Supplement 12-1

What is the Relationship Between Replicative Cell Senescence & Functional Tissue Senescence.

Addition to Biology of Aging 3e by Robert Arking

Relationship between stress & telomeres?

von Ziglincki

psychological stress alters telomere length

Relationship between telomere length/replicative senescence, cell function & tissue senescence?

heart

skin

other tissues

Psychological Stress Induces Oxidative Stress & Shortens Telomeres

Epel et al., PNAS 101: 17312, 2005

Perceived Stress Makes You Age!

This, when combined with data from glucocorticoid studies, provides the mechanistic basis for the findings of the MIDUS twin study

Senescence of the Heart is Related to Changes in Cell

Demographics Brought About By Changes in Stem Cell Activity

Lakatta, Circulation 107:490, 2003

Cardiac Myocyte Stem Cells Are Found In the Mouse Heart

Torella et al., Circulation Res. 94:514, 2004

Cardiac Stem Cells and Aging: mtOxDam in Cardiac Myocytes with Normal (WT) and Elevated (TG) Levels of IGF-

1

Rep Hyp Die

Torella et al., Circulation Res. 94:514, 2004

Rep Hyp Die

Cell Senescence Markers Increase in WT but not in TG

myocytes

Telomere lengths decrease significantly more in WT than in TG myocytes

Torella et al., Circulation Res. 94:514, 2004

Deaths >>Births

Function Lost

Deaths = Births

Function Kept

TG mice with elevated IGF-1 Have Larger Numbers of Functional Cardiac Stem Cells, & Their Demographic Balance

Keeps Their Heart Functional Longer Than in WT

Torella et al., Circulation Res. 94:514, 2004

Note difference in growth kinetics & the functional effects of these different growth strategies.

Is there a contradiction between these effects of elevated IGF 1 effects on the heart & the effects of decreased IGF 1 on longevity?

Summary of the Data

Note that localized high levels of IGF-1 also maintain regenerative capacity in skeletal muscle

ROS Limits the Lifespan of Hematopoetic Cells in vivo

HSC treated in vitro with a GSH depleter (BSO) have dose-dependent increases in ROS levels

This treatment has no effect on HSC’s ability to form colonies in vitro within 1 week after treatment

This treatment has an obvious effect on the HSC’s ability to form colonies in vivo 16 wks after being injected into a mouse.

Ito et al., Nature Medicine 12:445, 2006

One way to interpret the data of the prior slide is to make the reasonable assumption that the depletion of GSH brought about by BSO pre-treatment causes oxidative damage to the telomeres.

The damaged telomeres adversely affect the replicative potential of the HSC population. The damaged cells cannot effectively replicate in an otherwise permissive environment.

Skin Senescence

The Role of Senescent Cells in Skin Aging

Young --------------??----Old

Improbable Possible Probable

What Is The Relationship Between Cell Aging & Tissue Aging?

Fossel, Cells, Aging & Human Disease, Oxford 2005

Dimri et al., PNAS 92:9363, 1995

Early Passage Late Passage

pH 4, non-specific

pH 6.0, SA-β-Gal

SA-β-Gal stained cells in culture do not engage in scheduled DNA synthesis.

Young Young + Sun

Old, ++ staining Old, +++ staining

?

SA-β-Gal Staining in Human Skin of Different Ages

Dimri et al, 1995

Severino et al., ECR 257:162

Lack of Strong Correlation Between Age & Number of Stained Cells in Human Tissue Sections

Fetal

Young Adult, ~26 y

Old Adult, ~80 y

WI38 cells + Ox Stress

SA-β-Gal Staining is Induced by Oxidative Stress As Well As By Age (?)

Severino et al., 2000

? ?

? ?

Early Passage Cells

Late Passage Cells

SA-β-Gal Staining is Induced by Culture Conditions

Conclusion: SA-β-Gal Staining is a non-specific marker of senescent cells. It is a useful but not definitive marker.

Telomere Structure

Blasco, Nat. Rev. Gen. 6:611, 2005

Histone Modification Telomeres Yields Epigenetic Regulation of Cell Cycle

These complexes of altered histones and bound telomere binding proteins yield distinctive chromatin structures which can be detected by antibody staining. This allows one to determine the telomeric status of treated and control cells.

Cell Senescence In Aging Primate Skin As Assayed By Telomere Dysfunction

Herbig et al., Science 311:1257, 2006

Reconstruction of Skin From a Suspension of Skin Cells From a 15-Day Embryonic Mouse

<< Normal Human Skin

<<Human Skin Equiv. (grown in vitro from only keratinocytes & fibroblasts )

Carasco et al., Anat. Rec. 264:261, 2001

Not an exact imitation!

From Carasco et al, Anat. Rec. 264:261:2001

Reconstituted Human Skin Expresses Appropriate Cell Differentiation Antigens

DNA Content

The Cells in the HSE Appear to Go Through The Cell Cycle In A Normal Manner

Carasco et al., Anat. Rec. 264:261, 2001

Modified HSE protocol. Took dermal fibroblasts from culture with or without pTERT, and assayed their contribution to the phenotype of reconstituted skin.

Dissociated keratinocytes were mixed with aged and treated fibroblasts in culture chambers on nude mice.

Questions being asked:

1. Does telomerase treatment affect fibroblast function?

2. Does skin structure and function correlate with functional state of fibroblasts?

PD20 PD60

PD85 PD 110 hTERT

Resistance to Mechanical Blistering of Skin With Different Fibroblasts

Funk et al, ECR 258:270, 2000

<Laminin 5

<Collagen VII

Note that skin reconstituted with young passage fibroblasts (upper fig.) and fibroblasts expressing a telomerase transgene (lower fig.) both show hemidesmosomes linked into intermediate filaments within the epidermis are positioned directly across from regions containing collagen filaments (arrows).

Also note there was no EM photo of skin reconstituted with old passage fibroblasts, although the implication of the article is that the properties of young skin are associated with fibroblast cells expressing telomerase and certain patterns of gene expression.

From Funk et al ECR 258:271, 2000

FINE STRUCTURE IS RELATED TO FIBROBLAST CELL STATE?

Funk et al, ECR 258:270, 2000

Late Cells

Early Cells

Sen/young >> hTERT/young

Young/sen ~ = hTERT/sen