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Premature aging in mice lacking TR4 orphan receptor

Premature aging in mice lacking TR4 orphan receptor. Yi-Fen Lee, Ph.D. University of Rochester. What is aging?. Definition: an irreversible progressive functional decline and increase mortality over time.

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Premature aging in mice lacking TR4 orphan receptor

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  1. Premature aging in mice lacking TR4 orphan receptor Yi-Fen Lee, Ph.D. University of Rochester

  2. What is aging? • Definition: an irreversible progressive functional decline and increase mortality over time. • Symptoms: wrinkles, gray hair, and declines in strength, mental sharpness, reproductive ability, would healing, immunity, and increase in likelihood of mortality. • The average life expectancy for human has increased over the centuries with improvements in sanitation, health care, and food quality and availability. However, the maximum human lifespan has not grown much. • One of the most powerful ways to extend life is to reduce food intake while satisfying nutritional needs. Organisms ranging from yeast to mammals enjoy much longer (50% increase) and healthier lives if they consume just two-thirds of the calories they normally eat. Note: cutting back on calories may not require a lifelong commitment; mice whose caloric intake isn’t reduced until 19 month old still show the lifespan and health benefits after a couple month CR diet (PNAS, 101: 5524, 2004) Yi-Fen Lee

  3. In addition to CR diet, Geriatric mouse: Yoda (left), who passed away at the age of four years and 12 days, was the world's oldest mouse (U of Michigan ) without CR diet. geriatric mice are providing important clues about how genes and hormones affect the rate of human aging and risks of disease late in life. Yi-Fen Lee

  4. What genes involved in aging? • Genes involved in genomic stability • WRN (werner syndrome):DNA helicase • Ku80 • ERCC1 • Telomerase • p53 • Mitochondrial DNA • Mitochondria DNA polymerase • ROS • Catalase • Others • A-type almins. • Klotho Yi-Fen Lee

  5. What does aging occur? • Somatic mutation of aging (DNA damage) • Telomere loss theory • Mitochondria theory • Altered proteins and waste accumulation theory • Network theories of aging Yi-Fen Lee

  6. Testicular Orphan nuclear receptor 4 • TR4 was cloned from testis and prostate cDNA libraries and is highly homologous with TR2 orphan receptor. • TR4 belongs to a member of the nuclear receptor superfamily for which a ligand has not yet been found. • Ubiquitous expression, with higher expression level in the brain and testis • It is a transcriptional factor can recognizes and binds to AGGTCA-like direct repeat (DR) motif (with different spacing). • It can transactivate and suppress the activity of those genes which contain DR-(n) motif (n=1, 4, 3, 6) in promoter or enhancer regions. Yi-Fen Lee

  7. Generation of TR4 knockout mice by targeted gene disruption Yi-Fen Lee

  8. TR4 KO Mice Phenotypes: Growth retardation and abnormal maternal behavior in mice lacking Testicular orphan nuclear Receptor 4Proc Natl Acad Sci U S A. 2004 Oct 19;101(42):15058-63 Targeted inactivation of testicular nuclear orphan receptor 4 delays and disrupts late meiotic prophase and subsequent meiotic divisions of spermatogenesisMol Cell Biol. 2004 Jul;24(13):5887-99 Deficits in motor coordination with aberrant cerebellar development in mice lacking testicular orphan nuclear receptor 4Mol Cell Biol. 2005 Apr;25(7):2722-32 Priapism, reduced fertility, and growth retardation in mice lacking Testicular orphan nuclear Receptor 4paper in preparation Yi-Fen Lee

  9. TR4 KO mice, in general, have shorter lifespan (average < 12 month, compared with >2 years from wild type (TR4 wt) mice and more than 95% of the TR4 KO mice (67 out of 70 TR4 KO mice) won’t live over one year of age, without an obvious cause of death. at 6 month old, TR4 KO mice…. Yi-Fen Lee

  10. Aging phenotypes: Gray hair Normal albumin level reduced dermis thickness, subcutaneous adipose Skin: extramedullary haematopoiesis in the liver Yi-Fen Lee

  11. Kyphorsis: Reduced BMD Reduced trabeculae and cortical bone volume Yi-Fen Lee

  12. Osteoarthritis in TR4 KO mice WT KO Knee-joint space (arrows) narrowing with irregularity of joint surface and bony outgrowth (osteophytes) found in TR4 KO mice. WT KO s s * * Decreasing of superficial zone (s) cells, disruption of articular surface, and duplication (*) in transition zone were observed in TR4 KO mice. Yi-Fen Lee

  13. Premature Ovarian Failure (POF) in TR4KO mice KO WT A B C Corpora lutea Antral Pre-ovulation Pre-antral WT KO WT KO D Endometrium E Endometrium proliferate epithelia: thinner Grand formation Stroma not proliferate 100 X 100 X luma 1. No active estrus cycle (all stay in diestrus cycle) 2. Ovary: only pre-antral and small antral follicles; no pre-ovulation follicles, neither corpora lutea can be found 3. uterus atrophy Yi-Fen Lee

  14. Rapidly replicative cell senescence in TR4 KO MEF KO WT P2 P4 Yi-Fen Lee

  15. ROS is a source of chronic and persistent DNA damage in all cells and contribute to cell senescence. How cells respond to oxidative stress would determine the life span. Control H2O2treatment WT WT KO KO TR4KO MEF have higher ROS and H2O2-induced ROS Yi-Fen Lee

  16. A reduced ability to tolerate stresses is a hallmark of agingTest the resistant to other DNA-damage agent induced stress mM H2O2 100 10 50 250 150 200 Indeed, KO MEF cells are less resistant to the ROS-induces stress Yi-Fen Lee

  17. 1.1 1 0.9 wt Relative OD/MTT 0.8 ko 0.7 0.6 0.5 0 3 6 9 Gy Response to irradiation: KO MEF cells are less resistant to IR-induces stress Yi-Fen Lee

  18. To further confirm TR4 role in Anti-ROS defense system • Rescue TR4 KO by sending back TR4 pBabe-TR4 and pBabe-vector control were deliver back to TR4MEF cells and w/wo oxidative stress challenge and then measure the ROS status by flow cytometric quantification analysis Yi-Fen Lee

  19. ROS is higher in KO and restore TR4 back rescue Yi-Fen Lee

  20. Does TR4 directly involve in the anti-ROS defense system? • Regulate scavenger enzymes activities? • Catalase • Glutathione reductase; glutathione peroxidase • Superoxide dismutase (SOD) No obvious difference between TR4 KO vs wt B. Regulate non-enzyme low molecular weight antioxidants (LMWAs) activities Vitamin E (100nM) can lower down the endogenous or H2O2- induced ROS status in wt MEF cells, not in TR4 KO MEF cells Yi-Fen Lee

  21. TR4 protects DNA from ROS induced DNA break: • Fe++ + H2O2 oc sc . Fe+++ OH +:OH- + Supercoiled (SC) form of plasmid Open circle (OC) form of plasmid Yi-Fen Lee

  22. Abnormal mitochondria enlargement and proliferation in TR4 KO mice muscle Mitochondria Mitochondria ragged red fiber 15000X 5000x PS: we also found a significant decline ATP production in KO hepatocyte and MDF cells, compared with WT cells Yi-Fen Lee

  23. Growth retardation show in KO Indeed, we found that TR4KO mice has growth retardation and TR4-rescue growth retardation Yi-Fen Lee

  24. Accumulation of Single strand DNA break in TR4 KO MEF cells Yi-Fen Lee

  25. TR4 vs DNA repair? • UV-induced TCR pathway DS DNA break damage (end-joint) Puc19/BamHI (create DS and disrupt lacZ) Incubate with protein extracts from KO and wt Transform onto Amp/X-gal plate PCMXTR4 PCMX Note: GGR might not be involved Yi-Fen Lee

  26. Acceleration aging O2 OXPHOS Detoxification Mitochondria dysfunction Gpx, SOD, CAT Protective effect from DNA damage (-OH, ●OH, 1O2, -O2, NO) ROS double strand break NER Single strand DAN Break DNA damage DNA repair mice: grey hair, skin lordokyphosis, OP, OA, infertility. MEF: rapidly replicative senescence DNA replication cell proliferation Yi-Fen Lee

  27. Overall, our TR4 KO mice developed syndromes that imply the lost of TR4 might result in premature aging process • Shorter lifespan and slower growth rate • Skin atrophy; retarded hair regrowth, gray hair, and loss of hair in some; reduced sebaceous gland number • Mild, late-onset myopathy (atrophy, degenerated fiber, abnormal mito. proliferation) • Mild, late-onset reduced cardio-function (atrophy; the heart/body weight ratio: KO over 0.5) • Lordokyshosis; osteoporosis and osteoarthritis • Decline kidney function (reduced urine production and urine acidosis) • Premature ovarian failure (POF) • A rapidly replicative senescence in MEF cells. Yi-Fen Lee

  28. Molecular mechanism ? What signals alter in TR4 KO ? Irradiation stress Oxidative stress Genomic toxicity induced TR4 expression level Yi-Fen Lee

  29. In searching for the signals/molecules are responsible for TR4 KO accelerated aging process. A decreased Gadd45a gene was found in TR4KO muscle samples Gadd45a: 1. Stress (DNA-damaging agent or growth arrest signals) inducible in all mammalian cells 2. Regulated by p53, and p53-Gadd45a pathway control G2-M cell cycle progression 3. KO mice: Genomic instability in Gadd45a-deficient mice , 1999 Nature genetics 4. Control DNA repair Yi-Fen Lee

  30. Response to DNA-damage stress: IR Yi-Fen Lee

  31. Gadd45 a gene contains several DR3-motif which can be potentially bound and regulated by TR4 Reporter gene assay: CHIP assay demonstrated that TR4 can bind to C, D, and B regions , but not E region of DR3 motif Yi-Fen Lee

  32. Summary: Yi-Fen Lee

  33. UV: 100J/m2 8 1 3 0 Q 1: What signal control TR4? • Ligand? Some aging hormones? • Post-translational modification? • Phosphorylation ? TR4 * ß-Actin Acetyation ? SIRT 1 (Histone/protein deacetylase) Yi-Fen Lee

  34. Q2: Why we are still aged if TR4 works? • If TR4 is important to maintain the genomic stability, what is its expression level, activity or mutation ? during the aging? • Why do we age? Yi-Fen Lee

  35. Q 3:What happen to the mice if more TR4 ? • Mutation of the mouse klotho gene leads to a syndrome resembling ageingNature390, 45 - 51 (06 November 1997); Science, Vol 278, Issue 5340, 1013 , 7 November 1997 • Boosting Gene Extends Mouse Life SpanScience, Vol 309, Issue 5739, 1310-1311 , 26 August 2005 Long-life mice These mice, which overexpress the gene for Klotho, have celebrated their third birthdays. Yi-Fen Lee

  36. Q4: What’s role of TR4 in regulate cell transformation? • Replicative senescence as a causative factor to the tumorigenesis, how does TR4 correlate to cancer incidence/progression. • Backcross with p53 null mice • Backcross with Ras-Tsg mice • incubation prostate stroma from TR4 KO newborn pup with BHP in renal capsule implantation prostate cancer model. • ……..many to come Yi-Fen Lee

  37. Q 5: other millions questions… • Modulation of Radiation selectivity • A marker for DNA damage ? • A marker for aging ? • A marker for tumor ? • A sensor to stress response Yi-Fen Lee

  38. Aging-related disease will soon be future the biggest health problems …… OP, OA, brain degeneration, diabetes, cancer ……. Yi-Fen Lee

  39. Our goal is…. Delaying aging while maintaining good overall health Yi-Fen Lee

  40. Acknowledgements TR4 aging study group: Benson Liu Dr. Shin-Chiu Ho Su Liu Dr. Ray Wang Dr. John Yan Dr. Silvia Chen Orthopedics: Dr. Edward Puzas Yi-Fen Lee

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