Discussion 2 lectures 6-8

Discussion 2lectures 6-8 Ryan Klimczak February 5th, 2007

Lecture 6 - Cellular Senescence Lecture 7 - Telomeres Lecture 8 - Diseases of Aging

Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress Tory M. Hagen*, Jiankang Liudagger ,Dagger , Jens Lykkesfeldt§, Carol M. Wehrdagger , Russell T. IngersollDagger , Vladimir Vinarskydagger , James C. Bartholomew¶, and Bruce N. Amesdagger , et al. -ALCAR supplements improve membrane potential, caused in part by replenishment of carnitine, a betaine that shuttles fatty acids into the mitochondrion for beta-oxidation -Increases cardiolipin levels -Overall, improves metabolic function, improving substrate and electron flux -However, addition of higher levels of ALCAR can actually lead to increased ROS production from increased electron flow in the electron transport chain. -Rationale of using LA in synergy with ALCAR is its antioxidant potential. -Also improves mitochondrial bioenergetics as a cofactor for pyruvate dehydrogenase and aplha-ketoglutarate dehydrogenase -Shown to enhance glucose uptake as well. -The complementation of ALCAR and LA thus has a mutually beneficial effect on metabolic function.

Cellular Senescence What is it? Response of normal cells to potentially cancer-causing events

First description: the Hayflick limit Finite Replicative Life Span "Mortal" Infinite Replicative Life Span "Immortal" Proliferative capacity Number of cell divisions EXCEPTIONS Germ line Early embryonic cells (stem cells) Many tumor cells What happens when cells exhaust their replicative life span

What happens when cells exhaust their replicative life span REPLICATIVE SENESCENCE • Irreversible arrest of cell proliferation • (universal) • Resistance to apoptosis • (stem cells) • Altered function • (universal but cell type specific) SENESCENT PHENOTYPE

Cellular Senescence What causes it? (what causes the senescent phenotype?) Cell proliferation (replicative senescence) = TELOMERE SHORTENING DNA damage Oncogene expression Supermitogenic signals What do inducers of the senescent phenotype have in common?

Inducers of cellular senescence Cell proliferation (short telomeres) Potentially Cancer Causing DNA damage Oncogenes Strong mitogens Normal cells (mortal) Immortal cells (precancerous) Inducers of senescence Cell senescence Transformation Apoptosis Tumor suppressor mechanisms

Cellular Senescence An important tumor suppressor mechanism • Induced by potentially oncogenic events • Most tumor cells are immortal • Many oncogenes act by allowing cells to bypass • the senescence response • Senescence is controlled by the two most important • tumor suppressor genes -- p53 and pRB • Mice with cells that do not senesce die young • of cancer

Cellular Senescence An important tumor suppressor mechanism What does cellular senescence have to do with aging? • The senescent phenotype entails changes • in cell function • Aging is a consequence of the declining force • of natural selection with age

Aging before cell phones …… Modern, protected environment (very VERY recent) 100% Natural environment: predators, infections, external hazards, etc Survivors Most of human evolution AGE Antagonistic pleiotropy: Some traits selected to optimize fitness in young organisms can have unselected deleterious effects in old organisms (what's good for you when you're young may be bad for you when you're old)

Epithelial Cells Fibroblasts Senescent cells can strongly alter tissue microenvironments.May contribute to age-related declines in tissue structure and function, and age related disease

P53 p53 has many anti-cancer mechanisms:･ -It can activate DNA repair proteins when DNA has sustained damage. -It can also hold the cell cycle at the G1/S regulation point on DNA damage recognition -It can initiate apoptosis, the programmed cell death, if the DNA damage proves to be irreparable. .

PRB -pRb prevents the cell from replicating damaged DNA by preventing its progression through the cell cycle into its S or progressing through G1 -pRb binds and inhibits transcription factors of the E2F family. E2F transcription factors are dimers of an E2F protein and a DP protein. -The transcription activating complexes of E2 promoter-bindingﾐprotein-dimerization partners (E2F-DP) can push a cell into S phase.As long as E2F-DP is inactivated, the cell remains stalled in the G1 phase. -When pRb is bound to E2F, the complex acts as a growth suppressor and prevents progression through the cell cycle. The pRb-E2F/DP complex also attracts a protein called histone deacetylase (HDAC) to the chromatin, further suppressing DNA synthesis

Questions • What causes cellular senescence, what are the inducers and what do they have in common? • What is replicative senescence? • List 3 characteristics of the senescent phenotype. • What is the relationship between carcinogenesis, aging, and senescence? • Explain antagonistic pleiotropy

Why are telomeres important? Telomeres allow cells to distinguish chromosomes ends from broken DNA Stop cell cycle! Repair or die!! Homologous recombination (error free, but need nearby homologue) Non-homologous end joining (any time, but error-prone)

5' 3' 3' 5' 3' 5' 5' 5' 3' Ori DNA replication is bidirectional Polymerases move 5' to 3' Requires a labile primer Each round of DNA replication leaves 50-200 bp DNA unreplicated at the 3' end • The importance of telomeres (con’t) • Prevent chromosome fusion by non-homologous end joining • Provide a means for counting cell division • They resolve the end replication problem

Replicatively immortal cells bypass the restrictions telomeres impose by using the enzyme telomerase Enzyme (reverse transcriptase) with RNA and protein components Adds telomeric repeat DNA directly to 3' overhang (uses its own RNA as a template) Vertebrate repeat DNA on 3' end: TTAGGG Telomerase RNA template: AAUCCC

HOWEVER, CELLS THAT EXPRESS TELOMERASE STILL UNDERGO SENESCENCE (E.G., IN RESPONSE TO DNA DAMAGE, ONCOGENES, ETC.) Inducers of cellular senescence Strong mitogens/ stress Cell proliferation (short telomeres) DNA damage Oncogenes Potential Cancer Causing Events

The telomere hypothesis of aging Telomeres shorten with each cell division and therefore with age Short telomeres cause cell senescence and senescent cells may contribute to aging HYPOTHESIS: Telomere shortening causes aging and telomerase will prevent aging True or False?

SUMMARY Telomeres are essential for chromosome stability Telomere shortening occurs owing to the biochemistry of DNA replication Short telomeres cause replicative senescence (other senescence causes are telomere-independent) Telomerase prevents telomere shortening and replicative senescence The telomere hypothesis of aging depends on the cellular senescence hypothesis of aging

Discussion Questions: How do the looped structures of telomeres promote chromosomal stability? What is the correlation between aging, cellular senescence, and telomere length? Why do cells that express telomerase still undergo senescence?

Diseases of Aging

Disease may be viewed as a process that is : • Selective (i.e., varies with the species, tissue, organ, cell and molecule) • Intrinsic and extrinsic (I.e., may depend on environmental and genetic factors) • Discontinuous (may progress, regress, or be arrested) • Occasionally deleterious (damage is often variable, reversible) • Often treatable (with known etiopathology, cure may be available)

Causes of Death According to Age • All Races, Both Sexes • Ages: 65-84Ages: 85+ • 2003 2003 • 1. Heart disease (28.2%) 1. Heart Disease (36.2%) • 2. Cancer (27.7%) 2. Cancer (11.6%) • 3. COPD‡ (7.1%) 3. Stroke (9.4%) • 4. Stroke (6.6%)4. Alzheimer’s Disease (5.5%) • 5. Diabetes Mellitus (3.6%)5. Pneumonia/Influenza (4.6%) • 6. Pneumonia/Influenza (2.4%) 6. COPD‡ (4.4%) • 7. Alzheimer’s Disease (2.2%)7. Diabetes Mellitus (2.2%) • 8. Nephritis (1.9%) 8. Nephritis (2%) • 9. Accidents (1.9%) 9. Accidents (1.9%) • 10.Septicemia (1.5%) 10.Septicemia (1.4%) • - All others (17%) - All others (20.9%)

Diseases as a tool for studying aging:Syndromes in humans: having multiple characteristics of premature (early onset) of aging, oraccelerated (rapid progression) of aging Infantile Progeria: Hutchinson-Gilford SyndromeAdult onset progeria: Werner’s syndromeDown syndrome

Hutchinson-Gilford Progeria Syndrome: -Hutchinson-Gilford Progeria syndrome is an extremely rare genetic condition which causes physical changes that resemble greatly accelerated aging in sufferers. -Affects between 1 in 4 million (estimated actual) and 1 in 8 million (reported) newborns. Currently, there are approximately 40-45 known cases in the world. -Most people with progeria die around 13 years of age

-It is caused by mutations in a LMNA (Lamin A protein) gene on chromosome 1. • -Nuclear lamina is a protein scaffold around the edge of the nucleus that helps organize nuclear processes such as DNA and RNA synthesis. • -Lamin A cannot be produced and Prelamin A builds up on the nuclear membrane, causing a characteristic nuclear blebbing. This results in the premature aging symptoms of progeria, although the mechanism connecting the misshapen nucleus to the symptoms is not known. • The condition causes wrinkled skin, atherosclerosis and cardiovascular problems. Mental development is not affected. • The development of symptoms is comparable to aging at a rate six to eight times faster than normal, although certain age-related conditions do not occur. Specifically, victims show no neurodegeneration or cancer predisposition. The people diagnosed with this disease usually have fragile elderly-like bodies.

Werner Syndrome -The gene responsible for Werner syndrome (WRN) was identified (and found to be a member of the RecQ family of helicases. -The Werner protein is thought to perform several tasks in the cell, including the maintenance and repair of DNA. It also assists in making copies of DNA in preparation for cell division. Mutations in the WRN gene often lead to the production of an abnormally short Werner protein. -Some research suggests that this shortened protein is not sent to the nucleus, where it normally interacts with DNA. Evidence also suggests that the altered protein is broken down quickly in the cell, leading to a loss of Werner protein function. -Research into the biological role of the WRN protein is ongoing, but current evidence strongly suggests a role for WRN in the resolution of Holliday junctions. Roles in non-homologous end joining (NHEJ) and the restoration of stalled replication forks have also been suggested.

-Individuals with this syndrome typically grow and develop normally until they reach puberty. Following puberty, they age rapidly, so that by the time they reach age 40 they often appear as though they are several decades older. • Overall, people affected by Werner syndrome have thin arms and legs and a thick trunk. Affected individuals typically have a characteristic facial appearance described as "bird-like" by the time they reach their thirties. Patients with Werner sydrome also exhibit genomic instability and various age-associated disorders; these include cancer, heart disease, atherosclerosis, diabetes mellitus, and cataracts. However, not all characteristics of old-age are present in Werner patients; for instance, senility is not seen in individuals with Werner syndrome. People affected by Werner syndrome usually do not live past their late forties or early fifties, succumbing to death, often resulting from cancer or heart disease.

Discussion Questions: What are the two most common causes of death in individuals over the age of 50? What reasons underlie this trend? How do the symptoms of Hutchinson-Gilford Progeria Syndrome and Werner Syndrome mimic the characteristics of ‘normal’ aging? How are they different? Hypothesize how the genetic mutations responsible for these two syndromes lead to ‘accelerated aging’ phenotypes?

Discussion 2 lectures 6-8