Cellular Stress Responses, Cytoprotection and Aging Larry Hightower, Ph.D., FAAAS University of Connecticut Kirkwood, Nature, 2008
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 formsthat use an oxygen-based metabolism. Oxidation (oxidative stress) is one side of theredox reactions that drive our metabolism. Reduction (reductive stress) is the otherpart and both cause damage in cells.Our research goal is to promote healthy aging in humans. Our focus is not onincreasing 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
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 DiagnosisMorgan, Diab Care 23: 1103, 2000 6
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
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 AgingROS Hsp22 «X»? Mitochondria [ROS] Hsp70/Hsp40/sHspsDNA Hsp26 Lisosome/ Hsp23 microautophagy Nucleus ROS Aging ROS Hsp27 Proteasome
How to prevent?Young AgedMost of the following sequence of slides is from the laboratoryof Professor Robert Tanguay.
Flies overexpressing Hsp22 maintain their locomotor activity longer (healthy aging) 50507 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
Hsp22 overexpression (motorneurons) increases resistance to oxidative stress (paraquat) 100 90 +/D42-GAL4 EP(3)3247/D42-GAL4 80 70Survival (%) 60 50 40 30 20 10 0 2 40 60 80 Age of flies (days) Morrow et al. FASEB J, 2004
The main functions up regulated in the mitoproteome andmicroarray analysis are similar even if the analysis are not designed in the same wayFunctions 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
DmHSP22-expressing human fibroblasts live longer TIG-Vector B TIG-DmHsp22 Population doublings A DmHsp22 actin Days Wadhwa et al. J Biol Chem 2010
Dm Hsp22 in human cells DmHsp22 is functionally active in human cells1- causes lifespan extension of primary human fibroblasts2- 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 mice3- 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 C.elegansCysper JR and Johnson TE. J Gerontol A Biol Sci Med Sci. 2002 Mattson M. Ageing Res Rev. 2008
Examples of Hormesis: Calorie Restriction (CR) Control CR 37% of Control animals died due to age 13% of CR animals died due to ageColman RJ, et al. Science. (2009) 325, 201-204
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.elegansCysper JR and Johnson TE. J Gerontol A Biol Sci Med Sci. 2002
HBOT Project UCONN-OxyHealOxyCure 3000 DNA Microarray Technology
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
TRX-1 The target of rapamycin (TOR) Pathway regulates lifespan in a Broad range of organisms.
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 Keap1negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by theCUL3-ROC1 ligase and subsequent degradation by the proteasome.HO-1, Heme oxygenase catalyzes the oxidative degradation of heme into equimolaramounts of biliverdin, carbon monoxide, and free iron. HO-1 plays a cytoprotectiverole in modulating tissue responses to injury in pathophysiological states.TRX-1, thioredoxin-1 oxidoreductase, predominant role of Trx-1 to limit oxidativestress directly due to reactive oxygen species scavenging and by protein–proteininteraction with key signaling molecules.
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-1mTOR, 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 energystress. It is activated by low energy conditions and then inhibits TORC-1 activity andtriggers shift to catabolic metabolism. This shields organisms from metabolic overuseduring bad times.S6K, S6 kinase, regulator of translation initiation and elongation. Active mTOR resultsin the activation of S6K and stimulates both translation initiation and elongation. S6Kdeficient mice show an increased lifespan.
S6K1: 1.4-fold decrease 24hr following HBOTMouse model Science Oct 2009
• 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 TierneyDr. Charles GiardinaDr. George Perdrizet