Kirkwood, Nature, 2008

Cellular Stress Responses, Cytoprotection and Aging

Larry Hightower, Ph.D., FAAAS

University of Connecticut

Life expectancy around the world has increased steadily for nearly 200 years (5 hrs/day). The top countries for

longevity are shown here.

Kirkwood, Nature, 2008

Due to increased longevity and declining birth rates, we are living in a rapidly aging world

Petsko, Genome Biology, 2008

Longevity and ROS: Recent aging research using model organisms has focused on lifespan,

valuable in identifying key genes and proteins as well as generating theories.

• Longevity is influenced by environmental and genetics factors • Free radical theory of aging

“ Longevity is determined by the capacity of an organism to cope with random damages induced by radical oxygen species (ROS) ” Sohal, Free Rad Biol Med, 2002

• ROS are produced by the mitochondrial electron transport and by the Fenton

reaction (Fe2+ + H2O2 Fe3+ + OH˙+ OH-)

Our viewpoint: Aging is not a disease but rather it is a natural process for life forms

that use an oxygen-based metabolism. Oxidation (oxidative stress) is one side of the

redox reactions that drive our metabolism. Reduction (reductive stress) is the other

part and both cause damage in cells.

Our research goal is to promote healthy aging in humans. Our focus is not on

increasing lifespan directly, although this is a likely consequence.

Annual Number (in Thousands) of New Cases of Diagnosed Diabetes Among Adults Aged 18–79 Years, United States, 1980–2010 From 1980 through 2010, the number of adults in the United States aged 18–79 with newly diagnosed diabetes more than tripled from 493,000 in 1980 to over 1.7 million in 2010. The number of new cases of diabetes has increased since the early 1990s. From 2008 through 2010, the number of new cases of diagnosed diabetes has shown little change.

http://www.cdc.gov/diabetes/statistics/incidence/fig1.htm

6

Diabetes Reduces Years of

Life

Morgan, Diab Care 23: 1103, 2000

0

5

10

15

20

25

30

<35 35-44 45-54 55-64 65-74 75-84 85+

Years

Lost

Age at Diagnosis

Women

Men

Driscol, P. Chronic Wounds, 2008 Mediligence.com CAGR: compounded annual growth rate

Diabetic ulcers impede healthy aging

Chaperones, aging & diseases

- Chaperones and aging

- reduced HS response in aging

- chaperones important for protein refolding and/or degradation

- many age-related diseases, particularly neuronal diseases,are associated with protein misfolding, modification and aggregation

Core 2 Flavo

protein

Vb

α β

α

Υ

Atp6

p

Atp4

p

Atp8

p

Atp5

p

Atp7

p

3H+ AD

P

P

i H2O ATP

Intermembrane

space

Mitochondrial

matrix

Hsp23

Hsp27

Hsp26

Hsp22

ROS

ROS

ROS

Aging

Aging

[ROS]

ROS

ER

Mitochondria

Nucleus

DNA

ROS

Proteasome

Cytosol

Hsp70/Hsp40/sHsps Lisosome/ microautophagy

Tanguay and Morrow, 2008

« X » ?

Young Aged How to prevent?

Most of the following sequence of slides is from the laboratory

of Professor Robert Tanguay.

Drosophila Hsp22 overexpression

increases lifespan

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90 100 110 120

Time (days)

Surv

ival

(%)

+/actin-GAL4

EP(3)3247/actin-GAL4

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90 100 110 120

Time (days)

Surv

ival

(%)

+/D42-GAL4

EP(3)3247/D42-GAL4

62 82 68 90

Ubiquitous (actin) Motorneurons (D42)

32 % 32 %

Morrow et al. FASEB J, 2004

Flies overexpressing Hsp22 maintain their

locomotor activity longer (healthy aging)

0

10

20

30

40

50

40 60 80

Âge des mouches (jours)

Mou

ches

plu

s hau

te q

ue

7 c

m e

n 8

sec

on

des

(%

)

+/D42-GAL4

EP(3)3247/D42-GAL4

50

20

30

40

10

0

Flie

s ab

ove

7 c

m in

8 s

eco

nd

s (%

)

40 80 60

Age of flies (days)

+/D42-GAL4

EP(3)3247/D42-GAL4

Morrow et al. FASEB J, 2004

Hsp22 overexpression (motorneurons) increases resistance to oxidative stress

(paraquat)

0

10

20

30

40

50

60

70

80

90

100

2 40 60 80

Age of flies (days)

Su

rviv

al

(%)

+/D42-GAL4

EP(3)3247/D42-GAL4

Morrow et al. FASEB J, 2004

The main functions up regulated in the mitoproteome and microarray analysis are similar even if the analysis are not

designed in the same way

Functions up regulated in the mitoproteome analysis Functions up regulated in the microarray analysis

Mitochondrial function

Complex I

Complex V - ATP pump

Defense response

Proteolysis

Mitochondrial function

Complex I

Complex V - ATP pump

Protein biosynthesis

Defense response

Proteolysis

DmHsp22

actin

A

B

Po

pu

lati

on

do

ub

lin

gs

Days

TIG-Vector

TIG-DmHsp22

DmHSP22-expressing human fibroblasts

live longer

Wadhwa et al. J Biol Chem 2010

Dm Hsp22 in human cells

DmHsp22 is functionally active in human cells

1- causes lifespan extension of primary human fibroblasts 2- increases malignant properties of human cancer cells - malignant transformation of MCF-7 breast cancer cells - higher transformation (colony forming assay) - higher mobility (invasive assay) - enhanced motility (wound scratch/invasion assay) - tumor formation in nude mice 3- increases resistance to drugs

Molecular Causes of Aging

Reactive Oxygen Species

Nutritional Glucose

Errors in biochemical

processes

Molecular Damage

HBOT

Hormesis • a process in which exposure to a low dose of a chemical agent or environmental

factor that is damaging at higher doses induces an adaptive beneficial effect on

the cell or organism

Mattson M. Ageing Res Rev. 2008 Cysper JR and Johnson TE. J Gerontol A Biol Sci Med Sci. 2002

C.elegans

Examples of Hormesis: Calorie Restriction (CR)

Colman RJ, et al. Science. (2009) 325, 201-204

Control CR

37% of Control animals died due to age

13% of CR animals died due to age

Examples of Hormesis: HBOT

HBOT protects against toxic oxygen exposure in C.elegans

Cysper JR and Johnson TE. J Gerontol A Biol Sci Med Sci. 2002

HBOT

2.72 atm

8hrs

12-16hrs HBOT

2.72 atm

20-24hrs

Age (days)

Cell Culture Model:

Human Microvascular Endothelial Cell Line: HMEC-1

Protection Against Oxidative Stress

48h 16h 4h

Cells

plated

HBOT/

100% O2

t-bOOH MTT

HBOT Project

UCONN-OxyHeal

OxyCure 3000 DNA Microarray Technology

Keap1

SH SH

Nrf2

Cul3 Nrf2

Ub

Ub

Ub

Ub

Ub

Proteasome

ARE

sMAF

CBP/p300

TARGET GENES •Antioxidants •Xenobiotic metabolism •Glutathione homeostasis •DNA damage recognition •Proteasome function •Inhibition of inflammation

Nrf2 Signaling pathway

nucleus

cytoplasm

The target of rapamycin (TOR)

Pathway regulates lifespan in a

Broad range of organisms.

TRX-1

Nrf-2 Antioxidant Pathway

ROC1, named ROC for RING of cullins, a ubiquitin ligase. We suggest that Keap1

negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the

CUL3-ROC1 ligase and subsequent degradation by the proteasome.

HO-1, Heme oxygenase catalyzes the oxidative degradation of heme into equimolar

amounts of biliverdin, carbon monoxide, and free iron. HO-1 plays a cytoprotective

role in modulating tissue responses to injury in pathophysiological states.

TRX-1, thioredoxin-1 oxidoreductase, predominant role of Trx-1 to limit oxidative

stress directly due to reactive oxygen species scavenging and by protein–protein

interaction with key signaling molecules.

Nrf-2 transcription factor

is a crucial regulator of cellular redox

homeostasis through its capacity to induce

the expression of enzymes which detoxify

reactive oxygen species, and expression

of other antioxidant proteins. TRX-1

HO-1

TRX-1

HO-1

mTOR1 Signaling

mTOR, the mammalian form of TOR, target of rapamycin, an immunosuppressive drug.

Inhibition of TOR signaling pathway can extend lifespan.

The mTOR pathway integrates signals from nutrients, energy status and growth factors to

regulate many processes, including autophagy, ribosome biogenesis and metabolism.

AMPK, an AMP-dependent kinase, the ancient and central sensor of cellular energy

stress. It is activated by low energy conditions and then inhibits TORC-1 activity and

triggers shift to catabolic metabolism. This shields organisms from metabolic overuse

during bad times.

S6K, S6 kinase, regulator of translation initiation and elongation. Active mTOR results

in the activation of S6K and stimulates both translation initiation and elongation. S6K

deficient mice show an increased lifespan.

“TOR is absolutely essential for developmental

growth, but upon completion of development

it causes aging and age‐related diseases.”

Blagosklonny and Hall, Aging 2009.

Science Oct 2009

S6K1: 1.4-fold

decrease 24hr

following HBOT

Mouse model

• HBO treated cells are protected against

lethal oxidative and heat stresses.

• HBO increases expression of

antioxidant and cytoprotective genes

– HMOX1, MT, HSP, TXN, HIF1α, Nrf2

• Potential to stimulate healthy aging,

decrease toxicity after major surgery for

aged individuals, and as a preventative

therapy for other age-related diseases

(i.e.diabetes)

Acknowledgements:

Dr. Cassandra Tierney

Dr. Charles Giardina

Dr. George Perdrizet