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Genetic abnormalities in amyloid precursor protein (APP) are associated with Down's syndrome and familial Alzheimer's disease where hallmark plaques contain A beta peptides derived from APP. Both APP and its derivatives are implicated in neurodegenerative processes and may play important physiological and pathophysiological roles in synaptic function. Here, we show that young APP23 transgenic mice overexpressing human APP with the Swedish double mutation display altered novelty seeking behavior before the age of plaque onset. Using short interfering RNA (siRNA) targeted against APP, we investigate the direct contribution of APP and its derivatives to this behavioral deficit. After validating siRNAs targeting human APP in vitro, siRNAs were infused directly into the brain of APP23 mice for 2 weeks. Behavioral analysis shows that infusion of siRNA targeted against APP completely reverses increased exploratory activity in APP23 mice. Collectively, these data suggest that excessive APP and/or its derivatives, causes a hyperactive phenotype in APP23 mice when placed in a novel environment, which is fully reversible and not linked to plaque deposits.

In ischemic stroke, cytosolic death pathways are activated in injured neurons destined to die. Neuronal injury is modulated by cell surface receptors, among which the tumor necrosis factor receptor family obtained particular interest. Cytokine response modifier A (CrmA) is a cowpox virus-derived caspase inhibitor, which interferes with the so-called death-inducing signaling complex, thereby blocking receptor-mediated apoptosis. To elucidate CrmA's therapeutic potential in ischemic stroke, we characterized a transgenic mouse line expressing CrmA under a Thy1 promoter, which we subjected to intraluminal middle cerebral artery (MCA) occlusion. Using in situ hybridization histochemistry and Western blots, we show that the crmA gene integrated into chromosome 8 of the mouse genome, CrmA being expressed in the cerebral cortex and striatum. Although robustly expressed, transgenic CrmA did not influence ischemic injury, both when relatively long-lasting (90 min) and mild (30 min) MCA occlusions were imposed. As such, neither infarct volume, brain swelling or neurological deficits following 90-min ischemia, nor disseminated neuronal injury or caspase-3 activation following 30-min ischemia were influenced by CrmA. Our data argue against a therapeutic effect of CrmA in ischemic stroke.

Spatial learning and memory deficits in the APP23 transgenic mice have mainly been studied using the Morris water maze (MWM). However learning in the MWM relies on swimming abilities and may be confounded by the stressful nature of this test. We have therefore assessed spatial learning and memory in 12-month-old APP23 using a dry-land maze test developed by Barnes. Mice were given daily learning trials for a total of 41 successive days. After a 12-day interval the mice were re-tested for 4 additional days in order to examine the spatial memory retention. Immediately following this phase, reversal learning was examined for 13 additional days by moving the escape tunnel to the opposite position. During the initial learning phase, APP23 mice showed a significantly longer latency to find the escape tunnel as well as an increased number of errors compared to non-transgenic littermates. These deficits appeared to be due to a delay in switching from a "no strategy" to a spatial strategy. Indeed, this same delay in the use of spatial strategy was observed in the reversal phase of the study. Our results suggest that impairments in APP23 mice in learning and memory maze tests may be due to a specific deficit in the use of spatial strategy.

Background: Previous experimental work using in situ zymography has shown very early increased plasminogen activation in ischemic regions after 3 h of ischemia with and without reperfusion. The objective of the present study was to evaluate the time course and extent of plasminogen activation in long-term permanent focal cerebral ischemia., Material and Methods: The middle cerebral artery in male Fisher rats was irreversibly occluded by electrocoagulation. Duration of ischemia was 48, 72, and 168 h. Occlusion was controlled in vivo by MRI at day 2. Plasminogen activation was detected by in situ zymography of 10 microm cryosections with an overlay containing plasminogen and the plasmin substrate caseine. Areas of plasminogen activation were compared to structural lesions (immunohistochemical loss of microtubule-associated protein 2; MAP 2)., Results: Compared to controls, increased plasminogen activation was observed in the basal ganglia and the cortex of the ischemic hemisphere after 48, 72, and 168 h (affected area of basal ganglia: 44.5+/-21.9, 70.1+/-2.3 and 66.6+/-2.8%, respectively; affected area of cortex: 63.4+/-9.8, 67.7+/-0.7 and 64.0+/-3.7%, respectively). The duration of ischemia had no significant influence on the extent of plasminogen activation. Areas of increased plasminogen activation significantly overlapped with and exceeded areas of MAP 2 loss (P<0.005)., Discussion: Permanent focal cerebral ischemia leads to increased plasminogen activation in ischemic regions. This plasminogen activation remains elevated at persistent levels over days. It may contribute to extracellular matrix (ECM) disruption, secondary hemorrhage, and brain edema in subacute stages of ischemic stroke.

Two patterns of transforming growth factor-beta1 (TGF-beta1) expression were identified in brains of normotensive rats following permanent occlusion of the middle cerebral artery (MCAO). First, a relative increase of TGF-beta1 mRNA by 37% was found at 12 h after MCAO in the ipsilateral cingulate cortex as compared to the homotopic contralateral region. The cingulate cortex is located distant from the ischemic territory. Treatment with the glutamate receptor antagonists MK-801 and NBQX did not reduce this expression (34% and 26% increase, respectively). Therefore, peri-infarct depolarization waves were probably not responsible for induction. Secondly, an increase of TGF-beta1 mRNA by 116% was found at 7 days after MCAO within infarcted tissue. This expression was not reduced by the glutamate receptor antagonists MK-801 (increase 140%) and NBQX (increase 137%), either. TGF-beta1 mRNA expression in the cingulate cortex at 12 h after MCAO is possibly mediated by neurons and astroglia and may support cell survival. Expression in the infarcted tissue at 7 days after MCAO is most likely related to the invasion of monocytes and may be involved in the downregulation of inflammatory events, in neoangiogenesis, and in formation of a glial scar around the infarct., (Copyright 1999 Elsevier Science B.V.)

We investigated the effects of 30-min heat shock on survival, DNA degradation, and nuclear morphology of primary rat cortical and hippocampal neurones. In cell cultures which were grown for 8 days in vitro (DIV), only a small portion of neurones showed apoptotic morphology after heat shock of 45 degrees C and typical DNA laddering was not detectable, despite the fact that nearly 50% of the neurones died within 24 h. The majority of the neurones presumably died by necrosis, as indicated by random DNA degradation. In neuronal cultures grown for 15 DIV, heat shock, however, resulted in DNA laddering, occurrence of apoptotic bodies and DNA strand breaks, typical of apoptosis. In these cultures, about 50% of the neurones showed apoptotic morphology following exposure to 45 degrees C in TUNEL and acridine orange staining, whereas glia were not affected in vitality. In addition we were interested whether the highly inducible member of the heat-shock protein family, HSP72, would be induced in apoptotic cells. Double staining for HSP72 and TUNEL revealed concomitant HSP72 induction and occurrence of DNA degradation only in very few neurones in 15-DIV cultures, which were growing adjacent to astrocytes. A clear association of the degenerative process and HSP72 expression, therefore, could not be established. These results demonstrate that environmental stress, such as heat shock, can induce apoptotic death in aged primary cultured neurones. The differentiation state and/or the presence of glial cell elements in the cultures appears to be an important factor for the occurrence of apoptotic features in cultured neurones.

We investigated the temporo-spatial expression of astrocyte glial fibrillary acidic protein (gfap) and sulfated glycoprotein 2 (sgp-2) mRNAs in comparison to 70-kDa heat shock protein (hsp70) mRNA by in situ hybridisation in rats subjected to permanent occlusion of the middle cerebral artery (MCA). Gfap mRNA started to increase in the cingulate cortex of the lesioned hemisphere 6 h after MCA occlusion and gradually spread over the lateral part of the ipsilateral cortex and the striatum from 12 h to 3 days, peaking at 3 days after MCA occlusion. Gfap mRNA also increased in the contralateral cingulate cortex and corpus callosum at 12 and 24 h. Hsp70 mRNA increased markedly in the ipsilateral cortex adjacent to the ischemic lesion, and slightly within the lesion area from 3 to 24 h and disappeared after 3 days. By 7 days, gfap and sgp-2 mRNAs were increased markedly in the peri-infarct area, and in the ipsilateral thalamus parallel with the delayed neuronal damage, whereas the widespread increase of gfap mRNA in the ipsilateral hemisphere declined. Post-occlusion treatment with the glutamate receptor antagonists MK-801 and NBQX slightly attenuate the induction of gfap but did not qualitatively affect the topical expression pattern. Within the cingulate cortex MK-801 treatment resulted in a significant decrease of the signal intensity at all survival times, reflecting most likely an attenuation of lesion-induced spreading depression like depolarization waves by MK-801. The area of hsp70 expression was reduced by both MK-801 and NBQX, most likely reflecting the decrease of the lesion area by both treatment regimens. Our study thus revealed an early and widespread increase of gfap mRNA in the non-ischemic area including the contralateral hemisphere starting between 3 and 6 h, and a delayed circumscribed expression in the peri-infarct border zone after 1 week. Comparison with the expression of hsp70 mRNA suggests that the absence of an early gfap mRNA induction in the peri-lesion zone reflects an impairment of astrocytic function which may be of importance for infarct growth during the early evolution of the pathological process.