Bile acids and the brain. Suggested pathogenetic mechanism in connection with formation of brain xanthomas in patients with CTX

Transcription

1 Bile acids and the brain. Suggested pathogenetic mechanism in connection with formation of brain xanthomas in patients with CTX Ingemar Björkhem, Marjan Shafaati, Ulla Andersson, Ute Panzenboeck, Magnus Hansson, Shoshana Shpitzen, Vardiella Meiner, Wolfgang Slatter, Eran Leitersdorf

2 CYP27A1 CYP27A1 CYP27A1 Namn Efternamn 17 juli

3 CYP27A1 CYP27A1 Namn Efternamn 17 juli

4 Cerebrotendinous Xanthomatosis (CTX) CTX is caused by mutations in the CYP27A1 gene. The clinical picture includes xanthomas of the brain, skin, and tendons. The xanthomas in the brain may cause neurological disturbances (ataxia) and early dementia Biochemically the patients have markedly reduced levels of 27- hydroxycholesterol in the circulation, normal or low cholesterol levels, increased levels of cholestanol, markedly increased levels of abnormal 25-hydroxylated C27-bile alcohols Namn Efternamn 17 juli

5 Lack of CYP27A1 activity in humans leads to cerebrotendinous xanthomatosis Namn Efternamn 17 juli

6 The sterol 27-hydroxylase is antiatherogenic Namn Efternamn 17 juli

7 Treatment of CTX with chenodeoxycholic acid is effective, even xanthomas in the brain may reduced in size This means that the lack of CYP27A1 mediated flux of 27-oxygenated metabolites of cholesterol can not be the key mechanism behind the formation of xanthomas A mouse model with a disrupted Cyp27 gene does not develop xanthomas. In contrast to patients with CTX, Cyp27-/- mice do not have increased levels of cholestanol Namn Efternamn 17 juli

8 OH T HO 14 C H HO 14 C Rat T/ 14 C = 1.25 T/ 14 C = 1.02 Healthy man T/ 14 C = 1.74 T/ 14 C = 1.25 CTX- patient T/ 14 C = 1.54 T/ 14 C = 0.35 Namn Efternamn 4 april Skrede, S., Björkhem, I., et al J.Clin.Invest. 75 (1985) S Namn Efternamn 17 juli

9 HO O OH OH O O OH HO H Bile Acids Cholestanol Namn Efternamn 417 april juli

10 Comparison CTX with Cyp27-/- mice TX-patients 19-50x x 17-24x x 5-9x yp27 -/- mice 4.4x 1.8x Namn Efternamn 17 juli

11 Inhibition of CYP7A1 by bile acids in the normal situation Feed-back inhibition Bile acids Namn Efternamn 17 juli

12 Metabolic consequences of a defective CYP27A1 enzyme Bile acids Namn Efternamn 17 juli

13 Metabolic consequences of a defective CYP27A1 enzyme Namn Efternamn 17 juli

14 Metabolic consequences of a defective CYP27A1 enzyme Namn Efternamn 17 juli

15 Cholesterol Cholestanol 7-alpha-OH-4-cholesten-3-one Namn Efternamn 17 juli

16 Blood brain barrier model experiments pbcecs on filter insert apical ( blood ) 7αOH-Cholestenone Cholesterol 7αOH-Cholesterol Cholestanol 27OH-Cholesterol 15 basolateral ( brain ) % Transfer of Steroid (Mean +/- SD; n=3) Time (h) Namn Efternamn 17 juli

17 Production of Cholestanol from 7α-hydroxy-4-cholesten-3-one 0,45 Cholestanol/Cholesterol 0,30 0,15 Neuroblastoma Astrocytoma Microglia 0, Time hour Namn Efternamn 17 juli

18 O OH O OH O O HO H Namn Efternamn 17 juli

19 27-hydroxylation O OH 27-hydroxylation O OH 27-hydroxylation O 27-hydroxylation O 27-hydroxylation HO H Namn Efternamn 17 juli

20 27-hydroxylation O OH 27-hydroxylation O OH 27-hydroxylation O 27-hydroxylation O 27-hydroxylation HO H Namn Efternamn 17 juli

21 Why is the accumulation of cholestanol accompanied by accumulation of cholesterol? Cholestanol is less effective as a HMG CoA reductase inhibitor than cholesterol. Dilution of the cholesterol pool with cholestanol may thus increase cholesterol synthesis. There may be some direct conversion of cholestanol into cholesterol, due to a relatively low substrate specificity of the lathosterol 5alpha reductase There may be an upregulation of cholesterol synthesis caused by 7 alpha hydroxy-4-cholesten-3-one Namn Efternamn 17 juli

22 Incubation of Astrocytes with 7-alpha-hydroxy-4- cholesten-3-one 0,35 0,3 Cholestanol/Cholesterol Lathosterol/Cholesterol 0,25 Ratio 0,2 0,15 0,1 0, ug 0,5 ug 1 ug 2 ug 4 ug 8 ug 7-alpha-hydroxy-4-cholesten-3-one Namn Efternamn 17 juli

23 Incubation of MDM with 7-alpha-hydroxy-4-cholesten-3-one. Effects on mrna levels of key genes husrebp1c hsrebp2 h-hmgcoar HuACAT Control 0,5ug/mL 1ug/mL 2ug/mL 0,1 7-alpha hydroxy-4-cholesten-3-one Namn Efternamn 17 juli

24 Conclusions Accumulation of cholestanol in patients with CTX is likely to be of critical importance for the formation of xanthomas Most probably this accumulation is a consequence of the marked accumulation of 7 alpha hydroxylated bile acid intermediates which are precursors for cholestanol Namn Efternamn 17 juli

25 One of the bile acid intermediates, 7 alpha hydroxy-4- cholesten-3-one passes the blood-brain barrier very efficiently and is converted into cholestanol in the brain It seems likely that also the accumulation of cholesterol in the brain xanthomas is secondary to the flux of 7 alpha hydroxy-4- cholesten-3-one into the brain Namn Efternamn 17 juli

26 SREBP regulation of cholesterol synthesis N C N Golgi S2P SCAP Mature SREBP S1P N C SCAP Insig Sterols N N ER Activates transcription Nucleus Namn Efternamn 17 juli

27 Sterol structure and effect on HMG CoA reductase in mouse Namn Efternamn 17 juli

28 Accessory systems that may be affected in the CTX patient Substrate transport Electron transport StarD5? Cholesterol Adrenodoxin? Adrenodoxin reductase? CYP27A1 e - Mitochondria Namn Efternamn 17 juli

29 [ 2 H 6 ]7α-hydroxy-4-cholesten-3-one m/z 388 Detector response [ 2 H 6 ]cholestanol m/z 466 [ 2 H 6 ]4-cholesten-3-one m/z 390 Time Namn Efternamn 17 juli

30 Testing the hypothesis that the increased accumulation of cholestanol in the brain of patients with CTX is due to a flux of 7alpha-hydroxy-4-cholesten-3-one into the brain Daily intraperitoneal injections of 7alpha-hydroxy-4-cholesten-3-one, 200 ug, in a wild type mice and a Cyp27-/- mice for 10 weeks % Cholestanol of total sterol fraction Liver Brain Cyp27 -/- mouse 1 11 Cyp27 +/+ mouse 1 2 Untreated Cyp27+/+ 1 1 Namn Efternamn 17 juli

31 Incubation of MDM with Cholestanol 25 Lathosterol x 100 Cholestanol 20 Ratio ug 2 ug 5 ug 10 ug 20 ug Cholestanol Namn Efternamn 17 juli

32 Production of 4-cholesten-3-one from 7α-hydroxy-4- cholesten-3-one 0,15 4-cholesten-3- one/cholesterol 0,10 0,05 Neuroblastoma Astrocytoma Microglia 0, Time hour Namn Efternamn 17 juli

33 Increase in plasma levels of intermediates in cholestanol synthesis in patients with CTX 19-50x x 17-24x x 5-9x Björkhem et al. Hepatology 7 (1987) Namn Efternamn 17 juli

34 CYP27A1 is a widely expressed mitochondrial enzyme It belongs to the cytochrome p450 enzyme family. CYP27A1 is expressed in most tissues. In the liver it has an important role for bile acid synthesis. Requires NADPH, O 2, adrenodoxin, and adrenodoxin reductase for enzymatic activity. Transfer of substrate into the mitochondria may be a limiting factor for enzymatic activity. Namn Efternamn 17 juli

35 0.08 Cholestanol:Cholesterol α-Hydroxy-4-cholesten-3-one Added to Medium (µg/ml) Namn Efternamn 17 juli

36 Incubation of Astrocytes with Cholestanol 0,600 0,500 latho x 100/Cholesterol Cholestanol/Cholesterol 0,400 Ratio 0,300 0,200 0,100 0, ug 20ug 40ug Cholestanol Namn Efternamn 17 juli