␤-Secretase (BACE, Asp-2) is a transmembrane aspartic proteinase responsible for cleaving the amyloid precursor protein (APP) to generate the soluble ectodomain sAPP␤ and its C-terminal fragment CTF␤. CTF␤ is subsequently cleaved by ␥-secretase to produce the neurotoxic͞synaptotoxic amyloid-␤ peptide (A␤) that accumulates in Alzheimer's disease. Indirect evidence has suggested that amyloidogenic APP processing may preferentially occur in lipid rafts. Here, we show that relatively little wild-type BACE is found in rafts prepared from a human neuroblastoma cell line (SH-SY5Y) by using Triton X-100 as detergent. To investigate further the significance of lipid rafts in APP processing, a glycosylphosphatidylinositol (GPI) anchor has been added to BACE, replacing the transmembrane and C-terminal domains. The GPI anchor targets the enzyme exclusively to lipid raft domains. Expression of GPI-BACE substantially up-regulates the secretion of both sAPP␤ and amyloid-␤ peptide over levels observed from cells overexpressing wild-type BACE. This effect was reversed when the lipid rafts were disrupted by depleting cellular cholesterol levels. These results suggest that processing of APP to the amyloid-␤ peptide occurs predominantly in lipid rafts and that BACE is the rate-limiting enzyme in this process. The processing of the APP695 isoform by GPI-BACE was up-regulated 20-fold compared with wild-type BACE, whereas only a 2-fold increase in the processing of APP751/770 was seen, implying a differential compartmentation of the APP isoforms. Changes in the local membrane environment during aging may facilitate the cosegregation of APP and BACE leading to increased ␤-amyloid production.

The amyloidogenesis occurring in Alzheimer's disease represents a fundamental membrane-related pathology involving a membrane-bound substrate metabolized by integral membrane proteases (secretases). Thus, the amyloid-beta peptide (Abeta), which accumulates extracellularly as plaques in the brains of Alzheimer's disease patients, is derived by sequential proteolytic cleavage of the integral transmembrane amyloid precursor protein (APP). Beta-Secretase or BACE-1 (beta-site APP cleaving enzyme) is a transmembrane aspartic protease responsible for the first of these cleavage events, generating the soluble APP ectodomain sAPPbeta, and a C-terminal fragment CTFbeta. CTFbeta is subsequently cleaved by the ?gamma-secretase complex, of which presenilin is the catalytic core, to produce Ass. A variety of studies indicate that cholesterol is an important factor in the regulation of Ass production, with high cholesterol levels being linked to increased Ass generation and deposition. However, the mechanism(s) underlying this effect are unclear at present. Recent evidence suggests that amyloidogenic APP processing may preferentially occur in the cholesterol-rich regions of membranes known as lipid rafts, and that changes in cholesterol levels could exert their effects by altering the distribution of APP-cleaving enzymes within the membrane. Rafts may be involved in the aggregation of Ass and also in its clearance by amyloid-degrading enzymes such as plasmin or possibly neprilysin (NEP).

Mutations in leucine-rich repeat kinase 2 (LRRK2) are currently the most common genetic cause of familial late-onset Parkinson disease, which is clinically indistinguishable from idiopathic disease. The most common pathological mutation in LRRK2, G2019S LRRK2, is known to cause neurite retraction. However, molecular mechanisms underlying regulation of neurite length by LRRK2 are unknown. Here, we demonstrate a novel interaction between LRRK2 and the Rho GTPase, Rac1, which plays a critical role in actin cytoskeleton remodeling necessary for the maintenance of neurite morphology. LRRK2 binds strongly to endogenous or expressed Rac1, while showing weak binding to Cdc42 and no binding to RhoA. Co-expression with LRRK2 increases Rac1 activity, as shown by increased binding to the p21-activated kinase, which modulates actin cytoskeletal dynamics. LRRK2 constructs carrying mutations that inactivate the kinase or GTPase activities do not activate Rac1. Interestingly, LRRK2 does not increase levels of membrane-bound Rac1 but dramatically changes the cellular localization of Rac1, causing polarization, which is augmented further when LRRK2 is co-expressed with constitutively active Rac1. Four different disease-related mutations in LRRK2 altered binding to Rac1, with the G2019S and R1441C LRRK2 mutations attenuating Rac1 binding and the Y1699C and I2020T LRRK2 mutations increasing binding. Co-expressing Rac1 in SH-SY5Y cells rescues the G2019S mutant phenotype of neurite retraction. We hypothesize that pathological mutations in LRRK2 attenuates activation of Rac1, causing disassembly of actin filaments, leading to neurite retraction. The interactions between LRRK2 and Rho GTPases provide a novel pathway through which LRRK2 might modulate cellular dynamics and contribute to the pathophysiology of Parkinson disease. Parkinson disease (PD)2 is the most common neurodegenerative movement disorder affecting nearly 1% of elderly over 65 years old. Idiopathic PD may result from a combination of factors including age, genetic predisposition, environmental toxins, and neuroinflammation (1, 2). Mutations in LRRK2 are the most common genetic cause of PD and also contribute to many sporadic cases of PD (3, 4). At least 20 mutations identified to date in LRRK2 cause autosomal-dominant PD, accounting for ϳ7% of all familial cases (4, 5). The missense mutation, G2019S, in the kinase domain of LRRK2 is by far the most common of the LRRK2 mutations associated with PD, and disease associated with the G2019S mutations is clinically indistinguishable from idiopathic disease (6).The Lrrk2 gene of 51 exons encodes a large, multidomain protein that includes an ankyrin repeat and leucine-rich repeat regions (6). The kinase domain of LRRK2 shares homology with receptor-interacting protein kinases and mixed lineage kinases (3, 7). LRRK2 also contains a Ras-of-complex (ROC) domain that exhibits GTPase activity (8,9). In between these two domains lies a C-terminal of Ras complex (COR) domain. Each of these domains is thought to be important for bindi...

This communication integrates the purported role of cholesterol and statins in Alzheimer's disease (AD) with recent data. Meta-analysis of association studies relevant to AD indicates that apolipoprotein (apo)E4 is the only cholesterol-related polymorphism that shows clear association with AD. This suggests that the effect of apoE4 on the pathophysiology of AD occurs via a mechanism that is not directly related to cholesterol, such as fibrillization of Abeta. Despite the lack of genetic association, cholesterol and statins clearly modulate amyloid precursor protein (APP) processing in cell culture and animal models. Statins appear to act by a pleiotropic mechanism, involving both cholesterol (via lipid rafts) and isoprenylation. The pleiotropic mechanism of statin action clarifies conflicting data from clinical studies, where statins exert an action on Abeta and AD that might be dose dependent because of actions on both cholesterol and isoprenylation. Reduced isoprenylation can also inhibit inflammation. Our own studies of brains from Alzheimer subjects +/- statins indicate that statins inhibit inflammation in humans but might not reduce cerebral Abeta load. These results suggest that the primary action of statins in humans with AD might be to reduce inflammation rather than decrease Abeta load.

Background:Evidence from biochemical, epidemiological and genetic findings indicates that cholesterol levels are linked to amyloid-β (Aβ) production and Alzheimer's disease (AD). Oxysterols, which are cholesterol-derived ligands of the liver X receptors (LXRs) and oxysterol binding proteins, strongly regulate the processing of amyloid precursor protein (APP). Although LXRs have been studied extensively, little is known about the biology of oxysterol binding proteins. Oxysterol-binding protein 1 (OSBP1) is a member of a family of sterol-binding proteins with roles in lipid metabolism, regulation of secretory vesicle generation and signal transduction, and it is thought that these proteins may act as sterol sensors to control a variety of sterol-dependent cellular processes. Results:We investigated whether OSBP1 was involved in regulating APP processing and found that overexpression of OSBP1 downregulated the amyloidogenic processing of APP, while OSBP1 knockdown had the opposite effect. In addition, we found that OSBP1 altered the trafficking of APP-Notch2 dimers by causing their accumulation in the Golgi, an effect that could be reversed by treating cells with OSBP1 ligand, 25-hydroxycholesterol. Conclusion:These results suggest that OSBP1 could play a role in linking cholesterol metabolism with intracellular APP trafficking and Aβ production, and more importantly indicate that OSBP1 could provide an alternative target for Aβ-directed therapeutic.

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FIGURE 9.3. Possible roles of apoE isoforms in amyloid metabolism. The apoE4 isoform accelerates the aggregation and deposition of Aβ fibrils, whereas the apo E2 and E3 isoforms promote clearance of Aβ via LRP.