Article

Large quantities of Abeta peptide are constitutively released during amyloid precursor protein metabolism in vivo and in vitro.

Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 03/2011; 286(18):15989-97. DOI: 10.1074/jbc.M110.191262
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
74 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The 99-residue transmembrane C-terminal domain (C99, also known as β-CTF) of the amyloid precursor protein (APP) is the product of the β-secretase cleavage of the full-length APP and is the substrate for γ-secretase cleavage. The latter cleavage releases the amyloid-β polypeptides that are closely associated with Alzheimer's disease. C99 is thought to form homodimers; however, the free energy in favor of dimerization has not previously been quantitated. It was also recently documented that cholesterol forms a 1:1 complex with monomeric C99 in bicelles. Here, the affinities for both homodimerization and cholesterol binding to C99 were measured in bilayered lipid vesicles using both electron paramagnetic resonance (EPR) and Förster resonance energy transfer (FRET) methods. Homodimerization and cholesterol binding were seen to be competitive processes that center on the transmembrane G700XXXG704XXXG708 glycine-zipper motif and adjacent Gly709. On one hand, the observed Kd for cholesterol binding (Kd = 2.7 ± 0.3 mol %) is on the low end of the physiological cholesterol concentration range in mammalian cell membranes. On the other hand, the observed Kd for homodimerization (Kd = 0.47 ± 0.15 mol %) likely exceeds the physiological concentration range for C99. These results suggest that the 1:1 cholesterol/C99 complex will be more highly populated than C99 homodimers under most physiological conditions. These observations are of relevance for understanding the γ-secretase cleavage of C99.
    Biochemistry 07/2013; 52(30). DOI:10.1021/bi400735x · 3.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Amyloid-β peptide (Aβ), the main component of Alzheimer's disease (AD) senile plaques, has been found to accumulate within the lysosomal compartment of AD neurons. We have previously shown that in differentiated SH-SY5Y neuroblastoma cells cultured under normal conditions, the majority of Aβ is localized extralysosomally, while oxidative stress significantly increases intalysosomal Aβ content through activation of macroautophagy. It is, however, not clear which cellular compartments contain extralysosomal Aβ in intact SH-SY5Y cells, and how oxidative stress influences the distribution of extralysosomal Aβ. Using confocal laser scanning microscopy and immunoelectron microscopy, we showed that in differentiated neuroblastoma cells cultured under normal conditions Aβ (Aβ40, Aβ42, and Aβ oligomers) is colocalized with both membrane-bound organelles (endoplasmic reticulum, Golgi complexes, multivesicular bodies/late endosomes, lysosomes, exocytotic vesicles and mitochondria) and non-membrane-bound cytosolic structures. Neuroblastoma cells stably transfected with AβPP Swedish KM670/671NL double mutation showed enlarged amount of Aβ colocalized with membrane compartments. Suppression of exocytosis by 5 nM tetanus toxin resulted in a significant increase of the amount of cytosolic Aβ as well as Aβ colocalized with exocytotic vesicles, endoplasmic reticulum, Golgi complexes, and lysosomes. Hyperoxia increased Aβ localization in the endoplasmic reticulum, Golgi apparatus, mitochondria, and lysosomes, but not in the secretory vesicles. These results indicate that in SH-SY5Y neuroblastoma cells intracellular Aβ is not preferentially localized to any particular organelle and, to a large extent, is secreted from the cells. Challenging cells to hyperoxia, exocytosis inhibition, or Aβ overproduction increased intracellular Aβ levels but did not dramatically changed its localization pattern.
    Journal of Alzheimer's disease: JAD 07/2013; 37(4). DOI:10.3233/JAD-122455 · 3.61 Impact Factor
  • Source
    [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.
    Neuroscience Letters 03/2014; 566. DOI:10.1016/j.neulet.2014.02.050 · 2.06 Impact Factor