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 The Hayflick limit (1962)

Hypothesis: Elimination of senescent cells has a rejuvenating affect on aged tissues Significance: Senescent cells accumulate with age at sites of age-related pathology

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

Pathways activated by BubR1 hypomorphism Tubulin p16 EyeAdipose tissue BubR1 +/+ BubR1 H/H p19 p21 p53

x BubR1 H/H p16 Ink4a -FKBP-Casp8 BubR1 H/H p16 Ink4a -FKBP-Casp8 BubR1 H/H p16 Ink4a -FKBP-Casp8 BubR1 H/H Aged mouse tissue Dysfunctional aged mouse tissue Healthy mouse tissue 1-month-old mouse Senescent cell Methalloproteinases, Cytokines, etc… Dysfunctional cell Functional cell Interbreeding Untreated (–AP) p16 Ink4a -FKBP-Casp8 BubR1 H/H Healthy mouse tissue 1-month-old mouse Treated (+AP) Experimental design

p16 Ink4a expression overlaps with the INK-ATTAC transgene BubR1 HH ; INK-ATTAC 3 GFP 5-month-old IAT FKBP-Casp8 rel. expression BubR1 H/H ;INK-ATTAC 3 INK-ATTAC 3 SkM IAT Eye Liver Heart SkM IAT Eye Liver Heart EGFP relative expression SkM IAT Eye Liver Heart p21 Pai1 IL-6 p16 GFP intensity % of cells BubR1 H/H ; INK-ATTAC 3 BubR1 H/H ; INK-ATTAC 3 GFP+ Relative expression IAT BubR1 H/H ; INK-ATTAC 3 GFP–

x BubR1 H/H p16 Ink4a -FKBP-Casp8 BubR1 H/H p16 Ink4a -FKBP-Casp8 BubR1 H/H p16 Ink4a -FKBP-Casp8 BubR1 H/H Aged mouse tissue Dysfunctional aged mouse tissue Healthy mouse tissue 1-month-old mouse Senescent cell Methalloproteinases, Cytokines, etc… Dysfunctional cell Functional cell Interbreeding Untreated (–AP) 9-months-old p16 Ink4a -FKBP-Casp8 BubR1 H/H Healthy mouse tissue 1-month-old mouse Treated (+AP) 9-months-old Experimental design

Senescent cell clearance delays age-related dysfunction H/H;ATTAC 3 +AP H/H;ATTAC 3 +AP H/H;ATTAC 3 –AP H/H;ATTAC 3 –AP 9-months-old females Thickness ( μ m) Cell diameter ( μ m) Fiber diameter ( μ m) IATDermisAdiposeGastroABD * ** * * * H/H;ATTAC 3 –AP H/H;ATTAC 3 +AP H/H;ATTAC 5 –AP H/H;ATTAC 5 +AP

H/H;ATTAC 3 –AP H/H;ATTAC 3 +AP H/H;ATTAC 5 –AP H/H;ATTAC 5 +AP Running time (s) Distance (m) * * Energy (J) * **

INK-ATTAC clears senescent cells SA- β -Gal H/H;ATTAC 3 –AP H/H;ATTAC 3 +AP H/H;ATTAC 5 –AP H/H;ATTAC 5 +AP – AP20187 INK-ATTAC 3 + Rosiglitazone SA-  -GAL + Rosiglitazone + AP20187 SA-  -GAL INK-ATTAC 3 There are fewer indicators of senescence in skeletal muscle and eye as well

Goals for my fellowship 1.Test whether removal of senescent cells from aged tissues has rejuvenating potential

x BubR1 H/H p16 Ink4a -FKBP-Casp8 BubR1 H/H p16 Ink4a -FKBP-Casp8 BubR1 H/H p16 Ink4a -FKBP-Casp8 BubR1 H/H Rejuvenated mouse tissue Dysfunctional aged mouse tissue Healthy mouse tissue 1-month-old mouse AP20187 treatment (clearance of senescent cells) 2 to 5-month- old mouse Premature aging 8-month-old mouse Normal cell Senescent cell Methalloproteinases, Cytokines, etc… Dysfunctional cell Functional cell Interbreeding

Goals for my fellowship 1.Test whether removal of senescent cells from aged tissues improves function 1.Determine if elimination of senescent cellsdelays or prevents tumorigenesis 1.Test whether removal of senescent cells from aged tissues improves function 1.Determine if elimination of senescent cellsdelays or prevents tumorigenesis

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

INK-ATTAC- targeting the cancer microenvironment x MMTV-rtTA;NeuNT MTB-TAN p16 Ink4a -FKBP-Casp8 MTB-TAN p16 Ink4a -FKBP-Casp8 MTB-TAN Dysfunctional aged mouse tissue Healthy mouse tissue Aging Normal cell Senescent cell Methalloproteinases, Cytokines, etc… Dysfunctional cell Interbreeding Moody et. al., Cancer Cell, 2002 Clear senescent cells, then activate the oncogene for 6 weeks with doxycycline water to induce mammary tumors

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