Rat Organotypic Hippocampal Cultures Research Articles

Protein kinase C beta in postischemic brain mitochondria

PKC is implicated in the regulation of mitochondrial metabolism. We examined the association of PKCβ with mitochondria and followed postischemic changes in its amount in mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant (CA2–4,DG) hippocampus in gerbil model of transient brain ischemia. Our observations suggest that transient ischemic episode induces a significant, rapid and long lasting increase of PKCβ in mitochondria in CA2–4,DG, which may bespeak neuroprotection. In organotypic hippocampal culture (OHC) model of neurodegeneration, PKCβ inhibition imposed over NMDA toxicity extended the death area beyond the CA1. These results suggest that PKCβ might have a protective effect against excitotoxic damage in rat OHC. The pull-down method and LC-MS/MS analysis revealed mitochondrial proteins that can bind directly with PKCβΙ. The proteins were parts of i) mitochondrial redox carriers forming the electron transport chain including ATP synthase and ii) MPTP: ANT and creatine kinase. PKCβ acting through mitochondrial proteins could play a role in protecting the cells from death by e.g. influencing ROS and ATP production after ischemia in CA2–4,DG region of the hippocampus.

Preconditioning effects of tumor necrosis factor-α and glutamate on calcium dynamics in rat organotypic hippocampal cultures

During cerebral ischemia, elevation of TNF-α and glutamate to pathophysiological levels in the hippocampus may induce dysregulation of normal synaptic processes, leading ultimately to cell death. Previous studies have shown that patients subjected to a mild transient ischemic attack within a critical time window prior to a more severe ischemic episode may show attenuation in the clinical severity of the stroke and result in a more positive functional outcome. In this study we have investigated the individual contribution of pre-exposure to TNF-α or glutamate in the development of ‘ischemic tolerance’ to a subsequent insult, using organotypic hippocampal cultures. At 6days in vitro (DIV), cultures were exposed to an acute concentration of glutamate (30μM) or TNF-α (5ng/ml) for 30min, followed by 24h recovery period. We then examined the effect of the pretreatments on calcium dynamics of the cells within the CA region. We found that pretreatment with TNF-α or glutamate caused in a significant reduction in subsequent glutamate-induced Ca2+ influx 24h later (control: 100.0±0.8%, n=7769 cells; TNF-α: 76.8±1.0%, n=5543 cells; glutamate: 75.3±1.4%, n=3859 cells; p<0.001). Antagonism of circulating TNF-α (using infliximab, 25μg/ml), and inhibition of the p38 MAP kinase pathway (using SB 203580, 10μM) completely reversed this effect. However glutamate preconditioning did not appear to be mediated by p38 MAP kinase signalling, or NMDAR activation as neither SB 203580 nor D-AP5 (100μM) altered this effect. Glutamate and TNF-α preconditioning resulted in small yet significant alterations in resting Ca2+ levels (control: 100.0±0.9%, n=2994 cells; TNF-α: 109.7±1.0%, n=2884 cells; glutamate; 93.3±0.8%, n=2899 cells; p<0.001), TNF-α's effect reversed by infliximab and SB 203580. Both TNF-α and glutamate also resulted in the reduction of the proportion (P) of responsive cells within the CA region of the hippocampus (control; P=0.459, 0.451≤x≥0.467, n=14,968 cells, TNF-α; P=0.40, 0.392≤x≥0.407, n=15,218; glutamate; P=0.388, 0.303≤x≥0.396, n=13,919 cells), and in the depression of the frequency of spontaneous Ca2+ events (vs. control: TNF-α: p>0.00001, D=0.0454; glutamate: p>0.0001, D=0.0534). Our results suggest that attenuation in resting Ca2+ activity and Ca2+ related responsiveness of cells within the CA region as a result of glutamate or TNF-α pre-exposure, may contribute to the development of ischemic tolerance.

Mesenchymal Stem Cell-Conditioned Medium Triggers Neuroinflammation and Reactive Species Generation in Organotypic Cultures of Rat Hippocampus

Cell therapy using bone marrow-derived mesenchymal stem cells (MSCs) seems to be a new alternative for the treatment of neurodegenerative diseases. Despite several promising results with their use, possible side effects are still unknown. In a previous work, we have shown that MSC-conditioned medium is toxic to hippocampal slice cultures and aggravates cell death induced by oxygen and glucose deprivation. In this work, we investigated whether the inflammatory response and/or reactive species formation could be involved in that toxicity. Rat organotypic hippocampal cultures were exposed for 24 h to conditioned medium from MSCs isolated from rat bone marrow. A marked glial activation was observed after exposure of cultures to MSC-conditioned medium, as evidenced by glial fibrillary acid protein (GFAP) and isolectin B(4) increase. Tumor necrosis factor-α and interleukin-6 levels were increased in the culture medium, and 2,7-dihydrodichlorofluorescein diacetate oxidation (indicating reactive species generation) and inducible nitric oxide synthase (iNOS) immunocontent were also higher after exposure of cultures to MSC-conditioned medium. Antioxidants (ascorbic acid and TROLOX(®)), N(ω)-nitro-l-arginine methyl ester hydrochloride, and anti-inflammatory drugs (indomethacin and dexamethasone) reduced cell death in hippocampal organotypic cultures after their exposure to MSC-conditioned medium. The results obtained here suggest that MSC-secreted factors trigger reactive species generation and neuroinflammation in organotypic cultures of hippocampus, introducing a note of caution in the use of these cells for neurological application.

Transcriptomics of Traumatic Brain Injury: Gene Expression and Molecular Pathways of Different Grades of Insult in a Rat Organotypic Hippocampal Culture Model

Traumatic brain injury (TBI) is the one of the most common forms of head trauma, and it remains a leading cause of death and disability. It is known that the initial mechanical axonal injury triggers a complex cascade of neuroinflammatory and metabolic events, the understanding of which is essential for clinical, translational, and pharmacological research. These can occur even in mild TBI, and are associated with several post-concussion manifestations, including transiently heightened vulnerability to a second insult. Recent studies have challenged the tenet that ischemia is the ultimate modality of tissue damage following TBI, as metabolic dysfunction can develop in the presence of normal perfusion and before intracranial hypertension. In order to elucidate the cellular and molecular changes occurring in TBI as a direct result of neuronal injury and in the absence of ischemic damage, we performed a microarray analysis of expressed genes and molecular interaction pathways for different levels of severity of trauma using an in-vitro model. A stretch injury, equivalent to human diffuse axonal injury, was delivered to rat organotypic hippocampal slice cultures, and mRNA levels following a 10% (mild) and 50% (severe) stretch were compared with controls at 24 h. More genes were differentially expressed following 10% stretch than 50% stretch, indicating the early activation of complex cellular mechanisms. The data revealed remarkable differential gene expression following mTBI, even in the absence of cell damage. Pathway analysis revealed that molecular interactions in both levels of injury were similar, with IL-1beta playing a central role. Additional pathways of neurodegeneration involving RhoA (ras homolog gene family, member A) were found in 50% stretch.

Determination of ζ-Potential and Tortuosity in Rat Organotypic Hippocampal Cultures from Electroosmotic Velocity Measurements under Feedback Control

Extracellular translational motion in the brain is generally considered to be governed by diffusion and tortuosity. However, the brain as a whole has a significant zeta-potential, thus translational motion is also governed by electrokinetic effects under a naturally occurring or applied electric field. We have previously measured zeta-potential and tortuosity in intact brain tissue; however, the method was tedious. In this work, we use a four-electrode potentiostat to control the potential difference between two microreference electrodes in the tissue, creating a constant electric field. Additionally, some alterations have been made to simplify our previous procedure. The method entails simultaneously injecting two 70 kDa dextran conjugated fluorophores into rat organotypic hippocampal cultures and observing their mobility using fluorescence microscopy. We further present two methods of data analysis: regression and two-probe analysis. Statistical comparisons are made between the previous and current methods as well as between the two data analysis methods. In comparison to the previous method, the current, simpler method with data analysis by regression gives statistically indistinguishable mean values of zeta-potential and tortuosity, with a similar variability for zeta-potential, -21.3 +/- 2.8 mV, and a larger variability for the tortuosity, 1.98 +/- 0.12. On the other hand, we find that the current method combined with the two-probe analysis produces accurate and more precise results, with a zeta-potential of -22.8 +/- 0.8 mV and a tortuosity of 2.24 +/- 0.10.

Inositol 1,4,5-triphosphate receptors and NAD(P)H mediate Ca 2+ signaling required for hypoxic preconditioning of hippocampal neurons

Exposure of neurons to a non-lethal hypoxic stress greatly reduces cell death during subsequent severe ischemia (hypoxic preconditioning, HPC). In organotypic cultures of rat hippocampus, we demonstrate that HPC requires inositol triphosphate (IP 3) receptor-dependent Ca 2+ release from the endoplasmic reticulum (ER) triggered by increased cytosolic NAD(P)H. Ca 2+ chelation with intracellular BAPTA, ER Ca 2+ store depletion with thapsigargin, IP 3 receptor block with xestospongin, and RNA interference against subtype 1 of the IP 3 receptor all blunted the moderate increases in [Ca 2+] i (50–100 nM) required for tolerance induction. Increases in [Ca 2+] i during HPC and neuroprotection following HPC were not prevented with NMDA receptor block or by removing Ca 2+ from the bathing medium. Increased NAD(P)H fluorescence in CA1 neurons during hypoxia and demonstration that NADH manipulation increases [Ca 2+] i in an IP 3R-dependent manner revealed a primary role of cellular redox state in liberation of Ca 2+ from the ER. Blockade of IP 3Rs and intracellular Ca 2+ chelation prevented phosphorylation of known HPC signaling targets, including MAPK p42/44 (ERK), protein kinase B (Akt) and CREB. We conclude that the endoplasmic reticulum, acting via redox/NADH-dependent intracellular Ca 2+ store release, is an important mediator of the neuroprotective response to hypoxic stress.

Conditioned medium from mesenchymal stem cells induces cell death in organotypic cultures of rat hippocampus and aggravates lesion in a model of oxygen and glucose deprivation

Cell therapy using bone marrow-derived mesenchymal stem cells (MSC) seems to be a new alternative for the treatment of neurological diseases, including stroke. In order to investigate the response of hippocampal tissue to factors secreted by MSC and if these factors are neuroprotective in a model of oxygen and glucose deprivation (OGD), we used organotypic hippocampal cultures exposed to conditioned medium from bone marrow-derived MSC. Our results suggest that the conditioned medium obtained from these cells aggravates lesion caused by OGD. In addition, the presence of the conditioned medium alone was toxic mainly to cells in the CA1, CA2 and CA3 areas of the hippocampal organotypic culture even in basal conditions. GABA stimulation and NMDA and AMPA receptors antagonists were able to reduce propidium iodide staining, suggesting that the cell death induced by the toxic factors secreted by MSC could involve these receptors.

Subregion-specific vulnerability to endoplasmic reticulum stress-induced neurotoxicity in rat hippocampal neurons

It is well known that in certain disease states, including ischemia and Alzheimer's disease, neurodegeneration occurs in the hippocampus and that vulnerability to neuronal death is area dependent. The present study investigated the mechanism of area-dependent vulnerability to neuronal death under endoplasmic reticulum stress conditions induced by tunicamycin (TM), using rat organotypic hippocampal cultures (OHC) and hippocampal slices. Analysis of propidium iodide uptake showed that TM-induced neuronal death in a concentration-dependent manner (20–80 μg/mL) and that the rank order of vulnerability among hippocampal subregions was dentate gyrus (DG) > CA1 > CA3. Results of immunohistochemistry using hippocampal slices also showed that procaspase-12-positive cells in area CA3 were significantly fewer than those in area CA1 and the DG. Moreover, procurement of neurons in areas CA1, CA3 and the DG by laser microdissection, followed by Western blot analysis, also revealed that the level of procaspase-12 in area CA3 was significantly lower than those in area CA1 and the DG. Pretreatment with z-ATAD-fmk, a cell-permeable caspase-12-selective inhibitor significantly attenuated the TM-induced increase of PI fluorescence in the CA1 and DG subregion but not in area CA3. These results suggest that TM elicits subregion-specific neuronal toxicity in OHC and that the vulnerability to TM-induced toxicity is at least partly dependent on the expression level of endogenous procaspase-12 in each area of the hippocampus.

Protective Effect of (4-Methoxybenzylidene)-(3-methoxynophenyl)amine against Neuronal Cell Death Induced by Oxygen and Glucose Deprivation in Rat Organotypic Hippocampal Slice Culture

Resveratrol (trans-3,4',5-trihydroxystilbene) is a natural phytoalexin found in grape skin, and has been suggested to be an antioxidant agent, an anticancer agent and a cardioprotective agent. In particular, recent experimental evidence has demonstrated that resveratrol exhibits neuroprotective effects in various assay systems. During the study on the resveratrol derivatives, we found that (4-methoxybenzylidene)-(3-methoxyphenyl)amine (MBMPA), which has blocked free phenolic groups, strongly protects neuronal cells against ischemic damage on a higher activity than resveratrol. The MBMPA potently reduced the level of neuronal cell death in an oxygen and glucose deprivation-exposed rat organotypic hippocampal slice culture. In addition, ATP depletion following the onset of oxygen and glucose deprivation in an adult hippocampal slice was blocked by the MBMPA treatment. These results suggest that MBMPA has a neuroprotective effect on an in vitro ischemia model, and may be useful for treating stroke.

Postnatal effect of embryonic neurogenesis disturbance on reelin level in organotypic cultures of rat hippocampus

Despite a delayed emergence of the symptoms, schizophrenia is thought to be a late consequence of early disturbances during development. Several reports have found decreased levels of reelin in the cortex and the hippocampus of postmortem brains of schizophrenic patients. In the rat, intraperitoneal injection of the anti-mitotic agent methylazoxymethanol (MAM) during intra-uterine development (embryonic day 17) induces cytoarchitectural abnormalities in the hippocampus and the cortex and behavioural changes reminiscent of positive, negative and cognitive symptoms of schizophrenia. We aimed to examine whether a transient prenatal disturbance of neurogenesis induces postnatal changes in the expression of reelin in the hippocampus. Cellular modifications were explored using hippocampal organotypic slice cultures, which allow for conservation of the in vivo cytoarchitecture. MAM effect on hippocampal neurogenesis was confirmed by birthdating experiments. After 3 weeks in vitro, reelin was expressed by calretinin-negative cells. The number of reelin-positive neurons was increased whereas the total neuron number was decreased in the stratum oriens in the E17 MAM-exposed animals as compared to the control group. Not only an increase in the number of cells expressing reelin was observed, but there was also a slight increase in reelin mRNA levels in hippocampal pyramidal cells of MAM-exposed animals. In contrast, there was no significant change in the dentate gyrus. These results show that transient prenatal disturbance of neurogenesis induces long-term modifications in specific areas of the hippocampus and in particular in the number of neurons expressing reelin. They also confirm the value of organotypic slices to study postnatal maturation in the hippocampus.

Mitogen-Activated Protein Kinase Signaling, Oxygen Sensors and Hypoxic Induction of Neurogenesis

In the adult nervous system, neuronal subpopulations sustain a hierarchical pattern of selective vulnerability to hypoxia. Hypoxia also activates quiescent neural progenitor cells (NPCs) resulting in their amplification and subsequent differentiation into neurons and glia. Use of rat organotypic hippocampal cultures facilitates examination of early signaling events in response to hypoxia and reoxygenation that result in neurogenesis. Cultures were exposed to hypoxia for up to 6 h followed by reoxygenation. CA1 neurons showed focal nuclear condensation by 2 h of hypoxia, but CA2 and CA3 neurons were spared. JNKs and c-Jun reached peak activation by 4 h, returning to basal levels by 6 h. Expression of oxygen sensors, hemoxygenase 2 and HIF1, were elevated by 30 min and 2 h, respectively. By 24 h of reoxygenation, there was proliferation of nestin-positive NPCs. With U0126, an upstream inhibitor of ERK activation, BrdU labeling was markedly reduced immunohistochemically as well as PCNA protein expression, suggesting a role for ERKs in the proliferation response. Immunohistochemically, antinestin detected NPCs and on Western blots reached peak levels by 24–48 h of reoxygenation. Proliferation and differentiation of endogenous NPCs in the area of neuronal loss further suggests that mechanisms potentially exist in vitro for replacement with functional neurons.

Isoflurane Preconditions Hippocampal Neurons against Oxygen–Glucose Deprivation

Isoflurane preconditions neurons to improve tolerance of subsequent ischemia in both intact animal models and in in vitro preparations. The mechanisms for this protection remain largely undefined. Because isoflurane increases intracellular Ca2+ concentrations and Ca2+ is involved in many processes related to preconditioning, the authors hypothesized that isoflurane preconditions neurons via Ca2+-dependent processes involving the Ca2+- binding protein calmodulin and the mitogen-activated protein kinase-ERK pathway. The authors used a preconditioning model in which organotypic cultures of rat hippocampus were exposed to 0.5-1.5% isoflurane for a 2-h period 24 h before an ischemia-like injury of oxygen-glucose deprivation. Survival of CA1, CA3, and dentate neurons was assessed 48 later, along with interval measurements of intracellular Ca2+ concentration (fura-2 fluorescence microscopy in CA1 neurons), mitogen-activated protein kinase p42/44, and the survival associated proteins Akt and GSK-3beta (in situ immunostaining and Western blots). Preconditioning with 0.5-1.5% isoflurane decreased neuron death in CA1 and CA3 regions of hippocampal slice cultures after oxygen-glucose deprivation. The preconditioning period was associated with an increase in basal intracellular Ca2+ concentration of 7-15%, which involved Ca2+ release from inositol triphosphate-sensitive stores in the endoplasmic reticulum, and transient phosphorylation of mitogen-activated protein kinase p42/44 and the survival-associated proteins Akt and GSK-3beta. Preconditioning protection was eliminated by the mitogen-activated extracellular kinase inhibitor U0126, which prevented phosphorylation of p44 during preconditioning, and by calmidazolium, which antagonizes the effects of Ca2+-bound calmodulin. Isoflurane, at clinical concentrations, preconditions neurons in hippocampal slice cultures by mechanisms that apparently involve release of Ca2+ from the endoplasmic reticulum, transient increases in intracellular Ca2+ concentration, the Ca2+ binding protein calmodulin, and phosphorylation of the mitogen-activated protein kinase p42/44.

The Putative Neuroprotective Role of Neuropeptide Y in the Central Nervous System

Neuropeptide Y (NPY) is one of the most abundant and widely distributed neuropeptides in the mammalian central nervous system (CNS). An overview of the distribution of the G-protein coupled NPY receptor family (Y(1), Y(2), Y(4), Y(5) receptors) in the brain is described. The coexistence of NPY with other neurotransmitters and its wide distribution in several brain areas predict the high importance of NPY as a neuromodulator. Thus, the effect of NPY on the release of several neurotransmitters such as glutamate, gamma-aminobutyric acid (GABA), norepinephrine (NE), dopamine, somastotatin (SOM), serotonin (5-HT), nitric oxide (NO), growth hormone (GH) and corticotropin releasing factor (CRF) is reviewed. A neuroprotective role for NPY under physiological conditions and during hyperactivity such as epileptic-seizures has been suggested. We have shown previously that NPY inhibits glutamate release evoked from hippocampal nerve terminals and has a neuroprotective effect in rat organotypic hippocampal cultures exposed to an excitotoxic insult. Moreover, changes in NPY levels have been observed in different pathological conditions such as brain ischemia and neurodegenerative diseases (Huntington's, Alzheimer's and Parkinson's diseases). Taken together, these studies suggest that NPY and NPY receptors may represent pharmacological targets in different pathophysiological conditions in the CNS.

Src tyrosine kinase signaling regulates microglial protection of neurons from excitotoxicity

Microglial activation is thought to be important in Alzheimer's disease (AD). However, signal transduction pathways mediating microglia activation are incompletely understood. Src tyrosine kinase is important in the regulation of a variety of cellular functions, including inflammation. We previously found that overexpressing the M-CSF receptor (M-CSFR; encoded by c-fms) on microglia leads to rescue of neurons from NMDA-induced excitotoxicity. We sought to determine if Src signaling mediates M-CSFR-induced neuroprotection. We examined the effects of M-CSFR overexpression on Src kinase levels and activity. We also tested whether LPS, a potent inflammatory stimulus, activated Src. To determine the specificity of M-CSFR activation of Src, we quantified the activity of the Src-family kinase Lyn. A mutagenesis approach was used to block M-CSFR-induced Src activation, and the effects on microglial neuroprotection were examined. We also tested whether a Src dominant negative (DN) construct would block M-CSFR-induced neuroprotection. Western analysis showed that microglial M-CSFR activation resulted in a greater than 2 fold increase in Src expression, whereas an enzymatic assay showed a greater than 3.5 fold increase in Src kinase activity. Microglia treatment with LPS did affect Src activity. M-CSFR activation had no effect on Lyn activity. In the M-CSFR, tyrosine position c-fmsY559 binds Src and induces activation by phosphorylation. Position c-fmsY807, the global c-fms activation site, indirectly affects Src activation. Using co-cultures consisting of microglia overexpressing the M-CSFR and rat organotypic hippocampal cultures subjected to NMDA-induced excitotoxicity, overexpression of the Y559F or Y807F c-fms mutant constructs in microglia resulted in a loss of neurprotection. Similarly, microglial expression of a Src DN form resulted in loss of M-CSFR-induced neuroprotection, and also inhibited microglial proliferation. Increasing the number of microglia expressing the Src DN form in co-culture only partially restored neuroprotection. Adding exogenous M-CSF also partially restored neuroprotection by Src DN-treated microglia in co-cultures. Our results show that the Src signal transduction pathway is important in microglial neuroprotection. Identification of downstream Src kinase targets in microglia could lead to new pharmacological treatments for AD (Funding: Alzheimer's Association and NIH MH57833).

Inhibition of T-Type Calcium Channels Protects Neurons from Delayed Ischemia-Induced Damage

Intracellular calcium increase is an early key event triggering ischemic neuronal cell damage. The role of T-type voltage-gated calcium channels in the neuronal response to ischemia, however, has never been studied. Using an in vitro model of ischemia-induced delayed cell death in rat organotypic hippocampal slice cultures, we show that T-type calcium channels inhibitors drastically reduce ischemic cell damage. Immunostaining studies reveal the existence of Ca(V)3.1 and Ca(V)3.2 types of low-voltage-activated calcium channels in rat organotypic hippocampal cultures. Low extracellular calcium (100 nM) or increase of intracellular calcium buffering ability by BAPTA-acetoxymethyl ester significantly reduced ischemia-induced neuronal damage. Pharmacological inhibition of the T-type calcium current by mibefradil, kurtoxin, nickel, zinc, and pimozide during the oxygen-glucose deprivation episode provided a significant protection against delayed neuronal death. Mibefradil and nickel exerted neuroprotective effects, not only if administrated during the oxygen-glucose deprivation episode but also in conditions of postischemic treatment. These data point to a role of T-type calcium currents in ischemia-induced, calcium-mediated neuronal cell damage and suggest a possible new pharmacological approach to stroke treatment.

JIP1 regulates neuronal apoptosis in response to stress

We examined if the relative expression of JNK-interacting protein 1 (JIP1) and phosphorylated c-Jun N-terminal kinase (JNK) regulates cell signaling and contributes to selective neuronal vulnerability in response to environmental stress. In clonal neuroblastoma cultures, stresses such as hypoxia, ischemia, Aβ peptides, and UV irradiation rapidly reduced JIP1 expression. JIP1 mRNA expression was also down-regulated by UV stress and was accompanied by increased JNK and c-Jun activation and cell death. JIP1 protein reduction was partially reversed both by inhibitors predominantly of caspase 3 and of the JNK pathway and resulted in significantly increased cell survival. Conversely, overexpression of JIP1 decreased both nuclear translocation of activated-JNK, and c-Jun phosphorylation induced by either UV irradiation, or the JNK upstream activators, MKK7 or MEKK1. Cell death was reduced about 50% compared to GFP-transfected controls. JIP1 overexpression did not facilitate either JNK expression or activation. In the normal, non-stressed human hippocampus and rat hippocampal organotypic cultures, JIP1 and JNK3 were inversely expressed with more JIP1 in CA2 and CA3 and less in CA1 neurons. In the human hippocampus, transient hypoxia/ischemia selectively spares neurons in CA2 and CA3 and induces death of neurons in the hippocampal CA1 subregion. In the cultures, ischemia reduced JIP1 expression and activated JNK, c-Jun, and caspase 3. Inhibitors of the JNK pathway, JNK activation directly and of caspase 3 activation each partially reversed these effects. Thus, under certain stress conditions, down-regulation of JIP1 expression makes neurons more susceptible to apoptosis, suggesting JIP may serve as an anti-apoptosis factor.

Neurogenesis response to hypoxia-induced cell death: map kinase signal transduction mechanisms

Hypoxic stress induces apoptosis of hippocampal CA1 neurons while selectively sparing those in CA2–3. Proliferation and differentiation of local stem cells may potentially replace lost neurons. We examined MAP kinase signaling regulation of these dual responses. Rat organotypic hippocampal cultures were exposed to hypoxia for up to 6 h followed by reoxygenation. JNKs and ERKs were maximally activated by 4 h, returning approximately to basal levels by 6 h. Apoptosis of CA1 neurons was maximal by 6-h hypoxia, although JNK activation had returned to basal levels. A neuroprotective protein, JNK-interacting protein 1 (JIP1), an inhibitor of JNK-mediated apoptosis, was reduced by 6-h hypoxia and markedly decreased by 24-h reoxygenation in CA1 neurons as was DENN/MADD, which also modulates JNK-mediated cell death. A second peak of ERK1 activation occurred at 24-h reoxygenation and declined to control levels by 48 h. Stem cells were detected by antinestin and cell proliferation confirmed with anti-PCNA immunohistochemistry and BrdU incorporation. With U0126, an inhibitor of ERK activation, BrdU labeling was strikingly reduced implicating ERKs in the proliferation response. Antidoublecortin (DCX), which detects neural progenitor cells, colabeled a subset of BrdU-positive cells that extended from the dentate granule neurons into CA1. Astrocytes were colabeled with BrdU. Thus, hypoxia concurrently triggered both JNK and ERK signaling, and with reoxygenation, ERK1 activation and stem cell proliferation followed by neuronal progenitor cell differentiation and targeted migration to the site of pyramidal neuronal loss.

A novel peptide modulates alpha7 nicotinic receptor responses: implications for a possible trophic-toxic mechanism within the brain.

The alpha7 nicotinic acetylcholine receptor (nAChR) plays a key role in neural development and neurodegeneration. Here, we identify a novel, modulatory receptor ligand, a 14-amino acid peptide (AEFHRWSSYMVHWK) derived from the C-terminus of acetylcholinesterase (AChE). In three different in vitro preparations, this 'AChE-peptide' is bioactive in a ligand-specific and concentration-dependent manner. First, it modulates acutely the effect of acetylcholine (ACh) on Xenopus oocytes transfected with human alpha7, but not alpha4/beta2, nAChR. The action persists when intracellular calcium is chelated with BAPTA or when calcium is substituted with barium ions. This observation suggests that intracellular Ca(2+) signals do not mediate the interaction between the peptide and nAChR, but rather that the interaction is direct: however, the intervention of other mediators cannot be excluded. Secondly, in recordings from the CA1 region in guinea-pig hippocampal slices, AChE-peptide modulates synaptic plasticity in a alpha-bungarotoxin (alpha-BgTx)-sensitive manner. Thirdly, in organotypic cultures of rat hippocampus, long-term exposure to peptide attenuates neurite outgrowth: this chronic, functional effect is selectively blocked by the alpha7 nAChR antagonists, alpha-BgTx and methyllycaconitine, but not by the alpha4/beta2-preferring blocker dihydro-beta-ethroidine. A scrambled peptide variant, and the analogous peptide from butyrylcholinesterase, are ineffective in all three paradigms. The consequences of this novel modulation of the alpha7 nAChR may be activation of a trophic-toxic axis, of relevance to neurodegeneration.

Alcohol‐Induced Neurodegeneration: When, Where and Why?

This manuscript reviews the proceedings of a symposium organized by Drs. Antonio Noronha and Fulton Crews presented at the 2003 Research Society on Alcoholism meeting. The purpose of the symposium was to examine recent findings on when alcohol induced brain damage occurs, e.g., during intoxication and/or during alcohol withdrawal. Further studies investigate specific brain regions (where) and the mechanisms (why) of alcoholic neurodegeneration. The presentations were (1) Characterization of Synaptic Loss in Cerebella of Mature and Senescent Rats after Lengthy Chronic Ethanol Consumption, (2) Ethanol Withdrawal Both Causes Neurotoxicity and Inhibits Neuronal Recovery Processes in Rat Organotypic Hippocampal Cultures, (3) Binge Drinking-Induced Brain Damage: Genetic and Age Related Effects, (4) Binge Ethanol-Induced Brain Damage: Involvement of Edema, Arachidonic Acid and Tissue Necrosis Factor alpha (TNFalpha), and (5) Cyclic AMP Cascade, Stem Cells and Ethanol. Taken together these studies suggest that alcoholic neurodegeneration occurs through multiple mechanisms and in multiple brain regions both during intoxication and withdrawal.

Bioactivity of a peptide derived from acetylcholinesterase in hippocampal organotypic cultures.

While the molecular basis underlying the non-classical actions of acetylcholinesterase (AChE) is presently unknown, a candidate peptide sequence located at the C-terminus of AChE (AChE-peptide) has recently been identified. This study explored the bioactivity of synthetic AChE-peptide using in vitro organotypic cultures of rat hippocampus. Neurotrophic effects, detected as increased neurite outgrowth from MAP-immunopositive neurones, were apparent using 1 h exposure to 1-10 nM AChE-peptide. As exposure time increased, cell death occurred as indicated by TdT-mediated dUTP biotin nick-end labelling (TUNEL). This process was accelerated at higher AChE-peptide concentrations, with lactate dehydrogenase (LDH) efflux observed following prolonged exposure to 1-10 microM AChE-peptide. Apoptotic cells were detected by Hoechst 33342 staining following 24 h application of 10 nM AChE-peptide. However, propidium iodide reactivity revealed a simultaneous loss of membrane integrity indicative of necrosis, suggesting that AChE-peptide induces cell death via a continuum of apoptotic and necrotic processes. Prolonged exposure to AChE-peptide also resulted in a concentration-dependent reduction in neurite outgrowth from MAP2-positive neurons, although immunohistochemical studies provided some evidence of differential responsiveness in GABAergic, cholinergic and somatostatin neurones. In addition, bioactivity was sequence specific since a scrambled AChE-peptide analogue, as well as the corresponding BuChE-peptide, was ineffective. In conclusion, the bioactivity associated with the AChE-peptide sequence may account for the non-cholinergic actions of AChE, whilst its neurotrophic-apoptotic-necrotic spectrum of action may be involved in the aetiology of neurodegenerative disorders such as Alzheimer's disease.