Question: Use this article and any other references of your choice to discuss th

Question

Question: Use this article and any other references of your choice to discuss the evidence of cholesterol asymmetry and its possible roles.
"Concentrate on the transverse asymmetry between membrane layers."

Article: Cholesterol assymetry in Synaptic plasma membrane.

Abstract

Lipids are essential for the structural and functional integrity

of membranes. Membrane lipids are not randomly distributed

but are localized in different domains. A common

characteristic of these membrane domains is their association

with cholesterol. Lipid rafts and caveolae are examples

of cholesterol enriched domains, which have attracted keen

interest. However, two other important cholesterol domains

are the exofacial and cytofacial leaflets of the plasma

membrane. The two leaflets that make up the bilayer differ

in their fluidity, electrical charge, lipid distribution, and active

sites of certain proteins. The synaptic plasma membrane

(SPM) cytofacial leaflet contains over 85% of the total SPM

cholesterol as compared with the exofacial leaflet. This

asymmetric distribution of cholesterol is not fixed or immobile

but can be modified by different conditions in vivo: (i)

chronic ethanol consumption; (ii) statins; (iii) aging; and (iv)

apoE isoform. Several potential candidates have been

proposed as mechanisms involved in regulation of SPM

cholesterol asymmetry: apoE, low-density lipoprotein

receptor, sterol carrier protein-2, fatty acid binding proteins,

polyunsaturated fatty acids, P-glycoprotein and caveolin-1.

This review examines cholesterol asymmetry in SPM,

potential mechanisms of regulation and impact on membrane

structure and function.

Keywords: aging, apolipoprotein E, asymmetry, caveolin,

cholesterol, lipid domains.

There is substantial interest in membrane lipid domains

across numerous areas of biology. The roles of lipid domains

in brain structure, function, and neurodegeneration are

certainly one of those areas as demonstrated by this special

issue. Rafts and caveolae are domains that have attracted

considerable attention. However, two other domains of the

membrane are actually the two leaflets of the bilayer as

pictured in Fig. 1. There are substantial differences in the

two leaflets including for example electrical charge, thickness,

fluidity, and lipid distribution (Schroeder 1985; Wood

et al. 2002). Lipids are asymmetrically distributed in the

membrane bilayer (Fig. 1). Phosphatidylcholine and sphingomyelin

are enriched in the brain exofacial or outer leaflet

of the synaptic plasma membrane. There is evidence that

sphingomyelin is not present in the cytofacial or inner leaflet

of synaptic plasma membrane (SPM) but that phosphatidylethanolamine

(PE), phosphatidylserine (PS) and phosphatidylinositol

are in abundance in the cytofacial leaflet. This

transbilayer or asymmetric distribution of phospholipids

contributes to the differences in the electrical charges of the

two leaflets. The exofacial leaflet is neutral or zwitterionic

and the cytofacial leaflet is more negatively charged because

of the enrichment of the anionic phospholipids, phosphatidylinositol

and PS. This difference in the electrical charge of

the two leaflets is associated with the accumulation of certain

cationic and anionic drugs in membranes (Sweet et al.

1987). Cationic drugs acted on the cytofacial leaflet and

anionic drugs affected the exofacial leaflet. Regulation of

phospholipid asymmetry is thought to involve the actions of

different protein translocases and transporters and that topic

has been recently reviewed (Bevers and Williamson 2010).

Cholesterol, a major lipid in membranes accounting for over

40 mol% of synaptic plasma membrane lipids (Wood et al.

1989a) is also asymmetrically distributed. The purpose of

this review is to discuss cholesterol asymmetry in brain

SPMs, its alteration under certain conditions, mechanisms

involved in its regulation and the role of cholesterol

asymmetry in membrane structure and function.

Cholesterol asymmetry in plasma membranes

The establishment of cholesterol asymmetry in biological

membranes was advanced by the earlier work of Schroeder

and colleagues using quenching of the fluorescent sterol

dehydroergosterol (DHE) by trinitrobenzene sulfonic acid. A

comprehensive review on DHE has been recently published

(McIntosh et al. 2008) and so a detailed discussion will not

be presented in the present review. Briefly, DHE is a natural

fluorescent sterol found in sponge and yeast and thus does

not have a bulky fluorophore attached to it as compared

for example with the commonly used cholesterol analog

22-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino-23,24-bisnor-

5-cholen-3-b-ol)-cholesterol. DHE is structurally and functionally

most similar to cholesterol as compared with other

cholesterol analogs. This fluorescent sterol is used in cell

culture, isolated tissue, real-time imaging in living cells and

administered in vivo. A caveat to using DHE is that the

commercial compound is chemically synthesized and it can

contain impurities such as oxidized sterols which can perturb

membrane structure and function necessitating steps to

remove such contaminants (McIntosh et al. 2008).

The mouse cytofacial leaflet of SPM contains substantially

more cholesterol as compared with the exofacial leaflet

(Wood et al. 1990; Igbavboa et al. 1996, 1997; Kirsch et al.

2003). The cytofacial leaflet contains approximately 85% of

total SPM cholesterol. That the cytofacial leaflet contains

more cholesterol than the exofacial leaflet was also observed

in fibroblasts (Incerpi et al. 1992), human erythrocytes

(Schroeder et al. 1991) and most recently in the plasma

membrane and the endocytic recycling compartment of a

Chinese hamster ovary cell line (Mondal et al. 2009). Those

findings indicate that cholesterol asymmetry is a property

shared by different cell types with the abundance of

cholesterol contained in the cytofacial leaflet. The greater

concentration of cholesterol in the cytofacial leaflet versus

the exofacial leaflet is associated with the fluidity of the two

leaflets. The SPM cytofacial leaflet is distinctly less fluid than

the exofacial leaflet (Wood et al. 2002). The large difference

in leaflet fluidity affects the ability of various molecules to

partition into membranes. For example, ethanol disorders the

exofacial leaflet but has little if any effect on the cytofacial

leaflet (Schroeder et al. 1988; Wood et al. 1989b; Bae et al.

2005).

Synaptic plasma membrane cholesterol asymmetry is not

static and it is altered by chronic ethanol consumption,

statins, aging and apolipoprotein E isoform. There was

approximately a twofold increase in cholesterol in the

exofacial leaflet of mice chronically administered ethanol

(Wood et al. 1990). The total amount of SPM cholesterol

(exofacial leaflet + cytofacial leaflet) was similar for the

ethanol and control groups. Not surprisingly, the fluidity of

the two leaflets in the ethanol group was altered. The

exofacial leaflet became less fluid and the cytofacial leaflet

became more fluid in SPM of the ethanol group. This

change in fluidity was consistent with the ethanol-induced

redistribution of cholesterol between the two leaflets.

Chronic administration of statins (simvastatin, lovastatin,

atorvastatin) altered cholesterol asymmetry in mouse SPM

(Burns et al. 2006). There was an increase in exofacial

leaflet cholesterol and a corresponding reduction in cytofacial

leaflet cholesterol. Those results are different from the

findings of an earlier study which showed that lovastatin

and pravastatin but not simvastatin reduced cholesterol in

the SPM exofacial leaflets of chronic drug-treated mice

(Kirsch et al. 2003).

Increasing age alters SPM cholesterol asymmetry (Igbavboa

et al. 1996). Mice 24–25 months of age had significantly

more cholesterol in the SPM exofacial leaflet (32% cholesterol)

as compared with mice 3–4 months of age (14%

cholesterol). Mice 14–15 months of age also had significantly

more cholesterol in the exofacial leaflet (24%) than the

younger age group. In young mice, the exofacial leaflet is

significantly more fluid than the cytofacial leaflet. This

asymmetry in fluidity was not observed in SPM of aged

mice, which may be due in part to the redistribution of

cholesterol between the two leaflets. However, other factors

must also contribute to the loss of differences in fluidity

between the two leaflets of aged mice.

Attenuation of cholesterol asymmetry has also been

observed in SPM of mice expressing human apolipoprotein

E4 (Hayashi et al. 2002). Both increasing age and the

apolipoprotein E4 allele are risk factors for Alzheimer’s

disease and changes in cholesterol asymmetry may contribute

to the pathophysiology associated with Alzheimer’s

disease. An active area of research has been on the

association between cholesterol abundance and the production

of the amyloid beta-protein (Ab) including the role

of lipid rafts and this topic is reviewed elsewhere in this

issue. A twofold increase or greater of cholesterol in the

exofacial leaflet observed in SPM of aged mice and mice

expressing human apoE4 could certainly impact on membrane

structure and function and contribute to Ab production.

What makes changes in cholesterol asymmetry

observed in aged mice or mice expressing human apoE4

so notable are that major alterations can occur in the

absence of total changes in SPM cholesterol abundance.

Regulation of membrane cholesterol asymmetry

The abundance of cholesterol in the exofacial leaflet is

strikingly less as compared with the cytofacial leaflet. An

explanation for cholesterol asymmetry has not been established.

There have been several potential candidates

proposed as mechanisms involved in regulation of SPM

cholesterol asymmetry as depicted in Fig. 2. Sphingomyelin

had been proposed earlier to be a factor in cholesterol

asymmetry (Slotte and Bierman 1988; Porn et al. 1991).

Hydrolysis of sphingomyelin in fibroblasts and Leydig tumor

cells caused movement of cholesterol from the cell surface to

the cell interior. Sphingomyelin accounts for approximately

2–4% of the total non-sterol SPM lipid and it is all contained

in the exofacial leaflet (Rao et al. 1993; Wood et al. 1993).

In erythrocytes, sphingomyelin is approximately 25% of total

non-sterol lipid and abundance in the exofacial leaflet was

between 82% and 100% (Roelofsen 1982). Erythrocyte

exofacial leaflet cholesterol is approximately 25% of total

membrane cholesterol (Schroeder et al. 1991). Increasing

sphingomyelin levels in the exofacial leaflet is associated

with increasing cholesterol content in that leaflet. Regulation

of cholesterol in the exofacial leaflet but not the cytofacial

leaflet may involve interaction of cholesterol and sphingomyelin

via binding, complex formation, or changes in

membrane structure such as fluidity and lipid packing. In

addition, sphingomyelin is a component of lipid rafts and the

influence of lipid rafts on cholesterol asymmetry and vice

versa are topics that have not been examined.

There is evidence that both apoE and one of its receptors,

the low-density lipoprotein receptor (LDLR) may contribute

to the maintenance of cholesterol asymmetry. SPM of mice

deficient in apoE had a twofold increase in exofacial leaflet

cholesterol as compared with wild type mice (Igbavboa et al.

1997). This large difference cannot be accounted for by

changes in the total amount of SPM cholesterol, which were

similar in SPM of both groups. It was observed in the same

study that mice deficient in the LDLR or deficient in both

apoE and LDLR also showed greater abundance of cholesterol

in the exofacial leaflet as compared with wild type mice.

ApoE is the major cholesterol transporter in brain, it is

primarily synthesized in astrocytes and it has been shown

that neurons receive some of their cholesterol from astrocytes

(Mauch et al. 2001). Cholesterol in neurons is unique in

contrast to phospholipids because it would appear that it is

not synthesized at the nerve terminal of the axon (Vance

et al. 1994). The nerve terminal including the SPM may

receive some astrocyte derived cholesterol, which is delivered

by apoE and taken up by LDLR and other family

members. This cholesterol may then be recycled to SPM. A

problem with this interpretation is that the SPM of the apoE

and LDLR deficient mice either singly or the doubleknockout

had levels of total SPM cholesterol which were

similar to wildtype mice. This observation does not support a

deficit in transporting cholesterol from astrocytes to the nerve

terminal.

Assuming that apoE and LDLR may contribute to the

maintenance of cholesterol asymmetry, the changes in the

null mice did not exceed 34% of cholesterol in the exofacial

leaflet. This finding would imply that additional factors are

involved in regulating cholesterol asymmetry. There are data

indicating that fatty acid composition may be contributors to

cholesterol asymmetry in plasma membranes. Plasma

membranes of L-cell fibroblasts which were fed serum

enriched in unsaturated fatty acids had approximately 70% of

cholesterol sequestered in the exofacial leaflet as compared

with 28% in the control exofacial leaflet (Sweet and

Schroeder 1988). Linking fatty acid composition with SPM

cholesterol asymmetry are data showing that SPM of apoEdeficient

mice was enriched in the highly unsaturated fatty

acid 22 : 6 (n–3) in both the sn-1 and sn-2 positions

particularly in diacyl-PE and PS (Igbavboa et al. 2002). PE

and PS are in abundance in the cytofacial leaflet and an

increase in the phospholipid molecular species containing

22 : 6 (n–3) may stimulate the transbilayer movement of

cholesterol directly or act on a putative protein that regulates

cholesterol asymmetry. For example, it was shown that

fibroblasts over-expressing live fatty acid binding protein had

more cholesterol in the exofacial leaflet as compared to

control cells (Woodford et al. 1993). Live fatty acid binding

protein is a cytosolic protein that binds both fatty acids and

cholesterol (Schroeder et al. 2008).

Two additional proteins that could play a role in

maintaining membrane cholesterol asymmetry are P-glycoprotein

(P-gp) and caveolin-1 (Garrigues et al. 2002; Igbavboa

et al. 2009). P-gp is a member of the ATP-binding

cassette transporter family of proteins having multiple

functions including multidrug resistance in certain types of

tumor cells (Schinkel 1997). P-gp is expressed in brain

(Spector 2010). It was reported that P-gp stimulated the

movement of cholesterol from the cytofacial leaflet to the

exofacial leaflet in vesicles prepared from DC-3F cells overexpressing

human P-gp using accessibility of cholesterol to

cholesterol oxidase to determine cholesterol distribution

(Garrigues et al. 2002). This translocation of cholesterol

was inhibited by a P-gp inhibitor. It also was concluded in

that study that the cytofacial leaflet contained more cholesterol

as compared with the exofacial leaflet, which is

consistent with findings in other cells types using an

entirely different method (DHE fluorescence) for determining

cholesterol asymmetry as discussed earlier in this

review. In that paper, it was proposed that P-gp might

possibly interact with caveolin-1 in increasing exofacial

leaflet cholesterol. There are data showing that P-gp

co-immunoprecipitates with caveolin-1 (Demeule et al.

2000). Caveolin-1 is a 22-kDa protein associated with

caveolae and this protein binds cholesterol and is thought to

be a key contributor to cholesterol homeostasis (Smart et al.

1994; Conrad et al. 1995; Murata et al. 1995; Uittenbogaard

and Smart 2000; Pol et al. 2001; Ito et al. 2002). We have

recently reported that perturbation of astrocytes by Ab1–42

induced movement of cholesterol and caveloin-1 from the

plasma membrane to the Golgi complex (Igbavboa et al.

2009). Effects of Ab1–42 on both cholesterol and caveolin-1

were inhibited by siRNA targeted to the caveolin-1 gene.

There was also a significant reduction of cholesterol and

caveolin in the Golgi complex of cells treated with only

siRNA. There is evidence that caveolin may recycle lipids

including cholesterol. One possibility is that cholesterol

cycles in and out of the cytofacial leaflet and that caveolin

may regulate cholesterol specifically in the cytofacial leaflet.

Excess cholesterol in the cytofacial leaflet may be transported

by caveolin or P-gp to the Golgi complex and other

organelles. Caveolin may have a transbilayer effect and

cycle cholesterol between the cytofacial and exofacial leaflet;

similar to proteins involved in maintaining phospholipid

asymmetry. A recent study found that caveolin-1 was

enriched in the cytofacial leaflet and it sequestered fatty

acids (Simard et al. 2010). As mentioned earlier (Sweet and

Schroeder 1988), treating cells with unsaturated fatty acids

altered cholesterol asymmetry and perhaps such changes

could involve caveolin-1 complexing with fatty acids.

An obvious conclusion regarding regulation of cholesterol

asymmetry is that a single mechanism does not appear to

account for the greater abundance of cholesterol in the

cytofacial leaflet as compared with the exofacial leaflet.

Instead, we hypothesize that multiple mechanisms are

involved which may include both proteins and lipids in

regulating the transbilayer distribution of cholesterol.

Cholesterol asymmetry and membrane function

It is well-established that cholesterol plays a major role in

both membrane structure and protein function (Yeagle 1989;

Levitan et al. 2010; Schroeder et al. 2010). How specific

changes in the distribution of cholesterol in the two leaflets

affect membrane function have not been extensively studied.

There is some evidence that plasma membrane functions

such as receptor-effector coupling, ion transporters, and

translocation of proteins across the plasma membrane may be

influenced by the transbilayer lipid environment including

cholesterol (Schroeder et al. 2001). Export of cholesterol out

of the cell to lipoprotein acceptors may be altered by changes

in cholesterol asymmetry (Mondal et al. 2009). It has been

reported that statin-induced redistribution of cholesterol was

associated with reduced Ab and b-C-terminal cleavage

product levels in contrast to changes in bulk cholesterol

levels in brain membranes (Burns et al. 2006). SPM of

chronic ethanol treated mice, which showed a doubling of

cholesterol in the exofacial leaflet, were resistant to perturbation

by ethanol indicative of neuronal tolerance. Changes

in cholesterol asymmetry could impact on the capacity of the

membrane to form domains such as lipid rafts and caveolae.

Lipid and protein composition of lipid rafts from mice

expressing human apoE4 differed from mice expressing

human apoE3 (Igbavboa et al. 2005) and as discussed earlier

apoE expressing mice had a greater percentage of SPM

cholesterol in the exofacial leaflet as compared with apoE3

mice (Hayashi et al. 2002). What is not evident is whether

changes in cholesterol asymmetry alters lipid rafts or in fact,

lipid rafts contribute to the transbilayer distribution of

cholesterol. Finally, a question, which has not been rigorously

addressed, is if changes in cholesterol asymmetry are

adaptive or conversely are such changes inimical to cell

membrane function. The argument could be made that the

changes observed in ethanol-treated mice may be adaptive,

that is, reducing partitioning of ethanol into the membrane

but changes in cholesterol asymmetry in SPM of aged mice

or mice expressing human apoE4 which were similar to that

of ethanol treated mice may not be adaptive. In the instance

of the ethanol-treated mice, it is reasonable to predict that

additional effects because of changes in cholesterol asymmetry

would be observed which might not be adaptive. It is

clear that much more research is needed to establish the

functional consequences of modifying the transbilayer distribution

of cholesterol in membranes.

Summary

Cholesterol is asymmetrically distributed in plasma membranes

including SPM. The cytofacial leaflet contains

approximately five- to sixfold more cholesterol than the

exofacial leaflet, which has both structural and functional

consequences. Cholesterol asymmetry is not static but is

altered by several different conditions both in vivo and

in vitro. Mechanisms regulating cholesterol asymmetry are

not well-understood but the available data lead to the

conclusion that multiple mechanisms may be involved. The

functional consequences of changes in SPM cholesterol

asymmetry include fluidity, alterations in lipid domains,

lateral and transbilayer diffusion, lipid packing, and protein

function. Of particular interest and the need for further

research is the relationship between cholesterol asymmetry

and the formation and function of lipid rafts and caveolae.

Acknowledgements

This work was supported in part by grants from the National

Institutes of Health AG-23524, AG-18357 and Department of

Veterans Affairs.

Explanation / Answer

It is necessary to clarify with what refers to the asymmetry of the distribution of cholesterol, the cell membrane is formed by a lipid bilayer and the top (or the one that faces outwards) is the one that contains a higher percentage of cholesterol . However, this can be altered by diseases or other pathological processes (which ends up altering the function of the membrane).
This same article cites the distribution of the phospholipids in the lipid bilayer so that on the inner side of the membrane we have a greater concentration of some while on the outer side of the membrane there are others that do not even exist on the inner side. This difference in concentration in phospholipids causes differences between cholesterol concentrations because this steroid does not interact in the same way with some phospholipids that with others. Moreover, this asymmetry in the distribution of phospholipids helps the membrane's physiology because on the outer side it is neutral while towards the internal side the higher percentage of anionic lipids causes it to have a slightly negative charge.
Since the functions of each organ differ, it has been found that these asymmetries are not the same in different tissues. A curious effect is that the content of cholesterol either on the outer or inner side of the membrane modifies the flexibility of the membrane, whereby erythrocytes and fibroblasts (which are different tissues than neuronal ones) require a lot of flexibility for the functions which they carry out so that here we have other evidence by which these tissues have a different pattern in their asymmetry.
Now regarding the pathologies that reinforce these theories. The consumption of alcohol alter the pattern of distribution of cholesterol, the chronic consumption of alcohol causes the cells to double the content of cholesterol in their membranes which makes them more rigid and do not have an adequate functioning. The use of statins and the engraftment also alter this pattern of asymmetry with which the fluidity of the membrane is also altered. A cell that does not need rigidity in its membrane does not capture nutrients adequately, as well as its physiology that is altered. Sclerosis in arteries is a consequence of this, so that the risk of suffering a cerebral vascular event is increased. Another thing in histological sections are "foam cells" which have an abundance of cholesterol and do not perform their function well. That is why the asymmetric distribution of cholesterol must be maintained in adequate percentages.

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