1. Cellular Stress Responses, Cytoprotection and Aging
Larry Hightower, Ph.D., FAAAS
University of Connecticut
Kirkwood, Nature, 2008

2. 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

3. Due to increased longevity and declining birth rates,
we are living in a rapidly aging world
Petsko, Genome Biology, 2008

4. 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.

5. 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
30 Life
25
20
Years
Women
15
Lost
10
Men
5
0
<35 35-44 45-54 55-64 65-74 75-84 85+
Age at Diagnosis
Morgan, Diab Care 23: 1103, 2000 6

7. Diabetic ulcers impede healthy aging Driscol, P. Chronic Wounds, 2008 Mediligence.com
CAGR: compounded annual growth rate

8. 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

9. Atp8
Intermembrane p
Atp4
space p
Atp6
p
Vb
Mitochondrial Core 2 Υ
Flavo Atp7
matrix protein p α α
β
Atp5
p
P AD 3H+ ATP H2O
i P
ROS
Cytosol
ROS
ER Aging
ROS Hsp22
«X»?
Mitochondria
[ROS]
Hsp70/Hsp40/sHsps
DNA Hsp26 Lisosome/
Hsp23
microautophagy
Nucleus ROS
Aging
ROS
Hsp27
Proteasome

10. How to prevent?
Young Aged
Most of the following sequence of slides is from the laboratory
of Professor Robert Tanguay.

11. Drosophila Hsp22 overexpression
increases lifespan
Ubiquitous (actin) Motorneurons (D42)
100 100
90 90
80
32 % 80 32 %
Survival (%)
70 70
Survival (%)
60 60
50 50
40 40
30 30
20 +/actin-GAL4 20 +/D42-GAL4
10 EP(3)3247/actin-GAL4 10 EP(3)3247/D42-GAL4
0 0
0 10 20 30 40 50 60 70 80 90 100 110 120 0 10 20 30 40 50 60 70 80 90 100 110 120
Time (days) Time (days)
62 82 68 90
Morrow et al. FASEB J, 2004

12. Flies overexpressing Hsp22 maintain their
locomotor activity longer (healthy aging)
5050
7 cm en 8 secondes (%)
Mouches plus haute que
Flies above 7 cm in 8 seconds (%)
+/D42-GAL4
+/D42-GAL4
4040 EP(3)3247/D42-GAL4
EP(3)3247/D42-GAL4
3030
2020
1010
00 40 60 80
40 60
Age of flies (days) 80
Âge des mouches (jours) et al. FASEB J, 2004
Morrow

13. Hsp22 overexpression (motorneurons)
increases resistance to oxidative stress
(paraquat)
100
90 +/D42-GAL4
EP(3)3247/D42-GAL4
80
70
Survival (%)
60
50
40
30
20
10
0
2 40 60 80
Age of flies (days)
Morrow et al. FASEB J, 2004

14. 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 Mitochondrial function
Complex I Complex I
Complex V - ATP pump Complex V - ATP pump
Defense response Protein biosynthesis
Proteolysis Defense response
Proteolysis

15. DmHSP22-expressing human fibroblasts
live longer
TIG-Vector
B TIG-DmHsp22
Population doublings
A
DmHsp22
actin
Days
Wadhwa et al. J Biol Chem 2010

16. 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

17. Molecular Causes of Aging
Reactive Oxygen Species
Nutritional Glucose
Errors in biochemical
processes
Molecular Damage
HBOT

18. 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
C.elegans
Cysper JR and Johnson TE. J Gerontol A Biol Sci Med Sci. 2002 Mattson M. Ageing Res Rev. 2008

19. Examples of Hormesis: Calorie Restriction (CR)
Control CR
37% of Control animals died due to age
13% of CR animals died due to age
Colman RJ, et al. Science. (2009) 325, 201-204

20. Examples of Hormesis: HBOT
HBOT 12-16hrs HBOT
2.72 atm 2.72 atm
8hrs 20-24hrs
Age (days)
HBOT protects against toxic oxygen exposure in C.elegans
Cysper JR and Johnson TE. J Gerontol A Biol Sci Med Sci. 2002

21. Cell Culture Model:
Human Microvascular Endothelial Cell Line: HMEC-1

22. Protection Against Oxidative Stress
48h 16h 4h
Cells HBOT/ t-bOOH MTT
plated 100% O2

23. HBOT Project
UCONN-OxyHeal
OxyCure 3000 DNA Microarray Technology

24. Nrf2 Signaling pathway
Proteasome
Ub
Ub
SH SH Ub Ub
Ub
Cul3 Nrf
Keap1
2
Nrf
2
cytoplasm
CBP/p300
nucleus
sMAF TARGET GENES
•Antioxidants
ARE
•Xenobiotic metabolism
•Glutathione homeostasis
•DNA damage recognition
•Proteasome function
•Inhibition of inflammation

25. TRX-1
The target of rapamycin (TOR)
Pathway regulates lifespan in a
Broad range of organisms.

26. Nrf-2 Antioxidant Pathway
Nrf-2 transcription factor
is a crucial regulator of cellular redox
homeostasis through its capacity to induce
the expression of enzymes which detoxify
HO-1
TRX-1
reactive oxygen species, and expression
of other antioxidant proteins.
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.

27. mTOR1 Signaling
“TOR is absolutely essential for developmental
growth, but upon completion of development
it causes aging and age‐related diseases.”
HO-1
Blagosklonny and Hall, Aging 2009.
TRX-1
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.

28. S6K1: 1.4-fold
decrease 24hr
following HBOT
Mouse model Science Oct 2009

29. • 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)

30. Acknowledgements:
Dr. Cassandra Tierney
Dr. Charles Giardina
Dr. George Perdrizet