PLEIOTROPIC MECHANISMS OF STATIN ACTION IN ALZHEIMER’S DISEASE

By

STEPHEN MARC OSTROWSKI

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation Adviser: Dr. Gary Landreth

Department of Neurosciences

CASE WESTERN RESERVE UNIVERSITY

January, 2008

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

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candidate for the ______degree *.

(signed)______(chair of the committee)

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(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS

TABLE OF CONTENTS ...... - 1 - LIST OF FIGURES ...... - 3 - ACKNOWLEDGEMENTS ...... - 4 - ABSTRACT...... - 5 - CHAPTER1: INTRODUCTION...... - 7 - ALZHEIMER’S DISEASE ...... - 8 - Clinical and Pathological Features of AD...... - 8 - Aβ and the Amyloid Hypothesis...... - 14 - Production of Aβ...... - 16 - Pathogenic Mechanisms of Aβ...... - 20 - STATINS ...... - 23 - Development of statins as choleseterol lowering agents...... - 24 - Efficacy, safety and specificity of statins...... - 28 - Cholesterol, Statins, and AD...... - 30 - Cholesterol-independent effects of statin action...... - 35 - Statins, Protein Isoprenylation, and Ras Superfamily GTPases...... - 38 - RESEARCH GOALS ...... - 44 - REFERENCES ...... - 47 - CHAPTER 2: STATINS REDUCE AMYLOID-BETA PRODUCTION THROUGH INHIBITION OF PROTEIN ISOPRENYLATION...... - 76 - ABSTRACT...... - 77 - INTRODUCTION ...... - 79 - EXPERIMENTAL PROCEDURES ...... - 83 - RESULTS ...... - 87 - DISCUSSION...... - 97 - FIGURES...... - 105 - REFERENCES ...... - 128 - CHAPTER 3: MONITORING STATIN INHIBITION OF PROTEIN ISOPRENYLATION WITH 2D GEL ELECROPHORESIS ...... - 137 - ABSTRACT...... - 138 - INTRODUCTION ...... - 138 - EXPERIMENTAL PROCEDURES ...... - 140 - RESULTS ...... - 142 - DISCUSSION...... - 143 - FIGURES...... - 145 - REFERENCES ...... - 157 - CHAPTER 4: DISCUSSION ...... - 159 - RESEARCH CONCLUSIONS...... - 160 -

- 1 - FUTURE DIRECTIONS ...... - 163 - REFERENCES ...... - 174 - BIBLIOGRAPHY...... - 182 -

- 2 - LIST OF FIGURES

CHAPTER 1 – Introduction Figure 1 48 Figure 2 49 Figure 3 50

CHAPTER 2 - Statins Reduce Amyloid-Beta Production through Inhibition of Protein Isoprenylation

Figure 1 108 Figure 2 110 Figure 3 112 Figure 4 114 Figure 5 116 Figure 6 118 Figure 7 120 Figure 8 122 Supplemental Figure A 124 Supplemental Figure B 126 Supplemental Figure C 128

CHAPTER 3 – Monitoring Statin Inhibition of Protein Isoprenylation with 2D Gel Electrophoresis Table 1 148 Figure1 149 Figure2 151 Figure3 153 Figure4 155 Figure5 157

- 3 - ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Gary Landreth. His enthusiasm for science is infectious.

I would like to thank the members of my thesis committee: Dr. Sophia Sundararajan, Dr. Evan Deneris, and Dr. Robert Miller. Their enthusiasm and support has been greatly appreciated.

The Landreth lab is truly a special and exciting place to work. I would like to thank each and every member of the lab, past and present, for making the environment a great place. It has been a joy and an honor to work with such a wonderful group of people.

To my Mom and Dad, and my brothers Mike and Bill, your love and encouragement means more to me then you will ever know. I would also like to thank all my friends that have been with me through the good times and bad times while I’ve been in Cleveland.

-4- Pleiotropic Mechanisms of Statin Action in Alzheimer’s Disease

Abstract

by

STEPHEN MARC OSTROWSKI

Epidemiological evidence suggests that long term treatment with the cholesterol- lowering HMG-CoA reductase inhibitors, or statins, decreases the risk for developing

Alzheimer’s Disease (AD). However, statin-mediated AD protection cannot be fully

explained by reduction of cholesterol levels. In addition to their cholesterol lowering

effects, statins act pleiotropically 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 moieties for normal protein localization

and function.

In neuroblastoma cell lines, treatment with statins at high doses inhibits the

membrane localization of Rho and Rab proteins, without affecting cellular cholesterol

levels. Importantly, we show for the first time that at low, physiologically relevant, doses

statins preferentially inhibit the isoprenylation of only a subset of GTPases. In addition,

-5- we employ 2D gel electrophoresis to directly monitor the isoprenylated versus non- isoprenylated forms of the proteins, a technique that will be useful for further studies of statin inhibition of protein isoprenylation.

The amyloid precursor protein (APP) is proteolytically cleaved to generate beta- amyloid (Aβ), 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 protein geranylgeranylation. Toxin A, a specific and robust inhibitor of Rho family GTPases, has no effects on APP trafficking, suggesting that statins act to inhibit the function of Rho family proteins. In addition, effects of statins on

APP trafficking and Aβ production are strikingly similar to those reported previously after the inhibition of Rab1b.

Moreover, inhibition of Rho family protein function reduces levels of APP C- terminal fragments (CTFs), resulting in decreased Aβ secretion. We demonstrate that the reductions of CTFs is due to novel regulaton of lysosomal dependent degradation. In summary, we show that statins selectively inhibit GTPase isoprenylation at clinically relevant doses, leading to reduced Aβ production in an isoprenoid-dependent manner.

These studies provide insight into the mechanisms by which statins may reduce AD pathogenesis.

-6-

CHAPTER1: INTRODUCTION

- 7 -

Alzheimer’s Disease

Alzheimer’s Disease (AD) is the leading cause of dementia in the elderly. While it is not only a devastating condition to the individual patients and their families, AD is a widespread condition that will strike up to 40% of those over the age of 85. This has tremendous implications for health care. There are currently 5.1 million suffering from

AD in the United States, and as our population ages this number will increase to 11-16 million by the year 2050. The current direct Medicare and Medicaid costs of AD are approximately $110 billion dollars a year, and the indirect costs of AD approach a similar number. It has been estimated that finding treatments that could delay disease onset or progression by only 5 years could reduce the burden of AD by up to 50%.

Genetic, biochemical, and pathological studies have led to the conclusion that aberrant generation of the amyloid-beta (Aβ) peptide plays a central role in AD pathogenesis. Significant advances have been made in elucidating the mechanisms by which Aβ is produced, as well as the pathophysiological consequences of aberrant Aβ production in the brain. Clinical trials are underway to test the efficacy of targeted anti-

Aβ therapies.

Clinical and Pathological Features of AD.

For much of history, dementia was often seen as a normal consequence of aging

However, by the turn of the 20th century, dementia had been defined as we know it today: the progressive decline in cognitive function due to damage or disease in the brain beyond what might be expected from normal aging. It was recognized that most

- 8 - dementia occurred in the elderly and was termed senile dementia. Vascular changes,

including reduction in vessel diameter, in the brain of demented patients lead to the

theory that senile dementia was attributed to abnormalities of circulation to the brain.

In 1901, Dr. Alois Alzheimer encountered Auguste D., a demented 51 year old

patient. His first interview with her, on the day of Mrs. D’s admission, illustrates the

profundity of her disease (Maurer, Volk et al. 1997):

- What is your name? - Auguste. - And your surname - Auguste. - What is your husband’s name - Auguste, I think. - I am asking for your husband’s name - Oh, my husband! - Are you married - To Auguste. - Are you Mrs. D[eter] - Yes, to Auguste. - How long have you been here? - Three weeks. - Examination of her case history, as provided by here husband, showed that nine

months prior to admission, Mrs. D began to suffer delusions that her husband was having

an affair. She began to suffer from loss of memory, which within two months caused

impairment of her ability to accomplish everyday activities and chores. Mrs. D often

appeared restless and would wander through the house for no reason. She started having

progressive paranoia of those around her, and her memory problems worsened to the

point where she could not remember the location of things that she had recently put away.

By the time of her admission, she could not remember objects shown to her several

minutes earlier and had great difficulty reading and writing. Her behavior became

unpredictable and hostile and she had to be isolated from other patients. As her disease

- 9 - worsened, she became withdrawn and dazed, and would lie constantly in her bed. She visibly lost weight, deteriorated physically, and died of pneumonia on April 6, 1906.

The autopsy showed profound cerebral atrophy with no visible macroscopic lesions. Dr. Alzheimer used silver impregnation techniques developed by Bielschowsky to examine the autopsy material (Bielschowsky 1902). Dr. Alzheimer described two characteristic lesions: 1) Intraneuronal neurofibrillary tangles, which he described: "In

the centre of an otherwise almost normal cell there stands out one or several fibrils due to

their characteristic thickness and peculiar impregnability". He found these neurofibrially

tangles in 1/4 to 1/3 of cortical neurons, and found that they were accompanied by

substantial neuronal loss, and 2) extracelluar “military foci”, later to be known as senile

or neuritic plaques: "Numerous small miliary foci are found in the superior layers. They

are determined by the storage of a peculiar material in the cortex". The “military foci”

had been seen previously in autoposy material from patients with epilepsy (Blocq 1892)

and older demented patients (Redlich 1898; Fischer 1907), but the case of Auguste D was

seen as special because the lesions were more marked and occurred in a younger patient.

In 1909 Persuni described 4 other cases with similar clinical and pathological features as

Auguste D (Perusini 1909). Overall, these cases were thought to represent a novel

clinicopathological entity, which Kraepelin gave the name Alzheimer’s disease (AD)

(Kraepelin 1910).

The prevalence of AD was long underestimated because of distinctions made

between early-onset (pre-senile) and dementia associated with the elderly (senile

dementia). AD pathology of plaques and tangles could be found in elderly demented

patients (Simchowicz 1910), but also in non-demented elderly patients (Gellerstedt 1933),

- 10 - and initial reports found little correlation between such pathology and clinical presentation of dementia (Rothschild 1937; Rothschild 1941). Thus, AD was used only to describe early-onset patients where the accelerated clinical course and pathology was unmistakable. It was not until the 1960’s that a strong correlation between clinical assessment of dementia and the pathological findings of AD was demonstrated (Roth,

Tomlinson et al. 1966; Blessed, Tomlinson et al. 1968). These studies lead to the recognition of Alzheimer’s disease as the leading cause of dementia in the elderly.

Auguste D presented with most of the distinguishing features of AD. Memory loss is a central clinical finding. Most AD patients present with subtle onset of memory loss that at first is often thought to be benign. Slowly memory loss begins to interfere with daily activities, including following instructions or remembering directions. As disease progresses, patients may become lost on walks or while driving. In the middle stages of the disease, the patient is unable to lead a normal life, gets easily lost and confused, and requires daily supervision. Late stages of the disease are particularly frightening, as patients may not recognize even close family members. In AD, deficits extend beyond memory loss as cognitive functions, such as speaking, reading, and writing, also become impaired. Eventually, help may be required for simple tasks such as eating or dressing. August D’s case is also striking in that she presented with non- cognitive features – such as hallucinations, delusions and behavioral deficits – which have only recently been appreciated as symptoms of AD-related dementia. Death usually results from malnutrition, infection or heart disease. The average course of AD duration is 8-10 years, though it may span 1-25 years.

- 11 - Clinical diagnosis of AD has improved, allowing physicians to eliminate other causes of dementia to give a diagnosis of “probable AD” with 85-90% accuracy.

Diagnostic imaging techniques are improving, with the ultimate hope of detecting early- stage disease (Demetriades 2002). However, other causes of dementia cannot always be ruled out, and as such AD remains pathological diagnosis, requiring the pathological findings of plaques and tangles as first described by Alzheimer in 1907.

Most Alzheimer’s disease cases have an onset >65 years of age and are termed sporadic or late-onset AD. However, approximately 6-8% of AD cases are experienced before 60, and these are characterized as early onset AD (Rocca et al., 1991). A significant subset of early onset AD is familial, following Mendelian autosomal dominant inheritance with almost complete penetrance. Mutations in amyloid precursor protein

(APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2), have been identified and account for the bulk of familial early onset AD (Tandon et al. 2000). In addition, individuals with

Trisomy 21 invariably develop AD pathology by the 3rd or 4th decade of life. The APP gene is located on chromosome 21, and trisomy of the APP gene is thought to significantly contribute to the AD pathology.

The strongest risk factor for sporadic Alzheimer’s disease is increasing age.

Prevalance rates (per 100 population) for the age groups 30 to 59, 60 to 69, 70 to 79, and

80 to 89 years have been reported as 0.02, 0.3, 3.2, and 10.8, respectively (Rocca,

Hofman et al. 1991), and between 20-40% of those over 85 will develop AD. The second most important risk factor for AD is a positive family history, suggesting a genetic component of AD. Genetic twin studies have demonstrated high heritability for

AD risk and age of onset, with the largest of these studies estimating heritability at 79%

- 12 - after accounting for shared environmental influences (Raiha, Kaprio et al. 1996; Gatz,

Fratiglioni et al. 2005; Gatz, Reynolds et al. 2006).

The apolipoprotein E gene (APOE) gene on human chromosome 19 has been

identified as a strong risk factor for AD. There are three isoforms of APOE: 2, 3, and 4,

with allelic frequencies in normal populations of approximately .05, .8, and.15,

respectively, though there are significant ethnic differences (Siest, Pillot et al. 1995;

Mastana, Calderon et al. 1998). The APOE3 allele is the most common allele and is

considered neutral with regard to AD risk. The risk of AD by 3-fold in APOE3/4

heterozygous individuals and 15 fold in APOE4/4 homozygous individuals (Okuizumi,

Onodera et al. 1994). APOE2 heterozygosity provides slight protection against AD

(Smith, Johnston et al. 1994; Talbot, Lendon et al. 1994). APOE explains some but not

all of the heritability of AD. Little progress has been made in genome wide scans for

other AD risk genes, suggesting a lack of other strong single genetic loci that modify AD risk (Pericak-Vance, Bass et al. 1998; Pericak-Vance, Grubber et al. 2000; Blacker,

Bertram et al. 2003).

Non-genetic risk factors also play an important role and might be the focus for interventions to reduce disease risk or delay disease onset. For example, exercise and increased levels of education and occupational attainment have been shown to be protective against AD (Stern, Gurland et al. 1994; Eggermont, Swaab et al. 2006). There is also evidence that patients taking non-steroidal anti-inflammatory drugs (NSAIDs) or the cholesterol-lowering statins are protected against AD, though the mechanisms behind these effects are unclear (Breitner 1996; Jick, Zornberg et al. 2000).

- 13 - Aβ and the Amyloid Hypothesis.

The presence of amyloid plaques in AD, coupled with biochemical studies on the

composition of neuritic plaques and powerful genetic evidence, has lead to the amyloid

hypothesis: accumulation of amyloid-beta (Aβ) in the brain is the primary force

driving AD pathogenesis. Michael Kidd and Robert Terry first used electron

microscopy to demonstrate that neuritic plaques are composed of an amyloid core (Kidd

1964; Terry, Gonatas et al. 1964). Amyloid is a general term for insoluble, extracellular,

proteinaceous deposits exhibiting beta-pleated sheet conformation that are deposited in

different tissues in several diseases. The first clue regarding the composition of the

neuritic plaques was found when a novel peptide, termed Aβ, was identified from

cerbrovascular amyloid deposits from both AD and Downs syndrome patients patients

(Glenner and Wong 1984; Glenner and Wong 1984). Aβ was later identified as the

primary component of amyloid plaque cores from the brain parenchyma of patients with

Downs Syndrome and AD (Masters, Simms et al. 1985). Aβ is generated predominantly

two forms: the 40 amino acid Aβ1-40 and the 42 amino acid Aβ1-42. While Aβ1-40 is about

10-fold more abundant, Aβ1-42 contains two hydrophobic C-terminal amino acids. The hydrophobic Aβ1-42 aggregates more easily and is thought to contribute more to the

disease state (Roher, Lowenson et al. 1993; Roher, Lowenson et al. 1993).

Aβ was found to be derived from the amyloid precursor protein (APP) (Kang,

Lemaire et al. 1987; Weidemann, Konig et al. 1989). APP is located on chromosome 21,

providing a mechanism by which the pathology of Down’s syndrome patients and

Alzheimer’s patients may be linked (Kang, Lemaire et al. 1987). This hypothesis was

given strength after a case report of a 78-year old patient with Down’s syndrome due

- 14 - partial trisomy of chromosome 21, but who only carried two copies of the APP gene, did

not develop AD clinically or pathologically (Prasher, Farrer et al. 1998). In addition,

recently 5 families with early onset familial Alzheimer’s Disease have been identified

that carry duplications of small subregions that duplicate the APP gene (Rovelet-Lecrux,

Hannequin et al. 2006).

Soon after the discovery of the APP gene, genetic mutations in APP were found from patients with early onset familial AD (FAD) (Goate, Chartier-Harlin et al. 1991;

Mullan 1991) . The APP Swedish double mutation is located near the β-secretase cleavage site and increases β-secretase cleavage and total Aβ production. Most other

FAD APP mutations are located near the gamma secretase site, and cause a preferential increase in the production of AB1-42. Soon after, novel genes encoding the presenilins,

PSEN1 and PSEN2, were identified from positional cloning strategies from FAD families

(Levy-Lahad, Wasco et al. 1995; Sherrington, Rogaev et al. 1995). Demonstration that

familial AD mutations in PS1 and PS2 preferentially enhance Aβ1-42 production from

APP in vivo and in vitro has strengthened the amyloid hypothesis (Scheuner, Eckman et al. 1996; Citron, Westaway et al. 1997; Xia, Zhang et al. 1997). Mouse models of AD have been generated by expression of mutant forms of human APP or PS, and these mice develop Alzheimer’s like plaque pathology (Hsiao, Chapman et al. 1996; Lamb, Bardel et al. 1999) .

A longstanding criticism of the amyloid hypothesis has been that some reports found little correlation between plaque number and the extent of AD dementia and the extent of plaque pathology. However, the degree of dementia in AD correlates very strongly with soluble Aβ levels (Lue, Kuo et al. 1999; McLean, Cherny et al. 1999;

- 15 - Naslund, Haroutunian et al. 2000). There is now little controversy over the central role of

Aβ production in the pathogenesis of Alzheimer’s Disease, though the mechanisms by which Aβ contributes to synaptic dysfunction and neuronal loss in AD.

Production of Aβ.

As Aβ has long been thought to be a driving force behind AD, much research has been done into the mechanisms by which Aβ is produced from amyloid precursor protein

(APP). APP is a type I (single pass) transmembrane protein with a large extracellular domain and short intracellular domain. The Aβ sequence is composed of 40-42 amino acids spanning the transmembrane and cytoplasmic domains of APP. The function of

APP holoprotein is unknown, but some evidence suggests that APP may have a role in signaling, neuronal development, and axonal trafficking (Reinhard, Hebert et al. 2005).

Aβ is formed from APP by sequential cleavage (Figure 1) (reviewed by Hardy and Selkoe 2002). APP is first cleaved near the exoplasmic surface by either the α- or β- secretases. The α- and β- cleavages are mutually exclusive with the beta cleavage occurring at the N-terminus of the Aβ sequence, and the alpha cleavage occurring within the Aβ sequence. These cleavages result in the release of the soluble peptide of 100-

120kD, termed soluble or secreted APP (sAPP) –α or –β, and a 10-12kD membrane bound stub, the C-terminal fragment (CTF) –α or –β. The CTFs can be subsequently cleaved by γ-secretase. γ-secretase cleavage of β-CTF results in generation of Aβ.

Cleavage by γ-secretase can occur at several closely located residues, resulting primarily in production 40 or 42 amino acid Aβ species. γ-secretase cleavage of α-CTF results in the production of a non-amyloidgenic p3 peptide.

- 16 - The alpha-secretase cleavage of APP occurs between Lys-16 and Leucine-17

(amino acids 612 and 613 of APP). This site occurs within the Aβ sequence, and thus

alpha-secretase cleavage is not compatible with the generation of Aβ. The α-secretase

cleavage of APP has been shown to occur both at the cell surface and in intracellular

compartments such as the Golgi. The cleavage of APP to release a soluble ectodomain is

similar to the cleavage of other substrates such as TNFα and L-selectin. Similar to

processing of these other substrates, α-secretase cleavage of APP occurs at a constitutive

level, where 10-30% of APP is cleaved, but can also be activated by protein kinase C and

other second messengers, whereby up to 80-95% of APP molecules undergo α-secretase

cleavage. (Desdouits et al. 1996). The protease responsible for cleavage of TNFα has

been characterized: tumor necrosis factor-α converting enzyme (TACE), a member of

the a disintegrin and metalloproteinase (ADAM) family proteins. This enzyme has been

shown to be required for stimulated sAPP shedding regulated by protein kinase C, but not

for constitutive sAPP shedding (Buxbaum, Liu et al. 1998). ADAM10 and

ADAM9/MDCD9 have been since been identified as the enzymes responsible for the

bulk of α-secretase cleavage (Koike, Tomioka et al. 1999; Lammich, Kojro et al. 1999).

It is thought that α- cleavage is a “good” cleavage that may be protective against AD. As

α-cleavage precludes the production of Aβ, α-cleavage might leave less substrate

available to beta-secretase, leading to decreased Aβ production (Hung, Haass et al. 1993).

In support of this model, overexpression of ADAM10 caused decreased Aβ production in

APP transgenic mice, leading to alleviation of cognitive deficits (Postina et al. 2004).

Alternatively, it has been shown that sAPPα may exert neuroprotective effects

independent of decreased Aβ production (Smith-Swintosky 1994; Bour 2004; Meziane

- 17 - 1999). Knockouts of ADAM10 and TACE mice are embryonic lethal, as these proteins

are involved in the processing of many substrates, so it is has not been possible to

exclude a role for other α-secretases in vivo.

The first step towards Aβ production is cleavage of APP by the β-secretase at the

N-terminus of the Aβ peptide sequence. Four separate research groups identified the same protein as the beta secretase: beta-site APP cleaving enzyme (BACE) (Hussain,

Powell et al. 1999; Sinha, Anderson et al. 1999; Vassar, Bennett et al. 1999; Yan,

Bienkowski et al. 1999). Both in vitro and in vivo gain and loss of function studies have unequivocally identified BACE1 as the primary beta-secretase activity (Cai, Wang et al.

2001; Luo, Bolon et al. 2001). BACE1 shows a higher binding affinity for Swedish APP, a mutation of APP that has long been known to result in enhanced β-secretase cleavage.

γ-secretase cleavage is necessary to release Aβ from the β-CTF.

The γ-cleavage site is within the predicted transmembrane region of APP.

Presenilin is an aspartyl protease that serves as the catalytic subunit of γ-secretase. It has

been since elucidated that γ-secretase is a complex composed of presenilin as well as 3

other essential proteins: Nicastrin, Pen2, and Aph1 (Wolfe 2006). Substrate recognition

is thought to occur by interaction of the Nicastrin ectodomain with the N-terminus of the

β-CTF, which is then passed between two presenilin fragments to a water containing

active site at which the catalytic aspirate residue resides. There are several isoforms of

Pen2, and Aph1, and it has been suggested that different length Aβ peptides may be

generated by different combinatorial forms of the γ-secretase complex (Jankow 2004).

However, recent evidence suggests that all isoforms of Aβ are generated by sequential

cleavage using the same enzyme complex.

- 18 - Localization and trafficking of APP and the secretases is critical for the

processing of APP (reviewed by Vetrivel and Thinakaran 2006). APP is trafficked

through the common secretory pathway, and is post-translationally modified as it passes

through the ER and Golgi. APP resides only transiently at the cell surface, as over 70%

of surface bound APP is internalized within one minute. APP is internalized by

endocytosis and is either targeted for degradation to late endosomes lysosomes, or

through recycling pathways to cell surface or to the ER/Golgi. ADAM10 is localized on

the cell surface and the Golgi compartments, consistent with evidence suggesting that α-

cleavage can occur both at the cell surface and the Golgi (Lammich, Kojro et al. 1999).

β-secretase activity occurs optimally at low pH, and BACE1 localized primarily in the

late Golgi/TGN and endosomal compartments, and is thought to cleave APP either during

exocytosis or during the endocytic/recycling steps (Yan, Han et al. 2001; Cole, Grudzien

et al. 2005). Presenilin and other γ-secretase components are localized primarily within

the early secretory pathway (Kim, Yin et al. 2004; Capell, Beher et al. 2005), and also to

late Golgi/TGN (Siman and Velji 2003). It is unclear if γ-secretase and β-secretase exist

in the same compartments, or if β-CTFs must be recycled back to γ-secretase containing

compartments after β-secretase cleavage. Recent evidence shows that γ-secretase, β- secretase and APP are localized to lipid-raft compartments, and it is thought that these compartments may be critical in the production of Aβ, though the techniques used could

not determine if the components were co-localized to the same subcellular compartments

(Vetrivel, Cheng et al. 2004; Vetrivel, Cheng et al. 2005).

- 19 - Pathogenic Mechanisms of Aβ.

The ultimate consequence of Aβ generation in AD is severe synaptic dysfunction

and neuronal cell loss, though the mechanisms by which this occurs are still controversial.

The bulk of reaearch has focused on the direct neurotoxicity of insoluble amyloid plaques.

The main reason for this is that amyloid plaques are one of the characteristic lesions in

the disease, and genetic evidence suggests that Aβ causes the disease. In addition, it has

been shown that plaques are surrounded by dystrophic neurons, suggesting that plaques

may be directly neurotoxic. In vitro studies have shown fibrillar Aβ to be neurotoxic, though concentrations typically used are in excess of physiologically relevant concentrations (Pike, Walencewicz et al. 1991; Pike, Walencewicz et al. 1991; Busciglio,

Lorenzo et al. 1992).

Many investigators do not believe that fibrillar Aβ is responsible for AD

pathogenesis. This is based mainly on the fact that there is relatively weak correlation

between Aβ plaque density and degree of clinical dementia (Terry, Masliah et al. 1991;

Dickson, Crystal et al. 1995), while there is robust correlation between soluble Aβ levels and synaptic loss and clinical deterioration of AD (Lue, Kuo et al. 1999; Naslund,

Haroutunian et al. 2000). In addition, in mouse models cognitive function and synaptic activity can be found in the absence of plaque formation.

In the human brain, soluble Aβ can exist as monomers, dimers, or other

oligomers up to 100kD (Kuo et al. 1990). Natural, cell derived Aβ oligomers alter

hippocampal synaptic plasticity and short term memory in rats. Aβ oligomers have been

shown to be neurotoxic in vitro, and physiological levels of oligomers can cause synaptic

loss in rat slice preparations (Shankar 2007). Injection of anti-Aβ antibodies into the

- 20 - brain improves cognitive function within a short timeframe where there is no detectable decrease in plaque load (Dodart et al. 2002). Recently, dodecameric (*56) Aβ species were detected at the first onset of spatial memory dysfunction in Tg2576 mice, while dimers and trimers were present in the absence of dysfunction (Lesne et al. 2006).

However previous studies demonstrated that other cognitive losses in Tg2576 occurred before the time frame of these experiments. Thus it is likely that multiple soluble Aβ

species may be responsible for cognitive dysfunction and neuronal loss.

While Aβ was first identified as the component of extracellular plaques, it was

soon discovered that Aβ exists intracellularly in the AD brain. Similarly, intracellular Aβ

has been found in animal models of AD (LaFerla, Green et al. 2007). Aβ1-42 is the

primary intracellular species, and some groups have shown N-terminal truncated species

that are particularly insoluble (Takahashi, Milner et al. 2002) (Gouras, Tsai et al. 2000).

Evidence has suggested that Aβ may be accumulated after intracellular production, or

through reuptake of extracellular Aβ. Intracellular Aβ is likely neurotoxic, causing

neuronal dysfunction and death through impairment of mitochondria or protesosomal

function (Tseng, Kitazawa et al. 2004; Gouras, Almeida et al. 2005). Intracelluar Aβ has

been linked to cognitive deficits that occur before amyloid plaque deposition (Billings,

Oddo et al. 2005; Oakley, Cole et al. 2006).

The neurofibrillary tangles (NFTs) first described by Alzheimer have been found

to be composed of aggregates of hyperphosporylated and abnormally phosphorylated tau

(Grundke-Iqbal, Iqbal et al. 1986). Tau is a microtubule-associated protein (MAP)

expressed abundantly in neurons and involved in microtubule assembly and stabilization.

While overexpression of tau alone does lead to tangle formation in mice, coexpression of

- 21 - tau with mutant APP and PS1 lead to tangle formation (Oddo, Caccamo et al. 2003; Oddo,

Caccamo et al. 2007). In addition, Aβ immunotherapy clears tau aggregates in these mouse models (Oddo, Billings et al. 2004). These studies suggest that amyloid production is responsible for tangle pathology

Tau pathology is present in number of neurodengenerative conditions. Mutations in Tau are the cause of frontal temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), providing evidence that tau dysfunction can lead to neurodegeneration in the absence of other pathogenic insults (von Bergen, Barghorn et al.

2001). NFTs may thus play a central role in AD pathogenesis. There is evidence that the pathogenicity of NFTs is through a combination of toxic gain of function and loss of normal tau function. Loss of tau function may disturb the normal structure and function of the cytoskeleton, leading to impaired axonal transport, synaptic dysfunction, and neuronal loss (Roy, Zhang et al. 2005). Support for this hypothesis came from recent studies which showed that the microtubule stabilizing drug, paclitaxel, can rescue neuronal loss in tau transgenic mouse models (Zhang, Maiti et al. 2005). Potential toxic gain of function mechanisms include direct physical obstruction of cellular processes

such as axonal transport or by sequestering proteins required for normal cellular function

(Ballatore, Lee et al. 2007). In triple transgenic mice expressing mutant APP, PSEN1 and

Tau, reducing Aβ and tau, but not Aβ alone restores cognitive deficts, suggesting that tau

and NFTs contribute to cognitive decline in AD (Oddo, Vasilevko et al. 2006).

Another characteristic pathological changes that occurs in Alzheimer's disease is

loss of acetylcholine (ACh) from both cholinergic and noncholinergic neurons of the

brain (Davies and Maloney 1976; Perry, Gibson et al. 1977). Acetylcholinesterase

- 22 - inhibitors such as Donepezil and galantamine, which raise the levels of ACh in the brain, have been shown to ease some of the memory and language deficits early in the disease.

More recent evidence suggests that these drugs may also slow disease progression

(Winblad, Kilander et al. 2006). However, it is not clear whether destruction of the cells that make acetylcholine is a cause or a consequence of Alzheimer's disease.

Considerable attention has been focused on the role of inflammatory mechanisms in the etiology of AD. Senile plaques are the site of local inflammatory response as evidenced by abundant plaque-associated reactive microglia (Itagaki, McGeer et al. 1989).

These activated microglia release a wide array of pro-inflammatory molecules, such as complement, cytokines, and acute phase reactants. Accumulated over many years, neuroinflammation acts to exacerbate the disease process and contribute to neuronal loss

(Akiyama, Barger et al. 2000). The anti-inflammatory NSAIDs have been shown to be protective against Alzheimer’s disease (Etminan, Gill et al. 2003), though some studies have suggested that the effect of NSAIDs may be mediated through alterations of Aβ production (Weggen, Eriksen et al. 2001). HMG-CoA reductase inhibitors or statins also exhibit anti-inflammatory properties that may contribute to their ability to protect against

AD (Cordle, Koenigsknecht-Talboo et al. 2005; Cordle and Landreth 2005).

Statins

Retrospective studies have suggested that patients treated with HMG-CoA reductase inhibitors, or statins, have up to a 70% reduced risk of for developing AD.

Statins target the rate limiting enzyme in de novo cholesterol biosynthesis, HMG-CoA

- 23 - reductase, and have been shown to be extremely effective in lowering plasma cholesterol

in humans, resulting in remarkable efficacy in primary and secondary prevention of

cardiovascular disease. As a result much focus has been placed on a potential role for

cholesterol in AD. However, the data supporting a role of cholesterol in AD is mixed,

and recent evidence suggests that some of the benefits of statins may be through their

pleotropic, cholesterol-independent mechanisms.

Development of statins as choleseterol lowering agents.

Cholesterol is essential for the function of all human cells, but it is quite clear that

elevated plasma cholesterol levels are a strong risk factor for cardiovascular disease.

Genetic studies of families with familial hypercholesterolemia by Dr. Carl Muller and Dr

Avedis K. Khachadurian showed the first clinical link between high blood cholesterol

and cardiovascular disease. Individuals homozygous for this disease have prodigous

levels of plasma choleseterol (800mg/dL; normal=160-260), and suffer heart attacks as

early as 5 years of age. The development of methods to measure cholesterol from plasma paved the way for epidemiological studies that have established a role for high plasma cholesterol as a general risk factor for cardiovascular disease (Kannel, Dawber et al. 1965;

Keys, Aravanis et al. 1966).

The clinical importance of cholesterol in cardiovascular disease has led to intense

study of mechanisms of cholesterol homeostasis. Cholesterol homeostasis is achieved

through regulation of cholesterol uptake, cholesterol biosynthesis, cholesterol conversion

to bile acids and excretion of bile acids. While fine tuning of celluar cholesterol levels can be mediated by local cholesterol synthesis, the liver plays a central role in regulating

- 24 - whole-body cholesterol homeostasis. Much of the cholesterol used by tissues originates

from the liver, which generates cholesterol either through de novo synthesis or

incorporation from dietary uptake (Dietschy, Turley et al. 1993). The liver secretes

cholesterol in into the plasma as low-density-lipoproteins (LDL-C) or their precursors

very low density lipoproteins (VLDL-C), which play a role in delivering cholesterol to

peripheral tissues, but also in returning excess cholesterol from the periphery to the liver

(Dietschy, Turley et al. 1993). The liver takes up lipoproteins from the periphery

primarily through the LDL receptor. While critical for normal lipid homeostasis, LDLs

can also accumulate in vessel walls to form atherosclerotic plaques, the primary risk

factor for cardiovascular disease.

Despite wide variations in dietary cholesterol, most animals are able to maintain

cholesterol at relatively constant levels, reflecting a balance between uptake from the diet

and de novo synthesis, which occurs primarily in the liver. In particular, increased uptake

of cholesterol from the diet results in decreased hepatic cholesterol synthesis. The

mounting clinical interest in cholesterol lead to the delineation of the steps of the

cholesterol synthesis pathway in a remarkable collaboration of organic chemistry,

enzymology and use of radioisotope labeling. The cholesterol biosynthetic pathway

consists of almost 30 steps (Fig 2). These contributions were recognized with the award of the 1964 Nobel Prize in Medicine to Dr. Kongrad Bloch and Dr. Feodor Lynen, though significant contributions were also made by Dr. John Cornforth and Dr. George Popjak.

Initial studies showed that mevalonate or its metabolites were not responsible for regulation of cholesterol synthesis (Bucher, Mc et al. 1959). Siperstein and colleagues examined in detail the steps proximal to mevalonate and showed that cholesterol

- 25 - biosynthesis was regulated at the step of conversion of 3-hydroxy-3-methylglutaryl-CoA

to mevalonate (Siperstein and Guest 1960; Siperstein and Fagan 1966). The enzyme responsible for this reaction, HMG-CoA reductase, had previously been isolated by biochemical techniques (Bucher, Overath et al. 1960), and the protein became molecularly characterized with the advent of molecular techniques in the 1980’s (Chin,

Luskey et al. 1982). HMG-CoA reductase contains an N-terminal domain with 8

transmembrane stretches that anchor it to the ER membrane, and a C-terminal catalytic

domain located within the cytoplasm. Interestingly, the mechanism by which cholesterol negatively regulates HMG-CoA reductase activity has only been recently characterized.

Cholesterol promotes the binding of two ER-membrane proteins Insig-1 and Insig-2 to

HMG-CoA reductase, that mediate interaction with gp78 ubiquitin ligase, resulting in ubiquitination and degradation of HMG-CoA reductase (Sever, Song et al. 2003; Sever,

Yang et al. 2003; Sever, Lee et al. 2004; Song, Sever et al. 2005).

The discovery that the downregulation of HMG-CoA reductase activity exists as a homeostatic feedback mechanism suggested that pharmalogical inhibition of this enzyme may represent an effective and safe target to lower cholesterol biosynthesis in vivo. With these thoughts in mind, Endo and colleagues began a search for compounds that could inhibit HMG-CoA reductase. Endo and colleagues screened through microbial broths that blocked the incorporation of C14 labeled acetate into lipids, but did not block incorporation H3 labeled mevalonate. The principle active components were isolated

from these broths, and compounds were directly assayed for inhibition of HMG-CoA

reducatase as assessed by inhibition of the incorporation of C14 labeled HMG-CoA into cholesterol. This ultimately resulted in the isolation of the compound ML-236B from the

- 26 - fungus Penicillin citrinum, later named mevastatin, the first identified competitive

inhibitor of HMG-CoA reductase.

The reactive group of mevastatin is a beta-hydroxy-delta-lactone ring. The ring

can be hydrolyzed to an open acid-form that is structurally similar structure to HMG-

CoA and has a 10,000 higher fold affinity for HMG-CoA reductase. Lovastatin, an

analogue of mevastatin, was isolated from Aspergillus terreus, and was the first statin

marketed for human use. Simvastatin and pravastatin are chemically modified and more

potent analogues of lovastatin. By 1990, simvastatin (Zocor), lovastatin (Mevacor), and

pravastatin (Pravachol) were FDA approved and on the market. Since then, the synthetic

compounds fluvastatin (Lescor), atorvostatin (Lipitor), and rosuvastatin (Crestor) have

been introduced.

Interestingly, the primary mechanism of statin action in vivo is not a result of

inhibition of de novo cholesterol biosynthesis in the liver, but instead increased clearance

of plasma LDL (and its precursors IDL and VLDL) by the liver. Statins inhibit the

activity of HMG-CoA reductase in the liver. This decreases intracellular levels of

metabolites of cholesterol, which through a homeostatic mechanism leads to the

upregulation of LDL receptors in the liver and increased clearance of LDL particles from the blood, leading to decreased plasma LDL cholesterol (LDL-C) levels. This primary

mechanism is reflected by the fact that in patients with receptor negative homozygous

familial hypercholesterolemia, who have no capacity to synthesize functional LDL

receptors, the ability of statins to reduce plasma cholesterol is severely attenuated.

However, there is evidence that statins act in part by decreasing de novo

cholesterol synthesis in the liver to limit production of LDL precursors.(Grundy and

- 27 - Vega 1985). This mechanism may be important for lowering of plasma triglycerides, and is also demonstrated by the fact that a subset of patients with receptor negative homozygous familial hypercholesterolemia have up to 25% reduction of LDL-C after statin treatment (Raal, Pilcher et al. 1997; Raal, Pilcher et al. 1997; Raal, Pappu et al.

2000)

Efficacy, safety and specificity of statins.

Statins are relatively safe and effective drugs for treating hypercholesterolemia.

Numerous clinical trials have demonstrated that statins are effective in secondary prevention of cardiovascular disease. Recent large meta-analysis suggests that statins are effective for protecting patients with a previous major cardiovascular event, regardless of baseline cholesterol levels (Baigent, Keech et al. 2005). Severe, acute side effects of statins are rare, and represented primarily by rhabdomyolysis that occurs in .03% of patients. In clinical trials, myopathy and liver toxicity have been reported to occur in up to 3% of patients. Importantly, the incidence of statin-induced rhabdomyolysis is higher in practice than in controlled trials in which high-risk subjects are excluded (Antons et al.

2006). In addition, some studies have suggested that statins may be associated with an increased risk of certain forms of cancer (Browning Martin Int J Cancer 2007). While statins have been shown to be beneficial in the primary prevention of cardiovascular disease for males age 30-69 that are at high risk, there is considerable controversy whether benefits of statin treatments outweigh risks in other groups (Davidson and

Robinson 2007; Silva, Matthews et al. 2007).

- 28 - Animal studies have shown that liver concentrations of statins reach

approximately 10-fold higher levels than those in peripheral tissues (Koga, Fukuda et al.

1992), due to extensive first pass metabolism by the liver after oral dosing. However,

active metabolites of statin are resecreted from the liver, and have been shown in mice to

reach extrahepatic tissues in mice at high enough concentrations to mediate HMG-CoA

reductase inhibition (Koga, Fukuda et al. 1992). The first studies published by Merck

describing the distribution of simvastatin and lovastatin in man, after one time 100mg dosing of C14 labeled drug, plasma concentrations peaked at >200nM active inhibitors

and >600nM of total radioactivity. Many other pharmacokinetic studies on statins have

reported much lower plasma drug levels of statin (Pentikainen, Saraheimo et al. 1992).

This is due to the fact that the techniques these studies used to measured drug only detect

certain forms of the drug, while the majority of active statin metabolites were not

detected. More recent studies which measure total inhibitory activity have recapitulated

the original Merck data and show that peak plasma concentrations of active inhibitors can

exceed 200nM (Prueksaritanont, Vega et al. 2001; Bergman, Murphy et al. 2004). In

addition, most pharmacokinetic studies use young, healthy patients, and it has been

shown that in elderly patients that drug concentrations are higher due to reduced drug clearance (Cheng, Rogers et al. 1992).

While the issue has not been well studied, there is direct evidence that statins can mediate HMG-CoA reductase inhibition in extrahepatic tissues, as measured by inhibition of cholesterol synthesis in PMBCs taken from humans treated with statins

(Keidar, Aviram et al. 1994). The toxicity of statins has been shown to be directly

proportional to plasma statin concentrations. However, extrahepatic statin effects may

- 29 - also explain beneficial pleiotropic effects of statins that will be described below (Liao

and Laufs 2005).

Cholesterol, Statins, and AD

Three retrospective clinical studies suggested that long term use of statins may

lead to a reduction of Alzheimer’s Disease risk by as much as 70% (Jick, Zornberg et al.

2000; Wolozin, Kellman et al. 2000; Rockwood, Kirkland et al. 2002). However, three

subsequent longitudinal observational studies found no effect of statins on AD risk, and

criticized the design of the initial studies (Li, Higdon et al. 2004; Reitz, Tang et al. 2004;

Zandi, Sparks et al. 2005). Other studies have replicated the initial data (Zamrini,

McGwin et al. 2004), and a recent exciting paper shows that protection against dementia is associated only with simvastatin use (Wolozin, Wang et al. 2007). Next year the

ADCS’s progression-slowing clinical trial of simvastatin will be completed and will help to clarify the role of statins in slowing or preventing AD.

As discussed above, ApoE, a cholesterol transport protein, has been identified as a strong genetic risk factor for AD. Coupled with a potential role of the cholesterol lowering statins in limiting AD pathogenesis, this has led to the hypothesis that elevated cholesterol may be a risk factors for Alzheimer’s disease. Initial in vitro studies demonstrated that depletion of cellular cholesterol can lead to reduced neuronal Aβ secretion (Simons, Keller et al. 1998; Frears, Stephens et al. 1999; Buxbaum, Geoghagen et al. 2001; Fassbender, Simons et al. 2001). A number of studies have demonstrated that

β- and γ- secretases and APP are co-localized to cholesterol-rich lipid raft domains in the plasma membrane (Cordy, Hussain et al. 2003; Vetrivel, Cheng et al. 2004; Urano,

- 30 - Hayashi et al. 2005; Hattori, Asai et al. 2006). Cholesterol depletion inhibits β- and γ-

secretase activities and Aβ production, likely by disruption of lipid-rafts (Wahrle, Das et

al. 2002; Ehehalt, Keller et al. 2003). Numerous studies have also shown that statins in

vitro increase α-secretase cleavage of APP (Bodovitz and Klein 1996; Racchi, Baetta et

al. 1997; Kojro, Gimpl et al. 2001; Cole, Grudzien et al. 2005). One potential mechanism

for this increased α-secretase cleavage is that APP is displaced from lipid rafts, providing

more substrate for the α-secretase, which does not localize to lipid rafts.

Treatment of mice with an inhibitor of acyl-CoA cholesterol acyltransferase

(ACAT) (Puglielli, Konopka et al. 2001), or the cholesterol lowering drug BMI15.766

(Refolo, Pappolla et al. 2001), which inhibits 7-dehydrocholesterol delta 7 reductase

(7DHC reductase), blocks amyloid pathology in mouse models. In addition, blockade of

intracellular cholesertol transport by Niemann Pick type C1 mutations, leading to

cholesterol accumulation in neurons, increases amyloid pathology (Burns, Gaynor et al.

2003). Overall these studies have support a role for cholesterol in AD.

On the other hand, the outcome of clinical studies have been mixed with regard to whether cholesterol is a risk factor for Alzheimer’s disease (Reviewed by Wook,

Igbavboa et al., 2005). Statins exert their strongest effects on plasma cholesterol levels.

Several retrospective studies have suggested that elevated serum cholesterol is a risk factor for AD (Kuo, Emmerling et al. 1998; Pappolla, Bryant-Thomas et al. 2003).

However, this correlation is not consistently observed, with some studies showing little or

no correlation (Romas, Tang et al. 1999; Launer, White et al. 2001), and others find an

inverse correlation (Knittweis and McMullen 2000) between plasma cholesterol and AD.

In addition, in the studies that suggest elevated plasma cholesterol as a risk factor for AD,

- 31 - the differences in plasma cholesterol between AD and control patients were quite modest.

Moreover, analysis of the cohort from the Framingham heart study, where cholesterol

was measured for over 5000 paitents over 50 years, (Tan, Seshadri et al. 2003), or from

the Honolulu-Asia Aging Study which followed cholesterol levels in 1027 men for over

26 years (Stewart, White et al. 2007), no correlations were found between plasma

cholesterol and AD risk. Similarly, data from mouse models supporting cholesterol as a

risk factor for amyloid plaque pathology is mixed, with some showing increased dietary

cholesterol increasing amyloid pathology (Refolo, Malester et al. 2000; Shie, Jin et al.

2002), while others show that increased cholesterol reduces or has no effect on Aβ levels

or plaque pathology (Howland, Trusko et al. 1998; George, Holsinger et al. 2004; Elder,

Cho et al. 2007).

The vast majority of the cholesterol in the brain is provided by de novo synthesis

(Flint 1863; Connor, Johnston et al. 1969; Li, Higdon et al. 2004). Metabolic labeling

studies utilizing either labeled lipoproteins or labeled free cholesterol have shown no

detectable transfer of cholesterol from the periphery to the CNS (Edmond, Korsak et al.

1991; Turley, Burns et al. 1996). The majority of cholesterol in the CNS is incorporated into myelin, and turnover of this pool occurs very slowly; the half-life of cholesterol in the brain is 5 years (Bjorkhem, Lutjohann et al. 1998). The remaining cholesterol is present primarily in the plasma membrane of neurons and glia. As the CNS synthesizes cholesterol at a rate greater than it is accrued, sterols must move from the CNS to the periphery. The primary mechanism for export of sterols is hydroxylation of cholesterol by the enzyme to CYP46a1 to produce 24S-hydroxycholesterol. 24S-hydroxycholesterol diffuses down its concentration gradient across the BBB to the periphery where it is

- 32 - ultimately excreted in bile. Interestingly, the CYP46a1 enzyme is expressed primarily in

metabolically active neurons such as pyramidal cells of the cortex and Purkinje cells of the cerebellum, and is not expressed as highly in white matter, and thus 24S- hydroxylation is not a primary mechanism for turnover of cholesterol in glia or myelin

(Xie, Lund et al. 2003). 24S-hydroxycholesterol can act as a signaling molecule to upregulate ApoE and promote efflux of cholesterol from glial cells (Abildayeva, Jansen et al. 2006).

Elevated brain cholesterol does not appear to be a risk factor for AD. Total cholesterol levels have been measured from brains of AD patients versus controls, and mixed reports of increased cholesterol, reduced cholesterol, or no cholesterol changes in samples from AD patients as compared to controls have been reported (Mason,

Shoemaker et al. 1992; Sparks 1997; Eckert, Cairns et al. 2000). Similarly, reports showing changes in CSF cholesterol levels in AD patients versus controls have been mixed (Mulder, Ravid et al. 1998; Demeester, Castro et al. 2000).

Many studies have demonstrated elevated 24S-hydroxycholesterol levels in the

CSF or plasma of AD patients (Lutjohann, Papassotiropoulos et al. 2000;

Papassotiropoulos, Lutjohann et al. 2002; Schonknecht, Lutjohann et al. 2002) and have often been cited to support a role of increased brain cholesterol levels as a factor in AD.

However, levels of 24S-hydroxycholesterol represent only the excess cholesterol

synthesized by neurons that needs to be excreted to the periphery, and increased 24S-

hydroxycholesterol does not necessarily reflect increased neuronal cholesterol levels.

Some authors have also suggested that 24S-hydroxycholesterol production is a

mechanism to remove cholesterol from membranes of dying neurons, and thus it might be

- 33 - increased under circumstances of neurodegeneration. Alternatively, in situations of

extensive neurodegeneration, one might expect decreased 24S-hydroxycholesterol to be a

result of decreased metabolic output from neurons. This is supported by the observation that in a mouse model of Picks disease which displays severe neurodegeneration (German,

Quintero et al. 2001) and in severely demented human patients, that plasma 24S- hydroxycholesterol levels were reduced (Papassotiropoulos, Lutjohann et al. 2000).

Interestingly, reduction in plasma 24S-hydroxycholesterol was observed in humans after treatment with simvastatin, lovastatin or pravastatin (Vega, Weiner et al.

2003). Numerous other studies demonstrate that simvastatin reduces 24S- hydroxycholesterol (Locatelli, Lutjohann et al. 2002; Simons, Schwarzler et al. 2002;

Lutjohann and von Bergmann 2003), while one study has shown that pravastatin does not

(Thelen, Lutjohann et al. 2006). Statin specificity may be important; in mice, simvastatin, but not pravastatin, inhibits brain cholesterol synthesis (Thelen, Rentsch et al. 2006), and simvastatin reaches higher concentrations in the brain than pravastatin (Johnson-Anuna,

Eckert et al. 2005). This is due to the increased hydrophobicity and blood-brain-barrier permeability of simvastatin as compared to pravastatin. Overall, these studies suggest that statins inhibit cholesterol biosynthesis in the brain, but the relevance of this to AD is unclear.

It is also possible that changes in cholesterol domains, and not bulk cholesterol levels, may be the link between cholesterol and AD. Cholesterol is not evenly distributed throughout the cell, as cholesterol is asymmetrically distributed between the cytoplasmic and exoplasmic faces of the membranes. This distribution is altered by ApoE isoforms, age, and statins (Igbavboa, Avdulov et al. 1997; Kirsch, Eckert et al. 2003). A recent

- 34 - report showed that simvastatin, lovastatin and atrovastatin blocked Aβ production in mice

(Burns, Igbavboa et al. 2006). The Aβ reduction was associated with changes in transfer

of cholesterol from cytofacial to exofacial membrane surface, and not changes in total

brain cholesterol.

Overall, the data suggesting a role for cholesterol in AD is mixed. As statins may

play a role in ameliorating AD, it is pertinent to investigate cholesterol-independent

mechanisms by which they may be mediating AD protective effects.

Cholesterol-independent effects of statin action.

Recently, it has been appreciated that statins can act through cholesterol- independent mechanisms. This has been sparked primarily by clinical findings suggesting that many of the cardiovascular protective effects of statins are independent of cholesterol lowering. Three main lines of evidence support this hypothesis: 1) The clinical benefits of statins are greater than can be explained simply by lipid lowering

(Liao and Laufs 2005). 2) Statins mediate some clinical benefits in a time frame faster than can be explained by lipid lowering (O'Driscoll, Green et al. 1997; Schwartz, Olsson et al. 2001). 3) Statins benefits are independent of baseline cholesterol or the degree of lipid lowering (Sacks, Pfeffer et al. 1996; Collins, Peto et al. 2002).

Animal studies support a role for cholesterol-independent mechanisms of statin action. Lipid homeostasis is very species specific and rodents do not carry significant amount of cholesterol as LDL-C. Statins, which act primarily to reduce LDL-C, generally have no effect on cholesterol levels in mice or rats. While this makes them poor models for cholesterol-dependent effects of statins, it makes them good models to

- 35 - study cholesterol-independent effects. In rodents, statins have been shown to improve

endothelial function, increased vasodiation, decrease inflammation and oxidative stress, and improve cardiac function (Landmesser, Engberding et al. 2004; Habibi, Whaley-

Connell et al. 2007; Monetti, Canavesi et al. 2007). Numerous studies have implicated a role for inhibition of Rho family proteins and subsequent enhancement of endothelial nitric oxide synthase (eNOS) or abrogation of NADPH oxidase function in mediating these effects of statins (Landmesser, Engberding et al. 2004; Otto, Fontaine et al. 2006)

Statins also have clinical benefit in CNS disorders such as ischemic stroke and

multiple sclerosis. Statins reduce the incidence of stroke (Corvol, Bouzamondo et al.

2003), despite extensive epidemiological evidence that cholesterol is not a risk factor for

ischemic stroke (Kannel, Castelli et al. 1971; 1982). Statins protection against stroke

may be due to improved endothelial function and actions on the fibrolynitic pathways

(Liao and Laufs 2005). Mouse studies first suggested that statins may have benefits in

multiple sclerosis through immunomodulation of T-cells (Youssef, Stuve et al. 2002), and

patients treated with 80mg simvastatin had a 44% reduction in lesion burden after 3

months (Vollmer, Lancet 2004). There is also evidence that statins act within the CNS to

benefit oligodendrocyte proliferation and survival through cholesterol independent

mechanisms (Paintlia, Paintlia et al. 2005; Miron, Rajasekharan et al. 2007).

Statins have been shown to be protective against AD, though it is unclear if these

effects are dependent upon choleseterol. We believe that statin inhibition of

isoprenylation in the brain may be responsible for protective effects of statins. Previously

our lab has shown that statins can inhibit Aβ induced microglial responses in vitro

(Cordle, Koenigsknecht-Talboo et al. 2005; Cordle and Landreth 2005).

- 36 - It is unclear if statins reach sufficient concentrations to mediate cholesterol- independent effects in the CNS. Brain localization of statins has been shown to be statin- specific, as only hydrophobic statins are readily able to cross the blood-brain-barrier

(BBB). Simvastatin and lovastatin are hydrophobic, while pravastatin and atorvastatin are hydrophilic. A recent study showed for the first time the concentrations of statins in the brains of mice (Johnson-Anuna, Eckert et al. 2005). These authors showed that simvastatin, lovastatin, and pravastatin can reach the brain at peak concentrations of

500nM, 300nM, and 100nM, respectively. Strikingly, levels simvastatin remained at

50nM in the brain 24 hours after treatment. Similar results were reported by a different group (Thelen, Rentsch et al. 2006). In fact these studies may underestimate access of statins to the brain, as they used LC/MS/MS approach that can only detect the prodrug and hydroxy acid forms of these drugs. The bulk of circulating HMG-CoA reductase inhibitory activity of simvastatin and lovastatin consists of metabolites not detected by these techniques. Only one human study has looked at statin levels in the CSF, and reported concentrations of lovastatin in the low nanomolar range. However, in this study might underestimate brains levels of statins for several reasons: 1) patients were treated with low doses of lovastatin, 2) measurements were made at 1hours after treatment, while lovastatin concentrations peak in the plasma at 4h, and 3) LC/MS/MS techniques that underestimate active inhibitory drug concentration were used (Botti, Triscari et al. 1991).

Strikingly, Johnson-Anuna and colleagues demonstrated that statins can mediate changes in gene expression the brains (Johnson-Anuna, Eckert et al. 2005). Simvastatin, lovastatin, and pravastatin all significantly reduced neuronal membrane cholesterol by about 10% after 21 days of treatment. In addition, all 3 statins exerted similar effects on

- 37 - the expression of a subset of 13 genes. Simvastatin had large non-overlapping effects on

the expression of a number of other genes, which could be explained by the fact that

simvastatin reaches the brain at higher concentrations than other statins. These studies

suggest that statins may reach the brain in sufficient concentrations to mediate

isoprenoid-dependent effects.

Statins, Protein Isoprenylation, and Ras Superfamily GTPases.

Statins have been primarily used as cholesterol lowering therapy. As mentioned

above, statins may mediate some of their effects through cholesterol independent

mechanisms. The primary mechanism by which statins can act is through depletion of

isoprenoids Isoprenoids are a class of compounds derived from isopentenyl

pyrophosphate, an intermediate in the cholesterol biosynthesis pathway, which contains a

basic five-carbon isoprene building block (Lynen 1958). The best studied isoprenoids are

the 15-carbon farnesyl pyrophosphate and 20-carbon geranylgeranyl pyrophosphate

groups. The C-15 isoprenoid farnesyl was first shown to modify yeast polypeptide

mating factors (Kamiya, Sakurai et al. 1978) . In vitro studies demonstrated that high

doses of statins (>1µM) can affect cell cycle progression, cell morphology, and differentiation (Quesney-Huneeus, Wiley et al. 1979; Habenicht, Glomset et al. 1980;

Schmidt, Glomset et al. 1982; Quesney-Huneeus, Galick et al. 1983; Fairbanks, Witte et

al. 1984; Maltese 1984). These effects of statins can be rescued by addition of

mevalonate, the precursor of isoprenoids, but not through restoration of exogenous cholesterol. This suggests that mevalonate or an isoprenoid metabolite, and not cholesterol, is responsible for these effects. The seminal work of Schmidt, Schneider,

- 38 - and Glomset, demonstrated that a metabolite of mevalonate was incorporated into

proteins, and that inhibition of this incorporation by statins was responsible for the statin-

dependent changes of cell morphology (Schmidt, Schneider et al. 1984).

Further studies showed that in the presence of mevastatin and radioactive

mevalonate, incorporation of radioactivity occurred primarily into 22-26kD proteins of

(Bruenger and Rilling 1986), as well as into nuclear membrane proteins of approximately

66kD (Maltese and Sheridan 1987). Further studies showed that the 66kD proteins were

the nuclear membrane Lamin proteins (Wolda and Glomset 1988). Lamins were

modified by farnesylation of a C-terminal cysteine residue (Farnsworth, Wolda et al.

1989). p21ras, an oncogene encoding a GTPase of 21 kD, was also shown to be modified

by a farnesyl group (Casey, Solski et al. 1989). Importantly, isoprenylation of these

proteins has important functional consequence, as transformation of cell lines expressing

oncogenic forms of Ras was blocked by treatment with statins, suggesting that

isoprenylation was required for Ras function (Schafer, Kim et al. 1989; Howland, Trusko

et al. 1998).

A series of papers demonstrated that Ras associates with membranes, and that this

membrane association was dependent upon a C-terminal CaaX motif (C=cysteine, a= aliphatic amino acid, x=any amino acid) (Willumsen, Christensen et al. 1984). The CaaX

motif is the target signal for farnesylation, and farnesylation of Ras is required for

membrane association and function of Ras (Hancock, Magee et al. 1989; Schafer, Kim et

al. 1989). A CaaX motif identified in the yeast a mating factor peptide and in the lamin

proteins is necessary for farnesylation (Magee and Hanley 1988; Kitten and Nigg 1991).

Initial studies showed that isoprenoids of two different sizes were incorporated into

- 39 - cellular proteins (Rilling, Bruenger et al. 1989). The second isoprenoid was

demonstrated to be the C20 geranylgeranyl group (Epstein, Lever et al. 1990; Farnsworth,

Gelb et al. 1990). This modification is the predominant isoprenoid modification in the cell, and it has since been estimated that up to 2% of cellular proteins are isoprenylated

(Epstein, Lever et al. 1991). One of the primary targets for geranylgeranylation are the

Rho family proteins (such as Rac, Rho, and Cdc42), and these proteins also carry a CaaX motif that is required for isoprenylation (Kinsella, Erdman et al. 1991). Isoprenylation of

Rho proteins has also been shown to be required for their membrane association and function (Figure 3). Rab proteins, which have a C-terminal double cysteine CC or CxC motif instead of CaaX (Khosravi-Far, Lutz et al. 1991), become geranylated on both C-

terminal cysteines (Farnsworth, Seabra et al. 1994), and isoprenylation is required for

Rab membrane localization and function (Rossi, Yu et al. 1991) (Figure 2).

The mechanisms of protein isoprenylation have been well studied, and it has been

demonstrated that isoprenylation is a critical modification required for the function of Ras

superfamily GTPases. The mammalian farnesyl transferase (FTase) is a heterodimer

consisting of two subunits: αF/GGI and βF (Reiss, Goldstein et al. 1990; Chen, Andres et al.

1991; Chen, Andres et al. 1991). The substrates of farnesyl transferase include Ras,

laminA, laminB, γ-subunit transducin, and rhodopsin kinase. The mammalian protein

geranylgeranyltranferase Type I (GGTase-I) enzyme is also a heterodimer, sharing the

same alpha subunit as FTaseI (αF/GGI), with a different beta subunit (βGGI). The primary

substrates for GGTase-I are Rho family proteins (such as Rac, Rho and Cdc42). The

GGTaseI and FTase enzymes β-subunits share 30% identity, though they are generally

quite selective for their substrates. The C-terminal residue of the CaaX motif confers

- 40 - specificity for FTase or GGTase I (Moores, Schaber et al. 1991). However, some

substrates such as K-Ras, RhoB and TC21 are substrates for both enzymes (Hicks,

Hartman et al. 2005).

Not surprisingly, some G proteins are better substrates for FTase-I than others.

The Km values for association of FTase with Ha-Ras, N-Ras, Ki-Ras4A, and Ki-Ras4b are 0.6 µM, 0.4 µM, 0.4 µM, and .03 µM, respectively (Zhang, Kirschmeier et al. 1997).

The differential affinities of substrates for FTase-I were manifested by differential sensitivity to a farnsesyltransferase inhibitors (FTIs), with the IC50 of the FTI SCH44342

against Ha-Ras, N-Ras, Ki-Ras4A, and Ki-Ras4B peptides were 0.18 µM, 0.4 µM, 0.7 µM, and 60 µM, respectively (Zhang, Kirschmeier et al. 1997). The affinities of different

substrates for GGTase-I have not been investigated.

Most Rab proteins carry a C-terminal CxC or CC motif, and the protein is

prenylated on both of these cysteines. The enzyme responsible for this modification is

geranylgeranyltransferase type II (GGTase-II). GGTase-II is composed of two subunits;

αGGII and βGGII, which have 30% identity with the FTase and GGTase-I subunits.

However, GGTase-II requires a cofactor, Rab Escort Protein 1 (Rep1), which is crucial

for binding of Rab proteins to the GGTase-II. While FTase and GGTase-I can recognize

and prenylate short peptides containing a CaaX motif, GGTase-II only recognizes intact

Rab proteins that are bound to Rep1 (Andres, Seabra et al. 1993). It has been shown that

the dual cysteine prenylation of Rabs is critical for proper membrane targeting and

function. Rab-CaaX chimeras, which are singly prenylated, become improperly localized

(Gomes, Ali et al. 2003). Little is known about the specificity of GGTase-II for different

- 41 - substrates, though it has been shown to have low affinity for the Rab27a substrate in

comparison to other Rabs.

After prenylation of cysteine, the three peptides after the cysteine (-AAX) are

cleaved by the enzyme Rce1 (Boyartchuk, Ashby et al. 1997), and the cysteine is then

carboxymethylated by the membrane-bound isoprenylcysteine-O-carboxyl

methyltransferase (Icmt) (Dai, Choy et al. 1998). All of these modifications occur in the

ER. These post-prenylation processing events are functionally important, particularly for the farnesylated Ras proteins. In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized, while the intracellular localization of geranylgeranylated Rho

GTPases were not perturbed (Michaelson, Ali et al. 2005). Exchanging geranylgeranyl

moieties for farnesyl groups on Ras proteins or vice versa on Rho proteins reversed the

differential sensitivities to Rce1 and Icmt deficiency, suggesting that postprenylation

CAAX processing is required for proper localization of farnesylated Ras, but not

geranygeranylated Rho proteins (Leung, Baron et al. 2006). It has been shown that

carboxymethylation increases the stability of Rho family proteins (Backlund 1997), and

carboyxmethylation may be required for some Rho and Rac dependent functions

(Papaharalambus, Sajjad et al. 2005).

Geranylgeranylated Rab proteins usually terminate in either CC or CxC. While both classes of proteins geranylgeranylated on both cysteines, only the CxC class has been shown to be carboxyl-methylated on the terminal cysteines in vivo, and the CC

proteins are not methylated, presumably because steric hindrance does not allow access

to Icmt. (Smeland et al. 1994, Bergo, Leung et al. 2001). Several Rab proteins, such as

Rab8 and Rab13, contain the C-terminal -CaaX motif, and while isoprenylated by

- 42 - GGTase-II, are processed post-isoprenylation identically to Ras and Rho proteins (Leung,

Baron et al. 2007).

The understanding of the mechanisms by which protein isoprenylation occurs has

coincided with increased knowledge and interest in the function of Rho family proteins.

The most prominent of these proteins, Rac, RhoA and Cdc42 were initially characterized for their ability to regulate the actin cytoskeleton (Nobes and Hall 1995). There are now over 15 known members of the Rho family and they have been more recently characterized for their ability to regulate not only cytoskeletal-related events such as cell adhesion and migration, but also a number of other process such as gene transcription, cell differentiation and cell proliferation (Takai, Sasaki et al. 2001). Rho family proteins have been shown to play a role in regulating inflammatory gene expression, and may play a role in microglial activation in AD (Cordle and Landreth 2005). Several reports have shown that Rho family proteins may play a role in regulating Aβ production (Zhou, Su et al. 2003; Desire, Bourdin et al. 2005).

The Rab proteins comprise a family of greater than 60 proteins, and represent the largest subfamily of the Ras superfamily of small GTPases. Rab proteins are important

regulators of organelle biogenesis and vesicle transport (Pereira-Leal and Seabra 2000).

They are conserved throughout evolution, from yeast to mammals (Pereira-Leal and

Seabra 2001). Each Rab protein has a specific intracellular localization and regulates a

specific membrane trafficking step However, transport between two membrane

compartments may be governed by more than one Rab member(Zerial and McBride

2001). Rab proteins have been shown to important in the trafficking of APP through the

- 43 - secretory and endocytic pathyways (Dugan, deWit et al. 1995; McConlogue, Castellano et al. 1996).

Research Goals

This project was designed to studying isoprenoid-dependent effects of statins on

APP processing to Aβ. While carrying out these studies we have come to the realization that some of the basic biology of statin inhibtion of protein isoprenylation is poorly understood. This shifted our focus to studying the dose-dependence and selectivity of statin action on protein isoprenylation, and to developing 2D gel techniques to directly monitor protein isoprenylation status.

Chapter 2 describes the bulk of our research efforts. We utilized two widely used in vitro cell models of APP processing: N2a murine neuroblastoma cells and H4 human neuroglioma cells. We demonstrate that in both cell types, that statin treatment at high doses inhibited the membrane association of all Ras superfamily GTPases tested. Statins had a robust affect on the trafficking of APP, leading to accumulation of APP within the cells and delayed maturation of APP as demonstrated by pulse-chase experiments. These effects were much more pronounced in N2a cells than H4 cells, suggesting cell-type specific effects of statin action. We show that C. difficile Toxin A, which specifically and robustly inhibits the function of Rho family proteins (Rho, Rac, and Cdc42), had no effects on the trafficking or maturation of APP. This suggests that the effects of staitns on APP trafficking were due to the inhibition of Rab family proteins, which are intimately associated in vesicular trafficking. Strikingly, statin-dependent effects on the

- 44 - trafficking of APP are similar to those previously reported for inhibition of Rab1b. It is likely that inhibition of Rab1b was responsible for statin effects on APP processing,

though statins inhibit the isoprenylation of a number of proteins, so the contribution of

other Rab proteins likely contribute to the observed effects.

In H4 cells, statin treatment led to a decrease of APP C-terminal fragment (CTF)

levels and decreased Aβ secretion. These effects were mimicked by Toxin A and

Clostridium botulinum C3 Exoenzyme, which specifically and robustly inhibits the

function of Rho A, B and C, but not Rac and Cdc42. This suggests that inhibition of

RhoA, B, and/or C was responsible for the statin-mediated reduction of CTF levels. In

addition, we show that the α- and β- cleavages of APP were not affected by statin or

Toxin A treatment. Instead, statin and Toxin A treatment led to increased degradation of

CTFs by a lysosomal-dependent mechanism.

Importantly, we demonstrate that simvastatin impacted APP accumulation and

CTF degradation at doses as low as 300nM and 500nM, respectively. We had previous

hints from the literature suggesting that some GTPases are better substrates than others

for isoprenylation (Zhang, Kirschmeier et al. 1997). Also, it has been shown that after

cells are treated with statins, and low doses of mevalonate were replaced, the mevalonate

was incorporated only into a subset of GTPases (Rilling, Bruenger et al. 1993). These

findings suggest that statins may preferentially inhibit a subset of GTPases, particularly at

low doses. We demonstrate that at 200nM of simvastatin that the membrane association

of Rac and Rab1b were inhibited, but the association of Rab4 and Rab5b were not. This

demonstrates that statins selectively inhibit a subset of GTPases at lower, clinically

relevant, doses.

- 45 - Chapter 3 describes recent efforts to use 2D gels to directly monitor protein

isoprenylation. It has been shown previously that the cysteine carboxymethylation that

occurs after isoprenylation of -CaaX isoprenylation motif proteins such as Rho and

Cdc42 changes the pI of the proteins, causing a basic gel-shift as assesed 2D gel electrophoresis (Backlund 1997). We demonstrate that statin inhibition of protein

isoprenylation, which precludes carboxymethlation, resulted in acidic isoelectric shifting

of the CaaX Rho proteins RhoA and Cdc42, and the CxC Rab proteins Rab4 and Rab6.

Rab11, which contains a CC isoprenylation motif, and does not become

carboxymethylated, did not display a gel mobility shift after statin treatment. 2D gel

electrophoresis provides a sensitive measure to directly measure protein isoprenylation status, and will be useful in future investigations of statin effects on protein isoprenylation.

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- 75 -

Chapter 2: Statins Reduce Amyloid-Beta Production through Inhibition of Protein Isoprenylation

- 76 -

ABSTRACT

Epidemiological evidence suggests that long term treatment with HMG-CoA reductase inhibitors, or statins, decreases the risk for developing Alzheimer’s 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 200nM, 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 (Aβ), 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 Aβ secretion. In summary, we show that statins selectively inhibit GTPase isoprenylation at clinically relevant doses, leading to reduced

Aβ production in an isoprenoid-dependent manner. These studies provide insight into the mechanisms by which statins may reduce AD pathogenesis.

- 77 - - 78 -

INTRODUCTION

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia in the elderly. The pathologic hallmarks of AD are extraneuronal senile plaques composed of β-amyloid (Aβ) fibrils and intraneuronal accumulations of hyper-phosphorylated tau (Glenner and Wong 1984; Masters, Simms et al. 1985). Aβ is generated by sequential proteolytic processing of the type I transmembrane protein, amyloid precursor protein (APP), by β- and γ-secretases (Sinha,

Anderson et al. 1999; Vassar, Bennett et al. 1999). Nascent APP is trafficked via the common secretory pathway and undergoes post-translational modifications including N- and O-glycosylation. Following delivery to the cell surface, APP is trafficked to late endosomes, and either recycled to the cell surface or degraded within the lysosome. APP is cleaved by the β- or α-secretase to generate either a C99 or C83 C-terminal fragment

(CTF), respectively. γ-secretase cleaves the C99 CTF to form Aβ, while cleavage of C83 results in the production of a non-amyloidgenic p3 fragment. β- and γ-secretase complexes are found in multiple cellular compartments including the ER, late-

Golgi/TGN, endosomes and plasma membrane, though there is significant debate with regard to the magnitude of APP processing within individual subcellular compartments and their quantitative contribution to Aβ production (Kinoshita, Fukumoto et al. 2003;

Kim, Yin et al. 2004; Tarassishin, Yin et al. 2004; Chyung, Raper et al. 2005). The

Rab subfamily proteins are critical for vesicular trafficking and have been shown to be involved in regulating Aβ production (Dugan, deWit et al. 1995; McConlogue,

Castellano et al. 1996). In particular, Rab1b mediates the transport of APP from the

- 79 - endoplasmic reticulum to the Golgi, where it undergoes glycosylation. Inhibition of

Rab1b function through expression of dominant negative forms of this G-protein resulted in impaired trafficking that was associated with inhibition of APP processing and Aβ production (Dugan, deWit et al. 1995; Maltese, Wilson et al. 2001). Similarly, Rab6 is involved in intra-Golgi trafficking of APP and inhibition of its function by expression of a dominant negative Rab6 leads to a significant reduction of Aβ generation (McConlogue,

Castellano et al. 1996). The Rho subfamily of small G-proteins, such as RhoA, Rac, and

Cdc42, while first recognized for regulating actin-based cytoskeleton rearrangement

(Nobes and Hall 1995), have been shown to be important elements in a variety of intracellular signaling pathways, including those involved with Aβ production (Zhou, Su et al. 2003; Desire, Bourdin et al. 2005).

Statins are widely prescribed drugs for treatment of hypercholesterolemia, and act to reduce plasma cholesterol levels by inhibiting the rate-limiting enzyme in the cholesterol biosynthetic pathway, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, preventing de novo synthesis of cholesterol (Endo 1992; Tobert 2003).

Epidemiological studies suggest that treatment with statins reduces the risk of developing

AD (Jick, Zornberg et al. 2000; Wolozin, Kellman et al. 2000; Rockwood, Kirkland et al.

2002). Most work has focused on the cholesterol lowering effects of statins, and statins can reduce Aβ production in vitro through lowering of cholesterol levels. This effect was postulated to result from the sensitivity of β- and γ- secretases to neuronal membrane cholesterol content (Simons, Keller et al. 1998; Kojro, Gimpl et al. 2001; Burns and Duff

2002; Wahrle, Das et al. 2002; Cordy, Hussain et al. 2003). Statins have been shown to decrease Aβ levels and plaque load in some animal models (Fassbender, Simons et al.

- 80 - 2001; Petanceska, DeRosa et al. 2002), however, it is unclear if lowering of cholesterol is responsible for the observed effects. Several lines of evidence suggest that lowering of cholesterol may not fully explain the protective effects of statins in AD. Notably, the

balance of clinical data does not strongly support elevated serum or brain cholesterol as a risk factor for AD (Wood, Igbavboa et al. 2005; Stewart, White et al. 2007), and results from animal studies with regard to the involvement of cholesterol in AD pathology is mixed (Howland, Trusko et al. 1998; Refolo, Pappolla et al. 2001; Shie, Jin et al. 2002;

George, Holsinger et al. 2004).

Statins exhibit pleiotropic effects through reduction of isoprenoid intermediates in the cholesterol biosynthetic pathway (Liao and Laufs 2005). The isoprenoids geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) are added to the C-termini of the Ras superfamily of small G-proteins, including Rho and Rab.

Isoprenoid modification is critical for facilitating GTPase interactions with cytoplasmic regulators, cellular membranes, and effectors (Zhang and Casey 1996). Thus, the ability of statins to reduce AD risk may arise from inhibition of protein isoprenylation.

Cholesterol-independent actions of the statins have already been demonstrated to be important for the clinical benefits of these drugs on cardiovascular disease (Bellosta,

Ferri et al. 2000; Bellosta, Ferri et al. 2000; Takemoto and Liao 2001), as well as in animal models of central nervous system diseases with an inflammatory component, including multiple sclerosis and ischemic stroke (Kwak, Mulhaupt et al. 2000; Youssef,

Stuve et al. 2002; Zamvil and Steinman 2002).

In cell culture, it has been shown that statin inhibition of GTPase isoprenylation causes these proteins to lose their normal membrane association and function (Cordle,

- 81 - Koenigsknecht-Talboo et al. 2005). However, the effects of statins on protein isoprenylation have not been well studied in neurons. In addition, reports of statin effects on Rab family proteins have been quite limited, though statins have been shown to modulate protein trafficking through inhibition of Rab protein function (Overmeyer and

Maltese 1992; Ivessa, Gravotta et al. 1997). As APP is trafficked by Rab-dependent mechanisms and perturbation of Rab function is associated with suppression of APP processing and (Dugan, deWit et al. 1995; McConlogue, Castellano et al. 1996; Maltese,

Wilson et al. 2001), we thought it important to examine the effects of statins on Rab isoprenylation, and whether modulation of Rab function by statins may perturb Aβ production.

The physiological levels of statins in the brain have only recently been determined.

Johnson-Anuna et al. reported simvastatin reaches concentrations of 300-500nM in the brains of mice (Johnson-Anuna, Eckert et al. 2005). Effects of statins at these lower, more clinically relevant, doses are not well documented. We report that, in neuronal cell types, statins inhibit the isoprenylation and membrane association of GTPases of the Rho and Rab family at doses of statins as low as 200nM. Importantly, we show that while at high doses statins universally impair GTPase function, at low doses statins preferentially impair the isoprenylation and membrane localization of only a subset of GTPases. These

GTPases may represent specific, clinically relevant targets of statin action. We also show that statins impact APP metabolism through Rab and Rho dependent mechanisms, leading to reduced Aβ production. In summary, we show that statins can selectively inhibit the isoprenylation of GTPases at physiologically relevant doses, and suggest that

- 82 - statins may act by cholesterol-independent mechanisms to lower Aβ production and limit

AD pathogenesis.

EXPERIMENTAL PROCEDURES

Materials and reagents. Simvastatin and lovastatin were purchased from Calbiochem

(La Jolla, CA) and prepared following the manufacturer’s instructions. The statins were converted to the active form by dissolving them in absolute EtOH, followed by the addition of 1M NaOH to a final concentration of 100mM. This solution was stored at -

20°C until use. Immediately before use, the statin solution was neutralized with 1M HCl, and diluted in vehicle (50% EtOH, 5mM HEPES, pH 7.2). Geranylgeranyl pyrophosphate triammonium salt and farnesyl pyrophosphate triammonium salt were purchased from Biomol (Plymouth Meeting, PA). Mevalonic acid was purchased from

Sigma-Aldrich and reconstituted in 100% ethanol. Clostridium difficile Toxin A was purchased from List Labs (Campbell, CA). Cell-permeable C3 exoenzyme was obtained from Cytoskeleton (Denver, CO). The Amplex Red Cholesterol Assay kit was purchased from Molecular Probes (Eugene, OR). Antibodies to APP (22c11) and the APP C- terminal fragment were purchased from Chemicon (Temecula, CA). Antibodies to Rac and Rab4 were obtained from Upstate (Waltham, MA). Antibodies to flotillin and calnexin were obtained from BD Bioscience (San Jose, CA). The antibody to GAPDH was obtained from Trevigen (Gaithersburg, MD). Antibodies to ERK2, β-Tubulin, RhoA,

Cdc42, Rab1b, Rab5b, and Rab6 were obtained from Santa Cruz Biotechnology (Santa

Cruz, CA). Antibodies 6E10 and 4G8 were purchased from Covance (Cumberland, VA).

- 83 - Peroxidase-conjugated secondary antibodies were purchased from GE Healthcare (UK).

Cell culture reagents were purchased from Invitrogen (Carlsbad, CA).

Cell culture. Mouse N2a (parental) neuroblastoma cells were obtained from American

Type Culture Collection (Manassas, VA). N2a.Swe cells were obtained from Dr. Gopal

Thinakaran (University of Chicago). APPsw-293 cells were obtained from Dr. Robert

Vassar (Northwestern University). N2a and APPsw-293 cells were cultured in 50%

Optimem/50% DMEM, 5% FBS (Hyclone, Logan, UT), and 1% Penicillin/Streptomycin.

H4.APPWT and H4.HPLAP neuroglioma cells were maintained in Optimem plus 4%

FBS, 1% penicillin/streptomycin and 1% Zeocin. H4.APPWT cells overexpress wild type human APP under an actin promoter (Murphy, Uljon et al. 2000). H4.HPLAP express endogenous levels of APP, but overexpress the human APP C-terminal fragment fused to human placental alkaline phosphatase. All cells were cultured at 37°C and 5% CO2.

Neuron culture. Primary cultures of cortical neurons were prepared from embryonic day

15–16 C57BL/6 mouse embryos as described (Brown and Goldstein 1980). Briefly, embryo cortices were dissected, and meninges were removed. Tissue was digested, mechanically dissociated, and suspended in neurobasal medium (B27 supplement, 100

µg/ml penicillin/ streptomycin, 0.5 mM glutamine, and 25 µM glutamate), and plated densely onto poly-D-lysine-coated 6-well plates (1x106 cells/well). Neurons were maintained under serum-free conditions in neurobasal medium with B27 supplement prior to drug treatment.

- 84 - Drug treatments. Cells were plated at 5x105 cells per well in 6-well plates, and allowed

to grow for 1 (for 48h treatment) or 2 days (for 12-24h Tx) before drug treatment. H4 cells were plated on poly-L-lysine-coated 6-well plates. Cells were then treated with the indicated compounds for 12-48h.

Western blotting. Cells were collected and lysed with radioimmunoprecipitation assay

(RIPA) buffer (1% Triton X-100, 20 mM Tris, pH 7.5, 100 mM NaCl, 40 mM NaF, 0.2%

SDS, 0.5% deoxycholate, 1 mM EDTA, 1 mM EGTA, and 1 mM Na3VO4). Lysates

were sonicated for 2 x 10 s on ice and cleared by centrifugation (16,000 x g, 15 min, 4°C).

Protein concentration was determined by the Bradford method (46). The samples were

boiled under reducing conditions then resolved on 9% SDS-PAGE gels or NuPage 4-12%

BisTris gels (Invitrogen) and transferred to polyvinylidene fluoride membranes. After blocking in a 5% milk or 5% normal goat serum solution, blots were incubated overnight

at 4°C with the indicated antibodies. Bands were visualized by incubation of blots with

anti-mouse, rabbit or goat HRP-conjugated secondary antibodies (1:1000; 90 min at room

temperature) and visualized by enhanced chemiluminescence (Pierce, Rockford, IL).

Protein loading was evaluated by probing with anti-ERK2 (1:3000) or anti-GAPDH

(1:5000) antibodies. Images were scanned using Adobe Photoshop and band intensities

quantified using Image-Pro Plus software package (Media Cybernetics, Inc., Silver

Springs, MD). Band densities were normalized for protein loading by comparison with

ERK2 or GAPDH band densities. Mean values, ±SEM, were calculated. Pair wise

comparisons were determined using the Tukey-Kramer post-hoc test.

- 85 - Quantification of Secreted Aβ Peptide Levels by ELISA. Following drug treatments, the

culture medium was collected, a protease inhibitor cocktail containing 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride was added, and medium was centrifuged at

16,000 x g for 15 min at 4 °C. Media from H4.APPWT cells were diluted 1:5 and assayed by ELISA specific for Aβ1-40 from Biosource/Invitrogen (Carlsbad, CA).

H4.APPWT cells do not produce detectable levels of Aβ1-42.

Membrane Localization and Western blotting for GTPases. N2a cells were plated into 6- well plates and 24 hours later were treated with simvastatin for 24 or 48h. Cellular fractionation was carried out as described previously (47). Briefly, following statin treatment the cells were lysed by incubation in relaxation buffer (100 mM KCl, 3 mM

NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, and 10 mM PIPES, pH 7.3) on ice for 15 min followed by 10 s of sonication. Cells were cleared by centrifugation at 500 x g for 5 min at 4 °C. The resulting supernatant was centrifuged for 1 h at 110,000 x g at 4 °C in a

Beckman-Coulter ultracentrifuge (SW50.1 rotor). The resulting supernatant was removed

(cytosolic fraction), and the membrane pellet was then resuspended in relaxation buffer

(membrane fraction). The protein concentration from each fraction was measured using the BCA protein assay from Pierce (Rockford, IL). Standard Western blotting procedures were used to separate the fractions and transfer them to polyvinylidene difluoride membranes. Blots were probed with antibodies for the individual GTPases, as well as markers for cytosolic (GAPDH, ERK2) and membrane (flotillin, calnexin) fractions.

- 86 - RESULTS

Selective effects of statin treatment on G-protein isoprenylation and abundance

We investigated whether statins act uniformly to inhibit the isoprenylation of members of the Rho and Rab families of G-proteins in N2a neuroblastoma cells. We initially monitored statin-mediated effects by decreased electrophoretic mobility of non- prenylated versus prenylated forms of GTPases as examined by SDS-PAGE. It has been reported previously that Ras and Rab (Sinensky, Beck et al. 1990; Overmeyer and

Maltese 1992; Ivessa, Gravotta et al. 1997), but not Rho family proteins (Cicha,

Schneiderhan-Marra et al. 2004) exhibit altered electrophoretic mobility depending upon protein prenylation status, though we have reported altered isoprenoid-dependent Rac electrophoretic mobility in some cell types (Cordle, Koenigsknecht-Talboo et al. 2005).

In statin-treated N2a cells we observed lowered mobility of Rab family proteins after statin treatment but not of the Rho family proteins Rac, Rho, or Cdc42 (Fig. 1A).

The statin-dependent change in electrophoretic mobility of these protein families likely reflects the fact that Rab family proteins have two geranylgeranyl moieties added to them, whereas Rho family proteins possess only one lipid moiety. We find that after exposure of N2a cells to high doses of statins (10μM), the Rab GTPases Rab1b, Rab4, Rab5, and

Rab6 are converted entirely into the lower mobility, non-prenylated species (Fig. 1A).

Statins reduced the levels of Rab6, requiring overexposure of the blots to visualize the unprenylated Rab6 band. Similar results were observed in H4 neuroglioma cells (data not shown). Provision of mevalonate allows restoration of the isoprenyl intermediate pools, without significant effects on cholesterol synthesis (Brown and Goldstein 1980;

Goldstein and Brown 1990; Simons, Keller et al. 1998; Fassbender, Simons et al. 2001;

- 87 - Kojro, Gimpl et al. 2001). Consistent with previous reports that show electrophoretic shifts are a result of loss of protein isoprenylation, we observe that changes in electrophoretic protein mobility caused by statin treatment were reversed upon provision of exogenous mevalonate, demonstrating that these effects are dependent upon protein isoprenylation (Fig. 1A). Statins do not alter cellular cholesterol content when serum is present in the media, as has been reported previously (data not shown) (Cole, Grudzien et al. 2005; Cordle and Landreth 2005).

One remarkable outcome of these experiments was the effect of the statins on

Cdc42. We find that in N2a cells, statins cause a dramatic increase in Cdc42 levels (Fig.

1A,B). Similar results were found in H4 cells (data not shown). Elevated Cdc42 levels were seen in N2a cells at doses of statins as low as 50nM (Fig. 1B). Cdc42 mRNA levels were not increased after statin treatment (data not shown), suggesting that isoprenylation may be required for the normal turnover of this protein.

Statins block the membrane association of Ras superfamily GTPases

The isoprenoid modification of the small GTPases is essential for their membrane localization. The membrane association of G-proteins provides a sensitive measure of their prenylation status. Disruption of membrane association of GTPases likely results in loss of protein function owing to their inability to associate with membrane bound effectors. We surveyed the effects of statin inhibition of isoprenoid synthesis on membrane association of newly synthesized Rho and Rab family G-proteins. N2a cells were treated with 10 µM simvastatin or lovastatin for 24h, in the presence or absence of mevalonate (Fig. 2A). Cells were subjected to biochemical fractionation to isolate

- 88 - membrane and cytosolic fractions. At this statin concentration both simvastatin and lovastatin robustly inhibited the membrane association of all GTPases tested, including the Rho family members Rac, RhoA, and Cdc42, and the Rab family members, Rab1b,

Rab4, Rab5b, and Rab6 (Fig. 2A). Provision of exogenous mevalonate restored membrane localization of all GTPases tested, establishing the reliance of GTPase membrane association on isoprenoids (Fig. 2A). Treatment of N2a cells with 500nM simvastatin robustly inhibited the membrane association of all GTPases tested (Fig. 2B).

Remarkably, until the recent report by Johnson-Anuna and colleagues, the concentrations of statins within the brain were unknown (Johnson-Anuna, Eckert et al.

2005). Simvastatin treatment of mice results in brain levels of 300-500nM (Johnson-

Anuna, Eckert et al. 2005). However, the analysis of the effects of statins on protein isoprenylation in vitro has typically employed much higher dosages and little is known about drug effects at physiologically significant concentrations. Protein isoprenylation is directly related to cellular isoprenoid pool size, and at low isoprenoid pool sizes, isoprenoids are primarily incorporated into a subset of GTPases (Rilling, Bruenger et al.

1993). This suggests that statins may differentially alter protein isoprenylation depending on statin dose, owing to dose-dependent effects on cellular isoprenoid pool size. We tested whether statins might selectively inactivate GTPases within the physiological dose range. Strikingly, if physiologically appropriate levels of simvastatin were used to treat the cells we observed a selective effect on the membrane association of the G-proteins.

Rab4 and Rab5b membrane localization was not significantly changed by 200nM simvastatin treatment. Rac, and Rab1b localization were decreased after 200nM simvastatin treatment (Fig. 2C). Rac localization was the most dramatically affected, and

- 89 - quantification of the data showed that membrane association of Rac was significantly reduced by 40% after 200nM simvastatin treatment (Fig. 2D).

Statins and Toxin A increase synthesis of APP in N2a.Swe cells, but not in other cell types

Statins have well documented cholesterol-dependent effects on APP processing and Aβ generation (Fassbender, Simons et al. 2001; Kojro, Gimpl et al. 2001; Burns and

Duff 2002; Wahrle, Das et al. 2002; Cordy, Hussain et al. 2003), however the isoprenoid- dependent effects of statins on APP processing have not been well studied (Cole,

Grudzien et al. 2005; Pedrini, Carter et al. 2005). We investigated the effect of statin inhibition of isoprenylation on APP metabolism and Aβ production.

We investigated whether statins affect the synthesis of APP, owing to a previous report that statins act to stimulate APP expression (Pedrini, Carter et al. 2005). We found that in murine N2a.WT cells, the synthesis of endogenous APP was not significantly increased after simvastatin treatment (Fig. 3A). These studies were extended to examine if statins have different effects on murine versus human APP synthesis. In human H4 neuroglioma cells which express either endogenous hAPP (H4.HPLAP) or hAPP overexpressed under an actin promoter (H4.APPWT), there was no change in APP synthesis after statin or Toxin A treatment (Fig. 3A). Similarly, in APPsw-293 cells, which overexpress hAPP under a CMV promoter, we detected no changes in APP synthesis after statin treatment, concurring with results previously published by Cole et al.

(Cole, Grudzien et al. 2005)(Fig. 3A). In addition, we saw no significant changes in APP levels after 24h treatment with simvastatin (10μM) (Fig. 3D), or Toxin A (data not shown)

- 90 - in primary cortical neurons isolated from wild-type C57BL/6 embryos. Thus, in a variety of cell types statins do not affect APP synthesis.

These findings are in contrast with those of Pedrini et al. who have reported that statins cause increased APP synthesis in N2a.Swe cells, which overexpress human

Swedish mutant APP under a CMV promoter, a cell model used widely to examine APP processing (Pedrini, Carter et al. 2005). We observed similar results, and show that statin treatment of APP in N2a.Swe cells increased the expression of APP, consistent with the previous report (Pedrini, Carter et al. 2005). Pulse-chase experiments confirmed that this was due to increased APP synthesis (Fig. 3B,C). In N2a.Swe cells, simvastatin caused increased APP synthesis at doses as low as 300nM (Fig. 3C). Toxin A, a robust and specific Rho family inhibitor often used to delineate the Rho family-dependent effects of statins (Voth and Ballard 2005), also increased APP synthesis in N2a.Swe cells. (Fig.

3B), while having no affect on APP synthesis in other cell types (Fig. 3A,B).

Interestingly, we demonstrate that increased APP synthesis led to a concomitant increase in sAPP and Aβ1-40 and Aβ1-42 production (Fig. 3E). Thus, in N2a.Swe cells we found that the predominant effect of statin treatment was to increase APP expression, sAPP and

Aβ secretion. These data indicate that inhibition of Rho family G-protein function resulted in a stimulation of APP expression in N2a.Swe cells, but not in other cell types.

Similar to observations by Pedrini et al. (Pedrini, Carter et al. 2005), we observed that statinspreferentially increased the secretion of sAPPα over total sAPP in N2a.Swe

(Fig. 3F). However, in H4.APPWT and APPSw-293 cells we observed no increases in either sAPP or sAPPα after statin treatment (Fig. 3F). In addition, we found that while

N2a.Swe produced roughly comparable levels of sAPP to H4.APPWT and APPSw-293

- 91 - cells, that N2a.Swe cells produced extremely low amounts of sAPPα (Fig. 3G). We conclude that in N2a.Swe cells that the increased expression of the hAPP transgene and specific upregulation of sAPPα by statins is unique to this specific cell line and is not representative of the response of other cells lines and primary neurons, to statin treatment.

Statins treatment alters APP trafficking through inhibition of Rab function

We examined the effects of statins on endogenous APP processing in N2a parental cells (N2a.WT). Treatment of the N2aWT cells with either simvastatin or lovastatin at high doses (10μM) for 24h led to a 3-4 fold increase in APP levels (Fig. 4A).

As shown above, there were no effects on APP synthesis in this cell type. We performed pulse-chase experiments, and found that statins dramatically impaired APP maturation and led to accumulation of newly synthesized APP (Fig. 5A). Statin treatment also led to the accumulation of APP both α- and β- C-terminal fragments (CTFs) in N2a.WT cells

(Fig. 4A).

The statin-induced accumulation of APP and CTFs was completely reversed by addition of mevalonate, demonstrating that the effect of statins on APP accumulation was

HMG-CoA reductase dependent and due to reduction of isoprenoid intermediates (Fig.

4A). The accumulation of APP was reversed by GGPP, but not FPP, demonstrating that inhibition of protein geranylgeranylation by statins led to APP and CTF accumulation

(Fig. 4C). Toxin A had no effect on APP accumulation or maturation as detected by

Western blotting and pulse-chase experiments in either N2a or H4 cells (Fig. 5A,B and

7A). These data demonstrate that inhibition of protein geranylgeranylation by statins led to APP accumulation, but specific inhibition of Rho family proteins had no effect on APP

- 92 - accumulation. These data suggest that accumulation of APP by statins was due to

inhibition of Rab family protein function. These findings are consistent with previous

reports of effects of dominant negative forms of Rab1b on APP processing and A

generation (9,11).

Treatment of N2a cells with statins for 24h led to an approximate 30-40%

reduction in secreted Aβ (Fig. 4B). Reduction of Aβ secretion by statins was reversed by addition of mevalonate, showing that this effect was isoprenoid-dependent. In addition,

statins did not change the levels of sAPP generated by these cells. Interestingly, the

accumulation of APP and the decrease of Aβ secretion was observed at statin doses as

low as 300nM (Fig. 4D). Overall, these data demonstrate that statin treatment led to the

accumulation of APP through inhibition of protein geranylgeranylation, but independent

of Rho family inhibition. We conclude that as APP is trafficked within the cell through

Rab-dependent mechanisms, it is likely that inhibition of Rab isoprenylation by statins

alters APP trafficking leading to APP accumulation.

Inhibition of Rho by statins or Toxin A decreases CTF levels and reduces Aβ

secretion

In primary neurons, treatment with simvastatin or lovastatin for 24h resulted in no

detectable changes in APP levels (Fig. 3D). We conclude that there are likely cell-type

specific effects of statin actions, and thought it relevant to look at statin actions in other cell types. We examined APP processing in two human neuroglioma H4 cell lines: 1)

H4.APPWT, which overexpresses wild type human APP under an actin promoter, and 2)

H4.HPLAP, which express endogenous human APP, but overexpresses the human APP

- 93 - C-terminal C100 fragment. We show that while H4.APPWT cells expressed approximately 5-10 fold more APP than H4.HPLAP, both cell lines expressed similar levels of CTFs, suggesting that in H4.HPLAP cells that α- and β-cleavage of full-length

APP does not make a quantitatively significant contribution to CTF generation (Fig. 6A).

We examined the effects of statins on APP accumulation in H4 cells. Interestingly, simvastatin and lovastatin treatment caused a slight increase in APP accumulation in these cells (Fig. 6B). Pulse-chase analysis demonstrated that simvastatin, but not Toxin

A, caused the accumulation of newly synthesized APP, excluding the role of Rho family proteins in APP accumulation (Fig. 5B). This accumulation of APP by statins was rescued by mevalonate and GGPP, but not FPP, indicating that the statin-induced APP accumulation was dependent on inhibition of protein geranylgeranylation (Fig. 6B,D).

Similar to effects seen in N2a cells, statin treatment in H4 cells led to accumulation of

APP, likely through Rab dependent mechanisms, but these effects were much smaller in magnitude than those observed in N2a cells.

Surprisingly, statin treatment led to reduced levels of APP -CTFs in H4 cells.

The non-amyloidgenic C83 fragment is generated from α-secretase cleavage of APP, while the amyloidgenic C99 fragment is generated from β-secretase cleavage. Reduction of CTF levels may thus represent a mechanism of reducing Aβ production. Statin- mediated reduction of CTF levels was rescued by mevalonate and GGPP, but not FPP, and was thus dependent upon inhibition of geranylgeranylated proteins (Fig. 6B,C,D).

Toxin A treatment also caused a decrease of both α- and β-CTF levels (Fig. 7A). Toxin

A inhibits Rho family proteins, while not affecting Rab function, indicating that decreases in CTF levels after statin treatment were due to inhibition of Rho protein

- 94 - function. Toxin A treatment also decreased CTF levels in N2a.WT and APPsw-293 cells

(data not shown). In addition, CTF levels were reduced by treatment of cells with C3

exoenzyme, an inhibitor specific to RhoA,B and C (Fig. 7C).

Interestingly, while Toxin A reduced both α- and β-CTFs, statin treatment at

higher doses (10μM) decreased α- but not β-CTFs. Statin or Toxin A treatment led to a

statistically significant reduction of α-CTF levels by 50% (Fig 6C,7A). We hypothesized

that at higher statin doses, that Rab dependent effects on APP and CTF accumulation masked the Rho-dependent effect of statins on reduction of β-CTFs. Treatment with lower doses of statins (0.5-1μM) for longer times (5 days) decreased both α and β CTFs, while having no effects on APP accumulation (Fig. 6F). These data suggest that at high doses, Rab-dependent effects on APP and CTF accumulation, as seen in N2a cells, overlap with Rho dependent effects on decreased CTF levels, while at lower statin doses

Rho-dependent effects predominate.

Importantly, statin and Toxin A-mediated reduction of CTF levels were observed in both H4.APPWT and H4.HPLAP cells, suggesting that the ability of these agents to decrease CTF levels occurred at steps following APP cleavage that resulted in the formation of CTFs (H4.APPWT) or expression from the C100 transgene (H4.HPLAP)

(Fig. 6B-E and 7A). In addition, secretion of total sAPP and sAPPα did not change in

H4 cells after simvastatin or Toxin A treatment (Fig. 6C and 7A). These data support our conclusion that inhibition of Rho by statins or Toxin A did not affect the generation of

CTFs via APP cleavage, but instead increased their subsequent metabolism.

We examined effects of statins and Toxin A on Aβ secretion in H4 cells. Both statin and Toxin A treatment significantly reduced Aβ secretion by about 30% (Fig. 6G

- 95 - and7B). Ov erall, our data demonstrates that inhibition of the Rho family members RhoA,

B, and/or C by statins, Toxin A and C3 exoenzyme resulted in decreased CTF levels,

leading to decreased production of Aβ.

Statin and Toxin A mediated decrease of CTF levels is due to lysosomal degradation

We observed that statin and Toxin A-mediated G-protein inactivation leads to

decreased CTF levels in H4 cells and that this is likely due to metabolism of CTFs after they have been generated. We hypothesized that after the CTFs are produced, statin or

Toxin A treatment stimulated the proteolytic degradation of CTFs. CTFs have been

shown to be degraded within the lysosome (Caporaso, Gandy et al. 1992; Knops,

Lieberburg et al. 1992; Tsuzuki, Fukatsu et al. 1994) as well as by the proteasome

(Nunan, Shearman et al. 2001; Nunan, Williamson et al. 2003). To our knowledge,

signaling pathways that may regulate CTF degradation pathways have not been described.

Treatment of H4.HPLAP cells with the proteasomal inhibitor MG132 led to

significant increases in CTF levels with little change in APP levels, demonstrating that

CTFs are degraded by the proteasome (Fig. 8A). Chloroquine is a weak base that impairs

lysosomal function by increasing lysosomal pH (Ohkuma and Poole 1978). Treatment of

H4.HPLAP cells with the lysosomal inhibitor chloroquine increased CTF levels with

little change in APP levels, demonstrating that CTFs are also degraded by the lysosome

(Fig. 8A). We treated H4 cells with Toxin A or statins in the presence or absence of

MG132 or chloroquine. MG132 treatment did not impair Toxin A mediated CTF

degradation (Fig. 8A,B). Chloroquine blocked both Toxin A and statin-mediated

reductions of CTF levels (Fig. 8A,B). We conclude that statins and Toxin A, through

- 96 - inhibition of Rho family proteins, increased degradation of CTFs through a lysosomal

dependent mechanism.

DISCUSSION

The primary focus of this study was to determine the mechanistic basis of the

salutary effects of statins on AD risk. We focused on the pleiotropic actions of these

drugs since there is little compelling evidence that statin action in AD is a result of their

cholesterol lowering actions. Moreover, relatively little is known about how isoprenoids might participate in disease pathogenesis. One important confound in the investigation of

statin action in the brain has been that the concentration of statins that are achieved in the

brain after oral administration was unknown. It was only recently reported that the

physiologically relevant statin levels in the brain are in the 300-500 nM range, and thus

much of the literature concerning the biological effects of these drugs may not reflect

their actions in vivo. This study has two principal conclusions. First, we demonstrate that at physiologically relevant concentrations statins selectively inactivate only a subset of

G-proteins. Secondly, we find that the inhibition of Rab family isoprenylation results in

inhibition of APP trafficking and Aβ production while inhibition of members of the Rho family suppresses Aβ production through catabolism of APP CTFs through the lysosome.

This study provides a detailed analysis of the effects of statin-mediated inhibition

of GTPase isoprenylation on APP processing. This represents a departure from most previous investigations, which have examined the role of cholesterol depletion by statins on Aβ production. In order to focus on isoprenylation-dependent effects of statins, we

utilized well described properties of the isoprenoid and cholesterol pathways. In cell

- 97 - culture, statins inhibit de novo cholesterol synthesis, but cellular cholesterol levels are maintained from lipoprotein uptake from serum in media (Cole, Grudzien et al. 2005;

Cordle and Landreth 2005). Moreover, we have verified that the statin-mediated changes were due to reduction in protein isoprenylation by demonstrating complete reversal of statin effects upon provision of exogenous mevalonate at concentrations that do not affect cholesterol synthesis (Brown and Goldstein 1980; Goldstein and Brown 1990; Simons,

Keller et al. 1998; Fassbender, Simons et al. 2001; Kojro, Gimpl et al. 2001), or through provision of GGPP.

Statins selectively inhibit protein isoprenylation at physiologically relevant doses.

We observe that high doses of statins robustly inhibit the membrane association

(Fig. 2A) and electrophoretic mobility shifts of all Rab proteins examined (Fig. 1A), suggesting almost complete loss of protein isoprenylation. These results illustrate that statins, when used at concentrations typically employed in in vitro studies (Beck, Hosick et al. 1990; Leonard, Beck et al. 1990; Sinensky, Beck et al. 1990; Overmeyer and

Maltese 1992; Ivessa, Gravotta et al. 1997; Cole, Grudzien et al. 2005; Cordle and

Landreth 2005; Pedrini, Carter et al. 2005), inhibit the function of a large number of isoprenylated proteins. This makes mechanistic examination of statin effects extremely difficult, owing to the broad array of proteins that are affected by drug treatment.

Moreover, the effects of high dose statin treatment on protein isoprenylation may not be representative of statin effects at physiologically relevant concentrations.

We demonstrate, to our knowledge for the first time, that physiologically relevant concentrations of statins preferentially inhibit the membrane association and

- 98 - isoprenylation of a distinct subset of GTPases. The rationale for our experiments were based on a study reported by Rilling and colleagues (Rilling, Bruenger et al. 1993) who found that the isoprenylation status of the individual G-proteins exhibited isoform- dependent sensitivity to alteration of the size of the intracellular isoprenoids pools. We provide direct evidence for preferential inhibition of isoprenylation of a subset of G- proteins by statins. We demonstrate that after treatment with statins in a physiologically relevant dose range (200nM), that the membrane associations of a subset of GTPases (e.g.

Rac and Rab1b) are robustly inhibited, while the membrane associations of other

GTPases (e.g. Rab4 and Rab6) are unchanged (Fig. 2C,D). We conclude that only a subset of GTPases are biologically relevant targets of statin action.

The basis of the selectivity of statin action is unclear. It has been shown that primary sequence differences in Ras and Rab isoforms cause them to interact uniquely with the transferase enzymes, resulting in different reactivity with the enzyme (Zhang,

Kirschmeier et al. 1997; Larijani, Hume et al. 2003). It is likely that individual GTPases have intrinsic properties that regulate how efficiently they are isoprenylated by the individual transferases. A more detailed analysis will be needed to assess the magnitude of these differences and what role these mechanisms play in regulating the sensitivity of

GTPase isoprenylation to statin treatment.

Statins increase synthesis of APP only in N2a.Swe cells

We observed that in most cell lines and primary neurons that statins and Toxin A do not affect the synthesis of APP (Fig 3A). These observations confirm results published by Cole et al. (Cole, Grudzien et al. 2005), but conflict with those of Pedrini

- 99 - who found that statins increase APP expression in N2a.Swe cells. The present study

resolves this controversy. We demonstrate that statins and Toxin A increased APP synthesis by 2-2.5 fold in N2a.Swe cells through inhibition of Rho family proteins (Fig.

3B,C). These data indicate that inhibition of Rho proteins upregulate synthesis from this

promoter in N2a.Swe cells. Our data suggests that N2a.Swe cells respond to statin

treatment in an atypical manner and this is a caution in the use of these cells to study

statin effects on APP processing or sAPPα shedding.

Statins accumulate APP through inhibition of Rab protein function

While Aβ has been shown to be processed in various intracellular compartments,

the bulk of evidence suggests that Aβ is produced primarily in the TGN and recycling

compartments (Vassar, Bennett et al. 1999; Kim, Leem et al. 2001; Maltese, Wilson et al.

2001). Targeting APP away from the TGN with an ER retention motif, or by

pharmacologically blocking Golgi trafficking, decreases Aβ production (Dugan, deWit et

al. 1995; Kouchi, Kinouchi et al. 1998; Sudoh, Hua et al. 2000; Iwata, Tomita et al. 2001;

Maltese, Wilson et al. 2001; Khvotchev and Sudhof 2004), and conversely targeting APP

to the TGN increases Aβ production (Sudoh, Hua et al. 2000; Iwata, Tomita et al. 2001).

Rab proteins are involved in APP trafficking, and in particular Rab1b mediates ER-Golgi

trafficking (Dugan, deWit et al. 1995; Maltese, Wilson et al. 2001), suggesting that

inhibition of Rab function by statins may modulate APP trafficking and Aβ production.

In N2a.WT cells statin treatment led to the accumulation of newly synthesized

APP (Fig. 4) that was accompanied by the accumulation of CTFs and a 30-40%

reduction in Aβ (Fig. 4A,B). We conclude that impairment of APP trafficking in N2a

- 100 - cells causes accumulation of APP in subcellular vesicular compartments, leading to

reduced production of Aβ. Importantly, we observed these same effects on APP

accumulation and reduction of Aβ at low, physiologically relevant, statin doses (Fig. 4D).

Cole et al. reported that statins caused accumulation of APP in APPsw-293 cells and other cell types (Cole, Grudzien et al. 2005). We have extended these observations by demonstrating that statins impair maturation of APP, as assessed by pulse-chase analysis (Fig. 5). We also provide direct evidence that statins impair Rab protein isoprenylation, and provide data strongly suggesting that the cellular accumulation of

APP is due to impairment of Rab dependent mechanisms (Fig. 1 and 2). We observe that statin-mediated accumulation of APP is dependent upon protein geranylgeranylation (Fig.

4C). The observation that Toxin A, which selectively inhibits the Rho family proteins

(Rho, Rac, Cdc42) (Aktories and Just 1995), has no effect on statin-mediated APP accumulation, rules out a significant role for Rho family proteins on APP trafficking (Fig.

5). However, the Rho protein TC10 has been shown to be involved in CFTR (Cheng,

Wang et al. 2005) and GLUT4 (Chiang, Baumann et al. 2001) protein trafficking, and the sensitivity of TC10 to Toxin A is unknown. As Rho proteins are not responsible for the observed effects of statins on APP trafficking, it is likely that the effect is due to statin inhibition of Rab protein function.

The effects of statins on APP trafficking and Aβ production in N2a cells are strikingly similar to those previously reported after inhibition of Rab1b. Inhibition of

Rab1b by dominant negative forms of this G-protein has been shown to impair the

maturation of APP, as detected by pulse chase analysis, resulting in decreased Aβ secretion (Dugan, deWit et al. 1995; Maltese, Wilson et al. 2001). Interestingly, Rab1b

- 101 - also caused the retention of APP CTFs within the ER (Petanceska, DeRosa et al. 2002).

This suggests that Rab1b inactivation mislocalizes APP intracellularly, leading to

reduced Aβ production. Similarly, Rab6 function has been shown to reduce Aβ

production, however no effects were reported on APP trafficking or localization

(McConlogue, Castellano et al. 1996). Significantly, we show that Rab1b isoprenylation

is inhibited by statins at doses as low as 200nM (Fig. 1,2). As the effects of statin

treatment on APP trafficking are similar to those reported for Rab1b inhibition, it is likely

that inhibition of Rab1b is at least in part responsible for the observed effects. However, there are over 30 Rab family proteins involved in vesicular trafficking and it is probable that statins affect additional members of this family.

We demonstrate that in statin-treated N2a cells that APP accumulation is accompanied by decreased Aβ secretion. This finding conflicts with the report of Cole et

al. that following statin treatment of APPsw-293 cells, secreted Aβ levels are not

changed by statins, while intracellular Aβ levels are increased (Cole, Grudzien et al.

2005). Moreover, these authors report that statins increased β-CTF levels in APPsw-293

cells, but that α-CTF levels were not dramatically changed. We find that in N2a.WT

cells, both α- and β-CTF levels are increased by statin treatment. Thus, in different cell

types, statin treatment leads to differential processing of accumulated APP. It is

important to note that the experiments reported by Cole et al. utilized the Swedish mutant

APP, which is preferentially cleaved by β-secretase (Perez, Squazzo et al. 1996), and thus

may be processed differently than wild type APP. Overall, these data suggest that the

statin-mediated APP accumulation has cell type and transgene-dependent effects on Aβ

- 102 - production. We argue that cellular accumulation of APP by Rab- dependent mechanisms

may represent a mechanism by which statins limit Aβ production.

Statins cause lysosomal degradation of APP CTFs through inhibition of Rho family

proteins

We report that, in H4 cells, APP CTF levels are reduced after statin treatment

through inhibition of protein geranylgeranylation (Fig. 6B,D,E). Treatment of H4 cells

with Toxin A or C3 exoenzyme also resulted in decreased in CTF levels (Fig. 7A,C),

demonstrating that inhibition of RhoA, B and/or C is responsible for the observed effects.

RhoA inhibition has previously been shown to selectively decrease the production of

Aβ1-42 (Zhou, Su et al. 2003). This previous study utilized a dominant negative RhoA, while our study utilized C3 exoenzyme, which inhibits RhoA, B, and C. It is possible that inhibition of multiple Rho proteins is required for statin and toxin effects on CTF degradation.

It has been reported that APP and CTFs can be degraded within the lysosome and also by the proteasome, though the regulation of these processes is poorly understood

(Knops, Lieberburg et al. 1992; Tsuzuki, Fukatsu et al. 1994; Nunan, Shearman et al.

2001; Nunan, Williamson et al. 2003). We report that statin and Toxin A-mediated decreases in CTF levels are blocked by lysosomal inhibitors, but not proteasomal inhibitors (Fig. 8). Importantly, the reduction of CTF levels by statins and Toxin A is associated with a commensurate decrease in Aβ secretion (Fig. 6 and 7). Inducible degradation of CTFs within the lysosome may therefore represent not only a mechanism

- 103 - by which statins limit Aβ production, but also a novel therapeutic target for decreasing

Aβ production.

A significant outcome of this study is the recognition that at physiological doses statins are likely to affect the function of only a subset of GTPases. We report that statins can limit Aβ production through inhibition of Rho and Rab family proteins, suggesting mechanisms of statin action in AD. These findings represent potential mechanisms by which statin inhibition of protein isoprenylation may limit AD pathogenesis.

- 104 -

FIGURES

- 105 -

Figure 1 – Statin-dependent alteration of G-protein electrophorectic mobility and abundance. N2a cells were treated for A) 24h with simvastatin (10µM) or lovastatin

(10µM), in the presence or absence of mevalonate (250µM) or B) 6 days with 50-100nM simvastatin. SDS-PAGE was performed on cell lystates, followed by Western blotting using the indicated antibodies. Images shown are representative of at least 3 independent experiments.

- 106 -

Figure 1 – Statin-dependent alteration of G-protein electrophorectic mobility and abundance.

- 107 - Figure 2 – Statins inhibit the membrane association of G-proteins globally at higher doses, but selectively at lower doses. N2a cells were treated for A) 24h with either simvastatin (10µM), or lovastatin (10µM) in the presence or absence of mevalonate (250µM) B) 48h with simvastatin (500nM) or C) 72h with simvastatin

(200nM). Cytosolic and membrane fractions were prepared and Western blotting was performed using the indicated antibodies. Flotillin and calnexin are markers for the membrane fractions, while GAPDH and ERK mark the cytoplasmic fraction. Note that the expression of these markers was not affected by drug treatment. Images shown are representative of at least 3 independent experiments. C) Quantification of data from pooled experiments represented in Figure 2c (n=6, **p<0.01 compared with non-treated sample).

- 108 -

Figure 2 – Statins inhibit the membrane association of G-proteins globally at higher doses, but selectively at lower doses.

- 109 -

Figure 3 – Statins and Toxin A increase APP expression in N2a.Swe cells, but not

in other cell types. (A-C) Pulse-Chase Experiments A) N2a.WT, APPsw-293,

H4.HPLAP, and H4.APPWT cells were treated for 18h or 24h with simvastatin (10µM)

or 12 or 18h with Toxin A (500ng/mL). B) N2a.Swe cells were treated with simvastatin

(10µM) for 24h or Toxin A (500ng/mL) for 12h. C) N2a.Swe or N2a.WT cells were

treated with simvastatin at 300nM or 500nM for 48h. After treatment, cells were

changed to cystine/methionine-free media for 5 min, followed by media containing 35S labeled cystine methionine for 15 min. APP was immunoprecipitated from lysates.

Images shown are representative of at least 3 independent experiments. For N2a.Swe experiments (B) the APP bands were excised from the gels and incorporated radioactivity was quantified by scintillation counting (n=6, **p<0.01 compared with non-treated sample). (D-F) Western blots and ELISA D) Primary embryonic cortical neurons were treated for 24h in the presence or absence of simvastatin (10µM). E) N2a.Swe cells were treated with simvastatin (10µM) or Toxin A (500ng/mL) for 12h. F) Media was collected from N2a.WT, H4.APPWT, and APPsw-293 cells after treatment in the presence or absence of simvastatin (1µM) for 24h. G) Media was collected from non- treated cells. Western blotting was performed against sAPP and sAPPα. Images shown are representative of at least 3 independent experiments. Aβ1-40 and Aβ1-42 were

measured from media by ELISA and quantified (n=6, **p<.01 compared with non-

treated sample).

- 110 -

Figure 3 – Statins and Toxin A increase APP expression in N2a.Swe cells, but not in other cell types.

- 111 - Figure 4 – Statin, but not Toxin A, treatment leads to accumulation of APP in N2a

cells, perturbing APP processing and decreasing Aβ secretion. N2aWT cells A,B) were treated for 24h with simvastatin (10µM) or lovastatin (10µM) in the presence or

absence mevalonate (250µM) C) 24h with simvastatin (10µM) in the presence or absence of GGPP (1ug/mL) or FPP (1ug/mL) D) for 48h with simvastatin at doses of 300nM or

500nM. APP, CTF, and GAPDH were measured from lysates by Western blotting. sAPP levels were measured from the media by Western blotting. Images shown are

representative of at least 3 independent experiments. Aβ1-40 levels were measured in the

medium by ELISA and quantified (n=3-4, *p<0.05; **p<0.01 compared with non-treated sample).

- 112 -

Figure 4 – Statin, but not Toxin A, treatment leads to accumulation of APP in N2a cells, perturbing APP processing and decreasing Aβ secretion.

- 113 - Figure 5 - Statin, but not Toxin A, treatment leads to accumulation of APP in N2a

and H4 cells, perturbing APP processing. A) N2a.WT cells were treated for 18h with

Simvastatin or 12h with Toxin A. B) H4.APPWT cells were treated for 18h with

Simvastatin or 12h with Toxin A. Cells were pulsed for 15 min with 35S

cystine/methionine. One set of cells were immediately lysed (0 time point), while

remaining cells were incubated in fresh media for 1-3h before lysis. APP was

immunoprecipitated, resolved by SDS-PAGE, and audoradiograph was obtained. Images

shown are representative of at least 3 independent experiments. As APP synthesis is

increased in N2a.Swe cells, exposures are shown that are normalized for expression at the

0 time point. Quantification of 35S incorporated in APP at each timepoint in the presence

or absence of simvstatin or toxin A in shown in 5A. (n=3, *p<0.05 compared with non-

treated sample).

- 114 -

Figure 5 - Statin, but not Toxin A, treatment leads to accumulation of APP in N2a and H4 cells, perturbing APP processing.

- 115 - Figure 6 – Statins reduce CTF levels and Aβ production in H4 cells. Lysates and

media were collected from A) untreated H4.APPWT and H4.HPLAP cells. B,C) H4.APP

WT cells treated with simvastatin (10µM) or lovastatin (10µM) in the presence or

absence of mevalonate (250µM). Graph in right panel of 6c represents quantification of

α-CTF levels in H4.APPWT cells (n=3, *p<0.05;**p<0.01 compared with non treated

sample). D) H4.HPLAP cells treated with simvastatin in the presence or absence of

GGPP (0.5µM) or FPP (0.5µM); E) H4.HPLAP treated for 18h in the presence or

absence of simvastatin (10µM) for 18h. F) H4.HPLAP cells treated for 6 days with

simvastatin 500nM or 1µM. G) H4.APPWT cells treated with simvastatin (10µM) for

18h. APP, GAPDH and CTF levels from cellular lysates were evaluated by Western

blotting. Images shown are representative of at least 3 independent experiments. sAPP

levels were measured from conditioned cell media by Western blotting. Images shown

are representative of at least 3 independent experiments. G) Aβ1-40 levels were measure

from cell media by ELISA (n=8, **p<0.01 compared with non-treated sample). Aβ1-42 was not produced by H4 cells at detectable levels.

- 116 -

Figure 6 – Statins reduce CTF levels and Aβ production in H4 cells.

- 117 - Figure 7 – Toxin A treatment reduces CTF levels and Aβ production in H4 cells.

A) H4.APPWT or H4.HPLAP cells were treated in the presence or absence of Toxin A

(500ng/mL) for 12h. APP, GAPDH, and CTF levels were evaluated from cellular lysates by Western blotting. sAPP and sAPPα were evaluated from cell culture media by

Western blotting. Images shown are representative of at least 3 independent experiments.

Graph represents quantification of CTFα levels as measured by Western blotting (n=8,

*p<0.05;**p<0.01 compared with non-treated sample). B) H4.APPWT cells were treated for 12h with Toxin A (500ng/mL). Secreted Aβ1-40 levels were measured from conditioned cell media by ELISA and quantified (n=8, **p<0.01 compared with non- treated sample). C) H4.HPLAP cells were treated with C3 exoenzyme (10µg/mL) for

24h. APP, GAPDH, and CTF levels were measured by Western blotting. Images shown are representative of at least 3 independent experiments.

- 118 -

Figure 7 – Toxin A treatment reduces CTF levels and Aβ production in H4 cells.

- 119 - Figure 8 – Statin and Toxin A facilitate CTF lysosomal degradation. A)

H4.HPLAP cells were treated in the presence or absence of MG132 (10µM) or

chloroquine (20nM) with or without Toxin A (500ng/mL) for 12h, or simvastatin for 18h.

Cells were lysed and examined by SDS-PAGE followed by Western blotting to detect

APP, GAPDH and CTF. CTF blots are shown at long and short exposure lengths allowing appreciation of increases in CTF levels following inhibitor treatment, as well as the effects of Toxin A and statins on CTF levels in the presence of inhibitors. Images shown are representative of at least 3 independent experiments. B) Quantification of CTF levels (n=3-5, **p<0.01 compared with non-treated sample).

- 120 -

Figure 8 – Statin and Toxin A facilitate CTF lysosomal degradation.

- 121 -

Supplemental Figure A – Statins and Toxin A Increase APP mRNA expression in N2a.Swe Cells. N2a.Swe cells were treated for 12h or 24h in the presence or absence of simvastatin (10µM) or Toxin A (500ng/mL). mRNA was isolated and Real-time PCR was performed on a Bio-Rad iCycler. APP cycle thresholds were normalized against the values obtained for GAPDH. Graphs are representative of two independent experiments.

- 122 -

Supplemental Figure A – Statins and Toxin A Increase APP mRNA expression in N2a.Swe Cells.

- 123 -

Supplemental Figure B – Toxin A Decreases CTF levels in N2a, APPsw-293, and Sk- n-sh Cells. N2a.Swe, APPsw-293, or Sk-n-sh cells were treated with Toxin A (500ng/mL) for 12h or 18h. Cells were lysed and samples were examined by SDS-PAGE followed by Western blotting to detect GAPDH and CTF levels. Images shown are representative of at least 2 independent experiments.

- 124 -

Supplemental Figure B – Toxin A Decreases CTF levels in N2a, APPsw-293, and Sk- n-sh Cells.

- 125 - Supplemental Figure C – C3 Exoenzyme Decreases the Expression of RhoA, but not Rac. H4.HPLAP cells were treated in the presence or absence of C3 exoenzyme (5µg/mL) for 12h. Cells were lysed and lysates were examined by SDS-PAGE followed by Western blotting to detect Rac, RhoA, and GAPDH.

- 126 -

Supplemental Figure C – C3 Exoenzyme Decreases the Expression of RhoA, but not Rac.

- 127 -

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- 136 - Chapter 3: Monitoring Statin Inhibition of Protein Isoprenylation with 2D gel elecrophoresis

- 137 -

ABSTRACT

HMG-CoA reductase inhibitors, or statins, are widely prescribed drug for the treatment of hypercholesterolemia. Recently, cholesterol-independent benefits of statin action have been implicated in the beneficial properties of these drugs with respect to cardiovascular disease, multiple sclerosis, and Alzheimer’s Disease. Statins act through cholesterol-independent mechanisms to reduce intracellular pools of the isoprenoids geranylgeranylpyrophosphate (GGpp) and farnesylpyrophosphate (Fpp). GGpp and Fpp are covalently attached to cysteine residues at the C-terminus of the Ras superfamily

GTPases including the Rho and Rab family proteins. Isoprenylation is required for the proper function of these GTPases. Following isoprenylation, cysteine carboxymethylation occurs, which changes the isoelectric point (pI) of the protein. We describe a 2D gel and Western blotting approach to directly monitor the effects of statins on protein isoprenylation.

INTRODUCTION

Statins are widely prescribed drugs for treatment of hypercholesterolemia, and act to reduce plasma cholesterol levels by inhibiting the rate-limiting enzyme in the cholesterol biosynthetic pathway, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, preventing de novo synthesis of cholesterol (Endo 1992; Tobert 2003). Statins exhibit pleiotropic effects through reduction of isoprenoid intermediates in the cholesterol biosynthetic pathway (Liao and Laufs 2005). The isoprenoids geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) are added to the C-termini of the Ras superfamily of small G-proteins, including Rho and Rab family proteins.

- 138 - Isoprenoid modification is critical for facilitating GTPase interactions with cytoplasmic

regulators, cellular membranes, and effectors (Zhang and Casey 1996). Cholesterol-

independent actions of the statins have been demonstrated to be important for the clinical

benefits of these drugs (Bellosta, Ferri et al. 2000; Bellosta, Ferri et al. 2000; Takemoto

and Liao 2001).

Most studies have used membrane association of GTPases as a surrogate for

directly monitoring isoprenylation. This is due to the fact that direct monitoring of

isoprenylation using metabolic labeling techniques is technically challenging. We could

find no published reports that measure the incorporation of radioactive precursors

proximal to HMG-CoA reductase in the presence of statins. This is likely because

precursors like HMG-CoA, AcetoacetylCoA, and AcetylCoA are abundant and their

cellular pools cannot be readily depleted, making measurement of the radioactively

labeled species difficult.

Rho proteins carry a C-terminal CaaX isoprenylation motif, where C=cysteine,

a=aliphatic amino acid and X=any amino acid. Rab family proteins carry either a CxC or

CC C-terminal isoprenylation motif. Isoprenylation of all proteins occurs on the cysteine, and Rab proteins are isoprenylated on both cysteines in this motif. Rho family proteins are further processed by the prenyl protease, Rce1, to remove –AAX (Boyartchuk, Ashby et al. 1997), and the cysteine is then carboxymethylated by the enzyme isoprenylcysteine-

O-carboxyl methyltransferase (Icmt) (Dai, Choy et al. 1998). Rab family proteins are not

cleaved by Rce1, but CxC Rab proteins are carboxymethylated by Icmt (Bergo, Leung et

al. 2001). CC Rab proteins are not methylated, likely because the steric hinderence of

adjacent cysteines does not allow access to the Icmt enzyme. The carboxymethylation of

- 139 - GTPases is functionally important for GTPase function (Papaharalambus, Sajjad et al.

2005; Leung, Baron et al. 2006). In addition, the carboxymethylation masks an acidic

carboxyl group, causing a basic shift in the isoelectric point of the protein (Backlund

1997). For example, after inhibition of carboxymethylation, the pI of RhoA shifts from

6.35 to 5.9 and Cdc42 from 6.9 to 6.4 in human cells. As inhibition of protein

isoprenylation precludes the carboxymethylation of GTPases, we predicted that we could

directly monitor the effects of statins on protein isoprenylation through altered mobility

by 2D gel electrophoresis.

EXPERIMENTAL PROCEDURES

Materials and reagents. Simvastatin was purchased from Calbiochem (La Jolla, CA) and prepared following the manufacturer’s instructions. Simvastatin was converted to the

active form by dissolving in absolute EtOH, followed by the addition of 1M NaOH to a

final concentration of 100mM. This solution was stored at -20°C until use. Immediately

before use, the simvastatin solution was neutralized with 1M HCl, and diluted in vehicle

(50% EtOH, 5mM HEPES, pH 7.2). An antibody to Rab4 was obtained from Upstate

(Waltham, MA). Antibodies to Cdc42, Rab11 and Rab6 were obtained from Santa Cruz

Biotechnology (Santa Cruz, CA). An ntibody to RhoA was purchased from Cytoskeleton

(Denver, CO). Peroxidase-conjugated secondary antibodies were purchased from GE

Healthcare (UK). Cell culture reagents were purchased from Invitrogen (Carlsbad, CA).

- 140 - Cell culture. Mouse N2a (parental) neuroblastoma cells were obtained from American

Type Culture Collection (Manassas, VA). N2a cells were cultured in 50% Optimem/50%

DMEM, 5% FBS (Hyclone, Logan, UT), and 1% Penicillin/Streptomycin.

Isoelectric Focusing and Western blotting. Cells were collected and lysed in lysis buffer

(0.5% SDS, 25mM Tris, 2.5mM MgCl2, with protease and esterase inhibitors) for 5 minutes at 4°C, heated at 95 °C for 5 minutes with light vortexing, and cooled to room

temperature. Bovine pancreatic RNases and DNase (100ng/mL) were added for 15

minutes. Protein concentrations were measured by the Bradford method (Bradford 1976).

Lysates were precipitated overnight with 5x volume of ice cold acetone. Cells were

resuspended in IPG solubilization buffer (7M urea, 2M thiourea, 1%DTT, 1% Chaps, 1%

carrier ampholyates 5-8) at a concentration of 5g/mL. 200µL from each sample was

focused on 5-8 or 3-10 IPG strips to 32,000 volt hours. Focused strips were subjected to

alkylation and reduction, followed by running second dimension on Criterion 12% gels

(Biorad) and transfer to polyvinylidene fluoride membranes. After blocking in a 5% milk

or 5% normal goat serum solution, blots were incubated overnight at 4°C with the

indicated primary antibodies. Bands were visualized by incubation of blots with anti-

mouse, rabbit or goat HRP-conjugated secondary antibodies (1:1000; 90 min at room

temperature) and visualized by enhanced chemiluminescence (Pierce, Rockford, IL).

Images were scanned using Adobe Photoshop and band intensities quantified using

Image-Pro Plus software package (Media Cybernetics, Inc., Silver Springs, MD).

- 141 - RESULTS

Isoelectric Points of Rho and Rab Family GTPases.

We surveyed the literature to determine predicted and experimentally verified pI’s for mouse isoforms of GTPases of the Rho and Rab family (Table 1). This data allows us to verify the identity of spots detected by Western blotting.

Statins cause isoelectric 2D gel shift of CAAX Rho family proteins.

It has been shown that post-isoprenylation carboxymethlation of the CaaX containing proteins RhoA and Cdc42 causes a change in pI that can be detected using 2D gel electrophoresis (Backlund 1997). The change in isoelectric point occurs because the methylation masks a carboxyl residue, resulting in a shift to a more basic pI. We predicted that statins, which block protein isoprenylation, thus precluding carboxymethylation, resulting in an acidic gel shift. We treated N2a cells in the presence or absence of simvastatin (10µM) for 24h. Lysates were isoelectrically focused and subjected to SDS-PAGE followed by Western blotting using antibodies specific to the

CaaX containing Rho proteins RhoA and Cdc42. In non-treated cells both RhoA and

Cdc42 migrated at previously reported pI and molecular weight (Fig. 1 and 2).

Simvastatin caused an acidic isoelectric shift of both proteins, with the majority of both proteins being shifted after statin treatment (Fig 1 and 2). The migration and shifting of

RhoA was confirmed by using a second RhoA specific antibody (data not shown).

Statins cause isoelectric 2D gel shift of Rab CXC proteins but not CC Rab proteins.

- 142 - Rab proteins do not contain a –CaaX isoprenylation motif, but instead contain a

CxC or CC isoprenylation motif. CxC proteins have been shown to be

carboxymethylated, while CC proteins are not carboxymethylated. We predicted that

statins would block the methylation of CxC proteins, causing an acidic gel shift, while

statins would have no effect on the migration of CC proteins. N2a cells were treated for

24h in the presence or absence of simvastatin. Lysates were isoelectrically focused and

subjected to SDS-PAGE followed by Western blotting using antibodies specific to the

CxC proteins Rab4 and Rab6 and the CC protein Rab11. Statin treatment caused acidic

isoelectric shifting of Rab 4 and 6, but not of Rab 11 (Figures 3,4 and 5).

DISCUSSION

Statins may mediate some of their biological effects through inhibition of protein

isoprenylation. However, many of the effects of statins on protein isoprenylation are not well characterized, particularly in vivo, likely because there is an absence of methods for monitoring protein isoprenylation. In particular, metabolic labeling has not been reported for studying the effects on protein isoprenyaltion. It has been previously reported that carboxymethylation occurs after isoprenylation, resulting in altered isoelectric shifting of

the Rho –CaaX GTPases (Backlund 1997). We predicted that as carboxymethylation is

dependent upon isoprenylation, that statins will inhibit carboxymethylation, resulting in

acidic isoelectric shifting of GTPases.

We examined three classes of isoprenylated proteins: 1) The Rho family proteins

RhoA and Cdc42 that contain a –CaaX motif. 2) The Rab family proteins Rab4 and Rab6

that contain a –CxC motif, and 3) The Rab family protein Rab11 that contatins a -CC

motif. CaaX and CxC proteins have been previously shown to be carboxymethyled. In

- 143 - non-treated cells they migrate primarily as spot of one isoelectric point. After statin treatment, which blocks carboxymethylation, the proteins experience an acidic isoelectric shift. The Rab11 protein, which is not methylated, primarily runs as a single spot, and does not experience an isoelectric shift after statin treatment. Our data demonstrates that statins block carboxymethylation of Rho and Rab GTPases, which can be detected by altered isoelectric shifting. We believe that the use of 2D gels to detect such isoelectric shifts will be of great value in the study of statin effects on protein isoprenylation.

- 144 - FIGURES

- 145 - Protein Isoelectric Point Molecular Weight

Cdc42 6.16 21.259

RhoA 5.83 21.783

Rab4b 5.8 23.629

Rab5b 8.29 23.708

Rab6b 5.41 23.468

TABLE 1 – Molecular Weight and Isoelectric points of selected Rho and Rab GTPases.

- 146 - Figure 1 – Simvastatin treatment causes an acidic isoelectric shift of the –CaaX Rho family protein RhoA. N2a cells were treated for 24h with Simvastatin (10µM). Total cellular lysates were subject to isoelectric focusing followed by SDS-PAGE and Western blotting using a RhoA specific antibody.

- 147 -

Figure 1 – Simvastatin treatment causes an acidic isoelectric shift of the –CaaX Rho family protein RhoA.

- 148 -

Figure 2 – Simvastatin treatment causes an acidic isoelectric shift of the –CaaX Rho family protein Cdc42. N2a cells were treated for 24h with Simvastatin (10µM). Total cellular lysates were subject to isoelectric focusing followed by SDS-PAGE and Western blotting using a Cdc42 specific antibody.

- 149 -

Figure 2 – Simvastatin treatment causes an acidic isoelectric shift of the –CaaX Rho family protein Cdc42.

- 150 - Figure 3 – Simvastatin treatment causes an acidic isoelectric shift of the –CxC Rab family protein Rab4. N2a cells were treated for 24h with Simvastatin (10µM). Total cellular lysates were subject to isoelectric focusing followed by SDS-PAGE and Western blotting using a Rab4 specific antibody.

- 151 -

Figure 3 – Simvastatin treatment causes an acidic isoelectric shift of the –CxC Rab family protein Rab4.

- 152 -

Figure 4 – Simvastatin treatment causes an acidic isoelectric shift of the –CxC Rab family protein Rab6. N2a cells were treated for 24h with Simvastatin (10µM). Total cellular lysates were subject to isoelectric focusing followed by SDS-PAGE and Western blotting using a Rab6 specific antibody.

- 153 -

Figure 4 – Simvastatin treatment causes an acidic isoelectric shift of the –CxC Rab family protein Rab6.

- 154 - Figure 5 – Simvastatin treatment does not cause an isoelectric shift of the –CC Rab family protein Rab11. N2a cells were treated for 24h with Simvastatin (10µM). Total cellular lysates were subject to isoelectric focusing followed by SDS-PAGE and Western blotting using a Rab11 specific antibody.

- 155 -

Figure 5 – Simvastatin treatment does not cause an isoelectric shift of the –CC Rab family protein Rab11.

- 156 -

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inhibitors." Nat Rev Drug Discov 2(7): 517-26.

Youssef, S., O. Stuve, et al. (2002). "The HMG-CoA reductase inhibitor, atorvastatin,

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- 158 -

CHAPTER 4: DISCUSSION

- 159 - RESEARCH CONCLUSIONS

Alzheimer’s Disease (AD) is a devastating neurodegenerative disease and a

tremendous burden on our health care system. It has been estimated that a treatment that

delays disease onset or progression by as little as 5 years could reduce the burden of AD

by as much as 50%. AD has been well studied, and most researchers believe that the

aberrant production of amyloid beta (Aβ) is the underlying pathogenic cause of the

disease. Targeted anti-Aβ treatments are currently in clinical trials, and it is hoped that

they will help to ameliorate disease onset and progression. Notwithstanding, alternative

treatments for AD are of great interest. Retrospective studies have demonstrated that

patients taking the cholesterol lowering statins are at a reduced risk for developing AD.

However, it is not clear if the benefits of statins can be explained by their cholesterol-

lowering effects.

Cholesterol-independent effects of statins can be mediated by their ability to

deplete pools of intracellular isoprenoids such as farnesylpyrophosphate and

geranylgeranylpyrophosphate, which are hydrophobic carbon chains that are attached to

and required for the function of Ras superfamily GTPases, such as Rho and Rab. We

believe these isoprenoid-dependent effects of statins may be responsible for the

protective role of statins in AD. As such the main goal of this research was to investigate

the effects of statins on protein isoprenylation, and the effects of these isoprenoid-

dependent mechanisms of statin action in in vitro cell models of APP processing.

We show that inhibition of protein isoprenylation by statins causes the

accumulation of APP within the cell through inhibition of protein geranylgeranylation.

Toxin A, a specific and robust inhibitor of Rho family GTPases, has no effects on APP

- 160 - trafficking, suggesting that statins impact APP trafficking through the inhibition of Rab

family protein function. In addition, effects of statins on APP trafficking and Aβ production are strikingly similar to those reported previously after the inhibition of Rab1b

(Dugan, deWit et al. 1995). Inhibition of Rab6 function has been shown to reduce Aβ production, however no effects were reported on APP trafficking or localization

(McConlogue, Castellano et al. 1996). In sum, these data strongly suggest that inhibition

of Rab1b and/or other Rab family proteins by statins results in decreased Aβ production.

We believe that statin inhibition of Rab proteins has not received enough attention.

A Pubmed search of “statin and rab” yields 10 results, while “statin and rho” yields 289 results. Rab proteins are the largest subfamily of Ras GTPases, composing >60 members.

These proteins are expressed in all cell types and are critical mediators of vesicular trafficking within the cell. Rab family proteins subserve a number of cellular functions, and increased recognition that statins inhibit Rab function will have important impact on the investigation of the role that statins inhibition of protein isoprenylation plays in vivo.

We observed that inhibition of Rho family protein function by statins, C. difficile

Toxin A, or C. botulinum C3 exoenzyeme reduces the levels of APP C-terminal fragments, resulting in reduced Aβ secretion. We demonstrate that this is not due to decreased α- or β- cleavage of APP, but instead due to enhanced lysosomal dependent degradation. It had been shown previously that CTFs can be degraded via the lysosome

(Tsuzuki, Fukatsu et al. 1994), but ours is the first report that CTF degradation is a regulated process. Inducible degradation of CTFs within the lysosome may therefore represent not only a mechanism by which statins limit Aβ production, but also a novel therapeutic target for decreasing Aβ production.

- 161 - Similar to many previous studies, we require high doses of statins to achieve the

largest effects on decreased Aβ production. However, we show that statin can reduce Aβ

secretion from N2a cells at doses as low as 300nM. We also show that in H4 cells that

statins can reduce CTF levels at doses as low as 500nM. It is important to note that, in

mouse models, modest changes in Aβ levels can have dramatic effects on amyloid plaque

pathology. For example, Lehman and colleagues, who studied Aβ pathology in different

strains of mice, reported that B6 mice expressing an APP transgene exhibit plaque

deposition at 13.5 months. However, D2 mice, which have only 20% less Aβ levels,

show no plaque deposition even at 20 months (Lehman, Kulnane et al. 2003). Similarly,

inhibition of α-secretase activity increases Aβ production by about 25%, but increases plaque burden by over 300% (Postina, Schroeder et al. 2004).

In neuroblastoma cell lines, treatment with statins at high doses (10µM) inhibits the membrane localization of Rho and Rab proteins, without affecting cellular cholesterol levels. Most in vitro studies of statin effects on protein isoprenylation have utilized such high doses. This makes mechanistic examination of statin effects extremely difficult, owing to the broad array of proteins that are affected by drug treatment. Moreover, the effects of high dose statin treatment on protein isoprenylation may not be representative

of statin effects at physiologically relevant concentrations. It has been demonstrated that

different isoforms of Ras have differential affinities for the FTase enzyme, resulting in

differential sensitivity to a FTase inhibitor (Zhang, Kirschmeier et al. 1997). One would

expect that the different isoforms would also show the same differential sensitivity to

statin treatment. Rilling et al. demonstrated that, under conditions of low intracellular

mevalonate, mevalonate is incorporated only into a subset of GTPases, also suggesting

- 162 - specificity of protein isoprenylation (Rilling, Bruenger et al. 1993). We hypothesized

that isoform differences to statin treatment would be found if we profiled Rho and Rab

GTPases. Importantly, we show for the first time that at low, physiologically relevant,

doses statins preferentially inhibit the isoprenylation of only a subset of GTPases. Rac

and Rab1b have their membrane association decreased after 200nM simvastatin treatment,

while Rab5b and Rab4 are unaffected by this dose of simvastatin. We have also

employed 2D gel electrophoresis to directly monitor the isoprenylated versus non-

isoprenylated forms of isoprenylated GTPases. This technique will be useful for further

studies of statin inhibition of protein isoprenylation.

FUTURE DIRECTIONS

Targeting Rho Pathways to Decrease CTFs – A potential therapy for AD?

Statin inhibition of Rho protein function decreased CTF levels and Aβ secretion

in our cell culture models. This opens up the exciting possibility of therapeutically

targeting this pathway to reduce Aβ production and CTF degradation. We demonstrate that C3 exoenzyme, a toxin isolated from Clostridium botulinum that specifically inhibits

RhoA, B and C, reproduced the effects of statins on CTF degradation. Rho inhibition by

C3 exoenzyme has been shown to increase neurite outgrowth and regeneration in vitro

(Lehmann, Fournier et al. 1999). Expression of C3 using an adenovirus vector increased

regeneration of mature CNS neurons (Fischer, Petkova et al. 2004). A cell-permeable

form of the C3 exoenzyme has been developed and increased regeneration after optic

nerve lesion (Bertrand, Di Polo et al. 2007) and spinal cord injury (McKerracher and

- 163 - Higuchi 2006). The tools to specifically inhibit Rho in the CNS are available, and future

studies will determine if C3 treatment reduces AD pathology in mouse models.

A potential concern is that global Rho inhibition may interfere with critical

biological functions, resulting in toxicity that outweighs potential clinical benefit.

Targeting of pathways downstream of Rho will be less likely to have toxic side effects.

The most well known effector molecules of Rho include Rho-associated kinase (ROCK)

(Fujisawa K, Fujita A et al. 1996), Rhotekin (Reid, Furuyashiki et al. 1996), PKN-related kinases (PRK) (Watanabe, Saito et al. 1996), and Diaphanous (Watanabe, Madaule et al.

1997). In vitro studies will determine if targeting of any of these individual pathways can reduce CTFs and Aβ.

It has been reported in several studies that ROCK activation leads to altered cellular localization of lysosomes in cancer cells (Nishimura, Itoh et al. 2000; Nishimura,

Itoh et al. 2002; Nishimura, Itoh et al. 2003). It is unclear how altered localization of lysosomes may affect their function. Potentially, altered localization after ROCK activation leads to decreased lysosomal degradation of CTFs. ROCK inhibition might restore normal localization of lysosomes, leading to increased lysosomal processing of

CTFs and reduced Aβ production. ROCK inhibitors have been successfully used in animal models to promote regeneration of retinal ganglion axons (Sagawa, Terasaki et al.

2007) and to directly mediate neuroprotection against ischemia (Yamashita, Kotani et al.

2007).

RhoB regulates the endocytic recycling pathways (Li, Stupack et al. 1998;

Rondanino, Rojas et al. 2007). The downstream effectors of RhoB have been recently identified as members of the Diaphanous family, mDia1 and mDia2 (Fernandez-Borja,

- 164 - Janssen et al. 2005) . Diaphanous proteins are formin GTPase effector proteins that regulate actin nucleation and filament elongation (Krebs, Rothkegel et al. 2001; Geneste,

Copeland et al. 2002). RhoB and mDia2 interact on the surface of endosomes, and the actin dynamics controlled by RhoB and mDia2 are necessary for proper endocytic trafficking, though the mechanisms by which this occurs are unclear (Wallar, Deward et al. 2007). Endocytic trafficking is required for the the lysosomal degradation of CTFs, so

perturbation of these pathways by inhibition of mDia could explain the effects seen on

CTF degradation. Currently there are no known inhibitors of mDia function. Potentially,

small molecule or peptide inhibitors hat block interactions between mDia and Rho could

be isolated or designed. Design of small molecule inhibitors of protein-protein

interactions is an active area of research, and inhibitors have been isolated that inhibit

selectively inhibit certain protein-protein interactions (Best, Amezcua et al. 2004;

Horswill, Savinov et al. 2004).

Elucidation of the mechanisms by which statin inhibition of Rab proteins decreases

Aβ production

Our research has elucidated mechanisms by which statin inhibition of Rab family

proteins decrease Aβ production. However, there are still many unanswered questions.

We demonstrate that statins can inhibit Rab family proteins leading to perturbation of

APP trafficking. These effects were most pronounced in N2a cells and resulted in

reduced Aβ secretion. It is likely that this is due to mislocalization of APP from the

normal subcellular compartments in which Aβ is generated. However, the mechanisms

of these effects are still unknown. Subcellular fractionation or immunohistochemical

- 165 - techniques will determine to which compartments APP is being accumulated after statin treatment. It is possible that APP is accumulated in normal intracellular locations, or that inhibition of Rab function mislocalizes APP to inappropriate subcellular compartments.

An important question will be how APP mislocalization affects co-localization with and access to components of the α- β- and γ-secretases.

It will be interesting to examine the role of individual Rab family proteins in APP trafficking and Aβ production. Statin effects on APP trafficking are strikingly similar to those previously reported after Rab1b inhibition. However, statins inhibit the function of many Rab family proteins. It will be of particular importance to determine the role of

Rab family proteins that are inhibited by physiologically relevant doses of statins. This will allow ultimately allow the assessment of the mechanisms statin actions on APP trafficking at clinically relevant statin doses.

Do statins protect against AD through isoprenoid-dependent mechanisms?

The in vivo relevance of isoprenoid–dependent mechanisms of statin action in protecting against AD will be important to examine. Statins have been shown to reduce

Aβ levels and plaque pathology in guinea pig and mouse models (Fassbender, Simons et al. 2001; Burns, Igbavboa et al. 2006), though not in other studies using mouse models

(Park, Hwang et al. 2003). A recent study demonstrated that statin improve cognitive function while having no effects on Aβ levels (Li, Cao et al. 2006). However, it is unclear is these effects of statins on decreasing Aβ or improving cognitive function are mediated by cholesterol-dependent or isoprenoid-dependent mechanisms.

- 166 - A recent study has demonstrated that in vivo effects of statins on myocardial protection in mice are reversible by provision of GGpp, and thus dependent upon statin inhibition of protein isoprenylation (Bulhak, Roy et al. 2007). This is the first direct evidence that statins act in vivo by inhibiting protein isoprenylation. Similar experiments in mouse models of AD will delineate the roll of isoprenoid-dependent effects of statins in ameliorating amyloid pathology and cognitive improvement. These will be critical studies for assessing the relevance of isoprenoid-dependent actions of statins in AD.

Do statins act in the brain or in the periphery to protect against AD?

The ability of statins to inhibit isoprenylation in the brain has not been well studied. An important future direction will be to determine whether statins reach sufficient concentrations in the brain to mediate isoprenoid-dependent effects. We show that statins in vitro can mediate effects on protein isoprenylation at doses as low as

200nM. Analysis of protein isoprenylation status after statin treatment in different tissues has not been well studied. It will be important to analyze protein isoprenylation in the brain after statin treatment.

If statins act to inhibit isoprenylation in the brain, it will be relevant to determine whether statin effects on AD occur in the brain, or are a result of statin actions in the periphery. The prediction would be that simvastatin, which reaches the brain at higher concentrations than other statins, will more robustly inhibit protein isoprenylation in the brain. Evidence supporting a role for statin action in the brain was shown by Johnson-

Anuna et al. who demonstrated that statins can affect gene expression in the brains of mice (Johnson-Anuna, Eckert et al. 2005). It has also been demonstrated that simvastatin,

- 167 - but not pravastatin, can mediate effects in the CNS such as decreasing CSF Phospholipid transfer protein (PLTP) (Vuletic, Riekse et al. 2006), reducing CSF levels of phospho- tau-181 (p-tau181) in all subjects (Riekse, Li et al. 2006), and inhibiting brain cholesterol synthesis (Thelen, Rentsch et al. 2006).

If statins work in the brain to protect against AD, one would make the prediction that lipophilic statins would be more effective than hydrophilic statins. This is supported by a recent clinical study suggesting that only simvastatin protects against dementia

(Wolozin, Wang et al. 2007). Treatment of APP transgenic mice with lipophilic statins such as simvastatin or with a hydrophilic statin such as atorvastatin or pravastatin will help to resolve the role of statins in the brain. If lipophilic statins, but not hydrophilic statin, can ameliorate AD pathology or cognitive deficits in mouse models, this would strongly favor a role of direct statin action in the CNS. If all statins act equally to improve AD pathology or cognitive deficits, this would suggest at that statins are acting in the periphery.

Statins may not reach the brain in sufficient concentration to inhibit protein isoprenylation. Alternatively, the studies outlined above may show that statin effects on

AD pathogenesis are mediated in the periphery. If this is the case, there are two likely explanations for the ability of statin, acting in the periphery, to modulate AD pathogenesis: 1) Statins modulate the peripheral immune cell function or 2) Statins improve endothelial function.

Peripherally derived macrophages, microglia (Stalder, Ermini et al. 2005; Simard,

Soulet et al. 2006) and T-cells (Togo, Akiyama et al. 2002) have been shown to be recruited to the brain in AD. It is unclear exactly what role these cells play in AD

- 168 - pathogenesis. Peripheral derived microglia have been shown to associate with plaques,

and it has been suggested that they help to ameliorate plaque pathology (Simard, Soulet

et al. 2006). However, these microglia may also participate in detrimental inflammatory processes (Malm, Koistinaho et al. 2005). Statins can decrease infiltration of peripheral

leukocytes into the brain in models of EAE (Walters, Pryce et al. 2002; Greenwood,

Walters et al. 2003). If infiltrating microglia have a net detrimental effect on disease

progression, the ability of statins to limit infiltration could limit AD pathogenesis.

While it has been show that T cells enter the brain in AD, their role in disease

pathogenesis is unknown (Togo, Akiyama et al. 2002). Peripheral T-cells cross the BBB

by a Rho and ROCK dependent mechanism, and statins could thus reduce infiltration of

T-cells into the brain (Man, Ma et al. 2007). Alternatively, it has been demonstrated that

statin protection against multiple sclerosis is mediated through immunomodulation of T-

cells. Statins cause CD4+T cells to switch from Th1 (cell-mediated) to Th2 (humoral)

responses, and limiting disease progression in mouse EAE models. While Th1/Th2

balance has not been investigated in Alzheimer’s Disease there are several ways by

which such a bias may benefit AD pathogenesis. It has been shown that Th1, but not Th2

cells cause activation of brain microglia (Seguin, Biernacki et al. 2003). Therefore Th2

biasing by statins may decrease microglial inflammation driven by invading T-cells. In

addition, some reports suggest that T-cells are activated in Alzheimer’s disease and

participate in antibody-mediated removal of Aβ (Monsonego, Zota et al. 2003). In this

paradigm, Th2 bias would likely be beneficial in antibody-mediated removal of Aβ from

the brain.

- 169 - Statins have been shown to have effects on the vascular endothelium that ultimately augment cerebral blood flow, leading to neuroprotection of neurons against

cerebral ischemia.(Laufs, Endres et al. 2000; Amin-Hanjani, Stagliano et al. 2001; Zheng

and Chen 2007). It has been suggested that microcerebrovascular insufficiency may

contribute to Alzheimer’s Disease pathogenesis (de la Torre 2000) either by decreasing

neuronal viability or through increased production of Aβ (Bennett, Pappas et al. 2000).

Statin protection against AD may thus be mediated through improved endothelial

function and increased cerebral blood flow.

Do statins act on neurons to protect against AD?

The clinical data suggests that statins protect against AD. An important question

remains to be answered: Do statins exert their anti-AD effects in the brain? If the

experiments outlined above suggest that statins act in the brain to protect against AD, it

still remains to seen which cell types statin are acting upon. Our studies have focused on

the direct effects of statins on APP processing and Aβ production in in vitro cell models.

Importantly, it has been demonstrated that primary neurons process APP differently than

cell culture lines. An important future direction will be to analyze the effects of statins

on APP production in primary cortical neurons from transgenic APP models such as

Tg2576 or R.140 (Hsiao, Chapman et al. 1996; Lamb, Bardel et al. 1999). These

experiments will demonstrate the ability of statins to reduce Aβ production in primary

neurons by isoprenoid-dependent mechanisms.

Statins may mediate AD protection independent of effects on Aβ production.

Supporting this model, a recent study demonstrated that statins improve cognitive deficits

- 170 - in transgenic APP mice, but have no effects on Aβ levels (Li, Cao et al. 2006). It is thus

possible that statins act in other ways to protect against synaptic dysfunction and

neuronal loss in AD. Statins have been shown to be directly neuroprotective (Zacco,

Togo et al. 2003; Bosel, Gandor et al. 2005). Recent evidence has suggested that

neuroprotective effects of statins may occur through upregulation of the anti-apoptotic molecules Bcl-2 and reduction of the pro-apoptotic molecule Bax (Franke, Noldner et al.

2007; Johnson-Anuna, Eckert et al. 2007).

Statins might also work on non-neuronal cells in the brain. Statins have well-

studied anti-inflammatory properties, and it has been suggested that these properties may

be responsible for limiting microglial inflammation in AD (Cordle and Landreth 2005).

Microglial inflammation is thought to play a central role in AD pathogenesis, and statin

inhibition of microglial inflammation may be a key feature of statin protection against

AD.

Further Investigation into Statin Effects on Protein Isoprenylation in vivo

Clinical evidence and animal studies suggests that statins act independently of

cholesterol to mediate their effects in vivo (Liao and Laufs 2005). Statins have been

shown to decrease the migration of mononuclear cells taken from human patients treated

with statins (Frigerio, Gelati et al. 2006). In animal models, statins have also been shown

to block GTPase membrane localization in the heart (Laufs, Kilter et al. 2002; Bulhak,

Roy et al. 2007) and in lymph node cells (Dunn, Youssef et al. 2006). In addition, a

recent study has demonstrated decreased RhoA and ROCK activation in endothelial cells

after statin treatment (Ruperez, Rodrigues-Diez et al. 2007). However, only one study

- 171 - has demonstrated directly, through reversal of statin effects with GGpp, that statins act in

an isoprenoid dependent manner in vivo (Bulhak, Roy et al. 2007). Overall the data

supporting statin inhibition of protein isoprenylation in vivo is limited, likely because techniques for directly examining protein isoprenylation are unavailable. We believe that using 2D gel electrophoresis to directly assess protein isoprenylation will be a useful technique for addressing the question of in vivo effects of statins on protein

isoprenylation.

We demonstrate that at high doses (10 μM) statins inhibit the membrane

association of all GTPases examined. This corroborates many published reports that also

use such supraphysiological doses of statins. We demonstrate that statins can inhibit

protein isoprenylation and mediate their biological effects at doses as low as 200nM.

Most importantly, we demonstrate that statins preferentially inhibit a subset of GTPases

at lower statin doses. We believe that this could have tremendous implications with

regard to the analysis of statin actions in vivo. Just 2% of cellular proteins are modified

by isoprenylation, comprising a small group of about 200 isoprenylated proteins. The

majority of studies, including ours, focus only on a subset of these proteins.

Our data shows that at physiologically relevant doses, that statins will inhibit the

isoprenylation and function of only a subset of GTPases. A thorough screen of statin

effects on protein isoprenylation will identify the proteins that are most likely to be

affected by statins in vivo. Several recent studies have used isoprenoids fluorescently

labeled or labeled other techniques to measure incorporation of isoprenoids into proteins

(Chan Kim, Kho et al. 2005; Dursina, Reents et al. 2006). These assays have the

potential to be used for quantitative analysis of statin effects on protein isoprenylation.

- 172 - However, it is unclear if they will be able to overcome the hurdles that have limited the effectiveness of metabolic labeling techniques in answering these questions. Therefore, the use of 2D gels to directly monitor protein isoprenylation status may be of use in these studies.

An important feature of this thesis is the description of 2D gel techniques to

analyze protein isoprenylation. We also provide insight into the basic mechanisms by

which statins preferentially inhibit the isoprenylation of only a subset of GTPases at

clinically relevant doses. These observations will be important in the study of statin

effects, which is crucial as statins are among the most highly prescribed drugs in the

world. The main focus of this thesis is the isoprenoid dependent effects of statins on the

processing of APP to Aβ. We demonstrate novel mechanisms by which statins inhibit

Rho and Rab family proteins to decrease Aβ secretion. These studies provide insight into

the mechanisms by which statins may reduce AD pathogenesis.

- 173 -

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