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Brain cholesterol biosynthesis, a separate and distinct process from whole-body cholesterol homeostasis, starts during embryonic development. To gain a better understanding of the neuronal and glial contributions to the brain cholesterol pool, we studied this process in control, Dhcr7 -/- cell cultures. Our LC-MS/MS method allowed us to measure several different sterol intermediates and cholesterol during neuronal differentiation. We found that developing cortical neurons rely on endogenous cholesterol synthesis and utilize ApoE-complexed cholesterol and sterol precursors from their surroundings. Both developing neurons and astrocytes release cholesterol into their local environment. Our studies also uncovered that developing neurons produced significantly higher amounts of cholesterol per cell than the astrocytes. Finally, we established that both neurons and astroglia preferentially use the Bloch sterol biosynthesis pathway, where desmosterol is the immediate precursor to cholesterol. Overall, our studies suggest that endogenous sterol synthesis in developing neurons is a critical and complexly regulated homeostatic process during brain development.Dhcr24 -/- cell cultures. Our LC-MS/MS method allowed us to measure several different sterol intermediates and cholesterol during neuronal differentiation. We found that developing cortical neurons rely on endogenous cholesterol synthesis and utilize ApoE-complexed cholesterol and sterol precursors from their surroundings. Both developing neurons and astrocytes release cholesterol into their local environment. Our studies also uncovered that developing neurons produced significantly higher amounts of cholesterol per cell than the astrocytes. Finally, we established that both neurons and astroglia preferentially use the Bloch sterol biosynthesis pathway, where desmosterol is the immediate precursor to cholesterol. Overall, our studies suggest that endogenous sterol synthesis in developing neurons is a critical and complexly regulated homeostatic process during brain development.

Well-established cell culture models were combined with new analytical methods to assess the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7-DHC and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically active substances, and the effect of compounds on the cholesterol pathway was determined. Of some 727 compounds, over 30 compounds decreased 7-DHC in Dhcr7-deficient Neuro2a cells. The examination of chemical structures of active molecules in the screen grouped the compounds into distinct categories. In addition to statins, our screen found that SERMs, antifungals, and several antipsychotic medications reduced levels of 7-DHC. The activities of selected compounds were verified in human fibroblasts derived from Smith-Lemli-Opitz syndrome (SLOS) patients and linked to specific transformations in the cholesterol biosynthesis pathway.

During the development of the sympathetic nervous system, the p75 neurotrophin receptor (p75NTR) has a dual function: promoting survival together with TrkA in response to NGF, but inducing cell death upon binding pro or mature brain-derived neurotrophic factor (BDNF). Apoptotic signaling through p75NTR requires activation of the stress kinase, JNK. However, the receptor also undergoes regulated proteolysis, first by a metalloprotease, and then by gamma-secretase, in response to pro-apoptotic ligands and this is necessary for receptor mediated neuronal death (Kenchappa, R. S., Zampieri, N., Chao, M. V., Barker, P. A., Teng, H. K., Hempstead, B. L., and Carter, B. D. (2006) Neuron 50, 219-232). Hence, the relationship between JNK activation and receptor proteolysis remains to be defined. Here, we report that JNK3 activation is necessary for p75NTR cleavage; however, following release of the intracellular domain, there is a secondary activation of JNK3 that is cleavage dependent. Receptor proteolysis and apoptosis were prevented in sympathetic neurons from jnk3(-/-) mice, while activation of JNK by ectopic expression of MEKK1 induced p75NTR cleavage and cell death. Proteolysis of the receptor was not detected until 6 h after BDNF treatment, suggesting that JNK3 promotes cleavage through a transcriptional mechanism. In support of this hypothesis, BDNF up-regulated tumor necrosis factor-alpha-converting enzyme (TACE)/ADAM17 mRNA and protein in wild-type, but not jnk3(-/-) sympathetic neurons. Down-regulation of TACE by RNA interference blocked BDNF-induced p75NTR cleavage and apoptosis, indicating that this metalloprotease is responsible for the initial processing of the receptor. Together, these results demonstrate that p75NTR-mediated activation of JNK3 is required for up-regulation of TACE, which promotes receptor proteolysis, leading to prolonged activation of JNK3 and subsequent apoptosis in sympathetic neurons.

Cholesterol is a critical component of neuronal membranes, required for normal signal transduction. We showed previously that adult hippocampal neurons co-express high levels of cholesterogenic enzymes, and that their expression is under the control of the p75 neurotrophin receptor (p75NTR). Most of the cellular effects of p75NTR are mediated via interacting proteins, including neurotrophin receptor interacting factor (NRIF). In this study, we tested the hypothesis that p75NTR-dependent regulation of cholesterol and lipid biosynthesis genes is mediated by NRIF. We found that in vitro down regulation of NRIF expression decreased the mRNA for two main cholesterogenic enzymes, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr; EC 2.3.3.10) and 7-dehydrocholesterol reductase (Dhcr7; EC 1.3.1.21). Further analyses revealed that NRIF-dependent and Dhcr7-dependent transcriptional changes show a high degree of overlap, and that NRIF reduction resulted in reduced expression of sterol-sensing domain protein SCAP, followed by a decrease in mRNA levels of SRE-motif containing genes (HMGCR, FASN, SREBP2, S1P, and SQS1). Finally, a reduction in cholesterol biosynthesis-related gene expression was also observed in hippocampal tissue of mice with NRIF deletion. Our combined in vitro and in vivo studies suggest that hippocampal neuronal cholesterol biosynthesis is regulated through the p75NTR interacting factor NRIF.

The first dedicated step in de novo cholesterol biosynthesis begins with formation of squalene and ends with the reduction of 7-dehydrocholesterol by 7-dehydrocholesterol reductase (Dhcr7) into cholesterol, which is an essential structural and signaling molecule. Mutations in the Dhcr7 gene lead to Smith-Lemli-Opitz syndrome (SLOS), which is characterized by developmental deformities, incomplete myelination, and mental retardation. To understand better the molecular consequences of Dhcr7 deficiency in neuronal tissue, we analyzed the effect of cholesterol deficiency on the transcriptome in Neuro2a cells. Transient down-regulation of Dhcr7 by siRNA led to altered expression of multiple molecules that play critical roles in intracellular signaling or vesicular transport or are inserted into membrane rafts (e.g. Egr1, Snx, and Adam19). A similar down-regulation was also observed in stable Dhrc7-shRNA-transfected cell lines, and the findings were verified by qPCR. Furthermore, we investigated the Dhcr7-deficient and control cells for the expression of several critical genes involved in lipid biosynthesis. Among these, fatty acid synthase, sterol-regulatory element binding protein 2, SREBF chaperone, site-1 protease, and squalene synthase showed a significant down-regulation, suggesting that, in a neuronal cell line, Dhcr7 is a potent regulator of lipid biosynthesis. Importantly, the gene expression changes were present in both lipid-containing and cholesterol-deficient media, suggesting that intrinsic cholesterol biosynthesis is necessary for normal neuronal function and cannot be supplemented from extrinsic sources.

Normal brain function depends critically on cholesterol. Although cholesterol is synthesized locally in the adult brain, the precise anatomical localization of cholesterogenic enzymes is not known. Here we show that 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAred) and 7-dehydrocholesterol reductase (7dhcred), the first and last enzymes, respectively, in the cholesterol biosynthesis pathway, are co-expressed in neurons throughout adult murine brain. Co-localization is most prominent in cortical, hippocampal, and cholinergic neurons. Since adult hippocampal and cholinergic neurons express p75 neurotrophin receptors (p75NTR) we hypothesized that p75NTR regulates expression of cholesterogenic enzymes. Treatment of Neuro2a neuroblastoma cells or primary cerebellar cultures with siRNA downregulates p75NTR and decreases the expression level of HMG-CoAred and 7dhcred. Native neuroblastoma cell lines with differential expression of p75NTR differentially express 7dhcred; 7dhcred expression correlates with p75NTR expression. This suggests that, in p75NTR-expressing cells, p75NTR regulates cholesterol synthesis through regulation of HMG-CoAred and 7dhcred expression. The unexpected localization of cholesterogenic enzymes in adult neurons suggests that at least some adult neurons retain the ability to synthesize cholesterol.