Os mecanismos moleculares do olfato - IQ USPOs mecanismos moleculares do olfato: a deleção do gene...

Os mecanismos moleculares do olfato: a deleção do gene Ric-8b resulta em um camundongo anósmico

Olfactory system

Olfactory bulb

Olfactory epithelium

Olfactory cortex

Buck and Axel, 1991 Cell 65:175

Odorant receptors are expressed in the cilia of olfactory sensory neurons

COOH

NH3cilia

~1000 mouse OR genes~400 human OR genes

olfactory sensory neuron

A map for odorants in the brain

The odorant signaling pathway

Biochemistry. 5th edition.Berg JM, Tymoczko JL, Stryer L.New York: WH Freeman; 2002.

Von Dannecker, Mercadante & Malnic. J. of Neurosci. 25:3793 (2005)

Ric-8B interacts with Gαolf

Y2H screening of an OE cDNA library

Chan P, Gabay M, Wright FA and Tall GG, JBC (2011)

Ric-8B is a GEF (GTP Exchange fator)

Ric-8B amplifies signaling through Gαolf in HEK293 cells

GolfRic-8B

Golf

[AMPc] [AMPc]

Generation of a tissue specific Ric-8B KO using Cre-Lox

‘floxed’Ric-8B gene

OMP = Olfactory Marker Protein

Generation of a tissue specific Ric-8B KO using Cre-Lox

Western blot

In situ hybridization

Anti-digoxigenin antibodyconjugated with alkaline phosphatase

digoxigenin

probe

The Gαolf gene is transcribed in Ric-8B KO mice

The number of olfactory neurons is reduced in Ric-8B KO mice

Immunofluorescence

WT KO

Posi

tive

cells

(x10

-2)/m

m2

Calcium imaging

Calcium imaging

WT

cKO

Odorantes (Mix)

Hexanal

Heptanal

Eugenol

1-Heptanol

(±) Citronellal

1-Nonanol

S(-) Limonene

Acetophenone

(±) Carvone

Olfactory neurons from cKO mice are not responsive to odorants

Buried food test

Ric8-B cKO mice show impaired olfactory behavior

SUMMARY:

- Ric-8B is required for Gαolf expression in olfactory neurons;

- Ric-8B (and Gαolf) are required for survival of olfactory neurons;

- Ric-8B knockout mice are anosmic.

Department of BiochemistryUniversity of São PauloBettina Malnic bmalnic@iq.usp.brLúcia Armelin CorreaMaíra NagaiJoão Batista Plácido do Nascimento Artur Guazzelli Leme SilvaCleiton Fagundes MachadoCassandra Santantonio de LyraVictor Pereira de Sá Xavier

Past members:Adriana MercadanteLuiz E. C. Von DanneckerMarcela DavalosPedro A.F. GalanteJussara Michaloski SouzaDaniel Shikanai KerrDébora BrandtAna Carolina Bottura de BarrosDaniela Gonzalez-KristellerLuciana GutiyamaTiago JonasLuisa Kfouri Ribeiro

Maíra Nagai

ICB-UFRJJosé Garcia Ribeiro Abreu JuniorAlice Helena dos Reis Ribeiro

Cleiton Fagundes Machado

Hospital Sirio LibanêsAnamaria CamargoPedro AF Galante

Faculdade de Medicina VeterináriaUSPLuciano FelicioThiago Reis