Ken Rose

A point mutation in the ubiquitin-associated domain of SQSMT1 is sufficient to cause a Paget's disease-like disorder in mice

Mutations of SQSTM1 occur in about10% of patients with Pagets disease of bone (PDB), but it is unclear whether they play a causal role or regulate susceptibility to an environmental trigger. Here we show that mice with a proline to leucine mutation at codon 394 of mouse sqstm1 (P394L), equivalent to the P392L SQSTM1 mutation in humans, develop a bone disorder with remarkable similarity to PDB. The P394L mutant mice developed focal bone lesions with increasing age and by 12 months, 14/18 (77%) heterozygotes and 20/21 (95%) homozygotes had lesions, compared with 0/18 (0%) wild-type littermates (P< 0.001). Lesions predominantly affected the lower limbs in an asymmetric manner and were characterized by focal increases in bone turnover, with increased bone resorption and formation, disruption of the normal bone architecture and accumulation of woven bone. Osteoclasts within lesions were larger and more nucleated than normal and some contained nuclear inclusions similar to those observed in human PDB. Osteoclast precursors from P394L mutant mice had increased sensitivity to RANKL in vitro resulting in the generation of osteoclasts that were larger and more nucleated than those generated from wild-type littermates. There was increased expression of sqstm1, autophagy-related gene 5 (atg5) and light chain 3 gene (lc3) in osteoclast precursors and increased LC3-II protein levels in Bafilomycin-treated osteoclasts from P394L mutant mice compared with wild-type suggesting dysregulation of autophagy and enhanced autophagosome formation. These studies demonstrate that SQSTM1 mutations can cause a PDB-like skeletal disorder in the absence of an additional trigger and provide a new disease model for PDB.

Neurosteroid Hydroxylase CYP7B VIVID REPORTER ACTIVITY IN DENTATE GYRUS OF GENE-TARGETED MICE AND ABOLITION OF A WIDESPREAD PATHWAY OF STEROID AND OXYSTEROL HYDROXYLATION

The major adrenal steroid dehydroepiandrosterone (DHEA) enhances memory and immune function but has no known dedicated receptor; local metabolism may govern its activity. We described a cytochrome P450 expressed in brain and other tissues, CYP7B, that catalyzes the 7α-hydroxylation of oxysterols and 3β-hydroxysteroids including DHEA. We report here that CYP7B mRNA and 7α-hydroxylation activity are widespread in rat tissues. However, steroids related to DHEA are reported to be modified at positions other than 7α, exemplified by prominent 6α-hydroxylation of 5α-androstane-3β,17β-diol (A/anediol) in some rodent tissues including brain. To determine whether CYP7B is responsible for these and other activities we disrupted the mouse Cyp7b gene by targeted insertion of an IRES-lacZ reporter cassette, placing reporter enzyme activity (β-galactosidase) underCyp7b promoter control. In heterozygous mouse brain, chromogenic detection of reporter activity was strikingly restricted to the dentate gyrus. Staining did not exactly reproduce the in situ hybridization expression pattern; post-transcriptional control is inferred. Lower level staining was detected in cerebellum, liver, and kidney, and which largely paralleled mRNA distribution. Liver and kidney expression was sexually dimorphic. Mice homozygous for the insertion are viable and superficially normal, but ex vivo metabolism of DHEA to 7α-hydroxy-DHEA was abolished in brain, spleen, thymus, heart, lung, prostate, uterus, and mammary gland; lower abundance metabolites were also eliminated. 7α-Hydroxylation of 25-hydroxycholesterol and related substrates was also abolished, as was presumed 6α-hydroxylation of A/anediol. These different enzyme activities therefore derive from the Cyp7bgene. CYP7B is thus a major extrahepatic steroid and oxysterol hydroxylase and provides the predominant route for local metabolism of DHEA and related molecules in brain and other tissues.

cyp7b1 catalyses the 7alpha-hydroxylation of dehydroepiandrosterone and 25-hydroxycholesterol in rat prostate.

Dehydroepiandrosterone (DHEA) is the most prominent circulating steroid in humans, and it is a precursor for sex-steroid synthesis in peripheral tissues, including the prostate. Recently, enzyme-mediated pre-receptor metabolism has been recognized as a key step in determining steroid action in vivo. Hydroxylation of 3beta-steroids at the 7alpha-position has been reported in rat and human prostate to be a major inhibitory pathway to sex-steroid synthesis/action. However, the molecular identity of the enzyme responsible is so far unknown. We recently described a novel cytochrome P450 enzyme, cyp7b1, strongly expressed in the hippocampus of rodent brain, which catalyses the metabolism of DHEA, pregnenolone and 25-hydroxycholesterol to 7alpha-hydroxy products. In the light of this new enzyme, we have examined its possible role in 7alpha-hydroxylation conversion in rat prostate. NADPH-dependent 7alpha-hydroxylation was confirmed for 3beta-hydroxysteroids including DHEA and androstenediol, as well as 25-hydroxycholesterol. Kinetic analysis yielded an apparent K(m) of 14+/-1 microM for 7alpha-hydroxylation of DHEA in the prostate gland, a value similar to that recorded for recombinant cyp7b1 enzyme [13.6 microM; Rose, Stapleton, Dott, Kieny, Best, Schwarz, Russell, Bjoorkheim, Seckl and Lathe (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4925-4930]. The V(max) value of the prostate was 46+/-2 pmol/min per mg, and this activity was inhibited by clotrimazole, a P450-enzyme blocker. Moreover, RNA analysis (reverse-transcription PCR, Northern blotting and in situ hybridization) revealed a high expression of cyp7b1 mRNA in the rat prostate, restricted to the epithelium, suggesting that cyp7b1 catalyses oxysterol 7alpha-hydroxylation in the prostate gland.

Dehydroepiandrosterone (DHEA) metabolism in Saccharomyces cerevisiae expressing mammalian steroid hydroxylase CYP7B: Ayr1p and Fox2p display 17β-hydroxysteroid dehydrogenase activity

We have engineered recombinant yeast to perform stereospecific hydroxylation of dehydroepiandrosterone (DHEA). This mammalian pro‐hormone promotes brain and immune function; hydroxylation at the 7α position by P450 CYP7B is the major pathway of metabolic activation. We have sought to activate DHEA via yeast expression of rat CYP7B enzyme. Saccharomyces cerevisiae was found to metabolize DHEA by 3β‐acetylation; this was abolished by mutation at atf2. DHEA was also toxic, blocking tryptophan (trp) uptake: prototrophic strains were DHEA‐resistant. In TRP+ atf2 strains DHEA was then converted to androstene‐3β,17β‐diol (A/enediol) by an endogenous 17β‐hydroxysteroid dehydrogenase (17βHSD). Seven yeast polypeptides similar to human 17βHSDs were identified: when expressed in yeast, only AYR1 (1‐acyl dihydroxyacetone phosphate reductase) increased A/enediol accumulation, while the hydroxyacyl‐CoA dehydrogenase Fox2p, highly homologous to human 17βHSD4, oxidized A/enediol to DHEA. The presence of endogenous yeast enzymes metabolizing steroids may relate to fungal pathogenesis. Disruption of AYR1 eliminated reductive 17βHSD activity, and expression of CYP7B on the combination background (atf2, ayr1, TRP+) permitted efficient (>98%) bioconversion of DHEA to 7α‐hydroxyDHEA, a product of potential medical utility. Copyright

Chapter 2.2.3 Brain region-specific genes: the hippocampus

Publisher Summary The chapter discusses four approaches along with their merits and drawbacks, and possible means to the identification of genes and transcripts whose expression is restricted to the hippocampus, and the advantages and drawbacks of the techniques employed. Unexpectedly, each approach highlights a different and non-overlapping subset of genes, but the expression of at least some of these was largely if not exclusively restricted to the hippocampus. The most important method is differential hybridization. This yielded a gene, Cyp7b, whose expression is largely but not exclusively restricted to the hippocampal formation and encoding a novel enzyme metabolizing neurosteroids. The candidate gene family approach yielded a new serine protease, BSP1 with a highly restricted pattern of expression that permits exploitation in a transgenic context, and protein tyrosine phosphatase (PTP) gamma, whose restricted pattern of expression warrants further study.