Do Prion-Induced Changes in Membrane Cholesterol Trigger Neurodegeneration?

The transmissible spongiform encephalopathies (TSEs) are associated with the conversion of a host-encoded cellular protein (PrP^C) to alternatively folded, disease-associated isoforms (PrP^Sc) [1]. These diseases are unique in that the only identified component of the infectious agent is a self-replicating protein (PrP^Sc); which explains why such diseases are commonly called prion diseases (protein-only infectious agent). The accumulation of PrP^Sc is closely associated with the main pathological features of prion diseases; loss of synapses, activation of glial cells, neuronal loss and the spongiform degeneration of the brain [2]. However, the cellular and molecular mechanisms leading to neurodegeneration in these diseases are imperfectly understood. In this review, we outline developments implicating prion-induced changes in the levels of membrane cholesterol as a trigger of abnormal cell signaling, which leads to neurodegeneration.

Recent studies have demonstrated that prion infection has a significant impact on the biochemistry of neuronal cell membranes; it significantly increased the amounts of free cholesterol, but reduced the amounts of esterified cholesterol [3]. In addition, a highly significant correlation between the amounts of PrP^Sc and the concentration of free cholesterol in cell membranes was observed. Crucially, the effects of prion infection were not replicated by stimulating cholesterol biosynthesis. Stimulation of cholesterol production in uninfected neurons had the opposite effect to prion infection; it increased cholesterol ester formation without altering the amounts of free cholesterol. Such observations indicate that prion infection increased the capacity of cell membranes to solubilize free cholesterol. Cellular cholesterol is found as either free cholesterol in membranes, or as cholesterol esters that are stored in cytoplasmic droplets. A dynamic equilibrium between the pools of free cholesterol and cholesterol esters is tightly controlled by acyl-coenzyme A:cholesterol acyltransferase (ACAT), an endoplasmic reticulum (ER)-resident enzyme that esterifies free cholesterol with long-chain fatty acids [4]. Therefore, the combination of increased free cholesterol and reduced cholesterol esters observed in prion-infected cells may result from inhibition of ACAT, or from the sequestration of free cholesterol in microenvironments that avoid ACAT.

The capacity of cell membranes to solubilize free cholesterol is partly determined by its fatty acid composition. The high incidence of saturated fatty acids attached to glycosylphosphatidylinositol (GPI)-anchored proteins allows tight molecular packing and increases the solubilization of free cholesterol [5]. Since both PrP^Cand PrP^Scare linked to membranes via a GPI anchor [6], the formation of PrP^Sc can have a direct effect on the composition of cell membranes. One property of PrP^Sc molecules that is not shared by PrP^C is the propensity to self-aggregate. As a consequence, the formation of PrP^Scoligomers results in the clustering of GPI anchors in high densities. We propose that the increased density of saturated fatty acids within PrP^Sc-containing microdomains encourages the solubilization of free cholesterol. This hypothesis is consistent with observations that the addition of PrP^Sc with intact GPI anchors to uninfected neurons increased free cholesterol within cell membranes, while the addition of PrP^Sc with a GPI anchor lacking acyl chains had no effect. Similarly, the addition of synthetic GPI analogues containing two saturated acyl chains significantly increased the free cholesterol content of cell membranes, while GPI analogues containing either a single saturated acyl chain, or none at all, had no effect [Bate & Williams, Unpublished Data].

So what are the possible consequences of increased free cholesterol in cell membranes? Although free cholesterol is a component of normal cell membranes, the amounts of free cholesterol are increased up to fivefold in specialized microdomains within the plasma membrane that are commonly called lipid rafts [7]. Moreover, the formation of some of these lipid rafts is sensitive to changes in membrane cholesterol concentrations. Since GPI-anchored proteins, including PrP^C and PrP^Sc, are targeted to lipid rafts [8], the formation of PrP^Sc may alter conventional raft structure and the PrP^C–protein interactions that occur within lipid rafts. For example, PrP^C has been reported to bind to caveolin-1 [9] and neural cell adhesion molecule [10], proteins that reside within lipid rafts. It is unclear if these protein–protein interactions are affected following the conversion of PrP^C to PrP^Sc.

Currently, some lipid rafts are thought to act as membrane platforms in which signaling complexes assemble [11]. Since the formation of some of these rafts is sensitive to fluctuations in membrane cholesterol concentrations, prion-induced changes in free cholesterol levels within cell membranes may have profound effects on cell signaling. The unregulated activation of phospholipase A₂ (PLA₂) is now recognized as a key event in neurodegenerative diseases [12], including prion diseases [13]. Not only did prion-infected cells contain four-times more activated cytoplasmic PLA₂ (cPLA₂) than uninfected cells, but the amounts of activated cPLA₂ correlated with both the amounts of free cholesterol and with the amounts of PrP^Sc[3]. Immunoprecipitation studies demonstrated that cPLA₂ colocalized with PrP^Sc-containing lipid rafts, suggesting that the capture of free cholesterol by PrP^Sc was required for cPLA₂ activation. A causal relationship between cPLA₂ and PrP^Sc is consistent with reports that PLA₂ activation in prion-infected neuronal cells was cholesterol-sensitive [14] and that inhibition of cholesterol esterification increased free cholesterol, increased activation of PLA₂ and precipitated the death of prion-infected cells [15]. The activation of PLA₂ is the first step in the production of eicosanoids, docosanoids and platelet-activating factors, and although such molecules have a role in the maintenance of neuronal function, increased production of these factors causes synapse damage, glial cell activation and neuronal death. Although our studies focused on the PLA₂ pathway, many other signaling molecules, including the Src family tyrosine kinases [16], adenylyl cyclase [17] and the trimeric G-proteins [18], are also found in cholesterol-sensitive lipid rafts. The effect of prion-induced changes in free cholesterol levels on the activity of these signaling pathways is not currently known.

Abnormal cell signaling may not be the only pathological consequence of increased cholesterol. In neuronal cells, free cholesterol is readily oxidized into cytotoxic oxysterols [19]. Thus, increased free cholesterol may enhance neuronal death under oxidative conditions. Changes in the cholesterol content of membranes may also affect the activity of the Hedgehog family of tissue-patterning factors that are covalently modified by cholesterol [20]. Increasing the free cholesterol content of membranes may also affect membrane fluidity, endocytosis and intracellular trafficking of proteins [21]. Another affect of the sequestration of free cholesterol into PrP^Sc-containing lipid rafts may be depletion of free cholesterol from other cellular pools where it helps to stabilize the packing of sphingolipids, gangliosides and raft-associated proteins in the membrane. Thus, the sequestration of free cholesterol by PrP^Schas the potential to affect the function of many lipid raft-associated proteins. The receptors for acetylcholine, glutamate and γ-aminobutyric acid are found within lipid rafts and are affected by cellular cholesterol levels [22]. Since free cholesterol is also required for the formation and function of synapses [23], the sequestration of cholesterol by PrP^Sc may affect synaptic transmission. This hypothesis is supported by experimental data showing that prion-infected cells contain reduced amounts of synaptic proteins [24] and that the loss of synapses is characteristic of prion diseases [25].

Disturbances in membrane cholesterol are observed in other neurodegenerative conditions, including Alzheimer‘s and Parkinson‘s diseases [21]. The accumulation of amyloid-β peptides in Alzheimer‘s disease, and α-synuclein in Parkinson‘s disease, within cholesterol-rich domains in the brain is thought to play a central role in neuropathogenesis [26,27]. The similarities in the pathogenesis of such disorders and prion diseases raise the possibility that disturbed cholesterol metabolism may also contribute towards neurodegeneration in these diseases.

Much of what is known about the role of cholesterol in cell membranes is surmised from the changes in cells following cholesterol depletion. In particular, the statins, drugs that inhibit cholesterol biosynthesis, are increasingly being used to treat neurodegenerative conditions, including Alzheimer‘s disease [28], Parkinson‘s disease [29] and multiple sclerosis [30]. However, considering the number of neuronal processes that require cholesterol, the use of statins can be viewed as a crude pharmacological tool. Moreover, it is now clear that lipid rafts exist as heterogeneous subsets, each with different compositions and different functions [7]. These observations raise the intriguing possibility that drugs can be designed to alter the function of specific lipid raft subsets. Major future goals must surely include the development of novel therapeutics that are able to selectively target specific lipid rafts or raft functions.

We propose that the clustering of GPI anchors in prion diseases increases the solubilization of free cholesterol in specific membrane microdomains. This reduces the cycling of free cholesterol through the ER and exposure to ACAT, resulting in a reduction in cholesterol-ester formation. The increased amounts of free cholesterol in specific domains increase PLA₂ activation, a key event in prion-induced neurotoxicity. We further propose that similar perturbations in membrane cholesterol levels and altered cell signaling, albeit caused by different agents, may be a common factor in neurodegenerative diseases. Novel compounds that selectively disrupt prion, amyloid-β or α-synuclein-induced raft formation and activity may constitute the next generation of therapeutics for neurodegenerative diseases.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.