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The functional role of Optineurin. Formation. Theory Experiments Perspective. Optineurin (OPTN). a cytoplasmic protein ubiquitously expressed in brain , heart , skeletal muscle , placenta and kidney

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the functional role of optineurin

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

formation
Formation
  • Theory
  • Experiments
  • Perspective
optineurin optn
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)
optineurin optn1
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
optineurin optn2
Optineurin (OPTN)
  • Mutations in OPTN can:
  • causeglaucoma
  • Causeamytrophic lateral sclerosis (ALS)
  • (motorneurondiseasecausedbythedegeneration
  • ofneurons)
glaucoma
Glaucoma
  • Progressive opticneuropathywithlossofretinalganglioncells (RGCs)
  • Results in blindness, ifisleftuntreated
  • secondleadingcauseofblindness
  • Most common form (90%): Primary open-angelglaucoma (POAG)
primary open angle glaucoma poag
Primary open-angle glaucoma (POAG)
  • Genes associatedwith POAG: Myocilin, optineurinand WDR36
  • OPTN isparticularlylinkedto normal tensionglaucoma
  • normal visionvisionasitmightbeaffectedbyglaucoma
study of optineurin
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
conditional optineurin knock out mouse
ConditionalOptineurin Knock-Out Mouse

ES cells = embryonicstemcells

conditional optineurin knock out mouse1
ConditionalOptineurin Knock-Out Mouse

ES cells = embryonicstemcells

normal OPTN-sequence

conditional optineurin knock out mouse3
ConditionalOptineurin Knock-Out Mouse

+

+

=

“gefloxtes“ OPTN

optineurin knock out mouse
OptineurinKnock-Out Mouse

3000

1000

500

400

300

200

100

due to PCR youcan find thegenotypeofnewbornmice

control

Flox-PCR

Only KO ifthisgeneishomozygous!

knock out mouse
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

experiments arrestin tranducin
Experiments – Arrestin/Tranducin
  • ArrestinandTransducinhaveimportant
  • role in convertingthe light stimulus
  • eitherifitisdarkorbright, Arrestin
  • orTransducinislocated at OS/IS
  • transportmechanismneededtomove
  • theseproteinstotheothersegment
  • Photoreceptor
  • Question: does OPTN participate in thistransportmechanism?
experiments arrestin tranducin1
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
experiments sperm
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
experiments ctgf
Experiments – CTGF
  • Pre-experiment: CTGF-KO mice (4 weeksold)
  • Assumption: retinabecomesthinner due to CTGF overexpresion
  • But: retina not thinner!
  • → oldermice! (6 monthsold)
experiments ctgf1
Experiments – CTGF
  • 6 monthsold CTGF-KO micegetkilledandfixedwith PFA
  • watch at numberofaxonsandthicknessofretinawithelectronmicroscope
experiments ctgf2
Experiments – CTGF
  • Retina didn‘tbecomethinner!
experiments ctgf3
Experiments – CTGF
  • TUNEL-staining
  • blue: DAPI-staining
  • (normal nucleus)
  • green: dieingcell
  • Onlyifcellsaredieing, theretinabecomesthinner.
perspective
Perspective
  • Try tofigure out, if OPTN participates in thetransportmechanismofarrestin/tranducin
  • analyze different KO-miceforretinathickness
slide27

thankyoufor

yourattention!