Cellular Cholestorol Accumulation and Egress from ...

Helsinki University Biomedical Dissertations No. 120

Cellular Cholesterol Accumulation and Egress from

Endosomal Compartments

Matts D. Linder

Institute of Biomedicine,

University of Helsinki

Finland

Academic Dissertation

To be publicly discussed with the permission of the Medical Faculty of the University of Helsinki,

at the Women’s clinic, Helsinki University Central Hospital, Haartmaninkatu 2,

big lecture hall, on April 24th 2009, at 12 o’clock noon.

Helsinki 2009

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SUPERVISOR: Professor Elina Ikonen Institute of Biomedicine University of Helsinki Helsinki Finland REVIEWERS: Professor Markku Savolainen Department of Internal Medicine University of Oulu Oulu Finland Docent Jussi Jäntti Institute of Biotechnology University of Helsinki Helsinki Finland OPPONENT: Professor Arnold von Eckardstein Department for Clinical Chemistry University Hospital Zurich Zurich Switzerland ISBN 978-952-10-5469-3 (paperback) ISBN 978-952-10-5470-9 (PDF) ISSN 1457-8433 http://ethesis.helsinki.fi Helsinki 2009 Yliopistopaino

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TABLE OF CONTENTS

ORIGINAL PUBLICATIONS.......................................................................................................... 6

ABBREVIATIONS............................................................................................................................. 7

INTRODUCTION............................................................................................................................... 9

REVIEW OF THE LITERATURE ...............................................................................................11 1. PATHOPHYSIOLOGY OF ATHEROSCLEROSIS ...............................................................11 1.1 Atherogenic lipoproteins and macrophage foam cell formation ...........................................11 1.2 Macrophage reverse cholesterol transport ..............................................................................12

1.2.1 ABCA1 mediated lipid efflux ...............................................................................13

2. INTRACELLULAR CHOLESTEROL TRAFFICKING AND HOMEOSTASIS................15 2.1 Uptake and processing of LDL cholesterol in the endosomal system ..................................15 2.2 Regulation of cholesterol biosynthesis in the ER...................................................................16 2.3 Transcriptional regulation of cholesterol levels......................................................................17 3. NIEMANN-PICK TYPE C DISEASE......................................................................................18 3.1 Disease pathophysiology..........................................................................................................18 3.2 The NPC1 and NPC2 proteins .................................................................................................20

3.2.1 Structural features and sterol binding of NPC1.................................................20

3.2.2 Subcellular localization and trafficking of NPC1 ..............................................22

3.2.3 Structural features and lipid binding of NPC2...................................................22

3.2.4 Subcellular localization and trafficking of NPC2 ..............................................25

4. RAB GTPases IN MEMBRANE AND CHOLESTEROL TRAFFICKING .........................26 4.1 Rab GTPase function................................................................................................................26 4.2 Rab proteins in intracellular sterol trafficking ........................................................................27 4.3 The Rab8 GTPase.....................................................................................................................28

AIMS OF THE STUDY ...................................................................................................................31

METHODS.........................................................................................................................................32

RESULTS AND DISCUSSION ......................................................................................................33

1. Defective intracellular trafficking of NPC proteins in NPC disease (I) ...............................33 1.1 Effects of individual mutations on the NPC1 protein ............................................................33 1.2 Intracellular localization and regulation of the NPC2 protein in NPC1 cells.......................35

2. Complementation of Niemann-Pick type C disease by Rab overexpression (II)..............36 2.1 Use of filipin staining as a measure of cholesterol efflux from endosomal compartments .37 2.2 Rab8 restores sphingolipid trafficking in NPC fibroblasts ....................................................38 2.3 Rab8 is resistant to membrane retention during cholesterol loading ....................................38 2.4 Rab8 siRNA induces a cholesterol accumulation phenotype ................................................39

3. Rab8 regulates cholesterol processing in primary human macrophages (III)....................40

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3.1 Rab8 is expressed in macrophages present in atherosclerotic lesions in vivo......................40 3.2 Adenoviral overexpression of Rab8 in vitro increases ABCA1 dependent cholesterol efflux to apoA-I..........................................................................................................................................40 3.3 Rab8 RNAi inhibits cholesterol efflux and regulates ABCA1 trafficking in primary human macrophages....................................................................................................................................41

CONCLUSIONS AND FUTURE PROSPECTS..........................................................................44

ACKNOWLEDGEMENTS.............................................................................................................48

REFERENCES..................................................................................................................................50

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ORIGINAL PUBLICATIONS This thesis is based on the following articles, which are referred to in the text by their roman

numerals.

I Blom TS, Linder MD, Snow K, Pihko H, Hess MW, Jokitalo E, Syvänen AC and

Ikonen, E. Defective endocytic trafficking of NPC1 and NPC2 underlying infantile

Niemann-Pick type C disease. Hum Mol Genet. 2003 Feb 1;12(3):257-72.

II Linder MD, Uronen RL, Hölttä-Vuori M, van der Sluijs P, Peränen J and Ikonen E.

Rab8-dependent recycling promotes endosomal cholesterol removal in normal and

sphingolipidosis cells. Mol Biol Cell. 2007 Jan;18(1):47-56.

III Linder MD, Mäyränpää MI, Peränen J, Pietilä TE, Pietiäinen VM, Uronen RL,

Olkkonen VM, Kovanen PT and Ikonen E. Rab8 Regulates ABCA1 Cell Surface

Expression and Facilitates Cholesterol Efflux in Primary Human Macrophages.

Arterioscler Thromb Vasc Biol. 2009 Mar 19. [Epub ahead of print]

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ABBREVIATIONS

aa amino acid

ABCA1 ATP-binding cassette transporter A1

ACAT acyl-coenzyme A: cholesterol acyltransferase

acLDL acetylated LDL

apoA-I apolipoprotein A-I

BODIPY boron-dipyrromethene

ER endoplasmic reticulum

GDI guanosine nucleotide dissociation inhibitor

GSL glycosphingolipid

HDL high density lipoprotein

HMGR 3-hydroxy-3-methylglutaryl-CoA reductase

Insig Insulin-induced gene

LacCer lactosylceramide

Lamp lysosome-associated membrane protein

LBPA/BMP lysobisphosphatidic acid/bismonoacylglycerophosphate

LDL low density lipoprotein

LDLr low density lipoprotein receptor

LPDS lipoprotein-deficient serum

LAL lysosomal acid lipase

LXR liver X receptor

MDCK Madin-Darby canine kidney

mmLDL minimally modified LDL

MPR mannose-6-phosphate receptor

M6P mannose-6- phosphate

mRNA messenger RNA

NPC Niemann-Pick type C

NPC1 Niemann-Pick C 1 protein

NPC2 Niemann-Pick C 2 protein

NPC1L1 Niemann-Pick C 1 –like 1 protein

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oxLDL oxidated LDL

PM plasma membrane

RCT reverse cholesterol transport

RNA ribonucleic acid

SCAP SREBP cleavage activating protein

siRNA small interfering RNA

SR-A1 scavenger receptor A1

SREBP sterol regulatory element binding protein

SSD sterol-sensing domain

TGN trans-Golgi network

TfR transferrin receptor

7DHCR 7-dehydrocholesterol reductase

WB Western blot

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INTRODUCTION

Intracellular cholesterol homeostasis is a tightly regulated process aimed at balancing the flux of

cholesterol entering and exiting the cell. Defects in cellular cholesterol metabolism have been

linked to a variety of diseases ranging from atherosclerosis to Alzheimer’s disease. The

pathogenesis of these diseases are, however, quite complex, and it has been challenging to

pinpoint the exact cellular processes that lead to disease progression, although the relationship

between circulating cholesterol levels and atherosclerosis has been well documented. In general,

LDL particles in the circulation are clinically regarded as “bad cholesterol” since these particles

get entrapped in the vascular wall, leading to atherosclerosis. Circulating HDL particles are

conversely regarded as “good cholesterol” because of their ability to transport cholesterol from

peripheral tissues to the liver for secretion as bile salts.

During the progression of atherosclerosis, LDL cholesterol accumulates in the wall of arteries,

especially in places with high shear stress. Once inside the vessel intima, the LDL particles

undergo a series of chemical modifications either by enzymatic processes or through oxidation.

The modified LDL particles are then engulfed by macrophages, resulting in macrophage foam

cells. If the macrophage foam cells are not able to efflux the cholesterol back into the

bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of

cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly

dependent on the ABCA1 transporter which transfers cholesterol to extracellular apoA-I

particles, yielding nascent HDL particles. The nascent HDL particles are then further enriched

with cholesterol by the ABCG1 transporter. The process of atherosclerotic plaque development is

therefore to a large extent a cellular one, in which the capacity of the macrophages in handling

the excessive cholesterol load determines the progression of lesion development. Hence it is of

great importance to understand how cholesterol is transported within the cell. To achieve this we

have utilized the autosomal recessive cholesterol transport disease Niemann-Pick type C (NPC),

as a cellular model and tool to study the processes governing intracellular sterol transport.

In the first part of the study we characterized the mutations in an NPC1 disease patient and

studied the consequences of the individual mutations on the function of the NPC1 protein. A

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major finding was that mutations in the NPC1 protein cause an upregulation of NPC2 protein

gene transcription, lending further support to the theory that these two proteins might function in

concert to facilitate cholesterol egress from the endosomal system.

In the second part of the study we used NPC1 disease cells as a cellular model for cholesterol

accumulation, in order to identify individual Rab GTPases involved in cholesterol efflux from the

endosomal system. Using a combination of fluorescent microscopy and biochemical techniques

we identified Rab8 as a key protein in facilitating the transport of endocytosed LDL cholesterol

to extracellular acceptors. Knock-down of the Rab8 protein with RNAi resulted in intracellular

cholesterol accumulation, while overexpression of Rab8 alleviated the cholesterol storage

phenotype seen in NPC1 disease cells.

In the third part of the study we studied the role of Rab8 in macrophage foam cell formation and

cholesterol processing. We found that Rab8 is expressed in macrophage foam cells of

atherosclerotic lesions. Overexpression of Rab8 in primary human macrophages induced

ABCA1-mediated cholesterol efflux to apoA-I. Rab8 facilitated the efflux of cholesterol from

macrophage foam cells by regulating ABCA1 protein levels at the cell surface, as well as

facilitating cholesterol traffic to ABCA1 substrate pools.

REVIEW OF THE LITERATURE

1. PATHOPHYSIOLOGY OF ATHEROSCLEROSIS

1.1 Atherogenic lipoproteins and macrophage foam cell formation Although several proatherogenic factors have been identified, the level of circulating LDL

particles stands out as the most important determinant of lesion development. Atherosclerotic

plaques start out as fatty streaks in the intima of middle and large diameter arteries already at

early adulthood. The process of plaque formation is complex but lesions usually form at places

with high shear stress. LDL is deposited in the intimal matrix of the artery where it is modified to

yield so called minimally modified LDL (mmLDL) and oxidized LDL (oxLDL) (Schwenke and

Carew, 1989; Williams and Tabas, 1998). MmLDL can still be recognized by LDL receptors

while oxLDL is no longer recognized (Navab et al., 1996). Instead, oxLDL is taken up by

macrophages via different receptors, the most important of which are CD36 and scavenger

receptor A1 (SR-A1). The lesional macrophages are derived from infiltrating monocytes that are

attracted to the site by various chemotactic factors, which can be secreted by other cells in the

lesion, or can be components of oxLDL, e.g. lyso-phosphatidylcholine (lyso-PtdCho) (Hansson

and Libby, 2006; Quinn et al., 1987).

Blood-borne monocytes infiltrate the arterial intima and differentiate into macrophages, which

take up modified LDL particles. This is initially thought to serve as an atheroprotective function.

The internalized cholesterol is stored as cholesterol esters in cytoplasmic lipid droplets.

Macrophages are, however, not capable of downregulating the scavenger receptors on the plasma

membrane, which leads to continued scavenging of lipoprotein particles and expanding

intracellular cholesterol stores, giving a foamy appearance of the cytoplasm. The foam cells are

not able to efflux cholesterol at the same rate as uptake, leading to cell death, and the deposition

of cellular debris in the arterial intima (Figure 1).

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Figure 1 Macrophage foam cells in atherosclerotic lesion development. Circulating LDL

particles accumulate in the intima of arteries, and undergo chemical modification, resulting in

oxidized LDL (oxLDL). Blood-borne monocytes infiltrate the area and differentiate into

macrophages that scavenge the oxLDL particles. Macrophage foam cells undergo apoptosis

which results in a necrotic core, leading to further lesion development. Modified from (Glass and

Witztum, 2001).

1.2 Macrophage reverse cholesterol transport

Reverse cholesterol transport (RCT) is the process of net cholesterol flux from peripheral tissues

to the liver (Cuchel and Rader, 2006). In terms of atherosclerosis, the process of macrophage

RCT warrants the most attention, although conceivably RCT might also take place from other

cells in the atherosclerotic lesion. In terms of macrophage RCT, the combined role of the ATP-

binding cassette transporters ABCA1 and ABCG1 seem to be the most important. ABCA1 was

found to be mutated in Tangier disease (Bodzioch et al., 1999; Brooks-Wilson et al., 1999; Rust

et al., 1999) Although SR-B1 has also been implicated in cholesterol efflux in vitro it is not

involved in macrophage RCT in the mouse (Wang et al., 2007).


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1.2.1 ABCA1 mediated lipid efflux The primary function of ABCA1 is thought to be the lipidation of apolipoprotein A-I (apoA-I) at

the plasma membrane (Denis et al., 2008), although intracellular compartments may also be

involved (Cavelier et al., 2006; Hassan et al., 2008; Neufeld et al., 2004). However, the

contribution of intracellular apoA-I to total HDL production has been estimated to be only ~1.4%

of total HDL production (Denis et al., 2008). The precise mechanism of apoA-I lipidation is not

known. It has been shown in numerous studies that apoA-I interacts directly with ABCA1

(Chroni et al., 2004; Vedhachalam et al., 2004) and that the binding is essential for ABCA1

mediated lipid transfer to ABCA1 (Fitzgerald et al., 2002). However, there seems to be two

separate binding sites for apoA-I on the plasma membrane, a low- capacity binding site, thought

to represent direct binding to ABCA1, and a high-capacity binding site to membrane domains

generated through ABCA1 action (Hassan et al., 2007; Vedhachalam et al., 2007). The binding of

apoA-I to ABCA1 is relatively unspecific and apparently involves amphiphatic alpha-helices in

apoA-I (Fitzgerald et al., 2002; Vedhachalam et al., 2007). Accordingly, N- and C-terminal

domain mutants of apoA-I bind equally well to ABCA1. In contrast, insertion of apoA-I into the

plasma membrane domains requires intact hydrophobic C-terminal domain alpha-helices (Gillotte

et al., 1999; Saito et al., 2003; Saito et al., 2004). The lipidation process might involve the

transfer of phospholipids to apoA-I, generating nascent HDL particles, a process which is

defective in Tangier disease patients (von Eckardstein et al., 1998). ABCA1 might function by

flipping of phospholipid species from the inner to the outer leaflet (Alder-Baerens et al., 2005),

resulting in membrane bending and increased curvature, facilitating the binding and subsequent

lipidation of lipid poor apoA-I at the plasma membrane (Vedhachalam et al., 2007).

Binding of apoA-I to ABCA1 might initiate signaling responses via the Janus kinase 2,

stabilizing the protein on the cell surface (Tang et al., 2006; Tang et al., 2004). ABCA1 and

CDC42 are in close proximity of each other at the plasma membrane and Golgi compartment,

and these proteins co-immunoprecipitate (Tsukamoto et al., 2001). It was subsequently shown

that binding of apoA-I to ABCA1 also initiates signaling via CDC42 and phosphorylation of

PAK-1 and p54JNK, leading to actin polymerization (Nofer et al., 2006). There is evidence that

apoA-I interacts directly with ABCA1 since these proteins cross-link, indicating that they are in

close proximity (<7 Å) of each other (Chroni et al., 2004; Denis et al., 2004; Fitzgerald et al.,

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2004; Wang et al., 2000). Additionally, binding of apoA-I enhances the interaction between

ABCA1 and CDC42 (Nofer et al., 2006)


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2. INTRACELLULAR CHOLESTEROL TRAFFICKING AND HOMEOSTASIS

2.1 Uptake and processing of LDL cholesterol in the endosomal system Regulation of cellular cholesterol homeostasis is mainly achieved through balancing the amount

of cholesterol uptake, synthesis and efflux. Circulaing LDL particles containing cholesterol bind

to the LDL receptor at the cell surface. The LDL receptor complex is then internalized through

clathrin-coated pits. Once endocytosed, the LDL particles dissociate from the LDL receptor

which then recycles back to the cell surface (Matter et al., 1993). Cholesteryl esters in the LDL

particle are then hydrolyzed by lysosomal acid lipase (LAL) to yield free (unesterified)

cholesterol. Most of the cholesterol in the endocytic compartments is present in multivesicular

endosomes (multivesicular bodies, MVB) and recycling endosomes, ~60% and ~20%

respectively (Möbius et al., 2003). The internal membranes of MVBs contain the bulk of

cholesterol and are enriched in the endosome specific phospholipid LBPA/BMP

(lysobisphosphatidic acid / bismonoacyl-glycerophosphate) (Kobayashi et al., 1999). The free

cholesterol exits the endosomal compartments in a process that is dependent on the NPC1 and

NPC2 proteins. After exiting the endosomes, cholesterol is mainly distributed to the plasma

membrane and Golgi apparatus by yet unidentified pathways, before finally reaching the ER. The

ER is usually relatively cholesterol poor and cholesterol entering the ER is rapidly converted to

cholesteryl esters by acyl-coenzyme A: cholesterol acyltransferase (ACAT), for storage in lipid

droplets (Figure 2).

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Figure 2. Cholesterol uptake and compartmentalization. LDL particles bind to the LDL-receptor

at the plasma membrane, and are endocytosed through clathrin-coated pits. The LDL particle

then dissociates from the LDLr which is recycled back to the plasma membrane. The cholesterol

esters derived from the LDL particle is hydrolyzed in the endosomes to yield free cholesterol. The

free cholesterol is transported out of the endosomal compartments to the plasma membrane and

TGN before reaching the ER (dotted arrows). In the ER, the cholesterol is re-esterified by ACAT,

and stored in lipid droplets. (LDLr, LDL receptor; EE, early endosome; RE, recycling

endosome; MVB, multi vesicular body; LE, late endome; LY, lysosome; ER, endoplasmic

reticulum; LD, lipid droplet; TGN trans-Golgi network; N, nucleus )

2.2 Regulation of cholesterol biosynthesis in the ER

The ER is the main orchestrator of cellular cholesterol distribution and amount. However, the ER

is a relatively cholesterol poor organelle with a cholesterol content of only 1% of total cellular

cholesterol (Lange, 1991). The two main proteins mediating cholesterol regulation in the ER are


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HMG-CoA reductase (HMGR) and the Sterol response element binding protein (SREBP)

Cleavage Activating Protein (SCAP), which forms a complex with SREBP in the ER (Goldstein

et al., 2006). HMGR is the rate-limiting protein in de novo cholesterol biosynthesis, while SCAP

regulates SREBP processing. Both proteins are regulated by sterol binding to the sterol-sensing

domain (SSD) and the ER resident Insig proteins (Insig1 and Insig2). Rising cholesterol levels in

the ER induces regulatory mechanisms at both transcriptional and posttranscriptional levels.

Cholesterol binds directly to the SSD of SCAP, causing a conformational change in the SSD.

This causes SCAP to bind to Insig proteins, leading to retention of the SCAP/SREBP complex in

the ER, thus shutting down SREBP dependent gene transcription (Yang et al., 2002). HMGR is

also regulated by sterols, but the outcome of Insig binding is entirely different. Binding of

HMGR to Insigs leads to ubiquitinylation and subsequent degradation of HMGR in the

proteasome, thus reducing de-novo cholesterol synthesis (Espenshade and Hughes, 2007; Sever

et al., 2003).

2.3 Transcriptional regulation of cholesterol levels

There are two main transcriptional regulatory systems in the cell which work antagonistically to

balance the intake, de novo synthesis and efflux of cholesterol, namely the sterol regulatory

element proteins (SREBPs) and liver X receptors (LXRs). As described above, the SREBP

system is regulated by ER sterol levels through the SCAP protein. The role of SCAP is to

regulate the trafficking of SREBP to the Golgi. Upon cholesterol depletion in the ER, SREBP

traffics to the Golgi where it is activated through a proteolytic process by the site1 (S1P) and

site2 (S2P) proteases. The active transcription factor travels to the nucleus where it activates the

transcription of gene products which increase intracellular cholesterol levels, e.g. HMGR and the

LDL receptor (Goldstein et al., 2006). Conversely, activation of LXRs is mediated by oxysterols

leading to the transcription of gene products that facilitate cholesterol efflux from the cell, e.g.

the ABCA1 transporter (Tontonoz and Mangelsdorf, 2003).

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3. NIEMANN-PICK TYPE C DISEASE

3.1 Disease pathophysiology

Niemann-Pick type C disease is a severe lysosomal cholesterol-sphingolipid storage disorder.

The progress of the disease is varying with onset of symptoms ranging from early infancy to

adulthood. Typically, the disease presents with neurological regression and peripheral

manifestations due to massive cholesterol accumulation in the liver and spleen. Clinically the

patients present with hepatosplenomegaly, ataxia, dystonia, seizures, supranuclear gaze palsy,

and progressive dementia. Early-onset NPC disease is usually more severe and involves

peripheral tissues with hepatosplenomegaly often observed. In the brain, there is degeneration of

the brain stem and Purkinje neurons of the cerebellum (Walkley and Suzuki, 2004). Why only

parts of the brain and specific subsets of neurons are affected is not understood, but the process is

likely to involve both apoptosis and autophagy (Ko et al., 2005; Pacheco et al., 2007). A number

of biochemical defects have been described, but the traditional hallmark of the disease is

cholesterol accumulation in lysosomes combined with decreased esterification of cholesterol in

the endoplasmic reticulum (ER) (Blanchette-Mackie et al., 1988). NPC has therefore historically

been regarded as a cholesterol transport disorder. This is in contrast to Niemann-Pick types A and

B, which are caused by acid sphingomyelinase deficiency. Interestingly, there is concomitant

cholesterol accumulation in Niemann-Pick type A and B cells, and the depletion of cholesterol

from these cells restores trafficking of fluorescently tagged lactosyl ceramide (BODIPY-LacCer)

(Puri et al., 1999). These results emphasize the interdependence of cholesterol and sphingolipids

in lysosomal storage disorders. Accordingly, in NPC disease there is a concomitant sphingolipid

accumulation and decreased sphingomyelinase activity (Liscum and Klansek, 1998; Lloyd-Evans

et al., 2008). The pathophysiology of NPC disease is however quite complex and it is has been

challenging to pinpoint the precise pathological mechanism. Nevertheless it is likely that the

disease phenotype is the combined result of aberrant cholesterol and sphingolipid metabolism.


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Figure 3. Primary human fibroblasts from control and NPC1 patients. Cells are stained with

filipin which visualizes free cholesterol (blue). NPC1 cells accumulate large amounts of

cholesterol perinuclearly.

One of the main cellular perturbations is a defect in the regulatory responses to cholesterol

loading via the LDL pathway. Normally, cholesterol containing LDL particles taken up via the

LDL-receptor, where after the cholesterol exits the endosomal system and is transported to the

plasma membrane and to the ER, possibly via the trans-Golgi network (TGN). Superfluous

cholesterol is then re-esterified by the ER resident protein acyl-CoA acyltransferase (ACAT) for

storage in lipid droplets (Chang et al., 2001). In NPC disease cholesterol is trapped in aberrant

endosomes and fails to reach the ER (Blanchette-Mackie et al., 1988; Pentchev et al., 1985)

(Figure 3). This results in a relative cholesterol deficit in the ER, which is the primary sterol-

sensing organelle in the cell. As a result, there is a misregulation of the two major cholesterol

homeostasis regulatory machineries i.e. the SREBP and LXR pathways. Thus, de novo

cholesterol synthesis is not down-regulated, but rather increased (Liscum and Faust, 1987). Also

the cholesterol efflux pathways are not up-regulated appropriately, and the LDL-receptor fails to

down-regulate. The failure of down-regulation of the SREBP pathway can easily be attributed to

the relative lack of cholesterol in the ER. Concomitantly, there is defective generation of

oxysterols, the key activators of the LXR pathway (Frolov et al., 2003).

Control NPC1

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3.2 The NPC1 and NPC2 proteins

Niemann-Pick disease type C is comprised of two separate genetic complementation groups with

mutations in either of the NPC1 or NPC2 genes. Roughly 95% of disease cases are caused by

mutations in the NPC1 gene whilst the remaining cases are accounted for by mutations in the

NPC2 gene (Carstea et al., 1997; Naureckiene et al., 2000). The precise function of the two NPC

proteins has not been established. However, lack of either protein leads to a nearly identical

cellular phenotype, indicating that these proteins must perform similar functions in sterol egress

from the endosomal system. Also, the NPC2/NPC1 double knockout mouse model displays an

identical phenotype to that of NPC2 and NPC1 knockout models alone (Sleat et al., 2004). NPC2

is a cholesterol binding protein present in lysosomes. NPC1 has been suggested to function as a

transmembrane fatty acid transporter (Davies et al., 2000a), and more recently, as a sphingosine

transporter (Lloyd-Evans et al., 2008). The conclusive evidence for a specific transport function

for either of the proteins is however still lacking.

3.2.1 Structural features and sterol binding of NPC1 The gene mutated in the major complementation group was identified in 1997 through a

positional cloning strategy. The gene product, named NPC1, was found to be a large, 1278 amino

acid polytopic membrane protein with 13 transmembrane spans (Carstea et al., 1997) (Figure 4).

A number of interesting domains have been identified in the protein, including a putative sterol-

sensing domain (SSD) comprising of transmembrane loops three through seven (Davies and

Ioannou, 2000). This domain was originally identified in the SCAP (sterol regulatory element

binding protein (SREBP) cleavage activating protein) and hydroxymethylglutaryl (HMG)-CoA

reductase proteins (HMGR) (Osborne and Rosenfeld, 1998). Other proteins containing a SSD

include the Hedgehog morphogen receptor Patched and 7-Dehydrocholesterol reductase

(7DHCR) (Kuwabara and Labouesse, 2002), as well as Niemann-Pick type C 1 Like 1 (NPC1L1)

(Davies et al., 2000b). The function of the SSD has most extensively been studied in SCAP and

HMGR. The SSD is required for cholesterol dependent retention of SCAP in the ER, as well as

lanosterol-induced ubiquitinylation and degradation of HMGR (Nohturfft et al., 1999; Song et al.,

2005). The evidence regarding a specific sterol-sensing function of the SSD in the NPC1 protein


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is still inconclusive. NPC1 is capable of binding to a photoactivatable cholesterol analog,

azocholestanol, while mutations in the SSD (P691S, Y634C) abolish this binding (Ohgami et al.,

2004). In vitro [3H]-cholesterol binding to the purified SSD of SCAP has also been established,

but this was not inhibited by the corresponding Y298C mutation in SCAP (Radhakrishnan et al.,

2004). However this mutation in SCAP has previously been shown to inhibit the binding of

SCAP to Insig proteins involved in 25-hydroxycholesterol sensing in the ER (Radhakrishnan et

al., 2007; Yang et al., 2002). Also, mutations in NPC1, corresponding to gain of function

mutations in SCAP, resulted in increased cholesterol trafficking in the cell (Millard et al., 2005).

However, in a later study the sterol binding capacity of the NPC1 protein was limited to the N-

terminal domain (“NPC domain”) of purified NPC1 (Infante et al., 2008a). Mutations in the SSD

are none the less clinically significant, and mutations in the SSD abolish the cholesterol transport

function of NPC1 (Watari et al., 1999b). A critical role for the N-terminal domain in NPC1

function has also previously been shown, as well as for the C-terminal LLNF lysosome-targeting

motif (Watari et al., 1999b). Deletion of the C-terminal dileucine motif resulted in the retention

of the NPC1 protein in the ER. Site directed mutagenesis of cysteins (C63S, C74S/C75S and

C97S) resulted in non-functional NPC1 protein that localized to the surface of lysosomes (Watari

et al., 1999a).

Figure 4. Schematic representation of the NPC1 protein, The SSD is shown in red. Adapted from

(Davies and Ioannou, 2000).

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3.2.2 Subcellular localization and trafficking of NPC1 The NPC1 and NPC2 proteins are thought to reside mainly in late endocytic compartments. The

proteins show little co-localization, indicating that they are present in different subsets of

endosomes. NPC1 colocalizes with the late endosomal/lysosomal marker Lamp2, but not with

endocytosed LDL derived cholesterol or the mannose-6- phosphate receptor (MPR) (Neufeld et

al., 1999). NPC1 has also been shown to colocalize with LBPA, Rab7, and Rab9 (Higgins et al.,

1999; Puri et al., 1999; Zhang et al., 2001b). Drugs that induce lysosomal cholesterol

accumulation e.g. progesterone and U18666A redistributes NPC1 to cholesterol laden lysosomes

(Neufeld et al., 1999). In normal cells NPC1 is thought to only transiently localize to cholesterol

enriched organelles (Higgins et al., 1999; Kobayashi et al., 1999; Neufeld et al., 1999; Patel et al.,

1999; Watari et al., 1999a; Zhang et al., 2001b). Also the GM2 ganglioside localizes exclusively

with NPC1, but not with endocytosed cholesterol (Osborne and Rosenfeld, 1998).

Live cell microscopy using GFP-tagged NPC1 protein has revealed that NPC1 containing

endosomes are highly mobile. NPC1 function seems to be dually required for both lipid sorting

and tubular late endocytic trafficking (Ko et al., 2001). Also the motility of NPC1 containing

endosomes is modulated by the lipid composition of the membranes, and is inhibited by

cholesterol accumulation (Lebrand et al., 2002; Zhang et al., 2001a). NPC1-GFP containing

organelles undergo tubulation and fission with both anterograde and retrograde migrations along

microtubules. This movement also requires an intact SSD (Zhang et al., 2001a). Newly

synthesized NPC1-GFP was also seen by live cell microscopy to be transported from the ER via

the Golgi to late endosomes (Zhang et al., 2001a). NPC1 is ubiquitinylated upon cholesterol

depletion which might influence the trafficking of NPC1 within different subsets of endosomes

(Ohsaki et al., 2006).

3.2.3 Structural features and lipid binding of NPC2 The NPC2 gene product is a 151 amino-acid soluble lysosomal protein containing a 19 amino

acid signal peptide which is cleaved off to generate the active 132 amino-acid protein.


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Intracellular localization studies have shown that the protein resides both in Lamp1 positive and

Lamp1 negative endosomes, as well as the Golgi apparatus. The NPC2 protein is glycosylated

and contains a mannose-6-phosphate (M6P) post-translational modification conferring lysosomal

targeting through binding to the mannose-6-phosphate receptor (MPR). The NPC2 protein was

originally characterized as a cholesterol binding protein found in epididymal fluid named HE1

(human epididymis protein 1) (Okamura et al., 1999). As a result of a screening for M6P

containing proteins HE1 was found to localize to lysosomes. This allowed the identification of

HE1 as the second protein in NPC disease, and the HE1 protein was renamed NPC2

(Naureckiene et al., 2000).

Structural analysis of the bovine NPC2 protein and site-directed mutagenesis strategies have

shown the mechanism of cholesterol binding and identified functional domains in the protein.

The bovine orthologue of NPC2, EPV20 had previously been purified from bovine milk (Larsen

et al., 1997). The structure of the bovine homolog bNPC2 was elucidated by x-ray

crystallography, revealing an Ig-like -sandwich fold consisting of seven -strands arranged in

two -sheets (Friedland et al., 2003). Between the -sheets, three small cavities are formed with a

total volume of 158 Å3. The volume of a cholesterol molecule is 741 Å3, indicating that the -

sandwich needs to expand in order to accommodate the cholesterol molecule. The structure of

NPC2 bound to cholesterol-3-O-sulfate also supports this model (Xu et al., 2007). X-ray

crystallography of NPC2 bound to cholesterol sulfate indicates that the sterol is inserted into the

cholesterol binding pocket with the iso-octyl side chain inside the pocket, and the sulfate group at

position 3 of the A ring protruding from the entrance (Xu et al., 2007). Such an orientation would

explain why NPC2 has low affinity for oxysterols (Infante et al., 2008a). A mutational screen

based on evolutionally conserved residues highlights the importance of the cholesterol-binding

cavity (Ko et al., 2003) (Figure 5).

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Figure 5. Structure of the NPC2 sterol binding pocket. (A) Stereo view of the proposed sterol

binding cavity. Sidechains of residues lining the cavity are shown in stick representation. Side

chains required for cholesterol binding (Ko et al., 2003) are shown in red. (B) Space filling

model of cholesterol (geeen) docked in the proposed sterol-binding site. Reprinted from

(Friedland et al., 2003) with the permission of the publisher. Copyright (2003) National Academy

of Sciences, U.S.A.

The current evidence strongly favors a direct cholesterol shuttling model for NPC2 function.

Several studies have now established that NPC2 binds cholesterol (Friedland et al., 2003; Infante

et al., 2008a; Ko et al., 2003; Liou et al., 2006). NPC2 is able to extract and insert cholesterol

from membranes in vitro (Cheruku et al., 2006) as well as shuttle cholesterol between liposomal

vesicles (Babalola et al., 2007). NPC2 can also transfer cholesterol between the N-terminal

domain of NPC1 and liposomes in vitro (Infante et al., 2008b). NPC2 is also involved in Thymal

T cell selection by loading the MHC class I-like protein CD1d with the selecting

glycosphingolipid isoglobotrihexosylceramide (iGb3) in NKT cells (Schrantz et al., 2007).

Whereas the in vitro loading of iGb3 to CD1d required dimerization of NPC2, the transfer was

not affected by cholesterol binding.


25

3.2.4 Subcellular localization and trafficking of NPC2 The NPC2 protein resides mainly in the lumen of late endosomes/lysosomes. The intracellular

trafficking of NPC2 is governed by the mannose 6-phosphate receptor (MPR) owing to the

posttranslational addition of a mannose 6-phosphate moiety to the protein (Naureckiene et al.,

2000). The mannose 6-phosphate moiety is recognized by the MPR, which enables trafficking

between the Golgi apparatus and lysosomes as well as uptake from the PM. Depletion of the two

MPRs, MPR300 (cation-independent) and MPR46 (cation-dependent), in fibroblasts increases

the secretion of NPC2 from cells (Willenborg et al., 2005). NPC2 is abundantly present in

epididymal fluid and milk (Kirchhoff et al., 1996; Larsen et al., 1997), and can be secreted from

cells in the cholesterol bound form (Ko et al., 2003; Okamura et al., 1999). NPC2 is also secreted

from cultured glia cells, but does not associate with the major secretory sterol containing

lipoprotein particles. Also, overexpression of NPC2 in glia cells does not enhance sterol secretion

(Mutka et al., 2004). Secreted NPC2 can be re-endocytosed and is biologically active. Incubation

of NPC2 cells with medium preconditioned by incubation with WT cells complements the

cholesterol storage phenotype, but the complementation is inhibited by mannose-6 phosphate,

indicating that endocytosis by MPR is required (Naureckiene et al., 2000). However

overexpression of NPC2 mutants that are not properly secreted still complement the individual

transfected cells, indicating that secretion is not an absolute necessity (Ko et al., 2003).

NPC2 is glycosylated, appearing as two individual bands of 21 to 25 kDa in WB of human

fibroblast samples. After removal of N-linked glycans with PNGaseF (protein:N-glycosidase F)

the protein migrates at 18 kDa (Mutka et al., 2004). A mutational screen of the possible

asparagine linked (Asn, N) glycosylation sites revealed that only two of the sites, N58 and N135,

are utilized. The study also showed that glycosylation of Asn 58 is required for proper lysosomal

targeting of NPC2 (Chikh et al., 2004).

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4. RAB GTPases IN MEMBRANE AND CHOLESTEROL TRAFFICKING

4.1 Rab GTPase function Rab proteins are small GTPases belonging to the Ras superfamily of GTPases (Wennerberg et al.,

2005). Rab proteins function as molecular switches, regulating events such as vesicle trafficking,

motility, docking and fusion (Pfeffer, 2001; Zerial and McBride, 2001). Rab proteins cycle

between the cytosol and target membranes in an active GTP-bound state and an inactive GDP-

bound state. The switching to the active state is mediated by guanine nucleotide exchange factors

(GEFs), which mediate binding of GTP to the Rab protein. Inactivation is mediated by GTPase-

activating proteins (GAPs) which catalyze the intrinsic GTPase activity of the Rab protein,

resulting in hydrolysis of the bound GTP to GDP (Pfeffer, 2001; Segev, 2001). In the active GTP

bound state, Rab proteins insert into the target membrane by virtue of a prenylation motif located

in the C-terminal of the protein. Prenylation of Rab proteins is facilitated by Rab escort protein

(REP) which presents newly synthesized Rab protein to Rab geranylgeranyl transferase

(GGTase) (Andres et al., 1993). In the ‘soluble’ state Rab proteins are bound and sequestered in

the cytosol by GDI (guanosine nucleotide dissociation inhibitor) (Figure 6). Human cells express

two isoforms, GDI which is enriched in the brain and the ubiquitously expressed GDI (Alory

and Balch, 2001). The binding of Rab proteins to GDI is strongly dependent on prenylation as

well as the presence of GDP in the nucleotide binding site. Upon GDP- or GTP binding the Rab

proteins change conformation in the highly conserved ‘switch region’, which is recognized by

GDI. GDI can also extract Rab proteins from membranes (Sasaki et al., 1990; Ullrich et al.,

1993).

The targeting of Rab proteins to specific membranes is thought to be mediated by the C-terminal

‘hypervariable domains’ which are the most divergent amino acid stretches between different

Rab proteins (Chavrier et al., 1991). The association of the Rab protein to GDI is probably

modulated by the length of the hypervariable domain with affinities of 20-500nM Kd being

reported (Schalk et al., 1996; Shapiro and Pfeffer, 1995). The deposition of Rab proteins into the

target membrane is facilitated by GDI-displacement factors (GDF).


27

Figure 6. The Rab cycle. Rab GTPases are activated by guanine nucleotide exchange factors

(GEFs). The Rab proteins then bind to effector proteins which can for instance be molecular

motors. After fulfilling its task, the Rab protein is deactivated by GTPase activating proteins

(GAPs), and removed from the membranes by guanine nucleotide dissociation inhibitor (GDI).

Another round of action is initiated by GDI displacement factor (GDF) which facilitates the

deposition of Rab proteins into the target membrane.

4.2 Rab proteins in intracellular sterol trafficking Cholesterol efflux from the endosomal compartments seems to be mediated, at least in part, by

Rab protein directed membrane trafficking. It was initially shown that inhibition of Rab protein

function by microinjection of Rab-GDI inhibits cholesterol removal from endocytic

compartments (Höltta-Vuori et al., 2000). So far, the individual Rab proteins that have been

implicated in intracellular cholesterol trafficking localize to the endocytic compartments of the

cell (Table 1). Choudhury and colleagues showed that overxpression of dominant negative Rab7

or Rab9 inhibited the Golgi targeting of sphingolipids, and that overexpression of wild-type Rab7

or Rab9 reduced the cholesterol accumulation in NPC1 fibroblasts (Choudhury et al., 2002).

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Rab9 was subsequently shown to increase cholesterol efflux in two separate studies (Narita et al.,

2005) (Walter et al., 2003). In a recent study, the life span of Rab9 / NPC1-/- transgenic mice was

also found to be increased by ~ 22 % as compared to NPC1-/- mice (Kaptzan et al., 2009). The

dominant negative form of Rab4 also perturbs cholesterol recycling (Choudhury et al., 2004).

Overexpression of Rab11 affects endosomal cholesterol trafficking, resulting in the accumulation

of free cholesterol in recycling endosomes (Höltta-Vuori et al., 2002).

Table 1. Localization and function of Rab proteins which have been shown to affect cholesterol

transport in the endocytic compartments.

Rab Localization Function Reference

Rab4 Early and recycling

endosomes

Rapid endosomal recycling (van der Sluijs et al., 1992)

Rab7 Late endosomes,

lysosomes

Early to late endosomal transport,

Lysosome biogenesis

(Press et al., 1998) (Bucci

et al., 2000)

Rab9 Late endosomes,

trans Golgi network

Late endosome to Golgi transport (Lombardi et al., 1993)

Rab11 Recycling endosomes Recycling through the endocytic

recycling compartment (ERC)

(Ullrich et al., 1996) (Ren et

al., 1998)

4.3 The Rab8 GTPase Two isoforms, Rab8A (referred to here as Rab8) and Rab8b have been identified in humans.

Rab8 is ubiquitously expressed in tissues while Rab8b is predominantly expressed in the spleen,

testis and brain (Armstrong et al., 1996). There is an 83% sequence identity between Rab8A and

Rab8b, with the highest degree of divergence within the C-terminal hypervariable domain (Figure

7) (Armstrong et al., 1996). Rab8 differs from other Rab proteins in that it is geranylgeranylated

at a single site as opposed to the usual double geranylgeranylation. This is due to the presence of

a CaaL motif instead of the usual CC or CXC motifs (Casey and Seabra, 1996; Wilson et al.,

1998).


29

Figure 7. Bar representation of Rab8A and Rab8b. Guanine base binding sites are indicated in

black (aa 33; aa 119-125; aa 147-154). The hypervariable domain (aa 154-207) is indicated in

yellow. The C-terminal prenylation motif CVLL is composed of aa 204-207.

Rab8 affects cell shape, inducing cellular protrusions when overexpressed (Nachury et al., 2007)

(Armstrong et al., 1996; Peränen et al., 1996). In two separate studies Rab8 was found to regulate

primary cilium formation (Nachury et al., 2007; Yoshimura et al., 2007). Cilium formation was

also found to be dependent on the Rab8 specific GAP XM_037557 (Yoshimura et al., 2007).

Polarized transport by Rab8 is thought to be partly mediated by reorganization of actin and

microtubules (Ang et al., 2003; Peränen et al., 1996). Rab8 also regulates the actin based

movement of melanosomes to the PM in MDCK cells (Chabrillat et al., 2005), as well as vesicle

transport during neuronal outgrowth. Knock-down of Rab8 with siRNA in neurons results in

inhibition of anterograde vesicle movement and disrupts neuronal outgrowth (Huber et al., 1995).

In addition to Rab8, vesicle formation and transport to membrane protrusions is dependent on the

Rab8 specific GDP/GTP exchange factor Rabin8 (Hattula et al., 2002). Rab8 was shown to

mediate apical protein transport in intestinal ephitelial cells in a Rab8 -/- mouse model. In

intestinal epithelial cells Rab8 might control both direct protein transport from the TGN to the

apical membrane, or indirectly via the basolateral membrane. In any case, Rab8 deficiency causes

the mislocalization of apical proteins to the lysosomes, and formation of intracellular microvillus

inclusions, resulting in dysfunction of apical microvilli and malabsorbtion (Sato et al., 2007).

Reduced Rab8 expression was also found in a human microvillus inclusion disease patient with

an identical ultrastructural phenotype to the Rab8-/- mouse (Sato et al., 2007). Recently it was

also established that mutations in the MYOB5 gene, which encodes the Rab8 interacting

myosinVb motor protein (Roland et al., 2007), also causes microvillus inclusion disease in

humans (Muller et al., 2008).

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30

Rab8 is involved in both constitutive transport of proteins from the Golgi (Huber et al., 1993) and

basolateral vesicle transport in Madin-Darby canine kidney (MDCK) cells (Ang et al., 2003). In

MDCK cells, recycling endosomes might serve as intermediates for Golgi to PM transport, and as

a common sorting organelle for the endocytic and secretory pathways (Ang et al., 2004). The

polarized transport of proteins to the basolateral membrane is dependent on the AP-1B clathrin

adaptor complex, which directs clatrin-coated bud formation at the TGN, and is regulated by

Rab8 (Ang et al., 2003). The Rho GTPase CDC42 also selectively regulates the AP-1B pathway,

and dominant negative CDC42 causes selective missorting of AP-1B dependent cargo, indicating

that Rab8 and CDC42 might function in the same pathway (Ang et al., 2003). In addition, cell

morphogenesis is also altered by the Rab8 interacting, TNF -inducable protein FIP-2

(optineurin), linking Rab8 to huntingtin (Hattula and Peränen, 2000). Optineurin also links Rab8

to myosin VI, which acts as a motor for actin dependent transport of vesicles deriving from the

TGN (Au et al., 2007; Sahlender et al., 2005). Mutant huntingtin (mhtt), in turn inhibits Rab8

mediated post-Golgi trafficking, by delocalizing the optineurin/Rab8 complex from the Golgi

(Del Toro et al., 2009).

Table 2. Proteins which have been shown to interact directly with Rab8.

Protein Function Reference

Rabin8 Rab8 specific GEF (Hattula et al., 2002)

XM_037557 Rab8 specific GAP (Yoshimura et al., 2007)

Myosin Vb Molecular motor (Roland et al., 2007)

Rabaptin5 Regulator of endocytosis (Omori et al., 2008)

FIP-2/optineurin Vesicle trafficking (Hattula and Peränen, 2000)

31

AIMS OF THE STUDY

The aim of the first part of the study was to gain insight in to the function of the NPC1 and NPC2

proteins. Making use of a severe form of infantile NPC1 disease the goal was to identify

individual disease causing mutations in the NPC1 protein and to study the consequences of the

individual mutations at the cellular level. The other key goal of the study was to establish the

intracellular location of the NPC2 protein in normal and NPC1 fibroblasts in order to gain further

insight into the pathophysiology of NPC disease.

The aim of the second part of the study was to use the cholesterol accumulation found in NPC1

disease as a tool to identify proteins involved in the efflux of cholesterol from the endocytic

circuits. Proteins, primarily Rab GTPases, which reduce endosomal cholesterol accumulation in

NPC1 cells would then be further characterized in their ability to facilitate intracellular

cholesterol transport.

The aim of the third part of the study was to characterize the potential function of Rab8 in

cellular cholesterol efflux from primary human macrophages. Macrophage foam cells are key

elements in the development of atherosclerotic lesions and prime targets for pharmacological

intervention. It is therefore crucial to understand the intracellular processes leading to ABCA1-

mediated cholesterol efflux. The goal of this part of the study was therefore to investigate the role

of Rab8 in the physiological setting of macrophage foam cell formation.

METHODS

Method Publication

Cell culture I, II, III

SDS-PAGE, Western blot, ECL (enhanced chemiluminescence) I, II, III

Northern blot I

Quantitative real-time PCR III

Surface biotinylation I, III

Adenovirus production and infection III

Semliki Forest virus production and infection II

RNA isolation I, II

Biochemical cholesterol measurements II, III

Biochemical cholesterol ester measurements II, III

Direct cholesterol efflux measurements II, III

GDI extraction of Rab proteins in vitro II

Cell fractionation II

Recombinant protein production and purification I

Polyclonal antibody production I

Immunocytochemistry I, II, III

Immunohistochemistry III

RNA interference II, III

Transient transfection I, II, III

Electroporation III

RESULTS AND DISCUSSION

1. Defective intracellular trafficking of NPC proteins in NPC disease (I)

1.1 Effects of individual mutations on the NPC1 protein Since the discovery of the two separate complementation groups in NPC it has been speculated

that the two NPC proteins act along the same pathway and might even interact. In order to assess

this, we sought to determine the subcellular localization of both NPC proteins, and analyze the

effects of disease causing mutations in NPC1 on the distribution of the NPC2 protein. We

identified three mutations, two of which were causative of NPC disease, C113R and a c-terminal

deletion mutant (delC), and a benign polymorphism P237S. Interestingly, the P237S substitution

has previously been regarded as a disease causing mutation (Kaminski et al., 2002). The P237S

substitution was present in ~5% of the alleles in the Finnish and Swedish population samples

studied. If P237S were to be a disease causing mutation one would expect the incidence of NPC1

disease to be much higher in the Finnish and Swedish populations. Moreover, overexpression of

the NPC1 protein harboring the P237S substitution fully restored cholesterol trafficking at the

cellular level. Although our results rule out P237S as a disease causing mutation it is still possible

that the mutation has a more subtle phenotype not leading to NPC1 disease.

In the case of the C-terminal deletion the case is clear-cut, since it leads to an unstable protein

which is rapidly degraded. The C113R substitution, however, results in a non-functional protein

that is partially mistargeted. We found the C113R protein to localize mainly to the ER, Rab7-

negative endosomes and the plasma membrane. Retarded export from the ER was confirmed by

metabolic labeling of the newly synthesized protein with 35S-methionine followed by

immunoprecipitation and endoglycosidaseH (endoH) digestion. The C113R protein showed a

decrease in the EndoH resistant fraction indicating that a smaller fraction of NPC1-C113R

reached the Golgi compared to the WT protein. What might be the mechanism for the decreased

export of NPC1-C113R from the ER? The C113R substitution resides in a highly conserved

region of the 240 amino acid (aa) luminal N-terminus containing 13 cysteine residues. Amino

MATTS LINDER

34

acids 55 through 165 encompass the “NPC” domain, a 112 aa stretch with high conservation

between NPC1 orthologues (Watari et al., 1999a). There are 8 conserved cysteine residues within

this domain, and site directed mutagenesis of four of these cysteines resulted in an inactive

protein that was localized to the limiting membrane of cholesterol-laden lysosomes in NPC cells

(Watari et al., 1999a). Presumably, the cysteine at aa position 113 participates in disulfide bridge

formation contributing to the tertiary structure of the N-terminal domain. This region was

recently shown to mediate oligomerisation of the NPC1 protein as well as to bind to cholesterol

and 25-hydroxycholesterol (Infante et al., 2008a). Interestingly the stochiometry of sterol binding

indicates that one cholesterol or hydroxycholesterol molecule binds to one dimer of the NPC1

protein. Whether or not dimerization or oligomerization of the NPC1 protein is a requirement for

sterol binding has not been assessed. However, it does not seem that sterols are required for the

dimerization of the protein since an N-terminal mutant defective in sterol binding (NPC1-Q79A)

showed the same pattern of oligomerisation as the WT protein (Infante et al., 2008a) (Linder and

Ikonen, unpublished observations). Also the NPC1-C113R protein forms oligomers indicating the

presumable changes in the n-terminal conformation are not sufficient to inhibit oligomerisation of

the protein (Linder and Ikonen, unpublished observations).

Although the C113R mutation clearly causes a defect in maturation of the protein it is curious

that the protein is also mistargeted after leaving the Golgi. The putative dileucine endosomal

targeting motif resides in the C-terminal of the protein, and ablation of this motif causes the

retention and degradation of the protein in the ER (Watari et al., 1999a). One explanation could

be that the C113R substitution interferes with binding to accessory proteins required for the

proper targeting of the protein. Alternatively, sterol binding at the N-terminal is required for the

proper localization and function of the protein. As stated above, the Q79A mutation causes a

defect in sterol binding, whereby 25-HC binding is abolished but residual cholesterol binding

capacity is retained. As for the C113R substitution, the cholesterol binding capacity has not been

assessed.


35

1.2 Intracellular localization and regulation of the NPC2 protein in NPC1 cells

Characterization of the NPC2 protein has been hampered by the lack of high quality commercial

antibodies against NPC2. We generated polyclonal antibodies using recombinantly expressed

NPC2 fused to GST (amino acids 20-151, full length NPC2 w/o signal sequence) as an epitope.

The specificity of the antibody was confirmed by preincubation of the antibody with purified

recombinant protein prior to IF staining. Also the specificity has been confirmed by Western blot

using mouse NPC2 -/- samples (Mutka et al., 2004). In normal human fibroblasts we found the

endogenous NPC2 to have a punctate staining pattern. Double immunofluorescence staining

indicated that the majority of NPC2 was present in Lamp1-positive lysosomes. On the other

hand, in some cells, NPC2 was predominantly colocalized with gamma-adaptin, a marker for the

trans-Golgi network (TGN). Taken together, these results reconcile well with MPR regulated

shuttling of the NPC2 protein between the Golgi and endo/lysosomes. In contrast, NPC1 cells

showed an exclusively lysosomal localization of the NPC2 protein, indicating that there is a

sequesteration of the NPC2 protein in lysosomes. Consistent with this we saw a roughly 1.5-fold

increase in NPC2 protein by Western blotting. This might be a result of decreased degradation of

NPC2 or a compensatory upregulation of NPC2 gene transcription. Northern blot analysis

showed that the NPC2 transcript was significantly upregulated indicating that the increase in

NPC2 protein levels is due to a regulatory increase in NPC2 synthesis.

In the NPC1 fibroblasts the NPC2 protein was sequestered in the cholesterol-laden lysosomes

and very little Golgi like staining could be seen. In accordance with this there was also an

apparent redistribution of MPR to Lamp-1 positive organelles from the normally predominant

TGN localization. Transport of MPRs from late endosomes to the trans-Golgi is mediated by

Rab9 (Lombardi et al., 1993; Riederer et al., 1994) and the Rab9 effector TIP47 (Carroll et al.,

2001; Diaz and Pfeffer, 1998). Interestingly Rab9 function is perturbed in NPC1 fibroblasts

resulting in the accumulation of inactive Rab9 in late endosomes and lysosomal missorting and

degradation of MPR (Ganley and Pfeffer, 2006). It is therefore possible that NPC2 accumulates

as a result of defective Rab9 mediated transport steps. Whether or not this would have relevance

for the disease phenotype is unclear, since the putative site of NPC2 function is in the

MATTS LINDER

36

endo/lysosomal system, where NPC2 was shown to accumulate. In light of the increased NPC2

mRNA levels in NPC1 fibroblasts it would seem more likely that NPC2 is upregulated by a

compensatory mechanism as a result of the endosomal cholesterol accumulation. Alternatively,

transcription of NPC2 might fail to be downregulated due to the relative sterol deficiency in the

ER. It is therefore possible that the NPC proteins are transcriptionally regulated in concert to

maintain cholesterol efflux from the endo/lysosomal compartments, but an increase in one is not

sufficient to compensate for the loss of the other.

2. Complementation of Niemann-Pick type C disease by Rab overexpression (II)

Due to the diverse cellular pathology and the abundance of metabolites that accumulate in NPC

disease, it seems safe to argue that NPC is to a large extent a disorder of perturbed intracellular

membrane trafficking. Whatever the functions of the NPC proteins are and the nature of the

primary substrates, there is a gross stagnation of endosomal motility in NPC disease (Ko et al.,

2001; Zhang et al., 2001a). As of yet it has been difficult to pinpoint the exact nature of the

cellular lesion. It seems likely that the NPC2 protein is a bona fide cholesterol transporter,

arguing that in NPC2 disease cholesterol accumulation causes a malfunction in the endosomal

compartments. The case for NPC1 is less clear-cut. NPC1 has been proposed to function as a

transmembrane pump or transporter of yet unidentified substrates (Davies et al., 2000a), or as a

cholesterol transporter in tandem with NPC2 (Infante et al., 2008b).

Inactivation of the NPC1 proteins leads to the stagnation of late endosomal tubulovesicular

trafficking (Ko et al., 2001; Zhang et al., 2001a). This phenotype might be a result of impaired

function in select Rab GTPases, possibly through altered biophysical properties of the

membranes. Global inactivation of Rab GTPases with Rab-GDI causes retarded endosomal

cholesterol clearance in cultured human skin fibroblasts loaded with LDL (Höltta-Vuori et al.,

2000). Rab inhibition also disrupts complementation of NPC1 fibroblasts with overexpressed

NPC1 protein, indicating that this process is dependent on Rab proteins (Höltta-Vuori et al.,

2000). It was subsequently shown that overexpression of the late endosomal Rabs Rab7 and Rab9

was also able to complement the NPC1 phenotype. Of the recycling Rabs, only Rab4 has

previously been shown to complement the NPC1 phenotype whereas Rab11 does not. We


37

therefore reasoned that overexpression of Rab proteins in NPC fibroblasts would be suitable for

identifying novel pathways of cholesterol transport from the endosomal system.

2.1 Use of filipin staining as a measure of cholesterol efflux from endosomal compartments

The fluorescent antibiotic filipin has been used extensively to visualize free cholesterol in fixed

cells. Previous complementation studies on NPC cells have used filipin staining as a measure of

cholesterol clearance from cells. In the study we used two NPC1 cell lines and one control cell

line. The GM3123 (I1061T/P237S) cell line from ATCC has been used extensively by other

laboratiories for complementation and was therefore chosen. The F92-116 NPC1 and F92-99

control cell line have been previously characterized in (I). It was immediately apparent by filipin

staining that the cholesterol accumulation phenotype varies heavily between the GM3123 and

F92-116 cell lines, with much more prominent perinuclear accumulation in the F92-116 line. The

heterogeneity of filipin staining in the GM3123 line was also striking. This is noteworthy since

previous complementation studies using single cell analysis have utilized this cell line.

We initially determined if filipin could be used as a quantitative measure of cholesterol clearance

from endosomes in NPC cells. Since the stochiometry of filipin binding to cholesterol has not

been determined we measured the correlation between filipin intensity and cellular free

cholesterol amounts. We found that there is good correlation between cellular free cholesterol

amounts and filipin intensity, establishing that a decrease in filipin intensity should be a good

measure of complementation. Indeed overexpression of Rab4-GFP and Rab7-GFP, which have

been previously shown to complement the NPC phenotype, as well as Rab8-GFP decreased

whole cell filipin staining. Whole cell filipin intensity can only be used as a measure of loss of

free cholesterol i.e. through efflux or esterification, not for redistribution of free cholesterol

within the cell. Also, a decrease in whole cell filipin staining might also reflect cholesterol efflux

from other compartments than endosomes, e.g. the plasma membrane. Whole cell filipin intensity

is therefore not an optimal measure of moblilization of free cholesterol from the endosomal

system. To more precisely measure mobilization of free cholesterol from the storage

endo/lysosomes we stained the cells with anti-Lamp1 antibodies and used this as a marker for the

storage organelles. After image acquisition, the area under the Lamp1 positive organelles was

MATTS LINDER

38

determined by image analysis and scored for filipin intensity, after subtraction of the PM filipin

fluorescence. By specifically scoring the endosomal cholesterol content the difference between

control F92-99 and NPC GM3123 fibroblasts becomes more apparent. Using this setup we

showed that overexpression of GFP-tagged Rab4, Rab7, and Rab8 specifically reduced the

cholesterol content of endosomes in NPC cells.

2.2 Rab8 restores sphingolipid trafficking in NPC fibroblasts Previous studies have established the restoration of glycosphingolipid transport in parallel with

filipin clearance during Rab overexpression (Choudhury et al., 2002; Choudhury et al., 2004). To

test whether Rab8 also restored glycosphingolipid trafficking in NPC cells we studied the

distribution of the cholera toxin subunit B (CTxB). CTxB binds to GM1 ganglioside and is

normally transported to the Golgi upon internalization but is retained in the endocytic

compartment in NPC cells (Choudhury et al., 2002; Sugimoto et al., 2001). Fluorescently labeled

CTxB localized to punctuate cytosolic structures in GM3123 fibroblasts but in Rab8-GFP

overexpressing cells CTxB colocalized with the Golgi marker GM130, indicating that Rab8

restored proper glycosphingolipid trafficking in NPC1 cells.

2.3 Rab8 is resistant to membrane retention during cholesterol loading Membrane cholesterol loading, as seen in NPC disease, disrupts Rab function by interfering with

Rab membrane extraction by Rab-GDI (Choudhury et al., 2004; Ganley and Pfeffer, 2006;

Lebrand et al., 2002). In NPC cells, and upon acLDL loading of mouse peritoneal macrophages,

we observed an upregulation of the Rab8 protein. To investigate whether this increase in Rab8

was caused by sequestration of Rab8 in cholesterol enriched membranes, we incubated cellular

membranes with Rab-GDI. Incubation with 2µM purified GDI solubilized roughly 70% of

membrane bound Rab8 independent of cholesterol load. In contrast, Rab7 solubility was reduced

in NPC cells in accordance with previous reports (Lebrand et al., 2002). Apparently the

membranes that Rab8 resides in do not accumulate cholesterol in NPC cells, and therefore the

membrane extractability remains unaltered. Interestingly, the overall membrane extractability of


39

Rab8 seemed to be greater for Rab8 compared to Rab7. Rab8 is only geranylgeranylated at a

single site as opposed to the double prenylation of most other Rab proteins e.g. Rab7, which

might confer resistance to membrane sequestration during cholesterol loading. Also, the affinity

for Rab-GDI depends on the length of the hypervariable domain of the Rab protein. The high

membrane extractability of Rab8 might therefore help Rab8 to stay functional during cholesterol

enrichment of membranes.

2.4 Rab8 siRNA induces a cholesterol accumulation phenotype So far only global inactivation of Rab proteins with Rab-GDI has been shown to induce a

cholesterol storage phenotype, whereas RNAi of specific Rabs has been ineffective. Ganley and

coworkers showed that specific knock-down of Rab9 did not induce endosomal cholesterol

accumulation (Ganley and Pfeffer, 2006). It is therefore possible that overexpression of certain

Rab proteins stimulates cholesterol transport in redundant or marginal pathways. To evaluate if

Rab8 function is necessary for cholesterol trafficking in normal cells we knocked down the Rab8

protein using RNA interference. Transfection of primary human fibroblasts with Rab8 shRNA

resulted in an increase in intracellular cholesterol, as assessed by filipin staining, that partly

localized to Lamp1 positive organelles. The cholesterol accumulation was also accompanied by a

cholesterol efflux defect to apoA-I. Also, Rab8 RNAi resulted in decreased cholesterol

esterification, indicating that the mobilized cholesterol was not targeted to the ER.

MATTS LINDER

40

3. Rab8 regulates cholesterol processing in primary human macrophages (III)

3.1 Rab8 is expressed in macrophages present in atherosclerotic lesions in vivo

To assess if Rab8 might be beneficial in foam cell reduction we opted to work on primary human

macrophages. Initially, we conducted immunohistochemical staining of human coronary artery

sections of atherosclerotic lesions. Using a polyclonal antibody against Rab8 we found Rab8 to

be expressed in cells surrounding the necrotic core of the lesion. To confirm that these cells were

macrophages we double stained the sections with anti-Rab8 and anti-CD68 antibodies. The Rab8

expressing cells showed clear co-localization with CD68 indicating that the cells were indeed

macrophages. The results indicate that Rab8 might have a physiological role in the setting of

macrophage foam cell formation, but whether this is directly related to cholesterol metabolism

can not be determined from these experiments.

3.2 Adenoviral overexpression of Rab8 in vitro increases ABCA1 dependent cholesterol efflux to apoA-I

We used an adenoviral expression system to overexpress Rab8 in primary human macrophages.

Infection of macrophages with Rab8 induced marked morphological changes, as has previously

been shown in other cell types. The macrophages displayed multiple cell protrusions positive for

Rab8. To assess if these cells also showed an increased capacity to withstand foam cell formation

we incubated control (GFP) or Rab8-myc infected macrophages in the presence of acLDL and

apoA-I. Cells infected with Rab8 accumulated less total cholesterol as compared to the control

cells. Interestingly this decrease in total cholesterol could be accounted for by the total decrease

in cholesteryl esters indicating that the cholesteryl ester pool is mainly affected. This could be

due rapid recycling of cholesterol from the endosomes to the cell surface, thereby decreasing the

cholesterol pool that is available for re-esterification in the ER. Alternatively, Rab8 might

facilitate the mobilization of cholesterol from lipid droplets. Since the amount of free cholesterol

remained constant during Rab8 overexpression it would seem that the mobilized cholesterol is

readily effluxed from the cells, suggesting involvement of the ABCA1 transporter. In accordance

with this we saw a striking, 2.5-fold increase in the amount of ABCA protein in the Rab8


41

overexpressing cells. This increase of ABCA1 was unlikely to be caused by an increase in

ABCA1 transcription through e.g. LXR activation since we saw no significant upregulation of

the ABCA1 mRNA in quantitative real-time PCR. It is therefore logical to assume that Rab8

induces the stabilization of ABCA1 on the cell surface, and thereby retards the degradation of the

protein. Stabilization might occur through increased exposition of ABCA1 to extracellular apoA-

I through enhanced trafficking of ABCA1 to the PM. It has previously been shown that

degradation of ABCA1 is regulated by internalization of the protein, and that apoA-I decreases

internalization by stabilizing ABCA1 at the cell surface (Wang et al., 2003). Internalization of

ABCA1 is dependent on the C-terminal PEST sequence (Chen et al., 2005). Given the

established effects of Rab8 on actin remodelling it is also feasible that ABCA1 stabilization is an

actin-mediated effect. ABCA1 has been shown to interact with members of the syntrophin family

of proteins ( 1, 1 and 2) (Buechler et al., 2002; Munehira et al., 2004; Okuhira et al., 2005).

Overexpression of 1- syntrophin increases the half-life of ABCA1 and facilitates cholesterol

efflux (Munehira et al., 2004). Co-expression of 1-syntrophin and ABCA1 induces the

formation of ABCA1 clusters at the PM, stabilizing the ABCA1 protein and increasing efflux

capacity (Okuhira et al., 2005). The syntrophins link ABCA1 to the actin cytoskeleton by

forming a complex with the scaffolding protein utrophin. Rab8 might therefore facilitate

cholesterol efflux by providing the actin scaffold for stabilization of the

ABCA1/syntrophin/utrophin complex.

3.3 Rab8 RNAi inhibits cholesterol efflux and regulates ABCA1 trafficking in primary human macrophages

To establish if Rab8 is essential for cholesterol transport in primary human macrophages we used

RNA silencing to knock down the Rab8 protein. We previously showed that Rab8 siRNA in

primary human macrophages causes retention of cholesterol within the cells as assessed by filipin

staining (II). In the primary human macrophages we opted to biochemically assess the cholesterol

amounts in Rab8 knock-down and control cells. Two days after electroporation of the siRNA the

macrophages were loaded with 50 g/ml acLDL. This resulted in a marked, 3-fold increase in

total cellular cholesterol and an increase of the cholesteryl ester fraction from 3.5% to 45%.

MATTS LINDER

42

After acLDL loading the cells were incubated with 10 g/ml apoA-I for 18h. In control cells, the

total cellular cholesterol levels decreased by 40% during the incubation period, whereas the Rab8

depleted cells failed to efflux significant amounts of cholesterol. To confirm that the substrate

pool for efflux was derived from the endocytosed lipoprotein particles we labeled acLDL with 3H cholesteryl oleate. Scintillation counting of the cells after loading confirmed equal uptake of

the label. However after incubation with apoA-I, the Rab8 depleted cells failed to efflux the 3H cholesterol, in accordance with the previous results.

After endocytosis the cholesteroyl esters of the LDL particle are hydrolysed by lysosomal acid

lipase (LAL), and it is conceivable that Rab8 siRNA interferes with this step and therefore

inhibits cholesterol mobilization from the cells. This is, however, an unlikely scenario since Rab8

shRNA causes cholesterol accumulation in primary human fibroblasts as assessed by filipin

staining, and filipin only stains free cholesterol. There are a number of possibilities how Rab8

siRNA might interfere with cholesterol efflux. Firstly, Rab8 depletion might inhibit the

trafficking of recycling endosomes to the cell surface. In accordance, filipin staining of primary

human macrophages showed cholesterol accumulation in both Lamp1-positive and Lamp1-

negative organelles. This accumulation is separate from that seen in NPC1 disease, which is

primarily lysosomal in nature, indicating that Rab8 regulates a down-stream event in cholesterol

trafficking. As discussed above, one possibility is that Rab8 controls the stabilization of ABCA1

on the PM. To assess the effect of Rab8 on the plasma membrane localization of ABCA1 we

used RNA silencing to knock down Rab8, followed by surface biotinylation of ABCA1. In Rab8

siRNA treated cells there was a significant, 30% decrease in the fraction of ABCA1 on the

plasma membrane, indicating that Rab8 regulates the transport of ABCA1 to the PM or

stabilization of the protein on the PM.

It is possible that Rab8 controls the trafficking or recycling of ABCA1 to the cell surface. Due to

a lack of good antibodies the precise cellular localization of ABCA1 has been hard to pinpoint,

although it is clear through surface biotinylation experiments that a large portion of the protein

resides at the plasma membrane. By overexpressing GFP-tagged ABCA1 it has also been

suggested that ABCA1 resides in endosomal compartments. It seems that a part of this pool of

ABCA1 is destined for lysosomal degradation, whereas a part is recycled back to the PM


43

(Neufeld et al., 2001). Yet, the specific recycling route has not been identified. Since Rab8

affects the cell surface distribution of ABCA1 we studied whether Rab8 siRNA would also have

an effect on the intracellular distribution of ABCA1-GFP. Previous studies on ABCA1-GFP have

mostly been carried out in HeLa cells. In a study by Neufeld et al, ABCA-GFP was localized to

the plasma membrane and Lamp2-positive vesicles, but not to TfR-positive vesicles (Neufeld et

al., 2001). We also found ABCA1-GFP to localize to the PM in HeLa cells, mainly to cellular

lamellipodia like protrusions and to intracellular tubules, both colocalizing with 1 integrin. Rab8

siRNA caused a decrease in cellular protrusions in the ABCA1 expressing cells and retention of

ABCA1 and 1 integrin in intracellular tubular compartments, indicating that the trafficking of

b1 integrin and ABCA1-GFP are interconnected. In accordance with previous results (Neufeld et

al., 2001) we did not find ABCA1 to colocalize with TfR. However, in Rab8 siRNA treated cells

ABCA1 accumulated perinuclearily, in a partially TfR positive structure. Taken together, the

results indicate that Rab8 also regulates the intracellular trafficking of ABCA1.

CONCLUSIONS AND FUTURE PROSPECTS

In this study (I) we have identified a mutation in the NPC1 protein that disrupts the intracellular

trafficking of the protein, leading to a severe infantile form of NPC disease. The study

demonstrates the importance of the correct localization of NPC1 within the cell. Also, the

regulation of NPC2 gene transcription seems to be dependent on proper NPC1 function

indicating that these proteins might be functionally linked. Of the various domains in the NPC1

protein, the SSD is of outstanding interest. There is high sequence homology within this domain

between SCAP, HMGR, NPC1L1 and NPC1 (Kuwabara and Labouesse, 2002). Therefore, a

crucial challenge will be to establish whether the SSD performs a similar sterol-sensing function

in NPC1 as has been described for SCAP and HMGR, and if so, to establish the sterols that

regulate NPC1 function.

The lessons learned from NPC1 might also have impact on our understanding of the regulation of

other SSD containing proteins, e.g. NPC1L1. Conversely, mutational and functional analysis of

the SSD within NPC1L1 might shed light on the function of this domain within NPC1. It has

already been established that sterols regulate the intracellular trafficking of NPC1L1. Upon sterol

depletion NPC1L1 translocates from the endocytic recycling compartment to the plasma

membrane where it facilitates cholesterol uptake. It would be interesting to study whether the

trafficking of NPC1L1 is dependent on an intact SSD. A recent study also showed that

cholesterol induces the internalization of NPC1L1 in intestinal cells, and that the cholesterol

lowering drug ezetimibe blocks this internalization (Ge et al., 2008). It would therefore be of

interest to determine if ezetimibe added to cells in vitro could block NPC1 mediated

tubulovesicular trafficking. If so, this could suggest that the function of NPC1 could be the

generation of vesicle budding sites as proposed by Yiannis Ioannou (Ioannou, 2005). In this

model NPC1 would function by flipping membrane lipids such as fatty acids or sphingosine,

thereby facilitating an outward bending membrane curvature. However, any theory on the

function of NPC1 should also take in to account the cholesterol binding properties of the protein.

In analogy to NPC1L1 internalization, cholesterol might also serve as an activating trigger for

NPC1 function. A regulatory role for cholestrerol binding could explain why an NPC1 N-

terminal mutant with lack of 25-hydroxycholesterol binding and severely attenuated cholesterol


45

binding had no apparent cellular phenotype (Infante et al., 2008a). It is feasible that the residual

cholesterol binding capacity is sufficient to serve a regulatory function. The bulk cholesterol

transport would then be mediated by NPC2 to specific membrane domains created by NPC1.

NPC2 is capable of transferring cholesterol from the N-terminal domain of NPC1 to acceptor

membranes (Infante et al., 2008b). It is also possible that NPC2 could work in the opposite way,

transferring cholesterol from inner membranes to NPC1 at the limiting membrane. In this model

cholesterol transfer from inner membranes could be facilitated by LBPA/BMP, which has been

shown to increase the sterol transfer capacity of NPC2 when present in the donor membrane

(Cheruku et al., 2006).

To date, most studies aimed at elucidating the function of NPC1 have focused on transient

transfection to revert the accumulation phenotype. An alternative approach could be the acute

knock-down of NPC1 or NPC2, to assess which metabolite(s) accumulate first. This might help

to resolve the long standing hen-and-egg issue that has puzzled NPC researchers, i.e. which

comes first, cholesterol or sphingolipid accumulation. However, such a study should also take in

to account the sterol status of the cell, as well as the inherent difference in the sensitivities

between assays for cholesterol and sphingolipid accumulation.

Our studies (II, III) are the first to identify a specific Rab-GTPase localized outside the

endosomal system that affects the recycling of cholesterol from the endosomes to the PM. We

have also shown that Rab8 is involved in the cellular trafficking of ABCA1. It is clear that the

processes governing ABCA1-mediated cholesterol efflux to apoA-I are highly complex,

involving intracellular signaling cascades and cytoskeleton reorganization. Given the role of

Rab8 in inducing cell polarity and actin as well as microtubule reorganization, a likely scenario is

that Rab8 promotes cholesterol efflux by creating platforms for cholesterol efflux at the leading

edge of the cell. Although the contribution of endocytosed ABCA1/apoA-1 to cellular cholesterol

efflux remains to be resolved, it is quite clear that ABCA1 is regulated post-transcriptionally

through endocytosis and recycling of the protein. According to our results Rab8 has a role in this

process, but the precise mechanism of Rab8 function remains to be established. We have shown

that knock-down of Rab8 causes the retention of ABCA1 within the cell in 1 integrin and TfR

receptor positive structures. It remains to be established whether ABCA1 containing vesicles

MATTS LINDER

46

utilize Rab8 directly, e.g. for recruitment of myosin motors, or if the effect is secondary e.g.

through altered actin or microtubule organization.

Our results also suggest that Rab8 recycles cholesterol from the endosomal system back to the

plasma membrane. The next challenge will therefore be to identify the precise recycling

pathway(s) regulated by Rab8 other Rab proteins involved in this process. The strategy adopted

in this study i.e. transfection of select Rab proteins and single cell assessment of filipin intensity

is quite labor intensive. In order to identify the Rab proteins involved in intracellular cholesterol

trafficking there is therefore a need for high throughput approaches, for instance, utilization of

siRNA libraries with automated analysis. Alternatively, a Rab-GAP library such as the one used

to identify Rab-GTPases involved in primary cilium formation could be used (Yoshimura et al.,

2007). The challenge of these approaches is, however, to find a clear read-out for cholesterol

trafficking suitable for automated analysis, preferably in living cells. The filipin staining process

is not suitable for high throughput applications. To this end one might utilize fluorescent

cholesterol analogs to measure changes in uptake, intracellular distribution and efflux. One

candidate probe would be BODIPY-cholesterol which has been shown to behave similarly to

cholesterol in normal and storage-disease cells (Höltta-Vuori et al., 2008).

Finally, it would be interesting to carry out in vivo studies in mice. The Rab8-/- mice described

by Sato and colleagues could be used for such studies, for instance to assess the serum

lipoprotein particle composition during low and high cholesterol diet (Sato et al., 2007).

Alternatively, generation of transgenic mice between Rab8-/- and LDLr-/- (or apoE-/-) mice

might be used to assess the effect of Rab8 deficiency on atherosclerotic plaque development.

Also, bone marrow transplants from Rab8 KO mice to LDLr -/- mice might be used to directly

assess the contribution of Rab8 in foam cell development.

Macrophage RCT measurements in vivo could also be carried out by adenoviral infection of

macrophages with Rab8, and metabolic labeling with [3H]-cholesterol in vitro, followed by

intraperitoneal injection of infected macrophages. RCT could then be measured by the

appearance of the [3H]-tracer in the plasma, liver and feces (Tanigawa et al., 2007). Together


47

these approaches should determine the overall contribution of Rab8 in macrophage RCT and

atherosclerotic lesion development.

ACKNOWLEDGEMENTS

I would like to take this opportunity to thank the numerous people that have contributed to the

success of this work.

Special thanks go out to my supervisor, Professor Elina Ikonen, for her optimism and

encouragement.

This work would, of course, not have been possible without the generous help of my colleagues

within and outside the laboratory. I would like to thank all the members of the Ikonen Lab for

their friendship and support. Anna Uro is warmly thanked for her help. Likewise, I would like to

thank Titta Blom, Maarit Hölttä-Vuori, Vilja Pietiäinen, Aino-Liisa Mutka and Riikka-Liisa

Uronen for their contributions. I also wish to thank Seija Puomilahti, Liisa Arala and Birgitta

Rantala for their much needed assistance. Additionally, I would like to thank Docent Johan

Peränen for so generously sharing constructs and antibodies. Professor Ismo Virtanen, Professor

Christian Ehnholm, Adjunct Professor Vesa Olkkonen and Adjunct Professor Matti Jauhiainen

have also been very generous in sharing materials and know-how.

I would like to thank the former and current heads of the departments at which the work has been

conducted. Professors Christian Ehnholm and Leena Palotie at the National Institute for Health

and Welfare (formerly National Public Health Institute); Professor Mart Saarma, director of the

Institute of Biotechnology, University of Helsinki. Also, Professor Ismo Virtanen and Professor

Esa Korpi of the Institute of Biomedicine, University of Helsinki, are acknowledged for

providing excellent facilities and working environments.

I would also like to thank the members of my thesis committee group, Docent Ismo Ulmanen and

Docent Mikko Frilander, for their advice.

Docent Jussi Jäntti and Professor Markku Savolainen are warmly thanked for reviewing the

thesis.

49

I also wish to acknowledge the continuing support of my friends, with special thanks to Ron

Liebkind for helpful discussions.

I am very grateful to my family, especially my parents Birgitta and Ewert for their continuous

support, as well as my sister Nina for all her help.

Carina and my son Cedric are warmly thanked for their support and understanding.

Financial support from Finska Läkaresällskapet, the Finnish Foundation for Cardiovascular

Research, Oskar Öflunds Stiftelse, Medicinka understödsföreningen Liv och Hälsa and Helsinki

Biomedical Graduate School is gratefully acknowledged.

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