in adult mice, both lines (Gpx4wt/cys and Gpx4wt/ser (8)) were then cross-bred with TAM-inducible Rosa26CreERT2 mice (Gpx4flox/flox;Rosa26CreERT2 referred to as PZ (148)). This strategy would thus allow to bypass the early death of Gpx4cys/cys mice and to perform studies in adult animals. First, the frt-flanked neomycin phosphotransferase gene was removed from the transgenic alleles by mating Gpx4wt/cys (F3) and Gpx4wt/ser (F5) mice with Rosa26_Flpe animals expressing Flpe recombinase (Fig. 27A). Positive animals were backcrossed with C57BL/6J animals and finally mated with Gpx4flox/flox;Rosa26CreERT2 mice yielding Gpx4flox/cys;Rosa26CreERT2, Gpx4flox/ser;Rosa26CreERT2 and Gpx4flox/wt;Rosa26CreERT, mice, respectively (further referred to as Gpx4fl/cys, Gpx4fl/ser, Gpx4fl/wt). At the age of two months, CreERT2-mediated deletion of the loxP-flanked Gpx4 allele was induced by TAM-injection. Reportedly, induced PZ animals died around two weeks after TAM-injection due to massive kidney proximal tubular cell death and acute renal failure (ARF) (148).
Figure 27: Adult animals survive expressing only a Cys Gpx4 allele. (A) Breeding scheme describing the mating steps of Gpx4wt/ser or Gpx4wt/cys with a mouse strain expressing Flpe recombinase under the control of the Rosa26 promotor in order to remove the neomycin phosphotransferase gene (neo). Genotyping of positive animals was performed with a PCR that detects the wt region yielding either a product of 256 bp (wt) or two products of 256 bp and 310 bp (tg). In the final breeding step, Gpx4wt/ser or Gpx4wt/cys animals with a successful removal of the neo gene were mated with a strain expressing a loxP-flanked Gpx4 allele and an inducible Cre
recombinase under the control of the Rosa26 promotor yielding Gpx4flox/flox;Rosa26CreERT2, Gpx4flox/cys;Rosa26CreERT2, Gpx4flox/ser;Rosa26CreERT2 and Gpx4flox/wt;Rosa26CreERT, mice, respectively (further referred to as Gpx4fl/cys, Gpx4fl/ser, Gpx4fl/wt). Animals were genotyped by a PCR yielding one band with 310 bp (fl/cys or fl/ser) or two bands with 256 bp and 310 bp (fl/wt) (Yellow triangle
= Frt site; red triangle = loxP site). (B) A Kaplan-Meier survival analysis showed that Gpx4fl/ser animals died between 12-14 days after TAM injections, whereas Gpx4fl/cys animals survive like the wt controls (Statistical analysis was assessed using Mantel-cox test ****P<0.0001). (C) Immunoblot analysis of kidney tissue derived from Tam-induced Gpx4wt/ser, Gpx4wt/wt or Gpx4wt/cys animals showed a loss of GPX4 expression in Gpx4wt/ser compared to Gpx4wt/cys mice. (D) Immunohistochemical and histopathological analysis of kidneys derived from TAM injected animals showed signs of ARF only in Gpx4fl/ser animals. H&E staining revealed proteinacous casts in renal tubular cells (arrows). Besides massive increase of TUNEL-positivety was present while the same sections were negative for active caspase 3 (act. caspase 3) staining. Partial loss of GPX4 expression was detected in kidneys isolated from Gpx4fl/ser mice (Scale bars left panel = 20 µm; other panels = 100 µm).
While induced Gpx4fl/ser mice resembled the phenoptye of PZ mice including proteinaceous casts in tubular cells and widespread cell death of tubular renal cells, the mice had to be sacrificed around 12-14 days after TAM-injection (Fig.
27 B,D). By stark contrast, Gpx4fl/cys mice survived throughout the entire observation period of 40 days without any signs of ARF (Fig. 27B,D). Western blot analysis of GPX4 expression in kidney samples derived from Tam-induced Gpx4fl/ser, Gpx4fl/cys Gpx4fl/wt animals revealed a decrease in GPX4 expression in Gpx4fl/ser animals compared to Gpx4fl/cys mice (Fig. 27C), which is in line with the immunostainings, where reduced levels of GPX4 were observed in the kidney of Gpx4fl/ser animals (Fig. 27D). These findings demonstrate that the Cys GPX4 mutant form allows mouse survival and can substitute for wt GPX4 in in adult kidney, an organ that is otherwise the most sensitive organ requiring GPX4 expression (148)
5 Discussion
The trace element Se was discovered by Jöns Jacob Berzelius 200 years ago (1). First suspected to be a toxic compound in the sixties of last century, it was then later recognized as an essential trace element for mammals (2,5).
Meanwhile, it is widely accepted that Se exerts its main function in biological systems as an integral part of the 21st amino acid Sec (19). Both, the biosynthesis and the decoding mechanism of Sec are now known to be highly complex and energetically very costly. On one hand, Sec first needs to be synthesized in several consecutive steps on its cognate tRNA[Ser]Sec, and on the other hand the decoding at the opal codon UGA necessitates a series of co-factors acting in cis and trans to allow for a successful co-translational incorporation of Sec in the nascent polypeptide chain (7).
Despite the recognition that Se in form of Sec is essential for mammalian life (116), the question concerning the actual advantage of selenothiol over thiol-based catalysis of many selenoenzymes has remained unanswered to large extent. Moreover, it is still unclear why many organisms, such as some eu- and archaebacteria, protozoon, nematodes, fly, birds, mammals and fish express selenoproteins, whereas other including yeast and higher plants use cysteine-containing homologs instead. In addition, it is still unknown why some organisms, including mammals, rely on selenoenzymes for proper embryogenesis, fertility, tissue development and protection of somatic tissues from cell death, whereas others including nematodes and flies apparently do not depend on selenoproteins for basic life. Among the 24 (25) selenoproteins expressed in mammals four of them have been identified to be of vital importance for murine embryonic development and survival. These are TXNRD1, TXNRD2, SELENOT and GPX4.
SelenoT KO mice die during early embryogenesis (222) for unknown reasons and Txnrd1 and Txnrd2 KO mice die during early embryogenesis at E8.5 - E9.5 and around midgestation (E13.5), respectively (76,77). Systemic KO of Gpx4 (E7.5) (149,150,152) causes embryonic lethality almost at the same stage as the simultaneous loss of all selenoproteins due to a KO of the Sec-specific tRNA gene Trsp (E6.5) (116), suggesting that GPX4 might represent the most important and limiting selenoprotein in mammals. Although the mechanism of embryonic death of Gpx4-/- mice is still not elucidated, it is well accepted that
GPX4 protects cells from ferroptosis in vivo and in vitro by controlling specific lipid oxidation events (147,148).
5.1 Homozygous expression of the Ser variant of GPX4 leads to