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United States - English. Importantly, this effect was visible only in wt slices Figure 3b , upper panel and not in slices from APP-KO animals Figure 3b , lower panel. Cell death was visualized microscopically by PI staining. Whole slices are shown in the upper panel of each genotype; magnification: A representative area in the CA1 region of the hippocampus as depicted in the white square is shown in the lower panel of each genotype; magnification: To further substantiate our findings, we subsequently used hippocampal neurons derived from APP-KO and wt mice.
Again, these results could be verified by quantification of western blot data Figure 4e.
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Akt kinase activity was measured in vitro as outlined in Materials and Methods. Values were normalized to serum-treated controls dashed line. GAPDH was used as a loading control. Both graphs show the respective blot quantification normalized to serum-treated controls dashed line. Fluorescence signals measured in an on-cell-Western with unpermeabilized cells were normalized to background staining. Blot quantification values were normalized to serum-treated controls dashed line. We also applied an on-cell-Western approach to quantify their cell surface expression after transfection in comparison with wt control cells.
As shown in Figure 6c , cell surface expression levels of both constructs were comparable with those detected in wt cells. Recently, Jimenez et al. Future studies will have to address this important topic of APP biology in more detail. It is well established that APP and APLPs can form homo- and heterodimers, arguing for a functional connection between these molecules.
This would suggest a role for APP in tyrosine kinase-mediated signal transduction. They also provide novel mechanistic insights into the physiologic function of APP in limiting neuron damage and death in response to neurotoxic stress conditions, as well as the loss of this function during brain aging. APP C-terminal domain. All other biochemicals and chemicals were provided in analytical grade purity from Roth Karlsruhe, Germany or Sigma-Aldrich. Organotypic hippocampal slice cultures were prepared from P4 to 6 transgenic and non-transgenic littermate mouse pups as described in Stoppini et al.
Broken or otherwise lesioned slices were discarded. Culture medium was changed every 2—3 days. For cell death experiments, the culture medium was replaced with glucose-free neurobasal A medium without HS full medium as control after 5—6 days in vitro.
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PI stain 0. The asparagine residue at position of APP glycosylation site was replaced with serine by site-directed mutagenesis. The yeast was cultured in buffered glycerol complex medium with peptone. The proteins were isolated with Ni-sepharose beads or Ni-affinity chromatography HPLC from the medium and eluted with imidazole. Their biologic, neuroprotective activity was verified in several control experiments with different cell lines under proteasomal stress or serum deprivation data not shown. Cell death was quantified based on plasma membrane permeabilization. Following collection, cells were stained with 0.
Organotypic hippocampal slices were incubated with 0. The assay quantitates ATP levels, an indicator of metabolically active cells, photometrically with a fluorescence plate reader. Additionally, we applied the live-dead cell staining kit II from PromoKine Heidelberg, Germany according to the manual. Briefly, endogenous levels of pAkt were immunoprecipitated from whole-cell extracts with immobilized pAkt Ser mAb bead conjugate overnight.
On-cell-Western assays were performed to evaluate cell surface expression of APP. The experiment was performed by adding primary antibody 22C11, 1: All experiments were repeated at least three times yielding similar results. Image adjustments such as changes of contrast and brightness were applied equally across the entire image. Zheng H , Koo EH. Biology and pathophysiology of the amyloid precursor protein.
Mol Neurodegener ; 6: Functions of the APP gene family in the nervous system: Exp Brain Res ; Muller UC , Zheng H. Physiological functions of APP family proteins. Cold Spring Harbor Perspect Med ; 2: Selkoe DJ. Alzheimer's disease. Cold Spring Harbor Perspect Biol ; 3: Endres K , Fahrenholz F. Regulation of alpha-secretase ADAM10 expression and activity. The physiological functions of the beta-amyloid precursor protein APP. Fahrenholz F.
Alpha-secretase as a therapeutic target. Curr Alzheimer Res ; 4: Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members.
EMBO J ; Homo- and heterodimerization of APP family members promotes intercellular adhesion. Expression of amyloid precursor protein, tau and presenilin RNAs in rat hippocampus following deafferentation lesions. Brain Res ; Experimental brain injury induces expression of amyloid precursor protein, which may be related to neuronal loss in the hippocampus.
J Neurotrauma ; Upregulation of amyloid precursor protein messenger RNA in response to traumatic brain injury: Exp Neurol ; Amyloid precursor protein promotes post-developmental neurite arborization in the Drosophila brain. Amyloid precursor proteins are protective in Drosophila models of progressive neurodegeneration. Neurobiol Dis ; The neuroprotective domains of the amyloid precursor protein, in traumatic brain injury, are located in the two growth factor domains. Amyloid precursor proteins protect neurons of transgenic mice against acute and chronic excitotoxic injuries in vivo.
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Neuroscience ; Neurotrophic and neuroprotective effects of hAPP in transgenic mice. Ann NY Acad Sci ; Secreted forms of beta-amyloid precursor protein protect against ischemic brain injury. J Neurochem ; Hypersensitivity to seizures in beta-amyloid precursor protein deficient mice. Cell Death Differ ; 5: Furukawa K , Mattson MP. Secreted amyloid precursor protein alpha selectively suppresses N -methyl- D -aspartate currents in hippocampal neurons: Goodman Y , Mattson MP. Secreted forms of beta-amyloid precursor protein protect hippocampal neurons against amyloid beta-peptide-induced oxidative injury.
Neuroprotective secreted amyloid precursor protein acts by disrupting amyloid precursor protein dimers. J Biol Chem ; Suppression of cyclin-dependent kinase 5 activation by amyloid precursor protein: J Neurosci ; Regulation of gene expression by the amyloid precursor protein: Cell Death Differ ; The amyloid precursor protein protects PC12 cells against endoplasmic reticulum stress-induced apoptosis. Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the beta-amyloid precursor protein.
Neuron ; Schubert D , Behl C. The expression of amyloid beta protein precursor protects nerve cells from beta-amyloid and glutamate toxicity and alters their interaction with the extracellular matrix.