Wulf Paschen

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.

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 Alzheimers 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.

Endoplasmic Reticulum Stress

Abstract:  Stress is the imbalance of homeostasis, which can be sensed even at the subcellular level. The stress‐sensing capability of various organelles including the endoplasmic reticulum (ER) has been described. It has become evident that acute or prolonged ER stress plays an important role in many human diseases; especially those involving organs/tissues specialized in protein secretion. This article summarizes the emerging role of ER stress in diverse human pathophysiological conditions such as carcinogenesis and tumor progression, cerebral ischemia, plasma cell maturation and apoptosis, obesity, insulin resistance, and type 2 diabetes. Certain components of the ER stress response machinery are identified as biomarkers of the diseases or as possible targets for therapeutic intervention.

Endoplasmic reticulum: a primary target in various acute disorders and degenerative diseases of the brain.

Changes in neuronal calcium activity in the various subcellular compartments have divergent effects on affected cells. In the cytoplasm and mitochondria, where calcium activity is normally low, a prolonged excessive rise in free calcium levels is believed to be toxic, in the endoplasmic reticulum (ER), in contrast, calcium activity is relatively high and severe stress is caused by a depletion of ER calcium stores. Besides its role in cellular calcium signaling, the ER is the site where membrane and secretory proteins are folded and processed. These calcium-dependent processes are fundamental to normal cell functioning. Under conditions of ER dysfunction unfolded proteins accumulate in the ER lumen, a signal responsible for activation of the unfolded protein response (UPR) and the ER-associated degradation (ERAD). UPR is characterized by activation of two ER-resident kinases, PKR-like ER kinase (PERK) and IRE1. PERK induces phosphorylation of the eukaryotic initiation factor (eIF2alpha), resulting in a shut-down of translation at the initiation step. This stress response is needed to block new synthesis of proteins that cannot be correctly folded, and thus to protect cells from the effect of unfolded proteins which tend to form toxic aggregates. IRE1, on the other hand, is turned after activation into an endonuclease that cuts out a sequence of 26 bases from the coding region of xbp1 mRNA. Processed xbp1 mRNA is translated into the respective protein, an active transcription factor specific for ER stress genes such as grp78. In acute disorders and degenerative diseases, the ER calcium pool is a primary target of toxic metabolites or intermediates, such as oxygen free radicals, produced during the pathological process. Affected neurons need to activate the entire UPR to cope with the severe form of stress induced by ER dysfunction. This stress response is however hindered under conditions where protein synthesis is suppressed to such an extent that processed xbp1 mRNA is not translated into the processed XBP1 protein (XBP1(proc)). Furthermore, activation of ERAD is important for the degradation of unfolded proteins through the ubiquitin/proteasomal pathway, which is impaired in acute disorders and degenerative diseases, resulting in further ER stress. ER functioning is thus impaired in two different ways: first by the direct action of toxic intermediates, produced in the course of the pathological process, hindering vital ER reactions, and second by the inability of cells to fully activate UPR and ERAD, leaving them unable to withstand the severe form of stress induced by ER dysfunction.

Lactate and pH in the brain: association and dissociation in different pathophysiological states.

Abstract: Brain tissue pH and lactate content were measured in rats under three different experimental conditions, namely: (1) during complete global cerebral ischemia; (2) after reversible near‐complete cerebral ischemia; and (3) in experimental brain tumors. At the end of the experiments brains were frozen with liquid nitrogen. A series of 20‐μm thick coronal sections was prepared in a cryostat and then used for the regional determination of tissue pH (umbelliferone technique) and tissue lactate (bioluminescent technique). In addition, tissue samples were taken for the quantitative measurement of brain lactate (enzymatic fluorometric technique). The relationship between lactate content and tissue pH was different for each of the three experimental models studied: only after short‐term global cerebral ischemia did an increase in the lactate content correlate with a decrease in tissue pH (r= 0.94; p < 0.001). A highly significant increase in the lactate content (p < 0.001) was accompanied by physiological pH values (6.96 ± 0.08 in comparison to 6.97 ± 0.04 in controls) during recirculation after transient cerebral ischemia and in brain tumors even by an alkaline pH shift. In view of these observations the term “lactacidosis” should not be used without measuring both the lactate content and the pH. The observed dissociation between pH and lactate is due to the fact that both parameters are regulated independently. During anaerobiosis the main source of proton production is ATP hydrolysis rather than glycolysis. It is, therefore, suggested that the terms “acidosis” and “lactosis” should be used instead of “lactacidosis.”

Regional changes of blood flow, glucose, and ATP content determined on brain sections during a single passage of spreading depression in rat brain cortex.

Hemodynamic and biochemical substrate changes are associated with cortical spreading depression (CSD). Regional methods were used to measure blood flow, and glucose and ATP concentrations in intact brain sections in rats undergoing a single passage of cortical spreading depression. Changes were expressed as the percentages of the contralateral homotopic area of the unaffected cortex. A depression in tissue ATP content preceded the negative DC potential shift and ATP was reduced by 12% (P less than 0.01) despite unaltered blood flow and glucose concentration. When the negative shift of DC potential reached its maximum, glucose content decreased to 72% of control (P less than 0.01) and was accompanied by a further ATP decrease to 54%. When the cortical steady potential declined, blood flow was elevated twofold (P less than 0.01). The ATP content gradually returned to normal; however, cortical glucose concentrations remained at 55% of control values. The relationship of blood flow and glucose and ATP concentration with other known changes during spreading depression are discussed. With the advantage of higher resolution the provided techniques may be a useful tool for studies on hemodynamic and biochemical changes of other pathophysiologic conditions.

Relationship between Calcium Accumulation and Recovery of Cat Brain after Prolonged Cerebral Ischemia

The relationship between brain tissue calcium content and postischemic electrophysiological and metabolic recovery was investigated in 18 adult normothermic cats, 12 of which were submitted to 1 h of complete ischemia and 3 h of recirculation. Six animals served as controls. Functional recovery was estimated by recording the electrocorticogram (ECoG) and evoked potentials, and biochemical recovery by regional evaluation of ATP, glucose, and pH in intact brain sections. One group of animals was treated with the calcium antagonist flunarizine (0.1 mg/kg i. v., followed by continuous i. v. infusion of 0.1 mg/kg/h during the recirculation phase); another group did not receive this treatment. Evoked potentials in all six untreated animals (and in four also, spontaneous ECoG activity) returned after ischemia. In the animals with ECoG activity, biochemical recovery was homogeneous, as indicated by a return toward normal of regional tissue ATP and glucose content. In one animal without ECoG activity, several small regions were present in which energy metabolism was impaired. In regions with biochemical recovery, brain tissue calcium significantly increased by ∼35% (controls, 0.330 ± 0.045; ischemia, 0.447 ± 0.194 μg/mg protein; means ± SD). Changes were accompanied by a parallel increase in sodium (controls, 7.72 ± 1.92; ischemia, 10.50 ± 2.47 μg/mg protein), a slight decrease of potassium (controls, 29.52 ± 0.85; ischemia, 27.66 ± 2.30 μg/mg protein), and an increase of tissue pH (controls, 7.10 ± 0.096; ischemia, 7.307 ± 0.083). In regions without biochemical recovery, pH fell to 6.288 ± 0.157, and calcium content was 0.602 ± 0.235, sodium content 11.70 ± 4.60, and potassium content 23.00 ± 3.91 μg/mg protein. Treatment with the calcium antagonist flunarizine did not reduce tissue calcium content, nor did it improve functional or metabolic recovery after ischemia: three of six treated animals exhibited ECoG activity, one showed only evoked potentials, and two showed no recovery at all. It is concluded that postischemic accumulation of calcium in brain tissue cannot be prevented by the calcium antagonist flunarizine. However, the observed increase of calcium did not interfere with the early postischemic electrophysiological and biochemical recovery. Its pathophysiological importance, therefore, may be associated with more delayed postischemic disturbances.

Polyamine Changes in Reversible Cerebral Ischemia

Abstract: Putrescine, spermidine, and spermine levels were measured in the cortex, caudoputamen, and hippocampus of rats during 30 min of severe forebrain ischemia (induced by occlusion of both carotid and vertebral arteries) and subsequent recirculation. During ischemia, polyamine levels did not change significantly. During postischemic recirculation, however, putrescine levels dramatically increased whereas those of spermine and spermidine did not change, with the exception of the severely damaged caudoputamen, where the concentration declined after 24 h. The increase of putrescine is explained by postischemic activation of ornithine decarboxylase and inhibition of S‐adenosylmethionine decarboxylase. It is suggested that the accumulation of putrescine during postischemic recirculation may be responsible for the delayed neuronal death occurring after ischemia.

A topographic quantitative method for measuring brain tissue pH under physiological and pathophysiological conditions

A technique was developed for the quantitative regional assessment of brain pH by a modification of the umbelliferone method. Twenty micron thick sections were brought into contact with umbelliferone-soaked paper strips and the fluorescence (450 nm) following excitation at 370 nm and 340 nm was recorded photographically. The 340 nm excitation image was subtracted from 370 nm picture, using a computerized image-processing system. Regional pH values were measured in rats and cats under normal and ischemic conditions.

Transient Global Cerebral Ischemia Induces a Massive Increase in Protein Sumoylation

A new group of proteins, small ubiquitin-like modifier (SUMO) proteins, has recently been identified and protein sumoylation has been shown to play a major role in various signal transduction pathways. Here, we report that transient global cerebral ischemia induces a marked increase in protein sumoylation. Mice were subjected to 10 mins severe forebrain ischemia followed by 3 or 6 h of reperfusion. Transient cerebral ischemia induced a massive increase in protein sumoylation by SUMO2/3 both in the hippocampus and cerebral cortex. SUMO2/3 conjugation was associated with a decrease in levels of free SUMO2/3. After ischemia, protein levels of the SUMO-conjugating enzyme Ubc9 were transiently decreased in the cortex but not in the hippocampus. We also exposed HT22 cells to arsenite, a respiratory poison that impairs cytoplasmic function and induces oxidative stress. Arsenite exposure induced a marked rise in protein sumoylation, implying that impairment of cytoplasmic function and oxidative stress may be involved in the massive post-ischemic activation of SUMO conjugation described here. Sumoylation of transcription factors has been shown to block their activation, with some exceptions such as the heat-shock factor and the hypoxia-responsive factor, where sumoylation blocks their degradation, and the nuclear factor-κB (NF-κB) essential modulator where sumoylation leads to an activation of NF-κB. Because protein sumoylation is known to be involved in the regulation of various biologic processes, the massive post-ischemic increase in protein sumoylation may play a critical role in defining the final outcome of neurons exposed to transient ischemia.