1. Wnt ligands are involved in pre- and postsynaptic protein clustering and assembly at synapses and form functional new synapses.
2. Activation of Wnt signaling protects against Aβ toxicity in Alzheimer's disease by increasing neuronal survival, decreasing apoptosis, and stabilizing β-catenin levels. Inhibition of Wnt signaling can cause neuronal damage.
3. Wnt signaling interacts and forms a crosstalk with other pathways important in Alzheimer's like M1 muscarinic acetylcholine receptor activation and PPARγ activation, which may provide neuroprotective effects against Aβ toxicity.
2. INTRODUCTION
• ‘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
3. 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
5. Embryogenesis
Neurogenesis
Tissue Growth and Development
Unknown
Unknown
Unknown
Unknown
Logan and Nusse , Wnt signaling pathway in development and disease, Annu. Rev. Cell Dev. Biol. 2004. 20:781–810
7. • 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
11. PREsynaptic Assembly
• Drosophila – Wg induces synaptogenesis at NMJ
– Loss leads to reduction in synapses formation
• Mammals
– Wnt-7a increases levels of synaptic vesicle protein synapsin
I in developing cerebellar neurons
– Mutant mice show delay in synapsin I accumulation
– Wnt-3a, Wnt-7b also increase the number of excitatory
presynaptic puncta in hippocampal neurons
– Wnt-7a increases expression of α7-nAChR
– In mature CNS, Wnt-7a increases clustering of synaptic
vesicle proteins like synapsin I, synaptophysin, SV2, synaptotagmin
12. 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
13. 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?
14. 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
15. POSTsynaptic Assembly
• 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
17. 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
22. 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
23. Pathological cascade implicating deregulated
Wnt signaling in neurodegeneration
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
24. Consequences of loss of Wnt
signaling function
De Ferrari GV, Inestrosa NC. Wnt signaling function in Alzheimer's disease. Brain Res Brain Res Rev. 2000 Aug;33(1):1-12.
25. Wnt-3a protects hippocampal
neurons from Aβ neurotoxicity
o Control
• Wnt-3a
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
26. Wnt-3a protects hippocampal
neurons from Aβ induced apoptosis
-Aβ
+ 5 μM Aβ
Control
Wnt-3a
conditioned
medium
Control
vector
medium
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
27. 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
29. 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
30. Morphological changes
in 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
31. 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
32. 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
40. 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.
41. Control
I μM TGZ
5 μM Aβ
I μM GW
5 μM Aβ+ I μM GW
5 μM Aβ+ I μM TGZ
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 hippocampal
neurons and its activation prevents beta-amyloid neurodegeneration. Role of Wnt signaling. Exp Cell Res. 2005 Mar 10;304(1):91-104.
42. PPARγ agonists stabilize cytoplasmic β-catenin
and nuclear translocation in presence of Aβ
Control
5 μM Aβ
5 μM Aβ + 1 μM TGZ
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.
43. 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.
Talk about receptorsA recent survey of known components in or associated with canonical Wnt signaling pathways lists over 50 proteins (see www.stanford.edu/~rnusse/pathways/cell2.html). Due to this complexity, this review will focus only on well-established, core components of the pathway (for review, see Wodarz and Nusse, 1998; Peifer and Polakis, 2000). A major effector of the canonical Wnt signaling pathway is the transcription factor β-catenin (Armadillo in flies; Figure 1). In the absence of ligand, cytoplasmic β-catenin interacts with APC and Axin scaffold proteins and is a substrate for the kinases CKI and GSK3β. Phosphorylated β-catenin is then ubiquitinated and destroyed by the proteosome. When Wntligand binds to a Frizzled family receptor and a coreceptor of the LRP-5/6/arrow family, the APC/Axin/CK1/GSK3β destruction complex is inhibited, leading to the stabilization of β-catenin and its translocation to the nucleus where it interacts with TCF/LEF family transcription factors. In the absence of signal, TCF/LEF factors bind DNA at Wnt-responsive genes and interact with other factors (e.g. Groucho, histonedeacetylase) to repress transcription. β-catenin binding to TCF/LEF proteins provides a transcription activation domain so target gene expression is activated. NoncanonicalWnt signaling pathways in Drosophila and vertebrates are less well understood, but appear to function in a β-catenin independent manner to regulate processes such as convergent extension during vertebrate gastrulation, and the polarity of hairs, bristles and ommatidia in Drosophila (for review, see, Kuhl et al., 2000; Peifer and Polakis, 2000; Veeman et al., 2003). These noncanonical pathways have also been termed the Wnt/Calcium and Wnt/JNK pathways in vertebrates and the Wnt/planar cell polarity pathway (PCP) in flies. The similarity of these pathways to each other is under intense investigation, and a discussion of their molecular details is beyond the scope of this review. Briefly, activation of the Wnt/Calcium pathway involves Wnt binding to a Frizzled receptor, leading to release of intracellular calcium and the activation of enzymes such as CamKII and PKC (Kuhl et al., 2000). The Wnt/Jnk and PCP pathways appear to be similar in utilizing Frizzled receptors, Dishevelled, JNK and Rho family GTPases (Veeman et al., 2003). While Wnts are used in the vertebrate Wnt/Jnk pathway, no Wntligand has been identified for the Drosophila PCP pathway. Currently, the conservation of function of these pathways in C. elegans is unclear.
Function of Wnt in the central nervous system (CNS). Scheme summarizing the role of Wnt during CNS development. A,B: During early development, Wnt plays pivotal roles in axon and dendrites. A: In the axon, Wnt participates in processes such as axonal guide, axon attraction or retraction and growth cone (A). B: In dendrites, Wnt plays a role in dendritic branching. C,D: Later on, Wnts act as synaptogenic factors that regulates pre- and postsynaptic differentiation. Presynaptically, Wnt induces the clustering of several presynaptic vesicle proteins, structural presynaptic proteins, and presynaptic receptors (red circles; C); and postsynaptically, Wnt induces the clustering of the postsynaptic scaffold proteins PSD-95 in dendritic spines and glutamate receptors (red circles; D). In mature CNS, Wnt continued expressing and modulating synaptic function. Fig. 2. Wnt in synaptic function in mature hippocampal neurons. A: Scheme summarizing the Wnt-7a effects on neurotransmitter release. Wnt-7a induces the exocytosis and recycling of vesicles proteins. A possible mechanism involve adenomatouspolyposis coli (APC) protein, that in the presence of Wnt-7a ligand, dissociates the -catenin destruction complex and it associates to the 7-nicotinic acetylcholine receptor (7-nAChR). It is possible that APC functions as a cargo protein that interacts with microtulules to transport another protein. 7-nAChR localized in the plasma membrane can allow the entry of calcium to modulate the exocytosis of synaptic vesicles and finally to regulate the synaptic transmission. B: Wnt-5a increases synaptic transmission through a postsynaptic mechanism. New PSD-95 clusters are localized in dendritic spines, and they are formed through recruitment from the cytosolic PSD-95 pool. Activation of JNK by Wnt-5a is required for the clustering of PSD-95. Morever, glutamate receptors are anchored to the membrane and can explain the increase in the amplitudeof mEPSP. 96 FARI´AS ET AL.
*regulation in the patterning of synaptic connections
*indicates that Wnt-7a increases synaptic transmission by a presynaptic mechanism, probably involving an increase in neurotransmitter release.
FIGURE 8. Miniature EPSCs recorded in presence of tetrodotoxin (1 M) before and after continuous application of Wnt-7a. Shown are nine representative traces recording in control conditions at a holding potential of60 mV (A) and 30 min after the superfusion of Wnt-7a (B). C, increase of mean values of relative frequency of mEPSC (C) in both conditions without affecting the amplitude of mEPSC (D). E, cumulative probability plot of the mEPSCfrequency, recorded from a same cell during 90 s of sampling in control and after Wnt-7a treatment.
*Indicates the potentiation by Wnt-5a is due to ‘post’synaptic modulation of glutamatergic postsynaptic currents.
Pathological cascade implicating deregulated Wnt signaling in neurodegeneration. Through direct and indirect mechanisms such as Ab toxicity, both FAD mutations and genetic variations associated with SAD have been shown to deregulate Wnt signaling. The pathway indicated in red represents Wnt down regulation as second hit to abnormal Ab-induced cell cycle events, leading to programmed cell death. Shown in blue is the potential overactivation of Wnt signaling and its resultant effects due to FADPSEN mutations. In green, is shown the pathway that may link attenuation of the Wnt signal to tau hyperphosphorylation and associated neurodegeneration. Represented in black are other interactions leading to digression from the normal Wnt/b-catenin signal. When pathway is marked by a question mark, this association is either still debated or conflicting data are present concerning this interaction: NFTs, neurofibrillary tangles; Li+, lithium.
Fig. 1. Wnt-3a ligand protects survival of hippocampal neurons from Ab neurotoxicity. Six-day cultured hippocampal neurons (1X105 cells per well in a 96-well plate) were exposed to increasing Ab concentrations for 24 h in the presence of Wnt-3a conditioned media from stable HEK293/Wnt-3a cells (.) and control conditioned media from stable HEK293/empty vector cells (o). Cell viability was evaluated by the MTT assay. Results correspond to the mean F SE of three experiments carried out in triplicate. (Insert) Western blot for HEK 293/Wnt-3a-cells conditioned medium and control conditioned medium
Fig. 7. Wnt-3a protects hippocampal neurons from Ah induced apoptosis. (A) Five-day cultured hippocampal neurons in control Neurobasal media (a, b), Wnt-3a conditioned media (c, d) and control vector media (e, f) were exposed to 5 AMAh for 6 h (b, d, f). Apoptosis was evaluated by the TUNEL assay using an in situ cell death detection kit. (B) Apoptotic nuclei were counted by light microscopy and quantified in 10 fields from two independent preparations. The graph shows a mean F SE. *P < 0.001 compared with 5 AM Ah. (C) Inhibition of caspase-3 activation by Wnt-3a. 2 106 hippocampal neurons were incubated with 5 AMAh, 5 AMAh plus Wnt-3a media, or Wnt-3a conditioned media alone, during a 24-h period. Cells were lysed and 50 Ag aliquots of protein extracts were incubated with caspase-3 substrate (DEVD-pNA) for 12 h at 37jC and the reaction was recorded at 405 nm. The graph show a mean F SE for three experiments. *P < 0.05 compared with 5 AM Ah.
Fig. 5. Wnt/h-catenin signaling increases bcl-2 mRNA levels in primary rat hippocampal neurons. mRNA levels of bcl-2, engrailed-1, PrPc and the housekeeping genes actin and gapdh were analyzed by semiquantitative RT-PCR in primary neurons exposed to control, Wnt-3a or Frzb-1 conditioned media. (A) Wnt-3a causes a time dependent increase in bcl-2 mRNA levels that parallels those of the Wnt-target gene engrailed-1. (B) The Wnt antagonist Frzb-1 prevents bcl-2 and engrailed-1 mRNA increase induced by 24 h Wnt-3a co-treatment. Bottom pannel, densitometric analysis of normalized mRNA levels.
Fig. 4. Wnt-3a conditioned media prevents the loss of cytoplasmic h-catenin in hippocampal neurons. Six-day cultured hippocampal neurons were treated by 24 h with control vector conditioned media (a, b, c), Wnt-3a conditioned media (d, e, f), 5 AM Ah plus control vector conditioned media (g, h, i) and Wnt-3a conditioned media plus 5 AM Ah (j, k, l) and were analyzed by immunoflorescence for h-catenin and neurofilament (anti-NF-200) protein distribution. Neurons exposed to Ah displayed a somatic shrinkage plus dendritic dystrophy. On the other hand, neurons treated with Wnt-3a ligand plus Ah showed an almost intact morphology, with a well-developed branching of neurites. Red: neurofilament (a, d, g, j) Green: h-catenin (b, e, h, k), merge (c, f, i, l). Scale bar: 4 Am.
Fig. 3. Ah peptide induced a decrease, while Wnt-3a protected h-catenin levels in rat hippocampal neurons. (A) Six-day cultured hippocampal cells were incubated with 5 AM Ah for the indicated time periods, and the cytosolic fractions were analyzed for h-catenin with a polyclonal antibody H-102 (1:1000) (Santa Cruz Biotechnology) and for the internal control, tubulin (1:1000, h-tubulin polyclonal antibody, Santa Cruz Biotechnology). The graph shows the densitometric analysis of a representative experiment. Data corresponds to h-catenin/tubulin signal ratio, represented as arbitrary units (UA). (B) Wnt-3a protects against h-catenin destabilization induced by Ah. 1 106 cells were treated with 5 AM Ah for 24 h in the presence of Neurobasal media, Wnt-3a conditioned media or control vector conditioned media. Cytosolic protein was extracted and separated by 10% SDS-polyacrylamide gel electrophoresis. The cytosolic protein blot was probed with polyclonal antibodies against h-catenin and h-tubulin. The graph shows the densitometric analysis of three experiments. Data corresponds to mean F SE for h-catenin/tubulin signal ratio, represented as arbitrary units (UA). *P < 0.001 compared to Ah peptide alone. (C) Reverse Ah40 – 1 peptide does not induce h-catenin destabilization. 1 106 cells were treated with 5 AM Ah1–40 fibrils or Ah40 – 1 for 24 h in the presence of Neurobasal media before total h-catenin levels were analyzed in the neurons. (D) Lithium demonstrate better h-catenin stabilization in cells exposed to Ah than Wnt-3a. 1 106 cells were treated with 5 AM Ah for 24 h in the presence of Wnt-3a conditioned media obtained from transiently transfected Wnt-3a HEK 293 cells or control conditioned media from transfected HEK 293 control vector with or without 50 AM lithium. h-catenin and h-tubulin protein levels were analyzed by Western blot. The graph shows the densitometric analysis of three experiments. Data corresponds to mean F SE for h-catenin/tubulin signal ratio, represented as arbitrary units (UA).
Fig. 4 Morphological changes in cultured neurons induced by inhibition of Wnt and PI3K/Akt signaling. After 24 h incubation neurons exposed to sFRP2 (b), DKK1 (c) and wortmannin (d) show expansion of neurites and enlargement of cell bodies. A notable accumulation of tubulin staining is observed in apical processes (arrowheads). High magnifications of the zone marked by arrows from each photomicrograph are depicted in E to H. Control neurons are shown in A and E. Scale bar = 20 lM and for high magnifications = 10 lm. Representative immunocytochemistry from 4 independent experiments
Fig. 5 Increased PHF-1 Immunostaining by inhibition of Wnt and PI3K/Akt signaling. After 24 h exposure to sFRP2 (b, e) and wortmannin (c, f) the pattern of PHF-1 staining is highly increased and slight accumulation within enlarged cell bodies is observed (arrow). The degeneration of fiber bundles and neuronal branches is evident (arrowheads). High magnifications from each photomicrograph are depicted in (d–f). Control neurons are shown in (a) and (d). Scale bar = 20 lM and for high magnifications = 10 lm. Representative immunocytochemistry from 3 independent experiments performed in duplicate
Fig. 4. M1 mAChR activation rescues h-catenin from its reduction induced by Ah. Hippocampal neurons were exposed to 5 AM Ah in combined treatments with and without 10 AM AF267B, in the presence or absence of 10 nM PNZ for 4 h. (A) The cultures were observed with a confocal microscope. First lane, detection of h-catenin was assessed a rhodamine-conjugated second antibody. Second lane, detection of c-Jun by using a FITC-conjugated second antibody. Third lane, merged images of h-catenin and c-Jun immunostaining. (a–c) untreated neurons, (d–f) Ah treatment, (g–i) incubation with Ah plus AF267B, (j–l) neurons exposed to Ah plus AF267B in the presence of PNZ. In (i) the arrow shows yellow immunostaining corresponding to the combination of the red (rhodamine) and green (fluorescein) fluorochromes and in (f) and (l) the arrows show its absence. Data are the mean F SEM of three independent experiments. Scale bar = 10 AM. (B and C) Western blot of cytoplasmic and nuclear h-catenin, respectively, corresponding to neurons treated for 4 h; equal amount of proteins were used (50 Ag). Bars represent the mean F SEM from three independent experiments. In figures, *: P b 0.01.
Fig. 5. Target genes of the Wnt signaling are activated by M1 mAChR stimulation in hippocampal neurons exposed to Ah. Neuronal cultures were treated with 5 AM Ah, plus or minus 10 AM AF267B with or without 10 nM PNZ in combined treatments for 12 h. RNA was isolated and cDNAs were synthesized; then mRNA levels of engrailed-1 and cyclin-D1 were evaluated by RT-PCR analysis. In (A) engrailed-1 and in (B) cyclin-D1, each one with their respective normalized densitometric quantification with h-actin. Data are mean F SEM values of four independent experiments.
Fig. 3. Activation of PPARg receptors reverts the morphological changes induced by Ah peptide in hippocampal neurons. (A1) Control hippocampal neurons. (A2) Neurons exposed to troglitazone or GW (A3). Neurons exposed to Ah alone (5 AM for 10 h) displayed a somatic shrinkage plus dendritic dystrophy (A4) (white arrows). On the other hand, hippocampal neurons exposed to both Ah plus 1 AM troglitazone showed an almost intact morphology with a welldeveloped branching of neurites (A5). However, neurons exposed to Ah plus 1 AM GW-9662 (a PPARg antagonist) showed, besides a somatic shrinkage, adramatic reduction in the number of neurite per hippocampal neuron (white arrows) (A6). (B) The number of neurites of similar experimental conditions described in Awas quantified using an Image-Pro plus software (*P b 0.05). (C) Both the length and the area of the neurites were quantified in both control and troglitazone (TGZ)-treated cells using the Image-Pro plus software (*P b 0.05 for the length and **P b 0.001 for the area).
