Urule Igbavboa

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

Statins have been reported to lower the risk of developing Alzheimers 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 Alzheimers disease and lay the foundation for future studies.

Increasing Age Alters Transbilayer Fluidity and Cholesterol Asymmetry in Synaptic Plasma Membranes of Mice

Abstract: Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3–4‐, 14–15‐, and 24–25‐month‐old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)‐quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol‐to‐phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins.

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 Alzheimers disease. There is a growing body of evidence showing an association between cholesterol and Alzheimers 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 Alzheimers 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.

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 Alzheimers 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.

Distribution and fluidizing action of soluble and aggregated amyloid beta-peptide in rat synaptic plasma membranes.

The effects of soluble and aggregated amyloid β-peptide (Aβ) on cortical synaptic plasma membrane (SPM) structure were examined using small angle x-ray diffraction and fluorescence spectroscopy approaches. Electron density profiles generated from the x-ray diffraction data demonstrated that soluble and aggregated Aβ1–40 peptides associated with distinct regions of the SPM. The width of the SPM samples, including surface hydration, was 84 Å at 10 °C. Following addition of soluble Aβ1–40, there was a broad increase in electron density in the SPM hydrocarbon core ±0–15 Å from the membrane center, and a reduction in hydrocarbon core width by 6 Å. By contrast, aggregated Aβ1–40 contributed electron density to the phospholipid headgroup/hydrated surface of the SPM ±24–37 Å from the membrane center, concomitant with an increase in molecular volume in the hydrocarbon core. The SPM interactions observed for Aβ1–40 were reproduced in a brain lipid membrane system. In contrast to Aβ1–40, aggregated Aβ1–42intercalated into the lipid bilayer hydrocarbon core ±0–12 Å from the membrane center. Fluorescence experiments showed that both soluble and aggregated Aβ1–40 significantly increased SPM bulk and protein annular fluidity. Physico-chemical interactions of Aβ with the neuronal membrane may contribute to mechanisms of neurotoxicity, independent of specific receptor binding.

Amyloid β‐Peptides Increase Annular and Bulk Fluidity and Induce Lipid Peroxidation in Brain Synaptic Plasma Membranes

Abstract: Amyloid β‐peptides (Aβ) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of Aβ1–40 and Aβ25–35 on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer‐eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly (p≤ 0.02) by Aβ1–40. Similar effects on fluidity were observed for Aβ25–35 (p≤ 0.002). Increased fluidity was associated with lipid peroxidation. Both Aβ peptides significantly increased (p≤ 0.006) the amount of malondialdehyde in SPM. The addition of a water‐soluble analogue of vitamin E (Trolox) inhibited effects of Aβ on lipid peroxidation and fluidity in SPM. The fluidizing action of Aβ peptides on SPM may be due to the induction of lipid peroxidation by those peptides. Aβ‐induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.

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, Parkinsons disease, Alzheimers 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.

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

Inheritance of the apolipoprotein (apoE) ϵ4 allele is a risk factor for developing Alzheimers 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 ∼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.

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.