Youting Lin

Ghrelin protects against cell death of hippocampal neurons in pilocarpine-induced seizures in rats

Ghrelin, a 28-amino-acid peptide, is mainly secreted by the stomach. Evidence has shown ghrelin to have neuroprotective effects. However, whether ghrelin protects hippocampal neurons against cell death in pilocarpine-induced seizures is unknown. We used Nissl staining to show that ghrelin attenuated the neuronal loss caused by pilocarpine-induced seizures in the hippocampus. Ghrelin exerted the protective action through regulating the phosphatidylinositol-3-kinase and Akt pathway. Moreover, ghrelin treatment reversed the decreased ratio of Bcl-2 to Bax induced by seizures while inhibiting the activated caspase-3. Ghrelin can inhibit hippocampal neuronal damage caused by pilocarpine-induced seizures, which might have therapeutic value in seizures.

The role of p38 MAPK in valproic acid induced microglia apoptosis

Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, induces apoptosis in microglia, but the underlying mechanism by which microglia apoptosis in response to VPA is not yet known. In this study, we found that the mitochondrial pathway played an important role in VPA-induced apoptosis in both BV-2 microglia and mouse primary microglial cells. In addition, VPA increased the level of phospho-p38 mitogen-activated protein kinase (MAPK), but had no effects on phospho-ERK and phospho-JNK MAPKs. Moreover, p38 inhibitor SB203580 strongly inhibited VPA-induced apoptosis and caspase-3 activation. Taken together, our results clearly demonstrated that VPA could induce apoptosis of microglia via p38 MAPK and mitochondrial apoptosis pathway.

Autophagy is upregulated in rats with status epilepticus and partly inhibited by Vitamin E

Autophagy, a process of bulk degradation of cellular constituents through autophagosome-lysosomal pathway, is enhanced during oxidative stress. Whether autophagy is induced during status epilepticus (SE), which induces an excess production of reactive oxygen species (ROS) and leads to oxidative stress, is not established. We also sought to determine if pretreatment with Vitamin E reduced autophagy. We used pilocarpine to elicit SE in rats. The ratio of LC3 II to LC3 I and beclin 1 were used to estimate autophagy. We found that ratio of LC3 II to LC3 I and beclin 1 increased significantly at 2, 8, 16, 24 and 72 h, peaking at 24 h after SE onset. Pretreatment with Vitamin E partially inhibited autophagy by reducing LC3 II formation and de novo synthesis of beclin 1 at 24 h after seizures. These data show that autophagy is increased in rats with pilocarpine-induced SE, and Vitamin E have a partial inhibition on autophagy.

Vitamin E inhibits activated chaperone-mediated autophagy in rats with status epilepticus

Seizures and status epilepticus induce an excessive production of reactive oxygen species leading to oxidative stress. Vitamin E, a classic antioxidant, has a neuroprotective effect on rats with seizures by regulating reactive oxygen species production. The activity of chaperone-mediated autophagy, a selective pathway for the degradation of cytosolic proteins in lysosomes, is enhanced during oxidative stress. Whether chaperone-mediated autophagy is induced during status epilepticus is not established. To address this problem, we used pilocarpine to elicit status epilepticus in rats. Lysosome-associated membrane protein 2a was used to estimate chaperone-mediated autophagy. We showed that compared to control animals, lysosome-associated membrane protein 2a at lysosomal membranes increased significantly in rats at 8 h, 16 h, and 24 h after induction of status epilepticus, which directly correlated with chaperone-mediated autophagy activity. Since reactive oxygen species are believed to be important in the pathogenesis of status epilepticus and are essential for the process of chaperone-mediated autophagy, we also sought to determine if pretreatment with vitamin E reduced chaperone-mediated autophagy. Pretreatment with vitamin E reduced oxidative stress and partially inhibited chaperone-mediated autophagy in brain at 24 h after status epilepticus versus vehicle. Taken together, these data show that chaperone-mediated autophagy is increased in rats with pilocarpine-induced status epilepticus through upregulation of de novo synthesis of lysosome-associated membrane protein 2a. Antioxidants such as vitamin E may partially inhibit activated chaperone-mediated autophagy.

Mitochondrial DNA damage and the involvement of antioxidant defense and repair system in hippocampi of rats with chronic seizures.

In this study, we demonstrated a decreased level of mitochondrial DNA (mtDNA) with a large number of oxidized bases in hippocampi of rats with epilepsy induced by pilocarpine. In order to verify the underlying mechanism of mtDNA impairment, we detected the response of antioxidant defense system and mitochondrial base excision repair (mtBER) pathway. Superoxide dismutase2 (SOD-2) and glutathione (GSH) were significantly decreased in the experimental group, manifesting a decreased capacity of scavenging free radicals. Mitochondrial base excision repair (mtBER) pathway, which is the main repair pathway for the removal of oxidative base modifications, displayed unbalanced expression in epileptic group. DNA polymeraseγ (polγ) increased, while apurinic/apyrimidinic endonuclease (APE1), one of mtBER initiators, decreased in mitochondria in the chronic phase of epileptogenesis. In conclusion, mtDNA was impaired during chronic recurrent seizures, whereas the endogenous antioxidants and the mtBER pathway failed to respond to the elevated mtDNA damage.

Cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated acute epileptic neuronal death in vitro

Intense poly(ADP-ribose) polymerase-1 (PARP-1) activation was implicated as a major cause of caspase-independent cell death in the hippocampal neuronal culture (HNC) model of acute acquired epilepsy (AE). The molecular mechanisms are quite complicated. The linkage among neuronal death, cellular nicotinamide adenine dinucleotide (NAD) levels, apoptosis-inducing factor (AIF) translocation, SIRT1 expression and activity were investigated here. The results showed that PARP-1 over-activation caused by Mg²⁺-free stimuli led to cellular NAD depletion which could block AIF translocation from mitochondria to nucleus and attenuate neuronal death. Also, SIRT1 deacetylase activity was reduced by Mg²⁺-free treatment, accompanied by elevated ratio of neuronal death, which could be rescued by NAD repletion. These data demonstrated that cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated epileptic neuronal death in the HNC model of acute AE.

Role of PI3K/Akt in diazoxide preconditioning against rat hippocampal neuronal death in pilocarpine-induced seizures.

Diazoxide (DZ), a highly selective opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has neuroprotective effects. However, the mechanism of DZ protecting hippocampal neurons against cell death in pilocarpine-induced seizures is unknown. In this study, we investigated DZ attenuating neuronal loss caused by pilocarpine-induced seizures in rat hippocampus. DZ inhibited seizure-induced change in phospho-Akt expression, translocation of apoptosis-inducing factor (AIF), release of cytochrome c (CytC) and caspase-3 activation, which could be abolished by preincubation with 5-hydroxydecanoic acid, an inhibitor of mitoK(ATP). In addition, wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K), attenuated the translocation of AIF, CytC release and caspase-3 activation after seizures. DZ could reduce neuronal death induced by seizures in hippocampus by suppressing the translocation of AIF, CytC release and the activation of caspase-3 via the PI3K/Akt pathway.

Poly(ADP-ribose) polymerase inhibition protects epileptic hippocampal neurons from apoptosis via suppressing Akt-mediated apoptosis-inducing factor translocation in vitro

Inhibition of poly(ADP-ribose) polymerase (PARP) has been proposed to have a neuroprotective effect on hippocampal neurons in animal models of epilepsy. However, the mechanisms of PARP-mediated epileptic neuron apoptosis in vitro are still not thoroughly understood. Therefore, we investigated the effect of PARP inhibition and the underlying mechanisms in the hippocampal neuronal culture model of acquired epilepsy which is generally accepted as the neuronal culture model of spontaneous seizure discharge in vitro. As a result, PARP was activated and the administration of 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), an inhibitor of PARP, significantly decreased the percentage of neuron apoptosis induced by Mg(2+)-free treatment. Western blot and confocal laser scanning microscopy (CLSM) analysis showed that DPQ increased the phosphorylation of Akt and attenuated mitochondria-nucleus translocation of the apoptosis-inducing factor (AIF). Furthermore, wortmannin, an inhibitor of PI-3K, inhibited the translocation of AIF to the nucleus. The results of the present study demonstrated that the inhibition of PARP might have therapeutic value in seizure-induced hippocampal neuron damage in vitro via suppressing Akt-mediated AIF translocation.

Mitochondrial base excision repair pathway failed to respond to status epilepticus induced by pilocarpine

Oxidative damage to mitochondrial DNA (mtDNA) has been implicated as an important mechanism underlying mitochondrial deficiency in epileptic seizures. In focusing on the role of the DNA repair pathway, we determined the response of the mitochondrial base excision repair (mtBER) pathway in pilocarpine-induced status epilepticus (SE) in hippocampi of male Wistar rats. The expression of 8-oxoguanine DNA glycosylase (OGG1) and polymerase gamma (polgamma) was decreased at both the cellular mRNA and mitochondrial protein levels at 3, 9 and 25h after the onset of SE. The mRNA and protein levels of APE1 were maintained, but the mitochondrial protein level decreased at 3 and 9h and recovered at 25h. Therefore, the mtBER pathway failed to respond to SE induced by pilocarpine. The failure of mitochondrial import might be an important factor responsible for the lowered mtBER enzymes in mitochondria. We hypothesize that the down-regulation of mtBER enzymes may aggravate mtDNA damage and mitochondrial deficiency after the onset of SE.

Status epilepticus stimulates peroxisome proliferator-activated receptor γ coactivator 1-α/mitochondrial antioxidant system pathway by a nitric oxide-dependent mechanism.

Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1-α (PGC-1α) is a transcriptional coactivator identified as an upstream regulator of lipid catabolism, mitochondrial number and function. PGC-1α protects neurons against oxidative damage by inducing several members of the mitochondrial antioxidant system such as superoxide dismutase 2 (SOD2) and uncoupling protein 2 (UCP2). Its role in seizure-induced oxidative stress has not been studied. Here we showed that pilocarpine-induced status epilepticus (SE) stimulates the PGC-1α/mitochondrial antioxidant system signaling pathway in the rat hippocampus. Because nitric oxide (NO) is the key factor of mitochondrial biogenesis through the transcriptional induction of PGC-1α, we investigated whether NO is involved in activation of the PGC-1α/mitochondrial antioxidant system after SE. Treatment with the NO synthase (NOS) inhibitor N(G)-nitro-l-argininemethyl ester (l-NAME) attenuated the increased expression of the PGC-1α/mitochondrial antioxidant system after SE and enhanced oxidative stress. These results suggest that SE can induce the PGC-1α/mitochondrial antioxidant system signaling pathway, which may represent a protective mechanism against SE-induced oxidative stress. Furthermore, NO may positively regulate the mitochondrial antioxidant system by inducing PGC-1α in pilocarpine-induced SE.