RESUMO
The accumulation of amyloid-beta (Abeta) peptides in senile plaques is one of the hallmarks of Alzheimer's disease (AD) progression. The endocytic pathway has been proposed as a major subcellular site for Abeta generation while the compartments in which Abeta-degrading proteases interact with Abeta are still elusive. It was suggested that extracellular Abeta degradation may take place by plasma-membrane associated proteases or by extracellular proteases, among which insulin-degrading enzyme (IDE) is the most relevant. However, the mechanisms of IDE secretion are poorly understood. In the present study we used N2a cells to explore if IDE is indeed released through exosomes and the effect of exosomes release on extracellular levels of Abeta. We demonstrated that proteolytically-active plasma membrane associated-IDE is routed in living N2a cells to multivesicular bodies and subsequently, a major fraction is sorted to exosomes. We described that extracellular IDE levels decrease if the generation of multivesicular bodies is interfered and may be positively modulated by exosomes release under stress-induced conditions. Our results reinforce the relevance of functional IDE in the catabolism of extracellular Abeta.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/enzimologia , Exossomos/metabolismo , Insulisina/metabolismo , Via Secretória/fisiologia , Vesículas Transportadoras/enzimologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Animais , Transporte Biológico/fisiologia , Exossomos/química , Camundongos , Neuroblastoma/enzimologia , Neuroblastoma/metabolismo , Vesículas Transportadoras/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Insulin degrading enzyme (IDE) is implicated in the regulation of amyloid beta (Abeta) steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD). Although IDE sub-cellular localization has been well studied, the compartments relevant to Abeta degradation remain to be determined. RESULTS: Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM)-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-beta-cyclodextrin (MbetaCD), endogenous Abeta accumulated in the extracellular space and exogenous Abeta proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Abeta degradation in vitro. CONCLUSION: Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for insulin proteolysis. The concept that mis-location of Abeta degrading proteases away from DRMs may impair the physiological turn-over of Abeta in vivo deserves further investigation in light of therapeutic strategies based on enhancing Abeta proteolysis in which DRM protease-targeting may need to be taken into account.
RESUMO
Amyloid beta (Abeta) accumulates in the neuropil and within the walls of cerebral vessels in association with normal aging, dementia or stroke. Abeta is released from its precursor protein as soluble monomeric species yet, under pathological conditions, it self-aggregates to form soluble oligomers or insoluble fibrils that may be toxic to neurons and vascular cells. Abeta levels could be lowered by inhibiting its generation or by promoting its clearance by transport or degradation. Here we will summarize recent findings on brain proteases capable of degrading Abeta, with a special focus on those enzymes for which there is genetic, transgenic or biochemical evidence supporting a role in the proteolysis of Abeta in vivo.