Genotoxic Stress Abrogates Renewal of Melanocyte Stem Cells by Triggering Their Differentiation

1. Genotoxic Stress Abrogates Renewalof Melanocyte Stem Cells byTriggering Their Differentiation Ken Inomata,Takahiro Aoto,Nguyen Thanh Binh,Natsuko Okamoto, Shintaro Tanimura, Tomohiko Wakayama,Shoichi Iseki,Eiji Hara,Takuji Masunaga,Hiroshi Shimizu,and Emi K. Nishimura Cell 137, 1088–1099, June 12, 2009 Elsevier Inc. Presented by: Abhinav Parivesh M.Sc (Sem III), MHG

2. Stem Cells • Stem Cells are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. Stem Cell Embryonic Adult Somatic Germline HSC Muscle SC Melanocyte Stem Cells etc.

3. Hair Cycle

4. DNA Damage Response (DDR) Human DSB Replication fork arrest/SSB Sensors ATM ATM ATR-ATRIP ATR/ATRIP dimer monomer RPA MDC1 Claspin Mediators 53BP1 RSR,RAD17 9-1-1 complex TopBP1 TransducersChk2 Chk1 Effectorsp53,Brca1,Nbs1,FANCD2,Cdc25s….. Cell Cycle arrest BRCA1 Lahiri M

5. Some facts… • Stem Cell senescence and/or apoptosis are thought to be two major cellular mechanisms for stem cell depletion following DNA Damage. • MSC in Hair Follicles are continuously renewed in the Hair regeneration cycle.This cycle also involves melanocyte maturation,mediated by a lineage differentiating program involving MITF under the control of α-MSH. • Dct-lacZ+/KITlow melanoblasts can be used as markers for MSC lineage tracing. • Hair Graying is one of the most obvious sign of aging. Maintenance of MSC population becomes incomplete with aging, causing physiological hair graying.

6. The onset of hair graying is preceded by the appearance of ectopically pigmented melanocytes (EPMs), which have a dendritic morphology, in the niche, suggesting that MSCs are differentiated in the niche. • Premature hair graying is seen in progeroid syndromes such as Werner’s syndrome and Ataxia-telangiectasia (AT) as well as after ionizing radiation (IR) which damages DNA and causes DNA DSBs. • Inactivation of ‘‘caretaker genes’’,including ATM, causes genomic instability due to an inefficient DDR and results in progeria that segmentally mimics physiological aging

7. The paper talks about… • The cause(s) underlying the aging phenomenon of hair and the possible involvement of the DNA damage response in the appearance of EPMs have not been studied. This study examines the impact of DNA Damage in quiescent MSCs in vivo and the role of caretaker and gatekeeper genes in the determination of the fate of damaged stem cells as well as the involvement of DNA damage in the hair graying phenotype

8. Experimental Procedures • Transgenic and Knockout mice • Whole-Body X-Ray irradiation of mice • Immunohistochemical Analysis • TUNEL Assay • Senescence-Associated-β-Galactosidase Staining Assay • Electron Microscopy

9. Results

10. Morphological change of MSC to EPM after IR. Histochemical staining changes of DctlacZ/KIT marked MSC from colorless to brownish black because of melanogenesis after IR.

11. Depletion of MSCs because of irradiation as IR abrogates renewal of MSCs in the niche by inducing their differentiation into EPMs. Depigmentation of hair in subsequent hair cycles.

12. EPMs are induced due to exogenous (chemicals) as well as • endogenous (TTD mice) genotoxic agents. • As a result renewal of MSCs become defective,it gets depleted and • leads to hair graying in subsequent hair cycles.

13. EPMs appear • in the bulge at • Anagen IV,V • and disappear at • Anagen VI.

14. Epidermal melanocytes in hairless tail skin show no significant changes in cellular morphology, pigmentation and number after IR.

15. IR induces DDR and DNA-Damage foci formation. IR induced H2AX foci remain in KIT+ EPMs in the bulge area even in mid-anagen follicles (anagen IV) Not much difference in foci formation with/without telogen hair plucking

16. IR induces localisation of ATM from cytoplasm to nuclei. p ATM is induced only in response to IR and localised in nuclei only.

17. No increase in levels of cleaved caspase 3 or TUNEL positivity observed after IR. No SA-β-gal expression after IR in EPMs. • Apoptosis/Senescence is not likely to be the major early fate of DNA-damaged • MSCs. • p53 or p16INK4a-Rb pathways have been implicated in the fate determination of cells under genotoxic stress including IR.

18. p53 , p19ARF , p16INK4a , are not required for ectopic differentiation of MSCs in the niche to EPMs and resulting hair graying. IR-induced fate of MSCs is distinct from cellular senescence or apoptosis and is mediated by different signaling pathways.

19. IR irradiated KIT+ MSCs show prolonged expression of master regulator of melanocyte lineage,MITF, and downstream melanogenic enzymes Tyrosinase and Tyrosinase-related-protein. MSCs initiate differentiation in the niche to EPMs via the canonical signaling pathways for melanocyte lineage differentiation. Induction of EPMs is a specific event in activated MSCs at early anagen or is due to their niche microenvironment.

20. Typical stage II–III Melanosomes which are found only in melanin-synthesizing melanocytes but not in keratinocytes,were present in addition to abundant stage IV melanosomes in EPMs. This finding provides functional evidence of melanocyte maturation with EPMs and proves that MSCs in the niche undergo differentiation in response to IR to become pigment-producing dendritic melanocytes through melanosomal maturation.

22. ATM deficiency sensitizes MSCs to differentiate in the niche, resulting in premature hair graying. • ATM efficiently protects MSCs from differentiation at low doses of IR. • DNA-damage repair is inefficient in Atm-deficient mice as well as in vitro and that irreparable DNA-damage-induced signals may trigger the differentiation of damaged stem cells at the time of stem cell renewal to maintain the quality of the stem cell pool. • DDR is involved in the determination of the fate of MSCs, suggesting the existence of a ‘‘stemness checkpoint’’ to maintain the stem cell quality and to prevent hair graying.

23. Discussion

24. Genotoxic Stress Triggers a Melanocyte Differentiation Program in MSCs. • IR induces the prolonged expression of melanogenic genes downstream of MITF and melanosome maturation in MSCs, indicating that MSCs mature into EPMs in the niche in response to IR. • IR-induced melanogenesis in MSC progeny in the niche depends on the Mc1r-mediated melanogenesis pathway, which is commonly used for melanocyte maturation and eumelanin pigment synthesis. • p53 pathway is transiently activated in MSCs after DNA damage but is not required for the induction of EPMs in the niche. • the nodal point for damaged stem cell fate determination is located upstream of Mc1r-mediated melanogenic processes and is independent of the p53 pathway

25. Stemness Checkpoint • p53, p16INK4a, or p19ARF are not required for the commitment of MSCs to differentiate in the niche in response to DNA damage. • ATM and ATR deficiency have been shown to degrade tissue renewal capacity through stem cell depletion with or without exogenous genotoxic stress. • ATM efficiently protects MSCs from their differentiation in the niche by activating the downstream DDR pathways. • DNA-damage detection/repair machineries that serve as ‘‘caretakers’’ of the mammalian genome maybe functioning as a stemness checkpoint in some somatic stem cell systems.

26. Genotoxic Stress-Induced MSC DifferentiationUnderlies an Aging-Related Phenotype, Hair Graying

27. In a Nutshell…. • Genotoxic stress abrogates MSC renewal and triggers its differentiation into EPMs. • This leads to depletion of MSC population in subsequent hair cycles and onset of aging related phenotype- Hair Graying. • Genotoxic stress like IR induces prolonged expression of melanogenic genes downstream of MITF leading to formation of melanocytic microenvironment in niche which favors differentiation of MSCs to EPMs. • ATM acts as stemness checkpoint.

28. THANK YOU!