Astrocyte Progenitors Derived From Patients With Alzheimer Disease Do Not Impair Stroke Recovery in Mice.

BACKGROUND: Species-specific differences in astrocytes and their Alzheimer disease-associated pathology may influence cellular responses to other insults. Herein, human glial chimeric mice were generated to evaluate how Alzheimer disease predisposing genetic background in human astrocytes contributes to behavioral outcome and brain pathology after cortical photothrombotic ischemia. METHODS: Neonatal (P0) immunodeficient mice of both sexes were transplanted with induced pluripotent stem cell-derived astrocyte progenitors from Alzheimer disease patients carrying PSEN1 exon 9 deletion (PSEN1 ΔE9), with isogenic controls, with cells from a healthy donor, or with mouse astrocytes or vehicle. After 14 months, a photothrombotic lesion was produced with Rose Bengal in the motor cortex. Behavior was assessed before ischemia and 1 and 4 weeks after the induction of stroke, followed by tissue perfusion for histology. RESULTS: Open field, cylinder, and grid-walking tests showed a persistent locomotor and sensorimotor impairment after ischemia and female mice had larger infarct sizes; yet, these were not affected by astrocytes with PSEN1 ΔE9 background. Staining for human nuclear antigen confirmed that human cells successfully engrafted throughout the mouse brain. However, only a small number of human cells were positive for astrocytic marker GFAP (glial fibrillary acidic protein), mostly located in the corpus callosum and retaining complex human-specific morphology with longer processes compared with host counterparts. While host astrocytes formed the glial scar, human astrocytes were scattered in small numbers close to the lesion boundary. Aß (beta-amyloid) deposits were not present in PSEN1 ΔE9 astrocyte-transplanted mice. CONCLUSIONS: Transplanted human cells survived and distributed widely in the host brain but had no impact on severity of ischemic damage after cortical photothrombosis in chimeric mice. Only a small number of transplanted human astrocytes acquired GFAP-positive glial phenotype or migrated toward the ischemic lesion forming glial scar. PSEN1 ΔE9 astrocytes did not impair behavioral recovery after experimental stroke.

An arylthiazyne derivative is a potent inhibitor of lipid peroxidation and ferroptosis providing neuroprotection in vitro and in vivo.

Lipid peroxidation-initiated ferroptosis is an iron-dependent mechanism of programmed cell death taking place in neurological diseases. Here we show that a condensed benzo[b]thiazine derivative small molecule with an arylthiazine backbone (ADA-409-052) inhibits tert-Butyl hydroperoxide (TBHP)-induced lipid peroxidation (LP) and protects against ferroptotic cell death triggered by glutathione (GSH) depletion or glutathione peroxidase 4 (GPx4) inhibition in neuronal cell lines. In addition, ADA-409-052 suppresses pro-inflammatory activation of BV2 microglia and protects N2a neuronal cells from cell death induced by pro-inflammatory RAW 264.7 macrophages. Moreover, ADA-409-052 efficiently reduces infarct volume, edema and expression of pro-inflammatory genes in a mouse model of thromboembolic stroke. Targeting ferroptosis may be a promising therapeutic strategy in neurological diseases involving severe neuronal death and neuroinflammation.

Peripheral Administration of IL-13 Induces Anti-inflammatory Microglial/Macrophage Responses and Provides Neuroprotection in Ischemic Stroke.

Neuroinflammation is strongly induced by cerebral ischemia. The early phase after the onset of ischemic stroke is characterized by acute neuronal injury, microglial activation, and subsequent infiltration of blood-derived inflammatory cells, including macrophages. Therefore, modulation of the microglial/macrophage responses has increasingly gained interest as a potential therapeutic approach for the ischemic stroke. In our study, we investigated the effects of peripherally administered interleukin 13 (IL-13) in a mouse model of permanent middle cerebral artery occlusion (pMCAo). Systemic administration of IL-13 immediately after the ischemic insult significantly reduced the lesion volume, alleviated the infiltration of CD45+ leukocytes, and promoted the microglia/macrophage alternative activation within the ischemic region, as determined by arginase 1 (Arg1) immunoreactivity at 3 days post-ischemia (dpi). Moreover, IL-13 enhanced the expression of M2a alternative activation markers Arg1 and Ym1 in the peri-ischemic (PI) area, as well as increased plasma IL-6 and IL-10 levels at 3 dpi. Furthermore, IL-13 treatment ameliorated gait disturbances at day 7 and 14 and sensorimotor deficits at day 14 post-ischemia, as analyzed by the CatWalk gait analysis system and adhesive removal test, respectively. Finally, IL-13 treatment decreased neuronal cell death in a coculture model of neuroinflammation with RAW 264.7 macrophages. Taken together, delivery of IL-13 enhances microglial/macrophage anti-inflammatory responses in vivo and in vitro, decreases ischemia-induced brain cell death, and improves sensory and motor functions in the pMCAo mouse model of cerebral ischemia.

Loss of Cln5 leads to altered Gad1 expression and deficits in interneuron development in mice.

The Finnish-variant late infantile neuronal ceroid lipofuscinosis, also known as CLN5 disease, is caused by mutations in the CLN5 gene. Cln5 is strongly expressed in the developing brain and expression continues into adulthood. CLN5, a protein of unknown function, is implicated in neurodevelopment but detailed investigation is lacking. Using Cln5-/- embryos of various ages and cells harvested from Cln5-/- brains we investigated the hitherto unknown role of Cln5 in the developing brain. Loss of Cln5 results in neuronal differentiation deficits and delays in interneuron development during in utero period. Specifically, the radial thickness of dorsal telencephalon was significantly decreased in Cln5-/- mouse embryos at embryonic day 14.5 (E14.5), and expression of Tuj1, an important neuronal marker during development, was down-regulated. An interneuron marker calbindin and a mitosis marker p-H3 showed down-regulation in ganglionic eminences. Neurite outgrowth was compromised in primary cortical neuronal cultures derived from E16 Cln5-/- embryos compared with WT embryos. We show that the developmental deficits of interneurons may be linked to increased levels of the repressor element 1-silencing transcription factor, which we report to bind to glutamate decarboxylase (Gad1), which encodes GAD67, a rate-limiting enzyme in the production of gamma-aminobutyric acid (GABA). Indeed, adult Cln5-/- mice presented deficits in hippocampal parvalbumin-positive interneurons. Furthermore, adult Cln5-/- mice presented deficits in hippocampal parvalbumin-positive interneurons and showed age-independent cortical hyper excitability as measured by electroencephalogram and auditory-evoked potentials. This study highlights the importance of Cln5 in neurodevelopment and suggests that in contrast to earlier reports, CLN5 disease is likely to develop during embryonic stages.

Sulfosuccinimidyl oleate sodium is neuroprotective and alleviates stroke-induced neuroinflammation.

BACKGROUND: Ischemic stroke is one of the main causes of death and disability worldwide. It is caused by the cessation of cerebral blood flow resulting in the insufficient delivery of glucose and oxygen to the neural tissue. The inflammatory response initiated by ischemic stroke in order to restore tissue homeostasis in the acute phase of stroke contributes to delayed brain damage. METHODS: By using in vitro models of neuroinflammation and in vivo model of permanent middle cerebral artery occlusion, we demonstrate the neuroprotective and anti-inflammatory effects of sulfosuccinimidyl oleate sodium (SSO). RESULTS: SSO significantly reduced the lipopolysaccharide/interferon-γ-induced production of nitric oxide, interleukin-6 and tumor necrosis factor-α, and the protein levels of inflammatory enzymes including nitric oxide synthase 2, cyclooxygenase-2 (COX-2), and p38 mitogen-activated protein kinase (MAPK) in microglia, without causing cell toxicity. Although SSO failed to directly alleviate glutamate-induced excitotoxicity in murine cortical neurons, it prevented inflammation-induced neuronal death in microglia-neuron co-cultures. Importantly, oral administration of SSO in Balb/c mice subjected to permanent occlusion of the middle cerebral artery reduced microglial activation in the peri-ischemic area and attenuated brain damage. This in vivo neuroprotective effect of SSO was associated with a reduction in the COX-2 and heme oxygenase-1 immunoreactivities. CONCLUSIONS: Our results suggest that SSO is an anti-inflammatory and a possible therapeutic candidate in diseases such as stroke where inflammation is a central hallmark.

Loss of Cln5 causes altered neurogenesis in a mouse model of a childhood neurodegenerative disorder.

Neural stem/progenitor cells (NPCs) generate new neurons in the brain throughout an individual's lifetime in an intricate process called neurogenesis. Neurogenic alterations are a common feature of several adult-onset neurodegenerative diseases. The neuronal ceroid lipofuscinoses (NCLs) are the most common group of inherited neurodegenerative diseases that mainly affect children. Pathological features of the NCLs include accumulation of lysosomal storage material, neuroinflammation and neuronal degeneration, yet the exact cause of this group of diseases remains poorly understood. The function of the CLN5 protein, causative of the CLN5 disease form of NCL, is unknown. In the present study, we sought to examine neurogenesis in the neurodegenerative disorder caused by loss of Cln5 Our findings demonstrate a newly identified crucial role for CLN5 in neurogenesis. We report for the first time that neurogenesis is increased in Cln5-deficient mice, which model the childhood neurodegenerative disorder caused by loss of Cln5 Our results demonstrate that, in Cln5 deficiency, proliferation of NPCs is increased, NPC migration is reduced and NPC differentiation towards the neuronal lineage is increased concomitantly with functional alterations in the NPCs. Moreover, the observed impairment in neurogenesis is correlated with increased expression of the pro-inflammatory cytokine IL-1ß. A full understanding of the pathological mechanisms that lead to disease and the function of the NCL proteins are critical for designing effective therapeutic approaches for this devastating neurodegenerative disorder.

Retinal Degeneration In A Mouse Model Of CLN5 Disease Is Associated With Compromised Autophagy.

The Finnish variant of late infantile neuronal ceroid lipofuscinosis (CLN5 disease) belongs to a family of neuronal ceroid lipofuscinosis (NCLs) diseases. Vision loss is among the first clinical signs in childhood forms of NCLs. Mutations in CLN5 underlie CLN5 disease. The aim of this study was to characterize how the lack of normal functionality of the CLN5 protein affects the mouse retina. Scotopic electroretinography (ERG) showed a diminished c-wave amplitude in the CLN5 deficient mice already at 1 month of age, indicative of pathological events in the retinal pigmented epithelium. A- and b-waves showed progressive impairment later from 2 and 3 months of age onwards, respectively. Structural and immunohistochemical (IHC) analyses showed preferential damage of photoreceptors, accumulation of autofluorescent storage material, apoptosis of photoreceptors, and strong inflammation in the CLN5 deficient mice retinas. Increased levels of autophagy-associated proteins Beclin-1 and P62, and increased LC3b-II/LC3b-I ratio, were detected by Western blotting from whole retinal extracts. Photopic ERG, visual evoked potentials, IHC and cell counting indicated relatively long surviving cone photoreceptors compared to rods. In conclusion, CLN5 deficient mice develop early vision loss that reflects the condition reported in clinical childhood forms of NCLs. The vision loss in CLN5 deficient mice is primarily caused by photoreceptor degeneration.

Bexarotene targets autophagy and is protective against thromboembolic stroke in aged mice with tauopathy.

Stroke is a highly debilitating, often fatal disorder for which current therapies are suitable for only a minor fraction of patients. Discovery of novel, effective therapies is hampered by the fact that advanced age, primary age-related tauopathy or comorbidities typical to several types of dementing diseases are usually not taken into account in preclinical studies, which predominantly use young, healthy rodents. Here we investigated for the first time the neuroprotective potential of bexarotene, an FDA-approved agent, in a co-morbidity model of stroke that combines high age and tauopathy with thromboembolic cerebral ischemia. Following thromboembolic stroke bexarotene enhanced autophagy in the ischemic brain concomitantly with a reduction in lesion volume and amelioration of behavioral deficits in aged transgenic mice expressing the human P301L-Tau mutation. In in vitro studies bexarotene increased the expression of autophagy markers and reduced autophagic flux in neuronal cells expressing P301L-Tau. Bexarotene also restored mitochondrial respiration deficits in P301L-Tau neurons. These newly described actions of bexarotene add to the growing amount of compelling data showing that bexarotene is a potent neuroprotective agent, and identify a novel autophagy-modulating effect of bexarotene.

Inhibition of Excessive Oxidative Protein Folding Is Protective in MPP(+) Toxicity-Induced Parkinson's Disease Models.

AIMS: Protein misfolding occurs in neurodegenerative diseases, including Parkinson's disease (PD). In endoplasmic reticulum (ER), an overload of misfolded proteins, particularly alpha-synuclein (αSyn) in PD, may cause stress and activate the unfolded protein response (UPR). This UPR includes activation of chaperones, such as protein disulphide isomerase (PDI), which assists refolding and contributes to removal of unfolded proteins. Although up-regulation of PDI is considered a protective response, its activation is coupled with increased activity of ER oxidoreductin 1 (Ero1), producing harmful hydroperoxide. The objective of this study was to assess whether inhibition of excessive oxidative folding protects against neuronal death in well-established 1-methyl-4-phenylpyridinium (MPP(+)) models of PD. RESULTS: We found that the MPP(+) neurotoxicity and accumulation of αSyn in the ER are prevented by inhibition of PDI or Ero1α. The MPP(+) neurotoxicity was associated with a reductive shift in the ER, an increase in the reduced form of PDI, an increase in intracellular Ca(2+), and an increase in Ca(2+)-sensitive calpain activity. All these MPP(+)-induced changes were abolished by inhibiting PDI. Importantly, inhibition of PDI resulted in increased autophagy, and it prevented MPP(+)-induced death of dopaminergic neurons in Caenorhabditis elegans. INNOVATION AND CONCLUSION: Our data indicate that although inhibition of PDI suppresses excessive protein folding and ER stress, it induces clearance of aggregated αSyn by autophagy as an alternative degradation pathway. These findings suggest a novel model explaining the contribution of ER dysfunction to MPP(+)-induced neurodegeneration and highlight PDI inhibitors as potential treatment in diseases involving protein misfolding. Antioxid. Redox Signal. 25, 485-497.

Pyrrolidine dithiocarbamate activates the Nrf2 pathway in astrocytes.

BACKGROUND: Endogenous defense against oxidative stress is controlled by nuclear factor erythroid 2-related factor 2 (Nrf2). The normal compensatory mechanisms to combat oxidative stress appear to be insufficient to protect against the prolonged exposure to reactive oxygen species during disease. Counterbalancing the effects of oxidative stress by up-regulation of Nrf2 signaling has been shown to be effective in various disease models where oxidative stress is implicated, including Alzheimer's disease. Stimulation of Nrf2 signaling by small-molecule activators is an appealing strategy to up-regulate the endogenous defense mechanisms of cells. METHODS: Here, we investigate Nrf2 induction by the metal chelator and known nuclear factor-κB inhibitor pyrrolidine dithiocarbamate (PDTC) in cultured astrocytes and neurons, and mouse brain. Nrf2 induction is further examined in cultures co-treated with PDTC and kinase inhibitors or amyloid-beta, and in Nrf2-deficient cultures. RESULTS: We show that PDTC is a potent inducer of Nrf2 signaling specifically in astrocytes and demonstrate the critical role of Nrf2 in PDTC-mediated protection against oxidative stress. This induction appears to be regulated by both Keap1 and glycogen synthase kinase 3ß. Furthermore, the presence of amyloid-beta magnifies PDTC-mediated induction of endogenous protective mechanisms, therefore suggesting that PDTC may be an effective Nrf2 inducer in the context of Alzheimer's disease. Finally, we show that PDTC increases brain copper content and glial expression of heme oxygenase-1, and decreases lipid peroxidation in vivo, promoting a more antioxidative environment. CONCLUSIONS: PDTC activates Nrf2 and its antioxidative targets in astrocytes but not neurons. These effects may contribute to the neuroprotection observed for PDTC in models of Alzheimer's disease.

Protein disulfide isomerase in ALS mouse glia links protein misfolding with NADPH oxidase-catalyzed superoxide production.

Protein disulfide isomerase (PDI) is an oxidoreductase assisting oxidative protein folding in the endoplasmic reticulum of all types of cells, including neurons and glia. In neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), up-regulation of PDI is an important part of unfolded protein response (UPR) that is thought to represent an adaption reaction and thereby protect the neurons. Importantly, studies on animal models of familial ALS with mutant Cu/Zn superoxide dismutase 1 (SOD1) have shown that the mutant SOD1 in astrocytes or microglia strongly regulates the progression of the disease. Here, we found an early up-regulation of PDI in microglia of transgenic (tg) mutant SOD1 mice, indicating that in addition to neurons, UPR takes place in glial cells in ALS. The observation was supported by the finding that also the expression of a UPR marker GADD34 (growth arrest and DNA damage-inducible protein) was induced in the spinal cord glia of tg mutant SOD1 mice. Because mutant SOD1 can cause sustained activation of NADPH oxidase (NOX), we investigated the role of PDI in UPR-induced NOX activation in microglia. In BV-2 microglia, UPR resulted in NOX activation with increased production of superoxide and increased release of tumor necrosis factor-α. The phenomenon was recapitulated in primary rat microglia, murine macrophages and human monocytes. Importantly, pharmacological inhibition of PDI or its down-regulation by short interfering RNAs prevented NOX activation in microglia and subsequent production of superoxide. Thus, results strongly demonstrate that UPR, caused by protein misfolding, may lead to PDI-dependent NOX activation and contribute to neurotoxicity in neurodegenerative diseases including ALS.

Mutant SOD1 from spinal cord of G93A rats is destabilized and binds to inner mitochondrial membrane.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS). Mechanisms of mutant SOD1 toxicity are unknown, but increased SOD1 activity can boost production of reactive oxygen species (ROS) in the mitochondrial intermembrane space (IMS). Using non-reducing SDS-PAGE we found that in G93A-SOD1 rats the mutant SOD1 was prominently destabilized only in the diseased spinal cord, where this mutant enzyme was also up regulated in the IMS with increased ability to bind the inner membrane of isolated non-transgenic mitoplasts. These mitoplasts increased ROS production when exposed to mutant SOD1 from the spinal cord at the presymptomatic stage. The levels of disulfide-reduced SOD1 peaked at the end stage of the disease, whereas protein disulfide isomerase (PDI), a chaperone capable of rearranging disulfide bonds between cysteine residues of SOD1, was increased prior to the end stage. IMS binding and increased ROS production by destabilized SOD1 may contribute to mitochondrial damage in G93A-SOD1 rats.

Nuclear factor erythroid 2-related factor 2 protects against beta amyloid.

Nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates the up-regulation of cytoprotective genes via the antioxidant response element (ARE). In the pathogenesis of Alzheimer's disease (AD) current evidence supports the role of oxidative stress. Considering the protective role of Nrf2 against oxidative injury, we studied Nrf2 and Nrf2-ARE target genes in transgenic AD mice and tested whether Nrf2 could confer neuroprotection against amyloid-beta peptides (Abeta). Nrf2-ARE pathway was attenuated in APP/PS1 transgenic mouse brain at the time of Abeta deposition. Boosting the activity of the Nrf2-ARE pathway by tert-butylhydroquinone treatment or adenoviral Nrf2 gene transfer protected against Abeta toxicity. This neuroprotection was associated with increased expression of Nrf2 target genes and reduced phosphorylation of p66Shc, a marker of increased susceptibility for oxidative stress. The findings suggest that the Nrf2-ARE pathway may be impaired in AD and that induction of the Nrf2-ARE defence mechanism may prevent or delay AD-like pathology.

Deleterious role of superoxide dismutase in the mitochondrial intermembrane space.

This work demonstrates how increased activity of copper-zinc superoxide dismutase (SOD1) paradoxically boosts production of toxic reactive oxygen species (ROS) in the intermembrane space (IMS) of mitochondria. Even though SOD1 is a cytosolic enzyme, a fraction of it is found in the IMS, where it is thought to provide protection against oxidative damage. We found that SOD1 controls cytochrome c-catalyzed peroxidation in vitro when superoxide is available. The presence of SOD1 significantly increased the rate of ROS production in mitoplasts, which are devoid of outer membrane and IMS. In response to inhibition of respiration with antimycin A, isolated mouse wild-type mitochondria increased ROS production, but the mitochondria from mice lacking SOD1 (SOD1(-/-)) did not. Also, lymphocytes isolated from SOD1(-/-) mice produced significantly less ROS than did wild-type cells and were more resistant to apoptosis induced by inhibition of respiration. Moreover, an increased amount of the toxic mutant G93A SOD1 in the IMS increased ROS production. The mitochondrial dysfunction and cell damage paradoxically induced by SOD1-mediated ROS production may be implicated in chronic degenerative diseases.

Pyrrolidine dithiocarbamate activates Akt and improves spatial learning in APP/PS1 mice without affecting beta-amyloid burden.

Pyrrolidine dithiocarbamate (PDTC) is a clinically tolerated inhibitor of nuclear factor-kappaB (NF-kappaB), antioxidant and antiinflammatory agent, which provides protection in brain ischemia models. In neonatal hypoxia-ischemia model, PDTC activates Akt and reduces activation of glycogen synthase kinase 3beta (GSK-3beta). Because chronic inflammation, oxidative stress, and increased GSK-3beta activity are features of Alzheimer's disease (AD) pathology, we tested whether PDTC reduces brain pathology and improves cognitive function in a transgenic animal model of AD. A 7 month oral treatment with PDTC prevented the decline in cognition in AD mice without altering beta-amyloid burden or gliosis. Moreover, marked oxidative stress and activation of NF-kappaB were not part of the brain pathology. Instead, the phosphorylated form of GSK-3beta was decreased in the AD mouse brain, and PDTC treatment increased the phosphorylation of Akt and GSK-3beta. Also, PDTC treatment increased the copper concentration in the brain. In addition, PDTC rescued cultured hippocampal neurons from the toxicity of oligomeric Abeta and reduced tau phosphorylation in the hippocampus of AD mice. Finally, astrocytic glutamate transporter GLT-1, known to be regulated by Akt pathway, was decreased in the transgenic AD mice but upregulated back to the wild-type levels by PDTC treatment. Thus, PDTC may improve spatial learning in AD by interfering with Akt-GSK pathway both in neurons and astrocytes. Because PDTC is capable of transferring external Cu2+ into a cell, and, in turn, Cu2+ is able to activate Akt, we hypothesize that PDTC provides the beneficial effect in transgenic AD mice through Cu2+-activated Akt pathway.

Pyrrolidine dithiocarbamate inhibits induction of immunoproteasome and decreases survival in a rat model of amyotrophic lateral sclerosis.

Pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear transcription factor kappa-B (NF-kappaB) and an antioxidant, has beneficial effects in animal models of various diseases, including arthritis, brain ischemia, spinal cord injury, Alzheimer's disease, and Duchenne muscular dystrophy. Because inflammation and oxidative damage are also hallmarks of amyotrophic lateral sclerosis (ALS), we studied the effect of oral PDTC treatment on G93A-superoxide dismutase 1 (SOD1) transgenic (TG) rat model of human ALS and observed that PDTC treatment significantly decreases the survival. PDTC treatment evoked the end stage of the disease at 121 +/- 21 days, whereas untreated TG animals reached the end stage at 141 +/- 13 days (p < 0.01). The DNA binding activity of NF-kappaB was not altered in G93A-SOD1 TG rats by PDTC treatment. The copper concentration in the spinal cord was increased after PDTC treatment both in G93A-SOD1 TG and wild-type rats, suggesting that increased copper may enhance the neurotoxicity of mutant SOD1. The amount of ubiquitinated proteins were significantly higher and proteasomal activity was decreased in the spinal cords of PDTC-treated TG rats compared with other groups, suggesting that PDTC treatment decreases proteasome function. Immunoblotting and immunocytochemistry showed that the level of immunoproteasome but not constitutive proteasome was increased in glia of G93A-SOD1 TG rats along with disease development. PDTC treatment completely blocked the induction of immunoproteasome expression without affecting constitutive proteasome. These results suggest that PDTC acts as an immunoproteasome inhibitor in mutant SOD1 rats and that immunoproteasome may help the nervous system to cope with deleterious effects of SOD1-G93A mutation.

Aspirin inhibits p44/42 mitogen-activated protein kinase and is protective against hypoxia/reoxygenation neuronal damage.

BACKGROUND AND PURPOSE: Acetylsalicylic acid (ASA) is preventive against stroke and protects against focal brain ischemia in rats. We studied the mechanisms of the manner in which ASA provides neuroprotection against hypoxia/reoxygenation (H/R) injury. METHODS: Spinal cord cultures exposed to 20 hours of hypoxia followed by reoxygenation were treated with a vehicle, ASA or inhibitors of inducible nitric oxide synthase (iNOS), mitogen-activated protein kinases p38 MAPK and ERK1/2, or an N-methyl-d-aspartic acid (NMDA) receptor antagonist. Cell viability was assessed by LDH release measurement and cell counts. Prostaglandin production was measured by enzyme immunoassay, MAPK signaling by immunoblotting, and DNA binding of nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1) by electrophoretic mobility shift assay. RESULTS: One to 3 mmol/L ASA inhibited H/R-induced neuronal death when present during H/R but not when administered only for the reoxygenation period. Prostaglandin E2 production was very low and was not altered by ASA. The AP-1 and NF-kappaB DNA binding activities increased after H/R. ASA increased the H/R-induced AP-1 binding but had no effect on NF-kappaB binding. H/R induced a sustained ERK1/2 activation followed by neuronal death, whereas no changes in p38 or c-Jun N-terminal kinase were detected. ASA strongly inhibited this ERK1/2 activation. PD98059, an ERK1/2 inhibitor, was also neuroprotective, prevented H/R-induced ERK1/2 activation, and had no effect on NF-kappaB binding activity. Inhibition of NMDA receptors, iNOS, or p38 MAPK did not provide neuroprotection. CONCLUSIONS: Inhibition of the sustained activation of ERK1/2 may partially contribute to neuroprotection achieved by ASA against H/R injury.

Aspirin provides cyclin-dependent kinase 5-dependent protection against subsequent hypoxia/reoxygenation damage in culture.

Aspirin [acetylsalicylic acid (ASA)] is an anti-inflammatory drug that protects against cellular injury by inhibiting cyclooxygenases (COX), inducible nitric oxide synthase (iNOS) and p44/42 mitogen-activated protein kinase (p44/42 MAPK), or by preventing translocation of nuclear factor kappaB (NF-kappaB). We studied the effect of ASA pre-treatment on neuronal survival after hypoxia/reoxygenation damage in rat spinal cord (SC) cultures. In this injury model, COX, iNOS and NF-kappaB played no role in the early neuronal death. A 20-h treatment with 3 mm ASA prior to hypoxia/reoxygenation blocked the hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release from neurons. This neuroprotection was associated with increased phosphorylation of neurofilaments, which are substrates of p44/42 MAPK and cyclin-dependent kinase 5 (Cdk5). PD90859, a p44/42 MAPK inhibitor, had no effect on ASA-induced tolerance, but olomoucine and roscovitine, Cdk5 inhibitors, reduced ASA neuroprotection. Hypoxia/reoxygenation alone reduced both the protein amount and activity of Cdk5, and this reduction was inhibited by pre-treatment with ASA. Moreover, the protein amount of a neuronal Cdk5 activator, p35, recovered after reoxygenation only in ASA-treated samples. The prevention of the loss in Cdk5 activity during reoxygenation was crucial for ASA-induced protection, because co-administration of Cdk5 inhibitors at the onset ofreoxygenation abolished the protection. In conclusion, pre-treatment with ASA induces tolerance against hypoxia/reoxygenation damage in spinal cord cultures by restoring Cdk5 and p35 protein expression.