Significance of cytosolic cathepsin D in Alzheimer's disease pathology: Protective cellular effects of PLGA nanoparticles against ß-amyloid-toxicity.

BACKGROUND: Evidence suggests that amyloid ß (Aß) peptides play an important role in the degeneration of neurons during the development of Alzheimer's disease (AD), the prevalent cause of dementia affecting the elderly. The endosomal-lysosomal system, which acts as a major site for Aß metabolism, has been shown to exhibit abnormalities in vulnerable neurons of the AD brain, reflected by enhanced levels/expression of lysosomal enzymes including cathepsin D (CatD). At present, the implication of CatD in selective neuronal vulnerability in AD pathology remains unclear. METHODS: We evaluated the role of CatD in the degeneration of neurons in Aß-treated cultures, transgenic AD mouse model (that is 5xFAD) and post mortem AD brain samples. RESULTS: Our results showed that Aß1-42 -induced toxicity in cortical cultured neurons is associated with impaired lysosomal integrity, enhanced levels of carbonylated proteins and tau phosphorylation. The cellular and cytosolic levels/activity of CatD are also elevated in cultured neurons following exposure to Aß peptide. Additionally, we observed that CatD cellular and subcellular levels/activity are increased in the affected cortex, but not in the unaffected cerebellum, of 5xFAD mice and post mortem AD brains. Interestingly, treatment of cultured neurons with nanoparticles PLGA, which targets lysosomal system, attenuated Aß toxicity by reducing the levels of carbonylated proteins, tau phosphorylation and the level/distribution/activity of CatD. CONCLUSION: Our study reveals that increased cytosolic level/activity of CatD play an important role in determining neuronal vulnerability in AD. Additionally, native PLGA can protect neurons against Aß toxicity by restoring lysosomal membrane integrity, thus signifying its implication in attenuating AD.

Endosomal-Lysosomal Cholesterol Sequestration by U18666A Differentially Regulates Amyloid Precursor Protein (APP) Metabolism in Normal and APP-Overexpressing Cells.

Amyloid ß (Aß) peptide, derived from amyloid precursor protein (APP), plays a critical role in the development of Alzheimer's disease. Current evidence indicates that altered levels or subcellular distribution of cholesterol can regulate Aß production and clearance, but it remains unclear how cholesterol sequestration within the endosomal-lysosomal (EL) system can influence APP metabolism. Thus, we evaluated the effects of U18666A, which triggers cholesterol redistribution within the EL system, on mouse N2a cells expressing different levels of APP in the presence or absence of extracellular cholesterol and lipids provided by fetal bovine serum (FBS). Our results reveal that U18666A and FBS differentially increase the levels of APP and its cleaved products, the α-, ß-, and η-C-terminal fragments, in N2a cells expressing normal levels of mouse APP (N2awt), higher levels of human wild-type APP (APPwt), or "Swedish" mutant APP (APPsw). The cellular levels of Aß1-40/Aß1-42 were markedly increased in U18666A-treated APPwt and APPsw cells. Our studies further demonstrate that APP and its cleaved products are partly accumulated in the lysosomes, possibly due to decreased clearance. Finally, we show that autophagy inhibition plays a role in mediating U18666A effects. Collectively, these results suggest that altered levels and distribution of cholesterol and lipids can differentially regulate APP metabolism depending on the nature of APP expression.

The Effects of Extracellular Serum Concentration on APP Processing in Npc1-Deficient APP-Overexpressing N2a Cells.

Amyloid precursor protein (APP) is cleaved by a set of proteases including α-/ß-/γ- and recently identified η-secretases, generating C-terminal fragments (CTFs) of varying lengths and amyloid ß (Aß) peptides, which are considered to play a pivotal role in Alzheimer's disease (AD) pathogenesis. Cellular cholesterol content/distribution can regulate the production/clearance of APP metabolites and hence modify AD pathology. To determine the functional relation between endosomal-lysosomal (EL) cholesterol sequestration and APP metabolism, we used our recently developed mouse N2a-ANPC cells that overexpress Swedish mutant human APP in the absence of cholesterol-trafficking Niemann-Pick type C1 (Npc1) protein. Here, we report that neither increased levels nor EL cholesterol sequestration altered APP holoprotein levels but caused the intracellular accumulation of APP α-/ß-/η-CTFs and Aß1-40/42 peptides. The levels of APP-cleaved products increased as a function of extracellular serum concentration in N2a-ANPC cells, which are more vulnerable to death than the control cells. Additionally, we show that pH of the lysosomal vesicles in N2a-ANPC cells shifted to a less acidic range with increasing serum concentrations, thus making them less efficient functionally. Interestingly, the addition of cholesterol to the culture media not only increased the levels of cellular cholesterol and APP-cleaved products but also rendered the cells more vulnerable to toxicity. Collectively, our results suggest that extracellular cholesterol concentration in serum under conditions of Npc1 deficiency can influence intracellular cholesterol content/distribution and lysosomal efficacy, triggering the accumulation of toxic APP-cleaved products, eventually leading to cell death.