) no significant difference in neuronal injury between controls (no inhibitor) and inhibition of Na(+)-K(+)-ATP pump, voltage-gated Na(+) channel, ATP-sensitive K(+) channel, or reverse mode of Na(+)/Ca(2+) exchanger under hypoxia; however, 2) inhibition of NBCs with 500 microM DIDS did not cause hypoxic death in either cultured cortical neurons or hippocampal slices; 3) in contrast, inhibition of Na(+)/H(+) exchanger isoform 1 (NHE1) with either 10 microM HOE-642 or 2 microM T-162559 resulted in dramatic hypoxic injury (+95% for HOE-642 and +100% for T-162559 relative to normoxic control, P < 0.001) on treatment day 3, when no death occurred for hypoxic controls (no inhibitor). No further damage was observed by NHE1 inhibition on treatment day 5. We conclude that inhibition of NHE1 accelerates hypoxia-induced neuronal damage. In contrast, DIDS rescues neuronal death under hypoxia. Hence, DIDS-sensitive mechanism may be a potential therapeutic target.     

Change in Intracellular pH Causes the Toxic Ca<Superscript>2+</Superscript> Entry via NCX1 in Neuron- and Glia-Derived Cells

Brain hypoxia or ischemia causes acidosis and the intracellular accumulation of Ca²⁺ in neuron. The aims of the present study were to elucidate the interaction between intracellular pH and Ca²⁺ during transient acidosis and its effects on the viability of neuronal and glial cells. Intracellular Ca²⁺ and pH were measured using the fluorescence of fura-2 and 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester in neuroblastoma (IMR-32), glioblastoma (T98G), and astrocytoma (CCF-STTG1) cell lines. The administration of 5 mM propionate caused intracellular acidification in IMR-32 and T98G cells but not in CCF-STTG1 cells. After the removal of propionate, the intracellular pH recovered to the resting level. The intracellular Ca²⁺ transiently increased upon the removal of propionate in IMR-32 and T98G cells but not in CCF-STTG1 cells. The transient Ca²⁺ increase caused by the withdrawal of intracellular acidification was abolished by the removal of external Ca²⁺, diminished by a reduction of external Na⁺, and inhibited by benzamil. Transient acidosis caused cell death, whereas the cells were more viable in the absence of external Ca²⁺. Benzamil alleviated cell death caused by transient acidosis in IMR-32 and T98G cells but not in CCF-STTG1 cells. These results suggest that recovery from intracellular acidosis causes a transient increase in cytosolic Ca²⁺ due to reversal of Ca²⁺ transport via Na⁺/Ca²⁺ exchanger coactivated with Na⁺/H⁺ exchanger, which can cause cell death.     

Acidosis has opposite effects on neuronal survival during hypoxia and reoxygenation

To study the effect of extracellular acidosis on apoptosis and necrosis during ischemia and reoxygenation, we exposed human post-mitotic NT2-N neurones to oxygen and glucose deprivation (OGD) followed by reoxygenation. In some experiments, pH of the cell medium was lowered to 5.9 during either OGD or reoxygenation or both. Staurosporine, used as a positive control for apoptosis, caused Poly(ADP-ribose)-polymerase (PARP) cleavage and nuclear fragmentation, but no PARP cleavage and little fragmentation were seen after OGD. Low molecular weight DNA fragments were found after staurosporine treatment, but not after OGD. No protective effect of caspase inhibitors was seen after 3 h of OGD and 21 h of reoxygenation, but after 45 h of reoxygenation caspase inhibition induced a modest improvement in 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) cleavage. While acidosis during OGD accompanied by neutral medium during reoxygenation protected the neurones (MTT: 228 +/- 117% of neutral medium, p < 0.001), acidosis during reoxygenation only was detrimental (MTT: 38 +/- 25%, p < 0.01). We conclude that apoptotic mechanisms play a minor role after OGD in NT2-N neurones. The effect of acidosis on neuronal survival depends on the timing of acidosis, as acidosis was protective during OGD and detrimental during reoxygenation.     

Neuroprotective actions of PIKE-L by inhibition of SET proteolytic degradation by asparagine endopeptidase

Ischemia and seizure cause excessive neuronal excitation that is associated with brain acidosis and neuronal cell death. However, the molecular mechanism of acidification-triggered neuronal injury is incompletely understood. Here, we show that asparagine endopeptidase (AEP) is activated under acidic condition, cuts SET, an inhibitor of DNase, and triggers DNA damage in brain, which is inhibited by PIKE-L. SET, a substrate of caspases, was cleaved by acidic cytosolic extract independent of caspase activation. Fractionation of the acidic cellular extract yielded AEP that is required for SET cleavage. We found that kainate provoked AEP activation and SET cleavage at N175, triggering DNA nicking in wild-type, but not AEP null, mice. PIKE-L strongly bound SET and prevented its degradation by AEP, leading to resistance of neuronal cell death. Moreover, AEP also mediated stroke-provoked SET cleavage and cell death in brain. Thus, AEP might be one of the proteinases activated by acidosis triggering neuronal injury during neuroexcitotoxicity or ischemia.     

Conformational Changes in the Lower Palm Domain of ASIC1a Contribute to Desensitization and RFamide Modulation

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated cation channel that contributes to fear and pain as well as neuronal damage following persistent cerebral acidosis. Neuropeptides can affect acid-induced neuronal injury by altering ASIC1a inactivation and/or steady-state desensitization. Yet, exactly how ASIC1a inactivation and desensitization occur or are modulated by peptides is not completely understood. We found that regions of the extracellular palm domain and the β_11-12 linker are important for inactivation and steady-state desensitization of ASIC1a. The single amino acid substitutions L280C and L415C dramatically enhanced the rate of inactivation and altered the pH-dependence of steady-state desensitization. Further, the use of methanethiosulfonate (MTS) reagents suggests that the lower palm region (L280C) undergoes a conformational change when ASIC1a transitions from closed to desensitized. We determined that L280C also displays an altered response to the RFamide peptide, FRRFamide. Further, the presence of FRRFamide limited MTS modification of L280C. Together, these results indicate a potential role of the lower palm domain in peptide modulation and suggest RFamide-related peptides promote conformational changes within this region. These data provide empirical support for the idea that L280, and likely this region of the central vestibule, is intimately involved in channel inactivation and desensitization.     

Acidosis-Induced Dysfunction of Cortical GABAergic Neurons through Astrocyte-Related Excitotoxicity

Background

Acidosis impairs cognitions and behaviors presumably by acidification-induced changes in neuronal metabolism. Cortical GABAergic neurons are vulnerable to pathological factors and their injury leads to brain dysfunction. How acidosis induces GABAergic neuron injury remains elusive. As the glia cells and neurons interact each other, we intend to examine the role of the astrocytes in acidosis-induced GABAergic neuron injury.

Results

Experiments were done at GABAergic cells and astrocytes in mouse cortical slices. To identify astrocytic involvement in acidosis-induced impairment, we induced the acidification in single GABAergic neuron by infusing proton intracellularly or in both neurons and astrocytes by using proton extracellularly. Compared the effects of intracellular acidification and extracellular acidification on GABAergic neurons, we found that their active intrinsic properties and synaptic outputs appeared more severely impaired in extracellular acidosis than intracellular acidosis. Meanwhile, extracellular acidosis deteriorated glutamate transporter currents on the astrocytes and upregulated excitatory synaptic transmission on the GABAergic neurons. Moreover, the antagonists of glutamate NMDA-/AMPA-receptors partially reverse extracellular acidosis-induced injury in the GABAergic neurons.

Conclusion

Our studies suggest that acidosis leads to the dysfunction of cortical GABAergic neurons by astrocyte-mediated excitotoxicity, in addition to their metabolic changes as indicated previously.     

Mild acidosis enhances AMPA receptor-mediated intracellular zinc mobilization in cortical neurons

Overactivation of glutamate receptors and subsequent deregulation of the intraneuronal calcium ([Ca2+]i) levels are critical components of the injurious pathways initiated by cerebral ischemia. Another hallmark of stroke is parenchymal acidosis, and we have previously shown that mild acidosis can act as a switch to decrease NMDAR-dependent neuronal loss while potentiating the neuronal loss mediated by AMPARs. Potentiation of AMPAR-mediated neuronal death in an acidotic environment was originally associated only with [Ca2+]i dyshomeostasis, as assessed by Ca2+ imaging; however, intracellular dyshomeostasis of another divalent cation, Zn2+, has recently emerged as another important co-factor in ischemic neuronal injury. Rises in [Zn2+]i greatly contribute to the fluorescent changes of Ca2+-sensitive fluorescent probes, which also have great affinity for Zn2+. We therefore revisited our original findings (Mcdonald et al., 1998) and investigated if AMPAR-mediated fura-2 signals we observed could also be partially due to [Zn2+]i increases. Fura-2 loaded neuronal cultures were exposed to the AMPAR agonist, kainate, in a physiological buffer at pH 7.4 and then washed either at pH 7.4 or pH 6.2. A delayed recovery of fura-2 signals was observed at both pHs. Interestingly this impaired recovery phase was found to be sensitive to chelation of intracellular Zn2+. Experiments with the Zn2+ sensitive (and Ca2+-insensitive) fluorescent probe FluoZin-3 confirmed the idea that AMPAR activation increases [Zn2+]i, a phenomenon that is potentiated by mild acidosis. Additionally, our results show that selective Ca2+ imaging mandates the use of intracellular heavy metal chelators to avoid confounding effects of endogenous metals such as Zn2+.     

Acidosis Drives Damage-associated Molecular Pattern (DAMP)-induced Interleukin-1 Secretion via a Caspase-1-independent Pathway

The proinflammatory cytokine IL-1β is a key mediator of inflammatory responses that contribute to and exacerbate brain injury. IL-1β is synthesized by microglia in the brain as an inactive precursor (pro-IL-1β). Cleavage of pro-IL-1β to a mature form is stimulated by damage-associated molecular patterns (DAMPs). These DAMPs are sensed by a pattern recognition receptor called NLRP3, which forms an inflammasome, resulting in the activation of caspase-1 and cleavage of pro-IL-1β. To date, regulation of the inflammasome in culture has been studied under normal culture conditions, and it is not known how DAMPs signal under disease relevant conditions such as acidosis. Given the presence of acidosis in pathological states, our objective was to test the hypothesis that acidic conditions modify DAMP-induced IL-1β release from cultured primary mouse glial cells. When LPS-primed glial cells were stimulated with DAMPs under acidic conditions (pH 6.2), the predominant IL-1β form secreted was the 20-kDa rather than the 17-kDa caspase-1-dependent species. Lactic acidosis, induced by the addition of 25 mm lactic acid, also induced the release of 20-kDa IL-1β. This 20-kDa product was produced independently of NLRP3 and caspase-1 but was inhibited by the cathepsin D inhibitor pepstatin A. These data suggest that under disease relevant acidosis, DAMPs and lactic acid induce the secretion of IL-1β independently of the inflammasome. Therapeutic strategies directed to the inhibition of IL-1β processing should therefore consider alternative processing of IL-1β in addition to caspase-1-dependent processing.     

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.     

Protective Effects of Extracellular Acidosis and Blockade of Sodium/Hydrogen Ion Exchange During Recovery from Metabolic Inhibition in Neuronal Tissue Culture

Abstract: Acidosis is a universal response of tissue to ischemia. In the brain, severe acidosis has been linked to worsening of cerebral infarction. However, milder acidosis can have protective effects. As part of our investigations of the therapeutic window in our neuronal tissue culture model of ischemia, we investigated the effects of acidosis during recovery from brief simulated ischemia. Ischemic conditions were simulated in dissociated cortical cultures by metabolic inhibition with potassium cyanide to block oxidative metabolism and 2-deoxyglucose to block glycolysis. Lowering the extracellular pH (pH_e) to 6.2 during metabolic inhibition had no effect on injury, as measured by lactate dehydrogenase release from cultures after 24 h of recovery. Lowering the pH_e during the first hour of recovery, in contrast, had profound protective effects. When the duration of metabolic inhibition was lengthened to 30 min, most of the protective effects of the NMDA receptor antagonist MK-801 were lost. However, the protective effects of acidosis were unchanged. This suggested that the protective effects of extracellular acidosis could be due to more than blockade of NMDA receptors. Intracellular acidosis might be responsible. To test this, recovery of intracellular pH (pH_i) was slowed by incubation with blockers of Na⁺/H⁺ exchangers at normal pH_e. The two compounds tested, dimethylamiloride and harmaline, had protective effects when present during recovery from metabolic inhibition. Measurements of pH_i confirmed that the blockers slowed recovery from intracellular acidosis; more rapid pH_i recovery was correlated with injury. The protective effects of acidosis could be reversed by brief incubation with the protonophore monensin, which rapidly normalized pH_i. These results are the first demonstration of the protective effects of blocking Na⁺/H⁺ exchange in a model of cerebral ischemia. The protective effects of acidosis appear to arise either from suppressing pH-sensitive mechanisms of injury or from blocking sodium entry due to Na⁺/H⁺ exchange.     

Paralemmin-1, a modulator of filopodia induction is required for spine maturation

Dendritic filopodia are thought to participate in neuronal contact formation and development of dendritic spines; however, molecules that regulate filopodia extension and their maturation to spines remain largely unknown. Here we identify paralemmin-1 as a regulator of filopodia induction and spine maturation. Paralemmin-1 localizes to dendritic membranes, and its ability to induce filopodia and recruit synaptic elements to contact sites requires protein acylation. Effects of paralemmin-1 on synapse maturation are modulated by alternative splicing that regulates spine formation and recruitment of AMPA-type glutamate receptors. Paralemmin-1 enrichment at the plasma membrane is subject to rapid changes in neuronal excitability, and this process controls neuronal activity-driven effects on protrusion expansion. Knockdown of paralemmin-1 in developing neurons reduces the number of filopodia and spines formed and diminishes the effects of Shank1b on the transformation of existing filopodia into spines. Our study identifies a key role for paralemmin-1 in spine maturation through modulation of filopodia induction.     

Is neuroprotective efficacy of nNOS inhibitor 7-NI dependent on ischemic intracellular pH?

The purpose of this study was to test the hypothesis that the efficacy of 7-nitroindazole (7-NI), a selective neuronal nitric oxide (NO) synthase (NOS) inhibitor, is pH dependent in vivo during focal cerebral ischemia. Wistar rats underwent 2 h of focal cerebral ischemia under 1% halothane anesthesia. 7-NI, 10 and 100 mg/kg in 0.1 ml/kg DMSO, was administered 30 min before occlusion. Ischemic brain acidosis was manipulated by altering serum glucose concentrations. Confirmation of the effects of these serum glucose manipulations on brain intracellular pH (pH(i)) was confirmed in a group of acute experiments utilizing umbelliferone fluorescence. The animals were euthanized at 72 h for histology. 7-NI significantly (P < 0.05) reduced infarction volume in both the normoglycemic by 93.3% and hyperglycemic animals by 27.5%. In the moderate hypoglycemic animals, the reduction in infarction volume did not reach significance because moderate hypoglycemia in itself dramatically reduced infarction volume. We hypothesize that a mechanism to explain the published discrepancies on the effects of neuronal NOS inhibitors in vivo may be due to the effects by differences in ischemic brain acidosis on the production of NO.     

Stimulation of the plasma membrane Na+/H+ exchanger NHE1 by sustained intracellular acidosis. Evidence for a novel mechanism mediated by the ERK pathway

Activity of the Na+/H+ exchanger (NHE) isoform 1 (NHE1) is increased by intracellular acidosis through the interaction of intracellular H+ with an allosteric modifier site in the transport domain. Additional regulation is achieved via kinase-mediated modulation of the NHE1 regulatory domain. To determine if intracellular acidosis stimulates NHE1 activity solely by the allosteric mechanism, we subjected cultured neonatal rat ventricular myocytes (NRVM) with native NHE1 expression to intracellular acidosis (pHi approximately 6.6) for up to 6 min by transient exposure to NH4Cl and its washout in the presence of NHE inhibition (by zero [Na+]o or the NHE1 inhibitor cariporide) in HCO3- -free medium. After the desired duration of acidosis, NHE was reactivated (by reintroduction of [Na+]o or removal of cariporide), and the rate of recovery of pHi (dpHi/dt) was measured as the index of NHE activity. Regardless of the method used when intracellular acidosis was sustained for > or =3 min, subsequent NHE activity was significantly increased (>4-fold). Similar NHE stimulatory effects of sustained acidosis were observed in adult rat ventricular myocytes and COS-7 cells. Sustained (3 min) intracellular acidosis activated several NHE1 kinases in NRVM, in an in-gel kinase assay using as substrate a glutathione S-transferase fusion protein of the NHE1 regulatory domain. Detailed investigation of ERK and its downstream effector p90RSK, two putative NHE1 kinases, revealed time-dependent activation of both by intracellular acidosis in NRVM. Furthermore, inhibition of MEK1/2 by pretreatment of NRVM with two structurally distinct inhibitors, PD98059 (30 microM) or UO126 (3 microM), inhibited the activation of ERK and p90RSK and abolished the stimulation of NHE activity by sustained (3 min) intracellular acidosis. Our data show that not only the extent but also the duration of intracellular acidosis regulates NHE1 activity and suggest that the stimulatory effect of sustained intracellular acidosis occurs through a novel mechanism mediated by activation of the ERK pathway.     

A putative cation channel, NCA-1, and a novel protein, UNC-80, transmit neuronal activity in C. elegans

Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the α1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons.     

In vivo functional brain imaging and a therapeutic trial of l-arginine in MELAS patients

Background

Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is the most common type of mitochondrial disease and is characterized by stroke-like episodes (SEs), myopathy, lactic acidosis, diabetes mellitus, hearing-loss and cardiomyopathy. The causal hypotheses for SEs in MELAS presented to date are angiopathy, cytopathy and neuronal hyperexcitability. L-arginine (Arg) has been applied for the therapy in MELAS patients.

Scope of review

We will introduce novel in vivo functional brain imaging techniques such as MRI and PET, and discuss the pathogenesis of SEs in MELAS patients. We will further describe here our clinical experience with L-arg therapy and discuss the dual pharmaceutical effects of this drug on MELAS.

Major conclusions

Administration of L-arg to MELAS patients has been successful in reducing neurological symptoms due to acute strokes and preventing recurrences of SEs in the chronic phase. L-Arg has dual pharmaceutical effects on both angiopathy and cytopathy in MELAS.

General significance

In vivo functional brain imaging promotes a better understanding of the pathogenesis and potential therapies for MELAS patients. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.