PSD3 downregulation confers protection against fatty liver disease

Abstract

Fatty liver disease (FLD) is a growing health issue with burdening unmet clinical needs. FLD has a genetic component but, despite the common variants already identified, there is still a missing heritability component. Using a candidate gene approach, we identify a locus (rs71519934) at the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene resulting in a leucine to threonine substitution at position 186 of the protein (L186T) that reduces susceptibility to the entire spectrum of FLD in individuals at risk. PSD3 downregulation by short interfering RNA reduces intracellular lipid content in primary human hepatocytes cultured in two and three dimensions, and in human and rodent hepatoma cells. Consistent with this, Psd3 downregulation by antisense oligonucleotides in vivo protects against FLD in mice fed a non-alcoholic steatohepatitis-inducing diet. Thus, translating these results to humans, PSD3 downregulation might be a future therapeutic option for treating FLD.

Fig. 1. PSD3 minor allele protected against enhanced severity of histological liver damage in the LBC and PSD3 gene expression was higher in livers with FLD.

a–d, Histological liver damage stratified by PSD3 genotype. Bars show the prevalence expressed as percentage of the degree of the specified disease. Colour shading from white to black indicates increased disease severity. Histological damage was evaluated according to the different components of the NAS and hepatic fibrosis stage. Carriers of the PSD3 rs71519934 186T minor allele had less severe liver disease with lower degrees of steatosis (a), inflammation (b), ballooning (c) and fibrosis (d). The association was tested under an additive genetic model by an ordinal regression analysis adjusted for age, gender, BMI, recruitment centre and number of PNPLA3 I148M mutant alleles. Results were virtually the same after further adjustment for the other main genetic risk factors (TM6SF2 rs58542926 (E167K), MBOAT7 rs641738 and GCKR rs1260326 (L446P)) and environmental (presence of diabetes) variables influencing FLD (Supplementary Table 4). All the reported P values are two-sided with no adjustment for multiple testing. *Data available for ballooning, n = 1,805. e, Total PSD3 mRNA expression stratified by healthy and FLD livers. Liver PSD3 expression levels were higher in FLD subjects compared with healthy controls. f, Total NAT2 mRNA expression stratified by healthy and FLD livers. There was no difference in the NAT2 expression level based on the presence of FLD. e,f, Data are presented as mean and s.d. Two-sided P value was calculated by Mann–Whitney non-parametric test. Ctr, healthy control livers (n = 10); FLD, livers with fatty liver disease (n = 67). Source data

Fig. 2. PSD3 mRNA isoform expression in human liver and total PSD3 mRNA expression stratified by rs71519934 genotype.

a, Expression level of the different PSD3 mRNA isoforms in human liver tissue. To assess which isoform is the most abundant, the transcriptome from liver biopsies of a subset of 77 individuals from the Milan subgroup of the LBC was examined. Isoform 001 (identified as ENST00000327040 by Ensembl or as NP_056125 (isoform-a) by NCBI, 1,047 amino acids) had the highest expression, followed by isoform 008 (identified as ENST00000521841 by Ensembl, non-coding). Data are presented as mean and s.d. b, Total PSD3 mRNA expression stratified by rs71519934 genotype. There were no differences in PSD3 mRNA expression levels when stratified by genotype. c, Total NAT2 mRNA expression levels did not differ among PSD3 rs71519934 genotypes. b,c, Data are presented as mean and s.d. The two-sided P value was calculated by unadjusted linear regression. Values were log-transformed before entering the model. FPKM, fragments per kilobase of exon model per million reads mapped; 186L, homozygotes for the L allele (n = 42); L186T, heterozygotes (n = 29); 186T, homozygotes for the T allele (n = 6). Source data

Fig. 3. Primary human hepatocytes from a donor homozygous for the 186T allele had lower PSD3 protein and intracellular lipid levels compared with homozygous 186L hepatocytes. Primary human hepatocytes from donors homozygous for 186L or 186T, were cultured in 2D.

a, Intracellular neutral fat content visualized by ORO staining and quantified by Biopix iQ software v.2.3.1. Data are presented as mean and s.d. of the reported independent experiments (n = 5). Two-sided P value was calculated by Mann–Whitney non-parametric test. b, Cells were cultured in serum-free regular medium supplemented with 2% FBS, 10 µM OA or 25 µM OA for 48 h. Immunoblotting was performed with total cell lysates to detect PSD3 (NCBI: NP_056125, 1,047 amino acids) using a custom antibody. The bar graph shows the relative PSD3 amount calculated as PSD3/calnexin (CNX). c, Key genes involved in lipid metabolism that were differentially expressed between donors homozygous for 186T versus 186L obtained with RNA-seq. Data are presented as log₂(fold change) in expression and –log₁₀(P value) adjusted using the Benjamini and Hochberg’s approach for controlling the FDR. RU, relative units; VLDL, very low-density lipoprotein. Red Triglyceride synthesis; yellow VLDL secretion; blue Fatty acid oxidation; black cholesterol metabolism. Source data

Fig. 4. PSD3 downregulation lowered intracellular neutral fat content in primary human hepatocytes cultured in 2D and 3D spheroids.

Downregulation of endogenous PSD3 expression using siRNA in primary human hepatocytes cultured in 2D and 3D. For 2D culture, after attachment of cells in collagen-coated plates, cells were incubated with regular growth medium supplemented with 10 µM OA and transfected with negative control SCR siRNA or PSD3 siRNA for 48 h. a,b, Intracellular neutral fat content was visualized by ORO staining and quantified by Biopix iQ software v.2.3.1 in primary human hepatocytes carrying (a) the 186L allele (n = 5) or (b) the 186T allele (n = 4). Average PSD3 downregulation efficiency was ~80% as evaluated by quantitative retro transcription PCR analysed by the 2^−ΔΔCt method and western blotting for both donor types. For 3D culture of primary human hepatocytes, spheroids were generated by seeding 2,000 cells per well in a 96-well round-bottom flask, along with transfection mix in 100 µl of medium. For the generation of 186T allele spheroids, 5 nM of FMK-Z-VAD was added to support spheroid formation. After 24 h, additional growth medium was added to give a total volume of 200 µl per well. Fifty per cent of the total media was replenished with fresh media every 48 h. After 7 days of formation, spheroids were collected and 8-µM sections were subjected to ORO staining to visualize intracellular neutral fat content. c,d, Nuclei were stained with 4,6-diamidino-2-phenylindole and ORO staining was quantified by Image J, normalized to number of nuclei of primary human hepatocyte spheroids carrying (c) the 186L allele (n = 4) and (d) the 186T allele (n = 4). Average gene knockdown efficiency was ~50–60% as evaluated by quantitative retro transcription PCR analysed by the 2^−ΔΔCt method for both donor types. Cellular ATP levels (marker of viability) remained stable between the negative control SCR and PSD3 siRNA groups. Data are shown as mean ± s.d. of the reported independent experiments. Two-sided P values were calculated by Mann–Whitney non-parametric test comparing SCR siRNA versus PSD3 siRNA. RU, relative units; CNX, calnexin. Source data

Fig. 5. Downregulation of PSD3 resulted in partial loss of ARF6 activation.

Human hepatoma Huh7 cells were transiently transfected with negative control SCR siRNA, PSD3 siRNA or ARF6 siRNA. Forty-eight hours after transfection, the cell lysates were incubated with GGA3 PBD agarose beads that selectively isolate and pull down endogenous active ARF6 (ARF6–GTP). a, After precipitation, active ARF6–GTP was detected by immunoblotting using an anti-ARF6 antibody provided in the kit. Cells transfected with ARF6 siRNA were used as a control. The experiment was performed independently six times with similar results. Representative blot presented. b, Knockdown efficiency with ~60% reduction for PSD3 and ~75% for ARF6 as evaluated by quantitative retro transcription PCR analysed by the 2^−ΔΔCt method (n = 6). c, Relative ARF6–GTP (active) calculated as GTP–ARF6/calnexin (CNX) (n = 6). Data are shown as mean ± s.d. of the reported independent experiments. Two-sided P values calculated by Mann–Whitney non-parametric test. RU, relative units. Source data

Fig. 6. Liver Psd3 downregulation in mice fed a NASH-inducing diet reduced the severity of steatosis, inflammation and NAS, and liver collagen levels.

C57BL/6 male mice were fed a NASH-inducing diet for a total of 50 weeks and during the last 16 weeks, groups of mice were dosed via once weekly subcutaneous injections with saline (n = 10 animals), control GalNAc–ASO (n = 9 animals, 5 mg per kg body weight per week) or Psd3 GalNAc–ASO (n = 10 animals, 5 mg per kg body weight per week). a–d, Psd3 GalNAc–ASO reduced liver Psd3 mRNA expression levels (a) and was associated with reduced liver weight (b), total liver triglyceride content (c) and plasma ALT levels (d). e,f, Psd3 GalNAc–ASO treatment reduced liver Col1a1 protein levels (e) and liver lipid droplet number (f). g–j, Psd3 GalNAc–ASO treatment reduced the severity of steatosis (g), inflammation (h) and NAS (i), although there were no significant changes in liver fibrosis grade (j). a–f, Data are presented as mean ± s.d. Two-sided P values were calculated by one-way ANOVA Kruskal–Wallis non-parametric test followed by Dunn’s correction for multiple comparisons. Multiple comparisons were performed between the mean of each group and the mean of the control group (Ctr GalNAc–ASO). g–j, Data are presented as prevalence (%) of the specified disease’s degree. Colour shading from white to black indicates increased disease severity. Two-sided P values were calculated by using ordinal regression analyses. NS, non-significant. Source data

Fig. 7. Liver Psd3 downregulation in mice fed a NASH-inducing diet reduced liver cholesterol and circulating LDL cholesterol levels and reduced expression of hepatic genes involved in de novo lipogenesis.

C57BL/6 male mice were fed a NASH-inducing diet for a total of 50 weeks and during the last 16 weeks, groups of mice were dosed via once weekly subcutaneous injections with saline (n = 10 animals), control GalNAc–ASO (n = 9 animals, 5 mg per kg body weight per week) or Psd3 GalNAc–ASO (n = 10 animals, 5 mg per kg body weight per week). a–h, Psd3 GalNAc–ASO treatment did not affect body weight gain (a) but reduced liver cholesterol (b) and cholesteryl ester (c), plasma AST (d), total cholesterol (e) and LDL cholesterol levels (f). There were no effects on plasma triglyceride (g) or HDL cholesterol (h) levels. Hepatic mRNA was quantitated by digital gene expression profiling as described in the Methods and expressed as transcripts per million. i–n, Psd3 GalNAc–ASO treatment had no effect on hepatic Srebp1-c (i) but significantly reduced hepatic Acc1 (j), Fasn (k), Scd1 (l), αSma (m) and Ccl2 (n) mRNA expression levels. Data are presented as mean ± s.d. a–h, Two-sided P values were calculated by one-way ANOVA Kruskal–Wallis non-parametric tests followed by Dunn’s multiple comparisons tests. Multiple comparisons were performed by comparing each group with the control group (Ctr GalNAc–ASO). i–n, Two-sided P values were calculated by Mann–Whitney non-parametric tests. TPM, transcripts per million. Source data

Fig. 8. Liver Psd3 downregulation in mice fed a NASH-inducing diet improved liver histopathology.

C57BL/6 male mice were fed a NASH-inducing diet for a total of 50 weeks and during the last 16 weeks, groups of mice were dosed via once weekly with subcutaneous injections of saline (n = 10 animals), control GalNAc–ASO (n = 9 animals, 5 mg per kg body weight per week) or Psd3 GalNAc–ASO (n = 10 animals, 5 mg per kg body weight per week). Liver sections were stained with haematoxylin and eosin, ORO or picrosirius red according to standard procedures. Consecutive sections were immunohistochemically stained for Col1a1. Representative pictures are presented. Ctr, control. Source data