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Aging-induced MCPH1 translocation activates necroptosis and impairs hematopoietic stem cell function

Nature Aging volume 4, pages 510–526 (2024)Cite this article

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Abstract

DNA damage contributes to the aging of hematopoietic stem cells (HSCs), yet the underlying molecular mechanisms are not fully understood. In this study, we identified a heterogeneous functional role of microcephalin (MCPH1) in the nucleus and cytoplasm of mouse HSCs. In the nucleus, MCPH1 maintains genomic stability, whereas in the cytoplasm, it prevents necroptosis by binding with p-RIPK3. Aging triggers MCPH1 translocation from cytosol to nucleus, reducing its cytoplasmic retention and leading to the activation of necroptosis and deterioration of HSC function. Mechanistically, we found that KAT7-mediated lysine acetylation within the NLS motif of MCPH1 in response to DNA damage facilitates its nuclear translocation. Targeted mutation of these lysines inhibits MCPH1 translocation and, consequently, compromises necroptosis. The dysfunction of necroptosis signaling, in turn, improves the function of aged HSCs. In summary, our findings demonstrate that DNA damage-induced redistribution of MCPH1 promotes HSC aging and could have broader implications for aging and aging-related diseases.

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Fig. 1: Dysfunction of Mcph1 mildly disturbs hematopoietic homeostasis.
Fig. 2: Mcph1−/− HSCs exhibit accumulated DNA damage and severely compromised reconstitution capacity.
Fig. 3: MCPH1 antagonizes necroptosis by interacting with RIPK3.
Fig. 4: Dysfunction of RIPK3 cannot rescue DNA damage accumulation in HSCs upon Mcph1 deletion.
Fig. 5: MCPH1 translocates to the nucleus in response to DNA damage.
Fig. 6: KAT7 mediates MCPH1 nuclear translocation by direct acetylation of MCPH1 in response to DNA damage.
Fig. 7: HSCs are susceptible to necroptosis during aging.

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Data availability

RNA-seq data are deposited in the Gene Expression Omnibus under accession code GSE217101. Proteomics data from mass spectrometry are deposited in the ProteomeXchange Consortium through iProX65 under dataset identifier PXD048580. Further information and requests for resources and reagents should be directed to and will be fulfilled by J.W.

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Acknowledgements

We thank X. Wang (National Institute of Biological Sciences) for providing Ripk3−/− mice. We thank G. -H. Liu (Institute of Zoology, Chinese Academy of Sciences) for providing Kat7 cDNA. We thank S. -J. Chen (Shanghai Jiao Tong University School of Medicine) for providing the 32D cell line. This work was supported by grants Z200022, 82250002, 92249305 and 2018YFA0800200 to J.W. from the National Key R&D Program of China or the Beijing Municipal Science & Technology Commission and the National Natural Science Foundation of China.

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Authors and Affiliations

  1. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

    Hanqing He & Jianwei Wang

  2. School of Pharmaceutical Sciences, Tsinghua University, Beijing, China

    Hanqing He, Yuqian Wang, Baixue Tang, Chou Wu & Jianwei Wang

  3. Institute of Precision of Medicine, Peking University Shenzhen Hospital, Shenzhen, China

    Qiongye Dong

  4. Department of Hematology, Xuanwu Hospital, Capital Medical University, Beijing, China

    Wanling Sun

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  1. Hanqing He
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Contributions

Conceptualization, J.W.; Methodology, H.H., Y.W., B.T., Q.D. and C.W.; Investigation, H.H., Y.W., B.T., Q.D., C.W. and W.S.; Formal Analysis, J.W. and H.H.; Resources, J.W.; Writing, J.W.; Funding acquisition, J.W.; Supervision, J.W.

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Correspondence to Jianwei Wang.

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Extended data

Extended Data Fig. 1 Dysfunction of Mcph1 mildly disturbs hematopoietic homeostasis at young age.

(a) This histogram shows the expression of MCPH1 in various immune cells. Data were download from ASCOT. (b) The schematic diagram showing the targeting strategy to generate Mcph1flox mice. (c) The schematic diagram showing the breeding strategy to generate Mcph1−/− (Vav1-Cre; Mcph1flox/flox) mice. (d-j) 2-5 months old Mcph1−/− or WT mice were analyzed for hematopoietic progenitor cells and HSCs. (d-e) The histogram depicts the frequency (d) and the absolute numbers (e) of HSCs (CD34- CD150+ KSL). n = 7 mice per group. (f-g) The histogram shows the frequency (f) and the absolute numbers (g) of CMPs, GMPs, and MEPs. n = 7 mice per group. (h-i) The histogram shows the frequency (h) and the absolute numbers (i) of CLPs. n = 4 mice per group. (j) The scatter plots show the bone marrow (BM) cellularity of Mcph1−/− and WT mice. n = 7 mice per group. (k-n) 3-4 months old Mcph1−/− or WT mice were analyzed for thymocyte. (k) Representative flow cytometry of thymocytes for CD4/CD8 expression. (l) The scatter plots show the total cell number of thymus. (m-n) The scatter plots depict the percentage (m) and absolute numbers (n) of DN (CD4−CD8−), DP (CD4+CD8+), CD4+, and CD8+ T cells in thymus. n = 6-7 mice per group (WT: n = 7; Mcph1−/−: n = 6). (o-r) 3-4 months old Mcph1−/− or WT mice were analyzed for splenocytes. (o) Representative flow cytometry. (p) The scatter plots show the total cell number of the spleen. (q-r) The scatter plots depict the percentage (q) and absolute numbers (r) of myeloid, B, CD4+ T and CD8+ T cells in spleen. n = 6-7 mice per group (WT: n = 7; Mcph1−/−: n = 6). Two-tailed unpaired Student’s t-test was used for statistical analysis in l-n and q-r. Data are shown as mean ± SD in d-j, l-n and p-r.

Source data

Extended Data Fig. 2 Mcph1 dysfunction severely impairs the reconstitution capacity of HSCs.

(a-f) Freshly isolated 20 HSCs from 4-month old Mcph1−/− mice or age-matched WT mice were transplanted into lethally irradiated recipients together with 3 × 105 competitor cells. Chimera in peripheral blood was checked every month until the 3rd month. (a) This histogram displays the percentage of donor cell reconstitution in overall (CD45.2+), B (B220+), T (CD3+) and myeloid (Mac-1+) cell every month after transplantation. (b and d) The scatter plots show the percentage of donor cell reconstitution in bone marrow (b) and HSC (d). (c) This histogram displays the lineage distribution in donor-derived bone marrow at the 3rd month. (e) This histogram displays the percentage of donor cell reconstitution in erythroid progenitors (Ter119+) every month after transplantation. (f) the scatter plots show the percentage of donor cell reconstitution in erythroid progenitors of bone marrow. n = 6-7 mice per group in a and e (n =6 for WT, n =7 for Mcph1−/−). n = 6 mice per group in b, c, d, and f. Data were shown as mean ± SD. Two-tailed unpaired Student’s t-test was used for statistical analysis.

Source data

Extended Data Fig. 3 MCPH1 antagonizes necroptosis through interaction with RIPK3.

(a-b) 4 months old Mcph1−/− or WT mice were analyzed for cell death of fresh HSCs without ex vivo culture by Annexin-V and DAPI staining. (a) Representative flow cytometry. (b) Percentage of early stage of cell death (DAPI− Annexin-V+) in Mcph1−/− or WT HSCs. n = 4-5 mice per group (WT: n = 5; Mcph1−/−: n = 4). (c) The images show morphological alteration of WT and Mcph1−/− HSCs (3 months). The experiment was repeated 3 times. (d) Representative western blot shows the interaction between MCPH1 and RIPK3 in response to necroptotic stimuli. L929 cells stably expressing Flag-tagged MCPH1 were treated with T+Z for 1 hour. Cell lysates were immunoprecipitated with anti-Flag antibody, and western blot was performed with indicated antibodies. The experiment was repeated 3 times. (e) Interaction between MCPH1 and p-RIPK3 in HEK293T cells co-transfected with SFB-tagged MCPH1 and Myc-tagged RIPK3, demonstrated by co-immunoprecipitation using S beads and subsequent western blot analysis with specific antibodies. The experiment was repeated 3 times. (f) SFB-RIPK3 were transfected into HEK293T cells together with GFP-MLKL and Myc-MCPH1-WT or Myc-MCPH1-Δ624–882 constructs as indicated. Cells were subjected to immunoprecipitated with S beads and western blot analysis was performed with indicated antibodies. Representative western blot shows that MCPH1-WT but not MCPH1-Δ624-882 inhibits interaction between p-RIPK3 and MLKL. The experiment was repeated 3 times. (g) Co-immunoprecipitation assay in HEK293T cells transiently transfected with SFB- MCPH1 and either Myc-tagged wild-type or T231A-S232A RIPK3, revealing a reduced interaction between MCPH1 and the RIPK3T231A-S232A mutant, as indicated by western blot analysis. The experiment was repeated 3 times. (h) The mRNA expression of Ripk3 in aged (23 months) Mcph1−/− and WT HSCs. n = 3 independent experiments. (i) Representative western blot showing the expression of p-S6, S6, p-4EBP1, 4EBP1, and MCPH1 in aged (21–26 months) Mcph1−/− and WT HSCs. The experiment was repeated twice. Two-tailed unpaired Student’s t-test was used for statistical analysis in b. Data are shown as mean ± SD in b and h.

Source data

Extended Data Fig. 4 Dysfunction of Ripk3 partially rescues the deleterious impact of Mcph1 deletion on HSCs during short-term ex vivo culture.

(a-c) Freshly isolated KSL cells either from Ripk3−/− mice (2 months old) or age-matched WT mice were infected by lentivirus carrying Mcph1 shRNA or scramble control (shRNA expressing lentiviruses coexpressed GFP), and 72 hours later, FACS-purified 100 GFP+ CD48− Sca1+ cells were cultured in SFEM medium. 7 days later, the proliferation capacity was evaluated by examining the colony size and cell number. (a) Experimental design. (b) Representative images of ex vivo-cultured HSCs carrying the indicated shRNA on day 7. Scale bar, 50 μm. (c) The scatter plots depict the cell numbers of ex vivo-cultured HSCs carrying the indicated shRNA on day 7. Statistical significance was measured by two-way ANOVA followed by Sidak′s test for post hoc comparison. n = 18–20 independent experiments (n = 20 for WT: shCon, n = 19 for WT: shMcph1, n = 18 for Ripk3−/−: shCon, n = 20 for Ripk3−/−: shMcph1). Data were shown as mean±SD.

Source data

Extended Data Fig. 5 Nuclear translocation of MCPH1 in response to DNA damage.

(a-b) Cells were treated with 100 μM etoposide and 1 hour later, cell lysates were separated into cytoplasmic and nuclear fractions. The subcellular distribution of MCPH1 were analyzed by western blot. GAPDH and H3 were used to detect the purity of cytoplasmic and nuclear fractions respectively. (a) EL4 cells. (b) 32D cells. The experiment was repeated 3 times. (c) KSL cells were treated with 100 μM etoposide and 1 hour later, cell lysates were separated into cytoplasmic and nuclear fractions. The subcellular distribution of RIPK3 were analyzed by western blot. The experiment was repeated 3 times.

Source data

Extended Data Fig. 6 KAT7-dependent acetylation of MCPH1 facilitating nuclear translocation in response to DNA damage.

(a) HEK293T cells were either mock treated or treated with 100 μM etoposide with or without 1 μM KAT7 inhibitor for 1 hours. cell lysates were separated into cytoplasmic and nuclear fractions. The subcellular distribution of MCPH1 were analyzed by western blot. The experiment was repeated 3 times. (b) HEK293T cells were transiently transfected with SFB-tagged wild-type MCPH1 or indicated K/R mutants together with Myc-tagged KAT7. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. Representative western blot shows the acetylation level of wild-type or various K/R mutants of MCPH1. The experiment was repeated twice. (c) HEK293T cells were transiently transfected with SFB-tagged wild-type MCPH1 or MCPH1K348/350R, MCPH1K360/362R, MCPH1K364/365R, MCPH1K360/362/364/365R together with Myc-tagged KAT7. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. Representative western blot shows the acetylation level of wild-type or indicated mutants of MCPH1. The experiment was repeated 3 times. (d) HEK293T cells were transiently transfected with SFB-tagged wild-type MCPH1 or K348R-K350R-K360R-K362R mutant together with Myc-tagged KAT7. 24 hours later, cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. Representative western blot shows the interaction between MCPH1K348/350/360/362R and MCPH1 with KAT7. The experiment was repeated 3 times. (e) HEK293T cells were transiently transfected with SFB-tagged wild-type MCPH1 or K348R-K350R-K360R-K362R mutant together with Myc-tagged RIPK3. 24 hours later, cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. Representative western blot shows the interaction between MCPH1K348/350/360/362R and MCPH1 with RIPK3. The experiment was repeated twice. (f) MCPH1- and MCPH1K348R-K350R-K360R-K362R-expressed L929 cells were treated with etoposide + zVAD (100 μM etoposide, 20 μM zVAD) for 20 hours. Representative western blot shows the expression of p-MLKL, MLKL, p-RIPK3, RIPK3, and MCPH1 in WT or K348R-K350R-K360R-K362R mutant MCPH1 overexpressed cells upon etoposide + zVAD treatment. The experiment was repeated 3-4 times.

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Extended Data Fig. 7 Enhanced susceptibility of HSCs to necroptosis during aging.

(a) The images show morphological alteration of in Mlkl−/− and WT HSCs at young (3 months) and old (29 months) age. (b) The scatter plots depict the percentage of necroptotic cell death in Mlkl−/− and WT HSCs at young (3 months) and old (29 months) age. Data were shown as mean ± SD. Statistical significance was measured by two-way ANOVA followed by Tukey’s test for post hoc comparison. n = 3 independent experiments. (c-f) KSL cells were freshly isolated from young (5 months) or old (18 months) mice were infected by lentivirus carrying the cDNA of WT MCPH1 or MCPH1- K348R-K350R-K360R-K362R mutant (MCPH14KR), and 72 hours later, 2 × 104 GFP+ cells were sorted and injected into lethally irradiated recipients together with 2 × 105 competitor cells. Peripheral blood of recipient mice was evaluated every month until the 4th month. This histogram displays the percentage of GFP+ cell in overall (CD45.2+), B (B220+), T (CD3+) and myeloid (Mac-1+) cell every month after transplantation. n = 6-7 mice per group (n = 6 for Young: Vector, Young: MCPH1-WT, Old: Vector, and Old: MCPH1-WT; n = 7 for Young: MCPH1-4KR and Old: MCPH1-4KR), data were shown as mean ± SD. Two-tailed unpaired Student’s t-test was used for statistical analysis.

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Extended Data Fig. 8 Representative flow cytometry gating strategy used in this study.

(a) Representative flow cytometry showing the gating strategy used to isolate and analyze the hematopoietic stem and progenitor cell populations in mouse bone marrow presented on Figs. 1a,b,2a–h,3a–h,l,o,4a, b, j, k,5a, b,d–f,6n–o, F,7a–d, g–i, j, l and Extended Data Figs. 1d–h, 2a, d, 3a–c, h, i, 4a, b, 5c, 7a–f. (b) Strategy used to evaluate the the percentage of B, myeloid, CD4+ T, CD8+ T cells presented on Fig. 1d, e. (c-d) Strategy used to evaluate the the percentage (c) and lineage distribution (d) of test donor-derived cells (myeloid, B and T cells) presented on Fig. 2f–g, 4c–i, 7g, j, k, l, m, and Extended Data Figs. 2a–c, 7c–f. (e) Strategy used to evaluate the the percentage of test donor-derived erythroid progenitors (Ter119+) presented on Extended Data Fig. 2e, f.

Supplementary information

Supplementary Information

Working model

Reporting Summary

Supplementary Tables 1–3

Supplementary Table 1. GSEA gene list; Supplementary Table 2. Oligonucleotides; Supplementary Table 3. Materials;

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He, H., Wang, Y., Tang, B. et al. Aging-induced MCPH1 translocation activates necroptosis and impairs hematopoietic stem cell function. Nat Aging 4, 510–526 (2024). https://doi.org/10.1038/s43587-024-00609-z

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