<\/p>\n
HRZZ Research Projects<\/p>\n
(IP-06-2016)<\/p>\n
Research project proposal: “Exploring the borderland between neurodegeneration and neuroregeneration: identification of key molecules with proteomics and functional assays in the mammalian spinal cord”<\/p>\n
D<\/strong>efineRE<\/strong>genA<\/strong>geM<\/strong>ode<\/p>\n <\/p>\n Principal Investigator (PI): prof. Miranda Mladini\u0107 Pejatovi\u0107<\/strong><\/p>\n <\/p>\n One of the major challenges of modern biology concerns the inability of the adult mammalian central nervous system (CNS) to regenerate and repair itself after injury. Unlike the situation in adult mammals, lower vertebrates, such as fish and amphibians, and embryonal higher vertebrates can regenerate significant portion of their CNS. It is poorly understood why regenerative potential is lost with evolution and development and why it becomes very limited in adult mammals. Even though our understanding of molecular and cellular mechanisms promoting or inhibiting neuronal regeneration is expanding, it is still unclear what are the key differences between the neuronal systems that can and cannot regenerate, and how they can be manipulated as to revert the outcome. Our incomplete understanding of molecular events underlying neuronal development and regeneration is the main reason why neurodegenerative diseases and brain and spinal cord injuries are still mostly incurable today.<\/p>\n New achievements in the field suggest that differences in regeneration ability are due to alterations in several key attributes and not a single trait, and thus recent strategies employed to enhance regeneration in the mammalian CNS include the neutralization of potential growth inhibitory molecules combined with the transplantation of cells or materials that support axonal elongation and the delivery of factors that are known to promote axonal growth. These approaches are showing promising results, although have so far failed to produce robust axon regeneration. Moreover, the functional recovery was often limited and never enough to remedy neurological deficits. Full knowledge of the molecules that promote or prevent regeneration and the way they interact is far from complete.<\/p>\n A preferred model to study and reveal the cellular and molecular basis of regeneration is neonatal opossum (Monodelphis domestica<\/em>). Opossums are marsupials that are born at very immature stage with unique possibility to successfully regenerate spinal cord after injury in the first two weeks of their life. After that, the regenerative capacity is abruptly lost: at 14 days in cervical spinal segments and at 17 days in less mature lumbar spinal segments. Neonatal opossums represent the unique opportunity to achieve and study mammalian CNS that can regenerate, without a need of invasive intrauterine surgery of pregnant females (as necessary\u00a0for other mammalian laboratory animals, such as mice or rats). Moreover, the tiny neonatal opossum CNS can be maintained in culture in its entirety and this preparation is similar to the intact animal in its ability to regenerate.<\/p>\n In this project we will use neonatal opossum to study molecular and cellular properties of spinal tissue which has and has not the capacity to regenerate after injury, to pinpoint the key differences. First, we will use the comparative proteomic approach to identify the proteins differentially distributed in opossum spinal tissue with different regenerative capacities. The results will upgrade our previous data about the genes differentially expressed in the regenerating and non-regenerating opossum spinal cord tissue. The comparison of the data obtained by genomic and proteomic approach will allow the selection of the several most promising candidates controlling regeneration, to be tested in functional studies. The choice of the candidate molecules will be based on the hypothesis that changes in spinal radial glia \/ stem \/ progenitor cells and extracellular matrix are determinant for the regeneration loss during development. The activity of the candidate proteins will then be manipulated to reveal their role in regeneration in two different in vitro preparations: intact neonatal opossum spinal cord preparation (to easily visualize the axonal growth as to test regeneration capacity of the tissue) and primary spinal cell cultures (to easily genetically manipulate candidate molecules for testing their impact on regeneration-related stem cell properties). The selected candidate molecules will be tested simultaneously, in different combinations, to allow the alterations in several key attributes related to regeneration (stem cell activity and axonal growth promoting or inhibiting molecules).<\/p>\n This original interdisciplinary basic research project, unique in the choice of the animal model to be used, combines advanced techniques of biotechnology and molecular and cellular neurobiology to identify the molecular and cellular differences in mammalian CNS tissue that can and cannot regenerate. The results of the project would make substantial contribution to our understanding of neuronal regeneration in mammals, and also provide candidate targets for future novel therapeutic interventions for neurodegenerative disorders.<\/p>\n <\/p>\n <\/p>\n Istra\u017eivanje granice izme\u0111u neurodegeneracije i\u00a0neuroregeneracije: identifikacija klju\u010dnih molekula pomo\u0107u\u00a0proteomike i funkcionalnih testova na le\u0111noj mo\u017edini\u00a0sisavaca<\/p>\n D<\/strong>efineRE<\/strong>genA<\/strong>geM<\/strong>ode<\/p>\n Voditeljica projekta (PI): izv.prof.dr.sc. Miranda Mladini\u0107 Pejatovi\u0107<\/strong><\/p>\n Jedan od glavnih izazova s kojim je suo\u010dena suvremena neurobiologija je nemogu\u0107nost regeneracije sredi\u0161njeg \u017eiv\u010danog sustava (S\u017dS) odraslih sisavaca nakon ozljede. Za razliku od odraslih sisavaca, ni\u017ei kralje\u017enjaci kao \u0161to su ribe i vodozemci, te vi\u0161i kralje\u017enjaci tijekom embrionalnog razvoja imaju sposobnost obnove zna\u010dajnog dijela S\u017dS. Jo\u0161 uvijek nije jasno kako se i za\u0161to sposobnost regeneracije gubi tijekom evolucije i razvoja, te za\u0161to regeneracija kod odraslih sisavaca postaje iznimno ograni\u010dena. Iako se na\u0161e razumijevanje stani\u010dnih i molekularnih mehanizama koji poti\u010du ili inhibiraju regeneraciju zadnjih godina sve vi\u0161e produbljuje, jo\u0161 uvijek je nejasno koje su to klju\u010dne razlike izme\u0111u \u017eiv\u010danih sustava koji imaju i onih koji nemaju sposobnost regeneracije, te kako ih manipulirati sa ciljem da se promjeni ishod ozljede. Na\u0161e nepotpuno razumijevanje molekularnih doga\u0111aja koji su temelj razvoja i regeneracije \u017eiv\u010danog tkiva je glavni razlog za\u0161to su neurodegenerativne bolesti kao i ozljede mozga i le\u0111ne mo\u017edine danas jo\u0161 uvijek neizlje\u010dive.<\/p>\n Nova postignu\u0107a u ovom podru\u010dju istra\u017eivanja ukazuju da sposobnost regeneracije ovisi o nekoliko klju\u010dnih promjena, te stoga strategije koje nastoje pospje\u0161iti regeneraciju u S\u017dS sisavaca kombiniraju neutralizaciju potencijalnih inhibitora \u017eiv\u010danog rasta, s istovremenom transplantacijom stanica ili materijala koji omogu\u0107avaju rast aksona, te uvo\u0111enje molekularnih \u010dimbenika koji pozitivno utje\u010du na njihov rast. Rezultati ovih pristupa su obe\u0107avaju\u0107i, iako se do sada njima nije uspjelo potaknuti sna\u017enu regeneraciju aksona, a funkcionalni je oporavak<\/p>\n \u010desto ograni\u010den i nedovoljan za izlje\u010denje neurolo\u0161kih nedostataka. Na\u0161e znanje o molekulama koje poti\u010du ili sprje\u010davaju regeneraciju, te o na\u010dinu na koji one me\u0111usobno djeluju je zasad nepotpuno i nedovoljno.<\/p>\n Prikladan model za prou\u010davanje i otkrivanje stani\u010dne i molekularne osnove regeneracije je mladi oposum (Monodelphis domestica<\/em>). Oposumi su tobol\u010dari koji se ra\u0111aju nerazvijeni, pa tijekom prva dva tjedna svog \u017eivota posjeduju jedinstvenu sposobnost potpune regeneracije le\u0111ne mo\u017edine nakon ozljede. Nakon toga sposobnost regeneracije se naglo gubi: oko 14. dana u vratnom dijelu le\u0111ne mo\u017edine, te 17. dana u njenom manje razvijenom lumbalnom dijelu. Mladi oposum daje jedinstvenu priliku za istra\u017eivanje S\u017dS sisavaca koji ima sposobnost regeneracije, bez potrebe za invazivnim operacijama trudnih \u017eenki (kao \u0161to je potrebno kod ostalih laboratorijskih sisavaca kao \u0161to su mi\u0161evi ili \u0161takori). \u0160tovi\u0161e, cjeloviti S\u017dS novoro\u0111enog oposuma mo\u017ee se odr\u017eavati u kulturi, pri \u010demu se u potpunosti zadr\u017eava njegova sposobnost regeneracije.<\/p>\n U ovom projektu koristitimo mlade oposume za istra\u017eivanje molekularnih i stani\u010dnih svojstava tkiva le\u0111ne mo\u017edine koje ima sposobnost regeneracije nakon ozljede, te onog koje nema, kako bi se odredile klju\u010dne\u00a0razlike. Prvo \u0107emo uporabom komparativne proteomike odrediti bjelan\u010devine koje su razli\u010dito raspore\u0111ene u tkivu le\u0111nih mo\u017edina oposuma razli\u010ditih regenerativnih sposobnosti. Dobiveni \u0107e se rezultati nadograditi na prethodna istra\u017eivanja podnositeljice prijedloga vezane za gene razli\u010dito izra\u017eene u tkivu le\u0111ne mo\u017edine oposuma koje ima i koje nema sposobnost regeneracije. Usporedba podataka dobivenih pristupima genomike i proteomike omogu\u0107iti \u0107e odabir nekoliko najzanimljivijih molekula koje bi mogle imati va\u017enu ulogu u kontroli regeneracije i koje \u0107e se testirati dalje, kako bi se ta njihova uloga i dokazala. Izbor molekula koje bi mogle biti va\u017ene za regeneraciju temeljit \u0107e se na pretpostavci da su promjene u radijalnim glija\/mati\u010dnim\/praroditeljskim stanicama, te izvanstani\u010dnom matriksu le\u0111ne mo\u017edine klju\u010dne za gubitak sposobnosti regeneracije \u017eiv\u010danog tkiva tijekom razvoja. Aktivnost odabranih molekula eksperimentalno \u0107e se mijenjati kako bi se otkrilo imaju li one utjecaja na regeneraciju. Pri tome \u0107e se koristiti dva razli\u010dita in vitro <\/em>preparata: preparat cjelovite le\u0111ne mo\u017edine mladog oposuma odr\u017eavan u kulturi (kako bi se jednostavno vizualizirao rast aksona i testirala regenerativna sposobnost tkiva), te primarne stani\u010dne kulture le\u0111ne mo\u017edine (kako bi se genetski manipulirale odabrane molekule, te testirao njihov utjecaj na regenerativna svojstva mati\u010dnih i drugih stanica). Odabrane molekule ispitivat \u0107e se pojedina\u010dno ili u razli\u010ditim kombinacijama, kako bi se omogu\u0107ile promjene klju\u010dnih svojstava vezanih za regeneraciju (aktivnost mati\u010dnih stanica, aktivnost molekula koje poti\u010du ili inhibiraju rast aksona). Ovaj originalni interdisciplinarni temeljni istra\u017eiva\u010dki projekt, jedinstven u odabiru \u017eivotinjskog modela koji \u0107e biti kori\u0161ten, kombinira napredne tehnike biotehnologije i molekularne i stani\u010dne neurobiologije kako bi se identificirale klju\u010dne molekularne i stani\u010dne razlike u S\u017dS sisavaca koji imaju i onih koji nemaju sposobnost regeneracije. Rezultati ovog projekta mogli bi dati izniman doprinos na\u0161em razumijevanju regeneracije \u017eiv\u010danog tkiva sisavaca, te \u0107e pru\u017eiti potencijalne mete za razvoj budu\u0107ih novih strategija u lije\u010denju neurodegenerativnih poreme\u0107aja.<\/p>\n","protected":false},"excerpt":{"rendered":" HRZZ Research Projects (IP-06-2016) Research project proposal: “Exploring the borderland between neurodegeneration and neuroregeneration: identification of key molecules with proteomics and functional assays in the mammalian spinal cord” DefineREgenAgeMode Principal Investigator (PI): prof. Miranda Mladini\u0107 Pejatovi\u0107 One of the major challenges of modern biology concerns the inability of the adult mammalian central […]<\/p>\n","protected":false},"author":2,"featured_media":120,"parent":20,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/neuroreg.uniri.hr\/index.php?rest_route=\/wp\/v2\/pages\/41"}],"collection":[{"href":"https:\/\/neuroreg.uniri.hr\/index.php?rest_route=\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/neuroreg.uniri.hr\/index.php?rest_route=\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/neuroreg.uniri.hr\/index.php?rest_route=\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/neuroreg.uniri.hr\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=41"}],"version-history":[{"count":6,"href":"https:\/\/neuroreg.uniri.hr\/index.php?rest_route=\/wp\/v2\/pages\/41\/revisions"}],"predecessor-version":[{"id":191,"href":"https:\/\/neuroreg.uniri.hr\/index.php?rest_route=\/wp\/v2\/pages\/41\/revisions\/191"}],"up":[{"embeddable":true,"href":"https:\/\/neuroreg.uniri.hr\/index.php?rest_route=\/wp\/v2\/pages\/20"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/neuroreg.uniri.hr\/index.php?rest_route=\/wp\/v2\/media\/120"}],"wp:attachment":[{"href":"https:\/\/neuroreg.uniri.hr\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=41"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}