Levetiracetam protects hippocampal neurons in culture against hypoxia-induced injury

Many experimental studies indicate that some antiepileptic drugs possess neuroprotective properties in varied models of neuronal injury. Levetiracetam is a second-generation antiepileptic drug with a novel mechanism of action. In the present study, we evaluated the putative neuroprotective effect of levetiracetam on primary hippocampal cultures at seven day in vitro. Cell death was induced by incubation of neural cultures in hypoxic conditions over 24 hours. Neuronal injury was assessed by morphometric investigation of death/total ratio of neurons in light microscopy using Trypan blue staining and by evaluation of lactate dehydrogenase (LDH) release in the culture medium. Our results indicate that pre-conditioning of hippocampal cultures with high concentrations of levetiracetam (100 μM and 300 μM) protects neurons against hypoxia-induced death. Two-fold higher number of neurons remained viable as compared to control cultures without drug. Lack of neuroprotective action of the drug on hippocampal neural cultures was observed, when a low concentration (10 μM) of levetiracetam was used.     

Leptin protects hippocampal CA1 neurons against ischemic injury

Leptin is an adipose hormone with well characterized roles in regulating food intake and energy balance. A novel neuroprotective role for leptin has recently been discovered; however, the underlying mechanisms are not clearly defined. The purpose of this study was to determine whether leptin protects against delayed neuronal cell death in hippocampal CA1 following transient global cerebral ischemia in rats and to study the signaling mechanism responsible for the neuroprotective effects of leptin. Leptin receptor antagonist, protein kinase inhibitors and western blots were used to assess the molecular signaling events that were altered by leptin after ischemia. The results revealed that intracerebral ventricle infusion of leptin markedly increased the numbers of survival CA1 neurons in a dose-dependent manner. Infusion of a specific leptin antagonist 10 min prior to transient global ischemia abolished the pro-survival effects of leptin, indicating the essential role of leptin receptors in mediating this neuroprotection. Both the Akt and extracellular signal-related kinase 1/2 (ERK1/2) signaling pathways appear to play a critical role in leptin neuroprotection, as leptin infusion increased the phosphorylation of Akt and ERK1/2 in CA1. Furthermore, pharmacological inhibition of either pathway compromised the neuroprotective effects of leptin. Taken together, the results suggest that leptin protects against delayed ischemic neuronal death in the hippocampal CA1 by maintaining the pro-survival states of Akt and ERK1/2 MAPK signaling pathways.     

Dehydroepiandrosterone protects hippocampal neurons against neurotoxin-induced cell death: mechanism of action

Dehydroepiandrosterone (DHEA), an adrenal cortex hormone secreted in large quantities in humans, protects cells of the clonal mouse hippocampal cell line HT-22 against the excitatory amino acid glutamate (5 mM), and amyloid beta-protein (2 microM) toxicity in a dose-dependent manner with optimum protection obtained at 5 microM concentration of DHEA. The protective effects of DHEA appear to be specific in that other related steroids and metabolites of DHEA, such as 5-androstene-3beta,17beta-diol, etiocholan-3alpha-ol-17-one, etiocholan-3beta-ol-17-one, testosterone, and 5alpha-androstane-3, 17-dione, offered no protection even at 50 microM concentrations. In addition, using immunocytochemical techniques, we observed that 20 hr of treatment with 5 mM glutamate remarkably increased glucocorticoid receptor (GR) nuclear localization in neuronal cells. Interestingly, 5 microM DHEA treatment for 24 hr, followed by 5 mM glutamate treatment for 20 hr almost completely reversed the copious nuclear localization of GR observed by glutamate treatment alone. Results obtained suggest that DHEA protects hippocampal neurons, at least in part, by its antiglucocorticoid action via decreasing hippocampal cells nuclear GR levels.     

Apelin, an endogenous neuronal peptide, protects hippocampal neurons against excitotoxic injury

Several G protein-coupled receptors (GPCRs) mediate neuronal cell migration and survival upon activation by their native peptide ligands but activate death-signaling pathways when activated by certain non-native ligands. In cultured neurons, we recently described expression of the unique seven-transmembrane (7TM) -G protein-coupled receptor, APJ, which is also strongly expressed in neurons in the brain and various cell types in other tissues. We now demonstrate that the endogenous APJ peptide ligand apelin activates signaling pathways in rat hippocampal neurons and modulates neuronal survival. We found that (i) both APJ and apelin are expressed in hippocampal neurons; (ii) apelin peptides induce phosphorylation of the cell survival kinases AKT and Raf/ERK-1/2 in hippocampal neurons; and (iii) apelin peptides protect hippocampal neurons against NMDA receptor-mediated excitotoxicity, including that induced by human immunodeficiency virus type 1. Thus, apelin/APJ signaling likely represents an endogenous hippocampal neuronal survival response, and therefore apelin should be further investigated as a potential neuroprotectant against hippocampal injury.     

Fasudil protects hippocampal neurons against hypoxia-reoxygenation injury by suppressing microglial inflammatory responses in mice

Rho kinase (ROCK) may play an important role in regulating biological events of cells, including proliferation, differentiation and survival/death. Blockade of ROCK promotes axonal regeneration and neuron survival in vivo and in vitro, thereby exhibiting potential clinical applications in spinal cord damage and stroke. Our previous studies have demonstrated that Fasudil, a selective ROCK inhibitor, induced neuroprotection in vitro. Here we used an in vivo model of hypoxia/reoxygenation (H/R) injury to examine the neuroprotective effect of Fasudil, and explore its possible mechanism(s) in vivo. H/R resulted in the loss of hippocampal neurons, accompanied by increased apoptosis of neurons in hippocampus. The expression of ROCK II and activity of ROCK in the brain were increased after H/R, and located only in microglia, but not in astrocytes and neurons. The administration of Fasudil inhibited the activity of ROCK in brain tissue and cultured microglia, and protected hippocampal neurons against H/R injury. Further immunohistochemical analysis and cytokine determination revealed that Fasudil inhibited inducible nitric oxide synthase immunoreactivity in microglia and pro-inflammatory factors in brain tissue after H/R, which is consistent with the observation wherein Fasudil reduced the pro-inflammatory factors nitric oxide, IL-1β, IL-6 and TNF-, and increased anti-inflammatory factor IL-10 in cultured microglia under normoxic or hypoxic conditions. Our results indicate that inhibition of ROCK by Fasudil may represent a useful therapeutic perspective by inhibiting microglial inflammatory responses in the CNS.     

Dopamine protects neurons against glutamate-induced excitotoxicity

Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders including stroke, trauma and neurodegenerative disease. Loss of calcium homeostasis is a key mediator of glutamate-induced cell death. The neurotransmitter dopamine (DA) is known to modulate calcium signalling, and here we show that it can do so in response to physiological concentrations of glutamate. Furthermore, DA is able to protect neurons from glutamate-induced cell death at pathological concentrations of glutamate. We demonstrate that DA has a novel role in preventing delayed calcium deregulation in cortical, hippocampal and midbrain neurons. The effect of DA in abolishing glutamate excitotoxicity can be induced by DA receptor agonists, and is abolished by DA receptor antagonists. Our data indicate that the modulation of glutamate excitotoxicity by DA is receptor-mediated. We postulate that DA has a major physiological function as a safety catch to restrict the glutamate-induced calcium signal, and thereby prevent glutamate-induced cell death in the brain.     

A P-glycoprotein protects Caenorhabditis elegans against natural toxins.

P-glycoproteins can cause resistance of mammalian tumor cells to chemotherapeutic drugs. They belong to an evolutionarily well-conserved family of ATP binding membrane transporters. Four P-glycoprotein gene homologs have been found in the nematode Caenorhabditis elegans; this report describes the functional analysis of two. We found that PGP-3 is expressed in both the apical membrane of the excretory cell and in the apical membrane of intestinal cells, whereas PGP-1 is expressed only in the apical membrane of the intestinal cells and the intestinal valve. By transposon-mediated deletion mutagenesis we generated nematode strains with deleted P-glycoprotein genes and found that the pgp-3 deletion mutant, but not the pgp-1 mutant, is sensitive to both colchicine and chloroquine. Our results suggest that soil nematodes have P-glycoproteins to protect themselves against toxic compounds made by plants and microbes in the rhizosphere.     

YhbO protects cells against multiple stresses

YhbO is a member of the DJ-1/ThiJ/Pfp1 superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1. A yhbO-disrupted mutant of Escherichia coli is highly sensitive to oxidative, thermal, UV, and pH stresses, and the putative nucleophilic cysteine C104 of YhbO is required for stress resistance. These results suggest that YhbO affects a central process in stress management.     

Dimethyl sulphoxide protects cells against polypeptide toxins and poliovirus.

The effect of dimethyl sulphoxide and other cryoprotective compounds on the sensitivity of cells to polypeptide toxins and to poliovirus was tested. In the presence of these compounds, which all affect membrane fluidity, the cells were protected against the toxic proteins and against poliovirus. The large protection obtained was not due to reduced binding and endocytosis of the toxins. Apparently, the cryoprotective compounds interfere with the entry of toxins and of the poliovirus genome across the cell membrane.     

Lycopene protects against trimethyltin-induced neurotoxicity in primary cultured rat hippocampal neurons by inhibiting the mitochondrial apoptotic pathway

Lycopene is a potent free radicals scavenger with demonstrated protective efficacy in several experimental models of oxidative damage. Trimethyltin (TMT) is an organotin compound with neurotoxic effects on the hippocampus and other limbic structures and is used to model neurodegenerative diseases targeting these brain areas. Oxidative stress is widely accepted as a central pathogenic mechanism of TMT-mediated neurotoxicity. The present study investigated whether the plant carotene lycopene protects against TMT-induced neurotoxicity in primary cultured rat hippocampal neurons. Lycopene pretreatment improved cell viability in TMT-treated hippocampal neurons and inhibited neuronal apoptosis. Microfluorometric imaging revealed that lycopene inhibited the accumulation of mitochondria-derived reactive oxygen species (ROS) during TMT exposure. Moreover, lycopene ameliorated TMT-induced activation of the mitochondrial permeability transition pore (mPTP) and the concomitant depolarization of the mitochondrial membrane potential (ΔΨ_m). Consequently, cytochrome c release from the mitochondria and ensuing caspase-3 activation were markedly reduced. These findings reveal that lycopene protects against TMT-induced neurotoxicity by inhibiting the mitochondrial apoptotic pathway. The anti-apoptotic effect of lycopene on hippocampal neurons highlights the therapeutic potential of plant-derived antioxidants against neurodegenerative diseases.     

Prion protein protects human neurons against Bax-mediated apoptosis

The function of the cellular prion protein (PrP) is still poorly understood. We present here an unprecedented role for PrP against Bax-mediated neuronal apoptosis and show that PrP potently inhibits Bax-induced cell death in human primary neurons. Deletion of four octapeptide repeats of PrP (PrPDeltaOR) and familial D178N and T183A PrP mutations completely or partially eliminate the neuroprotective effect of PrP. PrP remains anti-apoptotic despite truncation of the glycosylphosphatidylinositol (GPI) anchor signal peptide, indicating that the neuroprotective form of PrP does not require the abundant cell surface GPI-anchored PrP. Our results implicate PrP as a potent and novel anti-apoptotic protein against Bax-mediated cell death.     

Iodoacetate protects hippocampal neurons against excitotoxic and oxidative injury: involvement of heat-shock proteins and Bcl-2.

Mild metabolic stress may increase resistance of neurons in the brain to subsequent, more severe insults, as demonstrated by the ability of ischemic pre-conditioning and dietary restriction to protect neurons in experimental models of stroke- and age-related neurodegenerative disorders. In the present study we employed iodoacetic acid (IAA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, to test the hypothesis that inhibition of glycolysis can protect neurons. Pre-treatment of cultured hippocampal neurons with IAA can protect them against cell death induced by glutamate, iron and trophic factor withdrawal. Surprisingly, protection occurred with concentrations of IAA (2-200 nM) much lower than those required to inhibit glycolysis. Pre-treatment with IAA results in suppression of oxyradical production and stabilization of mitochondrial function in neurons after exposure to oxidative insults. Levels of the stress heat-shock proteins HSP70 and HSP90, and of the anti-apoptotic protein Bcl-2, were increased in neurons exposed to IAA. Our data demonstrate that IAA can stimulate cytoprotective mechanisms within neurons, and suggest the possible use of IAA and related compounds in the prevention and/or treatment of neurodegenerative conditions.     

Uncovering multiple axonal targeting pathways in hippocampal neurons

Neuronal polarity is, at least in part, mediated by the differential sorting of membrane proteins to distinct domains, such as axons and somata/dendrites. We investigated the pathways underlying the subcellular targeting of NgCAM, a cell adhesion molecule residing on the axonal plasma membrane. Following transport of NgCAM kinetically, surprisingly we observed a transient appearance of NgCAM on the somatodendritic plasma membrane. Down-regulation of endocytosis resulted in loss of axonal accumulation of NgCAM, indicating that the axonal localization of NgCAM was dependent on endocytosis. Our data suggest the existence of a dendrite-to-axon transcytotic pathway to achieve axonal accumulation. NgCAM mutants with a point mutation in a crucial cytoplasmic tail motif (YRSL) are unable to access the transcytotic route. Instead, they were found to travel to the axon on a direct route. Therefore, our results suggest that multiple distinct pathways operate in hippocampal neurons to achieve axonal accumulation of membrane proteins.     

Mitochondrial peroxiredoxin-3 protects hippocampal neurons from excitotoxic injury in vivo

Mitochondria are involved in excitotoxic damage of nerve cells. Following the breakdown of the calcium-buffering ability of mitochondria, mitochondrial calcium overload induces reactive oxygen species (ROS) bursts that produce free radicals and open permeability transition pores, ultimately leading to neuronal cell death. In the present study, we focused on a mitochondrial antioxidant protein, peroxiredoxin-3 (Prx-3), to investigate the mechanism by which toxic properties of ROS were up-regulated in mitochondria of damaged nerve cells. Immunohistochemical analysis revealed that Prx-3 protein exists in mitochondria of rat hippocampus, whereas we found a significant decrease in Prx-3 mRNA and protein levels associated with an increase in nitrated proteins in the rat hippocampus injured by microinjection of ibotenic acid. Furthermore, in vivo adenoviral gene transfer of Prx-3 completely inhibited protein nitration and markedly reduced gliosis, a post-neuronal cell death event. Since mitochondrial Prx-3 seems to be neuroprotective against oxidative insults, our findings suggest that Prx-3 up-regulation might be a useful novel approach for the management of neurodegenerative diseases.     

Parkin protects against mitochondrial toxins and beta-amyloid accumulation in skeletal muscle cells

Mutations in the ubiquitin ligase-encoding Parkin gene have been implicated in the pathogenesis of autosomal recessive Parkinson disease. Outside of the central nervous system, Parkin is prominently expressed in skeletal muscle. We have found accumulations of Parkin protein in skeletal muscle biopsies taken from patients with inclusion body myositis, a degenerative disorder in which intramyofiber accumulations of the beta-amyloid peptide are pathognomonic. In comparing primary cultures of skeletal muscle derived from parkin knock-out and wild-type mice, we have found the absence of parkin to result in greater sensitivity to mitochondrial stressors rotenone and carbonyl cyanide 3-chlorophenylhydrazone, without any alteration in sensitivity to calcium ionophore or hydrogen peroxide. Utilizing viral expression constructs coding for the Alzheimer disease and inclusion body myositis-linked beta-amyloid precursor protein and for its metabolic byproducts A beta42 and C100, we found that parkin knock-out muscle cells are also more sensitive to the toxic effects of intracellular A beta. We also constructed a lentiviral system to overexpress wild-type Parkin and have shown that boosting the levels of parkin expression in normal skeletal muscle cultures provides substantial protection against both mitochondrial toxins and overexpressed beta-amyloid. Correspondingly, exogenous Parkin significantly lowered A beta levels. These data support the hypothesis that in myocytes parkin has dual properties in the maintenance of skeletal muscle mitochondrial homeostasis and in the regulation of A beta levels.     

Alpha-synuclein protects cerebellar granule neurons against 6-hydroxydopamine-induced death

The physiological role of alpha-synuclein, a protein found enriched in intraneuronal deposits characterizing Parkinson's disease, is debated. While its aggregation is usually considered linked to neuropathology, its normal function may be related to fundamental processes of synaptic transmission and plasticity. By using antisense oligonucleotide strategy, we report in this study that alpha-synuclein silencing in cultured cerebellar granule cells results in widespread death of these neurons, thus demonstrating an essential pro-survival role of the protein towards primary neurons. To study alpha-synuclein expression and processing in a Parkinson's disease model of neurotoxicity, we exposed differentiated cultures of cerebellar granule neurons to toxic concentrations of 6-hydroxydopamine (6-OHDA). This resulted in neuronal death accompanied by a decrease of the monomeric form of alpha-synuclein, which was due to both decreased synthesis of the protein and its increased mono-ubiquitination accompanied by nuclear translocation. The essential neuroprotective role of alpha-synuclein was confirmed by the fact that subchronic valproate treatment, which increases alpha-synuclein expression and prevents its nuclear translocation in cerebellar granule cells exposed to 6-OHDA, significantly protected these neurons from 6-OHDA insult. In agreement with the pro-survival role of alpha-synuclein in this model, subtoxic concentrations of alpha-synuclein antisense oligonucleotides, aggravated 6-OHDA toxicity towards granule neurons. Our results demonstrate that normal alpha-synuclein expression is essential for the viability of primary neurons and that its pro-survival role is abolished in 6-OHDA neurotoxic challenge. These results are relevant to more precisely define the role of alpha-synuclein in neuronal cells and to better understand its putative involvement in neurodegeneration.     

Squalestatin cures prion-infected neurons and protects against prion neurotoxicity

A key feature of prion diseases is the conversion of the normal, cellular prion protein (PrP(C)) into beta-sheet-rich disease-related isoforms (PrP(Sc)), the deposition of which is thought to lead to neurodegeneration. In the present study, the squalene synthase inhibitor squalestatin reduced the cholesterol content of cells and prevented the accumulation of PrP(Sc) in three prion-infected cell lines (ScN2a, SMB, and ScGT1 cells). ScN2a cells treated with squalestatin were also protected against microglia-mediated killing. Treatment of neurons with squalestatin resulted in a redistribution of PrP(C) away from Triton X-100 insoluble lipid rafts. These effects of squalestatin were dose-dependent, were evident at nanomolar concentrations, and were partially reversed by cholesterol. In addition, uninfected neurons treated with squalestatin became resistant to the otherwise toxic effect of PrP peptides, a synthetic miniprion (sPrP106) or partially purified prion preparations. The protective effect of squalestatin, which was reversed by the addition of water-soluble cholesterol, correlated with a reduction in prostaglandin E(2) production that is associated with neuronal injury in prion disease. These studies indicate a pivotal role for cholesterol-sensitive processes in controlling PrP(Sc) formation, and in the activation of signaling pathways associated with PrP-induced neuronal death.     

Brain-derived neurotrophic factor protects cultured rat hippocampal neurons from aluminum maltolate neurotoxicity

Aluminum is environmentally abundant but not an essential trace element. Although there is increasing evidence suggesting the implication of aluminum in the pathogenesis of Alzheimer's disease, it is still controversial. We found and report here that aluminum maltolate, a stable and hydrophilic aluminum complex, causes death of primary cultured rat hippocampal neurons in a time- and dose-dependent manner. Degenerated neurons were TUNEL-positive. Immunohistochemical detection of synapsin I and microtubule associated protein 2 revealed the synapse loss between neurons intoxicated by aluminum maltolate. To explore the mechanism underlying its neurotoxicity, we administered various pharmacological compounds prior to the application of aluminum maltolate, and found that brain-derived neurotrophic factor (BDNF) markedly attenuated the neurotoxicity. Furthermore, aluminum maltolate inhibited the elevation of intracellular calcium levels caused by BDNF. Our results suggest the involvement of BDNF in the molecular mechanism underlying neurotoxicity induced by aluminum maltolate.