Genetica dell invecchiamento: esistono geni che modulano la longevità

Transcript

1 Genetica dell invecchiamento: esistono geni che modulano la longevità

2 Invecchiamento nel lievito e nel verme

3 Il ruolo di Sir2 nell invecchiamento Dal lievito all uomo

4 Identificazione di Sir 2 nel lievito Delezione del gene: invecchiamento precoce Iper espressione del gene: estensione del ciclo replicativo (aumento della longevità)

5 Sir2 is an NAD dependent histone deacetylase SIR2 + NAD Nicotinamide + ADP Ribose NAD is not used as an electron acceptor, it is consumed in the reaction

6 Meccanismi molecolari di invecchiamento nel lievito

7 Accumulo di circoletti di rdna extracromosomale

8 Sir 2 nel lievito Nel lievito, l invecchiamento écausato dalla formazione di circoletti extracromosomali di DNA codificante originariamente per l RNA ribosomale. I geni per l RNA ribosomale sono presenti in copie multiple e vanno incontro a processi di ricombinazione con formazione di circoletti. Sir2 éuna istone deacetilasi (regolata dal metabolismo livelli di NAD+) che compatta la cromatina dei geni per l RNA ribosomale e ne riduce la ricombinazione.

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10 Collegamenti fra metabolismo ed invecchiamento La restrizione calorica (RC) allunga la vita in tutti gli organismi Nel lievito, mutazioni in SIR2 prevengono l estensione del life span determinato da RC RC: aumentati livelli di NAD+ (rapport NAD+/NADH a favore del NAD+), aumentata attivitá di SIR2, ridotta ricombinazione dei geni per l RNA ribosomale, invecchiamento rallentato. Il pathway di SIR2 é stato selezionato per aumentare la sopravvivenza in condizione di stress (scarso nutrimento)

11 Because it consumes NAD, SIR2 has been proposed to be the monitor that connects metabolism to aging High Calorie Diet NAD is converted to NADH & unavailable for SIR2 Caloric Restriction: Less NAD used in catabolism More available for SIR2 action

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13 SIR2 and extension of life span In yeast (replicative senescence) In C. elegans (post mitotic senescence) In mammals??? regulation of p53 activation by diet (CR)

14 Caloric restriction increases SIRT1 expression in a variety of rat tissues Cohen HY, et al. Science 305: , 2004.

15 Implicazioni pratiche

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17 Resveratrol increases NAD dependent HDAC activity Yeung F, et al. Embo J. 23: , 2004

18 Food Sources Found in grapes, wine, grape juice, and berries of Vaccinum species including blueberries, bilberries, and cranberries In grapes, resveratrol is found only in the skins The amount of resveratrol in grape skins varies with the grape cultivar, its geographic origin, and exposure to fungal infection PBRC 2005

19 Food Sources Also, the amount of fermentation time a wine spends in contact with grape skins is an important determinant of Its resveratrol content White and rose wines generally contain less resveratrol than red wines, which is because the skins are removed earlier during their production, lessening the amount that is extracted Red or purple grape juices may also be good sources of resveratrol PBRC 2005

20 The French Paradox Cardiovascular Disease The French Paradox was the observation that mortality from coronary heart disease is relatively low in France despite relatively high levels of dietary saturated fat and cigarette smoking This led to the idea that the regular consumption of red wine might provide additional protection from cardiovascular disease PBRC 2005

21 Total Resveratrol Content of Wines and Grape Juice Beverage Total resveratrol (mg/liter) Total resveratrol in a 5 ounce glass (mg) White wines (Spanish) Rose wines (Spanish) Red wines (Spanish) Red wines (global) Red grape juice (Spanish)

22 Il resveratrolo èla soluzione del French Paradox?

23 Il resveratrolo è attivo nel lievito stesso effetto della RC Risultati identici in C. elegans

24 Il resveratrolo aumenta la sopravvivenza in un piccolo vertebrato (N. furzeri)

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26 Il resveratrolo (attivatore di SIRT) ha un effetto antiinvecchiamento in modelli animali di dieta ipercalorica

27 Il resveratrolo porta il fegato di animali ipercalorici ad un profilo di espressione genica simile al fegato normale

28 Longevity Although resveratrol did increase the activity of the homologous human enzyme (Sirt1) in the test tube, whether or not resveratrol can extend the human lifespan is currently unknown And again, the resveratrol concentrations that were necessary to increase Sirt1 activity in the test tube were considerably higher than any concentration previously measured in human plasma PBRC 2005

29 Non prendiamo troppo alla lettera i giornali, neanche quelli scientifici!

30 Reasons why recommending a population-wide increase would be premature: 1. Little is known about the absorption and clearance of resveratrol, the identities of its metabolic products, or its effects on the liver 2. The research on resveratrol has focused on its short-term effects and has been dominated by in vitro studies on nonhuman models PBRC 2005

31 Reasons why recommending a population-wide increase would be premature: 3. Its role as a potentiator of breast carcinomas may significantly limit its use, even for its proven benefits 4. Its main dietary source is red wine, which is not only extremely variable, but possibly harmful to be recommending increased intakes of red wine to the population at this point PBRC 2005

32 In Conclusion At present, relatively little is known about the effects of resveratrol in humans PBRC 2005

33 Nel frattempo...

34 Nel frattempo... GlaxoSmithKline to acquire Sirtris Pharmaceuticals, a world leader in 'Sirtuin' research and development Issued Tuesday 22 April 2008, London, UK, Philadelphia PA, Cambridge, MA GlaxoSmithKline (NYSE: GSK) and Sirtris Pharmaceuticals Incorporated (Nasdaq: SIRT) announced today that they have entered into a definitive agreement pursuant to which GlaxoSmithKline will acquire Sirtris Pharmaceuticals for approximately USD720 million (or approx. GBP362 million) through a cash tender offer of USD22.50 (or approx. GBP11.33) per share.

35 Grandezza e longevità

36 Female mice were selectively bred from Institute for Cancer Research stock for differences in rate of body weight gain. Mice were selected for differential rates of growth either early (0 10 days) or later (26 56 days) in the first 2 months of life. Low body size well correlated with longer life span. Miller at al., 2000 Size vs. Lifespan

37 Plot of life span vs size for 15 Atchley mouse stocks

38 Ruolo del signaling IGF 1/Insulina Repair/Aging

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40 Igf1 knock out mice The single knock out Igf1r+/ mice lived an average of 26% longer than wild type mice. Female Igf1r+/ mice lived an average of 33% longer than wild type, Male Igf1r+/ mice lived an average of 16% longer.

41 Insulin receptor (Insr) loss Shortened lifespan in mice and humans Targeted knockout in adipose tissue produces 18% lifespan extension. Cre loxp used for the tissue specific knockout. Lower body fat observed, food consumption and metabolism normal.

42 Q I II AD P H + III Cy H + tc I H V 2 O V H + AC- Co A Cy tri c Ac id Cycle AC- Co A Cy tri c Ac id Cycle I Q e- I H Q + 2 I H e- II Cy t H + I c I O2 HO V AD V P H AT P ROS: 1. iduce damage of macromolecules (DNA) and cause cellular damage or cell death R e - Q H 2 NAD H PAT e - O 2 R R R NAD H 2. are implicated in the pathonesis of ageing and ageing-associated diseases

43 p66shc Un circuito di segnalazione presente nei mammiferi che collega l attivitá di p53 alla produzione di ROS ed all invecchiamento

44 CH2 PTB CH1 SH2 p66 Shc PTB CH1 SH2 p52 PTB CH1 SH2 p46

45 Homozygous mutation of p66shc prolongs life span in mice Cumulative Survival WT p6 6 +/ - p6 6 -/ Time (days)

46 Cellule p66 / sono resistenti allo stress ossidativo p66 interferisce con il rilascio di citocromo C dal mitocondrio

47 The absence of p66shc reduces early atherosclerosis in mice fed high fat diet Atherosclerotic lesions take place in arteries in sites where blood flow is not laminar. This influences the permeability of the EC barrier to LDL. LDL will be retained in the matrix were they undergo oxidation by ROS. Ox LDL are then take up by macrophages leading to foam cell formation. Male p66 / and wild type mice mice were fed with a 21% high fat diet for weeks. Atherosclerotic lesions were then evaluated by computer assisted image analysis and immunohistochemistry revealing oxidation specific epitopes of Ox LDL (mab EO6) Early lesion size (mm2) wt(nd) wt(hft) p66 \ (ND) p66 \ (HFD) wt p66 /

48 Mechanisms of p53-induced apoptosis Stress p53 STRESS OSSIDATIVO p53 P Ac Transcriptional activation of apoptotic genes Mitochondri a Cytochrome C releas e Caspase activatio n Apoptosis

49 p66shc regulate steady-state levels of intracellular ROS MEFs MAFs WT p66-/- autofluorescence DCF fluorescence % relative MF FL1-H WT p66-/- P3 P5 P7 FL1-H WT p66 -/- ECs % relative MF WT p66-/- p66-/- +p66

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51 Tutte le cellule invecchiano? Ci sono sottopopolazioni di cellule il cui invecchiamento ha più importanza?

52 Invecchiamento delle cellule staminali Le cellule staminali possono accumulare danni ed andare incontro ad invecchiamento/morte come tutte le altre cellule La perdita del pool di cellule staminali può portare alla perdita del tessuto derivante invecchiamento di cellule ad uno stadio differenziativo più avanzato ha un effetto via via più ristretto

53 Evidenze per la perdita delle cellule staminali hair graying dei topi

54 Anatomia cellulare dei follicoli/vibrisse del topo

55 Topi bcl2 / topi più vecchi topi più giovani

56 Invecchiamento del pelo La perdita delle cellule staminali melanocitarie precede la perdita dei melanociti differenziati La perdita del pool staminale (accelerata dalla delezione di bcl2) causa l ingrigimento del pelo per la perdita della progenie differenziata con l età Fenomeni analoghi possono avvenire in tutti i tessuti