2011 Brookdale Foundation Retreat Senescent cell clearance in

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)

Young tissue (fully functional) Old tissue (dysfunctional) Cell damage Normal (healthy) cell Senescent cell Dysfunctional 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

Bub. R 1 hypomorphic mice- a progeroid mouse model –/H 4% H/H 10% H/H +/– 30% +/– +/H 60% +/H +/+ Impaired wound healing Reduced adipose tissue Reduced dermal thickness +/+ 100% Bub. R 1 Lordokyphosis Gliosis Arterial stiffening Impaired respiration Infertility Bilateral cataracts Craniofacial dysphorisms 4 -5 fold reduced median and maximum lifespan Sarcopenia (muscle atrophy) Reduced stress tolerance

Pathways activated by Bub. R 1 hypomorphism Eye Bub. R 1+/+ Adipose tissue Bub. R 1 H/H Bub. R 1+/+ Bub. R 1 H/H p 16 p 19 p 21 p 53 Tubulin INK-ATTAC p 16 Ink 4 a promoter -2617 FKBP Caspase 8 -Flag IRES EGFP M M Cell membrane M M + AP 20187 (dimerization) FKBP Caspase 8 Myristoylation Inactive Caspase 8 Apoptosis

Experimental design Bub. R 1 H/H x p 16 Ink 4 a-FKBP-Casp 8 Interbreeding 1 -month-old mouse Healthy mouse tissue p 16 Ink 4 a-FKBP-Casp 8 Bub. R 1 H/H Untreated (–AP) Treated (+AP) Dysfunctional aged mouse tissue Aged mouse tissue Dysfunctional cell p 16 Ink 4 a-FKBP-Casp 8 Bub. R 1 H/H Methalloproteinases, Cytokines, etc… Senescent cell Functional cell

p 16 Ink 4 a expression overlaps with the INK-ATTAC transgene Bub. R 1 H/H; INK-ATTAC 3 GFP intensity p 16 5 -month-old IAT Relative expression IAT % of cells Bub. R 1 H/H; INK-ATTAC 3 Sk. M IAT Eye Liver Heart IL-6 Bub. R 1 H/H; INK-ATTAC 3 GFP– Bub. R 1 H/H; INK-ATTAC 3 GFP+ INK-ATTAC 3 p 21 Bub. R 1 HH; INK-ATTAC 3 Sk. M IAT Eye Liver Heart GFP EGFP relative expression FKBP-Casp 8 rel. expression INK-ATTAC 3 Pai 1 INK-ATTAC 3

Experimental design Bub. R 1 H/H x p 16 Ink 4 a-FKBP-Casp 8 Interbreeding 1 -month-old mouse Healthy mouse tissue p 16 Ink 4 a-FKBP-Casp 8 Bub. R 1 H/H Untreated (–AP) 9 -months-old Healthy mouse tissue Treated (+AP) Dysfunctional aged mouse tissue Dysfunctional cell 9 -months-old Aged mouse tissue p 16 Ink 4 a-FKBP-Casp 8 Bub. R 1 H/H Methalloproteinases, Cytokines, etc… Senescent cell Functional cell

Senescent cell clearance delays age-related dysfunction 9 -months-old females H/H; ATTAC 3 +AP H/H; ATTAC 3 –AP * 40 30 20 300 45 ** 250 200 150 10 50 0 0 IAT Inguinal adipose * 40 Fiber diameter (μm) 50 350 ** Thickness (μm) Cell diameter (μm) 60 * ** 35 * 30 H/H; ATTAC 3–AP 25 H/H; ATTAC 3 +AP 20 H/H; ATTAC 5 –AP 15 H/H; ATTAC 5 +AP 10 5 Dermis Adipose 0 Gastro ABD Abdominal

700 * 600 500 400 300 ** 160 Distance (m) Running time (s) 800 3. 5 180 ** 140 120 * 100 80 60 200 40 100 20 Running time (s) 2. 5 2 * H/H; ATTAC 3 +AP H/H; ATTAC 5 +AP 0. 5 0 Distance (m) H/H; ATTAC 3–AP H/H; ATTAC 5 –AP 1. 5 1 0 0 ** 3 Energy (J) 900 Energy expended (J)

INK-ATTAC clears senescent cells INK-ATTAC 3 + Rosiglitazone + AP 20187 SA-b-GAL INK-ATTAC 3 + Rosiglitazone – AP 20187 H/H; ATTAC 3 +AP H/H; ATTAC 5 –AP H/H; ATTAC 5 +AP SA-β-Gal H/H; ATTAC 3 –AP 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

Bub. R 1 H/H x p 16 Ink 4 a-FKBP-Casp 8 Interbreeding Healthy mouse tissue 1 -month-old mouse p 16 Ink 4 a-FKBP-Casp 8 Normal cell Bub. R 1 H/H Premature aging Dysfunctional aged mouse tissue 2 to 5 -monthold mouse p 16 Ink 4 a-FKBP-Casp 8 Bub. R 1 H/H Dysfunctional cell Senescent cell Methalloproteinases, Cytokines, etc… AP 20187 treatment (clearance of senescent cells) Rejuvenated mouse tissue 8 -month-old p 16 Ink 4 a-FKBP-Casp 8 mouse Bub. R 1 H/H Functional cell

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

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

INK-ATTAC- targeting the cancer microenvironment MMTV-rt. TA; Neu. NT MTB-TAN x p 16 Ink 4 a-FKBP-Casp 8 Interbreeding Healthy mouse tissue p 16 Ink 4 a-FKBP-Casp 8 MTB-TAN Normal cell Aging Dysfunctional aged mouse tissue p 16 Ink 4 a-FKBP-Casp 8 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

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