Death Of Primary Neurons Research Articles

Disruption of the LRRK2‐FADD Interface Using Constrained Peptides

ABSTRACTMutations in the gene encoding leucine‐rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD). The reduced penetrance of mutations in the LRRK2 gene has also led to variants appearing in seemingly sporadic forms of the disease. Kinase inhibition effectively blocks neuronal death and small‐molecule Class I inhibitors are proceeding through clinical trials in multiple PD cohorts. The toxic interaction between mutant LRRK2 and FADD lies downstream of its kinase activity and is required to induce neuronal death. The present study aimed to determine whether the FADD‐LRRK2 interface could be disrupted and what effects this may have on neuroprotection. A series of constrained peptides were designed to mimic the alpha‐helical protein interaction interface between the LRRK2 armadillo region and the death domain of FADD. These peptide‐based protein–protein interaction inhibitors significantly reduced this interaction and blocked apoptotic death of primary neurons expressing G2019S‐LRRK2. This work has identified novel constrained peptides that disrupt the LRRK2‐FADD interface and downregulate mutant LRRK2‐induced neuronal death in an allosteric manner, thereby providing a potential alternative therapeutic approach for PD.

Fabrication of neuroprotective silk-sericin hydrogel: potential neuronal carrier for the treatment and care of ischemic stroke

Ischemic stroke results in severe disabilities due to extensive cellular loss and the resulting impairment of brain functions. Current methods for regenerating brain tissue are ineffective. Stroke treatment requires innovative therapeutic techniques that are both safe and effective. For neuronal repair, a promising alternative is using a hydrogel-based tissue engineering technique that delivers neurotrophic cytokines and cells to injured sites. However, the limited encapsulation effectiveness, less in vivo cell survival ratio and cytokine loss make this strategy difficult to implement. We aim to design a biomaterial that can efficiently construct a matrix enriching the survival of cells and minimizing loss in vivo cytokines to overcome these constraints. We report the development of genipin conjugated sericin hydrogels (Gen-SH) with a high porous morphology and a moderate swelling rate utilizing sericin, a natural silk protein. In vitro, Gen-SH aids in the attachment and development of neurons. Our results indicate that sericin is inherently neuroprotective and neurotrophic, branching and publicizing axon extension and avoiding hypoxia-induced cell death in primary neurons. Notably, the breakdown products of Gen-SH inherit these capabilities, saving the expense of cytokines. Furthermore, we show that the Lkb1–Nuak1 pathway is required for this neurotrophic impact, whereas the Bcl-2/Bax protein ratio is necessary for the neuroprotective effect. Transplanted in vivo, Gen-SH has a high percentage of cell survival and promotes cell proliferation. Taking all this information into account, it's clear that Gen-SH can serve as a viable carrier for treatment and care for ischemic stroke healing, both in terms of delivering neuronal cells and protecting them from oxidative damage.

Prion peptide-mediated calcium level alteration governs neuronal cell damage through AMPK-autophagy flux

BackgroundThe distinctive molecular structure of the prion protein, PrPsc, is established only in mammals with infectious prion diseases. Prion protein characterizes either the transmissible pathogen itself or a primary constituent of the disease. Our report suggested that prion protein-mediated neuronal cell death is triggered by the autophagy flux. However, the alteration of intracellular calcium levels, AMPK activity in prion models has not been described. This study is focused on the effect of the changes in intracellular calcium levels on AMPK/autophagy flux pathway and PrP (106–126)-induced neurotoxicity.MethodsWestern blot and Immunocytochemistry was used to detect AMPK and autophagy-related protein expression. Flow cytometry and a TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay were used to detect the percentage of apoptotic cells. Calcium measurement was employed using fluo-4 by confocal microscope.ResultsWe examined the effect of calcium homeostasis alterations induced by human prion peptide on the autophagy flux in neuronal cells. Treatment with human prion peptide increased the intracellular calcium concentration and induced cell death in primary neurons as well as in a neuronal cell line. Using pharmacological inhibitors, we showed that the L-type calcium channel is involved in the cellular entry of calcium ions. Inhibition of calcium uptake prevented autophagic cell death and reduction in AMP-activated protein kinase (AMPK) activity induced by human prion peptide.ConclusionOur data demonstrated that prion peptide-mediated calcium inflow plays a pivotal role in prion peptide-induced autophagic cell death, and reduction in AMPK activity in neurons. Altogether, our results suggest that calcium influx might play a critical role in neurodegenerative diseases, including prion diseases.3p5PCBHnn1RWkUoaQCoeATVideo

Bioactive molecules isolated from olive pomace extract protect murine cortex neurons from NMDA‐mediated cell death

Here we demonstrate that olive pomace extracts contain bioactive molecules able to prevent primary neuron death induced by altered calcium homeostasis.We have observed previously that partially purified extracts counteracted calcium induced cell damages on human neuroblastoma (SK‐N‐BE) and mouse brain endothelioma (bEnd5) cell lines operating through the regulation of the cell dynamics that involve the calcium ions.It is well known that changes in [Ca2+]i are crucial events during signal transduction processes involved in several cellular physiological functions. Moreover, even a limited dysregulation in intracellular Ca2+ homeostasis is related to several acute and chronic diseases including neurodegenerations. In this context, we have now tested the effect of the bioactive molecules, detected in olive pomace, on primary cell cultures of murine cortex neurons, used as a cellular model. Accordingly, in order to induce a cytotoxic influx of Ca2+ through opening of the NMDA receptor, we treated neurons s with NMDA (N‐Methyl‐D‐Aspartic‐Acid) for 24 hour. When this cell treatment was carried out in the presence of preparations of the bioactive molecules, the effect of NMDA, evaluated as percentage of cell death, was significantly reduced. Moreover, following cell exposure to NMDA in the absence or the presence of the bioactive molecules, the [Ca2+]i was measured using different alternative techniques. The results obtained confirmed that these bioactive preparations act by molecular mechanisms that prevent the alteration of the intracellular calcium homeostasis mediated by NMDA. In conclusion, these bioactive molecules protect murine cortex neurons by reducing the toxic effects of [Ca2+]i, operated by the NMDA receptor. Further investigations are in progress to understand if the bioactive olive pomace derived molecules act directly on this receptor or on pathways activated downstream its opening. At present, the issue we give the highest priority is to identify the molecular nature of the relevant bioactive compounds. To solve this question we are carrying out mass spectrometry analyses on differently purified extracts.The ultimate aim is to achieve definitive conclusions about the potential practical applications for these natural molecules in their purified form. Indeed, providing comprehensive information about the structural and functional aspects of these molecules is crucial to propose new possible strategies for therapeutic interventions in neurodegenerations.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oleanolic acid protects against cognitive decline and neuroinflammation-mediated neurotoxicity by blocking secretory phospholipase A2 IIA-activated calcium signals

Neuroinflammation causes neurotoxic injury and underlies the pathogenesis of neurodegenerative disorders including Alzheimer's disease (AD). Astrocytes are the predominant immunoregulatory cells in AD. Oleanolic acid (OA) is a promising anti-inflammatory therapeutic agent that can ameliorate cerebral damage in ischemic environments, but its role in AD remains poorly elucidated. Here, preconditioning with OA inhibited the transcription and secretion of inflammatory cytokines IL-6, TNF-α, and IL-1β in amyloid-beta peptide (Aβ)-activated astrocytes. Moreover, OA ameliorated primary neuron death triggered by incubation in conditioned medium from Aβ-treated astrocytes. Furthermore, OA also suppressed Aβ-induced expression and production of group IIA secretory phospholipase A2 (sPLA2-IIA) in astrocytes. Supernatants supplemented with exogenous sPLA2-IIA reversed the protective role of OA against astrocyte activation-mediated neurotoxicity by suppressing cell viability and increasing LDH release, apoptosis, the contents of neurotoxic mediator arachidonic acid, and prostaglandin D2. Simultaneously, treatment with sPLA2 inhibitor aristolochic acid also counteracted neurotoxicity induced by Aβ-activated astrocytes through increasing cell viability, inhibiting cell apoptosis, and reducing the releases of arachidonic acid and prostaglandin D2. Additionally, OA restrained Ca2+ influx in neurons after incubation with supernatants from Aβ-activated astrocytes, which was abrogated by adding sPLA2-IIA. Activating Ca2+ signaling with BayK, an L-type Ca2 + channel agonist, reversed the beneficial role of OA against neurotoxicity induced by astrocyte activation-mediated inflammatory response. OA also ameliorated cognitive deficits in an adolescent rat model of Aβ-evoked AD. These findings confirm that OA abrogates neuroinflammation and subsequent neurotoxicity induced by conditioned media from Aβ-activated astrocytes in sPLA2-IIA mediated-calcium signals. Therefore, OA may protect neurons from injury caused by neighboring astrocyte activation in AD, indicating a promising therapeutic strategy against AD.

Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia.

Postcardiac arrest syndrome yields poor neurological outcomes, but the mechanisms underlying this condition remain poorly understood. Autophagy plays an important role in neuronal apoptosis induced by ischemia. However, whether autophagy is involved in neuron apoptosis induced by cardiac arrest has been less studied. This study found that TRPML1 participates in cerebral ischemic reperfusion injury. Primary neurons were isolated and treated with mucolipin synthetic agonist 1 (ML-SA1), as well as infected with the recombinant lentivirus TRPML1 overexpression vector in vitro. ML-SA1 was delivered intracerebroventricularly in transient global ischemia model. Protein expression levels were determined by western blot. Neurological deficit score and the infarct volume were analyzed for the detection of neuronal damage. We found that TRPML1 was significantly downregulated in vivo and in vitro ischemic reperfusion model. We also observed that TRPML1 overexpression or treatment with the ML-SA1 attenuated neuronal death in primary neurons and ameliorated neurological dysfunction in vivo. Our findings suggested that autophagy and apoptosis were activated after transient global ischemia. Administration of ML-SA1 before transient global ischemia ameliorated neurological dysfunction possibly through the promotion of autophagy and the inhibition of apoptosis.

Mutations in TGM6 induce the unfolded protein response in SCA35.

Spinocerebellar ataxia type 35 (SCA35) is a rare autosomal-dominant neurodegenerative disease caused by mutations in the TGM6 gene, which codes for transglutaminase 6 (TG6). Mutations in TG6 induce cerebellar degeneration by an unknown mechanism. We identified seven patients bearing new mutations in TGM6. To gain insights into the molecular basis of mutant TG6-induced neurotoxicity, we analyzed all the seven new TG6 mutants and the five TG6 mutants previously linked to SCA35. We found that the wild-type (TG6-WT) protein mainly localized to the nucleus and perinuclear area, whereas five TG6 mutations showed nuclear depletion, increased accumulation in the perinuclear area, insolubility and loss of enzymatic function. Aberrant accumulation of these TG6 mutants in the perinuclear area led to activation of the unfolded protein response (UPR), suggesting that specific TG6 mutants elicit an endoplasmic reticulum stress response. Mutations associated with activation of the UPR caused death of primary neurons and reduced the survival of novel Drosophila melanogaster models of SCA35. These results indicate that mutations differently impacting on TG6 function cause neuronal dysfunction and death through diverse mechanisms and highlight the UPR as a potential therapeutic target for patient treatment.

15-Deoxy-Δ12,14-prostaglandin J2 induced neurotoxicity via suppressing phosphoinositide 3-kinase

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) induces neuronal cell death via apoptosis independently of its receptors. 15d-PGJ2 inhibits growth factor-induced cell proliferation of primary astrocytes via down-regulating phosphoinositide 3-kinase (PI3K)-Akt pathway. Although 15d-PGJ2-reduced cell viability is accompanied with attenuation of the PI3K signaling in neuroblastoma, it has not been sufficiently clarified how 15d-PGJ2 induces cell death in primary neurons. Here, we found that 15d-PGJ2 exhibited neurotoxicity via inhibiting the PI3K signaling in the primary culture of rat cortical neurons. A PI3K inhibitor induced neuronal cell death regardless serum throughout maturation, confirming that PI3K is required for neuronal cell survival. The inhibitor disrupted neuronal cell bodies, shortened neurites thinly, damaged plasma membranes and activated caspase-3 similarly to 15d-PGJ2. Little additive or synergistic neurotoxicity was detected between 15d-PGJ2 and the PI3K inhibitor. A PI3K activator prevented neurons from undergoing the 15d-PGJ2-induced cell death in vitro. In vivo, the PI3K signaling is required for contextual memory retrieval, which was impaired by bilateral injection of 15d-PGJ2 into hippocampus. The activator suppressed the 15d-PGJ2-impaired memory retrieval significantly. In neurons as well as primary astrocytes and neuroblastomas, 15d-PGJ2 exhibited cytotoxicity via suppressing the PI3K-Akt pathway in vivo and in vitro.

A new procedure for amyloid β oligomers preparation enables the unambiguous testing of their effects on cytosolic and mitochondrial Ca2+ entry and cell death in primary neurons

Oligomers of the amyloid β peptide (Aβo) are becoming the most likely neurotoxin in Alzheimer’s disease. Controversy remains on the mechanisms involved in neurotoxicity induced by Aβo and the targets involved. We have reported that Aβo promote Ca2+ entry, mitochondrial Ca2+ overload and apoptosis in cultured cerebellar neurons. However, recent evidence suggests that some of these effects could be induced by glutamate receptor agonists solved in F12, the media in which Aβo are prepared. Here we have tested the effects of different media on Aβo formation and on cytosolic Ca2+ concentration ([Ca2+]cyt) in rat cerebellar and hippocampal cell cultures. We found that Aβo prepared according to previous protocols but solved in alternative media including saline, MEM and DMEM do not allow oligomer formation and fail to increase [Ca2+]cyt. Changes in the oligomerization protocol and supplementation of media with selected salts reported to favor oligomer formation enable Aβo formation. Aβo prepared by the new procedure and containing small molecular weight oligomers increased [Ca2+]cyt, promoted mitochondrial Ca2+ overload and cell death in cerebellar granule cells and hippocampal neurons. These results foster a role for Ca2+ entry in neurotoxicity induced by Aβo and provide a reliable procedure for investigating the Ca2+ entry pathway promoted by Aβo.

Cambinol, a novel inhibitor of neutral sphingomyelinase 2 shows neuroprotective properties.

Ceramide is a bioactive lipid that plays an important role in stress responses leading to apoptosis, cell growth arrest and differentiation. Ceramide production is due in part to sphingomyelin hydrolysis by sphingomyelinases. In brain, neutral sphingomyelinase 2 (nSMase2) is expressed in neurons and increases in its activity and expression have been associated with pro-inflammatory conditions observed in Alzheimer’s disease, multiple sclerosis and human immunodeficiency virus (HIV-1) patients. Increased nSMase2 activity translates into higher ceramide levels and neuronal cell death, which can be prevented by chemical or genetic inhibition of nSMase2 activity or expression. However, to date, there are no soluble, specific and potent small molecule inhibitor tool compounds for in vivo studies or as a starting point for medicinal chemistry optimization. Moreover, the majority of the known inhibitors were identified using bacterial, bovine or rat nSMase2. In an attempt to identify new inhibitor scaffolds, two activity assays were optimized as screening platform using the recombinant human enzyme. First, active hits were identified using a fluorescence-based high throughput compatible assay. Then, hits were confirmed using a 14C sphingomyelin-based direct activity assay. Pharmacologically active compounds and approved drugs were screened using this strategy which led to the identification of cambinol as a novel uncompetitive nSMase2 inhibitor (K i = 7 μM). The inhibitory activity of cambinol for nSMase2 was approximately 10-fold more potent than for its previously known target, silence information regulator 1 and 2 (SIRT1/2). Cambinol decreased tumor necrosis factor-α or interleukin-1 β-induced increases of ceramide and cell death in primary neurons. A preliminary study of cambinol structure and activity allowed the identification of the main structural features required for nSMase2 inhibition. Cambinol and its analogs may be useful as nSMase2 inhibitor tool compounds to prevent ceramide-dependent neurodegeneration.

Eriodictyol Attenuates β-Amyloid 25–35 Peptide-Induced Oxidative Cell Death in Primary Cultured Neurons by Activation of Nrf2

Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). Eriodictyol, a flavonoid isolated from the Chinese herb Dracocephalum rupestre, has long been established as an antioxidant. The present study was designed to investigate the effect of eriodictyol on β-amyloid 25-35 peptide (Aβ25-35)-induced oxidative cell death in primary neurons and to explore the role of the nuclear factor erythroid-2-related factor 2/antioxidant response element (Nrf2/ARE) pathway in this process. For this purpose, primary cultures of cortical neurons were exposed to 15μM Aβ25-35 in the absence or presence of eriodictyol (20, 40 and 80μM). The results revealed that Aβ25-35-induced cytotoxicity and apoptotic characteristics such as activation of JNK/p38 apoptotic signaling pathway were effectively attenuated by eriodictyol pretreatment. Eriodictyol treatment also resulted in an increase in Nrf2 protein levels and subsequent activation of ARE pathway genes in primary cultured neurons. The protective effects of eriodictyol were attenuated by RNA interference-mediated knockdown of Nrf2 expression. Taken together, these results clearly demonstrate that eriodictyol protects neurons against Aβ25-35-induced cell death partially through Nrf2/ARE signaling pathway, which further supports that eriodictyol might be a promising novel therapeutic agent for AD.

Antihelminthic benzimidazoles are novel HIF activators that prevent oxidative neuronal death via binding to tubulin.

Pharmacological activation of the adaptive response to hypoxia is a therapeutic strategy of growing interest for neurological conditions, including stroke, Huntington's disease, and Parkinson's disease. We screened a drug library with known safety in humans using a hippocampal neuroblast line expressing a reporter of hypoxia-inducible factor (HIF)-dependent transcription. Our screen identified more than 40 compounds with the ability to induce hypoxia response element-driven luciferase activity as well or better than deferoxamine, a canonical activator of hypoxic adaptation. Among the chemical entities identified, the antihelminthic benzimidazoles represented one pharmacophore that appeared multiple times in our screen. Secondary assays confirmed that antihelminthics stabilized the transcriptional activator HIF-1α and induced expression of a known HIF target gene, p21(cip1/waf1), in post-mitotic cortical neurons. The on-target effect of these agents in stimulating hypoxic signaling was binding to free tubulin. Moreover, antihelminthic benzimidazoles also abrogated oxidative stress-induced death in vitro, and this on-target effect also involves binding to free tubulin. These studies demonstrate that tubulin-binding drugs can activate a component of the hypoxic adaptive response, specifically the stabilization of HIF-1α and its downstream targets. Tubulin-binding drugs, including antihelminthic benzimidazoles, also abrogate oxidative neuronal death in primary neurons. Given their safety in humans and known ability to penetrate into the central nervous system, antihelminthic benzimidazoles may be considered viable candidates for treating diseases associated with oxidative neuronal death, including stroke.

Inhibition of autophagy and glycolysis by nitric oxide during hypoxia–reoxygenation impairs cellular bioenergetics and promotes cell death in primary neurons

Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons had the same basal cellular respiration in hypoxia as in normoxia, whereas NO-exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO-treated cells did not recover to prehypoxic levels. Hypoxia–reoxygenation in the presence of NO was associated with inhibition of autophagy, and the inability to recover during reoxygenation was exacerbated by an inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis, but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the medium or glycolysis was inhibited by 2-deoxy-d-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration that is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis, and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into the understanding of how autophagy and NO may play interactive roles in neuroinflammation-induced cellular damage, which is pertinent to our understanding of the pathology of neurodegenerative diseases in which excessive NO is generated.

80 : Novel multi-functional drugs for the treatment of neurodegenerative diseases

Oxidative stress (OS), glial activation and formation of plaques and tangles are believed to contribute to progressive neurodegeneration in Alzheimer’s disease (AD). None of the acetylcholinesterase (AChE) inhibitors currently in clinical use are able to reduce OS and inflammatory processes at doses used to treat AD. Therefore, there is a need for a drug which inhibits AChE and butyrylcholinesterase (BuChE), has anti-oxidant and anti-inflammatory activities. Indole propionic acid (IPA) prevents oxidative stress and death of primary neurons and neuroblastoma cells exposed to H2O2 or amyloid beta. Derivatives of indoline propionic acid (InPA) were found to be more potent anti-oxidants than those of IPA in cell culture. Therefore, a series of novel carbamates was synthesized based on the structure of InPA and their AChE, BuChE, anti-oxidant and anti-inflammatory activities were evaluated. Three compounds: Indoline-4-ylethyl methyl carbamate (AN-854), 3-(2-(methoxycarbonyl) ethyl) indolin-4-ylethyl methyl carbamate (AN-827) and 3-(2-(methoxycarbonyl) ethyl)-1-indoline-6-ylethyl methyl carbamate (AN-680) prevented cytotoxicity induced by OS and the release of cytokines from activated microglial cells at concentrations at, or lower than those inhibiting AChE. They inhibited AChE in rat brain and reduced lipid peroxidation and formation of nitrites in mouse brain after injection of lipopolysaccharide (LPS). When injected together with LPS they lowered the levels of COX-2 and pro-inflammatory cytokines (IL-6, IL-1β and TNF-α) in the brain and spleen. The mechanism of anti-inflammatory activity was evaluated in BV2 cells activated with LPS and shown to occur through reduction in phosphorylation of p38 and activation of the transcription factor AP-1. Conclusion: Indoline derivatives which display anti-oxidant and anti-inflammatory and cholinesterase inhibitory activities in vitro and in vivo, may have potential use for the treatment of AD and other neurodegenerative diseases.

WISP1 Neuroprotection Requires FoxO3a Post-Translational Modulation with Autoregulatory Control of SIRT1

As a member of the secreted extracellular matrix associated proteins of the CCN family, Wnt1 inducible signaling pathway protein 1 (WISP1/CCN4) is garnering increased attention not only as a potent proliferative entity, but also as a robust cytoprotective agent during toxic insults. Here we demonstrate that WISP1 prevents forkhead transcription factor FoxO3a mediated caspase 1 and caspase 3 apoptotic cell death in primary neurons during oxidant stress. Phosphoinositide 3-kinase (PI 3-K) and protein kinase B (Akt1) are necessary for WISP1 to foster posttranslational phosphorylation of FoxO3a and sequester FoxO3a in the cytoplasm of neurons with protein 14-3-3. Through an autoregulatory loop, WISP1 also minimizes deacytelation of FoxO3a, prevents caspase 1 and 3 activation, and promotes an effective neuroprotective level of SIRT1 activity through SIRT1 nuclear trafficking and prevention of SIRT1 caspase degradation. Elucidation of the critical pathways of WISP1 that determine neuronal cell survival during oxidative stress may offer novel therapeutic avenues for neurodegenerative disorders.

Evidence That the EphA2 Receptor Exacerbates Ischemic Brain Injury

Ephrin (Eph) signaling within the central nervous system is known to modulate axon guidance, synaptic plasticity, and to promote long-term potentiation. We investigated the potential involvement of EphA2 receptors in ischemic stroke-induced brain inflammation in a mouse model of focal stroke. Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and EphA2-deficient (EphA2−/−) mice by middle cerebral artery occlusion (MCAO; 60 min), followed by reperfusion (24 or 72 h). Brain infarction was measured using triphenyltetrazolium chloride staining. Neurological deficit scores and brain infarct volumes were significantly less in EphA2−/− mice compared with WT controls. This protection by EphA2 deletion was associated with a comparative decrease in brain edema, blood-brain barrier damage, MMP-9 expression and leukocyte infiltration, and higher expression levels of the tight junction protein, zona occludens-1. Moreover, EphA2−/− brains had significantly lower levels of the pro-apoptotic proteins, cleaved caspase-3 and BAX, and higher levels of the anti-apoptotic protein, Bcl-2 as compared to WT group. We confirmed that isolated WT cortical neurons express the EphA2 receptor and its ligands (ephrin-A1–A3). Furthermore, expression of all four proteins was increased in WT primary cortical neurons following 24 h of glucose deprivation, and in the brains of WT mice following stroke. Glucose deprivation induced less cell death in primary neurons from EphA2−/− compared with WT mice. In conclusion, our data provide the first evidence that the EphA2 receptor directly contributes to blood-brain barrier damage and neuronal death following ischemic stroke.

Role of Polyphosphate in Mitochondria: From Modification of Energy Metabolism to Induction of the Cell Death

Inorganic polyphosphate (polyP) is found in all living organisms ranging from bacteria to mammals. PolyP plays multiple physiological functions, which are distinct and dependent on the type of organism and the subcellular localization of the polymer. Recently we demonstrated that polyP levels are dependent on the cell metabolism and can be changed by mitochondrial substrates and inhibitors. We propose the existence of a feedback mechanism where polyP production and cell energy metabolism regulate each other. In order to investigate this we study the effect of polyP on mitochondrial oxygen consumption. We have found that application of short polyP (14 phosphate residues) or medium polyP (70 ortophosphates) significantly increase the level of respiratory coefficient by activation of state V3 and inhibition of V4 compare to control. Importantly, both short and medium polyP significantly reduced efficiency of oxidative phosphorylation (ADP/O ratio). It has previously been reported that polyP can modify membrane permeability for ions that can be a trigger for changes in mitochondrial metabolism. Furthermore polyP has been linked to activation of the mitochondrial permeability transition pore (mPTP) in different cells that also can be activated by calcium. Long, medium, and in lower degree, short polyP increase permeability of de-energised mitochondria for Ca2+. This effect was dependent on inhibitor of mPTP - cyclosporine A. We also found that long polyP (130 orthophosphates) caused cell death in primary neurons and astrocytes, while medium (70) polyP had a much smaller effect and short (14) did not cause any. Thus, polyP has a multiple action on mitochondrial function from modification of mitochondrial energy metabolism to stimulation of calcium permeability and cell death.

Loss of substance P and inflammation precede delayed neurodegeneration in the substantia nigra after cerebral ischemia

Focal cerebral ischemia leads to delayed neurodegeneration in remote brain regions. The substantia nigra (SN) does not normally show primary neuronal death after ischemic events affecting the striatum, but can exhibit delayed neuronal loss after the ischemic injury through mechanisms that are unknown. No data are available in mice showing acute post-stroke inflammation and remote injury in the SN. Substance P (SP), a mediator of neurogenic inflammation, is a key element of the striato-nigral circuitry, but alterations of SP in the SN have not been studied after acute striatal injury. Inflammation, a key contributor to neuronal death, is found in the SN after striatal ischemia, but it is unknown whether it precedes or occurs concomitantly with neuronal death. We hypothesised that focal striatal ischemia induces changes in SP levels in the SN and that inflammation precedes neuronal death in the SN. Using the middle cerebral artery occlusion model, we found a significant loss of SP in the ipsilateral SN 24h after striatal ischemia in mice. In the same area where SP loss occurs, significant glial and vascular activation, but no neuronal death, were observed. In contrast, a marked neuronal loss was observed within six days in the area of SP loss and inflammation. Our data suggest that focal loss of SP and early inflammatory changes in the SN precede remote neuronal injury after striatal ischemic damage. These observations may have important implications for motor impairment in stroke patients and indicate that striatal ischemia might facilitate Parkinson's disease development.

NAD+ administration decreases ischemic brain damage partially by blocking autophagy in a mouse model of brain ischemia

Nicotinamide adenine dinuleotide (NAD+) plays critical roles in multiple biological functions. Previous studies have indicated that NAD+ treatment decreases oxidative stress-induced death of primary neurons and astrocytes. Intranasal administration of NAD+ also reduces brain damage in a rat model of transient focal brain ischemia. However, the mechanisms underlying this protective effect remain unknown. In this study, we used a mouse model of brain ischemia to test our hypothesis that NAD+ decreases ischemic brain damage partially by preventing autophagy. Adult male mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 90min, and NAD+ was administered intraperitoneally (i.p.) immediately after reperfusion started. We found that administration with 50mg/kg NAD+ led to significant decreases in infarct size, edema formation, and neurological deficits at 48h after ischemia. NAD+ administration also significantly decreased brain ischemia-induced autophagy in the cortex and hippocampus. We further found that prevention of autophagy by 3-methyladenine (3-MA), a selective autophagy inhibitor, significantly reduced ischemic brain damage, suggesting an important role of autophagy in the ischemic brain injury in our animal model. Collectively, our findings have suggested that NAD+ administration decreases ischemic brain damage at least partially by blocking autophagy. This is the first suggested mechanism regarding the protective effects of NAD+ in cerebral ischemia, which further highlights the promise of NAD+ for treating brain ischemia.

Impedance measurement for real time detection of neuronal cell death

Detection of neuronal cell death is a standard requirement in cell culture models of neurodegenerative diseases. Although plenty of viability assays are available for in vitro applications, most of these are endpoint measurements providing only little information on the kinetics of cell death. Here, we validated the xCELLigence system based on impedance measurement for real-time detection of cell death in a neuronal cell line of immortalized hippocampal neurons (HT-22 cells), neuronal progenitor cells (NPC) and differentiated primary cortical neurons. We found a good correlation between impedance measurements and endpoint viability assays in HT-22 cells and NPC, for detecting proliferation, cell death kinetics and also neuroprotective effects of pharmacological inhibitors of apoptosis. In primary neurons we could not detect dendritic outgrowth during differentiation of the cells. Cell death in primary neurons was detectable by the xCELLigence system, however, the changes in the cell index on the basis of impedance measurements depended to a great extent on the severity of the insult. Cell death induced by ionomycin, e.g. shows as a fast paced process involving a strong cellular disintegration, which allows for impedance-based detection. Cell death accompanied by less pronounced morphological changes like glutamate induced cell death, however, is not well accessible by this approach. In conclusion, our data show that impedance measurement is a convenient and reliable method for the detection of proliferation and kinetics of cell death in neuronal cell lines, whereas this method is less suitable for the assessment of neuronal differentiation and viability of primary neurons.