Is it possible to improve memory function by upregulation of the cholesterol 24S-hydroxylase (CYP46A1) in the brain? PLoS One

Abstract We previously described a heterozygous mouse model overexpressing human HA-tagged 24S-hydroxylase (CYP46A1) utilizing a ubiquitous expression vector. In this study, we generated homozygotes of these mice with circulating levels of 24OH 30-60% higher than the heterozygotes. Female homozygous CYP46A1 transgenic mice, aged 15 months, showed an improvement in spatial memory in the Morris water maze test as compared to the wild type mice. The levels of N-Methyl-DAspartate receptor 1, phosphorylated-N-Methyl-D-Aspartate receptor 2A, postsynaptic density 95, synapsin-1 and synapthophysin were significantly increased in the hippocampus of the CYP46A1 transgenic mice as compared to the controls. The levels of lanosterol in the brain of the CYP46A1 transgenic mice were significantly increased, consistent with a higher synthesis of cholesterol. Our results are discussed in relation to the hypothesis that the flux in the mevalonate pathway in the brain is of importance in cognitive functions

Molecular mechanisms of transcriptional repression by the orphan receptor SHP

SHP (Small Heterodimer Partner) is an atypical orphan member of the mammalian nuclear receptor family that consists of only a putative ligand-binding domain and thus cannot bind DNA. The aim of this thesis was to investigate the molecular mechanisms of the transcriptionally inhibitory effect of SHP. This was achieved by analysis of structural motifs within SHP, by studying nuclear receptor interactions, by isolation of upstream target co-factors and by characterization of SHP mutations. In the first study, we provide evidence that SHP binds directly to the estrogen receptors a and beta via LXXLL-related motifs. Similar motifs, referred to as nuclear receptor (NR) boxes, are usually critical for the binding of co-activators to the ligand-regulated activation domain AF-2 within nuclear receptors. We demonstrate that SHP variants, carrying either interaction-defective NR-box mutations or a deletion of the repressor domain, have lost the capacity to inhibit agonist-dependent estrogen receptor activation. Our study suggests that SHP acts as a transcriptional co-regulator by inhibiting the activity of nuclear receptors via occupation of the co-activator binding surface and via active repression. However, active repression mechanisms have remained elusive and may involve factors distinct from known nuclear receptor co-repressors. In the second study, we describe the isolation of mouse EID1 (E1A-like inhibitor of differentiation 1) as the first co-inhibitor for SHP. We characterize the interactions between SHP and EID1 and identify two repression-defective SHP mutations that have lost the ability to bind EID1. We suggest histone acetyltransferases and histones as targets for EID1 action, and we propose that SHP inhibition of transcription involves EID1 antagonism of p300/CBP-dependent co-activator functions. Recent evidence suggests the existence of a larger family of EID1-related proteins. In the third study, we describe a third family member designated EID3 that is highly expressed in testis and shows homology to a region of EID1 implicated in binding to p300/CBP. We demonstrate that EID3 acts as a potent inhibitor of nuclear receptor transcriptional activity by a mechanism that is independent of direct interactions with nuclear receptors, including SHP. However, EID3 directly binds to the C-terminus of CBP, which has been implicated to act as the interaction surface for nuclear receptor co-activators. Consistent with this idea, EID3 prevents recruitment of CBP to a natural nuclear receptor-regulated promoter. Our study suggests that EID-family members act as inhibitors of p300/CBP-dependent transcription in a tissue-specific manner. In the fourth study, we characterize GPS2 (G-protein pathway suppressor 2), a subunit of the N-CoR/HDAC3 co-repressor complex, as an interaction partner for SHP. Specific interactions between GPS2 and SHP have been verified in vitro and in vivo and appear physiologically relevant as GPS2 mRNA is expressed in SHP target tissues. It is shown that GPS2 increases SHP-mediated repression of NR target genes by enhancing intrinsic repression of the SHP ligand-binding domain. Taken together, these results suggest that SHP recruits a conserved co-repressor complex to nuclear receptor target genes via direct interactions with the GPS2 subunit. SHP fulfils specific roles as inducible co-repressors in the feedback regulation of LRH-1 (liver receptor homologue 1) target genes involved in bile acid synthesis. In the fifth study, we have identified and characterized the homeodomain protein Prox-1 (Prospero-related homeobox 1) as an additional co-repressor for LRH-1. We show that Prox-1 binds to LRH-1 via two LXXLL motifs and represses the LRH-1 dependent expression of SHP. Our hypothesis is that Prox-1 may modulate LRH-1 target gene expression during development and under normal physiological conditions, whereas SHP may primarily serve as a metabolic sensor under conditions of increased metabolic activity such as a high-cholesterol diet

Functional conservation of interactions between a homeodomain cofactor and a mammalian FTZ-F1 homologue

Nuclear receptors are master regulators of metazoan gene expression with crucial roles during development and in adult physiology. Fushi tarazu factor 1 (FTZ-F1) subfamily members are ancient orphan receptors with homologues from Drosophila to human that regulate diverse gene expression programs important for developmental processes, reproduction and cholesterol homeostasis in an apparently ligand-independent manner. Thus, developmental and tissue-specific cofactors may be particularly important in modulating the transcriptional activities of FTZ-F1 receptors. In Drosophila, the homeodomain protein Fushi tarazu acts as a cofactor for FTZ-F1 (NR5A3), leading to the hypothesis that a similar type of homeodomain cofactor–nuclear receptor relationship might exist in vertebrates. In this study, we have identified and characterized the homeodomain protein Prox1 as a co-repressor for liver receptor homologue 1 (LRH1/NR5A2), a master regulator of cholesterol homeostasis in mammals. Our study suggests that interactions between LRH1 and Prox1 may fulfil roles both during development of the enterohepatic system and in adult physiology of the liver

A transcriptional inhibitor targeted by the atypical orphan nuclear receptor SHP

SHP (short heterodimer partner, NROB2) is an atypical orphan member of the mammalian nuclear receptor family that consists only of a putative ligand-binding domain and thus cannot bind DNA. Instead, SHP acts as a transcriptional coregulator by inhibiting the activity of various nuclear receptors (downstream targets) via occupation of the coactivator-binding surface and active repression. However, repression mechanisms have remained elusive and may involve coinhibitory factors (upstream targets) distinct from known nuclear receptor corepressors. Here, we describe the isolation of mouse E1A-like inhibitor of differentiation 1 (EID1) as a candidate coinhibitor for SHP. We characterize the interactions between SHP and EID1 and identify two repression-defective SHP mutations that have lost the ability to bind EID1. We suggest histone acetyltransferases and histones as targets for EID1 action and propose that SHP inhibition of transcription involves EID1 antagonism of CBP/p300-dependent coactivator functions

EID1 and EID3 block the recruitment of CBP to the ERα-bound pS2 promoter

<p><b>Copyright information:</b></p><p>Taken from "EID3 is a novel EID family member and an inhibitor of CBP-dependent co-activation"</p><p>Nucleic Acids Research 2005;33(11):3561-3569.</p><p>Published online 24 Jun 2005</p><p>PMCID:PMC1159117.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> MCF-7 cells cultured with oestradiol were transiently transfected with FLAG vector alone for control or with FLAG-EID1 and FLAG-EID3. Soluble chromatin was prepared and immunoprecipitated by using antibodies raised against rabbit IgG as negative control, EPα, CBP and FLAG. Immunoprecipitated DNA was PCR amplified with primers that span the –353 to –159 region of the pS2 promoter. Values were time-matched and normalized for the solvent (DMSO)

Is it possible to improve memory function by upregulation of the cholesterol 24S-hydroxylase (CYP46A1) in the brain?

We previously described a heterozygous mouse model overexpressing human HA-tagged 24S-hydroxylase (CYP46A1) utilizing a ubiquitous expression vector. In this study, we generated homozygotes of these mice with circulating levels of 24OH 30-60% higher than the heterozygotes. Female homozygous CYP46A1 transgenic mice, aged 15 months, showed an improvement in spatial memory in the Morris water maze test as compared to the wild type mice. The levels of N-Methyl-D-Aspartate receptor 1, phosphorylated-N-Methyl-D-Aspartate receptor 2A, postsynaptic density 95, synapsin-1 and synapthophysin were significantly increased in the hippocampus of the CYP46A1 transgenic mice as compared to the controls. The levels of lanosterol in the brain of the CYP46A1 transgenic mice were significantly increased, consistent with a higher synthesis of cholesterol. Our results are discussed in relation to the hypothesis that the flux in the mevalonate pathway in the brain is of importance in cognitive functions

Levels of postsynaptic proteins were increased in the hippocampus of CYP46A1 mice.

<p>Hippocampal samples from CYP46A1 and WT control mice were analyzed by Western blotting. NMDAR1, p-NMDAR2A and PSD95 levels were significantly increased in CYP46 mice. Data are shown as mean ± standard error of the mean (SEM) of immunoreactivity (OD x area of the band) normalizes by α-tubulin levels. N = 7 animal per group (***P<0.001, *P<0.01 compared to WT control mice).</p

Levels of presynaptic proteins were increased in the hippocampus of CYP46A1 mice.

<p>Hippocampal samples from CYP46A1 and WT control mice were analyzed by Western blotting. Synapthophysin and synapsin-1 levels were significantly increased in CYP46A1 mice, while no differences were found in Neun among the groups. Data are shown as mean ± standard error of the mean (SEM) of immunoreactivity (OD x area of the band) normalizes by α-tubulin levels. N = 7 animal per group (*P<0.01 compared to WT control mice).</p