ALKBH7 drives a tissue and sex-specific necrotic cell death response following alkylation-induced damage

Abstract

Regulated necrosis has emerged as a major cell death mechanism in response to different forms of physiological and pharmacological stress. The AlkB homolog 7 (ALKBH7) protein is required for regulated cellular necrosis in response to chemotherapeutic alkylating agents but its role within a whole organism is unknown. Here, we show that ALKBH7 modulates alkylation-induced cellular death through a tissue and sex-specific mechanism. At the whole-animal level, we find that ALKBH7 deficiency confers increased resistance to MMS-induced toxicity in male but not female mice. Moreover, ALKBH7-deficient mice exhibit protection against alkylation-mediated cytotoxicity in retinal photoreceptor and cerebellar granule cells, two cell types that undergo necrotic death through the initiation of the base excision repair pathway and hyperactivation of the PARP1/ARTD1 enzyme. Notably, the protection against alkylation-induced cerebellar degeneration is specific to ALKBH7-deficient male but not female mice. Our results uncover an in vivo role for ALKBH7 in mediating a sexually dimorphic tissue response to alkylation damage that could influence individual responses to chemotherapies based upon alkylating agents.

Figure 1

ALKBH7-deficient human or mouse cells are resistant to alkylation-induced cell death by necrosis. (a) Human ALKBH7 gene structure and location of genomic sequences targeted by sgRNA-1 (sg1) and sgRNA-2 (sg2) for CRISPR-Cas9 mutagenesis. (b) DNA sequence of ALKBH7 gene alleles in stable 293T cell lines generated by CRISPR mutagenesis with control-sg or ALKBH7-sg1 and sg2 RNAs. The WT sequence targeted for mutagenesis by each sgRNA is highlighted. (c) Immunoblot analysis of ALKBH7 levels in the indicated 293T cell lines. (d) Viability of control-sg and ALKBH7-sg cell lines after treatment with the indicated concentration of MMS or t-bu-OOH, as measured by live/dead cell staining with propidium iodide coupled with flow cytometry. (e) Immunoblot of extracts prepared from WT or Alkbh7^−/− MEF cells probed against mouse ALKBH7. (f) WT or Alkbh7^−/− MEF cells were treated with the indicated agents followed by viability analysis by vital cell stain. (g) Viability of WT or Alkbh7^−/− MEF cells after treatment with MNNG in the absence or presence of either z-vad-fmk or nec-1. (h) Viability of WT or Alkbh7^−/− MEF cells after treatment with TNF-α in the absence or presence of z-vad-fmk or nec-1. For (d), (f) and (g and h); error bars represent the S.D. of three, four and two independent experiments, respectively. *P<0.05; **P<0.01; ***P<0.001

Figure 2

Sex-specific effects of ALKBH7 deficiency on whole-body MMS-toxicity. (a) Approximate LD₅₀ of the indicated mouse strains in response to MMS. Four independent studies were performed. (b) Representative images (20X) of bone marrow samples extracted from femurs 24 h following MMS treatment (150 mg/kg). (c) Depletion of bone marrow is indistinguishable between WT and Alkbh7^−/− mice following MMS-induced damage. The percent bone marrow depletion was determined by pathological examination of bone marrow samples as shown in (b). (d) Ex vivo bone marrow clonogenic survival assays were performed using bone marrow isolated from WT (n=6), Alkbh7^+/− (n=6) and Alkbh7^−/−(n=6) mice. Data represent the mean and S.D. For each of the genotypes, three male and three female mice were used in the experiments

Figure 3

Alkbh7 deficiency confers partial resistance against MMS-induced toxicity in retina photoreceptors. (a) H&E stained representative sections of WT or Alkbh7^−/− retina treated with either vehicle control or 75 mg/kg MMS and harvested at day 7. INL, inner nuclear layer. (b) Relative damage of the ONL assessed by histopathologic examination. Samples were categorized by increasing intensity of retinal degeneration ranging from minimal to severe and analyzed by chi-squared test. (c) Alkylation-induced retinal degeneration as determined by the thickness of the ONL. The number of cells in the ONL were counted for 25 representative areas of four sections of H&E-stained retina of the indicated mouse strains 7 days post-injection with 75 mg/kg MMS. Plotted is the average number of cells per ONL for each individual mouse, the mean (WT=4.3, Alkbh7^−/−=5.9, and Aag^−/−=11.2) and S.D. for each genotype. *P<0.05; **P<0.01; ***P<0.001

Figure 4

Alkbh7^−/− mice display a sexually dimorphic response to alkylation-induced cerebellar degeneration. (a) H&E-stained cerebellar granule tissue showing the spectrum of responses in mice 24 h after mock treatment (control) or injection with 150 mg/kg MMS. The relative level of damage was scored by pathological classification of CGN cell death based upon the occurrence of pyknotic nuclei (yellow arrows). (b) Bar graph representing the relative percentage of cerebellar pyknosis of each mouse strain after alkylation exposure as scored in (a). (c) Quantification of cellular death in the cerebellar granular layer of the indicated male or female mouse strains 24 h post-treatment with 150 mg/kg MMS. The percent pyknotic nuclei represents the average of the number of pyknotic nuclei per total number of cells across 15 fields of H&E-stained brain sections of an individual mouse strain. The mean (males: WT=12.4, Alkbh7^−/−=5.3; females: WT=8.6, Alkbh7^−/−=12.2) and S.D. is represented for each genotype. *P<0.05; **P<0.01; ***P<0.001

Figure 5

Cellular sensitivity of isolated cerebellar neurons from WT or Alkbh7^−/− mice in response to MMS. (a) Procedure for isolating cortical granule neurons (CGNs) from 6- to 8-day-old mouse pups followed by MMS treatment and viability analysis by microscopy. (b) Viability of the indicated CGNs after treatment with vehicle MMS or MMS with the caspase inhibitor, z-vad-fmk. (c) Viability of the indicated CGNs after treatment with vehicle MMS or MMS with the PARP1/ARTD1 inhibitor, veliparib. Data presented are from biological replicates, where each data point is the average of 2–4 wells with each well imaged 6–7 times