Transient ischemia induces massive nuclear accumulation of SUMO2/3-conjugated proteins in spinal cord neurons.

OBJECTIVES: The objective of this study is to determine whether transient spinal cord ischemia activates small ubiquitin-like modifier (SUMO1-3) conjugation, a post-translational protein modification that protects neurons from ischemia-like conditions. METHODS: Mice were subjected to 8-12 min of spinal cord ischemia and 3-24 h of recovery using a newly developed experimental model. To characterize the model, activation of stress response pathways induced after spinal cord ischemia, previously observed in other experimental models, was verified by western blot analysis. Levels and subcellular localization of SUMO-conjugated proteins in spinal cords were evaluated by western blot analysis and immunohistochemistry, respectively. RESULTS: Following transient spinal cord ischemia, stress responses were activated as indicated by increased phosphorylation of eukaryotic initiation factor 2 (eIF2α), extracellular signal-regulated kinases (ERK1/2) and Akt. SUMO1 conjugation was not altered, but a selective rise in levels of SUMO2/3-conjugated proteins occurred, peaking at 6 h reperfusion. The marked activation of SUMO2/3 conjugation was a neuronal response to ischemia, as indicated by co-localization with the neuronal marker NeuN, and was associated with nuclear accumulation of SUMO2/3-conjugated proteins. CONCLUSION: Our study suggests that spinal cord neurons respond to ischemic stress by activation of SUMO2/3 conjugation. Many of the identified SUMO target proteins are transcription factors and other nuclear proteins involved in gene expression and genome stability. It is therefore concluded that the post-ischemic activation of SUMO2/3 conjugation may define the fate of neurons exposed to a transient interruption of blood supply, and that this pathway could be a therapeutic target to increase the resistance of spinal cord neurons to transient ischemia.

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 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 facilitate the search for drugs capable of blocking the pathological process directly at an early stage.

Changes in the phosphorylation of initiation factor eIF-2alpha, elongation factor eEF-2 and p70 S6 kinase after transient focal cerebral ischaemia in mice.

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-2alpha, 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-2alpha phosphorylation by immunohistochemistry. Transient MCA occlusion induced a persistent suppression of protein synthesis. Phosphorylation of eIF-2alpha 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-2alpha 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-2alpha phosphorylation. Dephosphorylation of eIF-2alpha was not paralleled by a rise in levels of p67, a glycoprotein that protects eIF-2alpha from phosphorylation, even in the presence of active eIF-2alpha kinase. Phosphorylation 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.

Response of neurons to an irreversible inhibition of endoplasmic reticulum Ca(2+)-ATPase: relationship between global protein synthesis and expression and translation of individual genes.

In the physiological state, there appears to be a regulatory link between endoplasmic reticulum (ER) Ca(2+) homoeostasis and the initiation of neuronal protein synthesis. Exposing neuronal cell cultures to thapsigargin (Tg), an irreversible inhibitor of sarcoplasmic/ER Ca(2+)-ATPase (SERCA), induced an almost complete suppression of protein synthesis, which recovered to approx. 60% of control 24 h after Tg exposure. This is an experimental model where the regulatory link between the initiation of protein synthesis and ER Ca(2+) homoeostasis recovers, despite an irreversible suppression of SERCA activity [Doutheil, Treiman, Oschlies and Paschen (1999) Cell Calcium 25, 419--428]. The model was used to investigate the relationship between transcription and translation of various stress genes that respond to conditions causing graded suppression of protein synthesis. Expression patterns revealed three groups of genes. The mRNA levels of genes responding to conditions of ER stress (grp78, grp94, gadd34 and gadd153) were increased up to 200-fold after Tg exposure, whereas those coding for ER-resident proteins (SERCA 2b and Bcl-2) were increased up to 6-fold in treated cultures, and those coding for cytoplasmic proteins (heat-shock protein 70 and p67) were increased only 2--4-fold. Analysis of translation of these mRNAs suggests an imbalance in the synthesis of apoptosis-inducing (GADD153) and tolerance-activating (GRP78 and Bcl-2) proteins after blocking of the ER Ca(2+) pump. The observation that the relationship between Tg-induced changes in mRNA and protein levels varied considerably for the various genes studied implies that translation of the respective transcripts is differently regulated under conditions causing graded suppression of global protein synthesis. Detailed analysis of the response of neuronal cells to transient disturbance of ER Ca(2+) homoeostasis may help to elucidate the mechanisms underlying neuronal cell injury in those neurological disorders in which an impairment of ER function is thought to contribute to the pathological process of deterioration.

Polyamine metabolism in brain tumours: diagnostic relevance of quantitative biochemistry.

OBJECTIVE: Activation of polyamine metabolism is closely associated with cellular proliferation. The purpose was to investigate whether the content of the polyamines putrescine, spermidine, and spermine, and the activity of the first metabolic key enzyme of polyamine metabolism, ornithine decarboxylase (ODC), represent biochemical markers of malignancy in brain tumours. METHODS: The concentration of putrescine, spermidine, and spermine, and the activity of ODC were biochemically quantified in tissue samples obtained during open microsurgery of 670 patients with brain tumours. Biochemical analysis and histopathological classification were carried out in serial tumour samples. RESULTS: The activity of ODC was very low in peritumoral non-neoplastic brain tissue (0.9 (SD 0.6) nmol/g/h). It was significantly higher in gliomas and it significantly increased with a higher grade of malignancy (grade I 2.7 (2.8) nmol/g/h, grade II 3.1 (4.0) nmol/g/h, grade III 5.7 (5.6) nmol/g/h, grade IV 10.6 (11.7) nmol/g/h). High enzyme activity was also found in medulloblastomas (25.5 (15.1) nmol/g/h), malignant lymphomas (52.1 (42.1) nmol/g/h), and metastases from carcinoma (14.9 (22.1) nmol/g/h). Lowest values were measured in epidermoid cysts (0.5 (0.2) nmol/g/h), craniopharyngiomas (1.2 (0.9) nmol/g/h), angioblastomas (1.6 (1.7) nmol/g/h), and neurinomas (2.0 (1.8) nmol/g/h). By contrast with ODC activity, polyamine concentrations did not correlate with the grade of malignancy. Correlation of regional biochemical and histomorphological data in rapidly growing neoplasms showed high enzyme activity in solid tumour parts and low activity in necrotic areas. CONCLUSIONS: Novel data relating ODC activation and polyamine concentrations to neuropathology is presented indicating that high ODC activity represents a biochemical marker of malignancy in brain tumours. This information is important for clinical and therapeutic investigations.

Peroxidative stress selectively down-regulates the neuronal stress response activated under conditions of endoplasmic reticulum dysfunction.

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 H(2)O(2) 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 Ca(2+)-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.

Dependence of vital cell function on endoplasmic reticulum calcium levels: implications for the mechanisms underlying neuronal cell injury in different pathological states.

The endoplasmic reticulum (ER) is a subcellular compartment playing a pivotal role in the control of vital calcium-related cell functions, including calcium storage and signalling. In addition, newly synthesized membrane and secretory proteins are folded and processed in the ER, reactions which are strictly calcium dependent. The ER calcium activity is therefore high, being several orders of magnitude above that of the cytoplasm. Depletion of ER calcium stores causes an accumulation of unfolded proteins in the ER lumen, a pathological situation which induces the activation of two highly conserved stress responses, the ER overload response (EOR) and the unfolded protein response (UPR). EOR triggers activation of the transcription factor NF kappa B, which, in turn, activates the expression of target genes. UPR triggers two downstream processes: it activates the expression of genes coding for ER-resident stress proteins, and it causes a suppression of the initiation of protein synthesis. A similar stress response is activated in pathological states of the brain including cerebral ischaemia, implying common underlying mechanisms. Depending on the extent and duration of the disturbance, an isolated impairment of ER function is sufficient to induce cell injury. In this review, evidence is presented that ER function is indeed disturbed in various diseases of the brain, including acute pathological states (e.g. cerebral ischaemia) and degenerative diseases (e.g. Alzheimer's disease). A body of evidence suggests that disturbances of ER function could be a global pathomechanism underlying neuronal cell injury in various acute and chronic disorders of the central nervous system. If that is true, restoration of ER function or attenuation of secondary disturbances induced by ER dysfunction could present a highly promising new avenue for pharmacological intervention to minimize neuronal cell injury in different pathological states of the brain.

Homocysteine-induced changes in mRNA levels of genes coding for cytoplasmic- and endoplasmic reticulum-resident stress proteins in neuronal cell cultures.

Elevated homocysteine levels have been suggested to contribute to various pathological states of the brain. However, the basic mechanisms underlying homocysteine-induced neurotoxicity have not yet been fully elucidated. In the present series of experiments, we investigated the effect of homocysteine on mRNA levels of genes coding for cytoplasmic- or endoplasmic reticulum-resident stress proteins. Primary neuronal cell cultures were exposed to different homocysteine levels for 1-24 h. Cell injury was evaluated using the MTT assay, protein synthesis was studied by measuring the incorporation of L-[4,5-3H]leucine into proteins, mRNA levels of hsp70, gadd153, grp78, and grp94 were evaluated by quantitative PCR, and changes in protein levels of hsp70, grp78 and grp94 were analyzed by immunoblotting. Exposure of cells to 5 or 10 mM homocysteine for 24 h induced marked cell injury (decrease of viability to 58 or 45% of control respectively). After 6 h treatment, gadd153, grp78 and grp94 mRNA levels increased markedly, but only when cells were exposed to levels of homocysteine high enough to induce cell injury. In addition, hsp70 mRNA levels and protein synthesis were significantly reduced. At earlier (1 or 3 h) or later (12 or 24 h) time intervals, homocysteine exposure induced a marked increase in mRNA levels of all genes studied. GRP78 and GRP94 protein levels were increased in cells exposed to 5 mM homocysteine for 24 h but not in cells exposed to 10 mM homocysteine. HSP70 protein levels, in contrast, were decreased in cells exposed to homocysteine for different periods. The expression of genes coding for ER-resident stress proteins is specifically activated under conditions of ER stress. The close relationship between the extent of cell injury and increase in grp78 mRNA levels suggests that ER dysfunction may contribute to the pathological process. The results imply that the ER is an intracellular target of homocysteine toxicity.

Effect of transient focal ischemia of mouse brain on energy state and NAD levels: no evidence that NAD depletion plays a major role in secondary disturbances of energy metabolism.

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 approximately 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.

Effect of nitric oxide on endoplasmic reticulum calcium homeostasis, protein synthesis and energy metabolism.

It has been suggested that nitric oxide (NO) may contribute to ischemia-induced cell injury. However, the mechanisms underlying NO toxicity have not yet been fully elucidated. In the present study, we investigated the effect of NO on the level of endoplasmic reticulum (ER) calcium stores, on ER Ca2+ pump activity, on protein synthesis, on concentrations of high-energy phosphates, and on gadd153 mRNA levels. Primary neuronal cells were exposed to the NO-donor (+/-)-S-Nitroso-N-acetylpenicillamine (SNAP) for 1 h, 2 h, 6 h or 24 h. The level of ER calcium stores was evaluated by measuring the increase in cytoplasmic calcium activity induced by exposing cells to thapsigargin, an irreversible inhibitor of ER Ca(2+)-ATPase; the activity of ER Ca(2+)-ATPase was determined by measuring a phosphorylated intermediate; SNAP-induced changes in gadd153 expression were evaluated by quantitative PCR; SNAP-induced changes in protein synthesis were investigated by measuring the incorporation of L-[4,5-3H]leucine into proteins, and changes in the levels of ATP, ADP, AMP were measured by HPLC. Exposing cells to SNAP for 1 h to 2 h induced a marked depletion of ER calcium stores through an inhibition of ER Ca(2+)-ATPase (to 58% of control), and a concentration-dependent suppression of protein synthesis which was reversed in the presence of hemoglobin, suggesting NO-related effects. ATP levels and adenylate energy charge were significantly decreased only when cells were exposed to the highest SNAP concentration for 6 h or 24 h, excluding significant effects of NO on the energy state of cells in the acute state, i.e. when ER calcium stores were already completely depleted and protein synthesis severely suppressed. In light of the regulatory role of ER calcium homeostasis in the control of protein synthesis, the results imply that the suppression of protein synthesis resulted from NO-induced inhibition of ER Ca(2+)-ATPase and depletion of ER calcium stores, and that NO-induced disturbances of energy metabolism are secondary to the effect of NO on ER calcium homeostasis. It is, therefore, concluded that ER calcium stores are a primary target of NO-toxicity.

Role of calcium in neuronal cell injury: which subcellular compartment is involved?

It is widely believed that calcium plays a primary role in the development of neuronal cell injury in different pathological states of the brain. Disturbances of calcium homeostasis may be induced in three different subcellular compartments, the cytoplasm, mitochondria or the endoplasmic reticulum (ER). The traditional calcium hypothesis holds that neuronal cell injury is induced by a marked increase in cytoplasmic calcium activity during stress (e.g., cerebral ischemia). Recently, this hypothesis has been modified, taking into account that under different experimental conditions the extent of cell injury does not correlate closely with calcium load or total calcium influx into the cell, and that neuronal cell injury has been found to be associated with both increases and decreases of cytoplasmic calcium activity. The mitochondrial calcium hypothesis is based on the observation that after a severe form of stress there is a massive influx of calcium ions into mitochondria, which may lead to production of free radicals, opening of the mitochondrial permeability transition (MPT) pore and disturbances of energy metabolism. However, it has still to be established whether drugs such as cyclosporin A are neuroprotective through their effect on MPT or through the blocking of processes upstream of MPT. The ER calcium hypothesis arose from the observation that ER calcium stores are depleted after severe forms of stress, and that the response of cells to disturbances of ER calcium homeostasis (activation of the expression of genes coding for ER resident stress proteins and suppression of the initiation of protein synthesis) resembles their response to a severe form of stress (e.g., transient ischemia) implying common underlying mechanisms. Elucidating the exact mechanisms of calcium toxicity and identifying the subcellular compartment playing the most important role in this pathological process will help to evaluate strategies for specific therapeutic intervention.

Recovery of neuronal protein synthesis after irreversible inhibition of the endoplasmic reticulum calcium pump.

In the physiological state, protein synthesis is controlled by calcium homeostasis in the endoplasmic reticulum (ER). Recently, evidence has been presented that dividing cells can adapt to an irreversible inhibition of the ER calcium pump (SERCA), although the mechanisms underlying this adaption have not yet been elucidated. Exposing primary neuronal cells to thapsigargin (Tg, a specific irreversible inhibition of SERCA) resulted in a complete suppression of protein synthesis and disaggregation of polyribosomes indicating inhibition of the initiation step of protein synthesis. Protein synthesis and ribosomal aggregation recovered to 50-70% of control when cells were cultured in medium supplemented with serum for 24 h, but recovery was significantly suppressed in a serum-free medium. Culturing cells in serum-free medium for 24 h already caused an almost 50% suppression of SERCA activity and protein synthesis. SERCA activity did not recover after Tg treatment, and a second exposure of cells to Tg, 24 h after the first, had no effect on protein synthesis. Acute exposure of neurons to Tg induced a depletion of ER calcium stores as indicated by an increase in cytoplasmic calcium activity, but this response was not elicited by the same treatment 24 h later. However, treatments known to deplete ER calcium stores (exposure to the ryanodine receptor agonists caffeine or 2-hydroxycarbazole, or incubating cells in calcium-free medium supplemented with EGTA) caused a second suppression of protein synthesis when applied 24 h after Tg treatment. The results suggest that after Tg exposure, restoration of protein synthesis was induced by recovery of the regulatory link between ER calcium homeostasis and protein synthesis, and not by renewed synthesis of SERCA protein or development of a new regulatory system for the control of protein synthesis. The effect of serum withdrawal on SERCA activity and protein synthesis points to a role of growth factors in maintaining ER calcium homeostasis, and suggests that the ER acts as a mediator of cell damage after interruption of growth factor supplies.

Disturbance of endoplasmic reticulum functions: a key mechanism underlying cell damage?

The endoplasmic reticulum (ER) plays a pivotal role in the folding and processing of newly synthesized proteins, reactions which are strictly calcium-dependent. Depletion of ER calcium pools activates a stress response (suppression of global protein synthesis and activation of stress gene expression) which is almost identical to that induced by transient ischemia or other forms of severe cellular stress, implying common underlying mechanisms. We conclude that disturbance of the ER functions may be involved in stress-induced cell injury. In our view, ER calcium homeostasis plays an important role in maintaining the physiological state in cells balanced between the extremes of growth arrest and cell death on the one hand, and uncontrolled proliferation on the other.

Effect of 3,4,5-trimethoxybenzoic acid 8-diethylamino-octyl ester (TMB-8) on neuronal calcium homeostasis, protein synthesis, and energy metabolism.

It has been suggested recently that disturbances of endoplasmic reticulum calcium homeostasis plays a major role in ischaemic cell injury of the brain. Depletion of endoplasmic reticulum calcium stores induces suppression of the initiation process of protein synthesis, a prominent feature of ischaemic cell damage. The benzoic acid derivative 3,4,5-trimethoxybenzoic acid 8-diethylamino-octyl ester (TMB-8), an established inhibitor of calcium release from endoplasmic reticulum, would be an ideal tool for elucidating the role of endoplasmic reticulum dysfunction in this pathological process. The present investigation was performed to study the effects of TMB-8 on neuronal metabolism (cytoplasmic calcium activity, ATP levels and protein synthesis) using hippocampal slices and primary neuronal cell cultures. In addition, we investigated whether the rise in cytoplasmic calcium activity and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion, is reversed by this agent. Exposure of neurones to TMB-8 (100 microM) induced a small transient increase in cytoplasmic calcium activity ([Ca2+]i), whereas a second dose of TMB-8 (200 microM) produced a marked and sustained rise in [Ca2+]i. The increase in [Ca2+]i evoked by blocking endoplasmic reticulum Ca(2+)-ATPase was only transiently suppressed and then aggravated by TMB-8. The dose-dependent suppression of protein synthesis by TMB-8, observed both in neuronal cultures and hippocampal slices, indicates that TMB-8 has a pathological effect on neuronal metabolism. This inhibition was not reversed after washing-off of the drug. TMB-8 did not reverse the inhibition of protein synthesis evoked by caffeine, which depletes endoplasmic reticulum calcium stores by activating the ryanodine receptor. The results indicate that TMB-8 is not a suitable investigative tool for blocking in neuronal cell cultures the depletion of endoplasmic reticulum calcium stores and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion.

Effect of induced tolerance on biochemical disturbances in hippocampal slices from the gerbil during and after oxygen/glucose deprivation.

To evaluate whether the state of tolerance is stable enough to be studied under in vitro conditions after induction by ischemic preconditioning in vivo, metabolic disturbances of hippocampal slices prepared from control and preconditioned gerbils were evaluated during and after oxygen/glucose deprivation (OGD). Slices were subjected to 5, 10 or 15 min OGD with or without 2h recovery. During the state of metabolic stress, changes in energy metabolism were identical in slices taken from control and preconditioned gerbils. Following OGD, however, recovery of protein synthesis was significantly improved in hippocampal slices of preconditioned animals, indicating that the effect of preconditioning on metabolic disturbances induced by transient OGD in vitro or transient ischemia in vivo is similar. It is suggested that the hippocampal slice preparation is an in vitro model suitable for the study of basic mechanisms underlying the induction of tolerance in vivo.

Changes in ornithine decarboxylase activity and putrescine concentrations after spinal cord compression injury in the rat.

Traumatic spinal cord injury results in direct physical damage to structures and the generation of local factors contributing to secondary pathogenesis. In the present study, we investigated changes in polyamine metabolism after spinal cord compression injury in the rat. This is a stress induced metabolic pathway, of which an activation may indicate both, secondary pathogenesis or induction of neuroprotective response. Ornithine decarboxylase (ODC) activity, the rate limiting step of polyamine synthesis, and levels of the diamine putrescine, the product of ornithine decarboxylase reaction, were analyzed in control (non-laminectomized) animals and at 2 and 4 h after laminectomy or compression injury at the L4 segmental level. ODC activity was significantly increased 4 h after laminectomy in L4 and in adjacent L3 and L5 segments and compression to L4 produced a further increase 4 h after injury as compared with the intact control group. Putrescine levels were likewise significantly elevated to the same extend in the laminectomized and injured cord as compared with the intact control group. These findings demonstrate increased ODC and putrescine levels in the laminectomized and traumatized spinal cord and suggest that laminectomy may be an important 'priming event' that contributes to secondary injury after spinal cord compression injury.

Ischemia-induced changes in 2'-5'-oligoadenylate synthethase mRNA levels in rat brain: comparison with changes produced by perturbations of endoplasmic reticulum calcium homeostasis in neuronal cell cultures.

2'-5' Oligoadenylate synthetase (OAS) expression is induced by interferon or viral infection of cells. To better understand ischemia-induced changes in gene expression and to elucidate the possible underlying mechanisms, changes in OAS mRNA levels were evaluated after 30 min four-vessel occlusion and 2, 4, 8 or 24 h recovery and compared to the temporal profile of changes in mRNA levels induced by a transient depletion of endoplasmic reticulum (ER) calcium stores in primary neuronal cell cultures. OAS mRNA levels dropped during early recovery both in vivo and in vitro. After 6 h recovery from ER calcium pool depletion, OAS mRNA levels increased to about 350% of controls and returned to control levels after 24 h of recovery. After 24 h recovery from ischemia, OAS mRNA levels rose to about 390% of controls in the hippocampus and striatum and to 210% of the control value in the cortex. It is concluded that transient ischemia place cells into an antiviral state, most pronounced in the hippocampus and striatum, and that disturbances of ER calcium homeostasis may contribute to this process.

Activation of MYD116 (gadd34) expression following transient forebrain ischemia of rat: implications for a role of disturbances of endoplasmic reticulum calcium homeostasis.

MyD116 is the murine homologue of growth arrest- and DNA damage-inducible genes (gadd34), a gene family implicated in growth arrest and apoptosis induced by endoplasmic reticulum dysfunction. The present study investigated changes in MyD116 mRNA levels induced by transient forebrain ischemia. MyD116 mRNA levels were measured by quantitative PCR. After 2 h of recovery following 30 min forebrain ischemia, MyD116 mRNA levels rose to about 550% of control both in the cortex and hippocampus. In the cortex, MyD116 mRNA levels gradually declined to 290% of control 24 h after ischemia, whereas in the hippocampus they remained high (538% of control after 24 h of recovery). To elucidate the possible mechanism underlying this activation process, MyD116 mRNA levels were also quantified in primary neuronal cell cultures under two different experimental conditions, both leading to a depletion of endoplasmic reticulum (ER) calcium pools. Changes in cytoplasmic calcium activity were assessed by fluorescence microscopy of fura-2-loaded cells, and protein synthesis (PS) was evaluated by measuring the incorporation of l-[4,5-3H]leucine into proteins. The first procedure, exposure to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase, produced a parallel increase in cytoplasmic calcium activity and a long-lasting suppression of PS, while the second, immersion in a calcium-free medium supplemented with the calcium chelator EGTA, caused a parallel decrease in cytoplasmic calcium levels and a short-lasting suppression of PS. Exposure of neurons to Tg induced a permanent increase in MyD116 mRNA levels. Exposure of cells to calcium-free medium supplemented with EGTA produced only a transient rise in MyD116 mRNA levels peaking after 6 h of recovery. The results demonstrate that depletion of ER calcium stores without any increase in cytoplasmic calcium activity is sufficient to activate MyD116 expression. A similar mechanism may be responsible for the increase in MyD116 mRNA levels observed after transient forebrain ischemia. It is concluded that those pathological disturbances triggering the activation of MyD116 expression after transient forebrain ischemia are only transient in the cerebral cortex but permanent in the hippocampus.

Disturbances of the functioning of endoplasmic reticulum: a key mechanism underlying neuronal cell injury?

Cerebral ischemia leads to a massive increase in cytoplasmic calcium activity resulting from an influx of calcium ions into cells and a release of calcium from mitochondria and endoplasmic reticulum (ER). It is widely believed that this increase in cytoplasmic calcium activity plays a major role in ischemic cell injury in neurons. Recently, this concept was modified, taking into account that disturbances occurring during ischemia are potentially reversible: it then was proposed that after reversible ischemia, calcium ions are taken up by mitochondria, leading to disturbances of oxidative phosphorylation, formation of free radicals, and deterioration of mitochondrial functions. The current review focuses on the possible role of disturbances of ER calcium homeostasis in the pathologic process culminating in ischemic cell injury. The ER is a subcellular compartment that fulfills important functions such as the folding and processing of proteins, all of which are strictly calcium dependent. ER calcium activity is therefore relatively high, lying in the lower millimolar range (i.e., close to that of the extracellular space). Depletion of ER calcium stores is a severe form of stress to which cells react with a highly conserved stress response, the most important changes being a suppression of global protein synthesis and activation of stress gene expression. The response of cells to disturbances of ER calcium homeostasis is almost identical to their response to transient ischemia, implying common underlying mechanisms. Many observations from experimental studies indicate that disturbances of ER calcium homeostasis are involved in the pathologic process leading to ischemic cell injury. Evidence also has been presented that depletion of ER calcium stores alone is sufficient to activate the process of programmed cell death. Furthermore, it has been shown that activation of the ER-resident stress response system by a sublethal form of stress affords tolerance to other, potentially lethal insults. Also, disturbances of ER function have been implicated in the development of degenerative disorders such as prion disease and Alzheimer's disease. Thus, disturbances of the functioning of the ER may be a common denominator of neuronal cell injury in a wide variety of acute and chronic pathologic states of the brain. Finally, there is evidence that ER calcium homeostasis plays a key role in maintaining cells in their physiologic state, since depletion of ER calcium stores causes growth arrest and cell death, whereas cells in which the regulatory link between ER calcium homeostasis and protein synthesis has been blocked enter a state of uncontrolled proliferation.