Ciljevi istraživanja:
Povišen izražaj gena p53 i Sesn2 u Rpl24-heterozigotnim miševima ukazuje da p53 regulira njihov fenotip putem regulacije mTOR-a i makroautofagije vjerojatno putem Sesn2.
Ciljevi su:
1. Odrediti uzrokuje li p53 patološki fenotip Rpl24-heterozigotnih miševa putem Sesn2.
2. Odrediti aktivnost i važnost makroautofagije u preživljavanju Rpl24-heterozigotnih miševa. 3. Razjasniti molekularne mehanizme putem kojih p53 i Sesn2 reguliraju patološke fenotipove u Rpl24-heterozigotnim embrijima.
Materijali i metode: U istraživanju je korišten miš s heterozigotnom mutacijom u genu za Rpl24, koja rezultira specifičnim fenotipom ovih miševa: slijepoćom, abnormalnosti skeleta, smanjenom masom i zavijenim repom (Bst, engl. Belly Spot and Tail). Izražaj Sesn2 utvrđen je u Rpl24-heterozigotnim embrijima na E10.5 korištenjem kvantitativne reakcije lančanom polimerazom u realnom vremenu (RT-PCR, engl. real time polymerase chain reaction). Da bih testirala mogućnost da p53 uzrokuje patološke fenotipove Rpl24-heterozigotnih miševa putem Sesn2, genetički sam u njima inaktivirala Sesn2. Da bih razlučila potencijalnu važnost makroautofagije i mTOR signalnog puta u Sesn2-ovisnom patološkom fenotipu Rpl24-heterozigotnih miševa, inaktivirala sam jedan alel Atg5, ključnog gena za makroautofagiju. Molekularne mehanizme regulacije preživljavanja Rpl24-heterozigotnih miševa razjasnila sam u modelu gladovanja u ranom poslijenatalnom periodu korištenjem Rpl24-heterozigotnih miševa u kojima je genetički inaktiviran p53, Sesn2 ili jedan alel Atg5.
Rezultati: Dokazano je da p53 uzrokuje kongenitalne malformacije u Rpl24-heterozigotnim miševima putem svog ciljnog gena, Sesn2, te da p53 omogućuje preživljavanje Rpl24-heterozigotnih miševa u ranom poslijenatalnom periodu putem regulacije makroautofagije, koja najvjerojatnije razgradnjom staničnih komponenti osigurava supstrate za sintezu glukoze neophodne za njihovo preživljavanje. p53 i Sesn2 neophodni su za preživljavanje Rpl24-heterozigotnih miševa u ranom poslijenatalnom periodu gladovanja. Međutim, mehanizmi putem kojih p53 i Sesn2 potiču preživljavanje Rpl24-heterozigotnih miševa barem su djelomično različiti.
Zaključci: Dokazano je da p53 uzrokuje kongenitalne malformacije u Rpl24-heterozigotnim miševima putem svog ciljnog gena, Sesn2, te su razjašnjeni molekularni mehanizmi kojima Sesn2 i p53 reguliraju patološke fenotipove u Rpl24-heterozigotnim miševima. Naši rezultati mogu pomoći razumijevanju mehanizama putem kojih p53 uzrokuje patološke promjene u ribosomopatijama, omogućiti bolji uvid u njihovu patogenezu i rezultirati otkrićem potencijalnih ciljeva za liječenje tih bolesti.Title: Mechanisms by which the p53 tumor suppressor mediates pathological manifestations in Rpl24-heterozygous mice
Objectives:
Upregulation of p53 and Sesn2 in Rpl24-heterozygous mice suggests that p53 regulates pathological manifestations of these mice through Sesn2-dependent regulation of autophagy and mTOR signalling pathway.
Our objectives are:
1. To determine if p53-dependent induction of Sesn2 causes pathological phenotype of Rpl24-heterozygous mice
2. To determine if autophagy is activated and important for the survival of Rpl24-heterozygous mice
3. To determine p53- and Sesn2-dependent molecular mechanisms of regulation of pathological phenotypes of Rpl24-heterozygous mice.
Material and Methods: Spontaneous heterozygous mutation in one allele of ribosomal protein l24 gene (Rpl24) in mice results in defects of the eye, skeleton, mass and coat pigmentation (Bst, Belly Spot and Tail). Sesn2 expression level was determined in Rpl24-heterozygous embryos at E10.5 using quantitative polymerase chain reaction (RT-PCR). To test the possibility that p53-dependent induction of Sesn2 plays a role in pathological phenotypes of Rpl24-heterozygous mice, I genetically inactivated Sesn2 in these mice. To dissect the importance of autophagy and mTOR signalling pathway in Sesn2-dependent pathological phenotype of Rpl24-heterozygous mice, I genetically inactivated one allele of Atg5, important autophagy gene. Molecular mechanisms of survival regulation in Rpl24-heterozygous mice were elucidated in the model of survival under fasting conditions using Rpl24-heterozygous mice with p53, Sesn2 or Atg5 inactivation.
Results: We demonstrate the key role of p53 target gene, Sesn2, in pathological phenotype of Rpl24-heterozygous mice. We show that p53 promotes survival of these mice in early postnatal period through regulation of autophagy, which provides substrates for gluconeogenesis necessary for survival of Rpl24-heterozygous mice. Both, p53 and Sesn2, are indispensable for survival of Rpl24-heterozygous mice in the early postnatal starvation period, but the mechanisms through which these two genes regulate survival of these mice are at least partially different.
Conclusions: We demonstrate the key role of p53 target gene, Sesn2, in pathological phenotype of Rpl24-heterozygous mice, and show the molecular mechanisms by which p53 and Sesn2 regulate these phenotypes.
Our findings may have important implications for understanding p53-dependent mechanisms underlying the pathogenesis of ribosomopathies and could result in the discovery of potential targets for treatment of these diseases
