Wnt Signaling Pathway and AD

Transcription

1 Center for Cell Regulation and Pathology Joaquín V. Luco (CRCP), Millennium Institute (MIFAB) Center for Aging and Regeneration (CARE). Wnt Signaling Pathway and AD Nibaldo C. Inestrosa European Union Funding for AD The ADIT Project, SIENABIOTECH Praha, 10 th March, 2009

2 Why Wnt Signaling is important to study? because it might has the key to understand Alzheimer s Neurodegeneration Tau kinase I is GSK-3b (Takashima et al 1993). Presenilin interacts with d-catenin (Kosik, 1997). b-catenin decreases almost 60% in the brain of AD (m PS1) patients (Yankner,1999) Li protects neurons from Ab neurotoxicity (Avila, 1999). DKK 1 increased in neurons treated with Ab (Caricasole, 2003) Inhibition of GSK-3b by Li, correlates with reduced tauopathy and neurodegeneration in vivo (Duff, 2005). Genetic Polymorphisms in GSK-3b & LRP5/6 genes ( ) Normal brain Tau Phosphorylation AD brain P P Intracellular Amyloid Formation

3 In 1999, we postulated that b-amyloid neurotoxicity results in the loss of function of Wnt Signaling, this suggested for the first time, that a single pathway can integrates the hallmark lesions of AD: Amyloid Deposits & Neurofibrillary Tangles GSK-3 activation Vicious cycle IntracellularAmyloid formation (PS1 dependent) b-catenin degradation no signaling loss of cell junctions Tau phosphorylation Apoptosis Extracellular Amyloid Loss of synapses, Neuronal cell death AD De Ferrari & Inestrosa; Brain Res. Rev. 33: 1-12, 2000)

4 Wnt Signaling Pathway WNT DKK1 Ab Frizzled R LRP 6 WNT Frizzled R LRP 6 Axin dishevelled dishevelled APC Axin GSK-3ß ß-catenin P P degradation APC GSK-3ß ß-catenin ß-catenin ß-catenin ß-catenin ß-catenin TCF/ Lef TCF/ Lef

5 Activation of the Wnt pathway prevents Ab-Neurotoxicity Ab + Wnt HMW Neurofilament Synapsin-1 Exp. Cell Res. 297: (2004) Prog. Neurobiol. 86: (2008)

6 Activation of Several Signaling Pathways Results in the Cross-Talk with the Wnt Pathway and Leads to Neuroprotection Against Ab-Toxicity Ab Wnt Fzd Nicotinic Agonist 7 AChR Muscarinic Agonist Plasma Membrane CK1 Dvl Axin Ca 2+ PKC m1achr IBU-PO anti-inflammatory plus anti-ache PS GSK-3b APC b-cat PPAR- Antidiabetic drug Endoplasmic Reticulum APP b-cat Antioxidant Wnt Wnt Target Target Genes Nucleus Farias et al., Neurobiol. Dis. 17: (2004) Inestrosa et al., Exp Cell Res. 304: (2005) Farías et al., Neurobiol. Dis. 18: (2005) Quintanilla et al J. Biol. Chem. 280: (2005)

7 Lithium and Rosiglitazone (RGZ) improve Flexibility Memory (Episodic-like memory) in double APP+PS-1 transgenic mice Double Transgenic mouse APPswe + PSEN1D E9 day 1 Tg Control day2 Tg Lithium ThS day3 Tg RGZ WT day 4 Aβ Starting point TREATMENT OF ANIMALS: 6 month-old. LiCl (3 meq/kg day IP). RGZ (3 mg/kg day Gavage). Treatment for 4 months starting at month 6, until month 10.

8 Lithium and RGZ did not change de number of Amyloid Plaques as studied by Thioflavine-S in the APP/PS-1 mice, but decrease the ratio of ThS Plaques / Ab Aggregates Tg Control Tg Li Tg RGZ Hippocampus Cortex ThS Area (pix 2 ) ThS Area (pix 2 ) Tg Control Tg Lithium Tg RGZ Hippocampus Mol. Psychiat. (2009) Submitted Tg Control Tg Lithium Tg RGZ Cortex Ratio ThS Plaque / Ab Agregates Control APP + PS-1 mice Li RGZ

9 Wnt components Lithium and RGZ increase Wnt components and decrease Ab oligomers in APP-PS1 mice Ab Oligomers Mol. Psychiat. (2009) Submitted

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11 Role of Wnt Signaling in Pre-Synaptic Function Wnt-7a, but not Wnt - 5a, Induces clustering of Synaptophysin Pre-Synaptic Differentiation Wnt Control sv Wnt 7a Pre-Synaptic Aggregation of Synaptic Vesicles Wnt 5a p-synaptophysin (puncta /10mm axon length) J. Biol. Chem. 283: (2008) 0 C Wnt 5a Wnt 7a

12 Wnt-7a increases the frequency of the mepsc A control B Wnt-7a 50pA 100ms C D Amplitude Frequency Control Wnt-7a E Acumulative Probability control Wnt-7a mepsc frequency (s ) -1 J. Biol. Chem. 283: (2008)

13 7-nAChRs and APC co-localized in pre-synaptic regions of hippocampal neurons exposed to Wnt-7a Synaptotag APC A7 AChR Merge 3.0 pcdna a b c d Clusters size (Relative units) Wnt-7a e f g h 0.0 pcdna Wnt-7a pcdna Wnt-7a pcdna Wnt-7a Synaptotag APC 7 nachr J. Neurosci. 27 (2007)

14 Wnt-7a APC 7 nachr Pre-synaptic terminal Ca ++ glutamate GABA Modulation of Synaptic Activity

15 stimulates Post-synaptic Differentiation sv PSD- 95 Control Wnt - 5a induces clustering of PSD-95 (using GFP-PSD-95) IUBMB Life 59: (2007)

16 increases both PSD-95 clusters in dendritic spines and field EPSC (at the Amplitude level) suggesting a post-synaptic effect. a d 160 PSD-95/ Phalloidin b c Control /sfrp PSD-95 clusters (number/100 mm neurite) Control / sfrp + anti- antibody J. Biol. Chem. (2009) In Press)

17 Increases Amplitude of the NMDA currents in two phases. sfrp, a Wnt antagonist, blocks the effect 2.0 pcdna 1.0 Normalized Amplitude sfrp Time (min) Wnt sfrp frizzled Time (min) Conditioned medium 1/5 2mM NBQX Membrane potential +40mV

18 Non-canonical Wnt pathways

19 BAPTA prevents the effect of on NMDA currents, indicating that Ca +2 entry is required for the Wnt effect Normalized Amplitude mM BAPTA Time (min) PKC inhibition prevents the early and JNK inhibition prevents the late effect of, indicating that the Wnt / Ca +2 and the Wnt / JNK pathways are implicated A 2.0 A nM Go6976 (PKC inhib) Time (min) 1.7mM TAT-TI- JIP (JNK inhib) Time (min)

20 Pre-Synaptic Modulation Canonical Wnt Signaling NMDA-R Ab Post-Synaptic Modulation Non-Canonical Wnt Signaling PKC Fzd PSD-95 JNK New Synapses Cytoskeleton

21 Ab oligomers decreased PSD-95 levels, however prevents such changes PSD-95 PSD-95 / Synapsin-1 / Phalloidin b-tubulin PSD-95 (% of control) Ab (mm) PSD-95 Synapsin-1

22 Normalized Amplitude Facilitation index R2-R1/R1 Y Axis Title induces the clustering of NMDA receptors in the postsynaptic region. Ab oligomers decrease both NMDA R and field EPSP amplitude in hippocampal slices. prevents these changes. 0.00E+000 control e e pA 25ms NR2B / Phalloidin NMDA R (puncta /10 mm neurite) Ab oligom Control Ab olig Ab olig E Normalized Amplitude 1.0 control 0 Wnt-7a X Axis Title Control Ab Ab co

23 Ab oligomers decrease: PSD-95, NMDA Receptors, Dendritic Spines, EPSPs, CONCLUSION But the activation of the Non-Canonical Wnt Signaling prevents such changes, including and a formylated hexapeptide NMDA R PSD-95 Fz R Ab PSD-95

24 Center for Cell Regulation and Pathology Joaquín V. Luco (CRCP), MIFAB & Center of Aging and Regeneration P. Catholic University of Chile Iván Alfaro Waldo Cerpa Margarita Dinamarca Ginny Farias Juan Godoy Catalina Grabowski Enrique Toledo Christian Bonansco & Marcos Fuenzalida Program of Neurosciences Universidad de Valparaiso (Chile) Andrés Barría Department of Physiology University of Washington, Seattle (USA) Jesús Avila Centro de Biología Molecular Severo Ochoa (Spain) Abraham Fisher & Gabi Amitai Israel Institute of Biological Research (Israel)