Aase Frandsen

Endoplasmic reticulum dysfunction – a common denominator for cell injury in acute and degenerative diseases of the brain?

Various physiological, biochemical and molecular biological disturbances have been put forward as mediators of neuronal cell injury in acute and chronic pathological states of the brain such as ischemia, epileptic seizures and Alzheimers or Parkinsons disease. These include over‐activation of glutamate receptors, a rise in cytoplasmic calcium activity and mitochondrial dysfunction. The possible involvement of the endoplasmic reticulum (ER) dysfunction in this process has been largely neglected until recently, although the ER plays a central role in important cell functions. Not only is the ER involved in the control of cellular calcium homeostasis, it is also the subcellular compartment in which the folding and processing of membrane and secretory proteins takes place. The fact that blocking of these processes is sufficient to cause cell damage indicates that they are crucial for normal cell functioning. This review presents evidence that ER function is disturbed in many acute and chronic diseases of the brain. The complex processes taken place in this subcellular compartment are however, affected in different ways in various disorders; whereas the ER‐associated degradation of misfolded proteins is affected in Parkinsons disease, it is the unfolded protein response which is down‐regulated in Alzheimers disease and the ER calcium homeostasis that is disturbed in ischemia. Studying the consequences of the observed deteriorations of ER function and identifying the mechanisms causing ER dysfunction in these pathological states of the brain will help to elucidate whether neurodegeneration is indeed caused by these disturbances, and will help to fascilitate the search for drugs capable of blocking the pathological process directly at an early stage.

Dantrolene Prevents Glutamate Cytotoxicity and Ca2+ Release from Intracellular Stores in Cultured Cerebral Cortical Neurons

Abstract: Using primary cultures of cerebral cortical neurons, it has been demonstrated that the antihyperthermia drug dantrolene completely protects against glutamate‐induced neurotoxicity. Furthermore, in the presence of extracellular calcium, dantrolene reduced the glutamate‐induced increase in the intracellular calcium concentration by 70%. In the absence of extracellular calcium, this glutamate response was completely blocked by dantrolene. Dantrolene did not affect the kinetics of [3H]glutamate binding to membranes prepared from similar cultures. These results indicate that release of calcium from intracellular stores is essential for the propagation of glutamate‐induced neuronal damage. Because it is likely that glutamate is involved in neuronal degeneration associated with ischemia and hypoxia, the present findings might suggest that dantrolene and possibly other drugs affecting intracellular calcium pools might be of therapeutic interest.

Excitatory Amino Acid-Mediated Cytotoxicity and Calcium Homeostasis in Cultured Neurons

Abstract: A large body of evidence suggests that disturbances of Ca2+ homeostasis may be a causative factor in the neurotoxicity induced by excitatory amino acids (EAAs). The route or routes by which an increase in intracellular calcium concentration ([Ca2+]i) is mediated in vivo are presently not clarified. This may partly reflect the complexity of intact nervous tissue in combination with the relative unspecific action of the available “calcium antagonists,” e.g., blockers of voltage‐sensitive calcium channels. By using primary cultures of cortical neurons as a model system, it has been found that all EAAs stimulate increases in [Ca2+]i but via different mechanisms. By using the drug dantrolene, it has been shown that 2‐amino‐3‐(3‐hydroxy‐5‐methylisoxazol‐4‐yl)propionate (AMPA) apparently exclusively stimulates Ca2+ influx through agonist‐operated calcium channels and voltage‐operated calcium channels. Increased [Ca2+]i due to exposure to kainate (KA) is for the major part caused by influx, as in the case of AMPA, but a small part of the increase in [Ca2+]i may be attributed to a release of Ca2+ from intracellular stores. Quisqualate (QA) stimulates Ca2+ release from an intracellular store that is independent of Ca2+ influx; presumably this store is activated by inositol phosphates. The increase in [Ca2+]i due to exposure to glutamate or N‐methyl‐d‐aspartate (NMDA) may be compartmentalized into three components, one of which is related to influx and the other two to Ca2+ release from internal stores. Only one of the latter stores is dependent on Ca2+ influx with regard to release of Ca2+, whereas the other is activated by some other second messengers or, alternatively, directly coupled to the receptor. In muscles dantrolene is known to inhibit Ca2+ release from the sarcoplasmic reticulum, and also in neurons dantrolene inhibits an equivalent release from one or more hitherto unidentified internal Ca2+ pool(s). By using this drug it has been possible to show to what extent these Ca2+ stores are involved in the toxicity observed subsequent to exposure to the EAAs. It turned out that dantrolene, even under conditions allowing Ca2+ influx, inhibited toxicity induced by QA, NMDA, and glutamate, whereas that induced by AMPA or KA was unaffected. In combination with the findings that dantrolene inhibited release from the intracellular stores activated by QA, NMDA, and glutamate, it may be concluded that Ca2+ influx per se is not the primary event causing toxicity following exposure to these EAAs in these neurons. However, it may certainly be involved in the cases of toxicity induced by AMPA and KA. Finally, it should be pointed out that this model only serves as a much simplified working hypothesis and that the situation in vivo is much more complex.

Direct Evidence That Excitotoxicity in Cultured Neurons Is Mediated via N-Methyl-D-Aspartate (NMDA) as well as Non-NMDA Receptors

Abstract: Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L‐glutamate, kainate (KA), N‐methyl‐D‐aspartate (NMDA), or RS‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolopropionate (AMPA). To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L‐aspartic acid β‐hydroxamate was added at 500 μM to the cultures that were exposed to glutamate. Each of these EAAs was able to induce neurotoxicity. It was not possible to reduce or prevent glutamate‐induced cytotoxicity by blocking only one of the glutamate receptor subtpes with either the NMDA receptor antagonist D‐(‐)‐2‐amino‐5‐phosphonopentanoate (APV) or with one of the specific non‐NMDA antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX). However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented. Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non‐NMDA glutamate agonists with no effect on NMDA‐induced cell death. Likewise, APV prevented NMDA‐induced cell death without affecting the KA‐ or AMPA‐induced neurotoxicity. It is concluded that EAA‐dependent neurotoxicity is induced by NMDA as well as non‐NMDA receptors.

Development of excitatory amino acid induced cytotoxicity in cultured neurons

The neurotoxicity of the excitatory amino acids (EAAs) l‐glutamate (l‐glu), l‐aspartate (l‐asp), N‐methyl‐d‐aspartate (NMDA), kainate (KA), quisqualate (QA) and RS‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolopropionate (AMPA) was followed as a function of development in primary cultures of cerebral cortex neurons and cerebellar granule cells. These two types of neurons express, respectively, glutamate receptor subtypes with sensitivity to all of these excitatory amino acids or only to glutamate and aspartate. None of the EAAs were toxic in cerebral cortex neurons at 2 days in culture, whereas at culture day 4 the neurons became sensitive to glutamate, at day 5 to KA followed by sensitivity to QA at day 6, and finally to NMDA, l‐asp and AMPA at day 7. The rank order of potency of the EAAs was in cerebral cortex neurons cultured for 12 days: l‐asp (ed50 = 0.5 μM) = l‐glu (ed50 = 1 μM) > AMPA(ed50 = 10 μM) > NMDA (ed50 = 65 μM) > QA = KA (ed50 = 100 μM). Cerebellar granule cells were insensitive to all of the EAAs at 3 and 5 days in culture but at day 8 the cells became sensitive to toxicity induced by l‐glu (ed50 = 70 μM) and l‐asp (ed50 = 30 μM). In order to determine Ed50 values for l‐asp and l‐glu accurately, media in these experiments also contained 500 μM of the glutamate uptake inhibitor l‐aspartate‐β‐hydroxamate. It is concluded that the EAA‐induced cytotoxicity is strictly related to the glutamate receptor subtype expression in the neurons. Furthermore, based on the differences in temporal development of toxicity of the different EAAs in cortex neurons, it is evident that all glutamate receptor subtypes independently mediate the cytotoxic action of EAAs. Finally, it seems justified to conclude that QA and AMPA do not have strictly analogous actions.

Glutamate stimulates the formation of N-acylphosphatidylethanolamine and N-acylphosphatidylethanolamine in cortical neurons in culture

Abstract The formation of anandamide N-armarachidonoylethanolamine, N-acylethanolamine, and N-acylphosphatidylethanolamine was studied in primary cultures of rat cortical neurons. The cells were incubated for 22 h with [14C]ethanolamine, [U-14C]arachidonic acid, [3H]arachidonic acid, [32P]phosphate, [14C]stearic acid, or [3H]myristic acid. The lipids from the cells and media were separated by thin layer chromatography. [14C]Ethanolamine labelling revealed two compounds (I and II), which on different thin layer chromatography systems migrated as N-acylethanolamine (0.06-0.55% of total radioactivity) and N-acylphosphatidylethanolamine (0.66-6.49% of total radioactivity), respectively. Compound II was also labelled with [32P]phosphate, and radioactive fatty acids. Treatment of compound II with phospholipase D (Streptomyces chromofuscus) resulted in two compounds, one comigrating as phosphatidic acid and the other as N-acylethanolamine. Compound I could be labelled with [14C]stearic acid and [3H]myristic acid, but not with [3H]- or [I4C]arachidonic acid. Exogenous [3H]anandamide was metabolised with a t 1 2 of 2.6 h. The labelling of the two compounds identified as N-acylethanolamine and N-acylphosphatidylethanolamine were more pronounced the older the culture. The neurotoxic amino acid, glutamate, stimulated within 2 h dose-dependently (ED50 = 40 μM) the formation of both compounds. It is suggested that N-acylethanol amine and N-acylphosphatidylethanolamine are formed in relation to the cytotoxicity induced by glutamate, and that these compounds may be markers of neurotoxicity. We could not detect any formation of anandamide using radioactive arachidonic acid.

Time and concentration dependency of the toxicity of excitatory amino acids on cerebral neurones in primary culture

The cytotoxicity of the glutamate receptor agonists, N-methyl-d-aspartate (NMDA), kainate (KA) and (RS)-?-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) on cultured cerebral cortex neurones was monitored as a function of exposure time and concentration by following the release into the culture medium of the cytoplasmic enzyme lactate dehydrogenase from the neurones. Chronic exposure of the cells to different concentrations of the agonists showed that AMPA was the most potent excitotoxin (ED(50) 10 ?M) followed in potency by NMDA (ED(50) 65 ?M) and KA (ED(50) 100 ?M). Experiments in which the neurones were exposed for different periods of time to fixed concentrations of the agonists showed that after short exposure times (1-3 min) cells survived for more than 24 h after removal of the agonists but after longer exposure times (5-10 min) cells survived for time periods ranging from 25 min to 6 h depending upon the exposure time and the nature of the agonist. The results of the latter experiments indicate that even short exposure times trigger processes in the cell membranes which even after removal of the excitotoxin will lead to neuronal death.

Characterization of Glutamate‐Induced Formation of N‐Acylphosphatidylethanolamine and N‐Acylethanolamine in Cultured Neocortical Neurons

Abstract: Glutamate‐induced formation of N‐acylethanolamine (NAE) and N‐acylphosphatidylethanolamine (NAPE) was studied in primary cultures of mouse neocortical neurons prelabeled with [14C]ethanolamine. The formation of these two lipids was dependent on the maturity of the cell culture; i.e., no glutamate‐induced formation was seen in 2‐day‐old cultures, whereas glutamate induced a pronounced formation in 6‐day‐old cultures. The calcium ionophore A23187 (2 µM) stimulated, within 2 h, formation of NAPE in 2‐day‐old cultures (fourfold) as well as in 6‐day‐old cultures (eightfold). Glutamate exerted its effect via NMDA receptors as seen by the inhibitory action of the NMDA‐selective receptor antagonists d‐(−)‐2‐amino‐5‐phosphonovalerate and N‐(1‐(2‐thienyl)‐cyclohexyl)piperidine and the lack of effect of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)/kainate‐receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). In 6‐day‐old cultures, exposure to NMDA (100 µM for 24 h) induced a linear increase in the formation of NAPE and NAE as well as a 40–50% neuronal death, as measured by a decrease in cellular formazan formation [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay]. The increase in NAPE and NAE could be detected earlier than the neuronal death. Neither cyclic AMP, cyclic GMP, nitric oxide, protein kinase C, nor peroxidation appears to be involved in the formation of NAPE and NAE, as assessed by the use of different pharmacological agents. Exposure to 5 mM NaN3 for 8 h resulted in a >80% decrease in the cellular MTT staining and a pronounced linear increase in the formation of NAE and NAPE (reaching 25–30% of total labeling). [14C]Anandamide was also formed in [14C]arachidonic acid‐labeled neurons exposed to NaN3. No NAPE formation was detected in A23187‐stimulated mouse astrocytes, rat Leydig cells and cardiomyocytes, and several other cells. These results suggest that the glutamate‐induced formation of NAPE and NAE was mediated by the NMDA receptor and the formation of these lipids may be associated with neuronal death.

Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca2+ by mobilization of cellular Ca2+ and activation of Ca2+ influx

SummaryThe Ca2+ sensitive fluorescent probe, fura-2 has been used to monitor cytosolic free calcium levels in mature primary cultures of cerebellar granule cells during exposure to L-glutamate and other excitatory amino acids: quisqualate (QA), kainate (KA) and N-methyl-D-aspartate (NMDA). Glutamate at micromolar concentrations produced a prompt and dose-related increase in the intracellular concentration of free Ca2+, ([Ca2+]i), whereas QA, KA and NMDA had no effect. This increase was also seen in the absence of extracellular Ca2+, suggesting that L-glutamate promotes mobilization of Ca2+ from intracellular stores. In the presence of extracellular calcium, the elevation of [Ca2+]i was, in part, mediated by an increase in the plasma membrane permeability to Ca2+. This Ca2+ influx was not affected by the Ca2+-channel antagonist 1-Verapamil. However, L-Verapamil did block the increase in [Ca2+]i seen after depolarization of the cells with potassium. The Ca2+ response elicited by glutamate was partially blocked by the excitatory amino acid antagonist glutamate diethyl ester (GDEE). Furthermore, glutamate stimulated the formation of inositol mono-, bis-, tris and tetrakisphosphates (IP1, IP2, IP3, and IP4) suggesting a role for these compounds for the increase in [Ca2+]i.

Characterization of a chemical anoxia model in cerebellar granule neurons using sodium azide: Protection by nifedipine and MK‐801

Induction of chemical anoxia, using sodium azide in cerebellar granule cells maintained in primary culture, was evaluated as an in vitro assay for screening of potential neuroprotective compounds. The purpose of this study was to evaluate sodium azide as an alternative to cyanide salts, compounds which, despite their unfavorable characteristics, are often used in assays for chemical anoxia. The viability of neuronal cultures after treatment with azide, with or without preincubation with calcium channel blockers, tetrodotoxin (TTX), or glutamate receptor antagonists, was monitored by subsequent incubation with the tetrazolium dye MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide), followed by isopropanol extraction and spectrophotometric quantification of cellularly reduced MTT. The azide‐induced degeneration of neurons was shown to be dependent on the concentration as well as on the duration of incubation with submaximal concentrations of azide. Incubation of the neurons with nifedipine, a blocker of L‐type voltage‐sensitive calcium channels (L‐VSCC), or with the noncompetitive N‐methyl‐D‐aspartate (NMDA) subtype glutamate receptor antagonist MK‐801, prior to addition of submaximal concentrations of azide, significantly attenuated azide‐induced neuronal death. Blockers of N‐type and Q‐type VSCC (o‐conotoxin MVIIA and MVIIC, respectively) and the P‐type VSCC blocker o‐agatoxin IVA had no effect in this assay. The sodium channel blocker TTX was without effect when added to neurons under depolarizing conditions, but potently and effectively protected cells when experiments were performed in a nondepolarizing buffer. The results show that chemical anoxia induced by incubation of cultured neurons with azide leads to detrimental effects, which may be quantitatively monitored by the capability of the cells to reduce MTT. This procedure is a suitable method for screening of compounds for possible protective effects against neuronal death induced by energy depletion. In addition, the results suggest involvement of L‐type VSCC as well as of glutamate receptors in the pathways leading to neuronal degradation induced by energy depletion in cerebellar granule neurons. This would further support the notion that these pathways might be important in neurodegeneration induced by cerebral ischemia or anoxia.