Adriana Maggi DOCENTE DI BIOTECNOLOGIE FARMACOLOGICHE CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO A

1. Adriana Maggi DOCENTE DI BIOTECNOLOGIE FARMACOLOGICHE CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO AA 2011/2012 Lezione 7

2. LE BASI BIOLOGICHE DELL’ INVECCHIAMENTO Invecchiamento e ambiente Invecchiamento e genetica Regolazione endocrina dell’invecchiamento

3. Non-Programmed Passive Aging Theories • Aging is a passive result of an organism’s inability to better resist fundamental deteriorative processes. • Aging serves no purpose, is not an adaptation, is not programmed. • Compatible with traditional evolutionary mechanics theory. Mammals needing more time for development needed a longer life span and therefore developed better maintenance and repair mechanisms that consequently delayed onset of age-related symptoms and diseases relative to shorter-lived mammals. • Poor fit to many other observations of humans, other mammals, and other organisms particularly those that die suddenly from apparent biological suicide following reproduction rather than from gradual deterioration (e.g. Octopus, salmon)

4. Programmed Active Aging Theories • Organisms are purposely designed and genetically programmed to age or otherwise limit life span because the deterioration and life span limitation serves an evolutionary purpose. • Aging is an adaptation, a purposeful design feature resulting from the evolution process. • Aging is the result of a potentially complex active aging mechanism or “life span management system.” • The mechanism could sense external conditions in order to adapt life span to local or temporary conditions and could operate by manipulating the maintenance and repair functions.

5. Aging Theory Status • “Main line” consensus of current gerontologists favors the passive theories. Earlier simple deterioration theories have little current scientific credibility in the biology community while still popular in the human-oriented (physician) community. • Some relatively recent discoveries appear to favor aging-by-design theories. • Efforts to explain aging based on traditional mechanics and efforts to explain other discrepancies with alternative mechanics cannot be simultaneously valid. Eventually there will be a unified theory.

6. “Non-Aging” Species • Some species have been identified that apparently do not age or have negligible senescence. Older individuals do not appear to be weaker, less agile, less reproductive, more susceptible to disease, or otherwise less fit than younger animals. (Ages of some wild animals can be determined by annual marks in scales or bones similar to tree rings.) • Some species with age of oldest recorded specimen: • Rougheye Rockfish 205 Years • Lake Sturgeon152 Years • Aldabra Tortise152 Years • Common U.S. Eastern Box Turtle is also long-lived (~100 years). • Non-aging species tend to defeat simple deterioration theories and suggest dramatically longer human life spans are possible.

7. QUALE APPROCCIO SEGUIRE PER LO STUDIO DELLE BASI MOLCOLARI DELL’INVECCHIAMENTO?

8. Aging Theories PlannedObsolescenceTheory Telomerase Theory of Aging The Neuroendocrine Theory The Free Radical Theory Mitochondrial Theory of Aging The Membrane Theory of Aging The Hayflick Limit Theory (The cell waste accumulation) Glycosylation Theory of Aging Immune system alterations

9. MALATTIE GENETICHE ESEMPI DI INVECCHIAMENTO PRECOCE

10. Progeriaand Werner Syndrome • Hutchinson-Guilford Progeria, a very rare human genetic disease, accelerates many symptoms of aging including atherosclerotic heart disease. Victims usually die by age 13. • Werner syndrome, another genetic disease, involves acceleration of most symptoms of aging including baldness, hair and skin conditions, heart disease, calcification of blood vessels, some cancers, cataracts, arthritis, diabetes, etc. Victims usually die by age 50. • These conditions suggest aging is centrally controlled such that a single genetic defect could result in proportionally accelerating all of the expressed symptoms. Central control suggests aging-by-design

11. Il gene LMNA checodifica per le laminineditipo A è statocaratterizzatonel 1993 e mappatosulcromosoma 1q21.2-q21.3 (Lin and Worman1993; Wydner et al. 1996). La prima malattiaumanacausatadamutazionidellalaminina A identificata per mezzo diclonaggioposizionale era autosomicadominante e determinava la distrofiamuscolare Emery-Dreifuss

12. Laminopatie • Lipoatrophy with diabetes and other features of • insulin resistance • Atypical lipodystrophy syndromes • Mandibuloacral dysplasia • Peripheral Nerve • Charcot-Marie-Tooth disease type 2B1 • Progeria Phenotype • Hutchinson-Gilford progeria syndrome • Atypical Werner Syndrome • Variant progeroid disorders • MandibuloacralDysplasi • Autosomaldominant (and rarely recessive) Emery-Dreifussmuscular dystrophy • Cardiomyopathy dilated 1A • Limb-girdle muscular dystrophy type 1B • Congenital muscular dystrophy • “Heart-hand” syndrome • Adipose Tissue • Dunnigan-type familial partial lipodystrophy

13. Funzionidellelaminine Capell and Collins Nature Reviews Genetics7, 940–952 (December 2006) | doi:10.1038/nrg1906

14. Charcot-Marie-Tooth Lipodistrofiafamiliare Displasiamandibulosacrale progeria

15. Hutchinson-Gilford progeria syndrome Una malattia autosomica dominante e sporadica e rarachedeterminainvecchiamentoprecoce: in genereilpazientemuore a 13 anni circa per patologiecardiache La base genetica per molticasidiquestapatologiaconsiste nellamutazionedellatripletta GGC in GGT nelcodone 608 dellalaminina A (LMNA) . Questodeterminal’insorgenzadi un sitodi splicing cripticoportaallasintesidiunaproteina con unadelezionedi 50 aa. La regionedeleta ha in se la sequenzariconosciutadaenzimiproteoliticichefannomaturare la Laminina. In mancanzadiquesta parte dellaproteina, questavienecarbossifarnesilata e siaccumula a livelloendocellulare e soprattutto a causadellafarnesilazione, nellamembrananucleare.

16. Invecchiamento e genetica Figure 1. Processing of lamin A in normal and HGPS cells Meshorer E., Gruenbaum Y. J. Cell Biol. 2008:181:9-13

17. La presenza di laminina mutata (progerin)altera le funzione della membrana nucleare, la sua permeabilità e la trascrizione genica .

19. Capell and Collins, Nature Reviews Genetics 2006.

20. The is a mouse model of progeria where the prelamin A is not mutated. Instead, the metallopeptidase ZMPSTE24, the specific protease that is required to remove the C-terminus of prelamin A, is missing.

23. INVECCHIAMENTO E PROGERIE I malatidiprogerie non mostrano: deficit cognitivi elevatilivelliematicidicolesterolo e LDL elevatilivellidiprot C reattiva Chegeneralmentesiaccompagnanoall’invecchiamento

25. SINDROME DI WERNER I portatoridimutazionichedterminano la sindromedi Werner manifestano molto precocemente: cataratta, arteriosclerosi, osteoporosi, diabetemellitoditipo II, tumoridioriginemesenchimale. Generalmente la causadimorte è infarto o tumori.

26. Sindromedi Werner Unapatologiaautosomicarecessiva La mutazione genica è a carico della DNA elicasi (cromosoma 8 braccio corto) che accorcia la lunghezza dei telomeri. La malattia si manifesta alla pubertà e i portatori della mutazione vivono fino circa 40 anni di età.

27. A. Topo I usually found in eukaryotes binds the 3’ end of the broken DNA strand, and removes (+) or (-) supercoils. As replicating DNA moves through the structure, the two parental strands (black) are separated by the helicase, while positive supercoiling is removed by the 3’ topoisomerase. B. A machine able to separate the daughter molecules at the end of replication is formed by a helicase (red) removing the last turns of parental DNA and a type II topoisomerase (green) untangling the daughter duplexes.

28. C. Nucleosome disruption. The positive supercoiling produced by the translocating helicase H (red) destabilizes the nucleosome, while a topoisomerase T (5’ or 3’ Topo I, or eukaryotic topo II, green) efficiently relaxes the negative supercoiling, reforming the normal duplex behind the helicase.

29. MAPPA DELLE MUTAZIONI IN PAZIENTI CON MALATTIA DI WERNER HRDC: Human Hematopoietic Cell Derived RnaCyclase

30. Le proteinecheinteragisconodirettamente con la RecQelicasicomprendono: Ku70/80, BLM, FEN1, TRF2, p53, polß, polδ, RAD51, RAD52, RAD54B, RPA, POT1, PCNA, PARP1, MRE11, e Cdc5L coinvolte in: Replicazione del DNA, Riparazione del DNA, Ricombinazione Metabolismodeitelomeri

31. Telomero = unaregionedi DNA ripetitivo al termine del cromosomanecessario per proteggere la parte terminale del cromosomastessoneiconfrontidienzimidegradativi o fusioni con cromosomivicini. telos (τέλος) “fine“ merοs (μέρος, root: μερ-) "parte". Con la replicazionedeicromosomiilterminaledeicromosomisi erode e la parte del telomerosiaccorcia. Un telomero in unacellagiovanepuo’ raggiungere le 15000 paiadibasi (ognidivisionesiraccorciadi 25-200 pb)

32. double stranded, a G-rich single strand forms a terminal 3' overhang Telomericrepeat-bindingfactor 1

33. protectionoftelomeres Telomerase is a ribonucleoprotein polymerase that maintains telmere ends by addition of the telomere repeat TTAGGG

34. INVECCHIAMENTO E SINDROME DI WERNER

35. REGOLAZIONE ENDOCRINA DELL’INVECCHIAMENTO

36. Drosophila melanogaster STUDIARE VERMI E INSETTI PER CAPIRE L’UOMO Coenorabditis elegans

37. CICLO VITALE DI C.ELEGANS adulto embrioni L4 Circa 3 giorni a 22°C L3 L1 L2

38. CICLO VITALE DI C.ELEGANS adulto embrioni L4 MANCANZA DI ALIMENTI STADIO DAUER L1

39. LARVA LARVA DAUER

41. Studio di processi biologici legati a una maggiore morbidità l’esempio dell’invecchiamento DAF 7 ( TGFbligand) DAF1 (IGF-R) DAF 4 (Type II TGFbR) AGE 1 (IP3-K) DAF 3, DAF 5 (SMAD prot) DAF 12 DAF 16* 3-keto-cholestenoic acid metabolite DAUER SIR2 (deacetilasi attiva di DAF 16) DAF9 (cytochrome C CYP27A1) * Proteine della famiglia FOXO coinvolte nel metab del glucosio

42. GH Insulin Insulin/IGF-1 Insulin/IGF-1 IGF-1 DAF 2 receptor IGF-1R 1R dFOXO TOR DAF 16/FOXO (adip. Tissue) TOR LONGEVITY LONGEVITY LONGEVITY germline germline

43. Invecchiamento e ambiente

44. EVOLUTION: LAND OF BIOLOGICAL EQUAL OPPORTUNITIES “EFFECTOR” SEXUAL REPRODUCTION “REGULATORS” NUTRIMENT AGE

45. EVOLUTION: LAND OF BIOLOGICAL EQUAL OPPORTUNITIES • FECUNDITY SHOULD BE DIRECTLY PROPORTIONAL TO NUTRIENT AVAILABILITY, but DEATH: A TOOL INDISPENSABLE TO ENSURE THE CONTINUATION OF THE SPECIE • HIGH NUTRIENT AVAILABILITY, FAVORING FECUNDITY, SHOULD SHORTEN THE LIFE SPAN AGE NUTRIENTS SEXUAL REPRODUCTION

46. AGING AS NECESSITY FOR THE CONTINUATION OF LIFE and AS A MEAN TO GIVE TO EACH INDIVIDUAL EQUAL POSSIBILITIES TO GIVE HIS GENETIC CONTRIBUTION TO THE NEXT GENERATION Intrinsicprogramforagingaiming at increasing the frailityof the organism: a biological clock(telomerslength, mitochondrialviability; DNA replicationerrors, loss of immune control and inflammation…) sex-dependent (male fecunditycannotbelimitedaswellas in females) Fertility-driven Extrinsic factors nutrition adaptable environment