Vulnerable CA1 Subfield Research Articles

Dysregulation of Amyloid-β Protein Precursor, β-Secretase, Presenilin 1 and 2 Genes in the Rat Selectively Vulnerable CA1 Subfield of Hippocampus Following Transient Global Brain Ischemia.

The interaction between brain ischemia and Alzheimer’s disease (AD) has been intensively investigated recently. Nevertheless, we have not yet understood the nature and mechanisms of the ischemic episodes triggering the onset of AD and how they influence its slow progression. The assumed connection between brain ischemia and the accumulation of amyloid-β (Aβ) peptide awaits to be clearly explained. In our research, we employed a rat cardiac arrest model to study the changes in gene expression of amyloid-β protein precursor (AβPP) and its cleaving enzymes, β- and γ-secretases (including presenilins) in hippocampal CA1 sector, following transient 10-min global brain ischemia. The quantitative reverse-transcriptase PCR assay demonstrated that the expression of all above genes that contribute to Aβ peptide generation was dysregulated during 30 days in postischemic hippocampal CA1 area. It suggests that studied Aβ peptide generation-related genes can be involved in AβPP metabolism, following global brain ischemia and will be useful to identify the molecular mechanisms underpinning that cerebral ischemia might be an etiological cause of AD via dysregulation of AβPP and its cleaving enzymes, β- and γ-secretases genes, and subsequently, it may increase Aβ peptide production and promote the gradual and slow development of AD neuropathology. Our data demonstrate that brain ischemia activates delayed neuronal death in hippocampus in an AβPP-dependent manner, thus defining a new and important mode of ischemic cell death.

Activation of calcium/calmodulin‐dependent protein kinase IV and peroxisome proliferator‐activated receptor γ coactivator‐1α signaling pathway protects against neuronal injury and promotes mitochondrial biogenesis in the hippocampal CA1 subfield after transient global ischemia

Delayed neuronal cell death occurs in the vulnerable CA1 subfield of the hippocampus after transient global ischemia (TGI). We demonstrated previously, based on an experimental model of TGI, that the significantly increased content of oxidized proteins in hippocampal CA1 neuron was observed as early as 30 min after TGI, followed by augmentation of PGC-1α expression at 1 hr, as well as up-regulation of mitochondrial uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2). Using the same animal model, the present study investigated the role of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and PGC-1α in delayed neuronal cell death and mitochondrial biogenesis in the hippocampus. In Sprague-Dawley rats, significantly increased expression of nuclear CaMKIV was noted in the hippocampal CA1 subfield as early as 15 min after TGI. In addition, the index of mitochondrial biogenesis, including a mitochondrial DNA-encoded polypeptide, cytochrome c oxidase subunit 1 (COX1), and mitochondrial number significantly increased in the hippocampal CA1 subfield 4 hr after TGI. Application bilaterally into the hippocampal CA1 subfield of an inhibitor of CaMKIV, KN-93, 30 min before TGI attenuated both CaMKIV and PGC-1α expression, followed by down-regulation of UCP2 and SOD2, decrease of COX1 expression and mitochondrial number, heightened protein oxidation, and enhanced hippocampal CA1 neuronal damage. This study provides correlative evidence for the neuroprotective cascade of CaMKIV/PGC-1α which implicates at least in part the mitochondrial antioxidants UCP2 and SOD2 as well as mitochondrial biogenesis in ischemic brain injury.

Transient oligemia is associated with long-term changes in binding densities of cortical inhibitory GABA A receptors in the rat brain

Recently, we could demonstrate in rats that a short transient oligemic period of only 20-minute duration, induced by systemic hypotension, resulted in a transient decline of spatial memory capacities without any histological damage over a subsequent period of 6 months. In our present study, we checked for more subtle alterations within the highly vulnerable hippocampal CA1 subfield using quantification of neuronal cell density and semi-quantitative analysis of the ischemia-sensitive protein MAP2. Since hippocampal excitatory and inhibitory neurotransmitter receptors are crucially involved in spatial memory processes, quantitative in vitro receptor autoradiography was performed using [ 3H]MK-801, [ 3H]AMPA, and [ 3H]muscimol for labeling of NMDA, AMPA, and GABA A receptors, respectively. Ligand binding values were analyzed in the ischemia-sensitive hippocampal formation and in the parietal cortex. Transient oligemia did not cause any reduction of the neuronal cell density in the vulnerable CA1 subfield nor did it cause any significant changes of MAP2 protein expression in the dendritic layers of CA1. After oligemia, hippocampal binding densities of NMDA, AMPA, and GABA A receptors remained unchanged in all subfields investigated. Similarly, there was no difference in cortical ligand binding values of the excitatory NMDA and AMPA receptors comparing oligemic to sham-operated control rats. In the parietal cortex, however, transient oligemia caused a significant reduction of [ 3H]muscimol binding values compared to controls. These findings demonstrate long-term changes in receptor binding densities even after short oligemic periods possibly reflecting part of the molecular basis leading to impaired cognitive abilities.

Redistribution of apoptosis promoters: XAF1, Smac/DIABLO and HtrA2 during transient forebrain ischemia/reperfusion

The neuropathology following brain injury after cerebral ischemia includes both, selective neuronal necrosis and apoptosis. Caspases are key regulators of apoptosis and are essential for the execution of cell death. Caspase activity is controlled through a wide range of regulatory mechanisms, including a family of genes encoding for inhibitors of apoptosis proteins (IAPs). Convincing anti-apoptotic activity has been shown for a subset of mammalian IAPs. XIAP, cIAP1, cIAP2. XIAP (X-linked inhibitor of apoptosis protein) is the best characterized mammalian IAP and is also the most potent and versatile regulator of cell death. Regulation of XIAP involves at least three recently discovered proteins: XAF-1 (XIAP-associated factor 1), Smac/Diablo (Second mitochondrial activator of caspase/direct IAP-binding protein with low pI) and HtrA2/Omi (high-temperature requirement serine protease A2 or Omi). XAF-1 is ubiquitously expressed in normal tissues and inhibits XIAP and localized in the nucleus. When co-expressed in cell lines, XAF-1 retains normally cytosolic localized XIAP in the nucleus. To investigate whether IAPs and their inhibitors XAF1, Smac/DIABLO, HtrA2 may play a role in the apoptotic machinery after ischemia, we analyzed dynamics in their expression pattern and in their localisation. Ischemia was performed in Wistar rats by clamping both common carotid arteries and reducing blood pressure to 40 mmHg for 10 min. Animals were decapitated at different times post-ischemia and processed for histology, immunohistochemistry and immunoblotting by using antibodies directed against XIAP-BIR2, Smac/DIABLO, HtrA2 and cleaved caspase-3 (P17). XAF-1 staining and colocalization with XIAP was seen in the selectively vulnerable CA1-subfield of the hippocampus. In damaged neurons, we found that XAF-1 colocalized with XIAP in the nuclei, suggesting that it sequestered XIAP, allowing the pathological apoptosis to proceed. Smac/Diablo is a mitochondrial protein, which binds to XIAP, antagonizing its anti-apoptotic activity. This role of XIAP is supported by our findings, as it colocalized after ischemia very well with Smac, and as these double-labelled cells presented signs of damage. HtrA2 is another mitochondrial protein released upon apoptotic stimuli that binds to XIAP and promotes cell death. The strong HtrA2 immunoreactivity around the nuclei of damaged neurons, that stained also for XIAP and Smac, supports a concerted action of these proteins after ischemia. Increased intensity of Smac and HtrA2 stainings presumably indicates release from mitochondria. Here presented data report to our knowledge for the first time an involvement of XAF-1 and HtrA2 in the apoptotic dynamics following global cerebral ischemia in rats.

Decreased expression and impaired function of muscarinic acetylcholine receptors in the rat hippocampus following transient forebrain ischemia

In this study, we investigated whether transient cerebral ischemia affects the function and molecular expression of specific muscarinic cholinergic receptors. Our results show that in contrast to the GABA-B and A1 adenosine receptor systems, the ability of muscarinic receptors to attenuate evoked excitatory responses at vulnerable CA1 synapses is significantly attenuated by 18 h following reperfusion. This attenuation in efficacy was restricted to the vulnerable CA1 subfield, as no significant change in muscarinic receptor-mediated attenuation of evoked responsiveness was observed within post-ischemic dentate granule cell synapses. Expression analysis revealed that the mRNA and immunoreactive protein levels for individual types of muscarinic receptors respond differently and uniquely to transient cerebral ischemia insult. Of particular interest is the m4 subtype of receptor, whose mRNA and protein expression levels were significantly diminished within the hippocampus by 12 and 24 h following reperfusion, respectively. As the m4 muscarinic receptor localizes to presynaptic terminals within the hippocampus, a decrease in its expression could account for the impaired functional responsiveness of the muscarinic receptor system following ischemic insult. Taken together, these results demonstrate that transient forebrain ischemia leads to dynamic alterations in the gene expression, protein prevalence, and functionality of muscarinic receptors in the post-ischemic hippocampus at times preceding the degeneration of the vulnerable neurons.

Altered expression levels of SEF-2 and p112 in the rat hippocampus after transient cerebral ischemia: identification by mRNA differential display.

The authors used mRNA differential display to identify genes whose expression levels are altered in the adult rat hippocampus 24 hours after global ischemia. At this time after challenge, the basic helix-loop-helix transcription factor, SEF-2, and the 26S proteasome complex subunit, p112, were identified as genes whose expression levels are decreased and increased, respectively, in the hippocampus. To determine the spatial and temporal patterns of expression change for each gene, the authors antisense in situ hybridization to paired brain sections of sham-operated and global ischemia-challenged rats at 6, 12, and 24 hours after reperfusion SEF-2 expression was not significantly altered from that of sham-operated controls in any hippocampal subfield at or before 12 hours after challenge. At 24 hours after ischemia, however, SEF-2 expression levels were significantly diminished in the vulnerable CA1 subfield, but not in the less vulnerable CA3 or dentate granule cell subfields. The proteasome p112 subunit gene displayed no change in expression levels at 6 hours after insult; however, an elevated expression was observed at 12 hours after challenge in the dentate granule cell subfield. By 24 hours after challenge, p112 expression was significantly elevated in both the CA1 and dentate granule cell subfields. These results demonstrate that a member of the basic helix-loop-helix family of transcription factors, SEF-2, and the major subunit of the 26S proteasome complex, p112, display altered gene expression in the hippocampus after transient cerebral ischemia.

Possible involvement of activator protein-1 DNA binding in mechanisms underlying ischemic tolerance in the CA1 subfield of gerbil hippocampus

Transcription factors are nuclear proteins with an ability to recognize particular nucleotide sequences on double stranded genomic DNAs and thereby modulate the activity of RNA polymerase II which is responsible for the formation of messenger RNAs in cell nuclei. Gel retardation electrophoresis revealed that transient forebrain ischemia for 5 min led to drastic potentiation of binding of a radiolabelled double-stranded oligonucleotide probe for the transcription factor activator protein-1, in the thalamus as well as the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of the gerbils previously given ischemia for 2 min two days before, which is known to induce tolerance to subsequent severe ischemia in the CA1 subfield. By contrast, ischemia for 5 min resulted in prolonged potentiation of activator protein-1 binding in the vulnerable CA1 subfield of the gerbils with prior ischemia for 5 min 14 days before, which is shown to induce delayed death of the pyramidal neurons exclusively in this subfield. Similar prolongation was seen with activator protein-1 binding in the vulnerable thalamus but not in the resistant CA3 subfield and dentate gyrus of the gerbils with such repeated ischemia for 5 min. Limited proteolysis by Staphylococcus aureus V8 protease as well as supershift assays using antibodies against c-Fos and c-Jun proteins demonstrated the possible difference in constructive partner proteins of activator protein-1 among nuclear extracts of the CA1 subfield obtained from gerbils with single, tolerated and repeated ischemia. These results suggest that de novo protein synthesis may underlie molecular mechanisms associated with acquisition of the ischemic tolerance through modulation at the level of gene transcription by activator protein-1 composed of different constructive partner proteins in the CA1 subfield. Possible participation of glial cells in the modulation is also suggested in particular situations.

Prolongation by bifemelane of potentiation of AP1 DNA binding in hippocampal CA1 subfield of gerbils with transient forebrain ischemia.

In eukaryotes, protein de novo synthesis is mainly under the control of transcription factors at the level of gene transcription in cell nuclei. Gel retardation electrophoresis was employed for determination of DNA-binding activity of the transcription factor activator protein-1 (AP1), which is a dimer between c-Fos and c-Jun protein families. Binding of a radiolabeled double-stranded oligonucleotide probe for AP1 was rapidly potentiated in the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of gerbils with forebrain ischemia for 5 min. Similarly marked potentiation was seen in the thalamus and the striatum, but not in the frontal cortex, following the recirculation of blood supply. The potentiation was transient in the vulnerable CA1 subfield, but was rather persistent in the thalamus and the striatum in addition to the resistant CA3 subfield and dentate gyrus. However, administration of the neuroprotective drug bifemelane (10 to 20 mg/kg, i.p.) resulted in prolongation of the potentiation of AP1 binding in the CA1 subfield up to 6 hr after ischemia, without significantly affecting that in other central structures. Limited proteolysis revealed that bifemelane induced expression of the AP1 consisting of constructive proteins different from those expressed in control animals in the CA1 subfield. These results suggest that bifemelane may protect neuronal cells against ischemic injuries through molecular mechanisms associated with prolongation of the potentiation of AP1 binding in the vulnerable CA1 subfield after ischemia.

Transient global ischemia alters NMDA receptor expression in rat hippocampus: correlation with decreased immunoreactive protein levels of the NR2A/2B subunits, and an altered NMDA receptor functionality.

We investigated the gene expression levels, the immunoreactive protein prevalence, and the functional activity of N-methyl-D-aspartate (NMDA) receptor complexes at early times after severe global ischemia challenge in rats. The mRNA expression levels for the NR2A and NR2B subunits of NMDA receptors changed to different degrees within different subregions of the hippocampus after reperfusion with respect to sham-operated control. No significant change in expression was observed in the vulnerable CA1 subfield at or before 6 h after challenge for either receptor subunit, although changes in expression in other hippocampal subfields were observed. At 12 and 24 h after challenge, significant decreases in expression for both subunits were found in the vulnerable CA1 subfield, as well as in other hippocampal regions. At the protein level, a significant decrease in the amount of NR2A/NR2B immunoreactivity in the total hippocampus was observed at both 6 and 24 h after reperfusion compared with sham control. Electrophysiological assessment of single-channel NMDA receptor activity in the CA1 subfield indicates that the main conductance state of NMDA receptor channels is maintained 6 h after challenge, although by 18-24 h after challenge, this main conductance state is rarely observed. The NMDA receptor component of the excitatory postsynaptic field potential was found to be significantly diminished from sham control 24 h after challenge, such that only approximately 10% of the sham response remained, but was not significantly altered from sham control at 6 h after challenge. These results indicate that decreases in the expression levels, the immunoreactive protein prevalence, and that alterations in the functionality of NMDA receptors occur in the hippocampus at early times after severe transient global ischemia.

Positive correlation between prolonged potentiation of binding of double-stranded oligonucleotide probe for the transcription factor AP1 and resistance to transient forebrain ischemia in gerbil hippocampus

Gel retardation electrophoresis revealed that binding of a radiolabelled double-stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 was markedly potentiated in the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of the gerbils with transient forebrain ischemia for 5 min, which is known to induce delayed death of pyramidal neurons exclusively in the CA1 subfield. The potentiation was transient in the vulnerable CA1 subfield, but persistent up to 18 h in the resistant CA3 subfield and dentate gyrus. However, no significant alteration was detected in endogenous levels of cyclic AMP response element binding protein phosphorylated at serine 133 in these three different hippocampal structures 3 h after the reperfusion. On the other hand, hypothermia during ischemia which is known to protect the CA1 subfield against ischemic damages, led to a prolonged elevation of the activator protein-1 binding up to 9 h after the reperfusion in this vulnerable subfield at least in part through expression of c-Fos protein. Moreover, activator protein-1 binding was significantly elevated in the CA1 subfield up to 12 h after forebrain ischemia for 2 min which is shown not to induce marked damages to the vulnerable subfield. These results suggest that prolonged elevation of DNA binding activity of activator protein-1 may be responsible for molecular mechanisms underlying the unique vulnerability and/or resistance of particular subfields to a transient ischemic insult in the gerbil hippocampus.

Clenbuterol protects mouse cerebral cortex and rat hippocampus from ischemic damage and attenuates glutamate neurotoxicity in cultured hippocampal neurons by induction of NGF

It has been shown previously that clenbuterol, a β 2-adrenergic receptor agonist, enhances NGF synthesis in adult rat brain. Since NGF is able to protect neurons against damage, we tried to find out whether clenbuterol can rescue cultured hippocampal neurons from excitotoxic damage by induction of NGF. The neuroprotective activity of clenbuterol on neurons in the vulnerable CA1 subfield of the hippocampus was tested in a rat model of transient forebrain ischemia. Additionally, in the mouse model of focal cerebral ischemia the ability of clenbuterol to reduce the infarct size was examined. Exposure of mixed neuronal/glial hippocampal cultures to clenbuterol (1 to 100 μM) enhanced significantly the content of NGF measured in the culture medium by two-site ELISA. The excitotoxic injury was induced in the same type of cells after 14 days in vitro by exposure to 1 MM l-glutamate for 1 h in serum-free medium. NGF itself (0.15 to 100 ng/ml) added to the growth medium 4 h before until 18 h after induction of injury (the point of glutamate-toxicity measurement), protected hippocampal neurons from excitotoxic damage. Clenbuterol (1 to 100 μM) provided similar neuroprotection as NGF under the same experimental conditions. The neuroprotective activity of clenbuterol (100 μM) against glutamate-induced damage in hippocampal cultures was blocked by anti-NGF monoclonal antibodies (0.5 μg/ml) added to the medium during the clenbuterol exposure, demonstrating that the neuronal rescue is mediated by NGF. Propranolol, a β-adrenergic receptor antagonist (10 μM) added 20 min before and kept in the medium during exposure of the cultures to clenbuterol (1 μM) reversed the neuroprotective activity, suggesting that the induction of NGF and neuroprotection caused by clenbuterol are mediated via β-adrenergic receptor activation. The capacity of clenbuterol to protect hippocampal neurons was also demonstrated in vivo in a rat model of transient forebrain ischemia. Clenbuterol (4 × 1 mg/kg) administered intraperitoneally increased the number of viable neurons in CA1 subfield of the rat hippocampus. Furthermore, clenbuterol (0.3 and 1 mg/kg, i.p. and 1 mg/kg, s.c.) reduced significantly the infarct area on the mouse brain surface after occlusion of the middle cerebral artery. The present data demonstrate that clenbuterol induces NGF synthesis in cultured hippocampal cells and protects hippocampal neurons from excitotoxic damage. The neuroprotective activity of clenbuterol is also demonstrated in vivo in two rodent models of cerebral ischemia. The results offer strong evidence that the neuroprotective activity of clenbuterol is caused by activation of β-adrenergic receptors and the subsequent increased expression of NGF.

Ischemic delayed neuronal death. A mitochondrial hypothesis.

A brief period of global brain ischemia causes cell death in hippocampal CA1 pyramidal neurons days after reperfusion in rodents and humans. Other neurons are much less vulnerable. This phenomenon is commonly referred to as delayed neuronal death, but the cause has not been fully understood although many mechanisms have been proposed. Hippocampal CA1 neuronal death usually occurs 3 to 4 days after an initial ischemic insult. Such a delay is essential for the mechanism of this type of cell death. Previous hypotheses have not well explained the reason for the delay and the exact mechanism of the cell death, but a disturbance of mitochondrial gene expression could be a possibility. Reductions of mitochondrial RNA level and the activity of a mitochondrial protein, encoded partly by mitochondrial DNA, occurred exclusively in CA1 neurons at the early stage of reperfusion and were aggravated over time. In contrast, the activity of a nuclear DNA-encoded mitochondrial enzyme and the level of mitochondrial DNA remained intact in CA1 cells until death. Immunohistochemical staining for cytoplasmic dynein and kinesin, which are involved in the shuttle movement of mitochondria between cell body and the periphery, also showed early and progressive decreases after ischemia, and the decreases were found exclusively in the vulnerable CA1 subfield. A disturbance of mitochondrial DNA expression may be caused by dysfunction of the mitochondrial shuttle system and could cause progressive failure of energy production of CA1 neurons that eventually results in cell death. Thus, the mitochondrial hypothesis could provide a new and exciting potential for elucidating the mechanism of the delayed neuronal death of hippocampal CA1 neurons.

Ornithine decarboxylase in reversible cerebral ischemia: an immunohistochemical study

Anesthetized Mongolian gerbils were subjected to 5-min ischemia and 8 h of recirculation. Vibratom sections were taken for studying changes in ornithine decarboxylase (ODC) immunoreactivity using an antiserum to ODC, and tissue samples were taken for measuring ODC activity. After 5-min ischemia and 8-h recirculation ODC activity increased 11.5-, 5.9-, and 7.9-fold in the cerebral cortex, striatum and hippocampus, respectively (P less than or equal to 0.05 to 0.01). In the cortex, striatum and hippocampus of control animals immunoreactivity was low but clearly above the detection limit. The reaction was confined to neurons. After 5-min ischemia and 8-h recirculation a sharp increase in immunoreactivity was observed confined to neurons, indicating that the postischemic activation of polyamine metabolism is a neuronal response to ischemia. The immunoreactivity was markedly increased in the perinuclear cytoplasm and the dendrites. In the striatum the density of neurons exhibiting a sharp increase in immunoreactivity was more pronounced in the lateral than in the ventral part. In the hippocampus a strong reaction was present in all subfields but the CA1 subfield was particularly affected. The present study demonstrates for the first time that biosynthesis of a protein is markedly activated during the first 24 h of recirculation after 5-min cerebral ischemia of gerbils even in the vulnerable CA1 subfield, in which the overall protein synthesis is sharply reduced at the same time. Studying polyamine metabolism after ischemia may, thus, provide new information about the basic molecular mechanisms responsible for the altered gene expression after metabolic stress.