Synaptic pathology is observed during hypoxic events in the central nervous system in the form of altered dendrite structure and conductance changes. These alterations are rapidly reversible, on the ...return of normoxia, but are thought to initiate subsequent neuronal cell death. To characterize the effects of hypoxia on regulators of synaptic stability, we examined the temporal expression of cell adhesion molecules (CAMs) in synaptosomes after transient middle cerebral artery occlusion (MCAO) in mice. We focused on events preceding the onset of ischemic neuronal cell death (< 48 h). Synaptosome preparations were enriched in synaptically localized proteins and were free of endoplasmic reticulum and nuclear contamination. Electron microscopy showed that the synaptosome preparation was enriched in spheres (≈650 nm in diameter) containing secretory vesicles and postsynaptic densities. Forebrain mRNA levels of synaptically located CAMs was unaffected at 3 h after MCAO. This is contrasted by the observation of consistent downregulation of synaptic CAMs at 20 h after MCAO. Examination of synaptosomal CAM protein content indicated that certain adhesion molecules were decreased as early as 3 h after MCAO. For comparison, synaptosomal Agrn protein levels were unaffected by cerebral ischemia. Furthermore, a marked increase in the levels of p-Ctnnb1 in ischemic synaptosomes was observed. p-Ctnnb1 was detected in hippocampal fiber tracts and in cornu ammonis 1 neuronal nuclei. These results indicate that ischemia induces a dysregulation of a subset of synaptic proteins that are important regulators of synaptic plasticity before the onset of ischemic neuronal cell death.
Confounding any genome-scale analysis of gene expression after cerebral ischemia is massive suppression of protein synthesis. This inefficient translation questions the utility of examining profiles ...of total transcripts. Our approach to such postischemic gene profiling in the mouse by microarray analysis was to concentrate on those mRNAs bound to polyribosomes. In our proof-of-principle study, polysomally bound and unbound mRNAs were subjected to microarray analysis: of the 1,161 transcripts that we found to increase after ischemia, only 36% were bound to polyribosomes. In addition to the expected increases in heat-shock proteins and metallothioneins, increases in several other bound transcripts involved in the promotion of cell survival or antiinflammatory behavior were noted, such as CD63 (Lamp3), Lcn2 (lipocalin-2), Msn (moesin), and UCP2 (uncoupling protein 2), all of which showed increases in cognate protein by Western blotting. The list of heretofore nonfunctionally annotated transcripts (RIKEN clones/ESTs) that increased appeared to be novel. How some transcripts are selected in ischemic brain for translation into protein, while others are rejected, is not clear. The length of the 5′-UTR in the ischemically induced transcripts that occur in the NCBI RefSeq database did not indicate any general tendency to be more than 200 nt, nor to be longer than the 5′-UTRs of the unbound transcripts. Thus, the presence of a complex 5′-UTR region with internal ribosome entry sites (IRES) or polypyrimidine tracts (TOP) does not appear to be the basis of selection for translation in ischemic brain.
: The transcription factor E2F1 is known to mediate apoptosis in isolated quiescent and postmitotic cardiac myocytes, and its absence decreases the size of brain infarction following cerebral ...ischemia. To demonstrate directly that E2F1 modulates neuronal apoptosis, we used cultured cortical neurons to show a temporal association of the transcription and expression of E2F1 in neurons with increased neuronal apoptosis. Cortical neurons lacking E2F1 expression (derived from E2F1 ‐/‐ mice) were resistant to staurosporine‐induced apoptosis as evidenced by the significantly lower caspase 3‐like activity and a lesser number of cells with apoptotic morphology in comparison with cortical cultures derived from wild‐type mice. Furthermore, overexpressing E2F1 alone using replication‐deficient recombinant adenovirus was sufficient to cause neuronal cell death by apoptosis, as evidenced by the appearance of hallmarks of apoptosis, such as the threefold increase in caspase 3‐like activity and increased laddered DNA fragmentation, in situ endlabeled DNA fragmentation, and numbers of neuronal cells with punctate nuclei. Taken together, we conclude that E2F1 plays a key role in modulating neuronal apoptosis.
Because of observations that cultured neurons from mice deficient in the transcription factor E2F1 exhibit resistance after treatment with a wide variety of cell-death inducers, the authors ...investigated whether resistance extended to a cerebral ischemic insult. No differences in cerebral blood flow or physiologic parameters were observed in the mutant E2F1 littermates after the focal ligation. After 2 hours of left middle cerebral artery occlusion and 1 day of reperfusion, a 33% smaller infarct (P < 0.05) was observed by 2,3,5-triphenyltetrazolium staining in the brains of E2F1-null mice compared with their E2F1+/+ and +/− littermates. A milder ischemic insult produced by 20 minutes of middle cerebral artery occlusion and 7 days of reperfusion produced a greater difference in the E2F1-null animals with a 71% smaller infarct (P < 0.001) compared to littermate controls. A decrease in neuronal damage after mild ischemia in E2F1-null mice was observed by immunohistochemical monitoring of the loss in neuronal-specific microtubule-associated protein 2 cytoskeletal protein and the appearance of nuclear DNA fragmentation by terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling. This decreased brain damage was evidenced by improved behavior in motor function of E2F1 −/− mice compared with their E2F1 +/+ littermates by 7 days of reperfusion. In an effort to address the underlying molecular mechanism of the resistance of E2F1-null mice, the expression of several downstream proapoptotic target genes (p73, Apaf1, Arf) of the E2F1 transcription factor was measured by quantitative polymerase chain reaction. Although an attenuated increase in Hsp68 mRNA was found in E2F1 −/− mice, no changes in the proapoptotic transcripts were found after ischemia, and a mechanistic inference was not possible. The authors conclude that the transcription factor E2F1 does modulate neuronal viability in brain after cerebral ischemia and corroborates the findings with cultured neurons.
Mammalian genomes are burdened with a large heterogeneous group of endogenous replication defective retroviruses (retrotransposons). Previously, we identified a transcript resembling a virus-like 30S ...(
VL30) retrotransposon increasing in mouse brain following transient cerebral ischemia. Paradoxically, this non-coding RNA was found bound to polyribosomes. Further analysis revealed that multiple retrotransposon species (
BVL-1-like and
mVL30-1-like) were bound to polyribosomes and induced by ischemia. These
VL30 transcripts remained associated with polyribosomes in the presence of 0.5 M KCl, indicating that
VL30 mRNA was tightly associated with ribosomal subunits. Furthermore, the profile of
BVL-1 distribution on polyribosomal profiles was distinct from those of translated and translationally repressed mRNA. Consistent with expectations, 5.0 kb
VL30 transcripts were detected in ischemic brain with a temporal pattern of expression that was distinct from c-
fos. Expression of
VL30 was localized in neurons using a combination of in situ hybridization and immunocytochemistry. 3′-RACE-PCR experiments yielded two unique sequences (
VL30x-1 and
VL30x-2) that were homologous to known VL30 genes. Phylogenetic analysis of
VL30 promoter sequence (U3 region) resulted in the identification of two large
VL30 subgroups.
VL30x-1 and
VL30x-2 were closely related and classified in a group that was distinct from the well-characterized VL30 genes
BVL-1 and
mVL30-1. The promoter regions of
VL30x-1 and
VL30x-2 did not possess the consensus sequences for either hypoxia or anoxia response elements, suggesting an alternative mechanism for induction. This is the first report that demonstrates ischemia-induced, neuronal expression of unique VL30 retrotransposons in mouse brain.
The E2F1 transcription factor modulates neuronal apoptosis induced by staurosporine, DNA damage and β‐amyloid. We demonstrate E2F1 involvement in neuronal death induced by the more physiological ...oxygen‐glucose deprivation (OGD) in mouse cortical cultures and by anoxia in mouse hippocampal slices. E2F1(+/+) and (−/−) cultures were comparable, in that they contained similar neuronal densities, responded with similar increases in intracellular calcium concentration (Ca2+i) to glutamate receptor agonists, and showed similar NMDA receptor subunit mRNA expression levels for NR1, NR2A and NR2B. Despite these similarities, E2F1(−/−) cultures were significantly less susceptible to neuronal death than E2F1(+/+) cultures 24 and 48 h following 120–180 min of OGD. Furthermore, the absence of E2F1 significantly improved the ability of CA1 neurons in hippocampal slices to recover synaptic transmission following a transient anoxic insult in vitro. These results, along with our finding that E2F1 mRNA levels are significantly increased following OGD, support a role for E2F1 in the modulation of OGD‐ and anoxia‐induced neuronal death. These findings are consistent with studies showing that overexpression of E2F1 in postmitotic neurons causes neuronal degeneration and the absence of E2F1 decreases infarct volume following cerebral ischemia.
Transient focal ischaemia was produced in rat right neocortex by temporary middle cerebral artery occlusion. DNA damage was visualized in situ in cells of this right hemisphere but not in the ...contralateral hemisphere. The extracted damaged DNA exhibited laddered fragmentation which is indicative of apoptotic degradation. The amount of DNA damage was quantified by an end-labelling technique and shown to increase with the duration of the ischaemic insult. We conclude that the neurodegeneration resulting from focal ischaemia has an apoptotic component.
Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia ...(NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3′-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.
Laddered DNA indicative of apoptosis was observed in the CA1 layer of hippocampus and in dorsolateral striatum following a global cerebral ischemic insult produced by transient two vessel occlusion ...in rats. The extent of this DNA damage was proportional to the duration of the ischemic episode. Breaks in DNA were demonstrated in situ in sections from post-ischemic brain in neurons of the hippocampal CA1 which undergo selective neuronal death but not in other cell types. It is concluded that there is an apoptotic component to selective neuronal death following global ischemia in rat brain.
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