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DFNA2 is a progressive deafness caused by mutations in the voltage-activated potassium channel KCNQ4. Hearing loss develops with age from a mild increase in the hearing threshold to profound deafness. Studies using transgenic mice for Kcnq4 expressed in a mixed background demonstrated the implication of outer hair cells at the initial phase. However, it could not explain the last phase mechanisms of the disease. Genetic backgrounds are known to influence disease expressivity. To unmask the cause of profound deafness phenotype, we backcrossed the Kcnq4 knock-out allele to the inbred strain C3H/HeJ and investigated inner and outer hair cell and spiral ganglion neuron degeneration across the lifespan. In addition to the already reported outer hair cell death, the C3H/HeJ strain also exhibited inner hair cell and spiral ganglion neuron death. We tracked the spatiotemporal survival of cochlear cells by plotting cytocochleograms and neuronal counts at different ages. Cell loss progressed from basal to apical turns with age. Interestingly, the time-course of cell degeneration was different for each cell-type. While for outer hair cells it was already present by week 3, inner hair cell and neuronal loss started 30 weeks later. We also established that outer hair cell loss kinetics slowed down from basal to apical regions correlating with KCNQ4 expression pattern determined in wild-type mice. Our findings indicate that KCNQ4 plays differential roles in each cochlear cell-type impacting in their survival ability. Inner hair cell and spiral ganglion neuron death generates severe hearing loss that could be associated with the last phase of DFNA2., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Parkinson's disease (PD) is a common neurodegenerative disease that is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The proapoptotic BH3-only protein Bim has been reported to be involved in dopaminergic neurodegeneration of experimental PD. However, an in situ expression profile of Bim in PD has not been performed, and the cell types of which Bim accounts for PD pathogenesis is unclear. Here, we report with in situ observations that Bim is transcriptionally induced in the dopaminergic neurons of the SNpc in 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. To investigate the precise role of Bim in the dopaminergic neurons in parkinsonian neuronal death, we obtained dopaminergic neuron-specific Bim null (Bim △Dat ) mice. Bim △Dat mice are shown to be resistant to MPTP-induced neurotoxicity, confirming that the induction of Bim in dopaminergic neurons is responsible for parkinsonian neurodegeneration. Furthermore, we demonstrated with dopaminergic neuron-specific c-Jun knockout (c-Jun △Dat ) that the transcriptional upregulation of Bim of nigral dopaminergic neurons was c-Jun-dependent and further validated the detrimental role of c-Jun in dopaminergic neurodegeneration. Together, these data specify that c-Jun-mediated Bim upregulation in nigral dopaminergic neurons contributes to parkinsonian neurodegeneration., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Elevated levels of cholesterol (hypercholesterolemia) and homocysteine (hyperhomocysteinemia, HHcy) in blood have been linked with the pathology of Parkinson's disease. However, the impact of their combined effect on brain is unknown. The present study aims to investigate the effect of HHcy on dopaminergic neurons in brain of mice with hypercholesterolemia. Mice were subjected to a high-cholesterol diet for 12 weeks to develop hypercholesterolemia, and were administered with homocysteine (250 mg/kg, b.w., i.p., 60 days) daily starting from 24th day of the high-cholesterol diet for induction of HHcy. The animals were subjected to Parkinsonian motor behavioral tests and sacrificed to estimate the levels of cholesterol, homocysteine and dopamine in brain, and to assess dopaminergic neuronal status. There occurred elevation in cholesterol and homocysteine levels in nigrostriatum of hypercholesterolemic animals with HHcy. Injection of homocysteine in hypercholesterolemic mice exacerbated the motor abnormalities as well as caused depletion of striatal dopamine level significantly, which was supported by a significant decrease in tyrosine hydroxylase (TH) immunoreactivity in striatum. While neither hypercholesterolemia nor HHcy caused significant changes in the number of TH-positive neurons, hypercholesterolemia in combination with HHcy resulted in a significant loss of nigral TH-positive neurons. The results highlighted the involvement of mitochondrial complex-I dysfunction with subsequent generation of hydroxyl radicals for the observed loss of midbrain dopamine neurons in animals receiving the combined treatment. Thus, the findings of the present study pointed out the combined effect of homocysteine and cholesterol toward dopamine neuronal dysfunctions, which has substantial relevance to Parkinson's disease., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is defined by senile plaques, tauopathy and neuronal cell death in specific area of the brain. Recent studies suggest that neurovascular dysfunction may be an integral part of AD pathogenesis, contributing to the onset and development of AD pathologies such as neuronal death, inflammatory response, and breakdown of blood-brain barrier (BBB). In addition, vascular complications caused by age-related metabolic diseases such as diabetes and high blood pressure have high incidence in development of dementia and AD. We previously reported that astrocytes, essential components of BBB, were chronically activated and some deteriorated in the brain of 5xFAD, an amyloid precursor protein/presenilin1 (APP/PS1) transgenic mouse model. Thus, it is rational to investigate if any vascular dysfunction is associated with considerable activation of astrocytes in APP/PS1 mouse model. In this study, we observed that cerebrovascular pathology was associated with large scale of reactive astrocytes and neurodegeneration in an Aβ plague-generating mouse model. Using 5xFAD mouse brains, we demonstrate damaged brain vessels and reduced expression of glucose transporter 1 (GLUT1), the main glucose transporter, and a tight junction protein zonula occludens-1 (ZO-1) of cerebrovascular endothelial cells. This vascular pathology was closely associated with astrocytic deterioration and neuronal loss due to buildup of Aβ plaques in 5xFAD mouse brains., (Copyright © 2018 IBRO. All rights reserved.)

Wobbler mice are experimental models for amyotrophic lateral sclerosis. As such they show motoneuron degeneration, motor deficits, and astrogliosis and microgliosis of the spinal cord. Additionally, Wobbler mice show increased plasma, spinal cord and brain corticosterone levels and focal adrenocortical hyperplasia, suggesting a pathogenic role for glucocorticoids in this disorder. Considering this endocrine background, we examined whether the glucocorticoid receptor (GR) modulator CORT 113176 prevents spinal cord neuropathology of Wobblers. CORT 113176 shows high affinity for the GR, with low or null affinity for other steroid receptors. We employed five-month-old genotyped Wobbler mice that received s.c. vehicle or 30 mg/kg/day for 4 days of CORT 113176 dissolved in sesame oil. The mice were used on the 4th day, 2 h after the last dose of CORT 113176. Vehicle-treated Wobbler mice presented vacuolated motoneurons, increased glial fibrillary acidic protein (GFAP)+ astrocytes and decreased glutamine synthase (GS)+ cells. There was strong neuroinflammation, shown by increased staining for IBA1+ microglia and CD11b mRNA, enhanced expression of tumor necrosis factor-α, its cognate receptor TNFR1, toll-like receptor 4, the inducible nitric oxide synthase, NFkB and the high-mobility group box 1 protein (HMGB1). Treatment of Wobbler mice with CORT 113176 reversed the abnormalities of motoneurons and down-regulated proinflammatory mediators and glial reactivity. Expression of glutamate transporters GLT1 and GLAST mRNAs and GLT1 protein was significantly enhanced over untreated Wobblers. In summary, antagonism of GR with CORT 113176 prevented neuropathology and showed anti-inflammatory and anti-glutamatergic effects in the spinal cord of Wobbler mice., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

The dysregulation of posttranslational modifications of the microtubule-associated protein (MAP) tau plays a key role in Alzheimer's disease (AD) and related disorders. Thus, we have previously shown that beta amyloid (Aβ)-induced neurotoxicity was mediated, at least in part, by tau cleavage into the tau 45-230 fragment. However, the mechanisms underlying the toxicity of tau 45-230 remain unknown. To get insights into such mechanisms, we first determined the subcellular localization of this tau fragment in hippocampal neurons. Tau 45-230 was easily detectable in cell bodies and processes extended by these neurons. In addition, cell extraction experiments performed using Triton X-100 and saponin showed that a pool of tau 45-230 was associated with the cytoskeleton and the cytoskeleton plus membrane-bound organelles, respectively, in cultured hippocampal neurons. Furthermore, they suggested that these associations were independent of the presence of full-length tau. We also assessed whether this tau fragment could alter axonal transport. Our results indicated that tau 45-230 significantly reduced the number of organelles transported along hippocampal axons. This altered axonal transport did not correlate with changes in the total number of organelles present in these cells or in motor protein levels. Together these results suggested that tau 45-230 could exert its toxic effects by partially blocking axonal transport along microtubules thus contributing to the early pathology of AD., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Mutations in the Pejvakin (Pjvk) gene cause autosomal recessive hearing loss DFNB59 with audiological features of auditory neuropathy spectrum disorder (ANSD) or cochlear dysfunction. The precise mechanisms underlying the variable clinical phenotypes of DFNB59 remain unclear. Here, we demonstrate that mice with conditional ablation of the Pjvk gene in all sensory hair cells or only in outer hair cells (OHCs) show similar auditory phenotypes with early-onset profound hearing loss. By contrast, loss of Pjvk in adult OHCs causes a slowly progressive hearing loss associated with OHC degeneration and delayed loss of inner hair cells (IHCs), indicating a primary role for pejvakin in regulating OHC function and survival. Consistent with this model, synaptic transmission at the IHC ribbon synapse is largely unaffected in sirtaki mice that carry a C-terminal deletion mutation in Pjvk. Using the C-terminal domain of pejvakin as bait, we identified in a cochlear cDNA library ROCK2, an effector for the small GTPase Rho, and the scaffold protein IQGAP1, involved in modulating actin dynamics. Both ROCK2 and IQGAP1 associate via their coiled-coil domains with pejvakin. We conclude that pejvakin is required to sustain OHC activity and survival in a cell-autonomous manner likely involving regulation of Rho signaling., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Efr3a has been found to be involved in the functional maintenance and structural degeneration of sensory and motor nervous tissues. Our previous data have suggested that Efr3a may be associated with the initiation of the degeneration of spiral ganglion neurons (SGNs). In this study, we used Efr3a knockdown (Efr3a KD) and Efr3a overexpression (Efr3a OE) mice to determine the role of Efr3a in age-related hearing loss. Measurements of hearing thresholds showed that Efr3a had little or no influence on the hearing threshold at all frequencies in adult mice, whereas in an early stage of senescence, Efr3a reduction resulted in better hearing function, especially at 10 and 12months of age. No significant differences were observed in hair cell loss among the three groups until 14months. The number of surviving hair cells in the OE mice was lower than that in the KD mice. As indicated by the density of SGNs in the upper basal turn, the Efr3a OE mice displayed earlier and more severe degeneration than the KD mice. In addition, the p-Akt levels in the cochlear spiral ganglions were higher in adult Efr3a KD mice than in WT and OE mice, although there was no difference in Akt expression among the three groups. Our study suggests that down-regulation of Efr3a might improve hearing function and alleviate the degeneration of SGNs in an early stage of senescence, probably via enhancing the basal expression of activated Akt., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Thrombin and activated protein C (aPC) bound to the endothelial protein C receptor (EPCR) both activate protease-activated receptor 1 (PAR1) generating either harmful or protective signaling respectively. In the present study we examined the localization of PAR-1 and EPCR and thrombin activity in Schwann glial cells of normal and crushed peripheral nerve and in Schwannoma cell lines. In the sciatic crush model nerves were excised 1h, 1, 4, and 7days after the injury. Schwannoma cell lines produced high levels of prothrombin which is converted to active thrombin and expressed both EPCR and PAR-1 which co-localized. In the injured sciatic nerve thrombin levels were elevated as early as 1h after injury, reached their peak 1day after injury which was significantly higher (24.4±4.1mU/ml) compared to contralateral uninjured nerves (2.6±7mU/ml, t-test p<0.001) and declined linearly reaching baseline levels by day 7. EPCR was found to be located at the microvilli of Schwann cells at the node of Ranvier and in cytoplasm surrounding the nucleus. Four days after sciatic injury, EPCR levels increased significantly (57,785±16602AU versus 4790±1294AU in the contralateral uninjured nerves, p<0.001 by t-test) mainly distal to the site of injury, where axon degeneration is followed by proliferation of Schwann cells which are diffusely stained for EPCR. EPCR seems to be located to cytoplasmic component of Schwann cells and not to compact myelin component, and is highly increased following injury., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Motor neuron (MN) diseases are characterized by progressive cell degeneration, and excitotoxicity has been postulated as a causal factor. Using two experimental procedures for inducing excitotoxic spinal MN degeneration in vivo, by acute and chronic overactivation of α-amino-3-hydroxy-5-methyl-4-isoxazoleacetic acid (AMPA) receptors, we characterized the time course of the neuropathological changes. Electron transmission microscopy showed that acute AMPA perfusion by microdialysis caused MN swelling 1.5h after surgery and lysis with membrane rupture as early as 3h; no cleaved caspase 3 was detected by immunochemistry. Chronic AMPA infusion by osmotic minipumps induced a slow degeneration process along 5days, characterized by progressive changes: endoplasmic reticulum swelling, vacuolization of cytoplasm, vacuole fusion and cell membrane rupture. Quantification of these ultrastructural alterations showed that the increase of vacuolated area was at the expense of the nuclear area. Caspase 3 cleavage was observed since the first day of AMPA infusion. We conclude that acute AMPA-induced excitotoxicity induces MN loss by necrosis, while the progress of degeneration induced by chronic infusion is slow, starting with an early apoptotic process followed by necrosis. In both the acute and chronic procedures a correlation could be established between the loss of MN by necrosis, but not by caspase 3-linked apoptosis, and severe motor deficits and hindlimb paralysis. Our findings are relevant for understanding the mechanisms of neuron death in degenerative diseases and thus for the design of pharmacological therapeutic strategies., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

The World Health Organization has predicted that by 2040 neurodegenerative diseases will overtake cancer to become the world's second leading cause of death after cardiovascular disease. This has sparked the development of several European and American brain research initiatives focusing on elucidating the underlying cellular and molecular mechanisms of neurodegenerative diseases. Connexin (Cx) and pannexin (Panx) membrane channel proteins are conduits through which neuronal, glial, and vascular tissues interact. In the brain, this interaction is highly critical for homeostasis and brain repair after injury. Understanding the molecular mechanisms by which these membrane channels function, in health and disease, might be particularly influential in establishing conceptual frameworks to develop new therapeutics against Cx and Panx channels. This review focuses on current insights and emerging concepts, particularly the impact of connexin43 and pannexin1, under neuroprotective and neurodegenerative conditions within the context of astrocytes., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

We previously reported that rolling Nagoya mice carrying a mutation in the α1 subunit of the Cav2.1 channel protective from ischemia- and kainate-induced neuronal damage. However, the protective effect of this mutation and its relationship to brain injury recovery have not been examined. To examine the relationship between Cav2.1 channel function and brain injury, we induced cryogenic brain damage in homozygous rolling Nagoya (rol/rol), control wild-type (+/+), ω-agatoxin IVA-pretreated +/+ (ω-aga +/+), and ω-agatoxin IVA-post-treated +/+ (ω-aga-post-treated +/+) mice. We measured the lesion area, blood brain-barrier permeability and performed immunohistochemistry and western blot analysis. The lesions of rol/rol and ω-aga +/+ mice were significantly smaller than those observed in +/+ mice at both day 1 and day 7 after injury. Similar results were shown in blood-brain barrier permeability. We observed more reactive astrogliosis in +/+ mice than in rol/rol or ω-aga +/+ mice. rol/rol and ω-aga +/+ mice had fewer degenerating cells due to cryogenic injury than did +/+ mice at both day 1 and day 7. ω-Aga-post-treated +/+ mice 24h after injury were sacrificed on day 7. The lesions were smaller in ω-aga-post-treated +/+ mice than those in vehicle-treated +/+ mice. We also examined phosphorylated p38 (pp38) at the injured site. ω-Aga-post-treated +/+ mouse brain slices showed weak pp38 signal; vehicle-treated +/+ mouse brain slices were pp38-positive. These findings demonstrate that the mutant Cav2.1 channel exerts a protective effect against cryogenic brain injury in rolling Nagoya mice. Our results indicate that inhibitors of the Cav2.1-dependent p38 signaling cascade would be useful as therapeutic agents in the treatment of brain injury., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

High levels of homocysteine (Hcy), known as hyperhomocysteinemia are associated with neurovascular diseases. H2S, a metabolite of Hcy, has potent anti-oxidant and anti-inflammatory activities; however, the effect of H2S has not been explored in Hcy (IC)-induced neurodegeneration and neurovascular dysfunction in mice. Therefore, the present study was designed to explore the neuroprotective role of H2S on Hcy-induced neurodegeneration and neurovascular dysfunction. To test this hypothesis we employed wild-type (WT) males ages 8-10 weeks, WT+artificial cerebrospinal fluid (aCSF), WT+Hcy (0.5 μmol/μl) intracerebral injection (IC, one time only prior to NaHS treatment), WT+Hcy+NaHS (sodium hydrogen sulfide, precursor of H2S, 30 μmol/kg, body weight). NaHS was injected i.p. once daily for the period of 7 days after the Hcy (IC) injection. Hcy treatment significantly increased malondialdehyde, nitrite level, acetylcholinestrase activity, tumor necrosis factor-alpha, interleukin-1 beta, glial fibrillary acidic protein, inducible nitric oxide synthase, endothelial nitric oxide synthase and decreased glutathione level indicating oxidative-nitrosative stress and neuroinflammation as compared to control and aCSF-treated groups. Further, increased expression of neuron-specific enolase, S100B and decreased expression of (post-synaptic density-95, synaptosome-associated protein-97) synaptic protein indicated neurodegeneration. Brain sections of Hcy-treated mice showed damage in the cortical area and periventricular cells. Terminal deoxynucleotidyl transferase-mediated, dUTP nick-end labeling-positive cells and Fluro Jade-C staining indicated apoptosis and neurodegeneration. The increased expression of matrix metalloproteinase (MMP) MMP9, MMP2 and decreased expression of tissue inhibitor of metalloproteinase (TIMP) TIMP-1, TIMP-2, tight junction proteins (zonula occulden 1) in Hcy-treated group indicate neurovascular remodeling. Interestingly, NaHS treatment significantly attenuated Hcy-induced oxidative stress, memory deficit, neurodegeneration, neuroinflammation and cerebrovascular remodeling. The results indicate that H2S is effective in providing protection against neurodegeneration and neurovascular dysfunction., (Copyright © 2013 IBRO. All rights reserved.)

Reactive astrogliosis, a feature of neuro-inflammation is induced by a number of endogenous mediators including cytokines. Despite interleukin-1 beta (IL-1β) stands out as the major inducer of this process, the underlying mechanism and its role on neuronal viability remain elusive. We investigated in human astrocytoma cells and the rat brain striatum, the role of the nuclear factor-kB (NF-kB) intracellular Ca(2+) concentration ([Ca(2+)]i) calmodulin (CaM) and extracellular regulated mitogen-activated protein kinases (ERK1/2) in IL-1β-induced expression of glial fibrillary acidic protein (GFAP) and neuronal apoptosis associated to a brain trauma. Cell data showed that IL-1β (1 ng/ml) increased NF-kB, pERK1/2 and GFAP expression. Nevertheless, further increase in IL-1β levels reversed progressively these responses. Preventing ERK1/2 activation with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]-butadiene antagonized IL-1β-induced GFAP expression while inhibiting selectively nuclear translocation of NF-kB with caffeic-acid phenethyl-ester down-regulated both ERK1/2 and GFAP expression induced by IL-1β. The GFAP response was also prevented by antagonizing selectively increase in [Ca(2+)]i, CaM activity or inducible nitric oxide synthase expression with respectively ryanodine plus 2-aminoethoxydiphenyl-borate, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride and N-[(3-(aminomethyl)-phenyl]methyl]-ethanimidamide dihydrochloride. Data in vivo supported these findings and showed that GFAP expression induced by IL-1β (50 ng/ml) correlated with attenuated glial scar formation and reduced neuronal apoptosis. Our data identified the NF-kB/Ca(2+)-CaM/ERK signaling pathway as a novel in vivo key regulator of IL-1β-induced astrogliosis which may represent a potential target in neurodegeneration., (Copyright © 2013. Published by Elsevier Ltd.)

The purpose of this paper is to study the effect of smilagenin (SMI) (PYM50028), a sapogenin compound originally identified from Chinese medicinal herb, on dopamine neurons and locomotor ability in aged rats. Experiments were carried out on young and aged Sprague-Dawley rats, which were daily administered with either SMI (18mg/kg/day) or vehicle (0.5% sodium carboxymethycellulose [CMCNa]). Open-field and rotarod performance tests revealed that behavioral ability was impaired in aged rats and was improved by oral administration of smilagenin. Immunohistochemical data showed that tyrosine hydroxylase (TH)-positive neuron numbers in the substantia nigra pars compacta (unbiased stereological counting) were altered with aging and were increased by smilagenin treatment. Likewise, the dopamine receptor density and the striatal dopamine transporter (DAT) density ((125)I-2β-carbomethoxy-3β-(4-iodophenyl)-N-(3-fluoropropyl) nortropane [(125)I-FP-CIT] autoradiography) were significantly lowered in aged rats as compared to young rats, and treatment with smilagenin significantly elevated the dopamine receptor and DAT density in aged rats. Furthermore, smilagenin enhances glial cell-derived neurotrophic factor (GDNF) release both in the striatum and midbrain. These results indicate a possible role of smilagenin in the treatment of age-related extrapyramidal disorders., (Copyright © 2013 IBRO. All rights reserved.)

Quinolinic acid (QA)-induced overactivation of N-methyl-d-aspartate receptors yields excitotoxicity, oxidative stress and mitochondrial dysfunction, which altogether contribute to trigger a wide variety of toxic pathways with biochemical, behavioral and neuropathological alterations similar to those observed in Huntington's disease. Noteworthy, in the brains of these patients, increased expression of heme oxygenase-1 (HO-1) levels can be found. It has been proposed that this enzyme can exert a dual role, as it can be either protective or deleterious to the CNS. While some evidence indicates that its overexpression affords cellular anti-oxidant protection due to decreased concentrations of its pro-oxidative substrate heme group, and increased bilirubin levels, other reports established that high HO-1 expression and activity may result in a pro-oxidizing atmosphere due to a release of Fe(2+). In this work, we examined the temporal evolution of oxidative damage to proteins, HO-1 expression, immunoreactivity, total activity, and cell death after 1, 3, 5 and 7 days of an intrastriatal QA infusion (240 nmol/μl). QA was found to induce cellular degeneration, increasing carbonylated proteins and generating a transitory response in HO-1 mRNA, protein content, and immunoreactivity and activity in nerve cells. In order to study the role of HO-1 in the QA-induced cellular death, the tin protoporphyrin IX (SnPP), a well-known HO inhibitor, was administered to rats (30 μmol/kg, i.p.). The administration of SnPP to animals treated with QA inhibited the HO activation, and exacerbated the striatal cell damage induced by QA. Our findings reveal a potential modulatory role of HO-1 in the toxic paradigm evoked by QA in rats. This evidence provides a valuable tool for further approaches on HO-1 regulation in neurotoxic paradigms., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

A compromised protein degradation machinery has been implicated in methamphetamine (MA)-induced neurodegeneration. However, the signaling mechanisms that induce autophagy and ubiquitin-proteasome system (UPS) dysfunction are not well understood. The present study investigates the contributions of protein kinase C delta (PKCδ)-mediated signaling events in MA-induced autophagy, UPS dysfunction, and cell death. Using an in vitro mesencephalic dopaminergic cell culture model, we demonstrate that MA-induced early induction of autophagy is associated with reduction in proteasomal function and concomitant dissipation of mitochondrial membrane potential (MMP), followed by significantly increased PKCδ activation, caspase-3 activation, accumulation of ubiquitin-positive aggregates and microtubule-associated light chain-3 (LC3-II) levels. Interestingly, siRNA-mediated knockdown of PKCδ or overexpression of cleavage-resistant mutant of PKCδ dramatically reduced MA-induced autophagy, proteasomal function, and associated accumulation of ubiquitinated protein aggregates, which closely paralleled cell survival. Importantly, when autophagy was inhibited either pharmacologically (3-MA) or genetically (siRNA-mediated silencing of LC3), the dopaminergic cells became sensitized to MA-induced apoptosis through caspase-3 activation. Conversely, overexpression of LC3 partially protected against MA-induced apoptotic cell death, suggesting a neuroprotective role for autophagy in MA-induced neurotoxicity. Notably, rat striatal tissue isolated from MA-treated rats also exhibited elevated LC3-II, ubiquitinated protein levels, and PKCδ cleavage. Taken together, our data demonstrate that MA-induced autophagy serves as an adaptive strategy for inhibiting mitochondria-mediated apoptotic cell death and degradation of aggregated proteins. Our results also suggest that the sustained activation of PKCδ leads to UPS dysfunction, resulting in the activation of caspase-3-mediated apoptotic cell death in the nigrostriatal dopaminergic system., (Published by Elsevier Ltd.)

The development of drugs that attenuate neurodegeneration is important for the treatment of Alzheimer's disease (AD). We previously found that smilagenin (SMI), a steroidal sapogenin from traditional Chinese medicinal herbs improves memory in animal models, is neither a cholinesterase inhibitor nor a glutamate receptor antagonist, but can significantly elevate the declined muscarinic receptor (M receptor) density. In this article, to clarify whether SMI represents a new approach for treating neurodegeneration disease, we first demonstrate that SMI pretreatment significantly attenuates the neurodegenerative changes induced by beta amyloid 25-35 (Aβ(25-35)) in cultured rat cortical neurons, including decreased cholinergic neuron number, shortened neurite outgrowth length, and declined M receptor density. Brain-derived neurotrophic factor (BDNF) protein levels in the culture medium were also decreased by Aβ(25-35) and significantly elevated by SMI. Parallel experiments revealed that when the trk receptors were inhibited by K252a or the action of BDNF was inhibited by a neutralizing anti-BDNF antibody, the effects of SMI on the Aβ(25-35)-induced neurodegeneration in rat cortical neurons were almost completely abolished. In the all-trans retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells, the BDNF transcription rate measured by a nuclear run-on assay was significantly suppressed by Aβ(25-35) and elevated by SMI, but the BDNF degradation rate measured by half-life determination was unchanged by Aβ(25-35) and SMI. Transcript analysis of the SH-SY5Y cells using quantitative RT-PCR (qRT-PCR) showed that the IV and VI transcripts of BDNF mRNA were significantly decreased by Aβ(25-35) and elevated by SMI. Taken together, we conclude that SMI attenuates Aβ(25-35)-induced neurodegeneration in cultured rat cortical neurons and SH-SY5Y cells mainly through stimulating BDNF mRNA transcription implicating that SMI may represent a novel therapeutic strategy for AD., (Copyright © 2012 IBRO. All rights reserved.)

This study examines the causes of hypothermia and rewarming injury in CA1, CA3, and dentate neurons in rat hippocampal slice cultures. Neuronal death, assessed with propidium iodide or Sytox fluorescence, Fluoro-Jade labeling, and Cresyl Violet staining, depended on the severity and duration of hypothermia. More than 6 h at temperatures less than 12 °C followed by rewarming to 37 °C (profound hypothermia and rewarming, PH/RW) caused swelling and death in large number of neurons in CA1, CA3, and dentate. During PH, [ATP] decreased and [Ca(2+)](I) and extracellular [glutamate] increased, with neuron rupture and nuclear condensation following RW. The data support the hypothesis that neuronal death from PH/RW is excitotoxic, due to ATP loss, glutamate receptor activation and Ca(2+) influx. We found that antagonism of N-methyl-D-aspartate (NMDA) receptors, but not 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid or metabotropic glutamate receptors, decreased neuron death and prevented increases in [Ca(2+)](I) caused by PH/RW. Chelating extracellular Ca(2+) decreased PH/RW injury, but inhibiting L- and T-type voltage-gated Ca(2+) channels, K+ channels, Ca(2+) release from the endoplasmic reticulum, and reverse Na(+)/Ca(2+) exchange did not affect the Ca(2+) changes or cell death. We conclude that the mechanism of PH/RW neuronal injury in hippocampal slices primarily involves intracellular Ca(2+) accumulation mediated by NMDA receptors that activates necrotic, but not apoptotic processes., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Excessive alcohol intake, characteristic of an alcohol use disorder (AUD), results in neurodegeneration as well as cognitive deficits that may recover in abstinence. Neurodegeneration in psychiatric disorders such as AUDs is due to various effects on tissue integrity. Several groups report that alcohol-induced neurodegeneration and recovery include a role for adult neurogenesis. Therefore, the initial purpose of this study was to investigate the effect of alcohol on the temporal profile of neural progenitor cells using the radial glia marker, vimentin, in a model of an AUD. However, striking vimentin expression throughout corticolimbic regions led, instead, to the discovery of a significant gliosis response in this model. Adult male rats were subjected to a 4-day binge model of an AUD and brains harvested for immunohistochemistry at 0, 2, 4, 7, 14, and 28 days following the last dose of ethanol. A prominent increase in vimentin immunoreactivity was apparent at 4 and 7 days post binge that returned to control levels by 14 days in the corticolimbic regions examined. Vimentin-positive cells co-labeled with glial fibrillary acidic protein (GFAP), which suggested that cells were reactive astrocytes. A second experiment supported that increased vimentin was not primarily due to alcohol withdrawal seizures and is more likely due to alcohol-induced cell death. As this gliosis was remarkably distinct in regions where cell death had not previously been reported in this model, adjacent tissue sections were processed for FluoroJade B staining for cell death. FluoroJade B-positive cells were evident immediately following the last ethanol dose as expected, but were significantly elevated in the hippocampal dentate gyrus and CA3 regions and corticolimbic regions from 2 to 7 days post binge. Intriguingly, vimentin labeling of astrogliosis is more widespread than FluoroJade B labeling of cell death, which suggests that 4-day binge ethanol consumption is more damaging than originally realized., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

We have previously demonstrated that glia maturation factor (GMF), a brain-specific protein, isolated, sequenced, and cloned in our laboratory, is a prominent mediator of inflammation in the CNS leading to the death of neurons. In the present study, we demonstrate, for the first time, a significant upregulation of the GMF protein in various regions of Alzheimer's disease (AD) brains compared with age-matched non-demented (ND) control brains. We analyzed AD and ND brain samples by quantitative enzyme-linked immunosorbent assay (ELISA) using a combination of highly specific monoclonal and polyclonal anti-GMF antibodies developed and characterized in our laboratory. For the comparison between ND controls and AD cases, we examined brain tissue from 12 ad cases (ages ranging from 78-92 years) and eight age-matched ND controls (ages ranging from 76-88 years). We observed a significant increase in GMF concentration in entorhinal cortex, parietal cortex, frontal cortex, occipital cortex, perirhinal cortex, and temporal cortex of AD patients. Our results clearly demonstrate that the GMF protein levels are significantly higher in all AD-affected brain regions than in ND controls. The immunohistochemistry analysis revealed co-localization of GMF with amyloid plaques (AP) and neurofibrillary tangles (NFTs) in AD brains. Our results imply that under conditions of neurodegeneration the expression of GMF is significantly upregulated., (Published by Elsevier Ltd.)

Neurons are highly susceptible to oxidative stress and oxidation of cytoskeletal proteins is considered one of the first steps of neurodegeneration. Protein glutathionylation is a key event in the redox regulation of protein function and constitutes a sensor of tissue oxidative stress in patho-physiological conditions. In this study, we analyzed for the first time tubulin glutathionylation and its relation to neurites degeneration. For this purpose, we exposed motoneuronal cells to the physiological oxidant glutathione disulfide (GSSG) and we analyzed the extent and morphology of axonal changes caused by protein glutathionylation in these cells. Then we studied the effect of glutathionylation on the distribution of stable and dynamic microtubules in the same cells. Our results indicate that oxidative stress conditions determined by an increased intracellular level of oxidized glutathione may cause an alteration of the cytoskeleton organization and function leading to axon degeneration. These findings might contribute to understand the sequence of pathogenic events involved in the axonal degeneration that characterizes many diseases of the nervous system associated with oxidative stress., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Parkinson's disease is associated with the loss of dopaminergic neurons in the substantia nigra and decreased striatal dopamine levels. We now report that caffeic acid phenethyl ester (CAPE), an active component of propolis, attenuated dopaminergic neurodegeneration and dopamine loss in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease. The neuroprotective effect of CAPE was associated with marked reductions in inducible nitric oxide synthase (iNOS) and caspase 1 expression. Additionally, CAPE inhibited MPP+-induced neurotoxicity in vitro and directly inhibited MPP+-induced release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Thus, CAPE may have beneficial effects in slowing or preventing the progression of Parkinson's disease and other neurodegenerative disorders., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Although nuclear factor-kappa B (NF-κB) is essential for neuron survival and its activation may protect neuron against oxidative-stresses or ischemia-induced neurodegeneration, NF-κB activation can contribute to inflammatory reaction and apoptotic cell death after brain injury and stroke. However, there are little data concerning the specific pattern of NF-κB phosphorylations in neuronal damage/survival induced by status epilepticus (SE). In the present study, NF-κB phosphorylation showed the cellular specific pattern in responses to SE. p52-S865, p52-Ser869, p65-Ser276, p65-Ser311, p65-Ser468, and p65-Ser529 NF-κB phosphorylation was significantly decreased in the CA1 and CA3 pyramidal cells vulnerable to SE, although neuronal specific nuclear antigen immunoreactivity was strongly detected. In contrast, p65-Ser536 NF-κB phosphorylation was enhanced in these neurons accompanied by TUNEL- and Fluoro-Jade B 244signals. These findings serve as the first comprehensive description of the cellular specific distribution of NF-κB phosphorylation in response to pilocarpine-induced SE in the rat hippocampus, and suggest that enhancement in p65-Ser536 NF-κB phosphorylation may be closely relevant to neuronal vulnerability to SE, while others may be involved in neuronal survival., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background and Purpose: Positron emission tomography (PET) studies in humans have used (11)C-flumazenil (FMZ) to assess neuronal viability after stroke. Here we aimed to study whether (11)C-FMZ binding was sensitive to neuronal damage in the acute phase following ischemia/reperfusion in the rat brain., Experimental Procedures: Transient (2 h followed by reperfusion) and permanent intraluminal middle cerebral artery occlusion was carried out. (11)C-FMZ binding was studied by PET up to 24 h after the onset of ischemia. Tissue infarction was evaluated post-mortem at 24 h. Immunohistochemistry against a neuronal nuclei specific protein (NeuN) was performed to assess neuronal injury., Results: No decrease in (11)C-FMZ binding was detected in the ipsilateral cortex up to 24 h post-ischemia in the model of transient occlusion despite the fact that rats developed cortical and striatal infarction, and neuronal injury was clearly apparent at this time. In contrast, (11)C-FMZ binding was significantly depressed in the ipsilateral cortex at 24 h following permanent ischemia., Conclusions: This finding evidences that (11)C-FMZ binding is not sensitive to neuronal damage on the acute phase of ischemia/reperfusion in the rat brain., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The transcription regulator, neuron-restrictive silencer factor (NRSF), also known as repressor element-1 silencing transcription factor (REST), plays an important role in neurogenesis and various neuronal diseases such as ischaemia, epilepsy, and Huntington's disease. In these disease processes, neuronal loss is associated with abnormal expression and/or localization of NRSF. Previous studies have demonstrated that NRSF regulates the effect of ethanol on neuronal cells in vitro, however, the role of NRSF in ethanol-induced neuronal cell death remains unclear. We generated nrsf conditional knockout mice using the Cre-loxP system to disrupt neuronal expression of nrsf and its truncated forms. At postnatal day 6, ethanol significantly increased the expression of REST4, a neuron-specific truncated form of NRSF, in the brains of wild type mice, and this effect was diminished in nrsf conditional knockout mice. The apoptotic effect of ethanol was pronounced in multiple brain regions of nrsf conditional mutant mice. These results indicate that NRSF, specifically REST4, may protect the developing brain from ethanol, and provide new evidence that NRSF can be a therapeutic target in foetal alcohol syndrome (FAS)., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neuron death due to deprivation of target-derived neurotrophic factors depends on protein synthesis regulated by transcription factor activity. We investigated the content and phosphorylation of activating transcription factor 2 (ATF-2) in axon-damaged retinal ganglion cells of neonatal rats. In the retina of neonatal rats, the ATF-2 protein is predominantly located in the nucleus of the ganglion cells. A gradual loss of the immunoreactivity for ATF-2 occurred after explantation. ATF-2 is phosphorylated early after explantation, with a peak within 3 hours, preceding the peak of cell death that occurs at 18 hours. Both the phosphorylation of ATF-2 and ganglion cell death were blocked by treatment with an inhibitor of c-Jun N-terminal kinase (JNK), whereas an inhibitor of p38 reduced only slightly the rate of ganglion cell death with no effect upon phosphorylation of ATF-2. Inhibitors of phosphatidyl inositol 3 kinase (PI-3K), protein kinase C (PKC) or extracellular regulated kinase (ERK) had no effect. Finally, the inhibitor of JNK blocked the upregulation of both c-Jun and Hrk in the GCL after retinal explantation. The data show that phosphorylation of ATF-2 by JNK is associated with retinal ganglion cell death after axon damage., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Interaction of genetic and environmental factors is likely involved in Parkinson's disease (PD). Mutations and multiplications of alpha-synuclein (α-syn) cause familial PD, and chronic manganese (Mn) exposure can produce an encephalopathy with signs of parkinsonism. We exposed male transgenic C57BL/6J mice expressing human α-syn or the A53T/A30P doubly mutated human α-syn under the tyrosine hydroxylase promoter and non-transgenic littermates to MnCl₂-enriched (1%) or control food, starting at the age of 4 months. Locomotor activity was increased by Mn without significant effect of the transgenes. Mice were sacrificed at the age of 7 or 20 months. Striatal Mn was significantly increased about three-fold in those exposed to MnCl₂. The number of tyrosine hydroxylase positive substantia nigra compacta neurons was significantly reduced in 20 months old mice (-10%), but Mn or transgenes were ineffective (three-way ANOVA with the factors gene, Mn and age). In 7 months old mice, striatal homovanillic acid (HVA)/dopamine (DA) ratios and aspartate levels were significantly increased in control mice with human α-syn as compared to non-transgenic controls (+17 and +11%, respectively); after Mn exposure both parameters were significantly reduced (-16 and -13%, respectively) in human α-syn mice, but unchanged in non-transgenic animals and mice with mutated α-syn (two-way ANOVA with factors gene and Mn). None of the parameters were changed in the 20 months old mice. Single HVA/DA ratios and single aspartate levels significantly correlated across all treatment groups suggesting a causal relationship between the rate of striatal DA metabolism and aspartate release. In conclusion, under our experimental conditions, Mn and human α-syn, wild-type and doubly mutated, did not interact to induce PD-like neurodegenerative changes. However, Mn significantly and selectively interacted with human wild-type α-syn on indices of striatal DA neurotransmission, the neurotransmitter most relevant to PD., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

A number of neurotoxin- and gene-based rodent models of acute neurodegeneration of nigrostriatal dopamine (DA) neurons are used to study Parkinson's disease (PD). The rapid degeneration achieved by many of these current models limits the capacity of the model to develop pathogenic mechanisms and display the various stages of motor degradation representative of the human Parkinsonian condition. Chronic rodent models have been the only ones to reproduce these characteristics, yet do not show correlated progress of DA loss with multiple stepwise behavioral deficits as seen in humans. In the present study, we have developed a progressive model of increasing DA loss and motor dysfunction via progressively increased administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), in the C57Bl/6J mouse. Mice were administered a daily (5 d/wk) dose of MPTP that increased weekly over the course of 4 weeks (4 mg/kg, 8 mg/kg, 16 mg/kg and 32 mg/kg). Each treatment group was tested for exploratory and motor behavioral changes after every week leading up to their final dose, as well as changes in tyrosine hydroxylase immunoreactivity (TH-ir) of the substantia nigra pars compacta (SNpc) and caudate putamen (CPu). We detected a 24% decrease in the mean number of TH-ir SNpc neurons/section after 1 week, and a 62% decrease after 4 weeks as compared to the vehicle group. CPu TH-ir began at a 35% loss after 1 week and increased to a 74% loss after 4 weeks compared to the vehicle group. CPu DA content showed an initial decrease of 20% after 1 week, and a final decrease of 70% following week 4 versus the vehicle group. Free-standing rears (versus wall-assisted rears, in a cylinder), decreased from 35% to 8% of total rears as the dose of MPTP increased from 4 mg/kg to 32 mg/kg, respectively. However, motor impairment as measured by a Parallel Rod Activity Chamber test was not significant until week 4 at 32 mg/kg compared to the vehicle group. The present study is the first to show stepwise progression of behavioral deficits which correlate with gradual dopaminergic decline in the nigrostriatal pathway. This progressive lesioning regiment may be appropriate for future investigation of pathogenic mechanisms and various intervention therapies in PD., (Published by Elsevier Ltd.)

A number of recent studies have indicated that accumulation of β amyloid (Aβ) peptides within neurons is an early event which may trigger degeneration of neurons and subsequent development of Alzheimer's disease (AD) pathology. However, very little is known about the internalization and/or subcellular sites involved in trafficking of Aβ peptides into the neurons that are vulnerable in AD pathology. To address this issue we evaluated internalization of fluoroscein conjugated Aβ1-42 (FAβ1-42) and subsequent alteration of endosomal-lysosomal (EL) markers such as cathepsin D, Rab5 and Rab7 in rat cortical cultured neurons. It is evident from our results that internalization of FAβ1-42, which occurred in a dose- and time-dependent manner, triggered degeneration of neurons along with increased levels and/or altered distribution of cathepsin D, Rab5 and Rab7. Our results further revealed that FAβ1-42 internalization was attenuated by phenylarsine oxide (a general inhibitor of endocytosis) and sucrose (an inhibitor of clathrin-mediated endocytosis) but not by antagonists of N-methyl-d-aspartate (NMDA) glutamate receptors. Additionally, inhibition of FAβ1-42 endocytosis not only protected neurons against toxicity but also reversed the altered levels/distributions of EL markers. These results, taken together, suggest that internalization of exogenous Aβ1-42, which is partly mediated via a clathrin-dependent process, can lead to degeneration of neurons, possibly by activating the EL system. Inhibition of FAβ endocytosis attenuated toxicity, thus suggesting a potential strategy for preventing loss of neurons in AD pathology., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Glaucoma is an age-related optic neuropathy involving sensitivity to ocular pressure. The disease is now seen increasingly as one of the central nervous system, as powerful new approaches highlight an increasing number of similarities with other age-related neurodegenerations such as Alzheimer's and Parkinson's. While the etiologies of these diseases are diverse, they involve many important common elements including compartmentalized programs of degeneration targeting axons, dendrites and finally cell bodies. Most age-related degenerations display early functional deficits that precede actual loss of neuronal substrate. These are linked to several specific neurochemical cascades that can be linked back to dysregulation of Ca(2+)-dependent processes. We are now in the midst of identifying similar cascades in glaucoma. Here we review recent evidence on the pathological progression of neurodegeneration in glaucoma and some of the Ca(2+)-dependent mechanisms that could underlie these changes. These mechanisms present clear implications for efforts to develop interventions targeting neuronal loss directly and make glaucoma an attractive model for both interrogating and informing other neurodegenerative diseases., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Dysregulation of Ca(2+) signaling following oxidative stress is an important pathophysiological mechanism of many chronic neurodegenerative disorders, including Alzheimer's disease, age-related macular degeneration, glaucomatous and diabetic retinopathies. However, the underlying mechanisms of disturbed intracellular Ca(2+) signaling remain largely unknown. We here describe a novel mechanism for increased intracellular Ca(2+) release following oxidative stress in a neuronal cell line. Using an experimental approach that included quantitative polymerase chain reaction, quantitative immunoblotting, microfluorimetry and the optical imaging of intracellular Ca(2+) release, we show that sub-lethal tert-butyl hydroperoxide-mediated oxidative stress result in a selective up-regulation of type-2 inositol-1,4,5,-trisphophate receptors. This oxidative stress mediated change was detected both at the transcriptional and translational level and functionally resulted in increased Ca(2+) release into the nucleoplasm from the membranes of the nuclear envelope at a given receptor-specific stimulus. Our data describe a novel source of Ca(2+) dysregulation induced by oxidative stress with potential relevance for differential subcellular Ca(2+) signaling specifically within the nucleus and the development of novel neuroprotective strategies in neurodegenerative disorders., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The lipid kinase PIK3C3 (also known as VPS34) regulates multiple aspects of endo-membrane trafficking processes. PIK3C3 is widely expressed by neurons in the CNS, and its catalytic product PI3P is enriched in dendritic spines. Here we generated a line of conditional mutant mouse in which Pik3c3 is specifically deleted in hippocampal and in small subsets of cortical pyramidal neurons using the CaMKII-Cre transgene. We found that Pik3c3-deficiency initially causes loss of dendritic spines accompanied with reactive gliosis, which is followed by progressive neuronal degeneration over a period of several months. Layers III and IV cortical neurons are more susceptible to Pik3c3-deletion than hippocampal neurons. Furthermore, in aged conditional Pik3c3 mutant animals, there are extensive gliosis and severe secondary loss of wild type neurons. Our analyses show that Pik3c3 is essential for CNS neuronal homeostasis and Pik3c3flox/flox; CaMKII-Cre mouse is a useful model for studying pathological changes in progressive forebrain neurodegeneration., (Copyright © 2010. Published by Elsevier Ltd.)

To elucidate whether interleukin-18 (IL-18) or interferon-γ (IFN-γ) participates in neurodegeneartion, we investigated the changes in IL-18 and IFN-γ systems within the rat hippocampus following status epilepticus (SE). In non-SE induced animals, IL-18, IL-18 receptor α (IL-18Rα), IFN-γ and IFN-γ receptor α (IFN-γRα) immunoreactivity was not detected in the hippocampus. Following SE, IL-18 immunoreactivity was increased in CA1-3 pyramidal cells as well as dentate granule cells. IL-18 immunoreactivity was also up-regulated in astrocytes and microglia/macrophages. IL-18Rα immunoreactivity was detected in astrocytes and microglia/macrophages. IFN-γ immunoreactivity was detected only in astrocytes within all regions of the hippocampus. IFN-γRα immunoreactivity was increased in neurons as well as astrocytes. Intracerebroventricular infusions of recombinant rat IL-18 or IFN-γ alleviated SE-induced neuronal damages, while neutralization of IL-18, IFN-γ or their receptors aggravated them, as compared to saline-infused animals. These findings suggest that astroglial-mediated IFN-γ pathway in response to IL-18 induction may play an important role in alleviation of SE-induced neuronal damages., (Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Phosphorylation of eukaryotic initiation factor-2 alpha (eIF2 alpha) is increased in Alzheimer's disease (AD) and this protein can be phosphorylated by several kinases, including double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), amino acids-regulated eIF2 alpha kinase (GCN2) and heme-regulated eIF2 alpha kinase (HRI). PKR and PERK especially are activated in the AD brain, and GCN2 is reported to increase presenilin-1 (PS1) activity. Okadaic acid (OA), a protein phosphatase-2A (PP2A) inhibitor, is known to increase tau phosphorylation, beta-amyloid (A beta) deposition and neuronal death, which are the pathological characteristics of AD. Here, we show that the phosphorylation of eIF2 alpha is increased and its kinases, PKR, PERK and GCN2 are activated in rat neurons by OA. Activating transcription factor (ATF4) which induces apoptosis in response to eIF2 alpha phosphorylation was increased and translocated to nuclei in OA-treated neurons. These results suggest that the successive events of activation of eIF2 alpha kinases and eIF2 alpha phosphorylation leading to ATF4 nuclear translocation may contribute to neuronal death. However, PKR inhibitors did not reduce eIF2 alpha phosphorylation or neuronal toxicity despite inhibiting PKR activity. These results suggest that PKR might not be the most responsible kinase for eIF2 alpha phosphorylation or cell death in PP2A-inhibited conditions such as AD., ((c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Various missense mutations were identified in TAR DNA-binding protein-43 (TDP-43) in patients with amyotrophic lateral sclerosis (ALS). To explore the toxic effect of mutant TDP-43, we generated stable transfection of wild-type and mutant TDP-43 in motor neuron-like cell line. We found that mutant TDP-43 induced mitochondrial dysfunction, oxidative damage and nuclear accumulation of nuclear factor E2-related factor 2 (Nrf2). Nrf2 is an indicator and modulator of oxidative stress and is known to promote the expression of phase || detoxification enzyme including heme oxygenase-1 (HO-1). However, HO-1 was down regulated in cells expressing the mutant TDP-43, and could not be restored by sulforaphane which is a known stimulator of Nrf2 and phase || detoxification enzyme, including HO-1. Nevertheless, sulforaphane reduced the level of lactate dehydrogenase and lipoperoxidation products in cells expressing TDP-43 mutant. However, sulforaphane could upregulate the expression of HO-1 and NAD(P)H/quinone oxidoreductase-1 (NQO-1) in cells transfected with the empty vector and the wild-type TDP-43. Thus, sulforaphane protected cells against mutant TDP-43 independent of Nrf2-antioxidant response element (ARE) pathway. How mutant TDP-43 reduces expression of HO-1 and prevents sulforaphane from activating Nrf2 signaling remains to be investigated., ((c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

It has been reported that an early activation of glial fibrillary acid protein (GFAP) in astroglial cells occurs simultaneously in peripheral nerves and spinal cord from the G93A SOD1 mouse model of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder. In ALS, the contribute to the pathological process of different cell types varies according to the disease stage, with a florid immune response in spinal cord at end stage disease. In this study, we have mapped in different anatomical sites the process of disease-induced functional perturbation from a pre-symptomatic stage using a marker of cellular distress expressed in neurons and glial cells, the activating transcription factor 3 (ATF-3), and applied large-scale gene expression analysis to define the pattern or transcriptional changes occurring in spinal cord from the G93A SOD1 rat model of ALS in parallel with ATF-3 neuronal activation. From the disease onset onward, transgenic lumbar spinal cord displayed ATF-3 transcriptional regulation and motor cells immunostaining in association with the over-expression of genes promoting cell growth, the functional integrity of cell organelles and involved in the modulation of immune responses. While spinal cord from the pre-symptomatic rat showed no detectable ATF-3 transcriptional regulation, ATF-3 activation was appreciated in large size neurofilament-rich, small size non-peptidergic and parvalbumin-positive neurons within the dorsal root ganglia (DRG), and in ventral roots Schwann cells alongside macrophages infiltration. This pattern of peripheral ATF-3 activation remained detectable throughout the disease process. In the G93A SOD1 rat model of ALS, signs of roots and nerves subtle distress preceded overt clinical-pathological changes, involving both glial cells and neurons that function as receptors of peripheral sensory stimuli from the muscle. In addition, factors previously described to be linked to ATF-3 activation under various experimental conditions of stress, become switched on in spinal cord from the end-stage transgenic rat model of ALS., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Doppel protein (Dpl) is a paralog of the cellular form of prion protein (PrP(C)). Its ectopic expression in the CNS elicits significant cerebellar Purkinje cell degeneration in some lines of PrP knockout mice. However, little is known about the Dpl-mediated neurodegenerative mechanism. To understand the molecular and intracellular pathways underlying Purkinje cell degeneration, here, we investigated the regulation of calcium-release channel protein, type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) gene in Ngsk mice. These knockout mice express high levels of Dpl and eventually develop cerebellar degeneration. We observed that the expression level of IP(3)R1 gene is reduced in the cerebella of Ngsk mice as early as 3 months of age compared with age-matched controls along with the reduction in DNA binding activity of nuclear factor of activated-T cells (NFAT) which is transcription factor of IP(3)R1. Notably, expression of PrP restored the reduced DNA binding activity of NFATc4 by Dpl. Reduced expressions of brain-derived neurotrophic factor (BDNF) and ionotropic glutamate receptor subtype 2 or B (GluR2), which are regulated by NFATc4, were also restored by PrP expression. In light of these findings, we suggest a mechanism for Dpl-mediated Purkinje cell degeneration linked to reduced gene expression of proteins related to neuronal activity. Decrease in IP(3)R1 gene expression may lead to functional deficits and ultimately death of Purkinje cells in Ngsk mice., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

p300/CREB binding protein-associated factor (PCAF) regulates gene expression by acting through histone acetylation and as a transcription coactivator. Although histone acetyltransferases were involved in the toxicity induced by amyloid-beta (Abeta) peptides, nothing is known about PCAF. We here analyzed the sensitivity of PCAF knockout (KO) mice to the toxic effects induced by i.c.v. injection of Abeta(25-35) peptide, a nontransgenic model of Alzheimer's disease. PCAF wild-type (WT) and KO mice received Abeta(25-35) (1, 3 or 9 nmol) or scrambled Abeta(25-35) (9 nmol) as control. After 7 days, Abeta(25-35) toxicity was measured in the hippocampus of WT mice by a decrease in CA1 pyramidal cells and increases in oxidative stress, endoplasmic reticulum stress and induction of apoptosis. Memory deficits were observed using spontaneous alternation, water-maze learning and passive avoidance. Non-treated PCAF KO mice showed a decrease in CA1 cells and learning alterations. However, Abeta(25-35) injection failed to induce toxicity or worsen the deficits. This resistance to Abeta(25-35) toxicity did not involve changes in glutamate or acetylcholine systems. Examination of enzymes involved in Abeta generation or degradation revealed changes in transcription of presenilins, activity of neprilysin (NEP) and an absence of Abeta(25-35)-induced regulation of NEP activity in PCAF KO mice, partly due to an altered expression of somatostatin (SRIH). We conclude that PCAF regulates the expression of proteins involved in Abeta generation and degradation, thus rendering PCAF KO insensitive to amyloid toxicity. Modulating acetyltransferase activity may offer a new way to develop anti-amyloid therapies., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

It has been reported that N-methyl-D-aspartate receptor (NMDAR)-triggered neurotoxicity is related to excessive Ca(2+) loading and an increase in nitric oxide (NO) concentration. However, the molecular mechanisms that underlie these events are not completely understood. NMDARs and neuronal NO synthase each binds to the scaffolding protein postsynaptic density (PSD)-93 through its PDZ domains. In this study, we determined whether PSD-93 plays a critical role in NMDAR/Ca(2+)/NO-mediated neurotoxicity. We found that the targeted disruption of the PSD-93 gene attenuated the neurotoxicity triggered by NMDAR activation, but not by non-NMDAR activation, in cultured mouse cortical neurons. PSD-93 deficiency reduced the amount of NMDAR subunits NR2A and NR2B in synaptosomal fractions from the cortical neurons and significantly prevented NMDA-stimulated increases in cyclic guanosine 3',5'-monophosphate and Ca(2+) loading in the cortical neurons. These findings indicate that PSD-93 deficiency could block NMDAR-triggered neurotoxicity by disrupting the NMDAR-Ca(2+)-NO signaling pathway and reducing expression of synaptic NR2A and NR2B. Since NMDARs, Ca(2+), and NO play a critical role during the development of brain trauma, seizures, and ischemia, the present work suggests that PSD-93 might contribute to molecular mechanisms of neuronal damage in these brain disorders., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Recent studies have found that liver X receptors (LXRs) agonists decrease brain inflammation and exert neuroprotective effect. The aim of this study was to examine the mechanisms of action of liver X receptor agonist GW3965 against brain injury following global cerebral ischemia in the rat. The 48 male SD (Sprague-Dawley) rats were randomly partitioned into three groups: sham, global ischemia (4-vessel occlusion for 15 min; 4VO) treated with vehicle and global ischemia treated with GW3965 (20 mg/kg, via i.p. injection at 10 min after reperfusion). The functional outcome was determined by neurological evaluation at 24 h post ischemia and by testing rats in T maze at 3 and 7 days after reperfusion. The rats' daily body weight, incidence of seizures and 72 h mortality were also determined. After Nissl staining and TUNEL in coronal brain sections, the numbers of intact and damaged cells were counted in the CA1 sector of the hippocampus. The expression of phosphorylated inhibitor of kappaB (p-IkappaBalpha), nuclear factor-kappaB (NF-kappaB) subunit p65, and cyclo-oxygenase-2 (COX-2) were analyzed with Western blot at 12 h after reperfusion. GW3965 tended to reduce 72 h mortality and the incidence of post-ischemic seizures. GW3965-treated rats showed an improved neuronal survivability in CA1 and a significant increase in the percentage of spontaneous alternations detected in T-maze on day 7 after ischemia. GW3965-induced neuroprotection was associated with a significant reduction in nuclear translocation of NF-kB p65 subunit and a decrease in the hippocampal expression of NF-kB target gene, COX-2. LXR receptor agonist protects against neuronal damage following global cerebral ischemia. The mechanism of neuroprotection may include blockade of NF-kappaB activation and the subsequent suppression of COX-2 in the post ischemic brain., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Degeneration of the noradrenergic neurons in the locus coeruleus (LC) is a major component of Alzheimer's (AD) and Parkinson's disease (PD), but the consequence of noradrenergic neuronal loss has different effects on the surviving neurons in the two disorders. Therefore, understanding the consequence of noradrenergic neuronal loss is important in determining the role of this neurotransmitter in these neurodegenerative disorders. The goal of the study was to determine if the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) could be used as a model for either (or both) AD or PD. Rats were administered DSP4 and sacrificed 3 days 2 weeks and 3 months later. DSP4-treatment resulted in a rapid, though transient reduction in norepinephrine (NE) and NE transporter (NET) in many brain regions receiving variable innervation from the LC. Alpha(1)-adrenoreceptors binding site concentrations were unchanged in all brain regions at all three time points. However, an increase in alpha(2)-AR was observed in many different brain regions 2 weeks and 3 months after DSP4. These changes observed in forebrain regions occurred without a loss in LC noradrenergic neurons. Expression of synthesizing enzymes or NET did not change in amount of expression/neuron despite the reduction in NE tissue content and NET binding site concentrations at early time points, suggesting no compensatory response. In addition, DSP4 did not affect basal activity of LC at any time point in anesthetized animals, but 2 weeks after DSP4 there is a significant increase in irregular firing of noradrenergic neurons. These data indicate that DSP4 is not a selective LC noradrenergic neurotoxin, but does affect noradrenergic neuron terminals locally, as evident by the changes in transmitter and markers at terminal regions. However, since DSP4 did not result in a loss of noradrenergic neurons, it is not considered an adequate model for noradrenergic neuronal loss observed in AD and PD., (Published by Elsevier Ltd.)

Angiogenesis and blood-brain-barrier (BBB) damage have been proposed to contribute to epileptogenesis and/or ictogenesis in experimental and human epilepsy. We tested a hypothesis that after brain injury angiogenesis occurs in the most damaged hippocampal areas with the highest need of tissue repair, and associates with formation of epileptogenic neuronal networks. We induced status epilepticus (SE) with pilocarpine in adult rats, and investigated endothelial cell proliferation (BrdU and rat endothelial cell antigen-1 (RECA-1) double-labeling), vessel length (unbiased stereology), thrombocyte aggregation (thrombocyte immunostaining), neurodegeneration (Nissl staining), neurogenesis (doublecortin (DCX) immunohistochemistry), and mossy fiber sprouting (Timm staining) in the hippocampus at different time points post-SE. As functional measures we determined BBB leakage (quantified immunoglobulin G (IgG) immunostaining), and hippocampal blood volume (CBV) and flow (CBF) in vivo (magnetic resonance imaging, MRI). The total length of hippocampal blood vessels was decreased by 17% at 2 d after status epilepticus (SE) induced by pilocarpine in adult rats (P<0.05 as compared to controls) which was not accompanied by alterations in hippocampal blood volume (BV) and flow (BF). Number of proliferating endothelial cells peaked at 4 d post-SE and correlated with an increase in vessel length (r=0.900, P<0.05). Vessels length had recovered to control level or even higher at 2 wk post-SE, angiogenesis being most prominent in the CA3 (128% as compared to that in controls, P<0.05), and was associated with increased BV (178% as compared to that in controls, P<0.05). Enlargement of vessel diameter in the hippocampal fissure was associated with thrombocyte aggregation in distal capillaries. BBB was most leaky during the first 4 d post-SE and increased IgG extravasation was observed for 60 d. Our data show that magnitude of endothelial cell proliferation is not associated with severity of acute post-SE neurodegeneration or formation of abnormal neuronal network. This encourages identification of molecular targets that initiate and maintain specific aspects of tissue reorganization, including preservation and proliferation of endothelial cells to reduce the risk of epileptogenesis and enhance recovery after brain injury., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Excess glutamate release and stimulation of post-synaptic glutamatergic receptors have been implicated in the pathophysiology of many neurological diseases. The hippocampus, and the pyramidal cell layer of the cornu ammonus 1 (CA1) region in particular, has been noted for its selective sensitivity to excitotoxic insults. The current studies examined the role of N-methyl-D-aspartate (NMDA) receptor subunit composition and sensitivity to stimulatory effects of the polyamine spermidine, an allosteric modulator of NMDA NR2 subunit activity, in hippocampal CA1 region sensitivity to excitotoxic insult. Organotypic hippocampal slice cultures of 8 day-old neonatal rat were obtained and maintained in vitro for 5 days. At this time, immunohistochemical analysis of mature neuron density (NeuN); microtubule associated protein-2(a,b) density (MAP-2); and NMDA receptor NR1 and NR2B subunit density in the primary cell layers of the dentate gyrus (DG), CA3, and CA1 regions, was conducted. Further, autoradiographic analysis of NMDA receptor distribution and density (i.e. [(125)I]MK-801 binding) and spermidine (100 microM)-potentiated [(125)I]MK-801 binding in the primary cell layers of these regions was examined. A final series of studies examined effects of prolonged exposure to NMDA (0.1-10 microM) on neurodegeneration in the primary cell layers of the DG, CA3, and CA1 regions, in the absence and presence of spermidine (100 microM) or ifenprodil (100 microM), an allosteric inhibitor of NR2B polypeptide subunit activity. The pyramidal cell layer of the CA1 region demonstrated significantly greater density of mature neurons, MAP-2, NR1 and NR2B subunits, and [(125)I]MK-801 binding than the CA3 region or DG. Twenty-four hour NMDA (10 microM) exposure produced marked neurodegeneration (approximately 350% of control cultures) in the CA1 pyramidal cell region that was significantly reduced by co-exposure to ifenprodil or DL-2-Amino-5-phosphonopentanoic acid (APV). The addition of spermidine significantly potentiated [(125)I]MK-801 binding and neurodegeneration induced by exposure to a non-toxic concentration of NMDA, exclusively in the CA1 region. This neurodegeneration was markedly reduced with co-exposure to ifenprodil. These data suggest that selective sensitivity of the CA1 region to excitotoxic stimuli may be attributable to the density of mature neurons expressing polyamine-sensitive NR2B polypeptide subunits.

Soluble forms of amyloid-beta (Abeta) have been considered responsible for cognitive dysfunction prior to senile plaque formation in Alzheimer's disease (AD). As its mechanism is not well understood, we examined the effects of repeated i.c.v. infusion of soluble Alphabeta(25-35) on peptidergic system and glial cells in the pathogenesis of AD. The present study aims to investigate the protective effects of memantine on Abeta(25-35)-induced changes in peptidergic and glial systems. Infusion of Alphabeta(25-35) decreased the level of immunoreactive somatostatin (SS) and substance P (SP) in the hippocampus prior to neuronal loss or caspase activation, which is correlated with the loss of spine density and activation of inducible nitric-oxide synthase (iNOS). Biochemical experiment with peptide-degrading enzymes, prolyl oligopeptidase (POP) and endopeptidase 24.15 (EP 24.15) activities demonstrated a concomitant increase with the activation of glial marker proteins, glial fibrillary acidic protein (GFAP) and CD11b in the Abeta-treated hippocampus. Double immunostaining experiments of EP 24.15 and GFAP/CD11b antibodies clearly demonstrated the co-localization of neuro peptidases with astrocytes and microglia. Treatment with memantine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist significantly attenuated Abeta(25-35)-induced changes of neuropeptides, their metabolizing enzymes, glial marker proteins, and activation of iNOS. Taken together, the data implies that memantine exerts its protective effects by modulating the neuropeptide system as a consequence of suppressing the glial cells and oxidative stress in AD model rat brain regions.

N-methyl-d-aspartate receptor antagonist drugs (NMDA-A), such as dizocilpine (MK801), induce long-lasting behavioral disturbances reminiscent to psychotic disorders in humans. To identify cortical structures affected by NMDA-A, we used a single dose of MK801 (10 mg/kg) that caused low and high neurodegeneration in intact and orchiectomized male rats, respectively. Degenerating somas (neuronal death) and axonal/synaptic endings (terminal degeneration) were depicted by a silver technique, and functionally affected cortical neuronal subpopulations by Egr-1, c-Fos, and FosB/DeltaFosB-immunolabeling. In intact males, MK801 triggered a c-Fos induction that remained high for more than 24 h in selected layers of the retrosplenial, somatosensory and entorhinal cortices. MK801-induced neurodegeneration reached its peak at 72 h. Degenerating somas were restricted to layer IV of the granular subdivision of the retrosplenial cortex, and were accompanied by suppression of Egr-1 immunolabeling. Terminal degeneration extended to selected layers of the retrosplenial, somatosensory and parahippocampal cortices, which are target areas of retrosplenial cortex. Induction of FosB/DeltaFosB by MK801 also extended to the same cortical layers affected by terminal degeneration, likely reflecting the damage of synaptic connectivity. In orchiectomized males, the neurodegenerative and functional effects of MK801 were exacerbated. Degenerative somas in layer IV of the retrosplenial cortex significantly increased, with a parallel enhancement of terminal degeneration and FosB/DeltaFosB-expression in the mentioned cortical structures, but no additional areas were affected. These observations reveal that synaptic dysfunction/degeneration in the retrosplenial, somatosensory and parahippocampal cortices might underlie the long-lasting impairments induced by NMDA-A.

Basal ganglia striosomes, or patches, are rich in mu opioid receptors (MOR) and form a three-dimensional labyrinth of cells that extend throughout the mid- and anterior striatum in mice. Though previous studies have suggested that striosomes could affect drug-induced motor output in rodents, the functional role of these compartmentalized MOR-rich striosomes is not well understood. To investigate any relationship between the striosomes and motor behavior we used the toxin dermorphin-saporin (DS) to selectively ablate MOR-rich striosomal cells. FVB mice were bilaterally infused with DS in the midstriatum alone or in the mid- and anterior striatum, and were tested on three motor tasks and in an open field. Two volume measurement procedures and stereological cell counts were used to confirm the induced pathology. Mice that received DS injections showed significantly smaller volumes (-26% to -44%) and fewer cells (-30% to -49%) in the striosome compartment compared to mice that received control injections of saline or saporin. Striosome pathology was greatest in the dorsolateral striatum. The extrastriosomal matrix was not significantly affected, resulting in an imbalance in the ratio of striosome-to-matrix cells. Behaviorally, toxin injections caused deficits on an accelerating rotarod task and the deficit was worse in mice that received mid and anterior injections than those that received midstriatal injections alone. However, DS-injected mice did not differ from control mice on other motor tasks. We conclude that the MOR-rich cells of the striosomes are necessary for optimal rotarod performance, including learning and/or improvement on the task.

Decreased availability or efficacy of neurotrophic factors may underlie an increased susceptibility of mesencephalic dopaminergic cells to age-related degeneration. Neuregulins (NRGs) are pleotrophic growth factors for many cell types, including mesencephalic dopamine cells in culture and in vivo. The functional NRG receptor ErbB4 is expressed by virtually all midbrain dopamine neurons. To determine if levels of the NRG receptor are maintained during aging in the dopaminergic ventral mesencephalon, expression of ErbB4 mRNA and protein was examined in young (3 months), middle-aged (18 months), and old (24-25 months) Brown Norway/Fischer 344 F1 rats. ErbB4 mRNA levels in the substantia nigra pars compacta (SNpc), but not the adjacent ventral tegmental area (VTA) or subtantia nigra pars lateralis (SNl), were significantly reduced in the middle-aged and old animals when compared to young rats. Protein expression of ErbB4 in the ventral midbrain was significantly decreased in the old rats when compared to the young rats. Expression of tyrosine hydroxylase (TH) mRNA levels was significantly reduced in the old rats when compared to young animals in the SNpc, but not in the VTA or SNI. TH protein levels in the ventral midbrain were also decreased in the old animals when compared to the young animals. These data demonstrate a progressive decline of ErbB4 expression, coinciding with a loss of the dopamine-synthesizing enzyme TH, in the ventral midbrain of aged rats, particularly in the SNpc. These findings may implicate a role for diminished NRG/ErbB4 trophic support in dopamine-related neurodegenerative disorders of aging such as Parkinson's disease.

Urokinase-type plasminogen activator receptor (uPAR) is functionally a pleiotropic mediator involved in cell adhesion, proliferation, differentiation and migration as well as in matrix degradation, apoptosis, and angiogenesis in cancer tissue. Comparable cellular alterations occur in the brain during post-injury tissue repair. As the first step to assess the role of uPAR in brain tissue remodeling, we tested a hypothesis that uPAR expression is altered in the hippocampus during epilepsy-related circuitry reorganization. Epileptogenesis was triggered by inducing status epilepticus (SE) with electrical stimulation of the amygdala in rats. To monitor the development of SE and the occurrence of spontaneous seizures animals were continuously video-EEG monitored until sacrificed (1, 2, 4 or 14 days after SE). The hippocampal expression of uPAR was studied with real time qPCR and immunohistochemistry. Double-immunohistochemistry and confocal microscopy were used to investigate the expression of uPAR in astrocytes, microglia and neurons. We show that in the normal hippocampus the expression of uPAR was low and confined to small population of astrocytes and interneurons. In animals undergoing SE, uPAR expression increased dramatically, peaking at 1 and 4 days after SE. According to double-immunohistochemistry, uPAR was highly expressed in parvalbumin positive interneurons in the hippocampus and dentate gyrus, and in a subgroup of somatostatin and neuropeptide Y positive hilar interneurons. Increased uPAR expression during post-injury phase supports its contribution to tissue remodeling in the brain. Surviving hilar interneurons that are known to be denervated due to loss of afferent inputs in post-SE brain provide a target for future studies to investigate the contribution of uPAR in reinnervation of these cells, and to identify the signaling cascades that mediate the effects of uPAR.

The transcription factor NF-kappaB is a key regulator of hundreds of genes involved in cell survival and inflammation. There is ample evidence that NF-kappaB is activated in cerebral ischemia, mainly in neurons. Despite its well known role as an antiapoptotic factor, in cerebral ischemia NF-kappaB contributes to neuronal cell death, at least if the ischemia is severe enough to lead to irreversible brain damage. In contrast, NF-kappaB also seems to be responsible for the preconditioning effect of a transient and sublethal ischemia, perhaps by dampening its own subsequent full activation. Among the five NF-kappaB subunits, RelA and p50 are responsible for the detrimental effect in cerebral ischemia. Activation of NF-kappaB signaling is mediated by the upstream kinase inhibitor of kappaB kinase and is triggered by hypoxia, reactive oxygen species, and several inflammatory mediators. Interestingly, the complex NF-kappaB signaling pathway provides drug targets at several levels. Modulation of NF-kappaB signaling has the potential to interrupt multiple inflammatory and apoptotic mechanisms through one specific molecular target.