24s-Hydroxycholesterol : studies on regulatory mechanisms behind its formation in the brain and its potential use as a marker for neurodegeneration

Cholesterol 24-hydroxylase (CYP46A1) belongs to the cytochrome P450 superfamily and is responsible for conversion of cholesterol to the oxysterol24S- hydroxycholesterol (24S-OHC). This structural modification allows24S-OHC to traverse the blood brain barrier and this pathway is the majorone for elimination of cholesterol from the mammalian brain. CYP46A1 isalmost exclusively located to neurons in the brain and retina. Thepromoter region of CYP46A1 shows classical hallmarks of a gene withputative housekeeping function. Oxidative stress was the only factorinitially shown to cause signicant increase in CYP46A1 reporteractivity.The aim of this thesis was to obtain a more profound knowledge aboutregulation of CYP46A1 and the regulatory importance of its product. Inaddition the possibility was investigated that the levels of 24S-OHC incerebrospinal fluid may be used diagnostically.In paper I we tested the possibility that epigenetic factors areimportant for CYP46A1 expression. We could demonstrate both in vivo andin vitro that the histone deacetylase inhibitors Valproate andTrichostatin A induce the expression of CYP46A1, essentiallyreprogramming non-neuronal cells to express CYP46A1 to the same level asthat found in adult neurons.In paper II we investigated the inhibitory effect of the antifungal drugVoriconazole on CYP46A1 in mice and we hypothesised that inhibition ofCYP46A1 may have a role in the reported side effects in connection withVoriconazole (neurological and visual disturbances). A decrease inconcentration of 24S-OHC in the mouse brain and a reduction in thelathosterol:cholesterol ratio, an index of brain cholesterol synthesis,was demonstrated.In paper III we investigated the effect of omega-3 polyunsaturated fattyacids in Syrian Hamsters. Enrichment of diet with omega 3-fatty acidsresulted in increased CYP46A1 mRNA levels in the brain.In paper IV we investigated the consequences of an overexpression ofCYP46A1. A transgenic mouse model overexpressesing human CYP46A1 wasdeveloped and characterized. Significant expression of the human CYP46A1protein was found in brain, testis and eye and the brain in whichcontained more than 10-fold higher levels than the other organs.Circulating levels of 24S-OHC were increased by 4-6 folds and the fecalexcretion of this steroid in free form was increased more than 20 fold.In the brain of the transgenic mice the total amount of CYP46A1 proteinwas increased 2-4 fold and had similar cellular distribution to theendogenous enzyme. The level of 24S-OHC in the brain was about doublecompared to the controls. It is known that 24S-OHC is an efficient ligandfor the liver X receptor (LXR) in vitro. The overproduction of 24S-OHCdid not significantly activate any LXR target genes in the brain, whilein the liver some of these genes were affected but not in the directionexpected in connection with LXR activation.Based on in vitro studies a regulatory link between 24S-OHC and APOE hasbeen suggested and addition of 24S-OHC stimulates secretion of APOE fromcultured astrocytes. In paper V we investigated if there is a correlationbetween the concentration of APOE and 24S-OHC in cerebrospinal fluid inpatients. A significant correlation was found in patients withAlzheimer s disease (AD) and mild cognitive impairment (MCI), but not inthe control group. In paper VI we investigated the possibility that thelevel of 24S-OHC in cerebrospinal fluid may be an early biomarker for AD.We could show that 24S-OHC is as sensitive as the standard diagnosticbiomarkers for AD (tau-protein, phospho-tau, and beta amyloid).Interestingly 24S-OHC appeared to be a more sensitive marker for MCI thanthe standard biomarkers. According to current concepts the levels ofcholesterol in critical neuronal membranes is of importance for thebalance the between α- and β- secretase pathway. An increased consumptionof this cholesterol by increased activity of CYP46A1 would be expected tofavour the α- secretase pathway and reduce amyloid formation. Under invitro conditions 24S-OHC seems to have a direct inhibitory effect onamyloid formation. The mouse model with overexpressed CYP46A1 presented in paper IV will be ideal to test the hypothesis that an upregulation ofCYP46A1 is neuroprotective and reduces amyloid formation. As shown inpaper I histone deacetylase inhibitors are potential drugs to reach thisgoal. High intake of omega-3 fatty acids seems to have someneuroprotective effects in humans and the results shown in paper III isconsistent with the possibility that part of this may be mediated by aneffect on CYP46A1. Inhibition of CYP46A1 would be expected to have anegative effect on the function of the central nervous system andpossibly also retina. The results of paper II demonstrated that the drugVoriconazole, with known negative side effects on CNS and visualfunction, inhibits the flux in the mevalonate pathway in the brain. Inpart this may be the consequence of inhibition of CYP46A1. Results arepresented to indicate that the levels of 24S-OHC in cerebrospinal fluidmay be used as diagnostic tool in connection with neurodegeneration(paper V and VI)