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2011 Brookdale Foundation Retreat Senescent cell clearance in healthspan extension Darren Baker

2011 Brookdale Foundation Retreat Senescent cell clearance in healthspan extension Darren Baker Assistant Professor of Biochemistry and Molecular Biology Brookdale Leadership in Aging Fellow 2011 Tuesday June 7, 2011. Mayo Clinic Kogod Center on Aging. Cellular senescence.

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2011 Brookdale Foundation Retreat Senescent cell clearance in healthspan extension Darren Baker

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  1. 2011 Brookdale Foundation Retreat Senescent cell clearance in healthspan extension Darren Baker Assistant Professor of Biochemistry and Molecular Biology Brookdale Leadership in Aging Fellow 2011 Tuesday June 7, 2011 Mayo Clinic Kogod Center on Aging

  2. Cellular senescence The Hayflick limit (1962)

  3. Young tissue (fully functional) Old tissue (dysfunctional) Cell damage Dysfunctional cell Normal (healthy) cell Senescent cell Methalloproteinases, Cytokines, etc… Significance: Senescent cells accumulate with age at sites of age-related pathology Hypothesis: Elimination of senescent cells has a rejuvenating affect on aged tissues

  4. BubR1 hypomorphic mice- a progeroid mouse model 100% 60% 30% 10% 4% Impaired wound healing Reduced adipose tissue Reduced dermal thickness H/H H/H –/H –/H +/H +/H +/+ +/+ +/– +/– BubR1 Lordokyphosis Gliosis Arterial stiffening Impaired respiration Bilateral cataracts Infertility Craniofacial dysphorisms 4-5 fold reduced median and maximum lifespan Sarcopenia (muscle atrophy) Reduced stress tolerance

  5. Pathways activated by BubR1 hypomorphism Eye Adipose tissue BubR1+/+ BubR1H/H BubR1+/+ BubR1H/H p16 p19 p21 p53 Tubulin INK-ATTAC p16Ink4a promoter Cell membrane FKBP Caspase 8-Flag IRES EGFP M M M M -2617 M M + AP20187 (dimerization) FKBP Myristoylation Inactive Caspase 8 Caspase 8 Active Caspase 8 Apoptosis

  6. Experimental design x BubR1H/H p16Ink4a-FKBP-Casp8 Interbreeding 1-month-old mouse 1-month-old mouse Healthy mouse tissue Healthy mouse tissue p16Ink4a-FKBP-Casp8 p16Ink4a-FKBP-Casp8 BubR1H/H BubR1H/H Untreated (–AP) Treated (+AP) Dysfunctional aged mouse tissue Aged mouse tissue Dysfunctional cell p16Ink4a-FKBP-Casp8 p16Ink4a-FKBP-Casp8 BubR1H/H BubR1H/H Senescent cell Methalloproteinases, Cytokines, etc… Functional cell

  7. p16Ink4a expression overlaps with the INK-ATTAC transgene INK-ATTAC3 BubR1H/H;INK-ATTAC3 INK-ATTAC3 FKBP-Casp8 rel. expression EGFP relative expression GFP BubR1HH; INK-ATTAC3 BubR1H/H; INK-ATTAC3 GFP– INK-ATTAC3 IAT IAT Eye IAT Eye Eye SkM SkM Liver SkM Liver Liver Heart Heart Heart BubR1H/H; INK-ATTAC3 BubR1H/H; INK-ATTAC3 GFP+ % of cells Relative expression IAT 5-month-old IAT GFP intensity p21 IL-6 p16 Pai1

  8. Experimental design x BubR1H/H p16Ink4a-FKBP-Casp8 Interbreeding 1-month-old mouse 1-month-old mouse Healthy mouse tissue Healthy mouse tissue p16Ink4a-FKBP-Casp8 p16Ink4a-FKBP-Casp8 BubR1H/H BubR1H/H Untreated (–AP) 9-months-old Treated (+AP) 9-months-old Dysfunctional aged mouse tissue Aged mouse tissue Dysfunctional cell p16Ink4a-FKBP-Casp8 p16Ink4a-FKBP-Casp8 BubR1H/H BubR1H/H Senescent cell Methalloproteinases, Cytokines, etc… Functional cell

  9. Senescent cell clearance delays age-related dysfunction 9-months-old females H/H;ATTAC3 +AP H/H;ATTAC3 –AP ** * * ** * ** * Fiber diameter (μm) Cell diameter (μm) Thickness (μm) H/H;ATTAC3–AP H/H;ATTAC3 +AP H/H;ATTAC5 –AP H/H;ATTAC5 +AP IAT Dermis Adipose Gastro ABD

  10. ** ** ** Running time (s) * Distance (m) H/H;ATTAC3–AP Energy (J) * * H/H;ATTAC3 +AP H/H;ATTAC5 –AP H/H;ATTAC5 +AP

  11. INK-ATTAC clears senescent cells INK-ATTAC3 INK-ATTAC3 + Rosiglitazone + Rosiglitazone – AP20187 + AP20187 SA-b-GAL SA-b-GAL H/H;ATTAC3 –AP H/H;ATTAC3 +AP H/H;ATTAC5 –AP H/H;ATTAC5 +AP SA-β-Gal There are fewer indicators of senescence in skeletal muscle and eye as well

  12. Goals for my fellowship • Test whether removal of senescent cells from aged tissues has rejuvenating potential

  13. x BubR1H/H p16Ink4a-FKBP-Casp8 Interbreeding Healthy mouse tissue 1-month-old mouse p16Ink4a-FKBP-Casp8 Normal cell BubR1H/H Premature aging Dysfunctional aged mouse tissue 2 to 5-month-old mouse p16Ink4a-FKBP-Casp8 Dysfunctional cell BubR1H/H Senescent cell Methalloproteinases, Cytokines, etc… AP20187 treatment (clearance of senescent cells) Rejuvenated mouse tissue 8-month-old mouse p16Ink4a-FKBP-Casp8 Functional cell BubR1H/H

  14. Goals for my fellowship • Test whether removal of senescent cells from aged tissues improves function • Determine if elimination of senescent cellsdelays or prevents tumorigenesis

  15. INK-ATTAC transgene- targeting cancer cells? Collado et. al., Nature, 2005

  16. INK-ATTAC- targeting the cancer microenvironment x MMTV-rtTA;NeuNT MTB-TAN p16Ink4a-FKBP-Casp8 Interbreeding Healthy mouse tissue p16Ink4a-FKBP-Casp8 MTB-TAN Normal cell Aging Dysfunctional aged mouse tissue p16Ink4a-FKBP-Casp8 MTB-TAN Dysfunctional cell Senescent cell Methalloproteinases, Cytokines, etc… Clear senescent cells, then activate the oncogene for 6 weeks with doxycycline water to induce mammary tumors Moody et. al., Cancer Cell, 2002

  17. Acknowledgements Jan van Deursen laboratory Tobias Wijshake Tyler Mann Kogod Center on Aging Jim Kirkland Tamar Tchkonia Tamar Pirtskhalava Nathan LeBrasseur

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