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Two different models of brain ischemia were used to examine the evoked changes in the tyrosine phosphorylation of NMDA receptor subunits 2A and 2B (NR2A and NR2B), as well as their interactions with non-receptor tyrosine kinases (NRTKs: FAK, PYK2 Src), and PSD-95 protein. Only short-term 5 min ischemia followed by 3 h reperfusion resulted in the elevated tyrosine phosphorylation of both investigated NMDA receptor subunits, but in contrast to previously published data, more pronounced in the case of NR2B. Concomitantly, an increased association of NR2B with FAK, PYK2, Src and PSD-95 has been observed. This sharp early reaction to brief ischemia was markedly attenuated during prolonged recovery (72 h) with almost complete return to control values. The initial recruitment of tyrosine kinases to NMDA receptor during the first 3 h of reperfusion is generally consistent with an active postischemic remodeling of PSD and may participate in the induction of the postischemic signal transduction pathway in gerbil hippocampus. In contrast, ischemia of longer duration (up to 30 min) caused an immediate decrease in the protein levels as well as tyrosine phosphorylation of both NR2A and NR2B subunits which was accompanied by the marked attenuation of the association with their investigated molecular partners--PSD-95 and NRTKs. This effect may be mimicked in vitro by Ca2+-dependent activation of endogenous calpains in purified PSD preparation suggesting irreversible deterioration of the synaptic signaling machinery during irreversible long-term ischemia.

Cerebral ischemia is known to modify the expression of genetic information in the brain. To complement this knowledge, in the present study we have estimated the expression of calcium- and phospholipid-dependent (classical) protein kinase C (c PKC) isoform mRNAs (alpha, beta1 and gamma) at different time following ischemia. Forebrain cerebral ischemia was performed on Mongolian gerbils by 5 minutes bilateral occlusion of common carotid arteries. At the pointed time the cytoplasmic RNA was extracted from hippocampus and the expression of PKC mRNA quantified by RT PCR technique using GAPDH expression as an internal standard. Results indicate that only one gamma isoform of cPKC mRNA expression becomes significantly modified in postischemic hippocampus. A transient increase up to 145% of control within the first 3 h was followed by its decline to 60-65% at a longer recirculation period. This lowered levels returned back to control at 72 h postischemic recovery. This result indicates that gamma PKC could be particularly sensitive to ischemic insult and would react in accordance with the other early signals determining ischemic outcome.

Cell adhesion to the extracellular matrix (ECM) functions as a survival factor and disruption of cell–ECM interaction can lead to cell death. Our previous study has demonstrated ischemia-induced enhancement of activity of extracellular metalloproteinases, which might result in the alteration of adhesive contact with ECM and affect the intracellular signaling pathway. The enzyme thought to play a major role in conveying survival signals from ECM to the cell interior is focal adhesion kinase (pp125FAK). In the present study, the temporal relation between activation of extracellular metalloproteinases (MMP-2 and MMP-9), degradation of extracellular matrix protein laminin and the expression of pp125FAK after 5 min of global ischemia in gerbil hippocampus were investigated. While significant activation of both investigated metalloproteinases occurred in the course of reperfusion, only changes in MMP-9 activity were correlated with degradation of laminin. These ischemia-induced extracellular events coincide temporarily with proteolytic modification of FAK protein and diminished level of its phosphorylated form, to about 50% of the initial value. These results are indicative of an involvement of ECM–pp125FAK signaling pathway in ischemia-induced neuronal degeneration.

Relatively mild ischemic insult can lead to delayed neuronal cell death in vulnerable brain regions. We provide evidence that the protein composition of the postsynaptic densities (PSD) undergoes rapid modification after 15 min postdecapitative as well as 5 min transient global ischemia. We observed a significant increase in cPKC and nPKC protein content in the postischemic PSD. Of the calcium-regulated PKC isoforms, the alpha and beta subtypes increase in PSD over ten times above the control values whereas gamma PKC, an isoform most abundant in the native PSD structure, shows relatively smaller changes under ischemic conditions. For the first time, the PSD membrane translocation of Ca(2+)-independent isoforms delta and epsilon is shown. The yield of the PSD protein preparation from the postischemic cortex was two times higher compared with control. This correlated with an abundant increase in electron density and changes in ultrastructure of PSD isolated from postischemic cortex. Also sections from CA1 gerbils hippocampus after transient ischemia showed persistent enlargement of postsynaptic densities up to 24 h of reperfusion. This was accompanied by elevation of the PSD/cytoskeleton-associated alpha, beta PKC immunoreactivity and other changes in neuronal and glial cell morphology typical of the early postischemic degeneration. Sustained changes in PKC composition and organization of postsynaptic membranes during and after ischemia may cause persistent alteration in synaptic transmission and subsequently contribute to delayed neuronal injury.

Paralytic tremor (pt) is a neurological sex-linked recessive mutation in rabbits which is characterized by a coarse body tremor and limb paresis. Morphological studies showed that this mutation affects CNS myelination. Although the number of oligodendrocytes is not reduced, myelination is slower, irregular and defective. We have made a biochemical and molecular analysis of 4-wk-old mutant and normal rabbits. The amount of myelin in the mutant represents only approximately 25% of the normal level. Radioimmunoassay for myelin basic protein showed a reduction to approximately 40% in pt whole-brain homogenate but the difference was not significant in purified myelin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of brain homogenates followed by immunoblotting showed that all major myelin proteins are affected by the pt mutation, although to different degrees. While most of the myelin proteins are reduced to approximately 60-80% of the normal level, an important reduction to approximately 30%, was measured for the proteolipid protein (PLP). In purified myelin, the difference in PLP concentration was significant while the other specific proteins were less affected. A similar reduction in myelin-protein gene expression was detected at the mRNA level. Sex-linked transmission, low concentrations of PLP and its specific mRNA in the CNS indicate that the pt mutation primarily affects the expression of the Plp gene.