Apolipoprotein E is associated with age-related risk for Alzheimer's disease and plays critical roles in Aβ homeostasis. We report that ApoE plays a previously unappreciated role in facilitating the proteolytic clearance of soluble Aβ from the brain. The endolytic degradation of Aβ peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Aβ degradation extracellularly by insulin degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Aβ degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Aβ degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Aβ load. GW3965 treatment also reversed contextual memory deficits. These data demonstrate a novel mechanism through which ApoE facilitates the clearance of Aβ from the brain and suggest that LXR agonists may represent a novel therapy for AD. Alzheimer's disease (AD) is characterized by the accumulation and deposition of Aβ peptides within the brain, leading to the perturbation of synaptic function and neuronal loss that typifies the disease (Tanzi and Bertram, 2005). Genetic analysis of familial forms of AD has established the centrality of APP processing and Aβ production to disease pathogenesis. Aβ peptides are normally produced by neurons in the brain and cleared through efflux into the peripheral Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inheritance of the apoE4 allele (4) increases the risk of developing Alzheimer's disease; however, the mechanisms underlying this association remain elusive. Recent data suggest that inheritance of 4 may lead to reduced apoE protein levels in the CNS. We therefore examined apoE protein levels in the brains, CSF and plasma of 2/2, 3/3, and 4/4 targeted replacement mice. These apoE mice showed a genotype-dependent decrease in apoE levels; 2/2 Ͼ3/3 Ͼ4/4. Next, we sought to examine the relative contributions of apoE4 and apoE3 in the 3/4 mouse brains. ApoE4 represented 30 -40% of the total apoE. Moreover, the absolute amount of apoE3 per allele was similar between 3/3 and 3/4 mice, implying that the reduced levels of total apoE in 3/4 mice can be explained by the reduction in apoE4 levels. In culture medium from 3/4 human astrocytoma or 3/3, 4/4 and 3/4 primary astrocytes, apoE4 levels were consistently lower than apoE3. Secreted cholesterol levels were also lower from 4/4 astrocytes. Pulse-chase experiments showed an enhanced degradation and reduced half-life of newly synthesized apoE4 compared with apoE3. Together, these data suggest that astrocytes preferentially degrade apoE4, leading to reduced apoE4 secretion and ultimately to reduced brain apoE levels. Moreover, the genotype-dependent decrease in CNS apoE levels, mirror the relative risk of developing AD, and suggest that low levels of total apoE exhibited by 4 carriers may directly contribute to the disease progression, perhaps by reducing the capacity of apoE to promote synaptic repair and/or A␤ clearance.

Recent epidemiological studies show a reduced prevalence of Alzheimer's disease (AD) in patients treated with inhibitors of cholesterol biosynthesis. Moreover, the cholesterol-transport protein, apolipoprotein E4, and elevated cholesterol are important risk factors for AD. Additionally, in vitro and in vivo studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein (APP) to beta-amyloid, the major constituent of senile plaques. Cholesterol plays a crucial role in maintaining lipid rafts in a functional state. Lipid rafts are cholesterol-enriched membrane microdomains implicated in signal transduction, protein trafficking, and proteolytic processing. Since APP, beta-amyloid, and the putative gamma-secretase, presenilin-1 (PS-1), have all been found in lipid rafts, we hypothesized that the recently identified beta-secretase, Asp2 (BACE1), might also be present in rafts. Here, we report that recombinant Asp2 expressed in three distinct cell lines is raft associated. Using both detergent and nondetergent methods, Asp2 protein and activity were found in a light membrane raft fraction that also contained other components of the amyloidogenic pathway. Immunoisolation of caveolin-containing vesicles indicated that Asp2 was present in a unique raft population distinct from caveolae. Finally, depletion of raft cholesterol abrogated association of Asp2 with the light membrane fraction. These observations are consistent with the raft localization of APP processing and suggest that the partitioning of Asp2 into lipid rafts may underlie the cholesterol sensitivity of beta-amyloid production.

Replacement of the central, para-substituted fluorophenyl ring in the γ-secretase inhibitor 1 (BMS-708,163) with the bicyclo[1.1.1]pentane motif led to the discovery of compound 3, an equipotent enzyme inhibitor with significant improvements in passive permeability and aqueous solubility. The modified biopharmaceutical properties of 3 translated into excellent oral absorption characteristics (~4-fold ↑ C(max) and AUC values relative to 1) in a mouse model of γ-secretase inhibition. In addition, SAR studies into other fluorophenyl replacements indicate the intrinsic advantages of the bicyclo[1.1.1]pentane moiety over conventional phenyl ring replacements with respect to achieving an optimal balance of properties (e.g., γ-secretase inhibition, aqueous solubility/permeability, in vitro metabolic stability). Overall, this work enhances the scope of the [1.1.1]-bicycle beyond that of a mere "spacer" unit and presents a compelling case for its broader application as a phenyl group replacement in scenarios where the aromatic ring count impacts physicochemical parameters and overall drug-likeness.

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We have previously reported that plasma apolipoprotein (apo) E-containing high density lipoprotein particles have a potent anti-platelet action, apparently by occupying saturable binding sites in the cell surface. Here we show that purified apoE (10 -50 g/ml), complexed with phospholipid vesicles (dimyristoylphosphatidylcholine, DMPC), suppresses platelet aggregation induced by ADP, epinephrine, or collagen. This effect was not due to sequestration of cholesterol from platelet membranes; apoE⅐DMPC chemically modified with cyclohexanedione (cyclohexanedione-apoE⅐DMPC) did not inhibit aggregation but nevertheless removed similar amounts of cholesterol as untreated complexes, about 2% during the aggregation period. Rather we found that apoE influenced intracellular platelet signaling. Thus, apoE⅐DMPC markedly increased cGMP in ADP-stimulated platelets which correlated with the resulting inhibition of aggregation (r ‫؍‬ 0.85; p < 0.01, n ‫؍‬ 10), whereas cyclohexanedione-apoE⅐DMPC vesicles had no effect. One important cellular mechanism for up-regulation of cGMP is through stimulation of nitric oxide (NO) synthase, the NO generated by conversion of Larginine to L-citrulline, binds to and activates guanylate cyclase. This signal transduction pathway was implicated by the finding that NO synthase inhibitors of distinct structural and functional types all reversed the anti-platelet action of apoE, whereas a selective inhibitor of soluble guanylate cyclase, 1H- platelets) than controls (0.18 ؎ 0.03; p < 0.05). In addition, hemoglobin which avidly binds NO also suppressed the anti-aggregatory effect, indicating that apoE stimulated sufficient production of NO by platelets for extracellular release to occur. We conclude that apoE inhibits platelet aggregation through the L-arginine:NO signal transduction pathway. Human apolipoprotein E (apoE)1 is a 299-residue protein of molecular mass 34 kDa found in the surface of circulating triglyceride-rich lipoproteins (very low density lipoprotein and chylomicrons, or their remnants) and certain HDL particles (1). Its major function is to mediate hepatic clearance of lipoproteins through interaction with two receptors, the low density lipoprotein or B,E receptor and an apoE-specific receptor, most probably the low density lipoprotein receptor-related protein (2). When the apoE polypeptide is dysfunctional or absent severe hyperlipidemia and atherosclerosis in humans or animal models ensues (1, 3-5). Although apoE is synthesized predominantly by the liver, macrophages also secrete apoE; this appears important for facilitating local cholesterol redistribution, for reverse cholesterol transport, and for restricting development of atherosclerotic lesions (6). Indeed, atherosclerosis in apoE-deficient (apoE Ϫ/Ϫ ) mice can be prevented by transplantation of normal murine bone marrow cells (5), by macrophage-specific expression of the human apoE transgene (7), or by adenovirus-mediated gene replacement (8).Recently, we proposed an additional anti-atherogenic role for apoE. We found that H...

The presenilin containing ␥-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. ␥-Secretase catalyzes the final step in the generation of A␤ 40 and A␤ 42 peptides from APP. Amyloid ␤-peptides (A␤ peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic A␤ peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide ␥-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits A␤ production with low nanomolar potency in cellular and cell-free assays of ␥-secretase function, and displaces a tritiated analog of GSI-953 from enriched ␥-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid A␤ levels, and a reversal of contextual fear-conditioning deficits that are correlated with A␤ load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dosedependent changes in plasma A␤ levels, confirming pharmacodynamic activity of GSI-953 in humans.This research was supported by Wyeth Research. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

The amyloid hypothesis states that a variety of neurotoxic ␤-amyloid (A␤) species contribute to the pathogenesis of Alzheimer's disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between A␤ production and clearance. Enzymes responsible for the degradation of A␤ are not well understood, and, thus far, it has not been possible to enhance A␤ catabolism by pharmacological manipulation. We provide evidence that A␤ catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain A␤ levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades A␤ oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain A␤. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain A␤ levels, restore long-term potentiation deficits in hippocampal slices from transgenic A␤-producing mice, and reverse cognitive deficits in these mice.Alzheimer ͉ plasminogen activator inhibitor ͉ tissue plasminogen activator A lzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of intracellular neuronal tangles and extracellular parenchymal and vascular amyloid deposits containing ␤-amyloid peptide (A␤). A␤ is a 39-to 42-aa peptide derived from the proteolytic processing of the amyloid precursor protein (APP) (1). The ''amyloid hypothesis'' of AD postulates a central causal role for A␤ in AD pathogenesis and is supported by genetic and physiological evidence. All known early onset familial AD mutations result in enhanced levels of cytotoxic A␤ species, amyloid plaque deposition, and dementia. Furthermore, A␤ peptide is reported to be neurotoxic and synaptotoxic in vitro and in vivo, inhibiting long-term potentiation (LTP), a physiological correlate of memory (2). Based on these observations, a number of strategies to reduce brain A␤ levels are being pursued as therapeutic approaches to treat AD (3, 4).If the amyloid hypothesis of AD is correct and A␤ levels are pivotal to disease etiology, then the balance between A␤ production and catabolism is likely to be a key determinant of disease progression. It has been suggested that insufficient clearance of A␤ may account for elevated A␤ levels in the brain and the accumulation of pathogenic amyloid deposits in sporadic AD (5). A number of proteases have been implicated in the proteolytic clearance of A␤ from the CNS, including neprilysin, insulin-degrading enzyme, endothelin converting enzyme, and plasmin (3, 6-8). The relative contribution of these enzymes to A␤ catabolism remains unclear, but each protease may play a significant role in the degradation and clearance of A␤, resulting in a slowing of A␤ accumulation and aggregation and u...