Sequestration of free cholesterol in cell membranes by prions correlates with cytoplasmic phospholipase A2 activation

<p>Abstract</p> <p>Background</p> <p>The transmissible spongiform encephalopathies (TSEs), otherwise known as the prion diseases, occur following the conversion of the normal cellular prion protein (PrP^C) to an alternatively folded isoform (PrP^Sc). The accumulation of PrP^Scwithin the brain leads to neurodegeneration through an unidentified mechanism. 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.</p> <p>Results</p> <p>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 PrP^Sc. 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 PrP^Sc, but not by PrP^C. 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 PrP^Scin ScGT1 cells. Furthermore, prion infection greatly increased both the phosphorylation of cPLA₂and prostaglandin E₂production.</p> <p>Conclusion</p> <p>Prion infection, or the addition of PrP^Sc, increased the free cholesterol content of cells, a process that could not be replicated by the stimulation of cholesterol synthesis. The presence of PrP^Scincreased solubilisation of free cholesterol in cell membranes and affected their function. It increased activation of the PLA₂pathway, previously implicated in PrP^Scformation and in PrP^Sc-mediated neurotoxicity. These observations suggest that the neuropathogenesis of prion diseases results from PrP^Scaltering cholesterol-sensitive processes. Furthermore, they raise the possibility that disturbances in membrane cholesterol are major triggering events in neurodegenerative diseases.</p

Transient visual responses reset the phase of low-frequency oscillations in the skeletomotor periphery.

We recorded muscle activity from an upper limb muscle while human subjects reached towards peripheral targets. We tested the hypothesis that the transient visual response sweeps not only through the central nervous system, but also through the peripheral nervous system. Like the transient visual response in the central nervous system, stimulus-locked muscle responses (\u3c 100 ms) were sensitive to stimulus contrast, and were temporally and spatially dissociable from voluntary orienting activity. Also, the arrival of visual responses reduced the variability of muscle activity by resetting the phase of ongoing low-frequency oscillations. This latter finding critically extends the emerging evidence that the feedforward visual sweep reduces neural variability via phase resetting. We conclude that, when sensory information is relevant to a particular effector, detailed information about the sensorimotor transformation, even from the earliest stages, is found in the peripheral nervous system

Specificity of cholesterol and analogs to modulate BK channels points to direct sterol–channel protein interactions

The activity (Po) of large-conductance voltage/Ca2+-gated K+ (BK) channels is blunted by cholesterol levels within the range found in natural membranes. We probed BK channel–forming α (cbv1) subunits in phospholipid bilayers with cholesterol and related monohydroxysterols and performed computational dynamics to pinpoint the structural requirements for monohydroxysterols to reduce BK Po and obtain insights into cholesterol’s mechanism of action. Cholesterol, cholestanol, and coprostanol reduced Po by shortening mean open and lengthening mean closed times, whereas epicholesterol, epicholestanol, epicoprostanol, and cholesterol trisnorcholenic acid were ineffective. Thus, channel inhibition by monohydroxysterols requires the β configuration of the C3 hydroxyl and is favored by the hydrophobic nature of the side chain, while having lax requirements on the sterol A/B ring fusion. Destabilization of BK channel open state(s) has been previously interpreted as reflecting increased bilayer lateral stress by cholesterol. Lateral stress is controlled by the sterol molecular area and lipid monolayer lateral tension, the latter being related to the sterol ability to adopt a planar conformation in lipid media. However, we found that the differential efficacies of monohydroxysterols to reduce Po (cholesterol≥coprostanol≥cholestanol>>>epicholesterol) did not follow molecular area rank (coprostanol>>epicholesterol>cholesterol>cholestanol). In addition, computationally predicted energies for cholesterol (effective BK inhibitor) and epicholesterol (ineffective) to adopt a planar conformation were similar. Finally, cholesterol and coprostanol reduced Po, yet these sterols have opposite effects on tight lipid packing and, likely, on lateral stress. Collectively, these findings suggest that an increase in bilayer lateral stress is unlikely to underlie the differential ability of cholesterol and related steroids to inhibit BK channels. Remarkably, ent-cholesterol (cholesterol mirror image) failed to reduce Po, indicating that cholesterol efficacy requires sterol stereospecific recognition by a protein surface. The BK channel phenotype resembled that of α homotetramers. Thus, we hypothesize that a cholesterol-recognizing protein surface resides at the BK α subunit itself

The influence of membrane cholesterol on GABAA currents in a acutely dissociated rat hippocampal neurones

Cholesterol has been shown to act as a modulator of many membrane proteins and this thesis extends these observations to the GABAA receptor. Since the neuroactive steroids are structurally related to cholesterol and are known to modulate the function of the GABAA receptor, it was hypothesised that membrane cholesterol might interact with the recognition site for neuroactive steroids on the GABAA receptor. Acutely dissociated rat hippocampal neurones were enriched with cholesterol by incubation with either liposomes containing cholesterol or methyl-β-cyclodextrin complexed with cholesterol. Omission of the cholesterol from these carrier systems resulted in depletion of the neuronal cholesterol. Enrichments to 235% of control and depletions to 54% of control could be achieved whilst maintaining neuronal viability. Electrophysiological responses of the GABAA receptor to GABA were observed with the whole-cell application of the patch clamp technique. Both depletion and enrichment of membrane cholesterol reduced the sensitivity of the GABAA receptor to GABA but only enrichment reduced the potency of the competitive antagonist bicuculline methochloride. The antagonistic effect of picrotoxinin was not affected by either enrichment or depletion of cholesterol. The effects of the GABAA potentiators propofol, flunitrazepam and pentobarbital sodium were enhanced by cholesterol enrichment and could also be enhanced by enrichment with epicholesterol. Cholesterol depletion did not affect these GABA modulators. The cholesterol dependence of the positive (pregnanolone, allopregnanolone and alphaxalone) and negative (pregnenolone sulphate) modulatory neuroactive steroids was quite distinct, the effect of all these steroids being reduced by cholesterol enrichment and enhanced by depletion. The results suggest that membrane cholesterol may act as an antagonist of the neuroactive steroids at their recognition sites. Epicholesterol seems to act as a partial agonist at this cholesterol site. Altogether, the evidence favours sites of action of the neuroactive steroids on the transmembrane segments of the GABAA receptor at the level of the outer leaflet of the membrane bilayer

Influence of membrane cholesterol on modulation of the GABA(A) receptor by neuroactive steroids and other potentiators

1. The influence of membrane cholesterol on some pharmacological properties of the GABA(A) receptor was investigated in acutely dissociated rat hippocampal neurones with whole cell patch clamp recording. The cholesterol levels were varied between 56% and 235% control using methyl-β-cyclodextrin as the cholesterol carrier. 2. Enrichment of neurones with cholesterol increased the effects of the non-steroidal GABA potentiators propofol, flunitrazepam and pentobarbitone. A similar result was obtained after pre-incubation of neurones with epicholesterol, the 3α-hydroxy isomer of cholesterol. 3. In contrast, the effects of the steroidal GABA potentiators pregnanolone and alfaxalone were reduced by cholesterol enrichment, but not by epicholesterol. Depletion of membrane cholesterol increased the potentiation of GABA by pregnanolone and alfaxalone but did not affect the non-steroidal potentiators. 4. The steroidal antagonist of GABA, pregnenolone sulphate, reduced the maximum response to GABA. This effect, also, was diminished in cholesterol-enriched neurones and enhanced in cholesterol-depleted neurones. 5. The effects of the cholesterol manipulations that were selective for the steroidal modulators of GABA are suggested to arise from direct interactions between membrane cholesterol and the GABA(A) receptor. The separate effects on the non-steroidal potentiators of GABA of cholesterol-enrichment or addition of epicholesterol to the neurones are suggested to be due to changes in membrane fluidity. 6. In view of the likely physiological modulation of GABA(A) receptors by endogenous neuroactive steroids and evidence of the in vivo lability of membrane cholesterol, the present observations may have physiological as well as pharmacological relevance