“Cholesterol, Alzheimer’s disease, prion disorders: a ménage a trios?” Pani et al, Curr Drug Targets (2010)

http://www.ncbi.nlm.nih.gov/pubmed/20450474

Aberrant folded proteins are hallmarks of amyloidogenic diseases. Examples are Alzheimer’s disease (AD) and prion-related disorders (PrD). These disorders, although clinically different, have the same underlying pathogenetic mechanism: an altered protein conformer with high beta-sheet structure content: the amyloid beta peptide (Abeta) in the case of AD, and the aberrant prion protein, PrPsc, in PrD. Although the molecular processes that cause these proteins to adopt non-native structures in vivo and become cytotoxic are still largely unknown, there is good reason to expect prion research to profit from advances in the understanding of AD, and vice versa. Growing evidence indicates that the various pathways of lipid/lipoprotein metabolism play a key role in AD and PrD pathophysiology. These findings clearly highlight the possible involvement of cholesterol in misfolded protein generation. In this review, we focus on recent studies which provide evidence that membrane domains, called lipid rafts, directly promote protein misfolding, and that this process takes place only if changes occur in the fine regulation of intracellular cholesterol. In addition, we discuss the implications of these results to introduce the concept that pharmacological interventions restoring cholesterol homeostasis could have potential preventive/therapeutic value against the progression of misfolding disorders. The aim of the review is to provide researchers with a general understanding of cholesterol’s involvement in protein folding/misfolding processes which maybe relevant for knowledge advancement regarding amyloidogenic proteins, and possible ways to prevent their pathological activity.

 

“Prions link Cholesterol to Neurodegeneration,” Science Daily (2008)

http://www.sciencedaily.com/releases/2008/02/080211195230.htm

ScienceDaily (Feb. 11, 2008) — Prion infection of neurons increases the free cholesterol content in cell membranes. A new study suggests that disturbances in membrane cholesterol may be the mechanism by which prions cause neurodegeneration and could point to a role for cholesterol in other neurodegenerative diseases.

Dr Clive Bate and colleagues from the Royal Veterinary College in the UK compared the amounts of protein and cholesterol in prion-infected neuronal cell lines and primary cortical neurons with uninfected controls. Protein levels were similar but the amount of total cholesterol (a mixture of free and esterified cholesterol) was significantly higher in the infected cell lines.

The cholesterol balance was also affected: the amount of free cholesterol increased but that of cholesterol esters reduced, suggesting that prion infection affects cholesterol regulation. The team attempted to reproduce the effects of prions on cholesterol levels, by stimulating cholesterol biosynthesis or by adding exogenous cholesterol. Both approaches resulted in increased amounts of cholesterol esters but not of free cholesterol.

The free cholesterol is thought to affect the function of the cell membranes and to lead to abnormal activation of phospholipase A2, an enzyme implicated in the depletion of neurons in prion and Alzheimer’s disease.

Studies have recently shown that the controlling cholesterol levels within the brain is critical in limiting the development of neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases, multiple sclerosis, and senile dementia. This study now gives far more specific insight into the kind of mechanisms at work. Dr Bate stated: “Our observations raise the possibility that disturbances in membrane cholesterol induced by prions are major triggering events in the neuropathogenesis of prion diseases”.

 

“Sequestration of free cholesterol in cell membranes by prions correlates with cytoplasmic phospholipase A2 activation,” Bate et al., BMC Biology (2008)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2270799/?tool=pubmed

BACKGROUND:

Since many neurodegenerative disorders including prion, Parkinson’s and Alzheimer’s diseases may be modified by cholesterol synthesis inhibitors, the effects of prion infection on the cholesterol balance within neuronal cells were examined.

RESULTS:

We report the novel observation that prion infection altered the membrane composition and significantly increased total cholesterol levels in two neuronal cell lines (ScGT1 and ScN2a cells). There was a significant correlation between the concentration of free cholesterol in ScGT1 cells and the amounts of PrPSc. This increase was entirely a result of increased amounts of free cholesterol, as prion infection reduced the amounts of cholesterol esters in cells. These effects were reproduced in primary cortical neurons by the addition of partially purified PrPSc, but not by PrPC. Crucially, the effects of prion infection were not a result of increased cholesterol synthesis. Stimulating cholesterol synthesis via the addition of mevalonate, or adding exogenous cholesterol, had the opposite effect to prion infection on the cholesterol balance. It did not affect the amounts of free cholesterol within neurons; rather, it significantly increased the amounts of cholesterol esters. Immunoprecipitation studies have shown that cytoplasmic phospholipase A2 (cPLA2) co-precipitated with PrPSc in ScGT1 cells. Furthermore, prion infection greatly increased both the phosphorylation of cPLA2 and prostaglandin E2 production.

CONCLUSION:

Prion infection, or the addition of PrPSc, increased the free cholesterol content of cells, a process that could not be replicated by the stimulation of cholesterol synthesis. The presence of PrPSc increased solubilisation of free cholesterol in cell membranes and affected their function. It increased activation of the PLA2 pathway, previously implicated in PrPSc formation and in PrPSc-mediated neurotoxicity. These observations suggest that the neuropathogenesis of prion diseases results from PrPSc altering cholesterol-sensitive processes. Furthermore, they raise the possibility that disturbances in membrane cholesterol are major triggering events in neurodegenerative diseases.

Increasing the free cholesterol content of membranes is thought to reduce membrane fluidity and subsequently affect the endocytosis and trafficking of proteins. Therefore, the formation of PrPSc may alter conventional lipid raft structure and the PrPC-protein interactions that occur within lipid rafts. For example, PrPC has been reported to bind to caveolin-1 [29] or N-CAM [30], proteins that reside within lipid rafts. It is unclear whether these protein-protein interactions are affected following the conversion of PrPC to PrPSc. The sequestration of free cholesterol into PrPSc-containing lipid rafts may deplete free cholesterol from other cellular pools where it helps to stabilise the packing of sphingolipids, gangliosides and raft-associated proteins in the membrane. This may affect the function of such proteins. For example, free cholesterol affects the formation and function of synapses [31]. Therefore, sequestration of cholesterol by PrPSc may affect synaptic transmission, a hypothesis supported by observations that ScGT1 cells contain altered amounts of synaptic proteins including synaptophysin [32] and that synapse damage is seen during the early stages of experimental prion diseases [6].

 

“Cholesterol and statins in Alzheimer’s disease: current controversies,” Fonseca et al., Exp Neurol (2010)

http://www.ncbi.nlm.nih.gov/pubmed/19782682

Alzheimer’s disease (AD) is the principal cause of dementia in older people, and accumulation of amyloid-beta (Abeta) peptide is a crucial event in AD pathogenesis. Despite opposite results found in literature, increased evidence posits that high cholesterol levels enhance the risk to develop AD. In fact, cholesterol metabolism and catabolism are affected in this neurodegenerative disorder. Since amyloid precursor protein (APP) processing and subsequent Abeta production are dependent on membrane cholesterol content and on levels of isoprenoid intermediates in the cholesterol biosynthesis pathway, changes in cholesterol might have different consequences on Abeta formation. These pieces of evidence support that inhibitors of cholesterol synthesis, like statins, could have a therapeutic role in AD. Many studies about the effect of statins use in AD show conflicting results; however, some authors explain it by the differences in administrated doses. Recent studies demonstrate that statins can efficiently decrease Abeta formation from APP and be neuroprotective against the peptide toxicity. Because of the high number of pleiotropic effects of statins, novel molecular mechanisms that account for the beneficial effect of these drugs on AD might be discovered in a near future.

 

“The protein Srebp2 drives cholesterol formation in prion-infected neuronal cells which may promote prion-dependent diseases,” InSciences organization (2009)

http://insciences.org/article.php?article_id=7602

Neuherberg, November 17, 2009. The regulating protein Srebp2 drives cholesterol formation, which prions need for their propagation, in prion-infected neuronal cells. With these findings, published in the current issue of the Journal of Biological Chemistry, scientists of Helmholtz Zentrum München and Technische Universität München anticipate new approaches in drug development to combat prion infection.

Prions are causing fatal and infectious diseases of the nervous system, such as the mad cow disease (BSE), scrapie in sheep or Creutzfeldt-Jakob disease in humans. Scientists of Helmholtz Zentrum München and Technische Universität München have now succeeded in elucidating another disease mechanism of prion diseases: The prion-infected cell changes its gene expression and produces increased quantities of cholesterol. Prions need this for their propagation.

Using microarrays developed in the lab of Dr. Johannes Beckers, Christian Bach and colleagues from Helmholtz Zentrum München and Technische Universität made a genome-wide analysis of gene activity in prion-infected and healthy cells. The researchers found over 100 genes which are differentially expressed in infected and healthy cells. This has serious consequences for the infected cells: “Several enzymes of cholesterol biosynthesis are affected“, explained Christian Bach, first author of the study. As a consequence, the cholesterol level rises in the infected cells.

The cause of this development is the increased activity of the regulating protein Srebp2. It switches on the genes that are involved in cholesterol biosynthesis and cellular uptake. To achieve this, Srebp2 binds to a special segment encoding the gene to be transcribed – the sterol regulatory element. This activates the gene, leading to the biosynthesis of the corresponding protein.

In every step of cholesterol biosynthesis Srebp2 switches on different genes, thus exactly controlling gene expression, i.e. the translation of gene information into the corresponding protein. If cholesterol concentration is elevated in a healthy cell, Srebp2 remains in its inactive form and does not bind to the sterol regulatory element. This control mechanism is obviously disturbed in the infected cells, causing increased cholesterol synthesis. “Remarkably, only neuronal cells react in this way – microglia cells exposed to prions do not increase their cholesterol production,” said Professor Hermann Schätzl of the Institute of Virology of Technische Universität München, who led the research together with Dr. Ina Vorberg. Further studies shall elucidate what role disturbed cholesterol regulation plays in neuronal cells for the development of prion diseases and shall thus point the way to new therapy approaches.

 

“Prion-Induced Activation of Cholesterogenic Gene Expression by a Sterol Regulatory Element Binding Protein (Srebp2) in Neuronal Cells,” Bach et al., Journal Biological Chemistry Vol 284, No. 45, pp 31260-31269 Nov 2009

http://www.jbc.org/content/284/45/31260.full

By performing microarray analysis on cultured neuronal cells infected with prion strain 22L, in the group of up-regulated genes we observed predominantly genes of the cholesterol pathway. Increased transcript levels of at least nine enzymes involved in cholesterol synthesis, including the gene for the rate-limiting hydroxymethylglutaryl-CoA reductase, were detected. Up-regulation of cholesterogenic genes was attributable to a prion-dependent increase in the amount and activity of the sterol regulatory element-binding protein Srebp2, resulting in elevated levels of total and free cellular cholesterol. The up-regulation of cholesterol biosynthesis appeared to be a characteristic response of neurons to prion challenge, as cholesterogenic transcripts were also elevated in persistently infected GT-1 cells and prion-exposed primary hippocampal neurons but not in microglial cells and primary astrocytes. These results convincingly demonstrate that prion propagation not only depends on the availability of cholesterol but that neuronal cells themselves respond to prions with specific up-regulation of cholesterol biosynthesis.

However, conflicting results exist as to whether prion infection leads to up-regulation (9, 11) or down-regulation (36) of cholesterol biosynthesis genes. Interestingly, up- or down-regulation appeared to be at least partially dependent on the time point of sampling during the infection process. In one study, the pre-clinical stage of prion infection had only marginal influence on cholesterogenic gene expression, whereas at terminal stages of disease, cholesterogenic transcripts decreased (36). In another study, pre-clinical animals infected with mouse-adapted ME7 scrapie prion strain displayed increased cholesterol biosynthesis gene expression in the central nervous system that decreased at terminal stages of the disease (11). Thus, cholesterol metabolism in the brain following prion infection appears to underlie dynamic changes that correlate with the disease state.

Brain cholesterol is almost exclusively synthesized locally, as the blood-brain barrier restricts import of plasma lipoproteins from peripheral circulation (58, 59). Cholesterol synthesis is mainly accomplished by astroglia, but neuronal cells also synthesize cholesterol at basal levels. Thus, the detected changes in cholesterol biosynthesis genes upon prion infection in vivo might reflect any of the following: (a) cell type-dependent differential expression; (b) disease progression-dependent differential expression; (c) neuronal cell loss-dependent differential expression; or (d) any combination thereof. Because microglial activation, mainly during clinical stages of disease, prominently influences transcript levels in mouse models of prion diseases (8, 10, 36, 56), it is possible that down-regulation of cholesterol biosynthesis during clinical prion disease reflects a glial response that masks neuronal up-regulation. This hypothesis is in agreement with our finding that exposure of microglial cells and primary astrocytes to prions in vitro either caused down-regulation of genes involved in cholesterol biosynthesis, e.g. Sc4mol and Srebf2, or left transcript levels relatively unaffected. Alternatively, neuronal cells in vivo might respond to prion infection dependent on the disease progression. Recent evidence shows that neuronal down-regulation of cholesterol biosynthesis can correlate with apoptosis (60). Neuronal loss is a hallmark of prion diseases, and several lines of evidence suggest that neurons infected with prions can undergo programmed cell death (61). Interestingly, recent studies on neuronal cultures demonstrated that de-regulation of intracellular cholesterol transport induced apoptotic cell death and was co-incident with decreased cholesterol biosynthesis transcripts. Notably, at earlier time points when no apoptosis was apparent, most cholesterol transcripts were increased, suggesting that damaged neurons might initially up-regulate cholesterol biosynthesis, potentially to compensate for cholesterol imbalances (60). Importantly, N2a cells can be persistently infected with prions and do not appear to undergo apoptosis, which could explain why N2a cells do not demonstrate decreased cholesterol biosynthesis transcripts upon prion infection. Similarly, primary hippocampal neurons exposed to 22L scrapie brain homogenate did not show decreased viability compared with primary neurons exposed to normal brain homogenate, at least not during the course of the experiment (data not shown). In summary, our results show that prions have the potential to alter the cholesterol homeostasis of cells in a cell type-specific manner.

 

“Monoacylated cellular prion protein modifies cell membranes, inhibits cell signalling, and reduces prion formation,” Bate et al., J Biol Chem (2011)

http://www.ncbi.nlm.nih.gov/pubmed/21212283

Prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into a disease related, protease-resistant isoform (PrP(Sc)). In these studies, a cell painting technique was used to introduce PrP(C) to prion-infected neuronal cell lines (ScGT1, ScN2a, or SMB cells). The addition of PrP(C) resulted in increased PrP(Sc) formation that was preceded by an increase in the cholesterol content of cell membranes and increased activation of cytoplasmic phospholipase A(2) (cPLA(2)). In contrast, although PrP(C) lacking one of the two acyl chains from its glycosylphosphatidylinositol (GPI) anchor (PrP(C)-G-lyso-PI) bound readily to cells, it did not alter the amount of cholesterol in cell membranes, was not found within detergent-resistant membranes (lipid rafts), and did not activate cPLA(2). It remained within cells for longer than PrP(C) with a conventional GPI anchor and was not converted to PrP(Sc). Moreover, the addition of high amounts of PrP(C)-G-lyso-PI displaced cPLA(2) from PrP(Sc)-containing lipid rafts, reduced the activation of cPLA(2), and reduced PrP(Sc) formation in all three cell lines. In addition, ScGT1 cells treated with PrP(C)-G-lyso-PI did not transmit infection following intracerebral injection to mice. We propose that that the chemical composition of the GPI anchor attached to PrP(C) modified the local membrane microenvironments that control cell signaling, the fate of PrP(C), and hence PrP(Sc) formation. In addition, our observations raise the possibility that pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases.

 

“Binding of prion proteins to lipid membranes, “ Critchley et al., Biochemical and Biophysical Research Communications 313 (2004) 559-567

http://www.tau.ac.il/lifesci/courses/molecular_biophysics/binding_of_prion_to_membrane.pdf

In conclusion, it is apparent that long-range ionic lipid-protein interactions play a crucial role in the association of PrP with lipid membranes and are likely to constitute the driving force for the initial binding.  However, once on the membrane surface short-range hydrophobic lipid-protein interactions take place.   These lipid-protein interactions can alter the protein conformation, which may result in a higher propensity for the association of PrP with other PrP molecules on the membrane surface.  Thus, the two-dimensional confinement of PrP molecules on a membrane surface, coupled to acidic conditions such as in endosomes, may provide a favourable environment for the conversion of PrP.