understanding deafness in the usher syndrome: a possible interaction between grxcr1 and the...

Understanding deafness in the Usher syndrome:

A possible interaction between Grxcr1 and the zebrafish

Usher complex

Jennifer M. Panlilio

Westerfield Lab

Roadmap I. Introduction to the Usher Syndrome and

mechanosensation

II. Grxcr1 as a possible protein partner of the Usher complex

III. Results: Characterization of Grxcr1 depletion phenotype in zebrafish

V. Future Directions

Overview of Usher Syndrome•Most common form of hereditary deafblindness in humans

•Symptoms include issues with hearing, balance and vision

•Three clinical subtypes:

Usher Type I Usher Type II Usher Type III

How Hearing Works: A look into the inner ear hair cells

Sound

MechanoreceptorStereocilia +Kinocilium

Cell body

Nerve fibers

Actin

Usher protein complex: Protein

Myosin VIIa

Harmonin

Cadherin 23

Protocadherin 15

Sans

Usherin

VLGR1b

Whirlin

Clarin-1

Hars-1

Finding proteins interacting with the Usher protein complex:

How are the Usher proteins transported?

How is the complex is assembled?

Grxcr1 as a candidate Glutaredoxin cysteine-rich 1 in Inner ear of mouse in p5

Frolenkov et al. Nature Reviews, 2004 Odeh et al., Journal of Human Genetics ,2010

Grxcr1: green Actin: red

Grxcr1 is present in the stereocilia

Karolyi et al. Human Molecular Genetics, 2003

There are phenotypic similarities in the inner ear between Grxcr1 mutant and the Usher mutants

Grxcr1 mouse mutant

Wild type mouse

Usher mutant (Myo7a) mouse

Grxcr1 as a candidate

Strategy

Characterization of grxcr1 expression pattern

Depletion of Grxcr1 to determine whether it is required in mechanoreceptor development

Effects of depletion of Grxcr1 on the Usher protein complex

grxcr1 expression patternWhere is grxcr1 expressed?

grxcr1 is expressed in the ears, eyes, brain and liver of 5 dpf larvae

Brain

Eyes

Inner Ear

Liver

Loss of Grxcr1 function

Exon 1 2 3 4

Exon 1 2 3 4Wt mRNA

Pre-mRNA

Splice blocker MO1-grxcr1

MO1-grxcr1 protein

Wt protein

Exon 1 2 3 4MO1-grxcr1 mRNA

Loss of Grxcr1 functionSplice blocker MO1-grxcr1

Exon 1 3 4

Exon 1 2 3 4Wt mRNA

2pre-RNA

Exon 1 2 3 4MO2-grxcr1 mRNA

Loss of Grxcr1 functionSplice blocker MO2-grxcr1

MO2-grxcr1 protein

Wt protein

Loss of Grxcr1 functionSplice blocker MO2-grxcr1

Non injected

MO-2 grxcr1

Defects in mechanoreceptor development following Grxcr1

depletion

Wild type MO2-grxcr1

MO2-grxcr1

Actin Staining

Usher protein complex: Protein

Myosin VIIa

Harmonin

Cadherin 23

Protocadherin 15

Sans

Usherin

VLGR1b

Whirlin

Clarin-1

Hars-1

Depletion of Grxcr1 on the Usher complex in 5 dpf larvae

Myo 7aa

Myo7aa seems to be less concentrated apically in morphants

Harmonin

Harmonin seems to be depleted in morphants

Wild type MO2- grxcr1

\

Conclusionsgrxcr1 is expressed in the eyes and inner ear like usher genes

Grxcr1 may play a role in mechanoreceptor development

Grxcr1 effects the localization of some Usher proteins

Future Directions

How does Grxcr1 affect the Usher complex?-via study of changes of localization with other known Usher proteins

Does Grxcr1 interact with the complex?-via Colocalization

-via Coimmunoprecipitation

AcknowledgementsMonte WesterfieldAurélie ClémentBerde Blanco-SanchezJudy PeirceJeremy WegnerSabrina ToroJennifer PhillipsRachel Rae Drake Marcel RockwellPeter O’DayAdam UngerNIHCD