tion-mass spectrometry (LC-ESI-MS) analysis. We hereby report the exact identity of 16 oxysterols and downstream metabolites, including cholestenoic acids, found in human CSF (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI68506DS1). The most abundant of these metabolites (19.48-0.40 ng/ml; Supplemental Figure 1) were 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7αH,3O-CA), 3β-hydroxycholest-5-en-26-oic acid (3β-HCA), and 2 newly identified metabolites in CSF, 3β,7α-diHCA and 3β,7β-dihydroxycholest-5-en-26-oic acid (3β,7β-diHCA). Precursors of these acids, including 26-HC and newly identified 7α,26-dihydroxycholesterol (7α,26-diHC; cholest-5-ene-3β,7α,26-triol) and 7α,26-dihydroxycholest-4-en-3-one (7α,26-diHCO), were also found, but at lower levels (0.15-0.03 ng/ml). Our results thus identified 4 novel oxysterol metabolites in human CSF that were downstream of 26-HC ( Figure 1A). 26-HC is metabolized via 7α,26-diHC and 7α,26-diHCO, or via 3β-HCA and 3β,7α-diHCA, to 7αH,3O-CA. While 26-HC can cross the blood-brain barrier (BBB) and enter the brain from the circulation (25), 7αH,3O-CA traverses the BBB and is exported from the brain (26). Very low levels of 24S-hydroxycholesterol (24S-HC; cholest-5-ene-3β,24S-diol), 25-hydroxycholesterol (25-HC; cholest-5-ene-3β,25-diol), and newly identified 7α,25-dihydroxycholesterol (7α,25-diHC; cholest-5-ene-3β,7α,25-triol) and 7α,25-dihydroxycholest-4-en-3-one (7α,25-diHCO) were also found in CSF (0.08-0.03 ng/ml).Reduced levels of 7α-hydroxylated cholestenoic acids in CSF and plasma/serum of human patients with SPG5. SPG5 presents with upper motor neuron signs and results from mutations in CYP7B1, encoding the oxysterol 7α-hydroxylase responsible for 7α-hydroxylation of side-chain oxidized sterols that is required for extrahepatic synthesis of 7αH,3O-CA and its precursor, 3β,7α-diHCA ( Figure 1A and ref. 18). In order to examine the pathogenic role of such mutations, we sought to identify alterations in oxysterol and cholestenoic acid profiles in CSF and plasma from these patients and then examine the biological activities of the altered metabolites. We first studied the CSF from 3 patients with SPG5

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C 27 and C 24 intermediates of the bile acid biosynthetic pathways with structures corresponding to 7␣-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ؎ 2.826 ng/ml, mean ؎ S.D., six subjects), 3␤-hydroxycholest-5-en-26-oic acid (

Liver X receptors (Lxrα and Lxrβ) are ligand-dependent nuclear receptors critical for ventral midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand selectively regulated the development of distinct midbrain neuronal populations. Whereas cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons, 24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class of highly selective and cell type-specific regulators of neurogenesis and neuronal survival. Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells, suggesting that Lxr ligands may thus contribute to the development of cell replacement and regenerative therapies for Parkinson's disease.

Control over progenitor proliferation and neurogenesis remains a key challenge for stem cell neurobiology and a prerequisite for successful stem cell replacement therapies for neurodegenerative diseases like Parkinson's disease (PD). Here, we examined the function of two nuclear receptors, liver X receptors (Lxralpha and beta) and their ligands, oxysterols, as regulators of cell division, ventral midbrain (VM) neurogenesis, and dopaminergic (DA) neuron development. Deletion of Lxrs reduced cell cycle progression and VM neurogenesis, resulting in decreased DA neurons at birth. Activation of Lxrs with oxysterol ligands increased the number of DA neurons in mouse embryonic stem cells (ESCs) and in wild-type but not Lxralphabeta(-/-) VM progenitor cultures. Likewise, oxysterol treatment of human ESCs (hESCs) during DA differentiation increased neurogenesis and the number of mature DA neurons, while reducing proliferating progenitors. Thus, Lxr ligands may improve current hESC replacement strategies for PD by selectively augmenting the generation of DA neurons.

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