TSPO mutations in rats and a human polymorphism impair the rate of steroid synthesis

Abstract

The 18 kDa translocator protein (TSPO) is a ubiquitous conserved outer mitochondrial membrane protein implicated in numerous cell and tissue functions, including steroid hormone biosynthesis, respiration, cell proliferation, and apoptosis. TSPO binds with high affinity to cholesterol and numerous compounds, is expressed at high levels in steroid-synthesizing tissues, and mediates cholesterol import into mitochondria, which is the rate-limiting step in steroid formation. In humans, the rs6971 polymorphism on the TSPO gene leads to an amino acid substitution in the fifth transmembrane loop of the protein, which is where the cholesterol-binding domain of TSPO is located, and this polymorphism has been associated with anxiety-related disorders. However, recent knockout mouse models have provided inconsistent conclusions of whether TSPO is directly involved in steroid synthesis. In this report, we show that TSPO deletion mutations in rat and its corresponding rs6971 polymorphism in humans alter adrenocorticotropic hormone-induced plasma corticosteroid concentrations. Rat tissues examined show increased cholesteryl ester accumulation, and neurosteroid formation was undetectable in homozygous rats. These results also support a role for TSPO ligands in diseases with steroid-dependent stress and anxiety elements.

Keywords: adrenal; cholesterol transport; gonads; lipid droplets; steroids; translocator protein.

Figure 1.. Genome editing of the rat Tspo locus using the CompoZr® optimized/customized KO ZFN (CKOZFN) technology.

(A) A diagram of the ZFN-optimized target region in the rat Tspo exon 3 region. The expected CKOZFN targeting area is indicated as a blue line. Three ZFN domains (ZF1, ZF2, and ZF3) linked to the Fokl nuclease (Fok1) domain on each side of the expected target area are indicated to form a ZFN homodimer binding to DNA. The 89-bp deletion in Rat5 is indicated in red. An intron sequence was introduced into the new mRNA, indicated by the first ‘GT’, and insertions were confirmed by sequencing (Supplementary Figure S1C). The indicated primers (CKOZFN-F and CKOZFN-R) were used for founder identification and genotype screening. (B) Sequencing confirmation of rat Tspo locus modification from the Rat5 founder. The 96-bp deletion disrupts the exon 3-intron 3 junction; the exon sequence is shown in capital letters, and the intron sequence is shown in lower-case letters. (C) Diagram of the CRAC-specific deletion by CompoZr® Custom ZFN technology within rat Tspo exon 4. The expected targeted area is indicated in blue as the CRAC; the actual deletion in Rat7 is indicated in red within exon 4 (166 bp were deleted within exon 4). The indicated primers (COMPOZr-1kbF and COMPOZr-1kbR) were used for founder and genotype screening. (D) Sequencing analysis confirmed that the rat Tspo locus was modified in the Rat7 founder. The 166-bp deletion within exon 4 was expected to generate a truncated exon 4. The TGA stop codon is highlighted in red. (E) Summary of the fertilized eggs injected with ZFN mRNAs, transferred at metaphase I, and the rare founders that were obtained. TSPO13, Rat5 with exon 3–intron 3 disruption (two founders obtained) and with a larger deletion (one founder obtained, not shown); TSPO17, Rat7 with a CRAC-specific deletion (one founder obtained). (F) Agarose gel image of the typical PCR products used for genotype screening of Rat5 with Tspo locus modification. WT, 362 bp; HE, two bands (362/273 bp); HO, 273 bp. (G) RT-PCR analysis of Rat5 vs. WT rat adrenal glands was used to detect mutated mRNA species. Red arrow, Rat5 mutated Tspo mRNA (the intron sequence was introduced and resulted in a transcript 10 bp shorter than WT Tspo mRNA or transcripts of increasing length up to the full intron sequence fused with exon 4). The 575-bp WT Tspo mRNA is indicated. The corresponding rat genotypes are indicated. (H) Agarose gel image of typical PCR products used for genotype screening of Rat7 with a mutated Tspo predicted to lack the CRAC domain. WT, 818 bp; HE, 818 bp; HO, 654 bp; n.s., nonspecific band. (I) RT-PCR of the Rat7 vs. WT rat adrenal glands was used to detect mutated mRNA species. Red arrow: mutated Rat7 Tspo mRNAs with a deletion in exon 4 were 411 bp, whereas WT Tspo mRNA species were 575 bp. The corresponding rat genotype (WT, HE, or HO) is indicated. (J) Immunoblot analysis of proteins extracted from homogenized adrenal gland tissues of Rat5 (left panel) and Rat7 (right panel). The rat genotype (WT, HE, and HO), TSPO, HPRT (loading control), and the protein ladder (kDa) are indicated.

Figure 2.. Effect of genome editing of rat Tspo on the PK 11195 binding and immunofluorescence staining.

(A and B) Representative optical bright-field images were used as controls to show the morphology of adrenal sections used for the binding assay. (C and D) Autoradiographic localization of PK 11195 in the adrenal glands from WT, Rat5, and Rat7. (C) Autoradiographic images of tissue incubation with 1.2 nmol/l [³H]-PK 11195 in 50 mmol/l Tris–HCl (pH 7.4) for 30 min at room temperature (22°C). [³H]-PK 11195 binding was analyzed by digital autoradiography using a Beta-Imager 2000 (Biospace Laboratory, Paris, France). Binding intensities are presented in false color using the ImageJ look-up table ‘royal’. (D) Autoradiographic images of tissue incubation as in C but additionally with cold PK 11195 to show nonspecific binding. (E) Saturation isotherm of [³H]-PK 11195-specific binding studies using 15 µg of protein extracted from adrenal glands of WT1, WT2, and TSPO KO Rat5-1 and Rat5-2. The K_D and B_max of PK 11195 binding to WT1 adrenal protein extract were 3.84 ± 1.62 and 84.95 ± 11.86, respectively, and for WT2, adrenal protein extract was 3.04 ± 0.96 and 60.40 ± 5.67, respectively. (F and G) IF staining of cryosections of Rat5 adrenal gland using rabbit polyclonal anti-TSPO Ab (NP155). Images were obtained using laser scanning confocal microscopy (A) and epifluorescence imaging using an inverted microscope (B). The rat genotype (WT, HE, and HO) is indicated. Scale bar = 100 µm.

Figure 3.. Effect of TSPO deletion on the accumulation of esterified cholesterol in rat models.

(A–J) ORO staining of adrenal glands from WT, HE, and HO rats. HO rats exhibited increased ORO staining of neutral lipids, which reflected esterified cholesterol in the steroidogenic tissues. (A–C) Male adrenal gland of Rat5; scale bar = 50 µm. (D–F) Male adrenal gland of Rat7; scale bar = 50 µm. (G–J) The distribution of esterified cholesterol in Rat5 and Rat7 testis. Esterified cholesterol was estimated using ORO staining of testis from Rat5 (G–H) and Rat7 (I–J). WT, HE, and HO rats are indicated, and the highlighted area is magnified below each panel. Lc, Leydig cell. Green arrows indicate lipid droplets; scale bar = 100 µm.

Figure 4.. Measurement of steroid biosynthesis in rat models.

(A and B) Circulating corticosterone levels in WT, HE, and HO Rat5 and Rat7 treated with and without ACTH. Plasma corticosterone levels from basal and ACTH-treated WT, HE, and HO Rat5 (A); Mann–Whitney U-test; **P < 0.01 (between ACTH-treated WT and HO; n = 10–13 animals per group). Plasma corticosterone levels from basal and ACTH-treated WT, HE, and HO Rat7 (B); Mann–Whitney U-test; P = 0.051 (between ACTH-treated WT and HO); n = 10–13 animals per group. (C and D) Circulating testosterone levels in Rat5 and allopregnanolone levels in Rat7 brains. Testosterone was measured using ELISA, and allopregnanolone was measured using LC–MS. (C) Basal testosterone levels in WT, HE, and HO from Rat5 (similar results were obtained in Rat7). Mann–Whitney U-test; *P < 0.05, **P < 0.01 (WT vs. HE or HO; n = 5–6 animals per group). (D) Relative amounts of allopregnanolone in Rat7 (WT, HE, and HO) cortex (not measurable in HO). n.d., not detectable; Student's t-test, n = 4. (E) Allopregnanolone in WT, HE, and HO Rat7 brains measured by UHPLC–QQQ MS. Representative chromatography of the allopregnanolone measurements from each Rat7 genotype: WT, HE, and HO. The reference standard is indicated.

Figure 5.. Effect of TSPO polymorphism on PK 11195/cholesterol binding in vitro.

Saturation isotherms of [³H]-PK 11195 and [³H]-cholesterol binding to reconstituted mouse TSPO WT and mutant (Ala/Thr) proteins. (A) [³H]-PK 11195 and [³H]-cholesterol-specific binding studies were performed using 200 ng of mouse WT TSPO. Inset, electron micrographs of WT TSPO proteoliposomes stained with 2% uranyl acetate after SDS elimination using biobeads. (B) 200 ng of mouse Ala147Thr mutant reconstituted TSPO was used to study specific binding with [³H]-PK 11195 and [³H]-cholesterol. Inset, electron micrographs of mutant TSPO proteoliposomes stained with 2% uranyl acetate after SDS elimination using biobeads. [³H]-PK 11195 concentrations varied from 0.1 to 15 nM; [³H]-cholesterol concentrations varied from 0.1 to 30 nM. Values shown represent the mean (SE) from three independent experiments. The extra sum-of-squares F-test was used to compare the fitting curves and the P-values presented (values of P < 0.05 are statistically significant to reject the null hypothesis).

Figure 6.. Humans carrying rs6971 (Ala147/Thr) polymorphism show reduced response to ACTH treatment.

(A) Western blot analysis of human TSPO expression in the human steroid-producing adrenocortical cell line H295R. TSPO recombinant protein in E. coli (E.-I, induced with IPTG; E.-UI, uninduced cell lysate). The mouse Leydig cell line MA-10 was used as a positive control, and HPRT was used as a loading control. Biotinylated protein ladder marker sizes are labeled in kDa. (B) Plasma cortisol concentrations in healthy male volunteers at 09:00 AM, with AA (n = 18), AT (n = 16), and TT (n = 11) polymorphisms. A gene-dose effect was not observed. AA, 304.9 (18.12) nmol/l; AT, 387.8 (24.4) nmol/l; TT, 365.6 (27.80) nmol/l (P = 0.0629, linear trend test). Values shown represent the mean (SE). (C) Plasma cortisol concentrations immediately before ACTH administration (at 11:00 AM) in healthy male volunteers with AA (n = 20), AT (n = 16), and TT (n = 11) polymorphisms. A gene-dose effect was observed. AA, 202.7 (16.7) nmol/l; AT, 244.5 (22.6) nmol/l; TT, 275.4 (30.6) nmol/l (*P = 0.029, linear trend test). Values shown represent the mean (SE). (D) Fold-change in plasma cortisol concentration after ACTH administration (250 µg, IV). A gene-dose effect was observed (fold-change): AA (n = 10) 2.75 (0.32); AT (n = 10) 2.17 (0.25); TT (n = 10) 1.85 (0.22) (*P = 0.023, linear trend test). Values shown represent the mean (SE).