1. Cholesterol 24-Hydroxylase: An Enzyme Turnover in the Brain"SPEAKER P.RAMESH Ph.D (Animal Biochemistry)

2. Introduction Cholesterol 24-Hydroxylase is a member of Cytochrome-p450 family (~500 a.a) Synonyms: Cytochrome P450 46A1, CP46, CYP46 Genes: Cyp46a1 (Mouse), CYP46A1 (Humans) Catalysis: Conversion of Cholesterol into Oxysterols Membranes of neurons in CNS (Brain & Spinal Cord) Exclusively present in the Brain & small amounts in testisand liver of the mouse 3. Cont. Responsible for majority of cholesterol turnover in theVetebrate Central Nervous System Expressed in neurons of hippocampal (learning), corticalneurons (memory) Disruption of the gene in mouse reduces both cholesterolturnover and synthesis in the brain No alteration in steady state levels of cholesterol in othertissues 4. Brain is a metabolically active tissueCont. Occupies 2% of body mass in humans and tissueconsumes 20% of resting oxygen Burns an estimated 4x1021 molecules of ATP/min ofthe day Mainly fueled by Glucose metabolism and on prolongedstarvation, it depends on ketonebodies Composed of lipids, including 20% of the bodys totalcholesterol content 5. Cholesterol Levels in Tissues Mouse Brain having cholesterol ~15mg/g tissueAdrenal gland ~19mg/g Lung ~6mg/g Cholesterol in the Brain exists in two pools One pool is largely containing ~70% (whitematter), conc. ~40 mg/g tissue Second pool is small having ~30% (gray matter),conc. ~8mg/g tissue 6. Cont.Origin of Brain Cholesterol Two sources: Exogenous (plasma lipoproteins)Endogenous (from Acetate) High vascularized tissues, (adrenal gland and liver) haveaccess to circulating lipoproteins (exogenous pathway) Brain exclusively depends on endogenous pathway Cholesterol is transported within the CNS in the form ofapo-E from Glial cells 7. Fig:1 Blood-Brain barrier prevents exchange with plasma Cholesterol 8. Cholesterol metabolism in the Brain Brain cholesterol located in myelin membranes ofoligodendrocytes & small amount in PM of neurons Cholesterol synthesis and accumulation as a function of Agedependent Synthesis are increased during active myelination after birth Cholesterol synthesis in the adult brain is larger than theaccumulation rate 9. Cont. Apo-E & membrane transporter of the ABC family areexpressed in CNS Apo-E containing lipoproteins from glial cells are bindto neuronal surface receptor & then uptake into it Cholesterol 24-hydroxylase catalyzes conversion ofcholesterol into 24s-hydroxycholesterol Interplay between glial & neuronal cells Regulates cholesterol homeostasis in brain 10. Cholesterol Turnover:Cont. It also catalyzes 24,25 and 24,27-dihydroxycholesterols in brain & cerebrospinal fluid of mice & other mammals Oxysterols is initially formed within the brain and then gain access to the circulation 11. Cont. In plasma, it associates with lipoproteins, which are clearedby the liver Mevalonate pathway (MP) synthesis cholesterol & nonsterolisoprenoids (Geranylgeraniol) responsible for learning Lacking 24-hydroxylase excrete cholesterol more slowly &tissue compensates by suppressing Mevalonate pathway Causes a severe deficiencies in spatial, associative, motorlearning & hippocampal long-term potentiation (LTP) 12. Metabolism of 24s-hydroxycholesterol:Cont. Having short half life & rapidly converted into Bile acids Infants have high levels in the circulation Cholesterol 7 & 7-hydroxycholesterols hydryoxlates 24s-hydroxycholesterol and 24,25 and 24,27 hydroxy-cholesterol in the liver In mouse CYP39 have a high 7hydroxylase activity 24s-hydroxycholesterol is a less efficient precursor for bile acids than 7-hydroxycholesterol Half of it is conjugated and eliminated in bile as such or as a conjugate of a27-OH cholesterol 13. Cont. Functions: Atherosclerosis, Apoptosis, Necrosis,Inflammation, Immune suppression, Development of Gall-stones Efficient suppressor of HMG-CoA reductase 14. Cont.24s-hydroxycholesterol as a marker: Marker for neurological and neurodegenerative diseases Brain-dead patients had a reduction in the conc. 50% In Multiple sclerosis appear to have increased levels in thecirculation Alzheimers disease (AD) had significantly reduced plasma levelsof the oxysterols Patients with AD were found to have increased levels 24s-OHcholesterol in cerebrospinalfluid and parallel with decreased levels in circulation 15. Cont.Cholesterol 27-hydroxylase (CYP27): It is present in all most all cells in the body Catalysis conversion of cholesterol into 27-OH cholesterol 27-OH cholesterol is further converted into 7-hydroxy-3-oxo-4-cholestenoic acid by Cholesterol71-hydroxylase (CYP71) in glial cells AD patients had increased levels of 27-OH cholesterol inbrain Alternatively for the high levels may be a reducedmetabolism 16. Alzheimers Disease (AD): It is neurological disease, characterized by death of neurons& even greater loss of synapses in the brain Neurons death seems to be deposition of -amyloid proteins Neurons with reduced arborization have less surface area &corresponds less plasma membrane Cholesterol catabolized through 2 main routes Can be esterified & stored within neurons as a cholesterol esters Oxidised at 24 or 27 to form 24-OHC & 27-OHC 17. Possible role of Oxysterols in AD:Cont. CYP46A1 are expressed in neurons & some astrocytes inthe normal brain CYP27A1 present in oligodendrocytes & absent in neurons In AD CYP46A1 shows prominent expression in astrocytes& around amyloid plaques Invitro studies shows that 24s-OHC & 27-OHC inhibits -amyloid proteins (A) Oxysterols also involved in signaling transduction pathway 18. Cont. Oxysterols have dual function: Both are cholesterol catabolites Ligand of the nuclear transcription factor LXR 24-OHC levels high in plasma of patients of AD Due to high levels its affect APP metabolism Processing of APP by & r-secretases produce A, takesplace in high cholesterol levels of lipid raft domains(Amyloid Protien Precursor) 19. Cont. Cholesterol esters increase A & inhibiting the LCATreduces A production Synthetic Oxysterol 22-OHC inhibits A production insome neuroblastoma cells Oxysterols inhibits APP processing in neurons through amechanism mediated by LXR Activation of LXR system is known to increase ABC(ATPase binding cassette) levels 20. Summary & Conclusion Cholesterol 24-hydroxylase is a highly conserved P450 that isexpressed in some, but not all, neurons of the brain Cholesterol24-hydroxylase converts cholesterol, into 24S-hydroxy-cholesterol, that diffuses from the brain and issubsequently metabolized by the liver Disruption of the mouse cholesterol 24-hydroxylase gene causesan 50% decrease in cholesterol turnover, which is compensatedfor by an equal decrease in the rate of de novo cholesterol synthesis 21. Cont. Cholesterol 24-hydroxylase knockout mice are deficient inspatial, associative, and motor learning, and have abnormal hippocampal LTP The defect in LTP can be reversed by geranylgeraniol, a poly-isoprenoid end product of the cholesterol biosynthetic pathway 22. Future Issues What role does cholesterol turnover play in the human brain? Are there naturally occurring mutations in the human cholesterol24-hydroxylase gene, and if so, what are their clinical consequences? What other pathways exist for cholesterol catabolism in the centralnervous system? Are these pathways present in the peripheral nervous system? What transport processes bring cholesterol to the enzyme in theendoplasmic reticulum membrane? 23. Fig: 24. Fig: Synthesis of A)24-hydroxy-cholesterol &B) cholesterol 25. Fig: Interaction between Astrocytes (glial cells) and Neuronal cells in Cholesterol Homeostatsis in the Brain 26. Fig: Flux of 24s-hydroxycholesterol from the Brain