Presented By

Nisha RizviGraduate Student

Department of PharmacologySIU School of Medicine

4/21/2010

• ‘Wnt’ = Wg (wingless) + Int = Wingless-type murine-mammary-tumor virus

integration site)

• Wg Gene originally identified as a recessive mutation affecting wing and

haltere development in Drosophila melanogaster.

• Subsequently characterized as segment polarity gene in Drosophila

melanogaster that functions during embryogenesis and also during adult

limb formation during metamorphosis.

• INT genes originally identified as vertebrate genes near several integration

sites of mouse mammary tumor virus (MMTV).

• Int-1 and Wg genes found to be homologous

INTRODUCTION

PHYSIOLOGICAL FUNCTIONS OF WNT SIGNALING

1. Embryogenesis

2. Adult tissue maturation and homeostasis

3. Pleiotropic: mitogenic stimulation, cell differentiation, cell fate

4. Nervous system:– Neural development, patterning and maintenance

• Neurogenesis of hippocampal stem cells (1)

• LTP (2)

– Synaptic plasticity (3)

– Neuroprotection (3)

(1) Lie, D. C., Colamarino, S. A., Song, H. J., De´sire´, L., Mira, H., Consiglio, A., Lein, E. S., Jessberger, S., Lansford, H., Dearie, A. R., and Gage, F. H. (2005) Nature 437, 1370–1375. (2) Tang, S. J. (2007) Synapse 61, 866–868. (3)Inestrosa, N. C., and Toledo, E. M. (2008) Mol. Neurodegener. 3, 9

HUMAN WNT PROTEINS

• WNT1

• WNT2, WNT2B

• WNT3, WNT3A

• WNT4

• WNT5A, WNT5B

• WNT6

• WNT7A, WNT7B

• WNT8A, WNT8B

• WNT9A, WNT9B

• WNT10A

• WNT10B

• WNT11

• WNT16

Ching W, Nusse R. A Dedicated Wnt Secretion Factor (2007) . Cell, 125 (3): 432-433

Unknown

Unknown

Unknown

Unknown

EmbryogenesisNeurogenesisTissue Growth and Development

Logan and Nusse , Wnt signaling pathway in development and disease, Annu. Rev. Cell Dev. Biol. 2004. 20:781–810

WNT SIGNALING

NCBI » Bookshelf » WormBook » Signal transduction » Wnt signaling

Canonical Wnt Signaling PathwayNon-Canonical Wnt Signaling Pathway

Habas and Dawid Journal of Biology 2005 4:2 doi:10.1186/jbiol22

• PART-1: Role of Wnt signaling in physiological

organization of synapses

• PART-2: Role of Wnt signaling in Alzheimer’s

disease --- Therapeutic implications

• PART-3: Crosstalk of Wnt signaling with

mAChR and PPARγ signaling --- Therapeutic

implications

PART 1

Physiological Effects

ROLE OF WNT SIGNALING COMPONENTS IN CNS

Farı´as GG, Godoy JA, Cerpa W, Varela-Nallar L, and Inestrosa NC. Wnt Signaling Modulates Pre- and Postsynaptic Maturation: Therapeutic Considerations. DEVELOPMENTAL DYNAMICS 239:94–101, 2010

Farı´as GG, Godoy JA, Cerpa W, Varela-Nallar L, and Inestrosa NC. Wnt Signaling Modulates Pre- and Postsynaptic Maturation: Therapeutic Considerations. DEVELOPMENTAL DYNAMICS 239:94–101, 2010

PREsynaptic Assembly

• Drosophila – Wg induces synaptogenesis at NMJ

– Loss leads to reduction in synapses formation

• Mammals

– Wnt-7a increases levels of synaptic vesicle protein synapsinI in developing cerebellar neurons

– Mutant mice show delay in synapsin I accumulation

– Wnt-3a, Wnt-7b also increase the number of excitatorypresynaptic puncta in hippocampal neurons

– Wnt-7a increases expression of α7-nAChR

– In mature CNS, Wnt-7a increases clustering of synapticvesicle proteins like synapsin I, synaptophysin, SV-2, synaptotagmin

Wnt-7a increases clustering of synaptic vesicle proteins

Cerpa W, Godoy JA, Alfaro I, Farı´as GG, Metcalfe MJ, Fuentealba R, Bonansco C, Inestrosa NC .Wnt-7a Modulates the Synaptic Vesicle Cycle and Synaptic Transmission in Hippocampal Neurons. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 9, pp. 5918–5927

PREsynaptic Function

• Regulate synaptic transmission and plasticity

• Wnt-7a mutants show a significant decrease in frequency of

postsynaptic mEPSC

• Wnt-7a increases frequency of mEPSCs and a decrease in

PPF*

– Mechanism of NT release unknown

– Possibly through interaction with synaptic vesicle proteins

– Ca2+ homeostasis?

Wnt-7a enhances synaptic transmission in hippocampus

Cerpa W, Godoy JA, Alfaro I, Farı´as GG, Metcalfe MJ, Fuentealba R, Bonansco C, Inestrosa NC .Wnt-7a Modulates the Synaptic Vesicle Cycle and Synaptic Transmission in Hippocampal Neurons. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 9, pp. 5918–5927

• Recruitment and clustering of scaffold proteins and receptors

to postsynaptic membrane to form functional synapses

• Loss of Wg in Drosophila glutamatergic NMJ--- aberrant

development of postsynaptic specializations

• Wnt-5a increases number of PSD-95 clusters in dendritic

spines

POSTsynaptic Assembly

Wnt-5a induces clustering of PSD-95 in mature hippocampal

neurons

POSTsynaptic Function

• Wnt-5a increases amplitude of fEPSP

– Mechanism of functional role in postsynaptic

region--- possibly through PSD-95 clustering

– Other components involved are still under study

Field Potential recording of a CA1 pyramidal cell after Wnt-5a treatment

SUMMARY OF PART 1

1. Wnt ligands are involved in pre- and post-

synaptic protein clustering and assembly

2. The new synapses formed are functional

PART 2

Role in Alzheimer’s Disease

Alzheimer’s Disease Research, A program of American Health Assistance Foundation. www.ahaf.org

WNT SIGNALING IN ALZHEIMER’S DISEASE

The role of Wnt signaling in neuronal dysfunction in Alzheimer's Disease. Nibaldo C Inestrosa and Enrique M Toledo. Molecular Neurodegeneration 2008, 3:9 doi:10.1186/1750-1326-3-9

Boonen RA, van Tijn P, Zivkovic D. Wnt signaling in AD: up or down, that is the question. Ageing Research Reviews; 8 (2009) 71–82

Pathological cascade implicating deregulated Wnt signaling in neurodegeneration

Consequences of loss of Wntsignaling function

De Ferrari GV, Inestrosa NC. Wnt signaling function in Alzheimer's disease. Brain Res Brain Res Rev. 2000 Aug;33(1):1-12.

Wnt-3a protects hippocampalneurons from Aβ neurotoxicity

Alvarez AR, Godoy JA, Mullendorff K, Olivares GH, Bronfman M, Inestrosa NC. Wnt-3a overcomes beta-amyloid toxicity in rat hippocampal neurons. Experimental Cell Research 297 (2004) 186– 196

• Wnt-3ao Control

Wnt-3a protects hippocampalneurons from Aβ induced apoptosis

Alvarez AR, Godoy JA, Mullendorff K, Olivares GH, Bronfman M, Inestrosa NC. Wnt-3a overcomes beta-amyloid toxicity in rat hippocampal neurons. Experimental Cell Research 297 (2004) 186– 196

Control

Wnt-3a conditioned

medium

Control vector

medium

-Aβ + 5 μM Aβ

Wnt-3a increases bcl-2 and eng-1 mRNA levels

Fuentealba RA, Farias G, Scheu J, Bronfman M, Marzolo MP, Inestrosa NC. Signal transduction during amyloid-β-peptide neurotoxicity: role in Alzheimer disease. Brain Research Reviews 47 (2004) 275– 289

Alvarez AR, Godoy JA, Mullendorff K, Olivares GH, Bronfman M, Inestrosa NC. Wnt-3a overcomes beta-amyloid toxicity in rat hippocampal neurons. Experimental Cell Research 297 (2004) 186– 196

Control

Wnt-3a

Aβ

Aβ + Wnt-3a

Neurofilament β-catenin Merge

Wnt-3a protected β-catenin levels

Alvarez AR, Godoy JA, Mullendorff K, Olivares GH, Bronfman M, Inestrosa NC. Wnt-3a overcomes beta-amyloid toxicity in rat hippocampal neurons. Experimental Cell Research 297 (2004) 186– 196

Morphological changesin cultured neurons

induced by inhibition of Wnt signaling

Inhibition of Wnt and PI3K Signaling Modulates GSK-3b Activity and Induces Morphological Changes in Cortical Neurons: Role of Tau Phosphorylation. Neurochem Res (2008) 33:1599–1609

Increased PHF-1 immunostaining by inhibition of Wnt signaling

Inhibition of Wnt and PI3K Signaling Modulates GSK-3b Activity and Induces Morphological Changes in Cortical Neurons: Role of Tau Phosphorylation. Neurochem Res (2008) 33:1599–1609

SUMMARY OF PART 2

1. Wnt activation by ligands increases neuronal survival and decreases Aβ-induced apoptosis

2. Wnt (3a) causes increased transcription of bcl2 and eng-1 target genes

3. Wnt (3a) is neuroprotective through stabilization of β-catenin (canonical)

4. Inhibition of Wnt signaling can cause drastic morphological changes in neurons possibly due to tau hyperphosphorylation– Possibly through increased GSK-3β activity

PART 3

Wnt Signaling crosstalk

Crosstalk with M1AChR

Basic Neurochemistry, 6th Ed

B-catenin C-Jun Merge

Untreated

Aβ

Aβ + AF267B

PNZ + Aβ + AF267B

Farías GG, Godoy JA, Hernández F, Avila J, Fisher A, Inestrosa NC. M1 muscarinic receptor activation protects neurons from B-amyloid toxicity. A role for Wnt signaling pathway. Neurobiology of Disease 17 (2004) 337– 348

M1 mAChR activation increases cytoplasmic and nuclear β-catenin

protein levels

Farías GG, Godoy JA, Hernández F, Avila J, Fisher A, Inestrosa NC. M1 muscarinic receptor activation protects neurons from B-amyloid toxicity. A role for Wnt signaling pathway. Neurobiology of Disease 17 (2004) 337– 348

Effect of M1AChR activation on target gene expression

Farías GG, Godoy JA, Hernández F, Avila J, Fisher A, Inestrosa NC. M1 muscarinic receptor activation protects neurons from B-amyloid toxicity. A role for Wnt signaling pathway. Neurobiology of Disease 17 (2004) 337– 348

SUMMARY SCHEME

Farías GG, Godoy JA, Hernández F, Avila J, Fisher A, Inestrosa NC. M1 muscarinic receptor activation protects neurons from B-amyloid toxicity. A role for Wnt signaling pathway. Neurobiology of Disease 17 (2004) 337– 348

Crosstalk with PPARγ signaling

Metabolic Syndrome and Alzheimer’s disease!!!

PPARγ agonists increase

hippocampal neuron survival in presence

of Aβ

Inestrosa NC, Godoy JA, Quintanilla RA, Koenig CS, Bronfman M. Peroxisome proliferator-activated receptor γ is expressed in hippocampal neurons and its activation prevents beta-amyloid neurodegeneration. Role of Wnt signaling. Exp Cell Res. 2005 Mar 10;304(1):91-104.

Inestrosa NC, Godoy JA, Quintanilla RA, Koenig CS, Bronfman M. Peroxisome proliferator-activated receptor γ is expressed in hippocampalneurons and its activation prevents beta-amyloid neurodegeneration. Role of Wnt signaling. Exp Cell Res. 2005 Mar 10;304(1):91-104.

Control I μM TGZ I μM GW

5 μM Aβ

5 μM Aβ+ I μM TGZ

5 μM Aβ+ I μM GW

PPARγ agonists protect hippocampal

neuron morphology in presence of Aβ

Inestrosa NC, Godoy JA, Quintanilla RA, Koenig CS, Bronfman M. Peroxisome proliferator-activated receptor γ is expressed in hippocampalneurons and its activation prevents beta-amyloid neurodegeneration. Role of Wnt signaling. Exp Cell Res. 2005 Mar 10;304(1):91-104.

PPARγ agonists stabilize cytoplasmic β-cateninand nuclear translocation in presence of Aβ

Control 5 μM Aβ 5 μM Aβ + 1 μM TGZ

PPARγ activation inhibits GSK-3β activity

Inestrosa NC, Godoy JA, Quintanilla RA, Koenig CS, Bronfman M. Peroxisome proliferator-activated receptor γ is expressed in hippocampal neurons and its activation prevents beta-amyloid neurodegeneration. Role of Wnt signaling. Exp Cell Res. 2005 Mar 10;304(1):91-104.

SUMMARY SCHEME

PERTINENT QUESTIONS

• Neuromodulators?

• Synthesis, storage and secretion of Wntligands?

• Isolation and purification?

• Therapeutic potential?

Thank you!

Questions???

Wnt Signaling in Cancer