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ICREA & Universitat Pompeu Fabra
Aging as a treatable condition:
New approaches to bring back
youthfulness to aged stem ce...
Increased risk of
tissue deterioration,
malfunction and
disease with aging
Disease, sarcopenia, frailty
Health
• Since 1900, lifespan has increased over 30 years
in men and women in western countries.
• So, in just 100 years, we have...
Frailty - Sarcopenia
Geriatric age
Geriatric age
Good mental condition
Muscle strength and
fitness
Healthy aging
Sarcopenia
• Sarco flesh (muscle)
• penia deficiency
Loss of skeletal muscles mass and
function (strength) with aging
Sarc...
40% loss of muscle mass between
20-70 years of age
6% decline in muscle mass at each
decade between 30-70 years
1.4 –2.5% ...
Loss of muscle regenerative capacity is
also maximal at geriatric age
(after 75-80 years)
Skeletal muscle,
regeneration and
aging
20000nm
Muscle fiber
Satellite cell
Pax7+Muscle fiber
ite cell
Basal lamina
10µm
Skeletal muscle and its stem cells
(satel...
Adult skeletal muscle homeostasis:
regeneration after injury
INJURY
Activation
Proliferation
Differentiation
Fusion
Self-r...
How do we study the regenerative
capacity of muscle stem cells in the lab ?
Injury by:
- Toxin injection
- Crushing
Formation of new fibers
In vivo model (mice): Regeneration
Muscle regeneration after one week
1 week2 days 4 days 3 weeks
Skeletal muscle has an high regenerative capacity
Skeletal muscle regenerative capacity
declines with aging
Extrinsic
influences
Parabiosis between 2 mice of different age:
old-young
Adapted from Conboy and Rando, Cell Cycle 2012
YOUNG MUSCLE OLD MUSCLE
Injury:
Efficient
proliferation/differentiation
Inj...
Major aims:
1. Understand muscle cell functions and
regenerative decline during physiological
aging, and especially at ger...
Exacerbated muscle regeneration defect in
geriatric mice, compared to old mice
CTX injury-
induced muscle
regeneration
Reduced regenerative capacity of geriatric satellite cells
after transplantation into young muscle
Satellite cell-intrinsi...
Geriatric age induces intrinsic alterations in muscle
stem cells that affect their regenerative functions,
which cannot be...
What are the satellite cell-intrinsic
alterations at geriatric age?
Question 1:
p16INK4a
is expressed in geriatric satellite cells
(but not in adult or old satellite cells) in muscle
homeostatic conditi...
“point of no-return”
p16INK4a
Pre-senescence
(not in quiescence)
G0
Irreversible
Arrest
p16INK4a
Proliferative pressure
Satellite cell
(Geriatric age)
R...
Senescent cells
A permanent cell cycle arrest
Cellular senescence
é Size
êProliferation
é p16, é p21
é DDR
é SA-βgal activ...
How do muscle stem cells lose their
regenerative capacity with aging?
- how do they become senescent
(express p16INK4a, et...
Transcriptomic/bioinformatic analysis
The quiescent state is enriched in
proteostatic pathways
Autophagy: a prevalent one
The process of (macro)autophagy in mammalian cells
N. Mizushima
Cold Spring Harbor Symposia on Quantitative Biology 2011
Questions:
Do satellite cells have autophagic activity in the
resting quiescence state?
If so, is autophagy altered in qui...
Analysis of the autophagy flux in quiescent satellite cells
GFP-LC3
-Baf
+Baf
-Baf
+Baf
YoungOld
YoungOld
+Baf
+Baf
GFP-LC...
Autophagy-lysosomal degradation
Quiescence
Senescence
Aging
Senescence
García-Prat et al., Nature 2016
Can autophagy be reactivated
in old satellite cells?
Rapamycin
- Rapamycin, by inhibiting mTOR, induces autophagy
Atg7 ove...
Rapamycin treatment of old GFP-LC3 mice (for 2 weeks)
restores basal autophagy in quiescent satellite cells
%∆GFP-LC3MFI(±...
Can autophagy reactivation
rescue the regenerative block
(and senescence entry) of old
(geriatric) satellite cells?
Reactivation of autophagy by Rapamycin treatment or Atg7
overexpression restores the expansion of geriatric satellite
cell...
Reinduction of autophagy rescues the proliferative
defect and reduces senescence in geriatric satellite cells
BrdU+
cells(...
Conclusion:
Autophagy decline contributes to the loss
of quiescence and entry into senescence
of aged satellite cells
Autophagy-lysosomal degradation
Quiescence
Senescence
Aging
Senescence
García-Prat et al., Nature 2016
Autophagy
Quiescence
Senescence
Aging
ROS
Accumulation of damaged organelles and mitochondria
p16INK4a
/ Mitophagy
INK4a l...
Senescence
Autophagy
Pre-senescence
Impaired autophagy
Quiescence
Stemness/Regenerative function
Aging
ProteotoxicityProte...
Autophagy / Proteostasis
Quiescence
Senescence
Aging
Caloric restriction
?
Senescence
Autophagy
Pre-senescence
Impaired autophagy
Quiescence
Stemness/Regenerative function
Aging
ProteotoxicityProte...
Anti-aging pills
today:
Science or
science fiction?
Matt Kaeberlein
Brian Kennedy
News and views - Nature 2009
Thank you!!!
Pedro Sousa-Victor *
Laura García-Prat *
Victoria Moiseeva
Pedro Maseres
Jessica Segalés
Sonia Alonso-Martín
...
83rd ICREA Colloquium 'Aging as a treatable condition: New approaches to bring back youthfulness to aged stem cells' by Pu...
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83rd ICREA Colloquium 'Aging as a treatable condition: New approaches to bring back youthfulness to aged stem cells' by Pura Muñoz-Cánoves

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New approaches to bring back youthfulness to aged stem cells
Aging is a nearly universal process affecting all tissues. Despite its constancy in our lives, aging remains mysterious at a fundamental level. Nevertheless, common hallmarks of aging across different species have been proposed offering an integrated view of the basic mechanisms of aging. Primary hallmarks include cell autonomous changes linked to epigenetic alterations, genomic instability, telomere attrition and loss of proteostasis (protein homeostasis), which are followed by antagonistic responses such us deregulated nutrient sensing, altered mitochondrial function and cellular senescence. Aging hallmarks converge in the exhaustion of stem cells, which provokes tissue regenerative decline. Skeletal muscle provides a stark example of this decline. Its stem cells sustain muscle regeneration throughout life but at advanced age they fail for largely undefined reasons. Several causes for this age-associated stem cell regenerative failure are emerging: decline in proteostatic quality-control mechanisms, metabolic alterations, entry into senescence and changes in the systemic (circulatory) environment. I will review our recent findings on how to improve the regenerative capacity of old stem cells by countering these age-associated alterations, with the ulterior idea that the aging process is malleable and that it is feasible to rejuvenate aged cells and tissues.

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83rd ICREA Colloquium 'Aging as a treatable condition: New approaches to bring back youthfulness to aged stem cells' by Pura Muñoz-Cánoves

  1. 1. ICREA & Universitat Pompeu Fabra Aging as a treatable condition: New approaches to bring back youthfulness to aged stem cells ICREA Colloquium Barcelona, October 9, 2018 Pura Muñoz-Cánoves
  2. 2. Increased risk of tissue deterioration, malfunction and disease with aging Disease, sarcopenia, frailty Health
  3. 3. • Since 1900, lifespan has increased over 30 years in men and women in western countries. • So, in just 100 years, we have extended human longevity quite significantly. • Maybe… we should pursue not just living longer, but better.
  4. 4. Frailty - Sarcopenia Geriatric age
  5. 5. Geriatric age Good mental condition Muscle strength and fitness Healthy aging
  6. 6. Sarcopenia • Sarco flesh (muscle) • penia deficiency Loss of skeletal muscles mass and function (strength) with aging Sarcopenia is associated to increased mortality and functional decline at advanced age
  7. 7. 40% loss of muscle mass between 20-70 years of age 6% decline in muscle mass at each decade between 30-70 years 1.4 –2.5% decline in muscle mass each year after 60 Changes in muscle mass with aging
  8. 8. Loss of muscle regenerative capacity is also maximal at geriatric age (after 75-80 years)
  9. 9. Skeletal muscle, regeneration and aging
  10. 10. 20000nm Muscle fiber Satellite cell Pax7+Muscle fiber ite cell Basal lamina 10µm Skeletal muscle and its stem cells (satellite cells) Quiescence
  11. 11. Adult skeletal muscle homeostasis: regeneration after injury INJURY Activation Proliferation Differentiation Fusion Self-renewal Quiescence
  12. 12. How do we study the regenerative capacity of muscle stem cells in the lab ?
  13. 13. Injury by: - Toxin injection - Crushing Formation of new fibers In vivo model (mice): Regeneration
  14. 14. Muscle regeneration after one week
  15. 15. 1 week2 days 4 days 3 weeks Skeletal muscle has an high regenerative capacity
  16. 16. Skeletal muscle regenerative capacity declines with aging Extrinsic influences
  17. 17. Parabiosis between 2 mice of different age: old-young
  18. 18. Adapted from Conboy and Rando, Cell Cycle 2012 YOUNG MUSCLE OLD MUSCLE Injury: Efficient proliferation/differentiation Injury: Non-efficient proliferation /differentiation Aging Rejuvenation (Heterochronic parabiosis) Aging and rejuvenation of skeletal muscle: Parabiosis model
  19. 19. Major aims: 1. Understand muscle cell functions and regenerative decline during physiological aging, and especially at geriatric age 2. Try to combat muscle stem cell aging and improve regeneration • Young: 2-3 months • Adult: 6-8 months • Old: 20-24 months • Geriatric: 28-32 months
  20. 20. Exacerbated muscle regeneration defect in geriatric mice, compared to old mice CTX injury- induced muscle regeneration
  21. 21. Reduced regenerative capacity of geriatric satellite cells after transplantation into young muscle Satellite cell-intrinsic alterations at geriatric age
  22. 22. Geriatric age induces intrinsic alterations in muscle stem cells that affect their regenerative functions, which cannot be fully rejuvenated by a young host environment
  23. 23. What are the satellite cell-intrinsic alterations at geriatric age? Question 1:
  24. 24. p16INK4a is expressed in geriatric satellite cells (but not in adult or old satellite cells) in muscle homeostatic conditions Transcriptomic / Bioinformatic analysis:
  25. 25. “point of no-return” p16INK4a
  26. 26. Pre-senescence (not in quiescence) G0 Irreversible Arrest p16INK4a Proliferative pressure Satellite cell (Geriatric age) Regeneration Growth factors Unable to proliferate (Through p16/Rb/E2F inhibition) Full senescence Sousa-Victor et al. Nature 2014 Geroconversion
  27. 27. Senescent cells A permanent cell cycle arrest Cellular senescence é Size êProliferation é p16, é p21 é DDR é SA-βgal activity éSASP (secreted factors) é ROS êTelomere length é Apoptosis resistance
  28. 28. How do muscle stem cells lose their regenerative capacity with aging? - how do they become senescent (express p16INK4a, etc)? Question 2: Which cellular processes are altered in geriatric muscle stem cells?
  29. 29. Transcriptomic/bioinformatic analysis The quiescent state is enriched in proteostatic pathways Autophagy: a prevalent one
  30. 30. The process of (macro)autophagy in mammalian cells N. Mizushima Cold Spring Harbor Symposia on Quantitative Biology 2011
  31. 31. Questions: Do satellite cells have autophagic activity in the resting quiescence state? If so, is autophagy altered in quiescent satellite cells with aging? GFP-LC3 transgenic mice LC3 (punta or fluorescence levels): as proxy for autophagosomes LC3
  32. 32. Analysis of the autophagy flux in quiescent satellite cells GFP-LC3 -Baf +Baf -Baf +Baf YoungOld YoungOld +Baf +Baf GFP-LC3 p<0.03 %∆GFP-LC3MFI(±Baf) Young Old 0 50 100 150 Bafilomycin: blocks lysosomal degradation Autophagic activity is impaired in quiescent satellite cells with aging
  33. 33. Autophagy-lysosomal degradation Quiescence Senescence Aging Senescence García-Prat et al., Nature 2016
  34. 34. Can autophagy be reactivated in old satellite cells? Rapamycin - Rapamycin, by inhibiting mTOR, induces autophagy Atg7 overexpression - Atg7, a crucial protein for autophagosome formation
  35. 35. Rapamycin treatment of old GFP-LC3 mice (for 2 weeks) restores basal autophagy in quiescent satellite cells %∆GFP-LC3MFI(±Baf) Rapamycin Control p<0.02 0 50 100 150 200 Old Control Rapamycin GFP-LC3 +Baf +Baf
  36. 36. Can autophagy reactivation rescue the regenerative block (and senescence entry) of old (geriatric) satellite cells?
  37. 37. Reactivation of autophagy by Rapamycin treatment or Atg7 overexpression restores the expansion of geriatric satellite cells in vivo YoungControlRapamycin Geriatric LV-Atg7 GFP Pax7 Dapi/Merge LV-Atg7 infection or rapamycin treatment C57BL6 mice SCID mice Cell Transplantation Analysis 4 and 28 days Young Control Rapamycin Geriatric LV-Atg7 %GFP+ cells/section p<0.008 p<0.008 p<0.007 0 50 100 150 LV-Atg7 infection or rapamycin treatment C57BL6 mice SCID mice Cell Transplantation Analysis 4 and 28 days Young Control Rapamycin Geriatric LV-Atg7 %GFP+ cells/section p<0.008 p<0.008 p<0.007 0 50 100 150 4 days after satellite cell transplantation
  38. 38. Reinduction of autophagy rescues the proliferative defect and reduces senescence in geriatric satellite cells BrdU+ cells(%) SA-ß-gal+ cells(%) 0 2 4 6 8 10 Young Control Rapamycin Geriatric LV-Atg7 p<0.001 p<0.001 p<0.001 0 10 20 30 40 p<0.005 p<0.05 p<0.05 BrdU+ cells(%) SA-ß-gal+ cells(%) 0 2 4 6 8 10 Young Control Rapamycin Geria LV-Atg7 p<0.001 p<0.001 p<0.001 0 10 20 30 40 p<0.005 p<0.05 p<0.05 p<0.001 p<0 SA-ß-gal+ cells(%) 0 20 40 60 RapamycinControl Human
  39. 39. Conclusion: Autophagy decline contributes to the loss of quiescence and entry into senescence of aged satellite cells
  40. 40. Autophagy-lysosomal degradation Quiescence Senescence Aging Senescence García-Prat et al., Nature 2016
  41. 41. Autophagy Quiescence Senescence Aging ROS Accumulation of damaged organelles and mitochondria p16INK4a / Mitophagy INK4a locus derepression García-Prat et al., Nature 2016
  42. 42. Senescence Autophagy Pre-senescence Impaired autophagy Quiescence Stemness/Regenerative function Aging ProteotoxicityProteostasis Rejuvenating strategies Systemic (blood) / niche (local) Autophagy reinduction Rapamycin Antioxidants Summary of strategies that increase muscle stem cell regenerative capacity
  43. 43. Autophagy / Proteostasis Quiescence Senescence Aging Caloric restriction ?
  44. 44. Senescence Autophagy Pre-senescence Impaired autophagy Quiescence Stemness/Regenerative function Aging ProteotoxicityProteostasis Rejuvenating strategies Systemic (blood) / niche (local) Autophagy reinduction Rapamycin Antioxidants Calorie restriction Senolytics Exercise (?) Summary of strategies that increase muscle stem cell regenerative capacity
  45. 45. Anti-aging pills today: Science or science fiction? Matt Kaeberlein Brian Kennedy News and views - Nature 2009
  46. 46. Thank you!!! Pedro Sousa-Victor * Laura García-Prat * Victoria Moiseeva Pedro Maseres Jessica Segalés Sonia Alonso-Martín Xiaotong Hong Laura Ortet Mercè Jardí Marta Flández Vera Lukesova William Roman Marina Raya Antonio Serrano Eusebio Perdiguero Salvador Aznar-Benitah Marco Sandri Marta Martínez-Vicente Esteban Ballestar Charles Keller Vittorio Sartorelli Michael Rudnicki Jun Hee Lee Thomas Braun / Johnny Kim Francesc Posas

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