Our previous study demonstrated that p75NTR regulates the expression of cholesterogenic enzymes in both Neuro2a cells and primary neuronal cultures (Korade et al. 2007
). However, p75NTR regulates a myriad of intracellular signals (Gentry et al. 2004
) and which of these would contribute to the control of genes for cholesterol biosynthesis is not clear. NRIF fits the profile of a potential effector for this action very well. First, NRIF and p75ICD directly interact and after gamma-secretase cleavage of the receptor NRIF translocates into the nucleus (Kenchappa et al. 2006
). Second, NRIF is a DNA-binding protein and as such it is potentially able to regulate the transcription of cholesterol biosynthesis genes. Finally, the anatomical distribution of the p75NTR, Dhcr7, and NRIF transcripts showed a great degree of overlap in the mouse brain (Kendall et al. 2003
), opening the possibility that this anatomical co-expression may be a basis for a functional interaction. These converging lines of evidence, in addition to the correlation between p75NTR and NRIF regulation of Dhcr7 (Korade et al. 2007
), suggested that NRIF itself would be a very potent transcriptional regulator of the cholesterogenic biosynthesis transcripts in the nervous system.
The results of our study confirmed our hypotheses, as we found that: (1) NRIF regulates transcription of the critical cholesterol biosynthesis genes, Hmgcr and Dhcr7 in Neuro2a neuroblastoma cell line; (2) Dhcr7 and NRIF down regulation have similar consequences on the expression of several signaling genes; (3) NRIF silencing itself leads to reduced expression of genes involved in sterol-regulated biosynthesis; (4) NRIF-dependent transcriptional regulation is present both in the absence and presence of exogenous cholesterol, suggesting that preservation of endogenous control over cholesterol biosynthesis in neurons cannot be replaced by exogenous sources; and (5) the regulatory role of NRIF on gene transcription is conserved across in vitro and in vivo experimental systems (Neuro2a and NRIF KO mice, respectively).
In addition to confirming our initial hypotheses, our findings have several noteworthy implications. Importantly, transcriptional regulation of the cholesterol biosynthesis genes is a novel role for NRIF. NRIF, being a nuclear transcription factor, may directly bind to the promoter of lipid genes. Based on the overlap between the expression profile of NRIF- and Dhcr7-deficient cells, this putative mechanism would suggest that Dhcr7, or a direct regulator of Dhcr7 is a target of NRIF. However, we acknowledge that NRIF regulation of lipid gene biosynthesis may involve a more complex set of cellular events, where NRIF would bind to a transcription complex that is several steps removed from regulation of cholesterol biosynthesis.
What could be the consequences of such transcriptional dysregulation of lipid genes? We speculate that the transcriptional disturbance of lipid biosynthesis genes by altered NRIF expression leads to a broader dysregulation of cellular homeostasis, and this is potentially a result of altered cholesterol biosynthesis and availability. Cholesterol is an essential building block of cell membranes and lipid rafts (Korade and Kenworthy 2008
), which are a critical place of receptor insertion, neurotrophin signaling, neurotransmitter release, and regulated intramembrane proteolysis of transmembrane proteins (Pike 2005
). Reduction in NRIF leads to reduced Dhcr7 levels, which in turn results in accumulation of 7-dehydrocholesterol (7DHC), which is inserted into the membranes instead of cholesterol (Keller et al. 2004
). However, although 7-dehydrocholesterol and cholesterol can both incorporate into membranes, they are structurally different, and this is likely to affect the structure and composition of the lipid rafts: it is known that the presence of 7-dehydrocholesterol in hippocampal membranes impairs ligand-binding activity of the serotonin 1A receptor (Singh et al. 2007
). Thus, the hypothesized p75NTR-NRIF-Dhcr7-lipid raft cascade is likely to be very important for neuronal homeostasis, and this hypothesis is supported by numerous literature reports: cholesterol content of rafts affects membrane structure, ion conductance/excitability, trafficking to and from the cell surface, size and number of postsynaptic receptor clusters, and neurotransmitter signaling through G-protein coupled receptors (Korade and Kenworthy 2008
). Furthermore, cholesterol is a substrate for biosynthesis of neurosteroids and oxysterols. Reduced “mature” cholesterol levels may result in the use of 7-DHC for neurosteroid synthesis, and it is not clear if the activity of the 7-DHC-generated neurosteroids are similar to those of “normal” neurosteroids.
Finally, as the NRIF-dependent expression changes we observed were present in both cholesterol-containing and cholesterol-free cell culture media, our findings suggest that the NRIF-regulated endogenous cholesterol biosynthesis is critical for neuronal homeostasis, and that external supplementation cannot counteract the detrimental effects of altered intrinsic neuronal cholesterol biosynthesis. This further underscores that through regulation of intrinsic neuronal cholesterol biosynthesis, NRIF may have an important role in mediating many signals impinging on neurons. This role of NRIF should be further investigated in the context of human disorders of cholesterol metabolism, such as Smith–Lemli–Opitz syndrome and Niemann–Pick Type C disease.