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The Functional Role of Optineurin in Glaucoma: Insights from Mouse Studies

This study explores the role of Optineurin (OPTN) in glaucoma using mouse models. Optineurin is a cytoplasmic protein expressed in ocular cells and implicated in vesicular trafficking and interferon regulation. Mutations in OPTN can lead to glaucoma and Amytrophic Lateral Sclerosis. The research involves experiments on knockout mice to investigate Optineurin's involvement in the transport mechanism of proteins like Arrestin and Transducin. The study also examines the effects of Optineurin on sperm and retinal health in mice. Through various experiments, the goal is to understand Optineurin's role in these processes, particularly in the context of glaucoma pathogenesis, aiming to shed light on potential therapeutic targets.

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The Functional Role of Optineurin in Glaucoma: Insights from Mouse Studies

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  1. The functionalroleofOptineurin Institut der Anatomie Fakultät für Humananatomie und Embryologie Universität Regensburg Biochemisches Praktikum im Jahr 2013 Betreuerin: Christiane Sippl 16.01.2014 Riedlberger Felix

  2. Formation • Theory • Experiments • Perspective

  3. Optineurin (OPTN) • a cytoplasmicproteinubiquitouslyexpressed in brain, heart, skeletalmuscle, placentaandkidney • expressed in severalocularcelltypes, e.g. retinalganglioncells, retinalpigmentepithelium, etc. • itsgenespans a ~37 kbgenomicregionandconsists 13 activeexonscodingfor a 577 aminoacidprotein (human)

  4. Optineurin (OPTN) • Optineurin was foundtobelocalized at Golgi apparatuswithinteractingproteins: Rab8, Huntingtinand Myosin VI • stabilizesmorphologyof Golgi apparatus, whereitisinvolved in vesiculartrafficking • Itisinvolved in a lotofprocesses, e.g. regulationofinterferon

  5. Optineurin (OPTN) • Mutations in OPTN can: • causeglaucoma • Causeamytrophic lateral sclerosis (ALS) • (motorneurondiseasecausedbythedegeneration • ofneurons)

  6. Glaucoma • Progressive opticneuropathywithlossofretinalganglioncells (RGCs) • Results in blindness, ifisleftuntreated • secondleadingcauseofblindness • Most common form (90%): Primary open-angelglaucoma (POAG)

  7. Primary open-angle glaucoma (POAG) • Genes associatedwith POAG: Myocilin, optineurinand WDR36 • OPTN isparticularlylinkedto normal tensionglaucoma • normal visionvisionasitmightbeaffectedbyglaucoma

  8. Study ofOptineurin • through "knockout mice“ (KO-Type) • The mouseorganismoffersparticularadvantages: • themouseis a mammalanditsdevelopment, body plan, physiology, behavioranddiseaseshavemuch in commonwiththoseofhumans • almost all (99%) mouse genes haveallogenics in human • Targetedmutagenesisbyhomologousrecombinatoinpossible

  9. ConditionalOptineurin Knock-Out Mouse ES cells = embryonicstemcells

  10. ConditionalOptineurin Knock-Out Mouse ES cells = embryonicstemcells normal OPTN-sequence

  11. ConditionalOptineurin Knock-Out Mouse + + =

  12. ConditionalOptineurin Knock-Out Mouse + + = “gefloxtes“ OPTN

  13. ConditionalOptineurin Knock-Out Mouse “gefloxtes“ OPTN

  14. ConditionalOptineurin Knock-Out Mouse

  15. ConditionalOptineurin Knock-Out Mouse

  16. OptineurinKnock-Out Mouse 3000 1000 500 400 300 200 100 due to PCR youcan find thegenotypeofnewbornmice control Flox-PCR Only KO ifthisgeneishomozygous!

  17. Knock-Out Mouse 3000 1000 500 400 300 200 100 another type of KO: CTGF-PCR (SV40) control This PCR shows, ifthemice will overexpressthe Protein CTGF CTGF will cause a higherintraocularpressure

  18. Experiments – Arrestin/Tranducin • ArrestinandTransducinhaveimportant • role in convertingthe light stimulus • eitherifitisdarkorbright, Arrestin • orTransducinislocated at OS/IS • transportmechanismneededtomove • theseproteinstotheothersegment • Photoreceptor • Question: does OPTN participate in thistransportmechanism?

  19. Experiments – Arrestin/Tranducin • Execution: • Mice (3 months)weredarkadaptedovernight • 4 KO mice, 4 WT mice • Next day different exposure time at 120 Lux (0, 5, 10, 30 min) • after killing: fix proteins in bodieswith PFA • stainingArrestinandTransducin

  20. Experiments – Arrestin/Tranducin Result:

  21. Experiments – Sperm • watch, if KO spermareaffected in anyway, causeofOPTN • isolationofsperm in theepididymides • watchspermwithphasecontrastmicroscopeandcellculture medium (medium = DMEM: high glucose, 37°C, pH = 7.4) • Result: nodifferences, bothtypesofspermsare mobile and fertil

  22. Experiments – CTGF • Pre-experiment: CTGF-KO mice (4 weeksold) • Assumption: retinabecomesthinner due to CTGF overexpresion • But: retina not thinner! • → oldermice! (6 monthsold)

  23. Experiments – CTGF • 6 monthsold CTGF-KO micegetkilledandfixedwith PFA • watch at numberofaxonsandthicknessofretinawithelectronmicroscope

  24. Experiments – CTGF • → • Retina didn‘tbecomethinner!

  25. Experiments – CTGF • TUNEL-staining • blue: DAPI-staining • (normal nucleus) • green: dieingcell • Onlyifcellsaredieing, theretinabecomesthinner.

  26. Perspective • Try tofigure out, if OPTN participates in thetransportmechanismofarrestin/tranducin • analyze different KO-miceforretinathickness

  27. thankyoufor yourattention!

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