S1C and D). of A production, on APP/A metabolism and its relation to neuronal viability. Levels of APP C-terminal fragments (-CTF/-CTF) and A peptides, but not APP mRNA/protein or soluble APP/APP, were increased in ANPC mouse brains and N2a-ANPC cells. These changes were accompanied by reduced clearance of peptides and an increased level/activity of -secretase, suggesting that accumulation of APP-CTFs is due to decreased turnover, whereas increased A levels may result from a combination of increased production and decreased turnover. APP-CTFs and A peptides were localized primarily in early-/late-endosomes and to some extent in lysosomes/autophagosomes. Cholesterol sequestration impaired endocytic-autophagic-lysosomal, but not proteasomal, clearance of APP-CTFs/A peptides. Moreover, markers of oxidative stress were increased in vulnerable brain regions of ANPC mice and enhanced -CTF/A levels increased susceptibility of N2a-ANPC cells to H2O2-induced toxicity. TH-302 (Evofosfamide) Collectively, our results show that cellular cholesterol sequestration plays a key role in APP/A metabolism and increasing neuronal vulnerability to oxidative stress in AD-related pathology. Introduction Alzheimer’s disease (AD), the most common form of dementia affecting the elderly, is characterized by the presence of intracellular neurofibrillary tangles, extracellular -amyloid (A)-containing neuritic plaques and loss of neurons in selected brain regions (1,2). Assimilated evidence indicates that accumulation of A may contribute to/trigger the loss of neurons and AD pathogenesis (3). These peptides are generated from the amyloid precursor protein (APP) which is processed by either non-amyloidogenic -secretase or amyloidogenic -secretase pathway (4). The -secretase cleaves APP within the Rabbit Polyclonal to ARTS-1 A domain, yielding soluble APP (sAPP) and a C-terminal fragment (-CTF) that is further processed by -secretase to generate A17C40/A17C42 fragments. Conversely, -secretase cleaves APP to generate soluble APP (sAPP) and an A-containing C-terminal fragment (-CTF), which is processed via -secretase to yield full-length A1C40/A1C42. While -secretase processing occurs mostly in the secretory pathway, the endosomalClysosomal (EL) system plays a critical role in the production of A peptides (4). Several studies show that cholesterol can influence APP processing and A generation. For example, an increase in cellular cholesterol up-regulates, whereas a decrease down-regulates, A TH-302 (Evofosfamide) generation in cultured neurons/cell lines (5,6). Moreover, a high-cholesterol diet has been reported to increase brain levels/deposition of A, whereas a low-cholesterol diet can decrease the levels/deposition of A in APP TH-302 (Evofosfamide) transgenic (Tg) mice (5,7). In contrast, some studies have reported that increased plasma cholesterol is associated with unchanged (8) or reduced (9) A levels, while lowering plasma cholesterol either does not affect (10) or elevates (11,12) brain A levels. Since plasma lipoproteins cannot cross the bloodCbrain barrier (13), it is important to determine how the cholesterol content of neurons influences the production/secretion of A-related peptides. Under normal conditions, cholesterol derived from astrocytes is taken up by neurons via receptor-mediated endocytosis and is delivered first TH-302 (Evofosfamide) to the EL system and then exported to other cellular compartments via a mechanism entailing the Niemann-Pick type C (NPC)-1 and -2 proteins (14C16). Although the overall cholesterol content isolated from Npc1-null mice is not higher than that of wild-type (WT) neurons, large amounts of cholesterol are sequestered in late-endosomes/lysosomes in cell bodies (17,18). Since the EL system is a major site of APP metabolism and exhibits marked changes in at risk neurons prior to A deposition in AD brains (4,19), it is important to determine how alterations in EL cholesterol levels can influence production and clearance of A peptides. Interestingly, lack of NPC1 protein has been shown to cause AD-like phenotype including A accumulation in human and mouse brains, while some recent studies have also reported altered expression of NPC1 in AD pathology (20C24). To better understand the functional link between NPC1 dysfunction, EL cholesterol sequestration and A metabolism, we have.