Mitotic Misregulation and Human Aging

1. Mitotic Misregulation and Human Aging Ly DH, Lockhart DJ, Lerner RA, and Schultz PG. 2000. Mitotic Misregulation and Aging. Science: 287(5462):2486. By Micaile Brown

2. Aging Characterized as the cumulative irreversible changes that take place in an organism over the life span following the attainment of maturity. Associated with gradual declines in the performance of most organs in the body. 1

3. Aging cont. • Why we age has intrigued mankind since the beginning of time. • The wide range in life-span of different species makes it clear that the genetic make-up of an animal plays a primary role. Why: - Flies: 90 days - Rats: 2 to 3 years - Dogs: 15 to 20 years - Horses: 40 to 45 years

4. Goal of Researchers: • Determination of how the potential life span of an individual is determined by genes. • Extensive studies of model systems (i.e. Drosophila and mice) have identified a number of processes thought to contribute to the aging phenotype.

5. Processes: • Oxidative damage associated with cellular metabolism. • Telomere shortening • Mitochondrial mutations • Chromosomal pathologies.

6. Oxidative Damage • Mitochondrial Respiratory System: - source of oxygen - produces free radicals as by-product • Elevation of by products cause oxidative damage to cells. • Research Shows: Aging cells have elevated oxidative stress and oxidative damage.

7. Telomere Shortening • Telomere: DNA sequences located at the ends of chromosomes. • Telomere hypothesis proposes that cells fail to divide due to shortening of the telomere regions of DNA during cell division. • Hence,……..Contributes to aging.

8. Chromosomal Pathologies • Mutations in chromosomes that contribute to the aging phenotype:

9. Mitochondrial Mutation • Mutations of the mitochondria caused by reactive oxygen species.

10. Purpose • To gain greater insights into the mechanisms that control life-span and age-related phenotypes. • Study consisted of studying gene regulation of normal and premature aging in actively dividing cells.

11. Procedures - Step 1: • Ten closely matched fibroblast cell lines were classified into 4 categories based on their chronological and diagnostic similarities

12. 4 Categories of Fibroblast Cells: • Normal Young (NY) • Normal Middle (NM) • Normal Old (NO) • Hutchinson-Gilford progeria (P)

13. Hutchinson-Gilford Progeria: -A rare genetic disorder, in which those who are affected display at a very early age, features typically associated with natural old age, such as, graying of hair, diminished subcutaneous fat, cardiovascular disease, and skeletal abnormalities.

14. Procedures - Step 2: • Determination of age related morphological changes: - Actively dividing early fibroblasts from each group were initially examined by phase contrast and fluorescence microscopy 2

15. Procedures - Step 3: • Flow activated cell sorting (FACS) analysis of each group of fibroblast cells.

16. Procedures - Step 4: • Examination of the transcriptional profiles (mRNA abundance) of the fibroblast cells: - Asynchronous and actively dividing cells and were cultured in vitro to about 60% confluency.

17. Step 4 cont. - Messenger RNA levels were analyzed with high density oligonucleotide arrays containing probes for more than 6000 known human genes. - Expression patterns for each age group (NM, NO, and P) were compared with NY fibroblasts (baseline). (Only those changes that were reproducible across all comparisons and all independent replicates were considered further.)

18. Results: Phase Contrast and Fluorescence Microscopy: • Each age group, the elliptical morphology characteristic of fibroblast nuclei was observed in the majority of cells. • However, in contrast to fibroblasts from NY and NM, whose nuclei appeared normal, the NO and P had a significant proportion of cells exhibiting aberrant nuclear morphology including multilobed nuclei and irregular nuclear boundaries. 3

19. NO and P populations also had a higher proportion of cells with multiple nuclei. - This is consistent with reports of age-dependent increases in micronucleation in human lymphocytes.

20. Results: Flow Activated Cell Sorting (FACS) • NY and NM groups revealed very similar populations of cells with 2N, S, and 4N DNA content. • However, NO and P fibroblasts showed higher percentages of 4N DNA content; which is consistent with the larger number of binucleated cells observed by microscopy. 4

21. FACS Results:

22. Results: Invitro Culturing for DNA content • 61 genes showed consistent expression level changes more than twofold between young and middle age. • More than half of these genes could be grouped into two functional classes:

23. 2 Functional Classes of The Genes: • #1 - genes whose products are involved in cell cycle progression (25%). • #2 - genes involved in maintenance and remodeling of extracellular matrix (ECM) (31%). 5

24. Cell Cycle Progression Genes: • All involved in mitosis. • Showed a down regulation between 2.6-12.5 fold.

25. Down Regulated Genes • **See Hand-Out**

26. Cell Cycle

27. Extracellular Matrix Remodeling Genes: • Up - regulation of the genes. • Examples: -Human macrophage metalloproteinase (HME) - Stromelysin 2 - Protease Inhibitors 6

28. Up-Regulation of Genes controlling enzymes: • Examples: Prostaglandin endoperoxidase synthase, endoperoxide synthase type II, cyclooxygenase 2, and endoperoxidase synthase. • Increased levels of such enzymes with age may affect a large number of physiological processes, such as platelet aggregation, muscle and kidney function, and bone formation.

29. Conclusions: • The purpose of this study was to gain greater insights into the mechanisms that control life-span and age related phenotypes. • Researchers did so by analyzing Messenger RNA levels in young, middle age, old age, and progeria patients. • This analysis revealed a common set of genes with altered expression levels.

30. Conclusion cont. • Some genes showed down- regulation in the varying aging groups, while other genes showed up-regulation. • These genes are principally involved in the G2-M phase of the cell cycle and in the remodeling of the ECM.

31. Conclusion cont. • A comparison of gene expression in old age and progeria also revealed disregulation of these same genes, as well as additional genes involved in DNA or RNA synthesis and processing • Hence, this study proposes that gene disregulations leads to chromosomal pathologies that result in misregulation of genes involved in the aging process.

32. References • Caleb EF and Tanzi RE. 2000. Genetics of aging. Science: 278(5337): 407. • Ly et. al. 2000. Mitotic misregulation and human aging. Science: 287(5462): 2486. • Marko et al. 1998. Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events. Journal of Cell Biology: vol. 141(6):1393-1406. • Walton et al. 2000. The musculoskeletal manifestations of Werner’s Syndrome. The Journal of bone and joint surgery: vol 82(B6): 885-888.

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