The beta-site APP-cleaving enzyme (BACE1) is a prerequisite for the generation of beta-amyloid peptides, which give rise to cerebrovascular and parenchymal beta-amyloid deposits in the brain of Alzheimer's disease patients. BACE1 is neuronally expressed in the brains of humans and experimental animals such as mice and rats. In addition, we have recently shown that BACE1 protein is expressed by reactive astrocytes in close proximity to beta-amyloid plaques in the brains of aged transgenic Tg2576 mice that overexpress human amyloid precursor protein carrying the double mutation K670N-M671L. To address the question whether astrocytic BACE1 expression is an event specifically triggered by beta-amyloid plaques or whether glial cell activation by other mechanisms also induces BACE1 expression, we used six different experimental strategies to activate brain glial cells acutely or chronically. Brain sections were processed for the expression of BACE1 and glial markers by double immunofluorescence labeling and evaluated by confocal laser scanning microscopy. There was no detectable expression of BACE1 protein by activated microglial cells of the ameboid or ramified phenotype in any of the lesion paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1-immunoreactive astrocytes in proximity to beta-amyloid plaques in the brains of aged Tg2576 mice and Alzheimer's disease patients.
The two proteases -secretase and ␥-secretase generate the amyloid  peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of -secretase cleavage instead of the -secretase enzyme itself. -Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited -secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit -secretase. Surprisingly, bepridil and amiodarone also modulated ␥-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target -and ␥-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.
Experimental transplantation of human umbilical cord blood (hUCB) mononuclear cells (MNCs) in rodent stroke models revealed the therapeutic potential of these cells. However, effective cells within the heterogeneous MNC population and their modes of action are still under discussion. MNCs and MNC fractions enriched (CD34(+)) or depleted (CD34(-)) for CD34-expressing stem/progenitor cells were isolated from hUCB. Cells were transplanted intravenously following middle cerebral artery occlusion in spontaneously hypertensive rats and directly or indirectly cocultivated with hippocampal slices previously subjected to oxygen and glucose deprivation. Application of saline solution or a human T-cell line served as controls. In vivo, MNCs, CD34(+) and CD34(-) cells reduced neurofunctional deficits and diminished lesion volume as determined by magnetic resonance imaging. MNCs were superior to other fractions. However, human cells could not be identified in brain tissue 29 days after stroke induction. Following direct application on postischemic hippocampal slices, MNCs reduced neural damage throughout a 3-day observation period. CD34(+) cells provided transient protection for 2 days. The CD34(-) fraction, in contrast to in vivo results, failed to reduce neural damage. Direct cocultivation of MNCs was superior to indirect cocultivation of equal cell numbers. Indirect application of up to 10-fold MNC concentrations enhanced neuroprotection to a level comparable to direct cocultivation. After direct application, MNCs migrated into the slices. Flow cytometric analysis of migrated cells revealed that the CD34(+) cells within MNCs were preferably attracted by damaged hippocampal tissue. Our study suggests that MNCs provide the most prominent neuroprotective effect, with CD34(+) cells seeming to be particularly involved in the protective action of MNCs. CD34(+) cells preferentially home to neural tissue in vitro, but are not superior concerning the overall effect, implying that there is another, still undiscovered, protective cell population. Furthermore, MNCs did not survive in the ischemic brain for longer periods without immunosuppression.
Whilst it is generally accepted that the activation of protein kinase C (PKC) increases amyloid precursor protein (APP) secretion in vitro, the role of PKC in the regulation of APP processing and beta-amyloid generation in vivo is still not well understood. In order to address this question, we established the animal model of neocortical microencephalopathy in guinea pigs caused by in utero treatment with methylazoxymethanol acetate, a DNA-methylating substance that eliminates proliferating cells of neuroepithelial origin. The induction of this neocortical malformation is accompanied by constitutive overactivation of PKC in the neocortex of the offspring. In the cortical and hippocampal tissues of juvenile microencephalic guinea pigs (postnatal day 30), we observed significant increases in basal (by 58% and 74%, respectively,) and phorbol ester-stimulated PKC enzyme activity (by 47% and 71%) as compared to age-matched control animals. In the same cortical/hippocampal preparations of methylazoxymethanol-treated animals, there was increased alpha-secretion of APP by 35% and 30% as measured by Western blot analysis using the antibody 6E10, whilst total APP secretion as well as APP mRNA expression remained unaltered. This upregulation of APP alpha-secretion was limited to brain areas that displayed elevated PKC activity. However, constitutive overactivation of neocortical PKC did not affect the generation of beta-amyloid peptides 1-40 or 1-42 as measured by ELISA, suggesting that only the alpha-secretase pathway of APP processing is affected by chronic PKC overactivation in vivo.
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