Impact of HMG-CoA reductase inhibition on brain pathology

Review

TRENDS in Pharmacological Sciences

Vol.28 No.7

Impact of HMG-CoA reductase inhibition on brain pathology Frauke Zipp1*, Sonia Waiczies1*, Orhan Aktas1, Oliver Neuhaus2, Bernhard Hemmer2, Burkhard Schraven3, Robert Nitsch4 and Hans-Peter Hartung2 1

Cecilie-Vogt-Clinic for Molecular Neurology, Charite´ – Universitaetsmedizin Berlin, and Max-Delbrueck-Center for Molecular Medicine, 10117 Berlin, Germany 2 Department of Neurology, Heinrich-Heine-University, 40225 Dusseldorf, Germany 3 Institute of Immunology, Otto von Guericke University, 39106 Magdeburg, Germany 4 Institute of Cell Biology and Neurobiology, Center for Anatomy, Charite´–Universitaetsmedizin Berlin, 10117 Berlin, Germany

Over the past two decades, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HMGCRIs), originally designed to lower cholesterol blood levels, have been found to affect GTPase signaling during normal intracellular tasks. This finding has prompted use of these drugs in pathological situations, where such signaling processes need to be manipulated. Here, we review recent progress on the outcome of modulating GTPase signaling after inhibition of protein prenylation by HMGCRIs. We also discuss current controversies over the direct implications of these cholesterol-lowering agents on cholesterolrich membrane lipid rafts and associated signaling. By reviewing these two different cellular events and the evidence from clinical studies, an overall assessment can be made of the concept of interfering with the HMG-CoA reductase pathway in different brain pathologies. We thereby provide a rational link between the benefit of applying HMGCRIs in brain pathologies, such as multiple sclerosis, Alzheimer’s disease and stroke, and the impact on signaling in specific cell types crucial to disease pathogenesis.

Introduction The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGCR) pathway is an important metabolic route that is present in almost every organism. It is a rich source of hydrophobic molecules including cholesterol and isoprenoids, which are important for several inter- and intracellular functions including hormonal communication, protein synthesis, cell membrane maintenance (via cholesterol synthesis) and protein lipid modifications (via isoprenoid synthesis). The transmembrane protein HMGCR, the key enzyme in the cholesterol pathway, regulates the rate-limiting step leading to mevalonate (Figure 1) and is antagonized by a group of drugs known as statins or HMGCR inhibitors (HMGRIs), which were originally developed to reduce cholesterol levels (Box 1). Thus, the cholesterol-rich membrane lipid rafts and subsequent signaling might be altered by these drugs. *

Corresponding author: Zipp, F. ([email protected]). Authors contributed equally. Available online 15 June 2007.

www.sciencedirect.com

The isoprenoids downstream of mevalonate are essential for switching on the function of GTPase (i.e. GTP-binding or G protein) molecules by post-translational lipid modifications – a process known as ‘isoprenylation’ (Box 2). GTPases are involved in many signaling events including those leading to inflammation. Indeed, the established role of HMGRIs in blocking cholesterol synthesis has been shown to extend to alterations in GTPase signaling. Likewise, the medical significance of the HMGCR pathway has also expanded beyond a direct role in cholesterol synthesis. In vascular conditions comprising lipid-dependent and inflammatory processes, HMGRIs could offer health benefits both through and beyond cholesterol reduction. Here, we attempt to link our knowledge of the therapeutic outcomes of HMGCRIs in the neurological diseases multiple sclerosis (MS), Alzheimer’s disease (AD) and stroke with molecular mechanistic evidence obtained from in vitro and in vivo experiments. Relevance of inhibiting HMGCR to CNS pathology The brain is the organ that is richest in cholesterol. Two per cent of the adult human brain is made up of cholesterol, which is mostly derived from local de novo synthesis. The outcome of manipulating cholesterol and isoprenoid synthesis with HMGCRIs, and thereby intracellular signaling, has thus been studied in various brain pathologies, including neuroinflammation. When applying HMGCRIs to diseases of the central nervous system (CNS), it is necessary to be aware that physical properties determine the capacity of these substances to cross the blood–brain barrier (BBB): whereas lipophilic HMGCRIs such as simvastatin and lovastatin easily reach the CNS compartment [1], more hydrophilic HMGCRIs such as fluvastatin and pravastatin do not cross the intact BBB and are not detectable in the brain parenchyma [2]. These properties should be taken into consideration when extrapolating information from in vitro studies to CNS pathological situations because the same drug levels might not be reached in vivo, although the BBB is at least transiently permeable in diseases such as MS and stroke. Furthermore, in vitro studies in rat hippocampal neuron cultures using different HMGCRIs have yielded ambiguous results: whereas lovastatin has been shown to disrupt

0165-6147/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2007.05.001

Review

TRENDS in Pharmacological Sciences

Vol.28 No.7

343

Figure 1. Chain of reactions in the HMGCR pathway leading to isoprenoid and cholesterol synthesis. HMG-CoA reductase (HMGCR) is the key and rate-limiting enzyme in the pathway. HMGCR inhibitors (HMGCRIs) displace HMGCR and inhibit the reduction of its natural substrate HMG-CoA to mevalonate. Subsequently, mevalonate is converted to isopentenyl pyrophosphate (Ipp), which isopentenylates tRNA such as selenocysteine (Sec)-tRNA and can be isomerized into dimethylallyl diphosphate (DMApp). These two isomers are the building blocks for the isoprenoids geranyl pyrophosphate (Gpp), farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (GGpp). Synthesis of Gpp and Fpp by Fpp synthase is targeted by biphosphonates, whereas the covalent attachment of Fpp and GGpp to GTP-binding protein (e.g. Ras, Rho and Rac) by prenyl transferases (farnesyl and geranylgeranyl transferases) is targeted by the respective prenyl transferase inhibitors FTI (farnesyl transferase inhibitor) and GGTI (geranylgeranyl transferase inhibitor). Other cholesterol-lowering agents, still under investigation, target enzymes downstream in the pathway: for example, squalene synthase (SQS) inhibitors target the synthesis of the cholesterol precursor squalene and oxidosqualene cyclase (OSC) inhibitors target lanosterol synthesis.

structural integrity [3], pravastatin used at even higher doses has been found to increase neurite extensions [4]. Differences in incubation conditions should also be taken into account when evaluating in vitro studies: whereas nanomolar concentrations of HMGCRIs protect primary neuron cultures from glutamate-mediated excitotoxic injury [5], micromolar concentrations induce apoptosis in neuronal cultures [6]. Similarly, short-time incubation of oligodendroyctes with HMGCRIs results in enhanced process extensions and differentiation, whereas prolonged incubation is detrimental to the same cultures [7]. Thus, when determining the significance of in vitro experiments for in vivo studies, both molecular properties and dosage should be www.sciencedirect.com

considered as important factors that determine the net effect in vivo. HMGCRIs have provided a new therapeutic concept for treating neuroinflammatory diseases because of their capacity to alter GTPase-mediated signaling relevant to inflammatory processes. The first evidence of the antiinflammatory property of HMGCRIs came from studies reporting an association between decreased natural killer cell cytotoxicity and cardiac transplantation success independent of altered cholesterol levels [8]. In antigen-presenting cells, HMGCRIs block the induced expression of major histocompatibility complex (MHC) class II antigens in vitro by suppressing the inducible promoter IV of the

344

Review

TRENDS in Pharmacological Sciences

Vol.28 No.7

Box 1. Statins

Box 2. Isoprenylation of GTPase molecules

In 1975 a Japanese scientist, Akira Endo, discovered HMGCR inhibitors (HMGCRIs) as natural products of some moulds [61]. Further studies by Endo led to the discovery and isolation of potent HMGCRIs of the mevastatin family. Since then, several other therapeutically active statins have been developed. These inhibitors can be subdivided into two groups: natural compounds derived from fungal fermentation (mevastatin, lovastatin, simvastatin and pravastatin), and fully synthetic compounds (fluvastatin, atorvastatin, rosuvastatin and pitavastatin). Some HMGCRIs are present as inactive lactone pro-drugs and are enzymatically hydrolyzed to their active hydroxy-acid form in vivo. HMGCRIs share a common moiety, which is structurally similar to HMG-CoA. This moiety enables them to bind competitively to HMGCR, displacing its natural substrate HMG-CoA and inhibiting its reduction to L-mevalonate.

GTPase molecules require GTP coupling and isoprenylation to become functionally active and to be capable of regulating their diverse cellular tasks. The latter post-translational modification (also known as prenylation) is required for the GTPase molecules to anchor to the plasma membrane, where they can transmit signaling messages efficiently. Isoprenylation occurs when isoprenoid products of the HMGCR pathways – namely farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (GGpp) – attach by covalent bonds to conserved CAAX motifs in the carboxyl terminus of the GTPases. This modification is brought about with the help of prenyl transferases (FTase or GGTase I), which help to transfer the isoprenoid fatty chain to these signaling molecules. Which isoprenoid attaches to which GTPase depends on the CAAX motif on the signaling molecule: C is cysteine, A is any aliphatic amino acid, and X is the amino acid that determines which prenyltransferase will transfer which isoprenoid to the target protein. For example, serine for H-ras or methionine for N-ras determines that Fpp attaches with the help of the prenyltransferase FTase, whereas leucine for RhoA determines that GGpp attaches with the help of the prenyltransferase GGTase I.

transactivator CIITA [9]. Also of relevance to CNS pathology, HMGCRIs interfere with the induction of tumor-necrosis factor-a (TNF-a) and inducible nitric oxide synthase (NOS) in astrocyte and microglia cultures [10]. Because damage processes including inflammation, oxidative stress and excitototoxic injury are thought to contribute to CNS pathologies such as MS, AD and stroke, these mechanistic insights might explain the possible protective effects of HMGCRIs in CNS pathology (Table 1).

Multiple sclerosis The pathogenesis of the neuroinflammatory disease MS is thought to be initiated by autoreactive T cells, which infiltrate the CNS and, in cooperation with other immune

Table 1. Relevance of inhibiting HMGCR to CNS pathologya CNS pathology Multiple sclerosis

HMGCRI treatment In vitro Atorvastatin blocks cell cycle and induces anergy in myelin-specific T cells [13,15] Lovastatin and simvastatin reduce immune cell (MS patients) migration through BBB endothelial cells [16] Simvastatin, lovastatin, mevastatin downregulate immune cell (MS patients) chemokine receptor and adhesion molecules; simvastatin diminishes MMP9 secretion [17]

Alzheimer’s disease

Simvastatin reduces b-amyloidmediated microglial neurotoxicity [26]

In vivo animal models Atorvastatin reduces disease score in EAE and prevents first relapse [13,14]; T cells from treated mice protected recipient mice from developing EAE [14] Atorvastatin downregulates MHC class II expression in CNS [14]

In vivo human Two explorative clinical studies (lovastatin in 7 RRMS patients [22] and simvastatin in 30 RRMS patients [21]) revealed possible benefits on disease activity in the MRI and no serious adverse events

Atorvastatin reduces inflammatory cytokines and promotes regulatory cytokines [13,14] Lovastatin enhances oligodendrocyte differentiation and remyelination [20]

Current larger-scale controlled clinical trials are ongoing

Simvastatin reduces cerebral levels of Ab but not total brain cholesterol levels in guinea pigs [24]

Pilot trial in 63 patients revealed possible clinical benefit of atorvastatin [27] Epidemiological study in 1364 patients revealed that previous HMGCRI treatment lowers risk of developing dementia [28] Meta-analysis of 7 independent trials did not show beneficial outcome [29] Phase III trial currently underway to clarify controversies in previous clinical studies

Simvastatin and lovastatin reduce infarct volume and increase blood flow through eNOS production in MCAo ischemia model [32] Simvastatin and atorvastatin increase neurotrophic factors and amplify brain plasticity and neurogenesis in MCAo ischemia model [36]

Lovastatin and simvastatin reduce monocyte CD11b and adhesion to endothelium ex vivo in hypercholesterolemic patients [35] Atorvastatin reduces risk of ischemic but not hemorrhagic stroke in patients with stroke or TIA history [31]

Neuronal cholesterol depletion by lovastatin and methyl-b-cyclodextrin reduces rate of Ab accumulation [23]

Stroke

lovastatin inhibits NOS and inflammation in astrocytes, microglia, macrophages [10] Simvastatin decreases tPA-induced MMP9 in cortical astrocytes [34] Nanomolar amounts of statins (rosuvastatin being most active) protect primary neurons from glutamate-mediated injury [5]

a Abbreviations: Ab, amyloid-b peptide; BBB, blood–brain barrier; EAE, experimental autoimmune encephalomyelitis; MCAo, middle cerebral artery occlusion; MMP9, matrix metalloproteinase-9; MRI, magnetic resonance imaging; NOS, nitric oxide synthase; RRMS, relapsing–remitting multiple sclerosis; TIA, transient ischemic attack; tPA, tissue plasminogen activator stroke therapy.

www.sciencedirect.com

Review

TRENDS in Pharmacological Sciences

cells including local microglia, then perpetuate the tissue destructive processes involving oxidative stress (Figure 2). The principal oxysterol 7-ketocholesterol induces microglia-mediated neuronal damage [11]. Thus, the anti-oxidative properties of HMGCRIs [12] represent a way of preventing cholesterol oxidation and formation of harmful oxysterol and thereby provide a rationale for attenuating inflammatory processes by HMGCR regulation. It was assumed that properties of HMGCRIs targeting the immune system outside the CNS would make these agents candidate therapies for chronic neuroinflammation. Indeed, the disease-ameliorating effect of HMGCRIs has been demonstrated in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE) [13,14]. In these in vivo studies, the HMGCRI atorvastatin reduced inflammation by diminishing the production of the inflammatory cytokines interferon-g, TNF-a, interleukin-12 (IL-12) and IL-2, while promoting the synthesis of regulatory cytokines including IL-4, IL-10 and transforming growth factor-b. In addition, in vivo treatment downregulated MHC class II expression in the CNS. It remains unclear whether HMGRIs directly block the migration of CNS antigenspecific cells into the brain or whether they act earlier at a more proximal step by regulating these cells outside the brain, as indicated by the following observations. First, in the mouse model T cells from HMGCRI-treated mice

Vol.28 No.7

345

protected recipient mice from developing EAE [14]. Second, in human T cells HMGCRIs block the cell-cycle in vitro [13] and render these cells anergic through extracellular-signal-regulated kinase (ERK)-mediated processes [15]. However, the capacity of HMGCRIs to reduce the permeability of BBB-derived endothelial cells and the migration of leukocytes, demonstrated in vitro, might also contribute to the salutary effects of this therapy [16]. HMGCRIs have indeed been shown to downregulate the expression of chemokine receptors and T-cell adhesion molecules and to diminish the secretion of matrix metalloproteinase-9 (MMP9) by peripheral immune cells derived from individuals affected with MS [17]. In MS, myelin and oligodendrocytes are among the main targets of immunemediated injury. HMGCRIs have been previously reported to inhibit oligodendrocyte differentiation and synthesis of myelin proteins in vitro [18,19]. In EAE, however, the beneficial effect of the HMGCRI lovastatin, which readily diffuses into the CNS compartment, has been linked to enhanced survival of oligodendrocyte progenitor cells and their differentiation to remyelinating oligodendrocytes in vivo [20]. Treatment with HMGCRIs in the mouse model of MS not only resulted in significant reductions in disease scores, but also prevented the incidence of first relapses after initiation of treatment [13,14]. This observation is significant for the

Figure 2. Impact of HMGCRIs on specific cell types relevant to the pathogenesis of MS. HMGCRIs target various cells of the immune system. Originally, decreased natural killer (NK) cell toxicity was reported in conjunction with successful transplantation. Later, induced MHC class II molecule (MHCII) expression was shown to be regulated by HMGCRIs in many antigen-presenting cells (APC) including B cells. T cells are also targeted directly by HMGCRIs independently of antigen presentation: for example, myelin-specific T cells are rendered anergic after blockade of the cell cycle and T-cell migration is impaired as a result of disrupted Rho function and decreased MMP9 secretion. HMGCRIs also target CNS-specific cell types including neurons and microglia. Neurorestorative properties of HMGCRIs are associated with ERK1 phosphorylation and inhibition of microglia-mediated inflammation (decreased release of inflammatory mediators and oxidation products), accounting for reduced neuroinflammation. www.sciencedirect.com

346

Review

TRENDS in Pharmacological Sciences

treatment of individuals suffering from relapsing–remitting MS. In two smaller open-label trials of HMGCRIs in individuals with MS, beneficial effects have been revealed on magnetic resonance imaging of the brain [21,22]. These promising data are currently undergoing evaluation in larger-scale controlled clinical trials. Alzheimer’s disease The neurodegenerative disease AD presents with a progressive deterioration of brain function, culminating in profound dementia. A histopathological hallmark of AD is the accumulation of abnormal amyloid, which has been associated with high intracellular cholesterol levels. In vitro, cholesterol depletion of cultured neurons after treatment with both HMGCRIs and methyl-b-cyclodextrin, which physically extracts membrane cholesterol, results in a reduced rate of b-amyloid accumulation [23]. This reduced accumulation has also been observed in vivo in guinea-pigs, which were found to show a strong reduction of amyloid-b peptides (Ab) in the CNS after treatment with HMGCRIs. Notwithstanding the beneficial role of HMGCRIs in this in vivo model, no changes in cholesterol levels were observed in the same compartment [24], suggesting that HMGCRIs provide benefits above and beyond cholesterol reduction. Indeed, microglia-mediated inflammatory responses are also thought to contribute to the progression of AD [25], and HMGCRIs reduce b-amyloid-mediated microglial neurotoxicity in vitro independently of cholesterol lowering through isoprenoid depletion [26]. The beneficial role of HMGCRIs in controlling microglial neurotoxicity provides a rationale for their therapeutic effects in AD. Indeed, in a placebo-controlled pilot trial with 80 mg atorvastatin in individuals with mild to moderate AD, this HMGCRI was significantly superior to placebo on some AD assessment scales [27]. Furthermore, in an epidemiological study of the effect of HMGCRIs on dementia, Jick et al. [28] observed that individuals aged 50 years and older who were prescribed HMGCRIs had a substantially lowered risk of developing dementia, independent of cholesterol status. In that study, however, AD was not distinguished from other forms of dementia, and its findings have been not confirmed in a recent meta-analysis of seven independent trials [29]. A phase III trial, the ‘CLASP-AD’ (cholesterol lowering agent to slow progression of Alzheimer’s disease) trial, is currently underway to clarify the inconsistencies evident in the previous clinical studies. Stroke Hypercholesterolemia is one risk factor, among others, for the development of atherosclerosis and thus stroke. Although it remains to be elucidated whether the therapeutic efficacy of HMGCRIs is due to their cholesterol-lowering properties [30], treatment with HMGCRIs does curtail the risk of stroke development. The 2006 SPARCL (stroke prevention by aggressive reduction in cholesterol levels) trial was designed to determine whether HMGCRIs reduce the risk of secondary stroke in individuals with a history of stroke or transient ischemic attacks, but with no known coronary heart disease in the 6 months before study entry. This study reported that HMGCRI treatment produced a www.sciencedirect.com

Vol.28 No.7

5-year absolute reduction in risk of developing a subsequent ischemic stroke (P = 0.03), in contrast to hemorrhagic stroke – the risk of which increased slightly [31]. Another potential mechanism underlying the beneficial role of HMGCRIs in stroke is the upregulation of endothelial NOS (eNOS) and thereby enhanced production of endothelial nitric oxide [32], which increases cerebral vascular tone and augments blood flow to ischemic regions. This mode of action, along with the anti-oxidative properties of HMGCRIs, might offer neuroprotection during stroke, because compromised brain perfusion and subsequent breakdown of energy supply will ultimately lead to neuronal death. A pathological hallmark of ischemic stroke lesions and atherosclerotic plaques is inflammation [25,33], which can be targeted by HMGCRIs [10]. In addition to their impact on microglia, HMGCRIs have been shown to decrease the production of MMP9 induced by tissue plasminogen activator in cortical astrocyte cultures [34]. Furthermore, in individuals with hypercholesterolemia, HMGCRIs diminish monocyte CD11b expression and cause a significant reduction in monocyte adhesion to endothelium ex vivo [35]. These anti-inflammatory properties represent possible mechanisms by which HMGCRIs protect the neurovascular unit and prevent brain injury. HMGCRIs also show direct neuroprotective properties in damage events in vitro: in contrast to micromolar concentrations [6], nanomolar amounts of HMGCRIs protect primary neuronal cultures from glutamate-mediated injury [5]. In vivo, HMGCRIs have been shown to increase levels of neurotrophic factors, thereby amplifying endogenous brain plasticity and neurogenesis in a rat model of ischemic stroke [36]. Beyond lipid-lowering towards altered cellular signaling In addition to their established role in lipid lowering, HMGCRIs have been gradually shown to control a wide range of cellular functions. Some research groups have attributed these functions to alterations in the cholesterol-rich membrane microdomains known as ‘lipid rafts’. Changes in cellular activities by HMGCRIs have, however, mostly been related to interference with signaling mediated by isoprenylated G proteins (Box 2), including both the heterotrimeric G protein and, more commonly, the small GTPases of the Ras superfamily. Although the role of Ras signaling in the immune system is relatively well established, its role in the CNS remains a subject of debate. Modulation of small GTPase signaling by HMGCRIs By targeting Ras isoprenylation, HMGCRIs are thought to block the evolutionarily conserved Ras–ERK signaling cascade, which is important for cell-cycle progression in various cell types including T cells, and thus to exert a beneficial anti-inflammatory effect. In neurons, however, the induction of Ras signaling presents a therapeutic strategy in neurodegenerative disorders, because it seems to be important for survival and neuroprotection (Box 3). The blocking of Ras isoprenylation by HMGCRIs, which is assumed to attenuate Ras effector signaling, would then prove detrimental. HMGCRIs have been shown, however, to promote Ras signaling in vitro in two investigations: one in the context of angiogenesis [37], and the other in T cells

Review

TRENDS in Pharmacological Sciences

Box 3. Ras signaling in neurons Neurons exit the cell cycle and switch on survival pathways, after prolonged and sustained Ras–ERK signaling, to avoid undergoing apoptosis [62]. Furthermore, uncontrolled Ras signaling in animals lacking neurofibromin (NF1), a negative regulator of Ras, delays apoptosis of sensory neurons during growth factor withdrawal [63]. Indeed, transgenic activation of Ras leading to selective ERK activation prevents neurodegeneration in vivo [64]. Moreover, ERK signaling has been shown to be important for ectodomain shedding of amyloid precursor protein (APP) into a cleaved soluble form (sAPPa), which is neuroprotective in the pathogenesis of AD [65]. The role of Ras signaling in brain plasticity remains ambiguous. In the amygdala, Ras signaling is important for synaptic events leading to memory consolidation [66]. By contrast, Ras deficiency increases long-term CA1 potentiation in the hippocampus, thereby implying that Ras has an adverse role in memory consolidation [67], and NF1-deficient mice with uncontrolled Ras signaling have spatial learning deficiencies [68]. Similarly, individuals with von Recklinghausen disease characterized by mutations in the NF1 gene have impaired cognitive functions [69]. These latter studies seem to implicate Ras signaling in impaired brain plasticity.

as described earlier [15]. These in vitro findings thus constitute mechanistic evidence for a beneficial role of HMGCRI-promoted Ras–ERK signaling in vivo. In experimental stroke, increased brain plasticity and restoration of neuronal function after HMGCRI administration in vivo might be attributable to increased Ras–ERK signaling in cortical neurons, as demonstrated in vitro [36]. The role of Ras in brain plasticity is, however, controversial (Box 3). Further in vivo experiments should aim at clarifying the mechanisms by which HMGCRIs might promote rather than inhibit Ras signaling in the CNS, notwithstanding decreased isoprenylation. HMGCRIs also target the Rac–stress kinase pathway and the Rab-dependent pathway of receptor endocytosis [38]. Whereas the Rab-family GTPases function as key regulators of intracellular trafficking, Rac belongs to the family of Rho GTPases, which are crucially involved in cytoskeletal organization. Rho signaling is usually triggered after interaction of immune cells with endothelial cells, thus leading to the necessary structural rearrangements in both cell types during tissue infiltration [39]. Indeed, treatment of brain endothelial cells with HMGCRIs in vitro inhibits Rho-mediated transendothelial T-cell migration [40]. Whereas Ras isoprenylation has been previously shown to be important for neurite outgrowth during neuronal differentiation in vitro [41], Rho isoprenylation seems to disturb neurite outgrowth in hippocampal neuronal cultures [4]. Rho GTPases also have essential roles in eNOS generation, thus promoting cerebral blood flow to ischemic regions of the brain [42]. In addition, by disrupting Rho-mediated cytoskeleton and inhibiting phagocytosis in vitro, HMGCRIs block the inflammatory actions of Ab-stimulated microglia [43]. By contrast, inhibition of HMGCR and geranylgeranyl pyrophosphate (GGpp) has been reported to enhance microglial activation in hippocampal slice cultures [44]. These controversial in vitro reports on the HMGCRI actions on Rho-mediated changes further accentuate the need to improve our understanding of the mechanism of action of these agents in vivo. Furthermore, it should be noted that activation of the GTP-GDP exchange factor for Rho might be mediated by, among others, the www.sciencedirect.com

Vol.28 No.7

347

GPCR-associated a subunit (Gsa) of the G12/13 family of heterotrimeric G proteins, which are also targeted by HMGCRIs. In sphincter muscle rings, HMGCRIs decrease membrane Gsa protein content and impair neurogenic relaxation, possibly by disrupted G-protein coupling [45]. In neuronal growth cones, however, HMGCRIs target the membrane localization of the bg-subunit and not Gsa [46]. Overall, in vitro studies on the impact of HMGCRIs on small GTPases greatly outweigh studies on heterotrimeric G proteins, particularly in the CNS. Influence of HMGCRIs on lipid raft signaling Cholesterol accounts for 20–25% of lipids in the plasma membrane, is important for membrane permeability, fluidity and interactivity with proteins [47], and is suggested to be the chief component of lipid microdomains, also known as lipid rafts, in the outer leaflet [48]. Lipid rafts have been proposed to be important signaling structures [49]. Because HMGCRIs block cholesterol synthesis, disruption of lipid raft integrity by these drugs has been investigated. Although in vitro studies of neuronal structures [50] and immune cells [51] report a disruption in lipid raft integrity after treatment with HMGCRIs, other studies show that HMGCRIs do not alter intracellular cholesterol levels or raft integrity [26,38]. We ourselves did not find alterations in lipid raft fractions of human peripheral mononuclear immune cells on HMGCR inhibition (F. Zipp et al., unpublished). Furthermore, treatment of mice with HMGCRIs does not alter the cholesterol content of T cells [52]. In the CNS, high brain cholesterol levels have been implicated in the pathogenesis of AD. Unfortunately, in vivo data on these levels after HMGCRI treatment are conflicting: whereas brain cholesterol levels in mice were reduced after treatment with 100 mg/kg of lovastatin [53], no reduction was observed in guinea pigs treated with much higher doses of simvastatin over the same period of time [24]. Notably, the cholesterol content of lipid rafts and subsequent signaling in vitro are altered when extreme conditions are introduced to the cultures, such as those following serum deprivation or physical extraction of membrane cholesterol [38]. Overall, studies investigating the mechanisms of action of HMGCRIs in the various brain pathologies have been fruitful, but in some cases they have yielded conflicting results. The impact of HMGCRIs on lipid rafts has been intensively researched, although so far only in vitro. It should also be kept in mind that the fundamental nature of these microdomains in receptor signaling has been questioned in recent years [54]. Because there are indications of a two-pronged role of GTPase signaling in the nervous system, the influence of HMGCRIs on GTPase signaling in neurons, which has so far been studied in vitro, also needs to be substantiated in vivo. Concluding remarks In addition to drugs that inhibit HMGCR, other therapeutic strategies have been designed to manipulate the cholesterol biosynthetic pathway. Inhibitors of the prenyltransferases FTase and GGTase I have already yielded promising results in cancer therapy and are now gaining attention in animal

348

Review

TRENDS in Pharmacological Sciences

models of autoimmunity including neuroinflammation [52]. In conditions where regulation of GTPases is not required, such as in lipid disorders without an inflammatory element, attempts have been made to develop lipid-lowering agents that target enzymes downstream in the HMGCR pathway, in particular enzymes beyond farnesyl pyrophosphate synthase. Such agents include inhibitors of oxidosqualene cyclases, which are enzymes that catalyze lanosterol synthesis. These inhibitors, however, have been associated with significant systemic toxicology in dogs and mice [55]. Another group of potent cholesterol blockers are the so-called squalene synthase (SQS) inhibitors, although, in contrast to HMGCRIs, tolerability of these drugs remains to be assessed thoroughly. Indeed, SQS is essential for development of the CNS: knockout mice show severe growth retardation and defective neural tube closure [56]. HMGCRdeficient mice are embryonic lethal, by contrast, suggesting that this enzyme has a crucial role during early development [57]. In normocholesterolemic dogs, HMGCRI treatment was found to result in neurotoxicity but no changes in brain cholesterol levels when given at a dose 180 times the maximum therapeutic dose in man [58]. Adverse reactions on application of HMGCRIs, including polyneuropathies, myopathy and autoimmune (mostly lupus-like) reactions, have been observed in individuals at a late stage of therapy. Myopathy is a relatively rare drug reaction (1 in 1000) and rhabdomyolysis is even rarer (1 in 10 000). The hepatoselectivity of these agents might be one reason for their relatively good tolerability. Two separate recent studies systematically reviewed public safety data on marketed HMGCRIs and concluded that HMGCRIs present a highly favorable benefit-to-risk ratio, although a threshold dose exists beyond which risks outweigh benefits [59,60]. HMGCRIs clearly show immune regulatory and antiinflammatory functions. Although evidence has been provided for their beneficial role in pathological conditions of the nervous system, their direct effects on CNS target cells – for those inhibitors that reach the CNS – has not been fully clarified. Owing to its contribution to cholesterol and isoprenoid synthesis, however, the HMGCR pathway is involved in fundamental processes in the nervous and immune system that are involved in CNS pathology in MS, AD and stroke. Accordingly, initial clinical trials in these diseases suggest the therapeutic potential of interfering with this pathway.

4

5

6

7 8 9 10

11

12

13

14

15 16

17 18

19 20

21 22 23

Acknowledgements The original work cited in this article was supported by the German Research Foundation (DFG: SFB 507 and SFB 650), the Federal Ministry of Education and Research (BMBF), and the Hertie Foundation for MS Research (IMSF), Goettingen. We thank A. Mason and A. Noon for reading this manuscript as native English speakers.

24

25

References 1 Botti, R.E. et al. (1991) Concentrations of pravastatin and lovastatin in cerebrospinal fluid in healthy subjects. Clin. Neuropharmacol. 14, 256– 261 2 Hamelin, B.A. and Turgeon, J. (1998) Hydrophilicity/lipophilicity: relevance for the pharmacology and clinical effects of HMG-CoA reductase inhibitors. Trends Pharmacol. Sci. 19, 26–37 3 Meske, V. et al. (2003) Blockade of HMG-CoA reductase activity causes changes in microtubule-stabilizing protein tau via suppression of www.sciencedirect.com

26

27

28

Vol.28 No.7

geranylgeranylpyrophosphate formation: implications for Alzheimer’s disease. Eur. J. Neurosci. 17, 93–102 Pooler, A.M. et al. (2006) The 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitor pravastatin enhances neurite outgrowth in hippocampal neurons. J. Neurochem. 97, 716–723 Zacco, A. et al. (2003) 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors protect cortical neurons from excitotoxicity. J. Neurosci. 23, 11104–11111 Tanaka, T. et al. (2000) Geranylgeranyl-pyrophosphate, an isoprenoid of mevalonate cascade, is a critical compound for rat primary cultured cortical neurons to protect the cell death induced by 3-hydroxy-3methylglutaryl-CoA reductase inhibition. J. Neurosci. 20, 2852– 2859 Miron, V.E. et al. (2007) Simvastatin regulates oligodendroglial process dynamics and survival. Glia 55, 130–143 Kobashigawa, J.A. et al. (1995) Effect of pravastatin on outcomes after cardiac transplantation. N. Engl. J. Med. 333, 621–627 Kwak, B. et al. (2000) Statins as a newly recognized type of immunomodulator. Nat. Med. 6, 1399–1402 Pahan, K. et al. (1997) Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages. J. Clin. Invest 100, 2671– 2679 Diestel, A. et al. (2003) Activation of microglial poly(ADP-ribose)polymerase-1 by cholesterol breakdown products during neuroinflammation: a link between demyelination and neuronal damage. J. Exp. Med. 198, 1729–1740 Suzumura, K. et al. (1999) Protective effect of fluvastatin sodium (XU-62320), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on oxidative modification of human low-density lipoprotein in vitro. Biochem. Pharmacol. 57, 697–703 Aktas, O. et al. (2003) Treatment of relapsing paralysis in experimental encephalomyelitis by targeting Th1 cells through atorvastatin. J. Exp. Med. 197, 725–733 Youssef, S. et al. (2002) The HMG-CoA reductase inhibitor, atorvastatin, promotes a Th2 bias and reverses paralysis in central nervous system autoimmune disease. Nature 420, 78–84 Waiczies, S. et al. (2005) Atorvastatin induces T cell anergy via phosphorylation of ERK1. J. Immunol. 174, 5630–5635 Ifergan, I. et al. (2006) Statins reduce human blood–brain barrier permeability and restrict leukocyte migration: relevance to multiple sclerosis. Ann. Neurol. 60, 45–55 Neuhaus, O. et al. (2002) Statins as immunomodulators: comparison with interferon-b1b in MS. Neurology 59, 990–997 Langan, T.J. and Volpe, J.J. (1987) Oligodendroglial differentiation in glial primary cultures: requirement for mevalonate. J. Neurochem. 48, 1804–1808 Sepp-Lorenzino, L. et al. (1994) Isoprenylated proteins in myelin. J. Neurochem. 62, 1539–1545 Paintlia, A.S. et al. (2005) HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis. FASEB J. 19, 1407–1421 Vollmer, T. et al. (2004) Oral simvastatin treatment in relapsing– remitting multiple sclerosis. Lancet 363, 1607–1608 Sena, A. et al. (2003) Therapeutic potential of lovastatin in multiple sclerosis. J. Neurol. 250, 754–755 Simons, M. et al. (1998) Cholesterol depletion inhibits the generation of b-amyloid in hippocampal neurons. Proc. Natl. Acad. Sci. U. S. A. 95, 6460–6464 Fassbender, K. et al. (2001) Simvastatin strongly reduces levels of Alzheimer’s disease b-amyloid peptides Ab42 and Ab40 in vitro and in vivo. Proc. Natl. Acad. Sci. U. S. A. 98, 5856–5861 Zipp, F. and Aktas, O. (2006) The brain as a target of inflammation: common pathways link inflammatory and neurodegenerative diseases. Trends Neurosci. 29, 518–527 Cordle, A. and Landreth, G. (2005) 3-Hydroxy-3-methylglutarylcoenzyme A reductase inhibitors attenuate b-amyloid-induced microglial inflammatory responses. J. Neurosci. 25, 299–307 Sparks, D.L. et al. (2005) Atorvastatin for the treatment of mild to moderate Alzheimer disease: preliminary results. Arch. Neurol. 62, 753–757 Jick, H. et al. (2000) Statins and the risk of dementia. Lancet 356, 1627– 1631

Review

TRENDS in Pharmacological Sciences

29 Zhou, B. et al. (2007) Prevention and treatment of dementia or Alzheimer’s disease by statins: a meta-analysis. Dement. Geriatr. Cogn. Disord. 23, 194–201 30 Sandercock, P. (2001) Statins for stroke prevention? Lancet 357, 1548– 1549 31 Amarenco, P. et al. (2006) High-dose atorvastatin after stroke or transient ischemic attack. N. Engl. J. Med. 355, 549–559 32 Endres, M. et al. (1998) Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc. Natl. Acad. Sci. U. S. A. 95, 8880–8885 33 Jander, S. et al. (2007) Imaging inflammation in acute brain ischemia. Stroke 38, 642–645 34 Wang, S. et al. (2006) Reduction of tissue plasminogen activatorinduced matrix metalloproteinase-9 by simvastatin in astrocytes. Stroke 37, 1910–1912 35 Weber, C. et al. (1997) HMG-CoA reductase inhibitors decrease CD11b expression and CD11b-dependent adhesion of monocytes to endothelium and reduce increased adhesiveness of monocytes isolated from patients with hypercholesterolemia. J. Am. Coll. Cardiol. 30, 1212–1217 36 Chen, J. et al. (2003) Statins induce angiogenesis, neurogenesis, and synaptogenesis after stroke. Ann. Neurol. 53, 743–751 37 Urbich, C. et al. (2002) Double-edged role of statins in angiogenesis signaling. Circ. Res. 90, 737–744 38 Ghittoni, R. et al. (2005) Simvastatin inhibits T-cell activation by selectively impairing the function of Ras superfamily GTPases. FASEB J. 19, 605–607 39 Etienne-Manneville, S. and Hall, A. (2002) Rho GTPases in cell biology. Nature 420, 629–635 40 Greenwood, J. et al. (2003) Lovastatin inhibits brain endothelial cell Rho-mediated lymphocyte migration and attenuates experimental autoimmune encephalomyelitis. FASEB J. 17, 905–907 41 Qiu, M.S. et al. (1991) ras isoprenylation is required for ras-induced but not for NGF-induced neuronal differentiation of PC12 cells. J. Cell Biol. 115, 795–808 42 Laufs, U. et al. (2000) Neuroprotection mediated by changes in the endothelial actin cytoskeleton. J. Clin. Invest 106, 15–24 43 Cordle, A. et al. (2005) Mechanisms of statin-mediated inhibition of small G-protein function. J. Biol. Chem. 280, 34 202–34 209 44 Bi, X. et al. (2004) Inhibition of geranylgeranylation mediates the effects of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors on microglia. J. Biol. Chem. 279, 48 238–48 245 45 Sari, R. et al. (2001) Impairment by lovastatin of neural relaxation of the rabbit sphincter of Oddi. Eur. J. Pharmacol. 432, 91–97 46 Nusse, O. and Neer, E.J. (1996) Localization of Gao to growth cones in PC12 cells: role of Gao association with receptors and Gbg. J. Cell Sci. 109 (Pt 1), 221–228 47 Munro, S. (2003) Lipid rafts: elusive or illusive? Cell 115, 377–388 48 van Meer, G. (2002) Cell biology. The different hues of lipid rafts. Science 296, 855–857 49 Horejsi, V. et al. (1999) GPI-microdomains: a role in signalling via immunoreceptors. Immunol. Today 20, 356–361 50 Kirsch, C. et al. (2003) Statin effects on cholesterol micro-domains in brain plasma membranes. Biochem. Pharmacol. 65, 843–856

www.sciencedirect.com

Vol.28 No.7

349

51 Hillyard, D.Z. et al. (2007) Statins inhibit NK cell cytotoxicity by membrane raft depletion rather than inhibition of isoprenylation. Atherosclerosis 191, 319–325 52 Dunn, S.E. et al. (2006) Isoprenoids determine Th1/Th2 fate in pathogenic T cells, providing a mechanism of modulation of autoimmunity by atorvastatin. J. Exp. Med. 203, 401–412 53 Eckert, G.P. et al. (2001) Differential effects of lovastatin treatment on brain cholesterol levels in normal and apoE-deficient mice. NeuroReport 12, 883–887 54 Nichols, B. (2005) Cell biology: without a raft. Nature 436, 638–639 55 Pyrah, I.T. et al. (2001) Toxicologic lesions associated with two related inhibitors of oxidosqualene cyclase in the dog and mouse. Toxicol. Pathol. 29, 174–179 56 Tozawa, R. et al. (1999) Embryonic lethality and defective neural tube closure in mice lacking squalene synthase. J. Biol. Chem. 274, 30 843– 30 848 57 Ohashi, K. et al. (2003) Early embryonic lethality caused by targeted disruption of the 3-hydroxy-3-methylglutaryl-CoA reductase gene. J. Biol. Chem. 278, 42 936–42 941 58 Berry, P.H. et al. (1988) Brain and optic system pathology in hypocholesterolemic dogs treated with a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Am. J. Pathol. 132, 427–443 59 Jacobson, T.A. (2006) Statin safety: lessons from new drug applications for marketed statins. Am. J. Cardiol. 97, 44C–51C 60 Law, M. and Rudnicka, A.R. (2006) Statin safety: a systematic review. Am. J. Cardiol. 97, 52C–60C 61 Endo, A. et al. (1976) Competitive inhibition of 3-hydroxy3-methylglutaryl coenzyme A reductase by ML-236A and ML-236B fungal metabolites, having hypocholesterolemic activity. FEBS Lett. 72, 323–326 62 Xue, L. et al. (2000) The Ras/phosphatidylinositol 3-kinase and Ras/ERK pathways function as independent survival modules each of which inhibits a distinct apoptotic signaling pathway in sympathetic neurons. J. Biol. Chem. 275, 8817–8824 63 Vogel, K.S. et al. (1995) Loss of neurofibromin results in neurotrophinindependent survival of embryonic sensory and sympathetic neurons. Cell 82, 733–742 64 Heumann, R. et al. (2000) Transgenic activation of Ras in neurons promotes hypertrophy and protects from lesion-induced degeneration. J. Cell Biol. 151, 1537–1548 65 Mills, J. et al. (1997) Regulation of amyloid precursor protein catabolism involves the mitogen-activated protein kinase signal transduction pathway. J. Neurosci. 17, 9415–9422 66 Brambilla, R. et al. (1997) A role for the Ras signalling pathway in synaptic transmission and long-term memory. Nature 390, 281–286 67 Manabe, T. et al. (2000) Regulation of long-term potentiation by H-Ras through NMDA receptor phosphorylation. J. Neurosci. 20, 2504–2511 68 Silva, A.J. et al. (1997) A mouse model for the learning and memory deficits associated with neurofibromatosis type I. Nat. Genet. 15, 281– 284 69 Moore, B.D., III et al. (2000) Brain volume in children with neurofibromatosis type 1: relation to neuropsychological status. Neurology 54, 914–920