Rat Hippocampal Cell Cultures Research Articles

P-510 - Idebenone attenuates amyloid β-peptide-induced cell death in rat hippocampal neuronal cell cultures

P-510 - Idebenone attenuates amyloid β-peptide-induced cell death in rat hippocampal neuronal cell cultures

Anti-apoptotic actions of cycloheximide: blockade of programmed cell death or induction of programmed cell life?

Cycloheximide (CHX), long recognized for its ability to inhibit protein synthesis, has been widely employed in studies of cell death to the extent that prevention of cell death by CHX has been used as prima facie evidence for a subtype of apoptosis called 'programmed cell death'. However, very rarely have investigators determined the effects of CHX on protein synthesis in their particular cell death paradigms. Recent findings are revealing alternative mechanisms of action of CHX that involve, ironically, stimulation of cytoprotective signalling pathways. For example, in embryonic rat hippocampal cell cultures CHX protects neurons against oxidative insults by a mechanism involving induction of neuroprotective gene products including Bcl-2. CHX induces increases in immediate early gene mRNA levels, and can activate several different kinases and transcription factors that are also activated by various insults and in response to anti-apoptotic growth factors. Concentrations of CHX that cause only a modest and/or transient decrease in over-all protein synthesis may prevent cell death by inducing cytoprotective signalling pathways ('programmed cell life'), whereas higher concentrations of CHX may prevent cell death by blocking the expression of 'death genes'. Establishing which of these anti-apoptotic mechanisms of action of CHX is operative in each cell death paradigm is clearly essential for proper interpretation of experimental results.

P-238 - Analysis of calcium waves observed in primary cultured rat hippocampal cells

P-238 - Analysis of calcium waves observed in primary cultured rat hippocampal cells

α1-Antichymotrypsin interaction with Aβ(1–42) does not inhibit fibril formation but attenuates the peptide toxicity

α1-Antichymotrypsin (ACT) is intimately associated with senile plaques in the Alzheimer's diseased (AD) brain. It was reported that ACT can promote the polimerization of Aβ(1–42) into amyloid filaments. It was suggested that neurotoxic amyloid deposits arise when β-peptide is induced to form fibrils by ACT or other amyloid-promoting factors (pathological chaperones) expressed in AD brain. However, it was reported recently that ACT can inhibit fibrillization of Aβ(1–40) and disaggregate pre-formed β-amyloid fibrils of this synthetic Aβ peptide. Our previous study [Aksenova et al., Neurosci. Lett., 411 (1996) 43–48] confirmed that ACT is able to inhibit Aβ(1–40) aggregation into fibrils, but it was shown that at the same time ACT does not change the peptide cytotoxicity. In this report we have observed that interaction of ACT with Aβ(1–42), unlike that for ACT-Aβ(1–40) interaction, does not prevent the formation of insoluble Aβ(1–42) aggregates, but completely blocks the peptide's toxicity in rat hippocampal cell cultures. These results are discussed in terms of the potential double role of peptide-protein interactions on Aβ aggregation and neurotoxicity.

α1-Antichymotrypsin interaction with Aβ(1–42) does not inhibit fibril formation but attenuates the peptide toxicity

α1-Antichymotrypsin (ACT) is intimately associated with senile plaques in the Alzheimer's diseased (AD) brain. It was reported that ACT can promote the polimerization of Aβ(1–42) into amyloid filaments. It was suggested that neurotoxic amyloid deposits arise when β-peptide is induced to form fibrils by ACT or other amyloid-promoting factors (pathological chaperones) expressed in AD brain. However, it was reported recently that ACT can inhibit fibrillization of Aβ(1–40) and disaggregate pre-formed β-amyloid fibrils of this synthetic Aβ peptide. Our previous study [Aksenova et al., Neurosci. Lett., 411 (1996) 43–48] confirmed that ACT is able to inhibit Aβ(1–40) aggregation into fibrils, but it was shown that at the same time ACT does not change the peptide cytotoxicity. In this report we have observed that interaction of ACT with Aβ(1–42), unlike that for ACT-Aβ(1–40) interaction, does not prevent the formation of insoluble Aβ(1–42) aggregates, but completely blocks the peptide's toxicity in rat hippocampal cell cultures. These results are discussed in terms of the potential double role of peptide-protein interactions on Aβ aggregation and neurotoxicity.

Stimulation of calcium uptake in cultured rat hippocampal neurons by 2,3,7,8-tetrachlorodibenzo- p-dioxin

This study examined the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (2,3,7,8-TCDD) and related compounds on the uptake of intracellular calcium ([Ca 2 +] i) in primary cultures of rat hippocampal neuronal cells. [Ca 2 +] i levels were detected and quantified by interactive laser cytometry with microscopic image analysis. Cells were noninvasively labeled with fluo-3/AM and all experiments were conducted on cultured rat hippocampal neurons 14 days in culture. Treatment of cell cultures with 2,3,7,8-TCDD (10–100 nM) resulted in a rapid concentration-dependent increase in [Ca 2 +] i associated with a decrease in mitochondrial membrane potential and activation of α-protein kinase C (α-PKC). In contrast, 1,2,3,4-TCDD, a weak Ah receptor agonist, had no effect on [Ca 2 +] i at concentrations as high as 10 μM and similar results were also observed for 2,2′,5,5′-tetrachlorobiphenyl. Maximal [Ca 2 +] i was observed within 30 s after addition of 2,3,7,8-TCDD and remained elevated (at higher concentrations) above resting levels for the duration of the experiment. This rapid increase in [Ca 2 +] i was blocked by addition of EDTA (2 mM) to the external medium or by pretreatment of the cells with the calcium channel antagonist nifedipine (10 μM). However, pretreatment of the cells with 100 μM cycloheximide failed to block calcium uptake in neuronal cells. These data indicate that rat hippocampal neuronal cells are responsive to 2,3,7,8-TCDD; however, the mechanism is not associated with altered gene transcription and may involve cellular membrane targets.

Morphology and Toxicity of Aβ-(1-42) Dimer Derived from Neuritic and Vascular Amyloid Deposits of Alzheimer's Disease

In the course of analyzing the chemical composition of Alzheimer's disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of Abeta peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by size exclusion chromatography in the presence of 80% formic acid or 5 guanidine thiocyanate, pH 7.4. The average ratio of monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic Abeta-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry. Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8-10-nm filaments, whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. The pathogenic effects of the dimeric Abeta-(1-40/42) were tested in cultures of rat hippocampal neuron glia cells. Only in the presence of microglia did the dimer elicit neuronal killing. It is possible that these potentially pathogenic Abeta-(1-40/42) dimers and trimers from Alzheimer's disease amyloid represent the soluble oligomers of Abeta recently described in Alzheimer's disease brains (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem., 271, 4077-4081).

α-1-Antichymotrypsin interaction with Aβ (1–40) inhibits fibril formation but does not affect the peptide toxicity

Recent studies have shown that senile plaque-associated or glial-derived proteins can prevent fibril formation of β-amyloid peptide (Aβ), while increasing the neurotoxicity of the latter (in the case of glutamine synthetase, apolipoprotein J or thrombin). α-1-Antichymotrypsin (ACT) is a glial-derived protein associated with senile plaques in the Alzheimer's brain. In this report we show that ACT, a minor protein component of β-amyloid deposits, is able to inhibit Aβ (1–40) aggregation into fibrils, but unable to modulate the toxicity of Aβ (1–40) in primary rat hippocampal cell cultures. These results are discussed in terms of the potential role of glial-derived proteins on Aβ aggregation and neurotoxicity.

Regulation of glucocorticosteroid receptor expression in rat hippocampal cell cultures by nerve growth factor

Dispersed hippocampal cells cultured in serum-free conditions were used to study the effects of nerve growth factor (NGF) on the expression of type I (mineralocorticosteroid or MR) and type II (glucocorticosteroid or GR) corticosteroid receptors. Cells, plated at a density of 1.2 x 10 6 cells/ml in 60 mm Petri dishes, were mainly identified as neurons (90–95%) and maintained for at least 2 weeks. A 7-day treatment with 10–50 ng NGF/ml induced a concentration-dependent decrease of GR binding (40% decrease) compared to entreated cells. In contrast, MR density was unaffected by a 7-day treatment with 50 ng NGF/ml. Data are discussed as possible direct and/or indirect effects of NGF at the level of both neuronal and glial cells.

PDGFs protect hippocampal neurons against energy deprivation and oxidative injury: evidence for induction of antioxidant pathways

Platelet-derived growth factors (PDGFs) and PDGF receptors are expressed in brain, where their functions are largely unknown. We tested the hypothesis that PDGFs play a role in promoting the survival of neurons exposed to metabolic and oxidative insults. Exposure of rat and mouse hippocampal cell cultures to glucose-deficient medium or the hydroxyl radical-promoting agent FeSO4 resulted in progressive neuronal loss. Pretreatment of cultures with PDGF-AA or PDGF-BB resulted in highly significant attenuation of glucose deprivation- and FeSO4-induced neuronal degeneration. In each injury paradigm the neuroprotective actions of the PDGFs were concentration dependent (3-100 ng/ml). In the case of glucose deprivation, significant protection was seen when cells were exposed to PDGFs prior to, or up to 8 hr following, the onset of glucose deprivation. Pretreatment with PDGFs was required for protection against FeSO4-induced oxidative injury. Western blot and immunocytochemical analyses demonstrated that cultured embryonic hippocampal neurons expressed both PDGF alpha- and beta-receptors. PDGFs induced tyrosine phosphorylation of several proteins including a band at 180 kDa, the molecular weight of PDGF receptors. Induction of peroxide accumulation in neurons by FeSO4 was attenuated in cultures pretreated with PDGFs, suggesting that PDGFs enhanced cellular antioxidant mechanisms. Measurements of anti-oxidant enzyme activities in control and PDGF-treated cultures showed that both PDGF-AA and PDGF-BB increased both catalase and glutathione peroxidase activities, and PDGF-AA also increased superoxide dismutase activities. These findings suggest that PDGFs, which are widely expressed in brain and induced in response to injury, may play roles in protecting neurons against metabolic and oxidative insults.

SB 201823-A antagonizes calcium currents in central neurons and reduces the effects of focal ischemia in rats and mice.

Excessive calcium entry into depolarized neurons contributes significantly to cerebral tissue damage after ischemia. We evaluated the ability of a novel neuronal calcium channel blocker, SB 201823-A, to block central neuronal calcium influx in vitro and to reduce ischemic injury in two rodent models of focal stroke. Patch-clamp electrophysiology and intracellular Ca2+ imaging in rat hippocampal and cerebellar neurons were used to determine effects on neuronal calcium channel activity. Middle cerebral artery occlusion was performed in Fisher 344 rats and CD-1 mice to determine the effects on rodent focal ischemic injury and neurological deficits. Cardiovascular monitoring in conscious rats was conducted to determine cardiovascular liabilities of the compound. In cultured rat hippocampal cells, calcium current measured at plateau was reduced by 36 +/- 8% and 89 +/- 4% after 5 and 20 mumol/L SB 201823-A, respectively. In cerebellar granule cells in culture, pretreatment with 2.5 mumol/L SB 201823-A totally prevented initial calcium influx and reduced later calcium influx by 50 +/- 2.5% after N-methyl-D-aspartate/glycine stimulation (P < .01). KCl depolarization-induced calcium influx also was reduced by more than 95%. In rats, a single treatment with 10 mg/kg IV SB 201823-A beginning 30 minutes after focal ischemia decreased (P < .05) hemispheric infarct by 30.4% and infarct volume by 29.3% and reduced (P < .05) forelimb deficits by 47.8% and hindlimb deficits by 36.3%. In mice, treatments with 10 mg/kg IP SB 201823-A beginning 30 minutes after focal ischemia significantly reduced infarct volume by 41.5% (P < .01). No blood pressure effects were observed with the therapeutic dose of the compound. These results indicate that the new neuronal calcium channel blocker SB 201823-A can block stimulated calcium influx into central neurons and can provide neuroprotection in two models of focal cerebral ischemia without affecting blood pressure. Data from several different studies now indicate that the neuronal calcium channel antagonists are a promising therapy for the postischemic treatment of stroke.

Opposing Actions of Thrombin and Protease Nexin‐1 on Amyloid β‐Peptide Toxicity and on Accumulation of Peroxides and Calcium in Hippocampal Neurons

Amyloid beta-peptide (A beta) is the principal component of neuritic plaques in the brain in Alzheimer's disease (AD). Recent studies revealed that A beta can be neurotoxic by a mechanism involving free radical production and loss of cellular ion homeostasis, thus implicating A beta as a key factor in the pathogenesis of AD. However, other proteins are present in plaques in AD, including the protease thrombin and protease nexin-1 (PN1), a thrombin inhibitor. We therefore tested the hypothesis that thrombin and PN1 modify neuronal vulnerability to A beta toxicity. In dissociated rat hippocampal cell cultures the toxicity of A beta was significantly enhanced by coincubation with thrombin, whereas PN1 protected neurons against A beta toxicity. A beta induced an increase in levels of intracellular peroxides and calcium. Thrombin enhanced, and PN1 attenuated, the accumulation of peroxides and calcium induced by A beta. Taken together, these data demonstrate that thrombin and PN1 have opposing effects on neuronal vulnerability to A beta and suggest that thrombin and PN1 play roles in the pathogenesis of neuronal injury in AD.

Apoptotic cell death of a temperature-sensitive central neuronal cell line

A neuron-like cell line HS-2, derived from a primary fetal rat (E17) hippocampal cell culture using the temperature-sensitive SV 40 large T antigen, exhibits flat shape and grows well in culture medium with 5% fetal calf serum (FCS) at the permissive temperature (PT, 33.5°C). At the non-permissive temperature (NPT, 38.5°C), many, but not all cells, have a neuronal shape with processes. The addition of dibutyryl-cAMP promotes the morphological changes in the cells to a neuron-like shape with long neurite-like processes and the cells exhibit neuron-specific enolase-and glutamic acid decarboxylase-immunoreactivity. Apoptotic cell death also occurs in these cultures at the NPT. DNA fragmentation and chromatin condensation that are characteristic of apoptosis occur within 8 h of being placed at the NPT. By 48 h after being placed at the NPT, the number of surviving cells decreases by 40% in the presence of 5% FCS. This cell line should be useful for investigating the mechanisms of ‘programmed cell death’ of neurons, which appears to occur during brain development and possibly in CNS degenerative diseases.

Neuroprotective sigma ligands interfere with the glutamate‐activated NOS pathway in hippocampal cell culture

We studied neuroprotective properties of 12 structurally different sigma site ligands in primary rat hippocampal cell cultures and analyzed whether they interfere with glutamate-induced activation of the nitric oxide synthase (NOS) pathway. Neurotoxicity was triggered with 1 mM glutamate on day 8 of culture. Cells were treated with various concentrations of the compounds for 7 days before glutamate exposure (prolonged pretreatment), or during glutamate exposure (acute treatment). Protection was seen after prolonged pretreatment (long-term protection) with sabeluzole, opipramole, haloperidol, ifenprodil, fenpropimorph, carbetapentane, and tiospirone, with pIC50s of 7.30, 7.15, 6.87, 6.68, 6.66, 6.39, and 6.34, respectively. There was no protection with PD 128298, 1,3-ortho-di-tolylguanidine, BMY-14802, (+)3-(3-hydroxyphenyl)-N-1(propyl) piperidine, or dextromethorphan. Upon acute treatment, only ifenprodil was protective. Interference of the drugs with glutamate activation of the NOS pathway was determined by measuring glutamate-activated cGMP formation and citrulline levels. Glutamate-activated cGMP formation was reduced by all neuroprotective sigma ligands after prolonged pretreatment but not after acute treatment. Sigma ligands added to cell culture lysate did not reduce citrulline formation, evidence that there was no direct effect on the NOS enzyme. We conclude that some but not all sigma ligands exert long-term protective properties against glutamate-induced neurotoxicity in primary hippocampal cultures, and that this protection is accompanied by attenuation of cGMP formation in the NOS pathway. However, inhibition of cGMP formation by itself appeared not sufficient for obtaining neuroprotective effects, as inhibition of glutamate-activated cGMP formation by N omega-nitro-L-arginine, haemoglobin, or PD128298 did not provide neuroprotection.

Attenuation of Traumatic Cell Death by an Adenosine A1 Agonist in Rat Hippocampal Cells

IN A RAT hippocampal cell culture, we studied the mechanism of adenosine-mediated neuroprotection in traumatic injury to neurons. When the processes and bodies of cells in culture were mechanically disrupted, neurons that were located at a distance from the damage site died. This secondary neuronal death is at least partially mediated by glutamate, because MK801, a specific N-methyl-D-aspartate glutamate channel blocker, diminished the toxic effect. Furthermore, cyclopentyl adenosine, a specific A1 adenosine receptor agonist that specifically attenuates synaptic release at the excitatory terminal, also blocked this trauma-mediated cell death. The dissemination of neurotoxicity from cell injury implies a release of a toxin by the dying cells. Consistent with this hypothesis, we found that neurotoxicity could be transferred to an uninjured neuronal culture by applying extracellular solution of the damaged culture to the healthy undamaged culture, as long as the fluid was transferred within 5 minutes. However, the glutamate concentrations in this medium were never higher than 20 nmol/L, suggesting that glutamate is not mediating the soluble and transferable toxicity. Consistent with this observation, the transferable neurotoxicity was not blocked by MK801 but was effectively blocked by cyclopentyl adenosine. Our observations suggest that traumatic cell death in culture is mediated by multiple mechanisms, including glutamate excitotoxicity.

Attenuation of traumatic cell death by an adenosine A1 agonist in rat hippocampal cells.

In a rat hippocampal cell culture, we studied the mechanism of adenosine-mediated neuroprotection in traumatic injury to neurons. When the processes and bodies of cells in culture were mechanically disrupted, neurons that were located at a distance from the damage site died. This secondary neuronal death is at least partially mediated by glutamate, because MK801, a specific N-methyl-D-aspartate glutamate channel blocker, diminished the toxic effect. Furthermore, cyclopentyl adenosine, a specific A1 adenosine receptor agonist that specifically attenuates synaptic release at the excitatory terminal, also blocked this trauma-mediated cell death. The dissemination of neurotoxicity from cell injury implies a release of a toxin by the dying cells. Consistent with this hypothesis, we found that neurotoxicity could be transferred to an uninjured neuronal culture by applying extracellular solution of the damaged culture to the healthy undamaged culture, as long as the fluid was transferred within 5 minutes. However, the glutamate concentrations in this medium were never higher than 20 nmol/L, suggesting that glutamate is not mediating the soluble and transferable toxicity. Consistent with this observation, the transferable neurotoxicity was not blocked by MK801 but was effectively blocked by cyclopentyl adenosine. Our observations suggest that traumatic cell death in culture is mediated by multiple mechanisms, including glutamate excitotoxicity.

Different amyloidogenic peptides share a similar mechanism of neurotoxicity involving reactive oxygen species and calcium

The amyloid β-peptide (Aβ) that accumulates as insoluble plaques in the brains of Alzheimer's victims can be neurotoxic, by a mechanism that may involve generation of reactive oxygen species (ROS) and destabilization of cellular calcium homeostasis. We now provide evidence that the mechanism of neurotoxicity of two other amyloidogenic peptides (APs), human amylin and β2-microglobulin, also involves induction of ROS and elevation of [Ca 2+] i. Human amylin, β2-microglobulin and Aβ1–40 all caused significant death of neurons in rat hippocampal cell cultures during 24–48h exposure periods. Rat amylin, a non-AP, was not neurotoxic. Each AP caused an elevation of rest [Ca 2+] i during a 20 h exposure period, and promoted a sustained elevation of [Ca 2+] i following exposure to glutamate which was significantly greater than controls. Each AP induced accumulation of ROS in neurons which preceded elevation of [Ca 2+] i. Several antioxidants, including propyl gallate, vitamin E and the spin-trapping compound N-tert-butyl-α-phenylnitrone attenuated the elevation of [Ca 2+] i and neurotoxicity induced by the peptides. The data indicate that different APs share a common mechanism of neurotoxicity involving free radical accumulation and destabilization of [Ca 2+] i homeostasis.

Measurements of exocytosis from single presynaptic nerve terminals reveal heterogeneous inhibition by Ca 2+-channel blockers

The effect of various Ca 2+-channel blockers on exocytosis has been studied at the level of single presynaptic terminals in rat hippocampal cell cultures. The fluorescence change of the styryl dye FM1–43 has been used as a measure of exocytosis during electrical stimulation. ω-Conotoxin GVIA (2–10 μM) completely inhibited exocytosis in ≈45% of the boutons in the field of view, while in ≈55% exocytosis was inhibited incompletely (by 38%). This heterogeneity in response of presynaptic boutons was not seen with isradipine (5 μM) or ω-agatoxin IVA (80 nM), which inhibited exocytosis by 23% and 17%, respectively. However, it was observed with a combination of all three blockers. Pre- and postsynaptic events could be separated in single synapses by measuring FM1–43 release and NMDA-induced changes in the intracellular Ca 2+ concentration independently.

Neurotrophin‐3 enhances neurite outgrowth in cultured hippocampal pyramidal neurons

Low density dissociated cultures of embryonic rat hippocampal cells were used to study the effects of neurotrophin-3 (NT-3) on neuronal morphogenesis. The results obtained indicate that NT-3 enhances neurite outgrowth and branching; this is a dose-dependent effect, detected in approximately 50% of the neurons, and prevented by K-252a, an inhibitor of the trk family of receptor protein kinases. NT-3 also accelerates the development of neuronal polarity, a phenomenon preceded by a dramatic accumulation of bundles of looped microtubules within axonal growth cones; these microtubule bundles contain tyrosinated, detyrosinated, and acetylated alpha-tubulin. Taken collectively, our data suggest that even though the basic shape of hippocampal neurons may be endogenously determined, critical aspects of their morphological development may be modulated by trophic factors such as NT-3. In addition, our observations suggest that at least some of the neuritogenic effects of NT-3 involve a stimulation of microtubule assembly and/or transport.

Secreted Forms of β-Amyloid Precursor Protein Protect Hippocampal Neurons against Amyloid β-Peptide-Induced Oxidative Injury

Alternative processing of the β-amyloid precursor protein (βAPP) can result in liberation of either secreted forms of βAPP (App ss), which may play roles in neuronal plasticity and survival, or amyloid β-peptide (Aβ), which can be neurotoxic. In rat hippocampal cell cultures Aβ1-40 caused a time- and concentration-dependent reduction in neuronal survival. App s695 and App s751 significantly reduced Aβ-induced injury in a concentration-dependent manner. Aβ caused an elevation of intracellular calcium levels ([Ca 2+] i) which was significantly attenuated by App ss. Aβ also caused induction of reactive oxygen species (measured using the oxidation-sensitive fluorescent dye 2,7-dichlorofluorescin) which was also attenuated by App ss. Aβ-induced neurotoxicity and elevations of [Ca 2+] i were attenuated by vitamin E, suggesting the involvement of free radicals in Aβ-induced loss of calcium homeostasis and neuronal injury. The App ss protected neurons against oxidative injury caused by exposure to iron. Taken together, the data indicate that Aβ kills neurons by causing free radical production and increased [Ca 2+] i. App ss can protect neurons against such free radical- and Ca 2+-mediated injury. These findings support the hypothesis that altered processing of βAPP contributes to neuronal injury in Alzheimer's disease.