Aging and the Circadian Clock: Lessons from Long-Lived Mice

Transcription

1 Aging and the Circadian Clock: Lessons from Long-Lived Mice Ruth Miskin Department of Biomolecular sciences Weizmann Institute of Science 22 th Biennial Meeting of The Israel Gerontological Society Tel-Aviv, February 2018

2 The Nobel Prize in Physiology or Medicine 2017 "for their discoveries of molecular mechanisms controlling the circadian rhythm". Jeffrey C. Hall U of Main Michael Rosbash Brandeis U Michael W. Young Rockefeller U

3 Drosophila The Nobel Circadian clocks exist in all light sensitive organisms, including plants, bacteria and mammals.

4 Biological Clocks Biological clocks are biochemical mechanisms generating periodic oscillations in living cells independently of external cues. The circadian clock generates cycles of about ( circa ) but not exactly 24 hours ( diem ) in order to synchronize metabolic functions and behavior with the geophysical time. Light input corrects the internal oscillator by a few minutes every day to synchronize with the geophysical time. The clock enables an organism to predict and prepare for daily challenges.

5 Against the clock Jet lag is characterized by a misalignment between internal circadian rhythms and local time caused by rapid travel across at least two time zones.

6 Circadian oscillators are controlled through a common mechanism: A negative transcriptional feedback loop

7 The Drosophila clock: A self sustained Transcription-Translation Feedback Loop The feedback regulation of period (PER) and timeless (TIM) negative genes. Additional proteins (e.g. CRY, DBT) are essential for the oscillations.

8 The molecular circadian clock in mammals: A cell autonomous, self sustained transcription-translation feedback loop AMPK NAD(P)H CLOCK E-box CACGTG BMAL1 Ac Transcriptional activation of Clock Controled Genes CRYs PERs Ac PERs CRYs CLOCK BMAL1 Ac E-box CACGTG Suppression LEPTIN, PAI-1 ATP ADP CKIe x Ac P P CRYs PERs ATP ADP x P P CRYs PERs NAD + SIRT1 CLOCK BMAL1 Ac CLOCK BMAL1 E-box CACGTG Relieve of Suppression E-box CACGTG Ac x NAM ADP-r The innate circadian cycle as measured in darkness (tau=period, ~24 hours) circa - about dies - day After Froy and Miskin, Aging (2010)

9 The molecular circadian clock in mammals NAD(P)H CLOCK E-box CACGTG BMAL1 Ac Transcriptional activation CRYs PERs Ac CRYs PERs CKIe ATP ADP x Ac P AMPK CRYs PERs ATP ADP P P P CLOCK BMAL1 Ac CLOCK BMAL1 Ac CLOCK BMAL1 E-box E-box E-box CACGTG CACGTG CACGTG Suppression Relieve of Suppression PAI-1 x NAD + NAM SIRT1 Ac CRYs ADP-r PERs x The innate circadian cycle (tau=period) as measured in darkness (~24 hours) After Froy and Miskin, Aging (2010) 2:7

10 Features of Circadian rhythms Period (t) in darkness phase shift amplitude arrhythmia

11 The hierarchy of the circadian clock in mammals: central and peripheral clocks SCN neurons Central Clock: SCN (suprachiasmatic nucleus) Light Autonomic innervation Humoral factors Indirectly by determining food intake Heart After Reppert et al, Nature (2002) 418:935 Peripheral Clocks Food intake Metabolism Locomotor activity Sleep-wake cycle Blood pressure Body temperature Cardiovascular activity Endocrine system Immunity

12 יש שעה לכל פעילות

13 Why heart attacks occur in the early morning? The human circadian system causes a morning peak in the circulating levels of PAI-1 (plasminogen activator inhibitor), an inhibitor of blood clot dissolution. PAI-1 belongs to the CCG s group, containing an E-box. Frank AJ et al, Blood (2014) 123:590

14 Against the clock Shift work is associated with an increased occurrence of metabolic disorders including diabetes, dyslipidemia and weight gain, and cognitive impairment and cancer. Jet lag is characterized by a misalignment between internal circadian rhythms and local time caused by rapid travel across at least two time zones.

15 Ageing leads to changes in circadian rhythmicity phase shift Period (t) in darkness amplitude arrhythmia Age-related changes in amplitude, phase shift, and/or period length have been described for hormonal rhythms, body core temperature, sleep-wakefulness, etc. These changes are paralleled by age-related alterations in the SCN. Rhythmicity is disrupted in old hamsters. Feta SCN implants restore higher amplitudes.

16 The circadian clock in a long-lived animal model

17 αmupa vs wild type (WT) female mice at 18 months of age WT (FVB/N) control ~25% are obese MUPA (transgenic) Obesity resistant

18 30-month-old mice

19 MUPA mice exhibit reduced body weight, reduced visceral fat, and resistance to obesity

20 Female αmupa fed ad libitum exhibit spontaneously reduced food intake span 36% 16% 23% 15% Miskin And Masos, J Gerontol A Biol Sci Med Sci (1997)

21 Female and male αmupa mice exhibit increased life-span 100% αmupa 50% WT 44% αmupa 62% WT 30% 6% Steckler R et al, J Gerontol A Biol Sci Med Sci (2015)

22 Hypothesis vs Observationsvypothesis vs Observation

23 Circadian patterns of food intake and body temperature are more pronounced in αmupa mice αmupa/15 WT WT αmupa αmupa Days after clock disruption αmupa/15 αmupa WT Froy O et al, Am J Physiol Endocrin Metab (2006)

24 mcry1 mcry1 mper2 Relative mrna levels mper2 mper1 mper1 mclock mclock αmupa mice show increased amplitudes in clock gene expression in the liver Froy and Miskin, Progress in Neurobiol (2007) WT MUPA CT Normal light CT Disrupted light

25 Food Intake (g / mouse) Circadian food consumption of young and aged mice Aged WT show a phase shift in circadian food intake. Aged MUPA maintain the youthful circadian pattern WT Both young (5 months) and old (18 month) mice eat 2.5 g/day young old 0 ZT MUPA Young (5 months) eat 1.9 g/day old (18 months) eat 2.0 g/day old young 0 ZT Froy and Miskin, Progress in Neurobiol (2007)

26 Age-induced changes in gene expression or behavior: amplitude reduction, phase shift, period length Period (t) in darkness phase shift amplitude arrhythmia

27 αmupa mice show a 24h period locomotor activity at both young and old ages Gutman R et al, Exp Gerontol (2011) 46:606

28 The circadian resonance hypothesis: Association between mammalian life-span and circadian period 9 strains of laboratory mice 13 primates 25 rodents Relationship between tau minus 24 and lifespan: Values for proximity of tau to 24 (tau minus 24) were significantly related to lifespan (75% failure) in (a), with strains with greater values having shorter lifespan. Multiple linear regression analysis confirmed a similar negative relationship between maximum life and tau minus 24 in (b) and (c). Wyse CA et al, Biol Lett (2010) 6:696

29 Features of the MUPA clock At young ages: 1. increased amplitudes in gene expression and behavior. 2. tau - 24h. At old ages: Maintaining the youthful feeding behavior and a 24h tau.

30 The circadian clock in the heart

31 Leading causes of death in the United States

32 Circadian rhythms play a crucial role in the cardiovascular system In humans Normal (heart rate, blood pressure) and adverse cardiovascular events do not occur at random times throughout the day. A peak of acute myocardial infarction (MI) occurs in the early morning (circadian increase in PAI-1). A diurnal pattern occurs for infarct size, stroke, sudden cardiac death etc.

33 Ligation of the Left Anterior Descending (LAD) coronary artery a model used to study cardiac damage after ischemia in-vivo LAD ligation Infarct area Echocardiography Infarct size

34 Circadian disturbance worsens outcome after myocardial infarction and exacerbates cardiac remodeling in heart disease Faisal J et al, Candian J of Cardiol (2015) 31:860

35 Cardiac aging is attenuated in αmupa mice Kaplan-Meier, P < 0.01 Levy E et al, PLoS One (2015) 10

36 Fractional Shortening (%) MUPA mice show Increased fractional shortening 24h after myocardial infarction ~ * WT αmupa * 35 ~ Sham MI Sham MI Young Old *p<0.05 αmupa compared to WT mice, ~p<0.05 sham compared to MI, n=6-8/ group. Levy E et al, PLoS One (2015) 10

37 Infarct Size/LV (%) MUPA mice show a Reduced Infarct Size TTC staining, 24h after myocardial infarction WT αmupa * * WT αmupa Young Old *p<0.05 αmupa compared to WT mice, n=6-8/group. Levy E et al, PLoS One (2015) 10

38 αmupa, the Clock and Longevity αmupa The first long-lived animal model found to show an improved circadian clock. The aged MUPA heart shows a youthful behavior. The aged MUPA circadian clock shows a youthful behavior. MUPA mice show a 24h tau at young and old ages. Is the circadian clock an upstream regulator of αmupa lifespan and health span?

39 Acknowledgements Ruth Miskin (Weizmann Institute of Science, Rehovot) J. Axelrod, O. Lachman, T. Masos, R. Abramovitz Heiner Westphal (NIH, Bethesda, USA) Oren Froy (Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot) H. Sherman, G. Bhargava, N. Chapnic, R. Cohen, R. Gutman, Y. Genzer Roee Gutman (MIGAL - Galilee Research Institute, Kiryat Shmona, Tel-Hai College, Upper Galilee, Israel). R. Steckler, A. Shabtay-Yanai, M. Pinsky, M. Rauch, S. Tamir Edith Hochhauser (Felsenstein - Beilinson Medical Research Center, Tel-Aviv University) E. Levi, R. Kornowski, G. Grienberg, Y. Cheporko, Ilana Fratty Arieh Gertler (Faculty of Agricalture) G. Solomon Oren Tirosh (Faculty of Agricalture) A. Aronis, Michal Pardo Betty Schwartz (Faculty of Agricalture Igor Zusman (Koret School of Veterinary Medicine, Rehovot) Shlomo Yahav (Agricultural Research Organization, Bet Dagan) Vadim Fraifeld (Faculty of Health Sciences, Ben Gurion University of the Negev) Hagai Yanai

40 The first observation of circadian behavior (Jean Jaques d Ortous, 1729): Leaves have an internal biological clock Mimosa plants have a cell autonomous clock that can maintain the biological rhythm even under constant darkness. constant darkness low their daily rhythm for several days, suggesting that Mimosa plants have a cell

41 The cyanobacterial periodosome a biochemical oscillator The first clock in evolution. Daylight is transduced through a cascade of phosphorylation and activation of several proteins, that transfer information through proteinprotein interactions. Late in the evening, binding of another protein leads to complex dissociation and ends the cycle. The periodosome controls the timing of cell division, metabolism, nitrogen fixation, photosynthesis, etc. Rpa A

42 Correct circadian period (tau) enhances growth and survival in Arabidopsis tau = 20.7h tau = Circadian system enhances chlorophyll content, photosynthetic carbon fixation and growth thus leading to improved survival and competitive advantage. Dodd AN et al, Science (2005) 309:630

43 Mutations affecting tau The tau mutation in casein kinase attenuates degradation of PERIOD in mice and hamster. It causes tau to shorten with ~2h for each copy of the mutant allel. Homozygous tau are unable to entrain to 24h LD cycles in the lab. Life-span is similar for homozygous and heterozygous tau mutants in darkness, but shorter in LD for heterozygous. Familial Advanced Sleep Phase Syndrome: A mutation in the human PERIOD2 gene interferes with casein kinase-induced degradation. This results in 4h advanced phase in rhythms of sleep, melatonin, and body temperature.

44 Clock gene expression in the liver under restricted feeding (RF) is rhythmic in normal and disrupted light Restricted feeding (RF) uncouples peripheral clocks from the SCN Food Food

45 High fat diet under restricted feeding does not increase body weight and fat depots Food was available for 4 h per day ( 4-8 h after light on) Hadas Sherman et al. FASEB J 2012;26:

46 The Fibrinolytic System Plasminogen activators (PAs) urokinase-type (upa), tissue-type (tpa) Plasminogen activator inhibitor- 1 (PAI-1) Plasminogen (inactive) Plasmin (active)

47 Miskin et al. (1999) Neurobiol Aging The αmupa Transgene

48 Transgenic upa mrna Expression in the Trigeminal Nucleus of the αmupa Brain Stem Miskin et al. (2005) Mech Age Dev

49 The Plasminogen Activator/Plasmin System

50 The hierarchy of the circadian clock in mammals: central and peripheral clocks SCN neurons Central Clock: SCN (suprachiasmatic nucleus) Light Autonomic innervation Humoral factors Indirectly by determining food intake Heart Food intake Metabolism Locomotor activity Sleep-wake cycle Blood pressure Body temperature Cardiovascular activity Endocrine system After Reppert et al, Nature (2002) 418:935 Peripheral Clocks

51 Biological clocks are biochemical mechanisms generating periodic oscillations in living cells independently of external cues. The circadian clock generates cycles of about ( circa ) but not exactly 24 hours ( diem ) in order to synchronize metabolic functions and behavior with the geophysical time. Light input corrects the internal oscillator by a few minutes every day to synchronize with the geophysical time in all light sensitive organisms, bacteria, plants, animals. The clock enables an organism to predict and prepare for daily challenges. Biological Clocks