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.     

RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA<Subscript>2</Subscript> Activation

Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A₂ (cPLA₂), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA₂. Inhibition of RhoA, Rho kinase and cPLA₂ significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA₂ activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA₂. The immunofluorescence staining showed that ROCK₁ or ROCK₂, two isoforms of Rho kinase, was co-localized with cPLA₂ in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK₁ or ROCK₂ bonded directly with cPLA₂ and phospho-cPLA₂. When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA₂ activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA₂ activation.     

The MLK Family Mediates c-Jun N-Terminal Kinase Activation in Neuronal Apoptosis

Neuronal apoptotic death induced by nerve growth factor (NGF) deprivation is reported to be in part mediated through a pathway that includes Rac1 and Cdc42, mitogen-activated protein kinase kinases 4 and 7 (MKK4 and -7), c-Jun N-terminal kinases (JNKs), and c-Jun. However, additional components of the pathway remain to be defined. We show here that members of the mixed-lineage kinase (MLK) family (including MLK1, MLK2, MLK3, and dual leucine zipper kinase [DLK]) are expressed in neuronal cells and are likely to act between Rac1/Cdc42 and MKK4 and -7 in death signaling. Overexpression of MLKs effectively induces apoptotic death of cultured neuronal PC12 cells and sympathetic neurons, while expression of dominant-negative forms of MLKs suppresses death evoked by NGF deprivation or expression of activated forms of Rac1 and Cdc42. CEP-1347 (KT7515), which blocks neuronal death caused by NGF deprivation and a variety of additional apoptotic stimuli and which selectively inhibits the activities of MLKs, effectively protects neuronal PC12 cells from death induced by overexpression of MLK family members. In addition, NGF deprivation or UV irradiation leads to an increase in both level and phosphorylation of endogenous DLK. These observations support a role for MLKs in the neuronal death mechanism. With respect to ordering the death pathway, dominant-negative forms of MKK4 and -7 and c-Jun are protective against death induced by MLK overexpression, placing MLKs upstream of these kinases. Additional findings place the MLKs upstream of mitochondrial cytochrome c release and caspase activation.     

Delayed Neuronal Death After Brief Histotoxic Hypoxia In Vitro

Abstract: The effect of three metabolic inhibitors—iodoacetate, potassium cyanide, and potassium arsenate—on neuronal viability was studied in primary rat cortical and hippocampal CA1 neuronal cultures. Iodoacetate (0.1 m M ) applied for 5 min to 8-day-old cultures resulted in delayed neuronal death within 3–24 h in cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by transient inhibition of energy metabolism to ∼40% and a permanent inhibition of protein synthesis to ∼50%. The inhibition of protein synthesis and the neuronal death were prevented by the free radical scavenger vitamin E but not by the glutamate antagonist MK-801. Removal of calcium during iodoacetate exposure could not protect against toxicity, and there was no increase of intracellular calcium concentration during and shortly after iodoacetate treatment. Cyanide and arsenate produced only partial neuronal degeneration, even at a dose of 10 m M . These observations demonstrate that brief exposure of neurons to low concentrations of iodoacetate produces a delayed type of neuronal death that is not mediated by either calcium or glutamate. The therapeutic effect of vitamin E points to a free-radical mediated injury and suggests that this type of pathology may also be involved in delayed neuronal death after transient energy depletion in vivo.     

Quantitative Analysis of Delayed Neuronal Death in the Hippocampal Subfields of SHRSP and SHR

Transient forebrain ischemia and reperfusion induces delayed neuronal death (DND) in the hippocampal Cornu Ammonis 1 (CA1) subfield of stroke-prone spontaneously hypertensive rat (SHRSP). The vulnerability to DND is potentially related to the genetic susceptibility to stroke in this strain. To elucidate the mechanism of DND in SHRSP, however, it is essential to establish a method for quantitative evaluation of DND, which is not available yet. Male SHRSPs and spontaneously hypertensive rats (SHRs) at 12 weeks of age were used in the experiment. The bilateral common carotid arteries were surgically occluded with aneurysmal clips for 10 min. The brain was taken out 7 days after the experiment of the transient ischemia, and was sliced into serial coronal sections. Quantitative estimation of the number of viable pyramidal cells in the CA1 and CA2/3 subfields was performed based on the stereology with a random and systematic sampling. The transient ischemia and reperfusion (TIR) significantly reduced the number of viable pyramidal cells in CA1 of SHRSP (61000 ± 20100 in TIR vs. 128500 ± 21900 in the sham-operation, P < 0.000001 by Student’s t-test), while no significant difference was observed in SHR (140300 ± 30800 in TIR vs. 128200 ± 16700 in the sham-operation, P = 0.35). Further analysis revealed a dorsal-ventral gradient in the distribution of DND in CA1 of SHRSP with the most severe change in the dorsal area. The quantitative measurement using a stereological method is useful in the precise evaluation of DND in SHRSP. This method can be applied in the studies of effects of medical treatments on the ‘ischemia/reperfusion’ insult.     

The Tyrosine Kinase p56lck Mediates Activation of Swelling-induced Chloride Channels in Lymphocytes

Osmotic cell swelling activates Cl⁻ channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56^lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (I_Cl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of I_Cl−swell is defective in p56^lck-deficient cells. Retransfection of p56^lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of I_Cl−swell. Addition of purified p56^lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56^lck washed into the cytoplasm activates I_Cl−swell in native and p56^lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56^lck, slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56^lck. We conclude that osmotic swelling activates I_Cl−swell in lymphocytes via the tyrosine kinase p56^lck.     

The p75 Neurotrophin Receptor Mediates Neuronal Apoptosis and Is Essential for Naturally Occurring Sympathetic Neuron Death

Abstract. To determine whether the p75 neurotrophin receptor (p75NTR) plays a role in naturally occurring neuronal death, we examined neonatal sympathetic neurons that express both the TrkA tyrosine kinase receptor and p75NTR. When sympathetic neuron survival is maintained with low quantities of NGF or KCl, the neurotrophin brain-derived neurotrophic factor (BDNF), which does not activate Trk receptors on sympathetic neurons, causes neuronal apoptosis and increased phosphorylation of c-jun. Function-blocking antibody studies indicate that this apoptosis is due to BDNF-mediated activation of p75NTR. To determine the physiological relevance of these culture findings, we examined sympathetic neurons in BDNF−/− and p75NTR−/− mice. In BDNF−/− mice, sympathetic neuron number is increased relative to BDNF+/+ littermates, and in p75NTR−/− mice, the normal period of sympathetic neuron death does not occur, with neuronal attrition occurring later in life. This deficit in apoptosis is intrinsic to sympathetic neurons, since cultured p75NTR−/− neurons die more slowly than do their wild-type counterparts. Together, these data indicate that p75NTR can signal to mediate apoptosis, and that this mechanism is essential for naturally occurring sympathetic neuron death.     

Plasmalogens Rescue Neuronal Cell Death through an Activation of AKT and ERK Survival Signaling

Neuronal cells are susceptible to many stresses, which will cause the apoptosis and neurodegenerative diseases. The precise molecular mechanism behind the neuronal protection against these apoptotic stimuli is necessary for drug discovery. In the present study, we have found that plasmalogens (Pls), which are glycerophospholipids containing vinyl ether linkage at sn-1 position, can protect the neuronal cell death upon serum deprivation. Interestingly, caspse-9, but not caspase-8 and caspase-12, was cleaved upon the serum starvation in Neuro-2A cells. Pls treatments effectively reduced the activation of caspase-9. Furthermore, cellular signaling experiments showed that Pls enhanced phosphorylation of the phosphoinositide 3-kinase (PI3K)-dependent serine/threonine-specific protein kinase AKT and extracellular-signal-regulated kinases ERK1/2. PI3K/AKT inhibitor LY294002 and MAPK/ERK kinase (MEK) inhibitor U0126 treatments study clearly indicated that Pls-mediated cell survival was dependent on the activation of these kinases. In addition, Pls also inhibited primary mouse hippocampal neuronal cell death induced by nutrient deprivation, which was associated with the inhibition of caspase-9 and caspase-3 cleavages. It was reported that Pls content decreased in the brain of the Alzheimer’s patients, which indicated that the reduction of Pls content could endanger neurons. The present findings, taken together, suggest that Pls have an anti-apoptotic action in the brain. Further studies on precise mechanisms of Pls-mediated protection against cell death may lead us to establish a novel therapeutic approach to cure neurodegenerative disorders.     

Dopamine Receptor Activation Reorganizes Neuronal Ensembles during Hippocampal Sharp Waves In Vitro

Hippocampal sharp wave (SW)/ripple complexes are thought to contribute to memory consolidation. Previous studies suggest that behavioral rewards facilitate SW occurrence in vivo. However, little is known about the precise mechanism underlying this enhancement. Here, we examined the effect of dopaminergic neuromodulation on spontaneously occurring SWs in acute hippocampal slices. Local field potentials were recorded from the CA1 region. A brief (1 min) treatment with dopamine led to a persistent increase in the event frequency and the magnitude of SWs. This effect lasted at least for our recording period of 45 min and did not occur in the presence of a dopamine D₁/D₅ receptor antagonist. Functional multineuron calcium imaging revealed that dopamine-induced SW augmentation was associated with an enriched repertoire of the firing patterns in SW events, whereas the overall tendency of individual neurons to participate in SWs and the mean number of cells participating in a single SW were maintained. Therefore, dopaminergic activation is likely to reorganize cell assemblies during SWs.     

Calpain I Activity and Its Relationship with Hippocampal Neuronal Death in Pilocarpine-Induced Status Epilepticus Rat Model

This study aims to establish pilocarpine-induced rat model of status epilepticus (SE), observe the activity of calpain I in the rat hippocampus and the subsequent neuronal death, and explore the relationship between calpain I activity and neuronal death in the hippocampus. Fifty-eight adult male Wistar rats were assigned randomly into either control group (n = 8) or epilepsy group (n = 50). SE was induced in the epilepsy group using pilocarpine. Before the injection, the rats were given atropine sulfate to reduce the side effect of pilocarpine. All rats in the seizure group were grouped into either SE or non-SE, depending on whether they developed convulsive seizures. The rats in SE group were treated with chloral hydrate to stop seizures after 60 min. Control animals were treated with the same dose of 0.9 % saline. All rats were monitored for seizures. At 24 h after SE, the rats’ left brain tissues were stained by HE and TUNEL. Neuronal necrosis and apoptosis in the hippocampal CA3 area were observed. Calpain I activity in the right hippocampus was also observed using western blotting. Eighty percent of the rats in the seizure group developed SE, of which 35 % died. No rat died in both the control and non-SE groups. At 24 h after SE, the number of HE-stained neurons decreased (SE group: 55.19 ± 8.23; control group: 102.13 ± 3.73; non-SE group: 101.2 ± 2.86) and the number of TUNEL-positive neurons increased (SE group: 4.91 ± 1.35; non-SE and control group: 0). No obvious changes were observed in the neurons of the control and non-SE group animals. The 76 kDa cleavage of calpain I (the average optical density ratio is 0.096 ± 0.015) emerged in the SE group. Neuronal death has a direct relationship with calpain I activity. There is high success rate and lower death rate for pilocarpine to induce SE. At 24 h after SE, activity of calpain I, neuronal necrosis and apoptosis increased in the hippocampus. Neuronal death has a direct relationship with calpain I activity, which suggests that calpain I plays an important role in neuronal damage during SE.     

Activation of Mitogen-Activated Protein Kinase by Epidermal Growth Factor in Hippocampal Neurons and Neuronal Cell Lines

Abstract: Epidermal growth factor (EGF) functions in a bimodal capacity in the nervous system, acting as a mitogen in neuronal stem cells and a neurotrophic factor in differentiated adult neurons. Thus, it is likely that EGF signal transduction, as well as receptor expression, differs among various cell types and possibly in the same cell type at different stages of development. We used hippocampal neuronal cell lines capable of terminal differentiation to investigate changes in EGF receptor expression, DNA synthesis, and stimulation of mitogen-activated protein (MAP) kinase by EGF before and after differentiation. H19-7, the line that was most representative of hippocampal neurons, was mitogenically responsive to EGF only before differentiation and increased in EGF binding after differentiation. MAP kinase was stimulated by EGF in both undifferentiated and differentiated cells, as well as in primary hippocampal cultures treated with either EGF or glutamate. These results indicate that the activation of MAP kinase by EGF is an early signaling event in both mitotic and postmitotic neuronal cells. Furthermore, these studies demonstrate the usefulness of hippocampal cell lines as a homogeneous neuronal system for studies of EGF signaling or other receptor signaling mechanisms in the brain.     

NADPH Oxidase NOX4 Mediates Stellate Cell Activation and Hepatocyte Cell Death during Liver Fibrosis Development

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2^−/−/p19^ARF−/−, Stat3^Δhc/Mdr2^−/−) and a model of experimental induced fibrosis (CCl₄) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes.     

Functions and Modulation of Neuronal SK Channels

Small conductance (SK) channels are calcium-activated potassium channels that, when cloned in 1996, were thought solely to contribute to the afterhyperpolarisation that follows action potentials, and to control repetitive firing patterns of neurons. However, discoveries over the past few years have identified novel roles for SK channels in controlling dendritic excitability, synaptic transmission and synaptic plasticity. More recently, modulation of SK channel calcium sensitivity by casein kinase 2, and of SK channel trafficking by protein kinase A, have been demonstrated. This article will discuss recent findings regarding the function and modulation of SK channels in central neurons.     

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.     

Neurotransmitter Modulation of Neuronal Calcium Channels

There are many different calcium channels expressed in the mammalian nervous system, but N-type and P/Q-type calcium channels appear to dominate the presynaptic terminals of central and peripheral neurons. The neurotransmitter-induced modulation of these channels can result in alteration of synaptic transmission. This review highlights the mechanisms by which neurotransmitters affect the activity of N-type and P/Q-type calcium channels. The inhibition of these channels by voltage-dependent and voltage-independent mechanisms is emphasized because of the wealth of information available on the intracellular mediators and on the effect of these pathways on the single-channel gating.