Heat stress reduces glutamate toxicity in cultured neurons without HSP70 expression

doi:10.1016/0006-8993(96)00572-0

Brain Research

Volume 729, Issue 2, 12 August 1996, Pages 273-276

https://doi.org/10.1016/0006-8993(96)00572-0 Get rights and content

Abstract

Consistent with known cytoprotective effects, mild heat stress reduced the vulnerability of cultured cortical neurons to glutamate-induced cell death. However, this neuroprotective effect occurred without expression of the inducible heat stress protein, hsp70, as detected by immunocytochemistry, immunoblotting, or metabolic labeling. Present data suggest that the anti -exeitotoxic effect of mild heat stress is mediated outside of the traditional hsp70 mechanism.

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Cited by (11)

Mild heat stress induces hormetic effects in protecting the primary culture of mouse prefrontal cerebrocortical neurons from neuropathological alterations
2018, IBRO Reports
Citation Excerpt :
It will substantially contribute to improve the health of general public directly by creating the awareness and indirectly by using the concept of hormesis while framing the government policies related to healthcare sector. Various studies have reported that mild heat stress exerts beneficial effects on the neurons by improving the functional ability, neurite outgrowth, neuroprotection and neuronal survival in various neurodegenerative disorders such as Alzheimer’s disease, Parkinson disease, Huntington’s disease etc. (Snider and Choi, 1996; Mattson and Cheng, 2006; Umschweif et al., 2014). The hormetic effects can be studied as a preconditioning hormesis and a postconditioning hormesis (Calabrese et al., 2007; Calabrese, 2016a, 2016b).
Hormesis is a dose response phenomenon of cells and organisms to various types of stressors. Mild stress stimulates prosurvival pathways and makes the cells adaptive to stressful conditions. It is a widely used fundamental dose-response phenomenon in many biomedical and toxicological sciences, radiation biology, health science etc. Mild heat stress is an easily applicable hormetic agent that exerts consistent results. In the present investigations mouse cerebrocortical prefrontal neurons from E17 mouse embryos were grown in the laboratory on poly-L-lysine coated glass cover slips. The cells from the mild heat stressed group were subjected to a hyperthermic stress of 38 °C for 30 min every alternate day (i.e. mild heat stress was repeated after 48 h) up to the sixth day. After completion of twenty four hours of the final i.e. third exposure of the mild heat stress, the neurons were fixed for the cytochemical studies of neurofibrillary tangles, senile plaques, lipofuscin granules and Nissl substance. There was highly significant decrease in the neuropathological alterations (viz. deposition of Neurofibrillary tangles, deposition of senile plaques, accumulation of Lipofuscin granules) in the neurons from the mild heat stressed group as compared to control. Moreover, the Nissl substance was significantly preserved in the mild heat stressed group as compared to control. The results indicate that the applied mild heat stress (38 °C for 30 min) exerts beneficial effects on the prefrontal cerebrocortical neurons by slowing down the neuropathological alterations, suggesting the hormetic effect of the mild heat stress.
A 72 kDa heat shock protein is protective against the selective vulnerability of CA1 neurons and is essential for the tolerance exhibited by CA3 neurons in the hippocampus
2002, Neuroscience
The correlation between the expression of a 72 kDa heat shock protein and vulnerability of hippocampal CA1, CA3, and dentate gyrus regions to glutamate toxicity was investigated using a highly specific antisense oligonucleotide technique. Glutamate (1 mM, 15 min) caused region-dependent neuronal damage in cultured hippocampal slices 24 h after exposure and the most severe damage was observed in CA1. When slices were heat-shocked (43.5°C, 30 min) before exposure to glutamate, neuronal damage in CA1 was attenuated. The strongest protection was observed when the interval between the heat shock and the exposure to glutamate was 3 days, which coincided with the maximal induction of a 72 kDa heat shock protein in neurons. When the expression of a 72 kDa heat shock protein was suppressed by the antisense oligonucleotide, the protective effect of the heat shock was completely inhibited. Glutamate itself also induced a 72 kDa heat shock protein in neurons, region-dependently, 24 h after the exposure. The signal of a 72 kDa heat shock protein in CA3 and dentate gyrus was significantly stronger than that in CA1. When the antisense oligonucleotide was applied, the damage in CA3 and dentate gyrus was exaggerated dose-dependently, and this effect was more remarkable in CA3 than in the dentate gyrus.
Based on these data, we concluded that: (i) a 72 kDa heat shock protein has a protective effect against the selective vulnerability of CA1 neurons, (ii) a 72 kDa heat shock protein is an essential factor for the tolerance exhibited by CA3 neurons, and (iii) dentate gyrus tolerance is based on mechanisms other than those mediated through a 72 kDa heat shock protein.
Cycloheximide reduces infarct volume when administered up to 6 h after mild focal ischemia in rats
2001, Brain Research
Citation Excerpt :
Proteins (10 μg) were denatured in Laemmli loading buffer and electrophoresed on a 10% SDS–polyacrylamide gel. Immunoblotting was performed as previously described [60] except that a chemiluminescent detection system was used (SuperSignal ECL, Pierce Chemical Co). Blots were probed with antibody specific for the inducible isoform of HSP70 (SPA-810, StressGen, 1:2000), β-tubulin (YOL1/34, Accurate Chemical Co., 1:1000), prostate apoptotis response gene 4 (PAR4, Santa Cruz R-334, 1:2500), apoptosis linked gene 2 (ALG-2, Transduction Laboratories, 1:1000), and Fas ligand/CD95L (FasL, Transduction Laboratories, 1:2500).
We have previously described a rodent model of brief (30 min) middle cerebral artery occlusion followed by reperfusion, in which infarction develops gradually, reaching completion more than 3 days after ischemia, accompanied by morphological, biochemical, and pharmacological evidence of apoptosis. In the present study, we tested the hypotheses that delayed administration of a protein synthesis inhibitor would be effective in reducing tissue injury in this slowly evolving ischemic infarction, and that efficacy of this treatment would wane with more prolonged ischemia. Focal cerebral ischemia was induced in Long–Evans rats by occlusion of the right middle cerebral artery. Infarction volume was analyzed using triphenyl tetrazolium chloride staining, and morphology was studied using hematoxylin and eosin stained sections. Following 30 min middle cerebral artery occlusion and reperfusion, the core ischemic region exhibited vacuolization in the neuropil by 36 h after ischemia, and infarction reached full size by 7 days after ischemia. Cycloheximide reduced infarct volume when given up to 6 h after ischemia. If the duration of ischemic insult was increased to 90 min, the therapeutic window for delayed cycloheximide was only 30 min. In permanent middle cerebral artery occlusion, cycloheximide was ineffective even when given prior to ischemia onset. After mild, but not severe, ischemic insults, cerebral infarction develops slowly and may be treatable with protein synthesis inhibitors, even when treatment is delayed for up to 6 h after the onset of ischemia.
Differential neuroprotection from human heat shock protein 70 overexpression in in Vitro and in Vivo models of ischemia and ischemia-like conditions
2001, Experimental Neurology
We previously showed that overexpressing the 70-kDa inducible heat shock protein in primary astrocyte cultures and in a rodent stroke model using viral vectors resulted in protection from ischemia and ischemia-like injury. However, viral transfection could potentially provoke a stress response itself; therefore, we examined whether transgenic mice constitutively expressing human heat shock protein 70 were protected from ischemic insults. Astrocyte cultures from brains of heat shock protein 70 transgenic mice were resistant to hydrogen peroxide injury in a dose-dependent fashion, but were less resistant to hypoglycemia and oxygen-glucose deprivation. Because hydrogen peroxide exposure and glucose deprivation are partially dependent on glutathione levels, we determined whether heat shock protein 70 transgenic cultures had altered glutathione levels under normal growth conditions. However, there was no significant difference in glutathione levels between heat shock protein 70 transgenic and wildtype astrocytes. Hippocampal, but not cortical neuron cultures from these same transgenic mice were also protected against oxygen-glucose deprivation and glutamate toxicity. In an in vivo model of permanent focal cerebral ischemia, there was no significant difference in infarct size assessed 24 h postinsult. These results suggest that heat shock protein 70 protects against some but not all kinds of central nervous system injury. The protective effects may be related to the nature and severity of the insults, as well as subpopulations of brain cells and dose-dependent effects of HSP70 overexpression.
Ischemic 'cross' tolerance in hypoxic ischemia of immature rat brain
1999, Brain Research
The phenomenon of ischemic tolerance has been closely associated with the expression of heat shock proteins but recently, stress tolerance not related to hsp72 has been reported. In the present study, we focused on ischemic tolerance induced by hypoxia and hyperthermia in neonatal rat brain and analyzed the expression of hsp72. In a neonatal rat model of hypoxic ischemia (H-I), preconditioning by whole-body hyperthermia or hypoxia was induced 24 h prior to the ischemia. Brain damage was histologically evaluated and the expressions of hsp72 were analyzed. Hyperthermic preconditioning at 41°C for 15 min, as well as hypoxic preconditioning with 8% hypoxia for 3 h, had almost complete neuroprotective effects. However, we failed to detect the expression of hsp72 in any of preconditioning. Only the H-I insult itself induced hsp72 in the dorsal striatum and slightly in the thalamus and the hippocampus. Hyperthermic preconditioning has neuroprotective effects which are comparable to hypoxic preconditioning in immature brain. The expression of hsp72 is not likely necessary for the ischemic tolerance in immature brain.
Ischaemic pre-conditioning in organotypic hippocampal slice cultures is inversely correlated to the induction of the 72 kDa heat shock protein (HSP72)
1999, Brain Research
In vivo, preconditioning with a sublethal insult can confer resistance to normally lethal episodes of cerebral ischaemia. This phenomenon has been linked with the induction of the 72 kDa heat shock protein (HSP72), but this has not been clearly demonstrated in vitro. We have used organotypic hippocampal slice cultures to investigate whether tolerance to lethal ischaemia is dependent on HSP72. Cultures were maintained in vitro for 14 days, and neuronal damage assessed using propidium iodide fluorescence. Prolonged neuronal HSP72 upregulation occurred following exposure to 30 min ischaemia, 45 min hypoxia and 1 μM kainate, but not 1 μM NMDA or 20 min ischaemia, all sublethal insults. Preconditioning with ischaemia, kainate or hypoxia 24 h prior to lethal ischaemia (45 min) was not protective, and when the delay was increased to 48 h, damage in the CA3 pyramidal cell region was significantly increased compared to cultures exposed to 45 min ischaemia alone. Preconditioning with 20 min ischaemia had no effect on the severity of ischaemic damage. Preconditioning with 1 μM NMDA significantly reduced neuronal damage produced by either 45 or 60 min ischaemia when the delay between insults was 48 h. NMDA pre-treatment also prevented neurotoxicity produced by glutamate (5–10 mM) but not NMDA (10–30 μM). These data suggest that in vitro, the increased expression of HSP72 following some sublethal insults should be considered as a marker of cell stress prejudicial to the survival of neurones subsequently exposed to ischaemia, while tolerance can be produced through mechanisms independent of HSP72 induction.

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Heat stress reduces glutamate toxicity in cultured neurons without HSP70 expression

Abstract

J. Neurosci. Methods

Neuroscience

Neuroscience

Mol. Brain Res.

Biochem. Biophys. Res. Commun.

Neuron

Hyperthermia protects against light damage in the rat retina

Science

bcl-2 is expressed in neurons that survive focal ischemic in the rat

Brain Res.