The metabolism of the amyloid precursor protein (APP) has been extensively investigated because its processing generates the amyloid-β-peptide (Aβ), which is a likely cause of Alzheimer disease. Much prior research has focused on APP processing using transgenic constructs and heterologous cell lines. Work to date in native neuronal cultures suggests that Aβ is produced in very large amounts. We sought to investigate APP metabolism and Aβ production simultaneously under more physiological conditions in vivo and in vitro using cultured rat cortical neurons and live pigs. We found in cultured neurons that both APP and Aβ are secreted rapidly and at extremely high rates into the extracellular space (2-4 molecules/neuron/s for Aβ). Little APP is degraded outside of the pathway that leads to extracellular release. Two metabolic pools of APP are identified, one that is metabolized extremely rapidly (t1/2;) = 2.2 h), and another, surface pool, composed of both synaptic and extrasynaptic elements, that turns over very slowly. Aβ release and accumulation in the extracellular medium can be accounted for stoichiometrically by the extracellular release of β-cleaved forms of the APP ectodomain. Two α-cleavages of APP occur for every β-cleavage. Consistent with the results seen in cultured neurons, an extremely high rate of Aβ production and secretion from the brain was seen in juvenile pigs. In summary, our experiments show an enormous and rapid production and extracellular release of Aβ and the soluble APP ectodomain. A small, slowly metabolized, surface pool of full-length APP is also identified.
"In an alternative pathway, APP is cleaved by ␣secretase instead of ␤-secretase, yielding sAPP␣ and APP-CTF␣, which upon ␥-secretase cleavage yields a harmless p3 product instead of A␤. ␤-and ␥-secretase processing most probably occur in intracellular compartments, since endocytosis of APP is required for the majority of the A␤ production . A recent study  showed that large amounts of A␤ were constitutively released from neurons . In addition, several studies suggest that intracellular A␤ might be more detrimental to the brain than secreted A␤   and that the intracellular A␤ concentrations are elevated in neurons at risk in AD brains . "
[Show abstract][Hide abstract] ABSTRACT: Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease. The molecular mechanism underlying this degeneration is not fully elucidated but one key player appears to be the synaptotoxic amyloid β-peptide (Aβ). The exact localization of the production of Aβ and the mechanisms whereby Aβ is released remain elusive. We have earlier shown that Aβ can be produced in crude synaptic vesicles and it has been reported that increased synaptic activity results in increased secreted but decreased intracellular Aβ levels. Therefore, we considered whether Aβ could be produced in synaptic vesicles and/or released through the same mechanisms as neurotransmitters in synaptic vesicle exocytosis. Small amounts of Aβ were found to be produced in pure synaptic vesicle preparations. We also studied the release of glutamate and Aβ from rat cortical nerve terminals (synaptosomes). We found that large amounts of Aβ were secreted from non-stimulated synaptosomes, from which glutamate was not released. On the contrary, we could not detect any differences in Aβ release between non-stimulated synaptosomes and synaptosomes stimulated with KCl or 4-aminopyridine, whereas glutamate release was readily inducible in this system. To conclude, our results indicate that the major release mechanism of Aβ from isolated nerve terminals differs from the synaptic release of glutamate and that the activity-dependent increase of secreted Aβ, reported by several groups using intact cells, is likely dependent on post-synaptic events, trafficking and/or protein synthesis mechanisms.
"Amyloid-β is generated by sequential cleavage of APP by β and γ-secretases. However, the likely predominant route of APP processing is via α-secretase [1,2], which not only precludes amyloid-β production but also generates secreted amyloid precursor protein alpha (sAPPα) [3,4]. This molecule interacts with the β-secretase, BACE1, and directly inhibits amyloid-β production . "
[Show abstract][Hide abstract] ABSTRACT: Background
Differential processing of the amyloid precursor protein liberates either amyloid-ß, a causative agent of Alzheimer’s disease, or secreted amyloid precursor protein-alpha (sAPPα), which promotes neuroprotection, neurotrophism, neurogenesis and synaptic plasticity. The underlying molecular mechanisms recruited by sAPPα that underpin these considerable cellular effects are not well elucidated. As these effects are enduring, we hypothesised that regulation of gene expression may be of importance and examined temporally specific gene networks and pathways induced by sAPPα in rat hippocampal organotypic slice cultures. Slices were exposed to 1 nM sAPPα or phosphate buffered saline for 15 min, 2 h or 24 h and sAPPα-associated gene expression profiles were produced for each time-point using Affymetrix Rat Gene 1.0 ST arrays (moderated t-test using Limma: p < 0.05, and fold change ± 1.15).
Treatment of organotypic hippocampal slice cultures with 1 nM sAPPα induced temporally distinct gene expression profiles, including mRNA and microRNA associated with Alzheimer’s disease. Having demonstrated that treatment with human recombinant sAPPα was protective against N-methyl d-aspartate-induced toxicity, we next explored the sAPPα-induced gene expression profiles. Ingenuity Pathway Analysis predicted that short-term exposure to sAPPα elicited a multi-level transcriptional response, including upregulation of immediate early gene transcription factors (AP-1, Egr1), modulation of the chromatin environment, and apparent activation of the constitutive transcription factors CREB and NF-κB. Importantly, dynamic regulation of NF-κB appears to be integral to the transcriptional response across all time-points. In contrast, medium and long exposure to sAPPα resulted in an overall downregulation of gene expression. While these results suggest commonality between sAPPα and our previously reported analysis of plasticity-related gene expression, we found little crossover between these datasets. The gene networks formed following medium and long exposure to sAPPα were associated with inflammatory response, apoptosis, neurogenesis and cell survival; functions likely to be the basis of the neuroprotective effects of sAPPα.
Our results demonstrate that sAPPα rapidly and persistently regulates gene expression in rat hippocampus. This regulation is multi-level, temporally specific and is likely to underpin the neuroprotective effects of sAPPα.
[Show abstract][Hide abstract] ABSTRACT: The amyloid cascade hypothesis, which posits that the deposition of the amyloid-β peptide in the brain is a central event in Alzheimer's disease pathology, has dominated research for the past twenty years. Several therapeutics that were purported to reduce amyloid-β production or aggregation have failed in Phase III clinical testing, and many others are in various stages of development. Therefore, it is timely to review the science underpinning the amyloid cascade hypothesis, consider what type of clinical trials will constitute a valid test of this hypothesis and explore whether amyloid-β-directed therapeutics will provide the medicines that are urgently needed by society for treating this devastating disease.
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