Bibliothek

Notizen: Abstract: We have used thapsigargin (TG), a specific, irreversible inhibitor of endoplasmic reticulum (ER) Ca2+-ATPases, and caffeine, an agonist of the ryanodine receptor, to study the effect of emptying of ER calcium stores on protein synthesis in neuronal cells. TG at 1 µM caused a permanent inhibition of protein synthesis in hippocampal slices from 3-week-old rats but no inhibition in slices prepared from 2-month-old animals. Caffeine at 10 mM caused a reduction of protein synthesis in both 3-week- and 2-month-old rats immediately after exposure, but complete recovery of protein synthesis occurred within 30 min after treatment. In neuronal cells, TG produced an almost complete inhibition of protein synthesis that was only partially reversed over a 24-h recovery period. TG did not significantly affect neuronal ATP levels or energy charge. Fifty percent inhibition of protein synthesis was achieved with ∼5 nM TG. Recovery of protein synthesis after TG treatment was significantly hindered when serum was omitted from the medium after TG exposure, suggesting that serum promotes recovery of ER calcium homeostasis. It is concluded that TG is a suitable tool for the study of the mechanisms of protein synthesis inhibition after transient cerebral ischemia. The possibility that disturbances in ER calcium homeostasis may contribute to the pathological process of ischemic cell death is discussed.

Notizen: Abstract: To elucidate whether the high sensitivity of gerbil compared with rat hippocampus to metabolic stress results from tissue-specific or hemodynamic factors, ischemia-induced metabolic disturbances [energy metabolism and protein synthesis rate (PSR)] were studied using the in vitro model of the hippocampal slice preparation. At the end of in vitro ischemia, ATP content was measured in individual slices with HPLC. In other groups of slices, PSR was measured after 120 min of recovery after in vitro ischemia. ATP breakdown was almost identical in rat and gerbil slices at all temperatures (37°C, 34°C, or 31°C) and periods of ischemia (5, 10, or 15 min) studied. In contrast to the identical rate of ATP depletion during ischemia, however, postischemic disturbances in PSR were significantly increased in gerbil slices compared with rat slices and this relationship was stable after different periods of ischemia and at different incubation temperatures. The results illustrate that the pattern of ischemia-induced disturbances observed in vivo can also be reproduced using the in vitro model of hippocampal slice preparation, as evidenced by the postischemic disturbance in PSR. It is concluded that comparison of the extent of metabolic disturbances in gerbil and rat hippocampal slices after transient in vitro ischemia may help to elucidate the mechanisms of ischemic cell damage.

Notizen: Abstract: Editing of mRNA in the coding region of the second transmembrane domain of glutamate receptor subunits GluR2, GluR5, and GluR6 involves a change of the base A in genomic DNA to the base G in mRNA as described in rat brain. To determine whether this reaction occurs in humans as well as rats, we studied RNA editing of GluR2 and GluR6 in human brain. We compared the extent of editing in controls and cases with Huntington's disease. To assay the extent of editing in brain RNA, first strand cDNA was amplified using the polymerase chain reaction yielding a product across the region of the second transmembrane spanning segment in which editing takes place in rats. The PCR product was incubated with the restriction enzyme BbvI, which recognizes the sequence GCAGC present in the nonedited sequence of the mRNA in subunits GluR2 and GluR6. Thus, BbvI cuts the nonedited version but leaves the edited version intact. As in the rat, the GluR2 subunit mRNA was completely edited in human brain. The GluR6 subunit was nearly completely edited in all gray matter structures investigated including cortex, striatum, thalamus, hippocampus, amygdala, and cerebellum with extent of editing ranging from 89% in the cerebellum to 95% in the cortex and striatum. No significant differences in the extent of RNA editing were apparent in control versus Huntington's disease brains. To compare the extent of editing in neurons and glia in the brain, editing in cerebral cortex (predominantly gray matter and thus neurons) was compared with editing in corpus callosum (white matter and thus nearly completely glial cells). In white matter, GluR2 was completely edited, whereas GluR6 was only ∼10% edited compared with ∼90% edited in gray matter. Thus, these studies indicate that RNA editing is seen in human brain as well as rat brain and that the extent of editing is similar in Huntington's disease compared with controls. The differences in editing in white matter for GluR6, but not for GluR2, suggest that different templates could be subject to different editing activities that undergo tissue-specific regulation.

Notizen: Abstract: Thirty minutes of insulin-induced reversible hypoglycemic coma (defined in terms of cessation of EEG activity) was produced in anesthetized rats. At the end of the hypoglycemic coma or after recovery for 3, 24, or 72 h induced by glucose infusion, the animals were reanesthetized and their brains frozen in situ. Two control groups were used: untreated controls without prior manipulations, and insulin controls, which received injections of insulin followed by glucose infusion to maintain blood glucose within the physiological range. The brains of these latter animals were frozen 3, 24, or 72 h after glucose infusion. Tissue samples from the cortex, striatum, hippocampus, and thalamus were taken to measure ornithine decarboxylase (ODC) activity, and putrescine and spermidine levels, as well as phosphocreatine (PCr), ATP, glucose, and lactate content. In addition, 20-μm thick coronal sections taken from the striatum and dorsal hippocampus were used for histological evaluation of cell damage and also stained for calcium. Insulin in the absence of hypoglycemia produced a significant increase in ODC activity and putrescine level but had no effect on the profiles of energy metabolites or spermidine. During hypoglycemic coma, brain PCr, ATP, glucose, and lactate levels were sharply reduced, as expected. Energy metabolites normalized after 3 h of recovery. In the striatum, significant secondary decreases in PCr and ATP contents and rises in glucose and lactate levels were observed after 24 h of recovery. ODC activity, and putrescine and spermidine levels were unchanged during hypoglycemic coma. After 3 h of recovery, ODC activity increased markedly throughout the brain, except in the striatum. After 24 h of recovery, ODC activity decreased and approached control values 2 days later. Putrescine levels increased significantly throughout the brain after reversible hypoglycemic coma, the highest values observed after 24 h of recovery (p≤ 0.001, compared with controls). After 72 h of recovery, putrescine levels decreased, but still significantly exceeded control values. Reversible hypoglycemic coma did not produce significant changes in regional spermidine levels except in the striatum, where an approximately 30% increase was observed after 3 and 72 h of recovery (p≤ 0.01 and p≤ 0.05, respectively). Twenty-four hours after hypoglycemic coma, intense calcium staining was apparent in layer III of the cerebral cortex, the lateral striatum, and the crest of the dentate gyrus. After 72 h of recovery, the intense calcium staining included also cortical layer II, the septal nuclei, the subiculum, and the hippocampal CA1-subfield. Changes in polyamine metabolism thus preceded the intense calcium staining in the brain. The results indicate that reversible hypoglycemic coma induces a sharp increase in putrescine level comparable to that observed previously after cerebral ischemia. We, therefore, conclude that the increase in putrescine content is an early biochemical marker of delayed neuronal cell necrosis irrespective of the pathogenesis of this injury. The possible role of polyamines in the manifestation of neuronal necrosis following hypoglycemic coma is discussed.

Notizen: Abstract: Biosynthesis and accumulation of the polyamines putrescine, spermidine, and spermine are closely associated with cellular growth processes. We examined polyamine levels and the activity of their first rate-limiting enzyme, ornithine decarboxylase (ODC), in stereotactically induced experimental gliomas of the rat brain 1 and 2 weeks after implantation. Regional ODC activity and polyamine levels were determined in the tumor and in the ipsi- and contralateral striatum, white matter, and cerebral cortex. In the tumor, both ODC activity and polyamine levels markedly increased with progressive tumor growth, as compared to those in the white matter of the opposite hemisphere. In the peritumoral brain tissue, ODC activity did not change, but there was a marked increase of putrescine and, to a lesser degree, of spermidine and spermine almost throughout the whole ipsilateral hemisphere. ODC activity, therefore, seems to be a reliable marker of neoplastic growth in the brain, which may be of use for new clinical concepts of the diagnosis and therapy of brain tumors. The more diffuse distribution of polyamines, however, may be associated with the formation and spreading of edema, which would explain some of the biological effects of tumors on distant brain tissue.

Notizen: Abstract: It has been proposed that NAD depletion resulting from excessive activation of poly(ADP-ribose) polymerase is responsible for secondary energy failure after transient cerebral ischemia. However, this hypothesis has never been verified by measurement of ATP and NAD levels in the same tissue sample. In this study, we therefore investigated the effect of transient focal cerebral ischemia on the temporal profiles of changes in the levels of energy metabolites and NAD. Ischemia was induced in mice by occluding the left middle cerebral artery using the intraluminal filament technique. Animals were subjected to 1-h ischemia, followed by 0, 1, 3, 6, or 24 h of reperfusion. During ischemia, ATP levels, total adenylate pool, and adenylate energy charge dropped to ∼20, 50, and 40% of control, respectively, whereas NAD levels remained close to control. Energy state recovered transiently, peaking at 3 h of recovery (ATP levels and total adenylate pool recovered to 78 and 81% of control). In animals subjected to reperfusion of varying duration, the extent of ATP depletion was clearly more pronounced than that of NAD. The results imply that depletion of NAD pools did not play a major role in secondary disturbances of energy-producing metabolism after transient focal cerebral ischemia. Changes in ATP levels were closely related to changes in total adenylate pool (p 〈 0.001). The high energy charge after 6 h of reperfusion (0.90 versus a control value of 0.93) and the close relationship between the decline of ATP and total adenylate pool suggest that degradation or a washout of adenylates (owing to leaky membranes) rather than a mismatch between energy production and consumption is the main causative factor contributing to the secondary energy failure observed after prolonged recovery.

Notizen: Abstract: Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase. Tg-induced rise in [Ca2+]i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca2+]i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c-fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c-fos mRNA levels (∼10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c-fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c-fos. Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.

Notizen: Mice were subjected to 60 min occlusion of the left middle cerebral artery (MCA) followed by 1–6 h of reperfusion. Tissue samples were taken from the MCA territory of both hemispheres to analyse ischaemia-induced changes in the phosphorylation of the initiation factor eIF-2α, the elongation factor eEF-2 and p70 S6 kinase by western blot analysis. Tissue sections from additional animals were taken to evaluate ischaemia-induced changes in global protein synthesis by autoradiography and changes in eIF-2α phosphorylation by immunohistochemistry. Transient MCA occlusion induced a persistent suppression of protein synthesis. Phosphorylation of eIF-2α was slightly increased during ischaemia, it was markedly up-regulated after 1 h of reperfusion and it normalized after 6 h of recirculation despite ongoing suppression of protein synthesis. Similar changes in eIF-2α phosphorylation were induced in primary neuronal cell cultures by blocking of endoplasmic reticulum (ER) calcium pump, suggesting that disturbances of ER calcium homeostasis may play a role in ischaemia-induced changes in eIF-2α phosphorylation. Dephosphorylation of eIF-2α was not paralleled by a rise in levels of p67, a glycoprotein that protects eIF-2α from phosphorylation, even in the presence of active eIF-2α kinase. Phosporylation of eEF-2 rose moderately during ischaemia, but returned to control levels after 1 h of reperfusion and declined markedly below control levels after 3 and 6 h of recirculation. In contrast to the only short-lasting phosphorylation of eIF-2a and eEF-2, transient focal ischaemia induced a long-lasting dephosphorylation of p70 S6 kinase. The results suggest that blocking of elongation does not play a major role in suppression of protein synthesis induced by transient focal cerebral ischaemia. Investigating the factors involved in ischaemia-induced suppression of the initiation step of protein synthesis and identifying the underlying mechanisms may help to further elucidate those disturbances directly related to the pathological process triggered by transient cerebral ischaemia and leading to neuronal cell injury.

Notizen: 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 Alzheimer's or Parkinson's 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 Parkinson's disease, it is the unfolded protein response which is down-regulated in Alzheimer's 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.

Notizen: Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Here, we investigated the effect of peroxide exposure on the expression of genes coding for cytoplasmic and endoplasmic reticulum (ER) stress proteins. Primary neuronal cell cultures were exposed to H2O2 for 6 h and mRNA levels of hsp70, grp78, grp94, gadd153 were evaluated by quantitative PCR. In addition, peroxide-induced changes in protein synthesis and cell viability were investigated. Peroxide treatment of cells triggered an almost 12-fold increase in hsp70 mRNA levels, but a significant decrease in grp78, grp94 and gadd153 mRNA levels. To establish whether peroxide exposure blocks the ER-resident stress response, cells were also exposed to thapsigargin (Tg, a specific inhibitor of ER Ca2+-ATPase) which has been shown to elicit the ER stress response. Tg exposure induced 7.2-fold, 3.6-fold and 8.8-fold increase in grp78, grp94 and gadd153 mRNA levels, respectively. However, after peroxide pre-exposure, the Tg-induced effect on grp78, grp94 and gadd153 mRNA levels was completely blocked. The results indicate that oxidative damage causes a selective down-regulation of the neuronal stress response activated under conditions of ER dysfunction. This down-regulation was only observed in cultures exposed to peroxide levels which induced severe suppression of protein synthesis and cell injury, implying a causative link between peroxide-induced down-regulation of ER stress response system and development of neuronal cell injury. These observations could have implications for our understanding of the mechanisms underlying neuronal cell injury in pathological states of the brain associated with oxidative damage, including Alzheimer's disease where the neuronal stress response activated under conditions of ER dysfunction has been shown to be down-regulated. Down-regulation of ER stress response may increase the sensitivity of neurones to an otherwise nonlethal form of stress.