Impaired geranylgeranyltransferase-I regulation reduces membrane-associated Rho protein levels in aged mouse brain.

Synaptic impairment rather than neuronal loss may be the leading cause of cognitive dysfunction in brain aging. Certain small Rho-GTPases are involved in synaptic plasticity, and their dysfunction is associated with brain aging and neurodegeneration. Rho-GTPases undergo prenylation by attachment of geranylgeranylpyrophosphate (GGPP) catalyzed by GGTase-I. We examined age-related changes in the abundance of Rho and Rab proteins in membrane and cytosolic fractions as well as of GGTase-I in brain tissue of 3- and 23-month-old C57BL/6 mice. We report a shift in the cellular localization of Rho-GTPases toward reduced levels of membrane-associated and enhanced cytosolic levels of those proteins in aged mouse brain as compared with younger mice. The age-related reduction in membrane-associated Rho proteins was associated with a reduction in GGTase-Iß levels that regulates binding of GGPP to Rho-GTPases. Proteins prenylated by GGTase-II were not reduced in aged brain indicating a specific targeting of GGTase-I in the aged brain. Inhibition of GGTase-I in vitro modeled the effects of aging we observed in vivo. We demonstrate for the first time a decrease in membrane-associated Rho proteins in aged brain in association with down-regulation of GGTase-Iß. This down-regulation could be one of the mechanisms causing age-related weakening of synaptic plasticity.

Cholesterol asymmetry in synaptic plasma membranes.

Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: (i) chronic ethanol consumption; (ii) statins; (iii) aging; and (iv) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, P-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function.

The interaction of beta-amyloid protein with cellular membranes stimulates its own production.

Gradual changes in steady-state levels of beta amyloid peptides (Abeta) in brain are considered an initial step in the amyloid cascade hypothesis of Alzheimer's disease. Abeta is a product of the secretase cleavage of amyloid precursor protein (APP). There is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Cleavage of APP strongly depends on membrane properties. Since Abeta perturbs cell membrane fluidity, the cell membrane may be the location where the neurotoxic cascade of Abeta is initiated. Therefore, we tested effects of oligomeric Abeta on membrane fluidity of whole living cells, the impact of exogenous and cellular Abeta on the processing of APP and the role of GM-1 ganglioside. We present evidence that oligoAbeta((1-40)) stimulates the amyloidogenic processing of APP by reducing membrane fluidity and complexing with GM-1 ganglioside. This dynamic action of Abeta may start a vicious circle, where endogenous Abeta stimulates its own production. Based on our novel findings, we propose that oligoAbeta((1-40)) accelerates the proteolytic cleavage of APP by decreasing membrane fluidity.

Regulation of the brain isoprenoids farnesyl- and geranylgeranylpyrophosphate is altered in male Alzheimer patients.

Post-translational modification of small GTPases by farnesyl- (FPP) and geranylgeranylpyrophosphate (GGPP) has generated much attention due to their potential contribution to cancer, cardiovascular and neurodegenerative diseases. Prenylated proteins have been identified in numerous cell functions and elevated levels of FPP and GGPP have been previously proposed to occur in Alzheimer disease (AD) but have never been quantified. In the present study, we determined if the mevalonate derived compounds FPP and GGPP are increased in brain grey and white matter of male AD patients as compared with control samples. This study demonstrates for the first time that FPP and GGPP levels are significantly elevated in human AD grey and white matter but not cholesterol, indicating a potentially disease-specific targeting of isoprenoid regulation independent of HMG-CoA-reductase. Further suggesting a selective disruption of FPP and GGPP homeostasis in AD, we show that inhibition of HMG-CoA reductase in vivo significantly reduced FPP, GGPP and cholesterol abundance in mice with the largest effect on the isoprenoids. A tentative conclusion is that if indeed regulation of FPP and GGPP is altered in AD brain such changes may stimulate protein prenylation and contribute to AD neuropathophysiology.

Cholesterol distribution, not total levels, correlate with altered amyloid precursor protein processing in statin-treated mice.

There are now a number of studies that suggest that cholesterol might regulate the processing of the amyloid precursor protein to form the neurotoxic peptide Abeta. This research has opened the possibility that cholesterol-lowering drugs might be efficacious as anti-Abeta drugs for use in Alzheimer's disease. The use of HMG-CoA reductase inhibitors (commonly called statins) in vitro and in vivo has proven them to be Abeta-lowering agents, however, the mechanism of action of these drugs is not yet known. One possible mechanism is that they reduce Abeta levels indirectly by reducing cholesterol in the central nervous system (CNS). In this study, we administered three different statins (simvastatin, lovastatin, and atorvastatin) to nontransgenic mice. We found that all three compounds had similar effects on Abeta, reducing both Abeta40 and Abeta42. The statins decreased beta-cleaved C-terminal fragment (CTF) although having no effect on alpha-CTF levels. However, the drugs did not have a similar effect on cholesterol in the CNS. Only lovastatin significantly reduced total cholesterol in isolated plasma membranes. As cholesterol is not distributed evenly in the plasma membrane, we examined bilayer distribution of cholesterol and found that all three statins caused CNS cholesterol to translocate from the cytofacial leaflet to the exofacial leaflet. This data suggests that cholesterol distribution and not total cholesterol levels may be important to Abeta production in the CNS.

Amyloid beta-protein interactions with membranes and cholesterol: causes or casualties of Alzheimer's disease.

Amyloid beta-protein (Abeta) is thought to be one of the primary factors causing neurodegeneration in Alzheimer's disease (AD). This protein is an amphipathic molecule that perturbs membranes, binds lipids and alters cell function. Several studies have reported that Abeta alters membrane fluidity but the direction of this effect has not been consistently observed and explanations for this lack of consistency are proposed. Cholesterol is a key component of membranes and cholesterol interacts with Abeta in a reciprocal manner. Abeta impacts on cholesterol homeostasis and modification of cholesterol levels alters Abeta expression. In addition, certain cholesterol lowering drugs (statins) appear to reduce the risk of AD in human subjects. However, the role of changes in the total amount of brain cholesterol in AD and the mechanisms of action of statins in lowering the risk of AD are unclear. Here we discuss data on membranes, cholesterol, Abeta and AD, and propose that modification of the transbilayer distribution of cholesterol in contrast to a change in the total amount of cholesterol provides a cooperative environment for Abeta synthesis and accumulation in membranes leading to cell dysfunction including disruption in cholesterol homeostasis.

Is hypercholesterolemia a risk factor for Alzheimer's disease?

There is considerable attention being given to the association of Alzheimer's disease and cholesterol homeostasis. To that end, some have suggested that elevated cholesterol levels are a risk factor for Alzheimer's disease. If elevated cholesterol is a risk factor for Alzheimer's disease, then it would be expected that patients with Alzheimer's disease would have elevated serum and brain cholesterol levels. Studies were reviewed that have examined cholesterol levels in Alzheimer's patients and control subjects, including prospective studies, and based on that review, the conclusion is reached that the majority of studies do not support elevated cholesterol levels in serum and brain as a risk factor for Alzheimer's disease. Alternative hypotheses are discussed, including cholesterol domains and subgroups of individuals with hypercholesteremia.

Brain membrane cholesterol domains, aging and amyloid beta-peptides.

Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. These domains consist of the exofacial and cytofacial leaflets, cholesterol pools, annular lipids, and lipid rafts. Membrane lipid domains have been proposed to be involved in a variety of different functions including e.g. signal transduction, lipid transport and metabolism, and cell growth. Membrane lipid domains have been identified in brain and can be modified by different experimental conditions, aging and certain neurodegenerative diseases. Recent data reveal the very interesting possibility that membrane lipid domains may be a target of Alzheimer's disease. There is a growing body of evidence showing an association between cholesterol and Alzheimer's disease, and cholesterol is a major component of membrane lipid domains. Here we discuss recent data on brain membrane lipid domains emphasizing the structural and functional role of cholesterol. In addition, lipid domains and aging, and the potential interaction of lipid domains and amyloid beta-peptides (Abeta) that are a major component of senile plaques in brains of Alzheimer's patients are considered. We propose that age changes in the asymmetric distribution of cholesterol in contrast to total or bulk cholesterol in neuronal plasma membranes provides a cooperative environment for accumulation of Abeta in plasma membranes and the accumulation of Abeta is due in part to a direct physico-chemical interaction with cholesterol in the membrane exofacial or outer leaflet.

Cholesterol is increased in the exofacial leaflet of synaptic plasma membranes of human apolipoprotein E4 knock-in mice.

Inheritance of the apolipoprotein (apoE) epsilon4 allele is a risk factor for developing Alzheimer's disease (AD). The purpose of the present study was to determine effects of apoE-isoforms on the transbilayer distribution of cholesterol in synaptic plasma membranes (SPM) using mice expressing human apoE3 and apoE4. Total SPM cholesterol levels did not differ among the wild-type and apoE3 and apoE4 knock-in mice. However, a striking difference was observed in the transbilayer distribution of SPM cholesterol. ApoE4 knock-in mice showed an approximately 2-fold increase in exofacial leaflet cholesterol compared with apoE3 knock-in mice and wild-type mice. The results of this study suggest that pathogenic effects of apoE4 on AD development could be closely linked to alteration of cholesterol distribution in SPM.

Lipid rafts of purified mouse brain synaptosomes prepared with or without detergent reveal different lipid and protein domains.

Lipid rafts have been proposed to be important in a variety of functions including lipid transport, signal transduction and cell growth. There is increasing evidence that lipid rafts may play a role in cell functions in brain. Lipid rafts are typically isolated using a detergent such as Triton X-100. There has been, however, data from non-brain tissue indicating that preparation of lipid rafts using a detergent may represent different raft domains as compared with non-detergent preparation. The purpose of the present study was to compare protein and lipid markers of lipid rafts using a highly purified mouse synaptosomal fraction and non-detergent and detergent methods. The lipid raft marker proteins, alkaline phosphatase and flotillin, and the lipid markers, cholesterol and sphingomyelin, were highly enriched in lipid rafts prepared with detergent as compared with the non-detergent fraction. Enrichment of Na(+),K(+)-ATPase was greater in the non-detergent lipid raft fraction as compared with lipid rafts prepared with detergent. Lipid rafts from the nerve terminal of neurons prepared with or without detergents may represent different membrane domains each with unique specialized functions.

Evaluation of prenylated peptides for use in cellular imaging and biochemical analysis.

Protein prenylation involves the addition of a farnesyl (C15) or geranylgeranyl (C20) isoprenoid moiety onto the C-terminus of approximately 2 % of all mammalian proteins. This hydrophobic modification serves to direct membrane association of the protein. Due to the finding that the oncogenic protein Ras is naturally prenylated, several researchers have developed inhibitors of the prenyltransferase enzymes as cancer therapeutics. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation in living cells. Using a combination of flow cytometry and confocal microscopy, we have shown that synthetic fluorescently labeled prenylated peptides enter a variety of different cell types. Additionally, using capillary electrophoresis we have shown that these peptides can be detected in minute quantities from lysates of cells treated with these peptides. This method will allow for further study of the enzymology of protein prenylation in living cells.

Statins, Bcl-2, and apoptosis: cell death or cell protection?

Statins have proven their effectiveness in the treatment of cardiovascular disease. This class of drugs has also attracted attention as a potential treatment for dissimilar diseases such as certain types of cancers and neurodegenerative diseases. What appears to be a contradiction is that, in the case of cancer, it has been suggested that statins increase apoptosis and alter levels of Bcl-2 family members (e.g., reduce Bcl-2 and increase Bax), whereas studies mainly using noncancerous cells report opposite effects. This review examined studies reporting on the effects of statins on Bcl-2 family members, apoptosis, cell death, and cell protection. Much, but not all, of the evidence supporting the pro-apoptotic effects of statins is based on data in cancer cell lines and the use of relatively high drug concentrations. Studies indicating an anti-apoptotic effect of statins are fewer in number and generally used much lower drug concentrations and normal cells. Those conclusions are not definitive, and certainly, there is a need for additional research to determine if statin repositioning is justified for noncardiovascular diseases.

AP-2ß regulates amyloid beta-protein stimulation of apolipoprotein E transcription in astrocytes.

Two key players involved in Alzheimer's disease (AD) are amyloid beta protein (Aß) and apolipoprotein E (apoE). Aß increases apoE protein levels in astrocytes which is associated with cholesterol trafficking, neuroinflammatory responses and Aß clearance. The mechanism for the increase in apoE protein abundance is not understood. Based on different lines of evidence, we propose that the beta-adrenergic receptor (ßAR), cAMP and the transcription factor activator protein-2 (AP-2) are contributors to the Aß-induced increase in apoE abundance. This hypothesis was tested in mouse primary astrocytes and in cells transfected with an apoE promoter fragment with binding sites for AP-2. Aß(42) induced a time-dependent increase in apoE mRNA and protein levels which were significantly inhibited by ßAR antagonists. A novel finding was that Aß incubation significantly reduced AP-2α levels and significantly increased AP-2ß levels in the nuclear fraction. The impact of Aß-induced translocation of AP-2 into the nucleus was demonstrated in cells expressing AP-2 and incubated with Aß(42). AP-2 expressing cells had enhanced activation of the apoE promoter region containing AP-2 binding sites in contrast to AP-2 deficient cells. The transcriptional upregulation of apoE expression by Aß(42) may be a neuroprotective response to Aß-induced cytotoxicity, consistent with apoE's role in cytoprotection.

Brain isoprenoids farnesyl pyrophosphate and geranylgeranyl pyrophosphate are increased in aged mice.

The mevalonate/isoprenoids/cholesterol pathway has a fundamental role in the brain. Increasing age could be associated with specific changes in mevalonate downstream products. Other than age differences in brain cholesterol and dolichol levels, there has been little if any evidence on the short-chain isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), as well as downstream lipid products. The purpose of the present study was to determine whether brain levels of FPP, GGPP and sterol precursors and metabolites would be altered in aged mice (23 months) as compared to middle-aged mice (12 months) and young mice (3 months). FPP and GGPP levels were found to be significantly higher in brain homogenates of 23-months-old mice. The ratio of FPP to GGPP did not differ among the three age groups suggesting that increasing age does not alter the relative distribution of the two isoprenoids. Gene expression of FPP synthase and GGPP synthase did not differ among the three age groups. Gene expression of HMG-CoA reductase was significantly increased with age but in contrast gene expression of squalene synthase was reduced with increasing age. Levels of squalene, lanosterol and lathosterol did not differ among the three age groups. Desmosterol and 7-dehydroxycholesterol, which are direct precursors in the final step of cholesterol biosynthesis were significantly lower in brains of aged mice. Levels of cholesterol and its metabolites 24S- and 25S-hydroxycholesterol were similar in all three age groups. Our novel find ings on increased FPP and GGPP levels in brains of aged mice may impact on protein prenylation and contribute to neuronal dysfunction observed in aging and certain neurodegenerative diseases.

Statins and neuroprotection: a prescription to move the field forward.

There is growing interest in the use of statins, HMG-CoA reductase inhibitors, for treating specific neurodegenerative diseases (e.g., cerebrovascular disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis) and possibly traumatic brain injury. Neither is there a consensus on the efficacy of statins in treating the aforementioned diseases nor are the mechanisms of the purported statin-induced neuroprotection well-understood. Part of the support for statin-induced neuroprotection comes from studies using animal models and cell culture. Important information has resulted from that work but there continues to be a lack of progress on basic issues pertaining to statins and brain that impedes advancement in understanding how statins alter brain function. For example, there are scant data on the pharmacokinetics of lipophilic and hydrophilic statins in brain, statin-induced neuroprotection versus cell death, and statins and brain isoprenoids. The purpose of this mini-review will be to examine those aforementioned issues and to identify directions of future research.

Simvastatin stimulates production of the antiapoptotic protein Bcl-2 via endothelin-1 and NFATc3 in SH-SY5Y cells.

The use of statins for the prevention or treatment of different neurodegenerative diseases has generated considerable interest albeit with some controversy. Mechanisms of statin-induced neuroprotection are not well understood. Recently, we reported that simvastatin stimulated neuronal gene expression and protein levels of the major antiapoptotic protein Bcl-2 in vivo and in vitro; suppression of Bcl-2 in SH-SY5Y cells reduced simvastatin neuroprotection; effects were independent of cholesterol and other products of the 3-hydroxy-3-methylglutaryl-CoA reductase pathway. Endothelin-1 (ET-1) can increase Bcl-2 abundance via the transcription factor nuclear factor of activated thymocytes (NFATc), and simvastatin was reported to increase ET-1 gene expression. We tested the hypothesis that simvastatin stimulation of Bcl-2 involves up-regulation of ET-1 and binding of NFATc to Bcl-2 promoter sites in SH-SY5Y human neuroblastoma cells. Simvastatin increased both intracellular and secreted ET-1 protein levels. Exogenous ET-1 increased Bcl-2 protein abundance, which was inhibited by ET-1 receptor antagonists. Simvastatin increased translocation of NFATc3 to the nucleus while reducing nuclear NFATc1 and having no effect on NFATc4. Endothelin-1 also increased NFATc3 levels in the nucleus, and this increase was inhibited by ET-1 receptor antagonists. Treatment of cells with simvastatin stimulated binding of NFATc3 to the Bcl-2 promoter. We report novel findings showing that up-regulation of Bcl-2 by simvastatin involves ET-1 and the transcription factor NFATc3. Discovering how statins can selectively alter a specific NFATc isoform that leads to an increase in an antiapoptotic protein will provide a new approach to understanding statin-induced neuroprotection and conditions outside the brain in which apoptosis contributes to pathophysiology.

Enlarging the scope of cell-penetrating prenylated peptides to include farnesylated 'CAAX' box sequences and diverse cell types.

Protein prenylation is a posttranslational modification that is present in a large number of proteins; it has been proposed to be responsible for membrane association and protein-protein interactions, which contribute to its role in signal transduction pathways. Research has been aimed at inhibiting prenylation with farnesyltransferase inhibitors based on the finding that the farnesylated protein Ras is implicated in 30% of human cancers. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation as it occurs in living cells. Here we describe the preparation of a series of farnesylated peptides that contain sequences recognized by protein farnesyltransferase. Using a combination of flow cytometry and confocal microscopy, we show that these peptides enter a variety of different cell types. A related peptide where the farnesyl group has been replaced by a disulfide-linked decyl group is also shown to be able to efficiently enter cells. These results highlight the applicability of these peptides as a platform for further study of protein prenylation and subsequent processing in live cells.

Simvastatin protects neurons from cytotoxicity by up-regulating Bcl-2 mRNA and protein.

Statins are most commonly prescribed to reduce hypercholesterolemia; however, recent studies have shown that statins have additional benefits, including neuroprotection. Until now, the mechanism underlying statin-induced neuroprotection has been poorly understood. Recent in vivo studies from our lab reported the novel finding that simvastatin increased expression levels of a gene encoding for a major cell survival protein, bcl-2 [Johnson-Anuna et al., J. Pharmacol. Exp. Ther.312 (2005) 786]. The purpose of the present experiments was to determine if simvastatin could protect neurons from excitotoxicity by altering Bcl-2 levels. Neurons were pre-treated with simvastatin and challenged with a compound known to reduce Bcl-2 levels and induce cell death. Simvastatin pre-treatment resulted in a significant reduction in cytotoxicity (lactate dehydrogenase release and caspase 3 activation) following challenge compared with unchallenged neurons. In addition, chronic simvastatin treatment significantly increased Bcl-2 mRNA and protein levels while challenge resulted in a significant reduction in Bcl-2 protein abundance. G3139, an antisense oligonucleotide directed against Bcl-2, abolished the protective effects of simvastatin and eliminated simvastatin-induced up-regulation of Bcl-2 protein. These findings suggest that neuroprotection by simvastatin is dependent on the drug's previously unexplored and important effect of up-regulating Bcl-2.

Chronic administration of statins alters multiple gene expression patterns in mouse cerebral cortex.

Statins have been reported to lower the risk of developing Alzheimer's disease; however, the mechanism of this potentially important neuroprotective action is not understood. Lowering cholesterol levels does not appear to be the primary mechanism. Statins have pleiotropic effects in addition to lowering cholesterol, and statins may act on several different pathways involving distinct gene expression patterns that would be difficult to determine by focusing on a few genes or their products in a single study. In addition, gene expression patterns may be specific to a particular statin. To understand the molecular targets of statins in brain, DNA microarrays were used to identify gene expression patterns in the cerebral cortex of mice chronically treated with lovastatin, pravastatin, and simvastatin. Furthermore, brain statin levels were determined using liquid chromatography/tandem mass spectrometry. These studies revealed 15 genes involved in cell growth and signaling and trafficking that were similarly changed by all three statins. Overall, simvastatin had the greatest influence on expression as demonstrated by its ability to modify the expression of 23 genes in addition to those changed by all three drugs. Of particular interest was the expression of genes associated with apoptotic pathways that were altered by simvastatin. Reverse transcription-polymerase chain reaction experiments confirmed the microarray findings. All three drugs were detected in the cerebral cortex, and acute experiments revealed that statins are relatively rapidly removed from the brain. These results provide new insight into possible mechanisms for the potential efficacy of statins in reducing the risk of Alzheimer's disease and lay the foundation for future studies.

A new role for apolipoprotein E: modulating transport of polyunsaturated phospholipid molecular species in synaptic plasma membranes.

Phospholipids and their acyl group composition are important in providing the proper membrane environment for membrane protein structure and function. In particular, the highly unsaturated phospholipids in synaptic plasma membranes in the CNS are known to play an important role in modulating receptor function and neurotransmitter release processes. Apolipoprotein E (apoE) is a major apolipoprotein in the CNS, mediating the transport of cholesterol, phospholipids and their fatty acids, particularly in reparative mechanisms during neuronal injury. This study was performed to determine whether deficiency in the apoE gene contributes to an alteration of the phospholipids in synaptic plasma membranes. Phospholipid molecular species were identified and quantitated by HPLC/electrospray ionization-mass spectrometry. Analysis of the different phospholipid classes in membranes of apoE-deficient and C57BL/6 J mice indicated no obvious differences in the distribution of different phospholipid classes but substantial differences in composition of phospholipid molecular species. Of special interest was the prevalence of phospholipids (phosphatidylcholine, diacyl-phosphatidylethanolamine, and phosphatidylserine) with 22:6n-3 in both the sn-1 and sn-2 positions of SPM and these phospholipid species were significantly higher in apoE-deficient mice as compared to control mice. Since polyunsaturated fatty acids in neurons are mainly supplied by astrocytes, these results revealed a new role for apoE in regulating polyunsaturated phospholipid molecular species in neuronal membranes.