Teresa Zalewska

Effect of brain ischemia on protein kinase C.

Abstract: We examined the influence of brain ischemia on the activity and subcellular distribution of protein kinase C (PKC). Two different models of ischemic brain injury were used: postdecapitative ischemia in rat forebrain and transient (6‐min) cerebral ischemia in gerbil hippocampus. In the rat forebrain model, at 5 and 15 min postdecapitation there was a steady decrease of total PKC activity to 60% of control values. This decrease occurred without changes in the proportion of the particulate to the soluble enzyme pools. Isolated rat brain membranes also exhibited a concomitant decrease of [3H]phorbol 12,13‐dibutyrate ([3H]PDBu) binding with an apparent increase of the ligand affinity to the postischemic membranes. On the other hand, the ischemic gerbil hippocampus model displayed a 40% decrease of total PKC activity, which was accompanied by a relative increase of PKC activity in its membrane‐bound form. This resulted in an increase in the membrane/total activity ratio, indicating a possible enzyme translocation from cytosol to the membranes after ischemia. Moreover, after 1 day of recovery, a statistically significant enhancement of membrane‐bound PKC activity resulted in a further increase of its relative activity up to 162% of control values. In vitro experiments using a synaptoneurosomal particulate fraction were performed to clarify the mechanism of the rapid PKC inhibition observed in cerebral tissue after ischemia. These experiments showed a progressive, Ca2+‐dependent, antiprotease‐insensitive down‐regulation of PKC during incubation. This down‐regulation was significantly enhanced by prior phorbol (PDBu) treatment. It can be postulated that in the ischemic gerbil hippocampus model as well as during phorbol stimulation of the synaptoneurosomal fraction in vitro, the common phenomenon was an initial PKC activation (translocation) followed by a subsequent enhancement of Ca2+‐dependent enzyme inhibition. Experimental data suggest that the inhibition is localized in the membrane compartment.

Transient forebrain ischemia modulates signal transduction from extracellular matrix in gerbil hippocampus.

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 (pp125(FAK)). 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 pp125(FAK) 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-pp125(FAK) signaling pathway in ischemia-induced neuronal degeneration.

The potential role of metalloproteinases in neurogenesis in the gerbil hippocampus following global forebrain ischemia.

Background Matrix metalloproteinases (MMPs) have recently been considered to be involved in the neurogenic response of adult neural stem/progenitor cells. However, there is a lack of information showing direct association between the activation of MMPs and the development of neuronal progenitor cells involving proliferation and/or further differentiation in vulnerable (Cornus Ammoni-CA1) and resistant (dentate gyrus-DG) to ischemic injury areas of the brain hippocampus. Principal Findings We showed that dynamics of MMPs activation in the dentate gyrus correlated closely with the rate of proliferation and differentiation of progenitor cells into mature neurons. In contrast, in the damaged CA1 pyramidal cells layer, despite the fact that some proliferating cells exhibited antigen specific characteristic of newborn neuronal cells, these did not attain maturity. This coincides with the low, near control-level, activity of MMPs. The above results are supported by our in vitro study showing that MMP inhibitors interfered with both the proliferation and differentiation of the human neural stem cell line derived from umbilical cord blood (HUCB-NSCs) toward the neuronal lineage. Conclusion Taken together, the spatial and temporal profiles of MMPs activity suggest that these proteinases could be an important component in neurogenesis-associated processes in post-ischemic brain hippocampus.

Functional alterations in endothelial NO, PGI2 and EDHF pathways in aorta in ApoE/LDLR−/− mice

Adequate endothelial production of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI₂) is critical to the maintenance of vascular homeostasis. However, it is not clear whether alterations in each of these vasodilatory pathways contribute to the impaired endothelial function in murine atherosclerosis. In the present study, we analyze the alterations in NO-, EDHF- and PGI₂-dependent endothelial function in the thoracic aorta in relation to the development of atherosclerotic plaques in apoE/LDLR⁻/⁻ mice. We found that in the aorta of 2-month-old apoE/LDLR⁻/⁻ mice there was no lipid deposition, subendothelial macrophage accumulation; and matrix metalloproteinase (MMP) activity was low, consistent with the absence of atherosclerotic plaques. Interestingly, at this stage the endothelium was already activated and hypertrophic as evidenced by electron microscopy, while acetylcholine-induced NO-dependent relaxation in the thoracic aorta was impaired, with concomitant upregulation of cyclooxygenase-2 (COX-2)/PGI₂ and EDHF (epoxyeicosatrienoic acids, EETs) pathways. In the aorta of 3-6-month-old apoE/LDLR⁻/⁻ mice, lipid deposition, macrophage accumulation and MMP activity in the intima were gradually increased, while impairment of NO-dependent function and compensatory upregulation of COX-2/PGI₂ and EDHF pathways were more accentuated. These results suggest that impairment of NO-dependent relaxation precedes the development of atherosclerosis in the aorta and early upregulation of COX-2/PGI₂ and EDHF pathways may compensate for the loss of the biological activity of NO.

Decrease of PKC precedes other cellular signs of calpain activation in area CA1 of the hippocampus after transient cerebral ischemia.

One of the specific features of severe brain injury is an activation of calcium-dependent proteolysis by calpains. We have observed a significant increase of activity as early as 3 h after the insult in a well defined model of delayed ischemic neuronal death in gerbil hippocampus. At 24 h, the enzymatic activity transiently normalized, then increased again, following the place and time of selective cellular death in the CA1 region of hippocampus. The enhanced postischemic proteolysis resulted in concomitant cleavage of calpain-specific endogenous substrates like protein kinase C (PKC), fodrin and microtubule-associated protein-2 (MAP2). These effects were also time-dependent and restricted to the vulnerable, CA1 pyramidal neurons-containing the dorsal part (DP) of the hippocampus. We have also characterized the postischemic changes of six different isoforms of PKC. The vulnerable dorsal part of the hippocampus, but not its relative resistant abdominal part (AbP), exhibited a loss of PKCalpha, beta, gamma, and delta isoforms as early as 3 h after ischemic insult. However, at this time, solely in the soluble fraction of homogenate. Later (72 h), a further loss of the enzyme proteins, comprised the particulate fraction as well and resulted in an about 50% decrease of total PKCs in the vulnerable DP region. In the case of PKCalpha, the immunostaining pattern showed, in addition to the disappearance of the enzyme from the injured area, an extensive translocation into nuclei of the survived, ischemia-resistant neurones. The early decreases of PKC isoforms in the cytosol paralleled the transient calpain activation at 3h postischemia but substantially preceded the proteolysis of any other classical calpain substrates, such as fodrin and MAP2, being evidenced not earlier than 48-72 h after the insult and restricted also to the vulnerable dorsal part. In conclusion, our results of the time-dependent effects of transient global cerebral ischemia on the calpain activity, levels and localization of its several substrates suggest, that calpain-mediated proteolysis is specifically involved in the early (induction) as well as in the late (execution) phases of delayed ischemic neuronal death in the CA1 hippocampus.

Ischemia-induced modifications of protein components of rat brain postsynaptic densities

Considering that postsynaptic densities (PSD) are a functionally active zone involved in excitatory synaptic transmission we evaluated the influence of global, postdecapitative cerebral ischemia of 15 min duration on characteristic protein constituents of PSD in rats. Ischemia induced changes in the assembly and function of calcium, calmodulin-dependent kinase II (CaMKII), calpains and a novel, 85 kDa/RING3 kinase but to different extents. CaMKII is translocated toward the PSD very rapidly and extensively after the first seconds of ischemia. Concomitantly, the total phosphorylating potency of this kinase with endogenous, as well as exogenous, substrates was elevated but to a lower extent than suggested by the increased protein content. Of the two brain-specific isoforms of calpain (mu and m), only recently recognized in PSD, the proteolytically activated, 76 kDa subunit of mu-calpain was significantly down-regulated after 15 min of brain ischemia. However, this effect is coupled with the decline of fodrin, the only calpain substrate that has been demonstrated to be a calpain target in vivo. Together, these findings may suggest that calpains, primarily activated by calcium in ischemic PSD, are subsequently degraded. A new observation is the relatively high phosphorylating activity of a novel, 85 kDa/RING3 kinase in the PSD which independently of other kinase systems, was greatly enhanced after ischemia. These data provide evidence that the signal transduction processes could be rapidly altered by short-term (15 min) brain ischemia due to changes in the assembly and function of PSD connected proteins.

Transient forebrain ischemia effects interaction of Src, FAK, and PYK2 with the NR2B subunit of N-methyl-D-aspartate receptor in gerbil hippocampus.

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.

Dual response of calpain to rat brain postdecapitative ischemia.

Calpains, Ca(2+)-dependent neutral proteinases (microM and mM Ca(2+)-sensitive), and their endogenous inhibitor calpastatin were examined in rat brain. Specific activity of m-calpain exceeded almost 10 times that of mu-calpain, and the both isoforms of calpain together with calpastatin were mainly located in the soluble fraction of homogenate. Acute postdecapitative ischemia of 15 min duration resulted in a gradual, time-dependent decrease of total mu-calpain activity (to 60% of control values) and in the moderate elevation of calpastatin activity (by 28%). The decrease of total mu-calpain activity coincided with its remarkable increase (above 300% of control values) in particulate fraction. In the case of m-calpain, the only observed effect of ischemia was its redistribution and, as a consequence, the elevation of activity in particulate fraction. The accumulation of breakdown products, resulting from calpain-catalyzed proteolysis of fodrin (as revealed by Western blotting) indicated activation of calpain under ischemia. The findings suggest that this rapid activation involves partial enzyme translocation toward membranes, and is followed (at least in acute phase) by mu-calpain downregulation and increased calpastatin activity.

Metabolic disturbances of synaptosomes isolated from ischemic gerbil brain

The effects of cerebral ischemia, induced for 10 min by bilateral common carotid ligation in the Mongolian gerbil, on the brain and synaptosomal content of phospholipids and free fatty acids were measured. Moreover, the incorporation of arachidonic acid and oleoyl-CoA into phospholipids, as well as the respiration and the accumulation of45Ca, norepinephrine, dopamine, choline, glutamate, and γ-aminobutyrate in the ischemic brain synaptosomal fraction were studied. Analyses of lipids showed a drop in phospholipids content with concomitant increase of lysocompounds and free fatty acids in ischemic cerebral cortex. Disturbances in lipid metabolism including rapid phospholipids hydrolysis and changes in the incorporation of arachidonic acid into inositol and choline phosphoglycerides were also shown in the synaptosomal fraction of ischemic brain. The uptake of neurotransmitter substances, expressed as a percent of control value, was reduced 21% for norepinephrine, 40% for dopamine, 20% for choline, 24% for glutamate and 13% for γ-aminobutyrate in ischemic synaptosomes. There was no significant effect of ischemia on synaptosomal respiration and45Ca uptake in both control and high potassium media. the inhibition of neurotransmitter uptake in ischemic brain synaptosomes may be caused by the disturbance of fatty acid metabolism.

Neonatal cerebral hypoxia-ischemia: involvement of FAK-dependent pathway

Focal adhesion kinase (FAK) is a non‐receptor tyrosine kinase thought to play a major role in transducing extracellular matrix (ECM)‐derived survival signals into cells. Thus, modulation of FAK activity may affect the linkage between ECM and signaling cascade to which it is connected and may participate in a variety of pathological settings. In the present study, we investigated the effect of neonatal cerebral hypoxia‐ischemia (HI) on levels and tyrosine phosphorylation of focal adhesion kinase and the interaction of this enzyme with Src protein tyrosine kinase and adapter protein p130Cas, involved in FAK‐mediated signaling pathway. The total amount of focal adhesion kinase as well as its phosphorylated form declined substantially to about 50% of the control between 24 and 48 h after the insult. Concomitantly a decreased association of FAK with its investigated molecular partners, Src kinase and p130Cas protein has been observed. This early response to brain hypoxia‐ischemia was attenuated during prolonged recovery with almost complete return to control values at 7 days. These data are indicative of an involvement of FAK‐dependent signaling pathway in the evolution of HI‐induced neuronal degeneration.