Salubrinal enhances eIF2α phosphorylation and improves fertility in a mouse model of Classic Galactosemia

Abstract

Loss of galactose-1 phosphate uridylyltransferase (GALT) activity in humans results in Classic Galactosemia, and the GalT-deficient (GalT^-/-) mouse mimics the patient condition. GalT^-/- ovaries display elevated endoplasmic reticulum (ER) stress marker, BiP, and downregulated canonical phosphatidylinositol 3-kinase (Pi3k)/protein kinase B (Akt) growth/pro-survival signaling. Numbers of primordial follicles are reduced in the mutants, recapitulating the accelerated ovarian aging seen in human patients. We previously found that oral administration of the compound Salubrinal (an eIF2α phosphatase inhibitor), resulted in reduction of ovarian BiP expression, rescued Pi3k/Akt signaling, and a doubling of primordial follicles in GalT^-/- adults. Here, we further characterized galactosemic stress in GalT^-/- mice versus wild-type (WT) controls, and examined whether Salubrinal treatment improved broader reproductive parameters. We assessed the expression levels of factors of the unfolded protein response (UPR), and found that BiP, phospho-Perk, and phospho-eIF2α were all elevated in GalT^-/- ovaries. However, neither IKK activation (NFκB pathway) nor alternative Xbp1 splicing downstream of ER membrane protein Ire1α activation was induced, suggesting an Xbp1-independent UPR in galactosemic stress. Moreover, Salubrinal treatment significantly increased the number of ovulated eggs in mutant animals after gonadotrophic superovulation. Salubrinal treatment also normalized estrus cycle stage lengths and resulted in significantly larger litter sizes than vehicle-treated mutants. Overall, we show that Salubrinal protects against galactosemia-induced primordial follicle loss in a fashion that includes suppressing the de-phosphorylation of eIF2α, and that intervention in this way significantly improves and extends ovarian function, fertility, and fecundity.

Keywords: Classic Galactosemia; Galactosemic stress; Perk branch of the unfolded protein response (UPR); Pi3K/Akt signaling; Salubrinal; Subfertility.

Figure 1.. Schematic representation of GALT-deficiency induced Subfertility phenotype in the mouse model.

Dietary and Endogenously produced galactose is phosphorylated to galactose- 1 phosphate (gal-1P) by galactokinase (GALK1). In the presence of galactose–1 phosphate uridylyltransferase (GALT), gal-1P reacts with UDP- glucose to form UDP-galactose and glucose- 1 phosphate. Gal-1P accumulation causes ER stress and ovarian dysfunction. Salubrinal inhibits eIF2α dephophorylation, potentially ameliorating galactosemic stress, and normalizes the ovarian functions.

Figure 2.. Galactosemic stress induced eIF2α.phosphorylation in GalT- deficient mouse ovaries.

a) Protein expression levels of UPR marker peIF2α in WT and GalT-deficient mice ovaries (n=5 for WT and n=7 for GalT^−/−. P values are derived from Student’s t-test analyses, **P = 0.008). b) Comparison of level of unspliced (Xbp1u) spliced (Xbp1s)) Xbp1 in the ovaries of mutant and WT controls. First two lanes contain PCR products from mouse cells +/− ER stress inducer (2 mM DTT) as controls. c) Comparison of protein levels of pIkB α and IkBα in mutant and WT ovaries (n=4 in each group). P values are derived from Student’s t-test analyses, P = 0.103 for Total IkBα and P = 0.345 for pIkBα).

Figure 3.. Role of active Perk in eIF2α phosphorylation in GalT-deficient mouse fibroblasts

a) Quantification of phospho-eIF2α and its downstream targets Atf4 and Chop in GalT^−/− fibroblasts in the presence/absence of 1 μM GSK2606414 or 2 μg/ml tunicamycin. (Representative image from three independent experiments using three different cell lines that yielded identical results.) b) Expression level of Atf4 in GalT^−/− fibroblasts in the presence/absence of 50 μM Salubrinal or 300 nM ISRIB. c) Protein expression levels of Atf4 in WT and GalT^−/− mice fibroblasts (n=3 in each group).

Figure 4.. Salubrinal treatment resulted in normalizing the aberrant pro survival & UPR signaling and more mature follicles in GalT- deficient mouse ovaries.

a) Salubrinal improved the ovulation in GalT-deficient mice (WT=Wild type, KO=GalT^−/−) (n=4 for each group). P values are derived from two-way ANOVA followed by Tukey HSD, *the data are significant (p < 0.05). b) Salubrinal reversed the aberrant Pi3K/Akt and UPR signaling in ovaries from gonadotrophin-stimulated GalT-deficient mouse. Graphical representations on the right (n=4 for each group. P values are derived from Student’s t-test analyses, *p ≤ 0.05, **p ≤ 0.005).

Figure 5.. Salubrinal effectively normalized reproductive cycle in GalT-deficient mice.

Salubrinal normalized he lengths of individual cycle stages to lengths that would be expected in young wild-type mice.

Figure 6.. Salubrinal normalized the Time to pregnancy and improved litter size in GalT-deficient mice.

a) Impact of Salubrinal treatment on litter size. b) Effects of Salubrinal treatment on Time to Pregnancy (TTP) (n=5 in untreated, n=6 in vehicle treated and n=7 in Salubrinal treated. (P values are derived from one-way ANOVA analysis using GraphPad Prism, *p ≤ 0.0005,) c) Impact of Salubrinal treatment on body weights of the pups at pre-weaning age. d) Effects of Salubrinal treatment on organ weights of the pups at post weaning age.