Statins reduce amyloid-beta production through inhibition of protein isoprenylation

Epidemiological evidence suggests that long term treatment with hydroxymethylglutaryl-CoA reductase inhibitors, or statins, decreases the risk for developing Alzheimer disease (AD). However, statin-mediated AD protection cannot be fully explained by reduction of cholesterol levels. In addition to their cholesterol lowering effects, statins have pleiotropic actions and act to lower the concentrations of isoprenoid intermediates, such as geranylgeranyl pyrophosphate and farnesyl pyrophosphate. The Rho and Rab family small G-proteins require addition of these isoprenyl moieties at their C termini for normal GTPase function. In neuroblastoma cell lines, treatment with statins inhibits the membrane localization of Rho and Rab proteins at statin doses as low as 200 nm, without affecting cellular cholesterol levels. In addition, we show for the first time that at low, physiologically relevant, doses statins preferentially inhibit the isoprenylation of a subset of GTPases. The amyloid precursor protein (APP) is proteolytically cleaved to generate beta-amyloid (Abeta), which is the major component of senile plaques found in AD. We show that inhibition of protein isoprenylation by statins causes the accumulation of APP within the cell through inhibition of Rab family proteins involved in vesicular trafficking. Moreover, inhibition of Rho family protein function reduces levels of APP C-terminal fragments due to enhanced lysosomal dependent degradation. Statin inhibition of protein isoprenylation results in decreased Abeta secretion. In summary, we show that statins selectively inhibit GTPase isoprenylation at clinically relevant doses, leading to reduced Abeta production in an isoprenoid-dependent manner. These studies provide insight into the mechanisms by which statins may reduce AD pathogenesis.     

Post-translational processing of beta-secretase in Alzheimer's disease

Beta-amyloid is released into the brains of Alzheimer's patients, where it aggregates and causes damage to neurons. It is cleaved proteolytically from a large transmembrane glycoprotein amyloid precursor protein by a membrane-bound protease, known as beta-secretase identified previously as the acid protease, Asp-2. We have shown previously that beta-secretase is up-regulated by increased intracellular cholesterol, and down-regulated by cholesterol biosynthesis inhibition. Here we show using mass spectrometry that discrete changes in the glycosylation and palmitoylation of beta-secretase occur when cells expressing it are treated with statins.     

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.     

Atorvastatin-induced activation of Alzheimer's alpha secretase is resistant to standard inhibitors of protein phosphorylation-regulated ectodomain shedding

Studies of metabolism of the Alzheimer amyloid precursor protein (APP) have focused much recent attention on the biology of juxta- and intra-membranous proteases. Release or 'shedding' of the large APP ectodomain can occur via one of two competing pathways, the alpha- and beta-secretase pathways, that are distinguished both by subcellular site of proteolysis and by site of cleavage within APP. The alpha-secretase pathway cleaves within the amyloidogenic Abeta domain of APP, precluding the formation of toxic amyloid aggregates. The relative utilization of the alpha- and beta-secretase pathways is controlled by the activation of certain protein phosphorylation signal transduction pathways including protein kinase C (PKC) and extracellular signal regulated protein kinase [ERK/mitogen-activated protein kinase (MAP kinase)], although the relevant substrates for phosphorylation remain obscure. Because of their apparent ability to decrease the risk for Alzheimer disease, the effects of statins (HMG CoA reductase inhibitors) on APP metabolism were studied. Statin treatment induced an APP processing phenocopy of PKC or ERK activation, raising the possibility that statin effects on APP processing might involve protein phosphorylation. In cultured neuroblastoma cells transfected with human Swedish mutant APP, atorvastatin stimulated the release of alpha-secretase-released, soluble APP (sAPPalpha). However, statin-induced stimulation of sAPPalpha release was not antagonized by inhibitors of either PKC or ERK, or by the co-expression of a dominant negative isoform of ERK (dnERK), indicating that PKC and ERK do not play key roles in mediating the effect of atorvastatin on sAPPalpha secretion. These results suggest that statins may regulate alpha-secretase activity either by altering the biophysical properties of plasma membranes or by modulating the function of as-yet unidentified protein kinases that respond to either cholesterol or to some intermediate in the cholesterol metabolic pathway. A 'phospho-proteomic' analysis of N2a cells with and without statin treatment was performed, revealing changes in the phosphorylation state of several protein kinases plausibly related to APP processing. A systematic evaluation of the possible role of these protein kinases in statin-regulated APP ectodomain shedding is underway.     

Independent inhibition of Alzheimer disease beta- and gamma-secretase cleavage by lowered cholesterol levels

The major molecular risk factor for Alzheimer disease so far identified is the amyloidogenic peptide Abeta(42). In addition, growing evidence suggests a role of cholesterol in Alzheimer disease pathology and Abeta generation. However, the cellular mechanism of lipid-dependent Abeta production remains unclear. Here we describe that the two enzymatic activities responsible for Abeta production, beta-secretase and gamma-secretase, are inhibited in parallel by cholesterol reduction. Importantly, our data indicate that cholesterol depletion within the cellular context inhibits both secretases additively and independently from each other. This is unexpected because the beta-secretase beta-site amyloid precursor protein cleaving enzyme and the presenilin-containing gamma-secretase complex are structurally different from each other, and these enzymes are apparently located in different subcellular compartments. The parallel and additive inhibition has obvious consequences for therapeutic research and may indicate an intrinsic cross-talk between Alzheimer disease-related amyloid precursor protein processing, amyloid precursor protein function, and lipid biology.     

The conflicting role of brain cholesterol in Alzheimer's disease: lessons from the brain plasminogen system

Retrospective clinical studies indicate that individuals chronically treated with cholesterol synthesis inhibitors, statins, are at lower risk of developing AD (Alzheimer's disease). Moreover, treatment of guinea pigs with high doses of simvastatin or drastic reduction of cholesterol in cultured cells decrease Abeta (beta-amyloid peptide) production. These data sustain the concept that high brain cholesterol is responsible for Abeta accumulation in AD, providing the scientific support for the proposed use of statins to prevent this disease. However, a number of unresolved issues raise doubts that high brain cholesterol is to blame. First, it has not been shown that higher neuronal cholesterol increases Abeta production. Secondly, it has not been demonstrated that neurons in AD have more cholesterol than control neurons. On the contrary, the brains of AD patients show a specific down-regulation of seladin-1, a protein involved in cholesterol synthesis, and low membrane cholesterol was observed in hippocampal membranes of ApoE4 (apolipoprotein E4) AD cases. This effect was also evidenced by altered cholesterol-rich membrane domains (rafts) and raft-mediated functions, such as diminished generation of the Abeta-degrading enzyme plasmin. Thirdly, numerous genetic defects that cause neurodegeneration are due to defective cholesterol metabolism. Fourthly, in female mice, the most brain-permeant statin induces neurodegeneration and high amyloid production. Altogether, this evidence makes it difficult to accept that statins are beneficial through acting as brain cholesterol-synthesis inhibitors. It appears more likely that their advantageous role arises from improved brain oxygenation.     

RAC1 inhibition targets amyloid precursor protein processing by gamma-secretase and decreases Abeta production in vitro and in vivo

beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.     

A specific inhibitor of cholesterol biosynthesis, BM15.766, reduces the expression of beta-secretase and the production of amyloid-beta in vitro

We have previously shown that statins reduce the production of amyloid-beta (Abeta) by both isoprenoid- and cholesterol-dependent mechanisms. These pathways contribute to the regulation of the dimerisation of BACE into its physiologically active form. Statins reduce cellular cholesterol levels by 20-40%; therefore, it is possible that the remaining cholesterol within the cell may play a significant role in the production of Abeta. Incubation of cells with the specific cholesterol biosynthesis inhibitor BM15.766 together with 50 micromol/L simvastatin and 400 micromol/L mevalonate reduced cellular cholesterol levels in a dose-dependent manner with increasing BM15.766 concentration (r = -0.9736, p = 0.0264). Furthermore, decreases in cellular cholesterol levels correlated with reductions in total Abeta production (r = 0.9683, p = 0.0317). A total of 2.5 micromol/L BM15.766 inhibited the dimerisation of BACE, whilst the expression of BACE monomer was reduced by 5 micromol/L BM15.766. BM15.766 treatment localised BACE predominantly within the Golgi, and reduced total BACE expression per cell. Similar changes were observed in the expression of the Golgi marker golgin-97, suggesting that reduced BACE expression may arise from a decrease in protein trafficking and an increase in degradation. By targeting cholesterol synthesis using specific cholesterol biosynthesis inhibitors, it is possible to reduce Abeta production without reducing protein isoprenylation.     

The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid beta peptide (Abeta), is generated from amyloid precursor protein (APP) by beta- and gamma-secretase-mediated cleavage. Because beta-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Abeta and grow normally, a beta-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel beta-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits beta-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Abeta production in vivo in the soluble fraction compared with vehicle, but the level of Abeta in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Abeta production in both the soluble and insoluble fractions. Our results indicate that the beta-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.     

Mechanisms of statin-mediated inhibition of small G-protein function

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been reported to reduce the risk of Alzheimer disease. We have shown previously that statins inhibit a beta-amyloid (Abeta)-mediated inflammatory response through mechanisms independent of cholesterol reduction. Specifically, statins exert anti-inflammatory actions through their ability to prevent the isoprenylation of members of the Rho family of small G-proteins, resulting in the functional inactivation of these G-proteins. We report that statin treatment of microglia results in perturbation of the cytoskeleton and morphological changes due to alteration in Rho family function. Statins also block Abeta-stimulated phagocytosis through inhibition of Rac action. Paradoxically, the statin-mediated inactivation of G-protein function was associated with increased GTP loading of Rac and RhoA, and this effect was observed in myeloid lineage cells and other cell types. Statin treatment disrupted the interaction of Rac with its negative regulator the Rho guanine nucleotide dissociation inhibitor (RhoGDI), an interaction that is dependent on protein isoprenylation. We propose that lack of negative regulation accounts for the increased GTP loading. Isoprenylation of Rac is also required for efficient interaction with the plasma membrane, and we report that statin treatment dramatically reduces the capacity of Rac to interact with membranes. These results suggest a mechanism by which statins inhibit the actions of Rho GTPases and attenuate Abeta-stimulated inflammation.     

Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor?

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.     

Characterization of palmitoylation of ATP binding cassette transporter G1: Effect on protein trafficking and function

ATP-binding cassette transporter G1 (ABCG1) mediates cholesterol efflux onto lipidated apolipoprotein A-I and HDL and plays a role in various important physiological functions. However, the mechanism by which ABCG1 mediates cholesterol translocation is unclear. Protein palmitoylation regulates many functions of proteins such as ABCA1. Here we investigated if ABCG1 is palmitoylated and the subsequent effects on ABCG1-mediated cholesterol efflux. We demonstrated that ABCG1 is palmitoylated in both human embryonic kidney 293 cells and in mouse macrophage, J774. Five cysteine residues located at positions 26, 150, 311, 390 and 402 in the NH₂-terminal cytoplasmic region of ABCG1 were palmitoylated. Removal of palmitoylation at Cys311 by mutating the residue to Ala (C311A) or Ser significantly decreased ABCG1-mediated cholesterol efflux. On the other hand, removal of palmitoylation at sites 26, 150, 390 and 402 had no significant effect. We further demonstrated that mutations of Cys311 affected ABCG1 trafficking from the endoplasmic reticulum. Therefore, our data suggest that palmitoylation plays a critical role in ABCG1-mediated cholesterol efflux through the regulation of trafficking.     

Accumulation and aggregation of amyloid beta-protein in late endosomes of Niemann-pick type C cells

There is growing evidence suggesting that cholesterol metabolism is linked to susceptibility to Alzheimer's disease by influencing amyloid beta-protein (Abeta) metabolism. However, the precise cellular linkage sites between cholesterol and Abeta have not yet been clarified. To address this issue, we investigated Niemann-Pick type C (NPC) model cells and NPC mutant cells, which showed aberrant cholesterol trafficking. We observed a remarkable Abeta accumulation in late endosomes of both NPC model cells and mutant cells where cholesterol accumulates and a significant accumulation in the NPC mouse brain. This Abeta accumulation was independent of its constitutive secretion and production through an endocytic pathway. In addition, it is characterized by a marked predominance of Abeta42 and insolubility in SDS, suggesting the presence of aggregated Abeta in late endosomes. Most importantly, Abeta accumulation is coupled with the cholesterol levels in late endosomes. Thus, late endosomes of NPC cells are a novel pool of aggregated Abeta42 as well as cholesterol, suggesting a direct interaction between aggregated Abeta and cholesterol.     

Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein

Elucidation of Abeta-lowering agents that inhibit processing of the wild-type (WT) beta-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of beta-secretase activity in regulated secretory vesicles that produce beta-amyloid (Abeta). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT beta-secretase site but not the rare Swedish mutant beta-secretase site. In vivo treatment of London APP mice, expressing the WT beta-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased Abeta40 and Abeta42, and reduced C-terminal beta-secretase fragment derived from APP by beta-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant beta-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce Abeta in an AD animal model expressing the WT beta-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents.     

Regulation of osteoclast differentiation by statins

HMG-CoA reductase inhibitor (statin) treatment is frontline therapy for lowering plasma cholesterol levels in patients with hyperlipidemia. In a few case studies, analysis of clinical data has revealed a decreased risk of fracture in patients on statin therapy. However, this reduction in the incidence of fracture is not always observed nor is it supported by an increase in bone density, which further complicates our understanding of the role of statins in bone metabolism. Thus, the precise role of statins in bone metabolism remains poorly understood. In this study, we examined the effect of statin treatment on osteoclastogenesis. Treatment with lovastatin resulted in a significant, dose-dependent decrease in the numbers of differentiated osteoclasts and decreased cholesterol biosynthesis activity with an EC(50) similar to that observed in freshly isolated rat or cultured human liver cells. Studies assessing the role of mevalonate metabolites in the development of the osteoclasts demonstrated that geranylgeraniol, but not squalene or farnesol was important for the development and differentiation of osteoclasts, implicating protein geranylgeranylation rather than protein farnesylation as a key factor in the osteoclast differentiation process. In conclusion, our data indicate that lovastatin inhibits osteoclast development through inhibition of geranylgeranylation of key prenylated proteins and that the bone effects of statins are at least partially due to their effects on osteoclast numbers.     

Statin therapy and angiogenesis

PURPOSE OF REVIEW: Clinical studies suggested that 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitor (statin) therapy has an additional cardiovascular protective activity that may function independently of the ability of statins to lower serum cholesterol. This paper reviews the available data on these effects and discusses the potential intracellular mechanisms involved. RECENT FINDINGS: Experimental studies have clearly shown that statins protect against ischaemia-reperfusion injury of the heart, and exert pro-angiogenic effects by stimulating the growth of new blood vessels in ischaemic limbs of normocholesterolemic animals. The mechanisms underlying these serum lipid-independent statin effects are not completely understood, but there is increasing evidence that statins improve endothelial function through molecular mechanisms that mediate an increase in endothelium-derived nitric oxide. Recent research has revealed a link between statins and the serine/threonine protein kinase Akt that regulates multiple angiogenic processes in endothelial cells. In contrast to these data, it has also been reported that higher doses of statins can inhibit endothelial cell migration and angiogenesis. SUMMARY: Statins have biphasic potential either to promote or inhibit angiogenesis. Low statin doses induce a pro-angiogenic effect through Akt activation and increase nitric oxide production, whereas high statin doses may decrease protein prenylation and inhibit cell growth. Notwithstanding, the clinical relevance of these serum lipid-independent effects is not fully understood. Further studies on the actions of statins on endothelial cells may lead to the identification of new pharmacological targets for the control of angiogenesis.     

ELISA analysis of beta-secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways

Limiting beta amyloid (Abeta) production could become an important therapeutic target in Alzheimer's disease (AD). Abeta is derived by the sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. A double missense mutation in APP found in a Swedish pedigree (APPsw) elevates Abeta40 and Abeta42 production. Abeta production and, therefore, beta-secretase cleavage of APPsw reportedly occur in the endoplasmic reticulum (ER), Golgi and endocytic compartments. However, the relative contribution of beta-secretase cleavage occurring in each compartment has not been determined. Experiments described here use a novel ELISA to measure the beta-cleaved product, APPswbeta. Using this ELISA, we provide new information regarding the relative amount of beta-secretase cleavage of APPsw that occurs in secretory and endocytic pathways. Using a dilysine retrieval motif to retain APPsw in the ER, we discovered that less than 15% of the beta-secretase cleavage was still detected. Experiments utilizing endocytosis-impaired mutants of APPsw revealed that little or no beta-secretase cleavage of APPsw appears to take place through endocytosis. Surprisingly, deletion of the entire cytoplasmic tail increased both APPswbeta and Abeta secretion, suggesting that protein interactions with this region normally impede beta-secretase cleavage. These results suggest that APPsw is cleaved by beta-secretase late in the secretory pathway.     

Pleiotropic effects of statin therapy: molecular mechanisms and clinical results

Statins inhibit the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is required for cholesterol biosynthesis, and are beneficial in the primary and secondary prevention of cardiovascular disease. Most of the benefits of statin therapy are owing to the lowering of serum cholesterol levels. However, by inhibiting HMG-CoA reductase, statins can also inhibit the synthesis of isoprenoids, which are important lipid attachments for intracellular signaling molecules, such as Rho, Rac and Cdc42. Therefore, it is possible that statins might exert cholesterol-independent or 'pleiotropic' effects through direct inhibition of these small GTP-binding proteins. Recent studies have shown that statins might have important roles in diseases that are not mediated by cholesterol. Here, we review data from recent clinical trials that support the concept of statin pleiotropy and provide a rationale for their clinical importance.     

The protease inhibitor, MG132, blocks maturation of the amyloid precursor protein Swedish mutant preventing cleavage by beta-Secretase

Amyloid (Abeta) peptides found aggregated into plaques in Alzheimer's disease are derived from the sequential cleavage of the amyloid precursor protein (APP) first by beta- and then by gamma-secretases. Peptide aldehydes, which inhibit cysteine proteases and proteasomes, reportedly block Abeta peptide secretion by interfering with gamma-secretase cleavage. Using a novel, specific, and sensitive enzyme-linked immunosorbent assay for the beta-secretase-cleaved fragment of the Swedish mutant of APP (APPSw), we determined that the peptide aldehyde, MG132, prevented beta-secretase cleavage. This block in beta-secretase cleavage was not observed with clasto-lactacystin beta-lactone and thus, cannot be attributed to proteasomal inhibition. MG132 inhibition of beta-secretase cleavage was compared with the serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). AEBSF inhibition of beta-secretase cleavage was immediate and did not affect alpha-secretase cleavage. With MG132, inhibition was delayed and it decreased secretion of alpha-cleaved APPSw as well. Furthermore, MG132 treatment impaired maturation of full-length APPSw. Both inhibited intracellular formation of the beta-cleaved product. These results suggest that peptide aldehydes such as MG132 have multiple effects on the maturation and processing of APP. We conclude that the MG132-induced decrease in beta-secretase cleavage of APPSw is due to a block in maturation. This is sufficient to explain the previously reported peptide aldehyde-induced decrease in Abeta peptide secretion.