Death In Rat Hippocampal Neurons Research Articles

Aging and amyloid \u03b2 oligomers enhance TLR4 expression, LPS-induced Ca2+ responses, and neuron cell death in cultured rat hippocampal neurons

BackgroundToll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer’s disease (AD). AD is associated to oligomers of the amyloid β peptide (Aβo) that cause intracellular Ca2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aβo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence.MethodsCa2+ imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aβo on cytosolic [Ca2+] and on apoptosis as well as on expression of TLR4.ResultsLPS increases cytosolic [Ca2+] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aβo increases TLR4 expression and enhances LPS-induced Ca2+ responses and neuron cell death.ConclusionsAging and amyloid β oligomers, the neurotoxin involved in Alzheimer’s disease, enhance TLR4 expression as well as LPS-induced Ca2+ responses and neuron cell death in rat hippocampal neurons aged in vitro.

1154 – Single prolonged stress triggers endoplasmic reticulum stress and endoplasmic reticulum associated death in rat hippocampal neurons

The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. The ER is highly sensitive to stresses that perturb cellular energy levels, the redox state or Ca 2+ concentration. ER stress and ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. We suspect that Single prolonged stress which is extensively used as an established animal model for PTSD triggers endoplasmic reticulum stress and endoplasmic reticulum associated death in rat hippocampal neurons. Grp94 (Glucose-regulated protein 94), is the ER-resident member of the heat-shock-protein 90 (Hsp90) family, highly expressed when ERS happened. Caspase-12 is a protease until now believed to play a central role in the initiation of ER stress-induced cell death. In present study, Changes of GRP94 (Glucose-regulated protein 94) and caspase-12 were detected by immunohistochemistry, reverse transcription-polymerase chain reaction and western blot. Changes of GRP94 and caspase-12 may contribute to the progress of molecular mechanism of PTSD.

Novel neuroprotective effect of cisternal and intra-cerebral magnesium sulfate solution infusion on delayed cerebral death in rat hippocampal neurons after transient global ischemia

Several neuroprotective strategies such as barbiturate coma and hypothermic therapies have been promising in experimental studies but had limited efficacies in clinical trials. Magnesium ion has vasodilator and neuroprotective properties as a natural calcium blocker. The neuroprotective effects of cisternal and intra-cerebral infusion of magnesium sulfate (MgSO4) solution were evaluated against delayed neuronal death (DND) of CA1 hippocampal pyramidal neurons, and the involvement of changes in cerebral blood flow (CBF) and metabolism were investigated. Osmotic pumps with various MgSO4 solutions were embedded in the Sprague Dawley rat backs on day 1 and either cisternal or intra-cerebral (left CA1) infusion started. Global ischemic insult for 10min was induced on day 3, and the numbers of surviving neurons and dead neurons in the CA1 region were evaluated by hematoxylin–eosin and Fluoro-Jade B staining, respectively, on day 7. The regional CBF and glucose utilization rate (CMRglc) were determined by autoradiographic methods using [14C]iodoantipyrine and [14C]deoxyglucose, respectively, on day 5 after the start of either cisternal or intra-cerebral infusion. In the cisternal infusion experiment, 100mmol/lMgSO4 solution significantly increased the number of surviving neurons and decreased the number of dead neurons. In the intra-cerebral infusion experiment, 1mmol/lMgSO4 and 10mmol/lMgSO4 solution significantly increased the number of surviving neurons and decreased the number of dead neurons in the infusion side in dose-dependent manner. Neither regional CBF or CMRglc changed significantly in the CA1 of the hippocampus. Pretreatment with either cisternal or intra-cerebral infusion with MgSO4 solution can provide neuroprotection against DND without changing the CBF and metabolism.

Methylglyoxal causes strong weakening of detoxifying capacity and apoptotic cell death in rat hippocampal neurons

The hippocampus is known to play a crucial role in learning and memory. Recent data from literature show that cognitive problems, common to aged or diabetic patients, may be related to accumulation of toxic α-oxoaldehydes such as methylglyoxal. Thus, it is possible that methylglyoxal could be, at least in part, responsible for the impairment of cognitive functions, and the knowledge of the mechanisms through which this compound elicits neuronal toxicity could be useful for the development of possible therapeutic strategies.We previously reported a high susceptibility of hippocampal neurons to methylglyoxal, through an oxidation-dependent mechanism. In the present study, we extend our investigation on the molecular mechanisms which underlie methylglyoxal toxicity, focusing on possible effects on expression and activity of glyoxalases, its main detoxifying enzymes, and glutathione peroxidase, as well as on the levels of reduced glutathione. We also investigate methylglyoxal-induced modulation of brain derived neurotrophic factor and proinflammatory cytokines.Our results show that methylglyoxal causes a dramatic depletion of reduced glutathione and a significant inhibition of both glyoxalase and glutathione peroxidase activities. Furthermore, methylglyoxal treatment seems to affect the expression of inflammatory cytokines and survival factors.In conclusion, our findings suggest that methylglyoxal-induced neurotoxicity occurs through the impairment of detoxification pathway and depletion of reduced glutathione. This, in turn, triggers widespread apoptotic cell death, occurring through the convergence of both mitochondrial and Fas-receptor pathways.

Δ 9-Tetrahydrocannabinol protects hippocampal neurons from excitotoxicity

Excitotoxic neuronal death underlies many neurodegenerative disorders. Because cannabinoid receptor agonists act presynaptically to inhibit glutamate release, we examined the effects of Win 55212-2, a full agonist at CB 1 receptors, and Δ 9-tetrahydrocannabinol (THC), a partial agonist, on the survival of neurons exposed to an excitotoxic pattern of synaptic activity. Reducing the extracellular Mg 2+ concentration ([Mg 2+] o) to 0.1 mM evoked an aberrant pattern of glutamatergic activity that produced synaptically mediated death of rat hippocampal neurons in culture. Neuronal viability was quantified with a multiwell fluorescence plate scanner equipped to detect propidium iodide fluorescence. Win 55212-2 (100 nM) and THC (100 nM) significantly reduced 0.1 mM [Mg 2+] o-induced cell death by 77 ± 11% and 84 ± 8%, respectively. Interestingly, the protection afforded by THC was not significantly different from that produced by Win 55212-2, suggesting that attenuation without a complete block of excitatory activity is sufficient for neuroprotection. The effect of prolonged drug exposure on the neuroprotection afforded by cannabinoid receptor agonists was also studied. When cultures were pretreated for 24 h with Win 55212-2 (100 nM) or THC (100 nM), inhibition of 0.1 mM [Mg 2+] o-induced toxicity was significantly reduced to 39 ± 19% and 45 ± 13%, respectively. Thus, desensitization of CB 1 receptors diminishes the neuroprotective effects of cannabinoids. This study demonstrates the importance of agonist efficacy and the duration of treatment on the neuroprotective effects of cannabinoids. It will be important to consider these effects on neuronal survival when evaluating pharmacologic treatments that modulate the endocannabinoid system.

Attenuation of zinc-induced neuronal death by the interaction of growth inhibitory factor with Rab3A in rat hippocampal neurons

We examined the protective effect of growth inhibitory factor (GIF) against zinc-induced neuronal death in rat hippocampal neurons. In an in vitro cell culture system, 300 μM Zn 2+ readily induced death of hippocampal neuronal cells, which was characterized by massive necrosis and a minor degree of apoptosis. Neither the addition of recombinant GIF nor Rab3A alone could rescue these cells from death. However, the combination of GIF with Rab3A could significantly enhance the survival of the hippocampal neurons. This result was supported by both Annexin -V FITC/propidium dual staining and chromosomal DNA analysis. These findings suggest that GIF may inhibit Zn 2+-induced neuronal death via its interaction with Rab3A.

Influence of cytosolic and mitochondrial Ca 2+, ATP, mitochondrial membrane potential, and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, induces both rapid necrotic and slow apoptotic death in rat hippocampal neurons. Low levels of extracellular glutamate (10 μM) shift the 3NP-induced cell death mechanism to necrosis, while NMDA receptor blockade results in predominantly apoptotic death. In this study, we examined the 3NP-induced alterations in free cytosolic and mitochondrial calcium levels, ATP levels, mitochondrial membrane potential, and calpain and caspase activity, under conditions resulting in the activation of apoptotic and necrotic pathways. In the presence of 10 μM glutamate, 3NP administration resulted in a massive elevation in [Ca 2+] c and [Ca 2+] m, decreased ATP, rapid mitochondrial membrane depolarization, and a rapid activation of calpain but not caspase activity. In the presence of the NMDA receptor antagonist MK-801, 3NP did not induce a significant elevation of [Ca 2+] c within the 24 h time period examined, nor increase [Ca 2+] m within 1 h. ATP was maintained at control levels during the first hour of treatment, but declined 64% by 16 h. Calpain and caspase activity were first evident at 24 h following 3NP administration. 3NP treatment alone resulted in a more rapid decline in ATP, more rapid calpain activation (within 8 h), and elevated [Ca 2+] m as compared to the results obtained with added MK-801. Together, the results demonstrate that 3NP-induced necrotic neuron death is associated with a massive calcium influx through NMDA receptors, resulting in mitochondrial depolarization and calpain activation; while 3NP-induced apoptotic neuron death is not associated with significant elevations in [Ca 2+] c, nor with early changes in [Ca 2+] m, mitochondrial membrane potential, ATP levels, or calpain activity.

Propentofylline protects β-amyloid protein-induced apoptosis in cultured rat hippocampal neurons

β-Amyloid protein 1–42 (β42) can induce apoptosis in the cultured hippocampal neurons, suggesting that it plays an important role in causing neurodegeneration in Alzheimer's disease. Recently, propentofylline, a synthetic xanthine derivative, has been reported to depress ischemic degeneration of hippocampal neurons in gerbils. The present study investigated whether or not propentofylline affected the β42-induced apoptosis of hippocampal neurons, and if so, which type of signaling machinery works in the neuroprotective action of propentofylline. Addition of propentofylline markedly attenuated the β42-induced cell death of rat hippocampal neurons. The amyloid protein certainly induced apoptosis in the cultured hippocampal cells revealed by nuclear condensation, caspase-3 activation and an increase of Bax. Intriguingly, propentofylline blocked both the apoptotic features induced by β42 and further induced an anti-apoptotic protein, Bcl-2, during a short time of incubation. The neuroprotective action of propentofylline was comparably replaced with dibutyryl cAMP (dbcAMP) and was completely suppressed by a low concentration of specific protein kinase A (PKA) inhibitor. Taken altogether, the data strongly suggest that the protection of propentofylline on the β42-induced neurotoxicity is caused by enhancing anti-apoptotic action through cAMP–PKA system. Propentofylline as a therapeutic agent to Alzheimer's disease is discussed.

Recombinant core protein fragment of phosphacan, a brain specific chondroitin sulfate proteoglycan, promote excitotoxic cell death of cultured rat hippocampal neurons

We investigated the role of phosphacan, a chondroitin sulfate proteoglycan that is constitutively expressed in the adult hippocampus, and recombinant core proteins of phosphacan in excitotoxic cell death of primary cultured rat hippocampal neurons. Phosphacan had no significant effect on excitotoxic neuronal death. Surprisingly, one of three recombinant proteins corresponding to N-terminal portions of phosphacan core protein dramatically promoted excitotoxic neuronal death. Moreover, the recombinant protein induced cell death of rat hippocampal neurons, even when neurons were not exposed to glutamate. These results suggest that proteolytic degradation of phosphacan and resultant core protein fragments may contribute to neuronal degeneration of hippocampal neurons in various neuropathological conditions.

Role of calpain- and interleukin-1 beta converting enzyme-like proteases in the beta-amyloid-induced death of rat hippocampal neurons in culture.

We investigated the potential role of different proteases in the death of cultured rat hippocampal pyramidal neurons induced by beta-amyloid (A beta) (25-35). Both A beta(25-35)- and staurosporine-induced death of these neurons appeared to involve apoptosis, as indicated using Hoechst 33342 and terminal dUDP nick end labeling staining, whereas NMDA-induced death appeared more complex. Two irreversible inhibitors of the interleukin-1 beta converting enzyme (ICE) and related proteases, Z-Val-Ala-Asp-CH2F and acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone, blocked neuronal death produced by A beta(25-35), staurosporine, and NMDA to differing extents. Furthermore, MDL 28,170, a selective inhibitor of the calcium-regulated protease calpain, also inhibited death induced by all agents. A beta(25-35) and staurosporine stimulated the breakdown of the protein spectrin, a calpain substrate. Spectrin breakdown was inhibited by MDL 28,170 but not by ICE inhibitors. Leupeptin was only effective in preventing NMDA-induced death. These results support the role of apoptosis in neuronal death due to A beta(25-35) treatment and also suggest a role for calcium-regulated proteases in this process.

Exposure of rat hippocampal neurons to amyloid β peptide (25–35) induces the inactivation of phosphatidyl inositol-3 kinase and the activation of tau protein kinase I/glycogen synthase kinase-3β

Exposure of rat hippocampal neurons to the peptide amyloid β (Aβ) (25–35) as well as Aβ (1–40) peptides enhances phosphorylation of tau to a paired helical filament (PHF)-state through activation of tau protein kinase I (TPK I)/glycogen synthase kinase-3β (GSK-3β) [Busciglio, J., Lorenzo, A., Yeh, J. and Yankner, B.A., Neuron, 14 (1995) 879–888; Takashima, A., Ishiguro, K., Noguchi, K., Michel, G., Hoshi, M., Sato, K., Takahashi, M., Hoshino, T., Uchida, T. and Imahori, K., Neurosci. Meeting Abstr., 671 (1995) 17]. In order to examine the effects of Aβ treatment on intracellular signaling mechanism, we have investigated the role of phosphatidyl inositol-3 (PI-3) kinase in the phosphorylation of tau. Aβ (25–35) exposure induced an inactivation of PI-3 kinase and an activation of TPK I/GSK-3β in rat hippocampal culture. Wortmannin, an inhibitor of PI-3 kinase, also activated TPK I/GSK-3β, leading to an enhancement of tau phosphorylation and neuronal death in hippocampal culture. These results suggest that Aβ (25–35) inhibition of PI-3 kinase results in the activation of TPK I/GSK-3β, the phosphorylation of tau, and resultant neuronal death in rat hippocampal neurons.