Identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in human cerebrospinal fluid and plasma.

Dihydroxyoxocholestenoic acids are intermediates in bile acid biosynthesis. Here, using liquid chromatography - mass spectrometry, we confirm the identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in cerebrospinal fluid (CSF) based on comparisons to authentic standards and of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic and 7α,x-dihydroxy-3-oxocholest-4-en-26-oic (where hydroxylation is likely on C-22 or C-23) based on exact mass measurement and multistage fragmentation. Surprisingly, patients suffering from the inborn error of metabolism cerebrotendinous xanthomatosis, where the enzyme CYP27A1, which normally introduces the (25 R)26-carboxylic acid group to the sterol side-chain, is defective still synthesise 7α,24-dihydroxy-3-oxocholest-4-en-26-oic acid and also both 25 R- and 25 S-epimers of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic acid. We speculate that the enzymes CYP46A1 and CYP3A4 may have C-26 carboxylase activity to generate these acids. In patients suffering from hereditary spastic paraplegia type 5 the CSF concentrations of the 7α,24- and 7α,25-dihydroxy acids are reduced, suggesting an involvement of CYP7B1 in their biosynthesis in brain.

Cholestenoic acids regulate motor neuron survival via liver X receptors.

Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3ß,7α-dihydroxycholest-5-en-26-oic acid (3ß,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3ß-hydroxy-7-oxocholest-5-en-26-oic acid (3ßH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3ß,7α-diHCA and 3ßH,7O-CA, 3ß-hydroxycholest-5-en-26-oic acid (3ß-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3ß-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3ß,7α-diHCA. Moreover, 3ß,7α-diHCA prevented the loss of motor neurons induced by 3ß-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death.