Pathophysiological Roles of Transient Receptor Potential (Trp) Channels and Zinc Toxicity in Brain Disease.

Maintaining the correct ionic gradient from extracellular to intracellular space via several membrane-bound transporters is critical for maintaining overall cellular homeostasis. One of these transporters is the transient receptor potential (TRP) channel family that consists of six putative transmembrane segments systemically expressed in mammalian tissues. Upon the activation of TRP channels by brain disease, several cations are translocated through TRP channels. Brain disease, especially ischemic stroke, epilepsy, and traumatic brain injury, triggers the dysregulation of ionic gradients and promotes the excessive release of neuro-transmitters and zinc. The divalent metal cation zinc is highly distributed in the brain and is specifically located in the pre-synaptic vesicles as free ions, usually existing in cytoplasm bound with metallothionein. Although adequate zinc is essential for regulating diverse physiological functions, the brain-disease-induced excessive release and translocation of zinc causes cell damage, including oxidative stress, apoptotic cascades, and disturbances in energy metabolism. Therefore, the regulation of zinc homeostasis following brain disease is critical for the prevention of brain damage. In this review, we summarize recent experimental research findings regarding how TRP channels (mainly TRPC and TRPM) and zinc are regulated in animal brain-disease models of global cerebral ischemia, epilepsy, and traumatic brain injury. The blockade of zinc translocation via the inhibition of TRPC and TRPM channels using known channel antagonists, was shown to be neuroprotective in brain disease. The regulation of both zinc and TRP channels may serve as targets for treating and preventing neuronal death.

Engineered Mesenchymal Stem Cells Over-Expressing BDNF Protect the Brain from Traumatic Brain Injury-Induced Neuronal Death, Neurological Deficits, and Cognitive Impairments.

Traumatic brain injury (TBI) causes transitory or permanent neurological and cognitive impairments, which can intensify over time due to secondary neuronal death. However, no therapy currently exists that can effectively treat brain injury following TBI. Here, we evaluate the therapeutic potential of irradiated engineered human mesenchymal stem cells over-expressing brain-derived neurotrophic factor (BDNF), which we denote by BDNF-eMSCs, in protecting the brain against neuronal death, neurological deficits, and cognitive impairment in TBI rats. BDNF-eMSCs were administered directly into the left lateral ventricle of the brain in rats that received TBI damage. A single administration of BDNF-eMSCs reduced TBI-induced neuronal death and glial activation in the hippocampus, while repeated administration of BDNF-eMSCs reduced not only glial activation and delayed neuronal loss but also enhanced hippocampal neurogenesis in TBI rats. In addition, BDNF-eMSCs reduced the lesion area in the damaged brain of the rats. Behaviorally, BDNF-eMSC treatment improved the neurological and cognitive functions of the TBI rats. The results presented in this study demonstrate that BDNF-eMSCs can attenuate TBI-induced brain damage through the suppression of neuronal death and increased neurogenesis, thus enhancing functional recovery after TBI, indicating the significant therapeutic potential of BDNF-eMSCs in the treatment of TBI.

Effects of Pyruvate Kinase M2 (PKM2) Gene Deletion on Astrocyte-Specific Glycolysis and Global Cerebral Ischemia-Induced Neuronal Death.

Ischemic stroke is caused by insufficient blood flow to the brain. Astrocytes have a role in bidirectionally converting pyruvate, generated via glycolysis, into lactate and then supplying it to neurons through astrocyte-neuron lactate shuttle (ANLS). Pyruvate kinase M2 (PKM2) is an enzyme that dephosphorylates phosphoenolpyruvate to pyruvate during glycolysis in astrocytes. We hypothesized that a reduction in lactate supply in astrocyte PKM2 gene deletion exacerbates neuronal death. Mice harboring a PKM2 gene deletion were established by administering tamoxifen to Aldh1l1-CreERT2; PKM2f/f mice. Upon development of global cerebral ischemia, mice were immediately injected with sodium l-lactate (250 mg/kg, i.p.). To verify our hypothesis, we compared oxidative damage, microtubule disruption, ANLS disruption, and neuronal death between the gene deletion and control subjects. We observed that PKM2 gene deletion increases the degree of neuronal damage and impairment of lactate metabolism in the hippocampal region after GCI. The lactate administration groups showed significantly reduced neuronal death and increases in neuron survival and cognitive function. We found that lactate supply via the ANLS in astrocytes plays a crucial role in maintaining energy metabolism in neurons. Lactate administration may have potential as a therapeutic tool to prevent neuronal damage following ischemic stroke.

The Protective Role of Glutathione on Zinc-Induced Neuron Death after Brain Injuries.

Glutathione (GSH) is necessary for maintaining physiological antioxidant function, which is responsible for maintaining free radicals derived from reactive oxygen species at low levels and is associated with improved cognitive performance after brain injury. GSH is produced by the linkage of tripeptides that consist of glutamic acid, cysteine, and glycine. The adequate supplementation of GSH has neuroprotective effects in several brain injuries such as cerebral ischemia, hypoglycemia, and traumatic brain injury. Brain injuries produce an excess of reactive oxygen species through complex biochemical cascades, which exacerbates primary neuronal damage. GSH concentrations are known to be closely correlated with the activities of certain genes such as excitatory amino acid carrier 1 (EAAC1), glutamate transporter-associated protein 3-18 (Gtrap3-18), and zinc transporter 3 (ZnT3). Following brain-injury-induced oxidative stress, EAAC1 function is negatively impacted, which then reduces cysteine absorption and impairs neuronal GSH synthesis. In these circumstances, vesicular zinc is also released into the synaptic cleft and then translocated into postsynaptic neurons. The excessive influx of zinc inhibits glutathione reductase, which inhibits GSH's antioxidant functions in neurons, resulting in neuronal damage and ultimately in the impairment of cognitive function. Therefore, in this review, we explore the overall relationship between zinc and GSH in terms of oxidative stress and neuronal cell death. Furthermore, we seek to understand how the modulation of zinc can rescue brain-insult-induced neuronal death after ischemia, hypoglycemia, and traumatic brain injury.

Acid Sphingomyelinase Inhibitor, Imipramine, Reduces Hippocampal Neuronal Death after Traumatic Brain Injury.

Traumatic brain injury (TBI) broadly degrades the normal function of the brain after a bump, blow, or jolt to the head. TBI leads to the aggravation of pre-existing brain dysfunction and promotes neurotoxic cascades that involve processes such as oxidative stress, loss of dendritic arborization, and zinc accumulation. Acid sphingomyelinase (ASMase) is an enzyme that hydrolyzes sphingomyelin to ceramide in cells. Under normal conditions, ceramide plays an important role in various physiological functions, such as differentiation and apoptosis. However, under pathological conditions, excessive ceramide production is toxic and activates the neuronal-death pathway. Therefore, we hypothesized that the inhibition of ASMase activity by imipramine would reduce ceramide formation and thus prevent TBI-induced neuronal death. To test our hypothesis, an ASMase inhibitor, imipramine (10 mg/kg, i.p.), was administrated to rats immediately after TBI. Based on the results of this study, we confirmed that imipramine significantly reduced ceramide formation, dendritic loss, oxidative stress, and neuronal death in the TBI-imipramine group compared with the TBI-vehicle group. Additionally, we validated that imipramine prevented TBI-induced cognitive dysfunction and the modified neurological severity score. Consequently, we suggest that ASMase inhibition may be a promising therapeutic strategy to reduce hippocampal neuronal death after TBI.

Combined Treatment of Dichloroacetic Acid and Pyruvate Increased Neuronal Survival after Seizure.

During seizure activity, glucose and Adenosine triphosphate (ATP) levels are significantly decreased in the brain, which is a contributing factor to seizure-induced neuronal death. Dichloroacetic acid (DCA) has been shown to prevent cell death. DCA is also known to be involved in adenosine triphosphate (ATP) production by activating pyruvate dehydrogenase (PDH), a gatekeeper of glucose oxidation, as a pyruvate dehydrogenase kinase (PDK) inhibitor. To confirm these findings, in this study, rats were given a per oral (P.O.) injection of DCA (100 mg/kg) with pyruvate (50 mg/kg) once per day for 1 week starting 2 h after the onset of seizures induced by pilocarpine administration. Neuronal death and oxidative stress were assessed 1 week after seizure to determine if the combined treatment of pyruvate and DCA increased neuronal survival and reduced oxidative damage in the hippocampus. We found that the combined treatment of pyruvate and DCA showed protective effects against seizure-associated hippocampal neuronal cell death compared to the vehicle-treated group. Treatment with combined pyruvate and DCA after seizure may have a therapeutic effect by increasing the proportion of pyruvate converted to ATP. Thus, the current research demonstrates that the combined treatment of pyruvate and DCA may have therapeutic potential in seizure-induced neuronal death.

The Inhibition of Zinc Excitotoxicity and AMPK Phosphorylation by a Novel Zinc Chelator, 2G11, Ameliorates Neuronal Death Induced by Global Cerebral Ischemia.

AMP-activated protein kinase (AMPK) is necessary for maintaining a positive energy balance and essential cellular processes such as glycolysis, gene transcription, glucose uptake, and several other biological functions. However, brain injury-induced energy and metabolic stressors, such as cerebral ischemia, increase AMPK phosphorylation. Phosphorylated AMPK contributes to excitotoxicity, oxidative, and metabolic problems. Furthermore, brain disease-induced release of zinc from synaptic vesicles contributes to neuronal damage via mechanisms including ROS production, apoptotic cell death, and DNA damage. For this reason, we hypothesized that regulating zinc accumulation and AMPK phosphorylation is critical for protection against global cerebral ischemia (GCI). Through virtual screening based on the structure of AMPK subunit alpha 2, we identified a novel compound, 2G11. In this study, we verified that 2G11 administration has neuroprotective effects via the blocking of zinc translocation and AMPK phosphorylation after GCI. As a result, we demonstrated that 2G11 protected hippocampal neurons against GCI and OGD/R-derived cellular damage. In conclusion, we propose that AMPK inhibition and zinc chelation by 2G11 may be a promising tool for preventing GCI-induced hippocampal neuronal death.

The Role of Zinc in Axon Formation via the mTORC1 Pathway.

Zinc is an essential micronutrient required for proper function during neuronal development because it can modulate neuronal function and structure. A fully functional description of zinc in axonal processing in the central nervous system remains elusive. Here, we define the role of intracellular zinc in axon formation and elongation, involving the mammalian target of rapamycin complex 1 (mTORC1). To investigate the involvement of zinc in axon growth, we performed an ex vivo culture of mouse hippocampal neurons and administrated ZnCl2 as a media supplement. At 2 days in vitro, the administration of zinc induced the formation of multiple and elongated axons in the ex vivo culture system. A similar outcome was witnessed in callosal projection neurons in a developing mouse brain. Treatment with extracellular zinc activated the mTORC1 signaling pathway in mouse hippocampal neuronal cultures. The zinc-dependent enhancement of neuronal processing was inhibited either by the deactivation of mTORC1 with RAPTOR shRNA or by mTOR-insensitive 4EBP1 mutants. Additionally, zinc-dependent mTORC1 activation enhanced the axonal translation of TC10 and Par3 may be responsible for axonal growth. We identified a promising role of zinc in controlling axonogenesis in the developing brain, which, in turn, may indicate a novel structural role of zinc in the cytoskeleton and developing neurons.

Administration of an Acidic Sphingomyelinase (ASMase) Inhibitor, Imipramine, Reduces Hypoglycemia-Induced Hippocampal Neuronal Death.

Severe hypoglycemia (below 35 mg/dL) appears most often in diabetes patients who continuously inject insulin. To rapidly cease the hypoglycemic state in this study, glucose reperfusion was conducted, which can induce a secondary neuronal death cascade following hypoglycemia. Acid sphingomyelinase (ASMase) hydrolyzes sphingomyelin into ceramide and phosphorylcholine. ASMase activity can be influenced by cations, pH, redox, lipids, and other proteins in the cells, and there are many changes in these factors in hypoglycemia. Thus, we expect that ASMase is activated excessively after hypoglycemia. Ceramide is known to cause free radical production, excessive inflammation, calcium dysregulation, and lysosomal injury, resulting in apoptosis and the necrosis of neurons. Imipramine is mainly used in the treatment of depression and certain anxiety disorders, and it is particularly known as an ASMase inhibitor. We hypothesized that imipramine could decrease hippocampal neuronal death by reducing ceramide via the inhibition of ASMase after hypoglycemia. In the present study, we confirmed that the administration of imipramine significantly reduced hypoglycemia-induced neuronal death and improved cognitive function. Therefore, we suggest that imipramine may be a promising therapeutic tool for preventing hypoglycemia-induced neuronal death.

Transient Global Ischemia-Induced Brain Inflammatory Cascades Attenuated by Targeted Temperature Management.

Sudden cardiac arrest leads to a significantly increased risk of severe neurological impairment and higher mortality rates in survivors due to global brain tissue injury caused by prolonged whole-body ischemia and reperfusion. The brain undergoes various deleterious cascading events. Among these damaging mechanisms, neuroinflammation plays an especially crucial role in the exacerbation of brain damage. Clinical guidelines indicate that 33 °C and 36 °C are both beneficial for targeted temperature management (TTM) after cardiac arrest. To clarify the mechanistic relationship between TTM and inflammation in transient global ischemia (TGI) and determine whether 36 °C produces a neuroprotective effect comparable to 33 °C, we performed an experiment using a rat model. We found that TTM at 36 °C and at 33 °C attenuated neuronal cell death and apoptosis, with significant improvements in behavioral function that lasted for up to 72 h. TTM at 33 °C and 36 °C suppressed the propagation of inflammation including the release of high mobility group box 1 from damaged cells, the activation and polarization of the microglia, and the excessive release of activated microglia-induced inflammatory cytokines. There were equal neuroprotective effects for TTM at 36 °C and 33 °C. In addition, hypothermic complications and should be considered safe and effective after cardiac arrest.

The Effects of Atorvastatin on Global Cerebral Ischemia-Induced Neuronal Death.

(1) Background and Purpose: Global cerebral ischemia-induced severe hypoxic brain damage is one of the main causes of mortality and long-term neurologic disability even after receiving early blood reperfusion. This study aimed to test the hypothesis that atorvastatin potentially has neuroprotective effects in global cerebral ischemia (GCI). (2) Methods: We performed two sets of experiments, analyzing acute (1-week) and chronic (4-week) treatments. For the vehicle (Veh) and statin treatments, 1 mL of 0.9% saline and 5 mg/kg of atorvastatin (ATOR) were administered orally. For histological analysis, we used the following staining protocols: Fluoro-Jade B and NeuN, 4-hydroxynonenal, CD11b and GFAP, IgG, SMI71, and vWF. Finally, we evaluated the cognitive function with a battery of behavioral tests. (3) Results: The GCI-ATOR group showed significantly reduced neuronal death, oxidative stress, inflammation, and BBB disruption compared with the GCI-Veh group. Moreover, the GCI-ATOR group showed decreased endothelial damage and VV proliferation and had significantly improved cognitive function compared with the GCI-Veh group in both models. (4) Conclusions: ATOR has neuroprotective effects and helps recover the cognitive function after GCI in rats. Therefore, administration of atorvastatin may be a therapeutic option in managing GCI after CA.

Reinforced-hydrogel encapsulated hMSCs towards brain injury treatment by trans-septal approach.

Encapsulated stem cells in various biomaterials have become a potentially promising cell transplantation strategy in the treatment of various neurologic disorders. However, there is no ideal cell delivery material and method for clinical application in brain diseases. Here we show silk fibroin (SF)-based hydrogel encapsulated engineered human mesenchymal stem cells (hMSCs) to overproduce brain-derived neurotrophic factor (BDNF) (BDNF-hMSC) is an effective approach to treat brain injury through trans-septal cell transplantation in the rat model. In this study, we observed SF induced sustained BDNF production by BDNF-hMSC both in 2D (9.367 ± 1.969 ng/ml) and 3D (7.319 ± 0.1025 ng/ml) culture conditions for 3 days. Through immunohistochemistry using α-tubulin, BDNF-hMSCs showed a significant increased average neurite length of co-cultured neuro 2a (N2a) cells, suggested that BDNF-hMSCs induced neurogenesis in vitro. Encapsulated BDNF-hMSC, pre-labeled with the red fluorescent dye PKH-26, exhibited intense fluorescence up to 14 days trans-septal transplantation, indicated excellent viability of the transplanted cells. Compared to the vehicle-treated, encapsulated BDNF- hMSC demonstrated significantly increased BDNF level both in the sham-operated and injured hippocampus (Hip) through immunoblot analysis after 7 days implantation. Transplantation of the encapsulated BDNF-hMSC promoted neurological functional recovery via significantly reduced neuronal death in the Hip 7 days post-injury. Using magnetic resonance imaging (MRI) analysis, we demonstrated that encapsulated BDNF-hMSC reduced lesion area significantly at 14 and 21 days in the damaged brain following trans-septal implantation. This stem cell transplantation approach represents a critical set up towards brain injury treatment for clinical application.

Zinc in the Brain: Friend or Foe?

Zinc is a trace metal ion in the central nervous system that plays important biological roles, such as in catalysis, structure, and regulation. It contributes to antioxidant function and the proper functioning of the immune system. In view of these characteristics of zinc, it plays an important role in neurophysiology, which leads to cell growth and cell proliferation. However, after brain disease, excessively released and accumulated zinc ions cause neurotoxic damage to postsynaptic neurons. On the other hand, zinc deficiency induces degeneration and cognitive decline disorders, such as increased neuronal death and decreased learning and memory. Given the importance of balance in this context, zinc is a biological component that plays an important physiological role in the central nervous system, but a pathophysiological role in major neurological disorders. In this review, we focus on the multiple roles of zinc in the brain.

Effects of Cerebrolysin on Hippocampal Neuronal Death After Pilocarpine-Induced Seizure.

Epilepsy is one of the most common and severe brain diseases. The exact cause of epilepsy is unclear. Epilepsy often occurs following brain damage, such as traumatic brain injury (TBI) and ischemia. Cerebrolysin is a porcine brain peptide that is a unique neurotropic and neuroprotective agent. Cerebrolysin has been reported to increase neuroprotective effects after TBI, ischemia, and other CNS diseases. However, the effects of cerebrolysin on seizures are not known. Therefore, this study aimed to investigate the effects of neuropeptide cerebrolysin on neuronal death in the hippocampus after a seizure. To confirm the effects of cerebrolysin, we used a pilocarpine-induced seizure animal model. Cerebrolysin (2.5 ml/kg, i.p., once per day for 7 days) was immediately injected after a seizure induction. After 1 week, we obtained brain tissues and performed staining to histologically evaluate the potentially protective effects of cerebrolysin on seizure-induced neuronal death in the hippocampus. We found that cerebrolysin decreased hippocampal neuronal death after a seizure. In addition, an increase in brain-derived neurotrophic factor (BDNF) was confirmed through Western blot analysis to further support our hypothesis. Therefore, the present study suggests that the administration of cerebrolysin can be a useful therapeutic tool for preventing neuronal death after a seizure.

Effects of Transient Receptor Potential Cation 5 (TRPC5) Inhibitor, NU6027, on Hippocampal Neuronal Death after Traumatic Brain Injury.

Traumatic brain injury (TBI) can cause physical, cognitive, social, and behavioral changes that can lead to permanent disability or death. After primary brain injury, translocated free zinc can accumulate in neurons and lead to secondary events such as oxidative stress, inflammation, edema, swelling, and cognitive impairment. Under pathological conditions, such as ischemia and TBI, excessive zinc release, and accumulation occurs in neurons. Based on previous research, it hypothesized that calcium as well as zinc would be influx into the TRPC5 channel. Therefore, we hypothesized that the suppression of TRPC5 would prevent neuronal cell death by reducing the influx of zinc and calcium. To test our hypothesis, we used a TBI animal model. After the TBI, we immediately injected NU6027 (1 mg/kg, intraperitoneal), TRPC5 inhibitor, and then sacrificed animals 24 h later. We conducted Fluoro-Jade B (FJB) staining to confirm the presence of degenerating neurons in the hippocampal cornus ammonis 3 (CA3). After the TBI, the degenerating neuronal cell count was decreased in the NU6027-treated group compared with the vehicle-treated group. Our findings suggest that the suppression of TRPC5 can open a new therapeutic window for a reduction of the neuronal death that may occur after TBI.

The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity.

Transient receptor potential melastatin 7 (TRPM7) is an ion channel that mediates monovalent cations out of cells, as well as the entry of divalent cations, such as zinc, magnesium, and calcium, into the cell. It has been reported that inhibitors of TRPM7 are neuroprotective in various neurological diseases. Previous studies in our lab suggested that seizure-induced neuronal death may be caused by the excessive release of vesicular zinc and the subsequent accumulation of zinc in the neurons. However, no studies have evaluated the effects of carvacrol and 2-aminoethoxydiphenyl borate (2-APB), both inhibitors of TRPM7, on the accumulation of intracellular zinc in dying neurons following seizure. Here, we investigated the therapeutic efficacy of carvacrol and 2-APB against pilocarpine-induced seizure. Carvacrol (50 mg/kg) was injected once per day for 3 or 7 days after seizure. 2-APB (2 mg/kg) was also injected once per day for 3 days after seizure. We found that inhibitors of TRPM7 reduced seizure-induced TRPM7 overexpression, intracellular zinc accumulation, and reactive oxygen species production. Moreover, there was a suppression of oxidative stress, glial activation, and the blood-brain barrier breakdown. In addition, inhibitors of TRPM7 remarkably decreased apoptotic neuron death following seizure. Taken together, the present study demonstrates that TRPM7-mediated zinc translocation is involved in neuron death after seizure. The present study suggests that inhibitors of TRPM7 may have high therapeutic potential to reduce seizure-induced neuron death.

Transient Receptor Potential Melastatin 2 (TRPM2) Inhibition by Antioxidant, N-Acetyl-l-Cysteine, Reduces Global Cerebral Ischemia-Induced Neuronal Death.

A variety of pathogenic mechanisms, such as cytoplasmic calcium/zinc influx, reactive oxygen species production, and ionic imbalance, have been suggested to play a role in cerebral ischemia induced neurodegeneration. During the ischemic state that occurs after stroke or heart attack, it is observed that vesicular zinc can be released into the synaptic cleft, and then translocated into the cytoplasm via various cation channels. Transient receptor potential melastatin 2 (TRPM2) is highly distributed in the central nervous system and has high sensitivity to oxidative damage. Several previous studies have shown that TRPM2 channel activation contributes to neuroinflammation and neurodegeneration cascades. Therefore, we examined whether anti-oxidant treatment, such as with N-acetyl-l-cysteine (NAC), provides neuroprotection via regulation of TRPM2, following global cerebral ischemia (GCI). Experimental animals were then immediately injected with NAC (150 mg/kg/day) for 3 and 7 days, before sacrifice. We demonstrated that NAC administration reduced activation of GCI-induced neuronal death cascades, such as lipid peroxidation, microglia and astroglia activation, free zinc accumulation, and TRPM2 over-activation. Therefore, modulation of the TRPM2 channel can be a potential therapeutic target to prevent ischemia-induced neuronal death.

Role of Excitatory Amino Acid Carrier 1 (EAAC1) in Neuronal Death and Neurogenesis After Ischemic Stroke.

Although there have been substantial advances in knowledge regarding the mechanisms of neuron death after stroke, effective therapeutic measures for stroke are still insufficient. Excitatory amino acid carrier 1 (EAAC1) is a type of neuronal glutamate transporter and considered to have an additional action involving the neuronal uptake of cysteine, which acts as a crucial substrate for glutathione synthesis. Previously, our lab demonstrated that genetic deletion of EAAC1 leads to decreased neuronal glutathione synthesis, increased oxidative stress, and subsequent cognitive impairment. Therefore, we hypothesized that reduced neuronal transport of cysteine due to deletion of the EAAC1 gene might exacerbate neuronal injury and impair adult neurogenesis in the hippocampus after transient cerebral ischemia. EAAC1 gene deletion profoundly increased ischemia-induced neuronal death by decreasing the antioxidant capacity. In addition, genetic deletion of EAAC1 also decreased the overall neurogenesis processes, such as cell proliferation, differentiation, and survival, after cerebral ischemia. These studies strongly support our hypothesis that EAAC1 is crucial for the survival of newly generated neurons, as well as mature neurons, in both physiological and pathological conditions. Here, we present a comprehensive review of the role of EAAC1 in neuronal death and neurogenesis induced by ischemic stroke, focusing on its potential cellular and molecular mechanisms.

An Inhibitor of the Sodium-Hydrogen Exchanger-1 (NHE-1), Amiloride, Reduced Zinc Accumulation and Hippocampal Neuronal Death after Ischemia.

Acidosis in the brain plays an important role in neuronal injury and is a common feature of several neurological diseases. It has been reported that the sodium-hydrogen exchanger-1 (NHE-1) is a key mediator of acidosis-induced neuronal injury. It modulates the concentration of intra- and extra-cellular sodium and hydrogen ions. During the ischemic state, excessive sodium ions enter neurons and inappropriately activate the sodium-calcium exchanger (NCX). Zinc can also enter neurons through voltage-gated calcium channels and NCX. Here, we tested the hypothesis that zinc enters the intracellular space through NCX and the subsequent zinc accumulation induces neuronal cell death after global cerebral ischemia (GCI). Thus, we conducted the present study to confirm whether inhibition of NHE-1 by amiloride attenuates zinc accumulation and subsequent hippocampus neuronal death following GCI. Mice were subjected to GCI by bilateral common carotid artery (BCCA) occlusion for 30 min, followed by restoration of blood flow and resuscitation. Amiloride (10 mg/kg, intraperitoneally (i.p.)) was immediately injected, which reduced zinc accumulation and neuronal death after GCI. Therefore, the present study demonstrates that amiloride attenuates GCI-induced neuronal injury, likely via the prevention of intracellular zinc accumulation. Consequently, we suggest that amiloride may have a high therapeutic potential for the prevention of GCI-induced neuronal death.

Zinc transporter 3 modulates cell proliferation and neuronal differentiation in the adult hippocampus.

The subgranular zone of the dentate gyrus is a subregion of the hippocampus that has two uniquely defining features; it is one of the most active sites of adult neurogenesis as well as the location where the highest concentrations of synaptic zinc are found, the mossy fiber terminals. Therefore, we sought to investigate the idea that vesicular zinc plays a role as a modulator of hippocampal adult neurogenesis. Here, we used ZnT3-/- mice, which are depleted of synaptic-vesicle zinc, to test the effect of targeted deletion of this transporter on adult neurogenesis. We found that this manipulation reduced progenitor cell turnover as well as led to a marked defect in the maturation of newborn cells that survive in the DG toward a neuronal phenotype. We also investigated the effects of zinc (ZnCl2 ), n-acetyl cysteine (NAC), and ZnCl2 plus 2NAC (ZN) supplement on adult hippocampal neurogenesis. Compared with ZnCl2 or NAC, administration of ZN resulted in an increase in proliferation of progenitor cells and neuroblast. ZN also rescued the ZnT3 loss-associated reduction of neurogenesis via elevation of insulin-like growth factor-1 and ERK/CREB activation. Together, these findings reveal that ZnT3 plays a highly important role in maintaining adult hippocampal neurogenesis and supplementation by ZN has a beneficial effect on hippocampal neurogenesis, as well as providing a therapeutic target for enhanced neuroprotection and repair after injury as demonstrated by its ability to prevent aging-dependent cognitive decline in ZnT3-/- mice. Therefore, the present study suggests that ZnT3 and vesicular zinc are essential for adult hippocampal neurogenesis.