1. MCB 135K Mid-Term I Review February 15, 2005 GSI: Jason Lowry

2. General Information Mid-Term I – Friday, February 18 th, 2005 –In Class Exam –Need Pen and Pencil –100 Points 30 Points Multiple Choice 20 Points True / False 50 Points Short Answer / Essay –We will provide scan-tron –Be prepared to place all personal belongings in aisle or at front of room before exam begins

3. Exam Material Demography, Comparative/Differential Aging, and Epidemiology Biomarkers and Functional Assessment Telomeres, Apoptosis, and Cellular Senescence Mitochondrial Decay, Oxidants, and Anti- Oxidants Evolutionary Theory of Lifespan, Yeast as a model for cellular lifespan Don’t Forget The Handouts!

4. Demography Be familiar with: –When does aging begin –Role of genome and environment on aging –What Demography is and how it is useful –How life expectancy has changed in the last 200 years and what has contributed the most to this change –How the population distribution has changed and what impact this might have for the future –Centenarians and aging What have we learned from these studies –How old was the oldest living documented person of each sex

5. Divisions of the Lifespan Prenatal Life Ovum: Fertilization - end 1st week Embryo: 2nd-8th week Fetus: 3rd-10 lunar month Neonatal Period Newborn: end of 2nd week Infancy: 3rd week-1st year Childhood: 2-15 years Adolescence: 6 yrs after puberty Postnatal Life Adulthood Prime & transition (20-65 yrs) Old age & senescence (65 yrs+)

6. Demography Statistical study of human populations: –Size and density distribution Vital Statistics: –Epidemiology: Births, deaths, diseases

7. Life expectancy at birth by sex, France 1806-1997

8. Proportion of population aged 0-14 versus 65+(In Italy)

9. Centenarians Generally good health –Escapers –Late onset of disease –Early disease that was overcome SSC (Semi-Super) –105+ SC (Super) –110+ Possible role of IGF-1 Receptor Oldest Female –122 years –Jeanne Calment Oldest Male –115 years –Christian Mortensen

10. Women and Longevity Probable Causes for increased longevity –Genetic –Environmental –Lesser Life Stress –Decreased Smoking –Protective Hormones –Better Protection Against Oxidative Damage

11. Comparative / Differential Aging Be familiar with: –What are the models for study –What does physiological assessment require –What are the physiological correlates with longevity and how do they correlate –What are the different models for studying rate of senescence –Handout

12. Figure 3.1: Comparative Maximum Life Spans **Detailed discussion of figure in the legend, pg. 26

13. Assessment of Physiological Age in Humans Physiological age depends on Physiologic competence: good to optimal function of all body systems & Health status: absence of disease Physiological age may or may not coincide with chronological age

15. Epidemiology Be familiar with: –What is epidemiology and why is it useful –Why is it thought that older people are at an elevated risk for disease –What are the major age associated causes of death –Understand why falls are a problem for the elderly

16. EPIDEMIOLOGY OF AGING THE STUDY OF THE AGE-RELATED DISTRIBUTION AND CAUSES OF DISEASE, DISABILITY, AND MORTALITY IN HUMAN POPULATIONS.

17. EPIDEMIOLOGY OF AGING ACCUMULATION OF ENVIRONMENTAL/BEHAVIORAL INSULTS. REDUCED IMMUNOLOGICAL SURVEILLANCE

18. EPIDEMIOLOGY OF AGING WHY IMPORTANT? – AGING OF THE HUMAN POPULATION –HEALTH AND VITALITY OF AN AGING POPULATION –QUALITY OF LIFE AND COST OF CARE

19. EPIDEMIOLOGY OF AGING MAJOR AGE-ASSOCIATED CAUSES OF DEATH –CANCER –CARDIOVASCULAR DISEASE –CHRONIC OBSTRUCTIVE PULMONARY DISEASE –DIABETES

20. EPIDEMIOLOGY OF AGING AGE-SPECIFIC COLORECTAL CANCER INCIDENCE RATES (Per 100,000 in population ) WM WF BM BF <65 20.4 14.7 25.3 20.4 65+ 408.0 269.3 385.8 286.1

21. EPIDEMIOLOGY OF AGING COGNITIVE FUNCTION Moderate/Severe Memory Impairment Male Female 65-69 5.3 3.8 85+ 37.3 35.0

22. EPIDEMIOLOGY OF AGING FALLS 30% OF PEOPLE AGED 65+ FALL EACH YEAR. 10-15% OF THOSE FALLS ARE CONSIDERED “SERIOUS/NON-FATAL” FALLS REPRESENT THE LEADING CAUSE OF ACCIDENTAL DEATH IN PEOPLE AGED 65 AND OLDER. FEAR OF FALLING IS A LEADING REASON FOR NOT ENGAGING IN PHYSICAL ACTIVITY.

23. EPIDEMIOLOGY OF AGING CAUSES OF FALLS IN THE ELDERLY - DIZZINESS - POOR COGNITIVE FUNCTION - VISION PROBLEMS - GENERAL FRAILTY - ENVIRONMENTAL HAZARDS

24. Biomarkers Be familiar with: –What a biomarker is –How a disease can be used as a tool to study aging –How genetic susceptibility can influence disease –Differences between disease and aging –Characteristics of progeria syndromes –Experimental models to study aging –Compression / Expansion of morbidity

25. Disease as a tool for the study of aging Sporadic cases of syndromes having multiple characteristics of premature (early onset, 20-30 years of age) or accelerated (rapid progression) aging occur in humans These conditions are grouped under the name of progeria. Examples of progeria syndromes are Werner’s syndrome (WS) and Hutchinson-Guildford syndrome

26. How genetic susceptibility may influence a disease: By itself By making the carrier more susceptible to disease By increasing the expression of a risk factor, or the risk factor may increase the genetic effects

27. Differences between WS & Aging WS Rare NO hypertension NO dementia Tissue calcifications Aging Universal Hypertension Dementia NO tissue calcifications

28. Werner’s Syndrome (pgs. 42-43) Shortness of stature Senile appearance Cataracts & graying of hair Skin changes (scleroderma) Joint deformities Atrophy of muscle & connective tissue Early cessation of menstruation Increased incidence of neoplasm

29. Down’s Syndrome (mongolism) Trisomy of chromosome 21 (mimicked in mice with introduction of chromosome 16 trisomy) Occurs in children born to older mothers Major symptoms: Cretinism (severe mental deficiencies) Accelerated aging & premature death

30. A theory of “compression” of morbidity (rectangularization of survivorship) curve

31. Functional Assessment Be familiar with: –What comprises geriatric assessment –What programs are used to test assessment and what are their parameters –Why do women have more disability –Compare/Contrast aging with physical inactivity Know some parameters –Aging vs. Disease

32. Geriatric Assessment Involves a multi-dimensional diagnostic process designed to qualify an elderly individual in terms of: Functional capabilities Disabilities Medical & Psychological characteristics A list of typical assessments is summarized in Table 3.3 For our discussion, we will consider particularly: Activities of Daily Living (ADL) Instrumental Activities of Daily Living (IADL) **See Table 3.4**

33. Assessment Programs include tests that are grouped into three categories: 1.Tests examining general physical health 2.Tests measuring ability to perform basic self care (ADLs) 3.Tests measuring ability to perform more complex activities (IADLs), reflecting the ability to live independently in the community

35. Figure 3. 6: % of persons 70 years & older having difficulty/inability to perform ADLs & IADLs With advancing age, 1) disability intensity increases in men & women; 2) disability intensity is higher in women than in men at the same age (esp. at later ages); 3) females live a longer average life span but live longer with disability

37. Aging is associated with increased incidence of: Diseases Accidents Stress

38. Aging should be differentiated from Disease Disease: is selective; it varies with the species, tissue, organ, cell molecule may depend on intrinsic & extrinsic factors is discontinuous (may progress, regress or be arrested) is occasionally deleterious, damage is often variable/reversible is often treatable with known cause(s)

39. Telomeres Be familiar with: –What telomeres are –Why telomeres are important –Consequence of telomere end-shortening –What telomerase is –The telomere hypothesis of aging

40. Telomeres Ends of linear chromosomes Centromere Telomere Repetitive DNA sequence (TTAGGG in vertebrates) Specialized proteins Form a 'capped' end structure

41. 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)

42. Why are telomeres important? Prevent chromosome fusions by NHEJ NHEJ Mitosis FUSION BRIDGE BREAKAGE Fusion-bridge-breakage cycles Genomic instability Cell death OR neoplastic transformation

43. Telomere Length (humans) Number of Doublings 20 10 Cellular (Replicative) Senescence Normal Somatic Cells (Telomerase Negative) Telomere also provide a means for "counting" cell division: telomeres shorten with each cycle Telomeres shorten from 10-15 kb (germ line) to 3-5 kb after 50-60 doublings (average lengths of TRFs) Cellular senescence is triggered when cells acquire one or a few critically short telomeres.

44. TELOMERASE: Key to replicative immortality 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

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

46. The telomere hypothesis of aging Telomere length is not related to life span (mice vs human; M musculus vs M spretus) Telomeres contribute to aging ONLY if senescent cells contribute to aging Telomerase protects against replicative senescence but not senescence induce by other causes

47. Apoptosis Be familiar with: –What apoptosis is –Why apoptosis is important –How apoptosis is regulated –The role of apoptosis in age related disease

48. APOPTOSIS Programmed cell death Orderly cellular self destruction Process: as crucial for survival of multi-cellular organisms as cell division

49. Forms of cell death Necrosis Apoptosis Mitotic catastrophe Passive Active Passive Pathological Physiological or Pathological pathological Swelling, lysis Condensation, Swelling, lysis cross-linking Dissipates Phagocytosed Dissipates Inflammation No inflammation Inflammation Externally induced Internally or Internally induced externally induced

50. STAGES OF APOPTOSIS Healthy cell DEATH SIGNAL Commitment to die (reversible) EXECUTION (irreversible) Dead cell (condensed, crosslinked) ENGULFMENT DEGRADATION

51. APOPTOSIS: important in embryogenesis Morphogenesis (eliminates excess cells): Selection (eliminates non-functional cells):

52. APOPTOSIS: important in embryogenesis Immunity (eliminates dangerous cells): Self antigen recognizing cell Organ size (eliminates excess cells):

53. APOPTOSIS: important in adults Tissue remodeling (eliminates cells no longer needed): Virgin mammary gland Late pregnancy, lactation Involution (non-pregnant, non-lactating) Apoptosis - Testosterone Prostate gland

54. APOPTOSIS: important in adults Tissue remodeling (eliminates cells no longer needed): Resting lymphocytes + antigen (e.g. infection)- antigen (e.g. recovery) Apoptosis Steroid immunosuppressants: kill lymphocytes by apoptosis Lymphocytes poised to die by apoptosis

55. APOPTOSIS: control Receptor pathway (physiological): Death receptors: (FAS, TNF-R, etc) FAS ligand TNF Death domains Adaptor proteins Pro-caspase 8 (inactive)Caspase 8 (active) Pro-execution caspase (inactive) Execution caspase (active) Death MITOCHONDRIA

56. APOPTOSIS: Role in Disease TOO MUCH: Tissue atrophy TOO LITTLE: Hyperplasia Neurodegeneration Thin skin etc Cancer Athersclerosis etc

57. Senescence Be familiar with: –What senescence is –What the types of senescence are –What causes senescence –Why senescence is important to aging and disease

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

59. Cellular Senescence What causes it? (what causes the senescent phenotype?) Cell proliferation (replicative senescence) = TELOMERE SHORTENING DNA damage Oncogene expression Supermitogenic signals

60. First description: the Hayflick limit Proliferative capacity Number of cell divisions Finite Replicative Life Span "Mortal" Infinite Replicative Life Span "Immortal" EXCEPTIONS Germ line Early embryonic cells (stem cells) Many tumor cells

61. 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

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

63. p53 and pRB proteins Nuclear proteins controlled by complex pathways (upstream regulators and downstream effectors) Control expression of other genes Halt cell cycle progression in response to inducers of senescence Crucial for allowing normal cells to sense and respond to senescence signals

64. 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 decling force of natural selection with age

65. Antagonistic pleiotropy Cellular senescence Selected for tumor suppression (growth arrest) Functional changes unselected, deleterious FUNCTIONAL CHANGES ASSOCIATED WITH CELLULAR SENESCENCE: Secretion of molecules that can be detrimental to tissues if not controlled e.g., senescent fibroblasts secrete proteases, growth factors, inflammatory cytokines

66. Mitochondrial Decay Be familiar with: –Mitochondria function/damage –Assays for oxidative damage –Other types of oxidative damage –Acetyl Carnitine (ALCAR) –R-  Lipoic Acid (LA) –Enzyme-cofactor interactions affected by oxidative damage in cells –Behavior of old rats fed ALCAR and LA –Micronutrient under-nutrition in Americans

67. Mitochondria O 2  O 2 -  H 2 O 2   OH  H 2 O, oxidative metabolism - Reaction intermediates are similar to products formed by radiation exposure. - 1-2% leakage of reactive oxygen species (ROS)

68. Assays for Oxidative Damage Products from Base Excision Repair (BER) and Nucleotide Excision Repair (NER) are secreted in the urine. - Estimated to be ~100,000 events/cell each day. - Frequency increases in older individuals Rat liver cells – Young ~24,000/cell Old ~67,000/cell

69. Other types of oxidative damage increase with age Protein Carbonylation (Stadtman) - Lipid oxidation (aldehydes "stuck" to proteins/enzymes) - age pigment -fluorescent lipid peroxides accumulate with age, can see fluorescence in tissues of older individuals (humans and rats) Mitochondria may deteriorate with time, old or damaged mitochondria may be destroyed by lysosomes in the cytoplasm.

70. As organisms age… - mitochondrial potential decreases. - oxidant leakage increases.

71. Acetyl Carnitine (ALCAR) –involved in mitochondrial membrane transport of long chain fatty acids into, and short and medium chain fatty acids out of the mitochondria. –Feeding ALCAR to aging rats is able to suppress or ameliorate many age-related changes in mitochondrial function and oxidant stress. –restores membrane potential –although oxidant leakage remains

72. R-  -Lipoic Acid (LA) – Potent Anti-oxidant – Lowers oxidants in old rats – Restores ascorbic acid levels (vitamin C) – Restores glutathione levels (another anti- oxidant)

73. Enzymes and Cofactors, changes in Km –Km is a measure of binding between an enzyme and substrate (or cofactor) –Many point mutations reduce the Km of enzymes for the enzyme cofactor (cofactors are often vitamins, B family vitamins in particular). –Increasing vitamin doses can push binding equilibrium toward enzyme-cofactor interaction, thereby overcoming the deleterious effects of point mutations on enzyme function

74. Are Enzyme-cofactor interactions affected by oxidative damage in cells? –Binding (Km) is reduced in older animals –Effect can be mimicked using purified protein and mixing with aldehydes (oxidized lipids). –This may indicate that loss of enzyme-cofactor binding with age is the result of accumulation of lipid oxidation products (aldehydes) that interfere with enzyme-cofactor binding. Feeding rats ALCAR and LA can reverse the loss in binding between an enzyme and cofactor

75. Behavior of old rats fed ALCAR and LA –Old rats are less active at night, and have decreased function in cognitive tests (hidden platform, skinner box) –Is actually cognitive effect because decrease in function occurs with both visual and audible cues. –ALCAR/LA can reverse age-associated losses in cognitive function. –Neurons in aged rats can be stained for oxidized guanine (a nucleotide) to indicate damage to nucleic acid.

76. Ambulatory Activity before and After Supplementation with Lipoic Acid (LA) + Acetyl- L-Carnitine (ALCAR) 0 200 400 600 800 + LA + ALCAR Old Young + LA + ALCAR * Distance Traveled (cm/hour/day) * # # vs. young vs. old *

77. Do americans get enough micronutrients? NO Iron deficiency, results in loss of complex 4 (part of the electron transport chain in the mitochonrial membrane). –Complex 4 loss results in increased oxidative stress. –Iron deficiency mimics neurodegeneration. Zinc deficiency –Increased DNA damage in zinc deficient cells (demonstrated by Comet assay) –Zn required as a cofactor for many enzymes, DNA repair and responses to DNA damage is reduced in Zn deficient cells due to loss of enzyme function involved in these processes. p53 function reduced, increased cancer risk?

78. Oxidants and Anti-Oxidants Be familiar with: –What is the Free Radical Theory of Aging –What is a free radical –What are the implications of free radicals on aging –How oxygen can be toxic –What the major oxidants are –What the major oxidant sources are –What are the major antioxidants –Free radical damage markers –Experimental Models

79. Evolutionary Theory of Lifespan Be familiar with: –Evolutionary theory of lifespan –Aging in nature –Disposable soma –Antagonistic Pleiotropy –Mutation Accumulation –Trait that correlate with longevity –Model systems that agree and disagree with theory

80. Yeast as a Model for Lifespan Be familiar with: –A molecular cause of yeast aging – SIR2 – Aging and genetic instability, in yeast and humans