Urokinase-Type Plasminogen Activator Triggers Wingless/Int1-Independent Phosphorylation of the Low-Density Lipoprotein Receptor-Related Protein-6 in Cerebral Cortical Neurons

Animals and reagents

We used 6-month-old male wild-type (Wt), and uPA (uPA^-/-) and plasminogen deficient (Plg^-/-) mice on a C57BL/6J background. The Institutional Animal Care and Use Committee (IACUC) of Emory University, Atlanta, GA, approved the animal experiments. A murine neuroblastoma cell line stably transfected with the APP-695 Swedish mutation (K595 N/M596 L) and herein referred to as N2asw, was a kind gift from Dr. G. Thinakaran (University of South Florida). Other reagents were recombinant murine uPA and its amino terminal fragment (ATF, devoid of proteolytic activity; Molecular Innovations, Novi, MI; Cat # MATF and MUPA, respectively), recombinant murine WIF-1 and Wnt3a (R & D Systems; Cat # 133-WF-050 and 1324-WN-010, respectively), dynabeads protein G immunoprecipitation kit, ultrapure agarose, Pierce BCA protein assay kit, sample buffer 5X, bovine serum albumin, penicillin/streptomycin, ProLong gold antifade, phalloidin DyLight 554, Hoechst, and ELISA kits for murine Aβ 1–40 and 1–42 peptides (ThermoFisher, Grand Island, NY; Cat # 10007D, 16500 - 500, 23225, 1859594, J64655, 15140-122, P36930, 21834, H3570, KMB3181 and KMB3441, respectively), triton X-100 and poly-l-lysine (Millipore, Burlington, MA; Cat # 9400 and P1399, respectively), oligomers of β-amyloid 1-42 (rPeptide, Watkinsville, GA; Cat # A - 1163), XAV-939 (Tocris Bioscience, Minneapolis, MN; Cat # 3748), Rneasy mini kit (Qiagen, Germantown, MD; Cat # 74106), protease inhibitors complete tablets EASYpack (Roche, Indianapolis, IN; Cat # 46931320001), RIPA buffer (TEKNOVA, Hillister, CA; Cat # R3792), iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA; Cat # 1708891), and the following antibodies: goat anti-mouse uPAR (R & D Systems; Cat # AF534), rabbit polyclonal LRP6 and mouse anti-β-actin (ThermoFisher; Cat # PA5-89161 and A1978, respectively), mouse anti-β-Catenin (BD Biosciences, San Jose, CA; Cat # 610153), monoclonal β-Catenin non-phosphorylated at Ser37/Thr41 (active β-Catenin), monoclonal anti-LRP6, and goat anti-mouse IgG HRP conjugate (Millipore; Cat # 05-665, MABS341, AP127P, respectively), mouse bassoon (Enzon, Farmingdale, NY; Cat # ENZ- ABS666-0200), rabbit anti-histone-H3, rabbit anit-PSD-95 and rabbit anti-phospho-LRP6 (Ser1490; Cell Signaling, Denver, MA; Cat # 4499, 2507 and 2568, respectively), sheep anti-uPAR (Molecular Innovations; Cat # SASMUPAR-306), IRDye 800CW donkey anti-rabbit, IRDye 800CW donkey anti-mouse, IRDye 680RD donkey anti-mouse, and IRDye 680RD donkey anti-rabbit (Li-Cor, Lincoln, NE; Cat # 926-32213, 929-32212, 926-68072, and 927-50000, respectively), and donkey anti-mouse Alexa Fluor 594, donkey anti-rabbit Alexa 594, goat anti-mouse Alexa Fluor 488, goat anti-rabbit Alexa Fluor 488, donkey anti-goat Alexa Fluor 488, and donkey anti-sheep IgG HRP conjugated (ThermoFisher; Cat # A21203, A21207, A11017, A11008, A11055, and A16041, respectively). PCR primers for Actb (forward 5’-ACTGGGACGACATGGAGAAG-3’, reverse 5’-GGGGTGTTGAAGGTCTCAAA-3’) and BACE1 (forward 5’-CTTTGTGGAGATGGTGGAC-3’, reverse 5’-GTTACTACTGCCCGTGTC-3’) were purchased from Integrated DNA Technologies (Coralville, IA).

Murine neuronal cultures

We cultured Wt cerebral cortical neurons from E16-18 mice as described [28]. Briefly, the cerebral cortex was dissected, transferred into Hanks’ balanced salt solution containing 100 units/ml penicillin, 100μg/ml streptomycin, and 10 mM HEPES, and incubated in trypsin containing 0.02% DNase at 37°C for 15 min. Tissue was triturated and the supernatant was re-suspended in GS21-supplemented neurobasal medium containing 2 mM l-glutamine, and plated onto 0.1 mg/ml poly-l-lysine-coated wells.

Preparation of nuclear extracts

Wt neurons incubated for 60 min with 5 nM of uPA or its ATF, or vehicle (control) were washed and centrifuged at 1,600 x g for 10 min. The pellet was resuspended in a hypotonic buffer containing 10 mM Hepes pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 1 mM DTT, and protease and phosphatase inhibitors, incubated in ice for 10 min, homogenized with a Pyrex homogenizer with 1 ml volume capacity and centrifuged for 10 min at 3,400 x g. The pellet was resuspended in a buffer containing 20 mM Hepes pH 7.9, 20% glycerol, 0.42 mM KCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT, and protease and phosphatase inhibitors before centrifugation for 30 min at 21,000 x g. Supernatants containing nuclear extracts were used for western blot analysis. To test the purity of these preparations cytoplasmic and nuclear extracts were immunoblotted with antibodies against histone-3 and GAPDH (data not shown).

Preparation of Aβ₁₄₂ oligomers

To prepare Aβ₁₄₂ oligomers [29], 80μM Aβ₁₄₂ monomers lyophilized in 1,1,1,3,3,3-Hexafluoro-2-Propanol were incubated overnight after resuspension to a 5 mM concentration in DMSO. Experiments were performed with final concentration of 100 nM.

Western blot analyses

The abundance of β-Catenin was studied in Wt cerebral cortical neurons treated for 30 min with 5 nM of uPA or a comparable volume of vehicle (control), or 24 h with 100 nM of soluble Aβ₁₄₂ oligomers in the presence of three doses of 5 nM of uPA or vehicle (control) administered at time 0, 12, and 23 h, or in nuclear extracts prepared from Wt cerebral cortical neurons treated during 60 min with 5 nM of uPA or its ATF, or in N2asw cells incubated during 15 min with 5 nM of uPA. The abundance of active β-Catenin was studied in Wt cerebral cortical neurons treated during 30 min with 5 nM of uPA. The abundance of LRP6 phosphorylated at Ser1490 (pLRP6) was studied in Wt cerebral cortical neurons incubated 15 min with 5 nM of uPA. Cells were then homogenized with RIPA buffer. Protein was loaded onto 4–15% gels (Bio-Rad) and transferred into a nitrocellulose membrane (Bio-Rad). Then, membranes were incubated with antibodies against either total β-catenin (1:2,000), or active β-catenin (1:1,000), or LRP6 phosphorylated at Ser1490 (1:1,000), or β-actin (1:50,000), or histone-3 (1:1,000). Membranes were then washed and incubated with either one of the following secondary antibodies prepared at a 1:10,000 dilution: IRDye 800CW donkey anti-rabbit, or IRDye 800CW donkey anti-mouse, or IRDye 680RD donkey anti-mouse, or IRDye 680RD donkey anti-rabbit, or IRDye 800CW goat anti-mouse IgG1. LI-COR Odyssey Fc reader and IMAGE STUDIO version 5.2 were used to detect and measure infrared signal (Li-Cor). Values were expressed as a ratio to β-actin and normalized to intensity detected in extracts prepared from untreated cells.

Polymerase chain reaction

RNA was extracted from Wt cerebral cortical neurons and N2asw cells treated 24 h with 5 nM of uPA or vehicle (control), and from Wt cerebral cortical neurons incubated during 24 h with vehicle (control) or 1μM of XAV-939, followed by 24 h of treatment with either 5 nM of uPA or vehicle (control). A subgroup of neurons was treated during 24 h with 5 nM of uPA’s ATF. cDNA was prepared using iScript cDNA Synthesis kit, and a MultiGene OptiMax Thermal Cycler [72°C×2 min; 30 cycles of 98°C×was used 10 s, 60°C×30 s, 72°C×20 s, and 72°C×10 min (Labnet International, Edison, NJ)] was used to detect a PCR product to detect either BACE1 or Actb with the primers described above in Animals and reagents. The LI-COR Odyssey Fc reader and IMAGE STUDIO ver 5.2 was used to detect and measure the amplified PCR product.

Immunocytochemistry

To study the effect of uPA on the synapse, Wt cerebral cortical neurons were treated during 24 h with 1μM of XAV-939 or vehicle (control), and then incubated for 24 h with 100 nM of soluble Aβ oligomers or vehicle (control), alone or in the presence of three doses of 5 nM of uPA administered at 0, 12, and 23 h. To study the effect of uPA on LRP6 phosphorylation at Ser1490, Wt cerebral cortical neurons were incubated 15 min with 5 nM of uPA or vehicle (control); or 30 min with 10μg/ml of WIF1 or vehicle (control) followed by 15 min of treatment with with 5 nM of uPA or 80μg/ml of Wnt3a; or 24 h with vehicle or 100 nM of soluble Aβ oligomers, followed by 15 min of treatment with vehicle, or 80μg/ml of Wnt3a, or 5 nM of uPA; or 15 min with vehicle (control), or 5 nM of uPA, or 6μg/ml of anti-uPAR antibodies, or a combination of either uPA or Wnt3a and anti-uPAR antibodies. To study the effect of uPA on the nuclear translocation of β-Catenin Wt cerebral cortical neurons were treated 60 min with vehicle (control) or 5 nM of uPA. To study the effect of uPA on BACE1 expression, Wt cerebral cortical neurons were treated 24 h with 5 nM of uPA or vehicle (control). At the end of each time point cells were fixed for 10 min with 4% paraformaldehyde, washed, permeabilized, blocked with 0.1% triton X-100 and 3% BSA in TBS, and incubated overnight at 4°C with either one of the following antibodies at a 1:100 dilution: mouse anti-bassoon, and/or rabbit anti-PSD-95, and/or rabbit anti-phospho-LRP6, and/or mouse anti- β-Catenin. Cells were then washed and incubated with either phalloidin DyLight 488 (1:1000) and/or Hoechst (1:5000), and either one of the following antibodies at a 1:500 dilution: donkey anti-mouse Alexa Fluor 594, donkey anti-rabbit Alexa 594, goat anti-mouse Alexa Fluor 488, goat anti-rabbit Alexa Fluor 488, or donkey anti-goat Alexa Fluor 488. Confocal images were obtained using a Fluoview FV10i automated confocal laser-scanning microscope (Olympus, Center Valley, PA) with a 60x lens UPLSAPO60XO NA 1.35, 1X pinhole (50 um), 512×512 pixels, speed 4.4 s/frame, scanning device 2 galvano-meter mirrors, XY resolution 300 nm, immersion oil IMMOIL F30CC. Pictures were deconvoluted using 64 iterations of CellSens dimension 1.17 software (Olympus). To quantify the nuclear translocation of β-Catenin, we used the CellSens dimension 1.17 software to delineate the nucleus in the Hoechst channel. Then, the area immunoreactive to anti β-Catenin antibodies was quantified, and values were expressed as percentage of nuclear β-Catenin immunoreactivity in control cells. The ImageJ puncta analyzer was used to quantify pLRP6- and bassoon/PSD-95-positive puncta in 50μm extensions of neurons in each experimental group.

ELISA

Brain cortices were harvested and processed following manufacturer’s instructions. Briefly, 100 mg of tissue was homogenized 8 times with 100μl of 5M guanidine-HCl/50 mM Tris at pH 8 and mixed at room temperature for 3 h. The lysate was diluted 10-fold with cold PBS plus protease inhibitors cocktail and centrifuged at 4°C 16,000 x g for 20 min. The supernatant was collected and diluted two times using the kit dilution buffer. N2asw cells were maintained in culture media containing 1:1 DMEM and Opti-MEM, 5% fetal bovine serum, 100 units/ml penicillin/streptomycin, 200 mM L-glutamine, and 0.2 mg/ml G418. Upon reaching 80-90% confluence, cells were plated onto 35 mm wells and treated for 24 h with either vehicle control or 5 nM of uPA. Then, 100μl of culture medium was used to measure the concentration of Aβ oligomers following manufacturer’s instructions. 100μl of brains or cells, or standards were incubated for 2 h in the antibody-coated plate. Wells were then washed four times with the kit wash buffer and incubated with the kit detector antibody for 1 h at room temperature. The plate was then incubated with anti-IgG HRP for 30 min at room temperature, and then washed again and treated during 30 min with 100μl of the kit chromogen buffer. The absorbance was read at 450 nm after addition of 100μl of the kit stop solution. Protein concentration was measured in cortices and cells, and values were expressed as pg of Aβ₄₀ or Aβ₄₂ per mg (tissue) or μg (cells) of protein.

Statistics

Statistical analysis was performed with student’s t-test and one- or two-way ANOVA with corrections, as deemed appropriate and described in each figure legend. p-values of < 0.05 were considered as significant.

UPA protects the synapse from the harmful effects of soluble Aβ by abrogating its inhibitory effect on the accumulation of β-catenin

First, we decided to study the effect of uPA on the expression of β-Catenin. We found that 30 min of treatment with 5 nM of uPA increases the abundance of β-Catenin in wild-type (Wt) cerebral cortical neurons (Fig. 1A, B). Because soluble Aβ decreases the abundance of β-Catenin [30], we studied its expression in Wt cerebral cortical neurons incubated during 24 h with 100 nM of soluble Aβ oligomers, alone or in the presence of 5 nM of uPA. Importantly, our previous studies indicate that with this experimental paradigm soluble Aβ does not induce neuronal death [19]. These experiments revealed that soluble Aβ decreases the abundance of β-Catenin in cerebral cortical neurons, and that this effect is abrogated by treatment with uPA (Fig. 1 C, D). To determine if uPA also has an effect of endogenously generated soluble Aβ on the abundance of β-Catenin, we studied its expression in a murine neuroblastoma cell line stably transfected with the APP-695 Swedish mutation (N2asw) that by rendering AβPP a more optimal substrate for BACE-1 increases the production of Aβ₄₀ and Aβ₄₂ [31]. Our data show that the low baseline level of β-Catenin in N2asw cells is increased after 15 min of treatment with 5 nM of uPA (Fig. 1E, F).

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Fig. 1

UPA protects the synapse from the harmful effects of soluble Aβ by reversing its inhibitory effect on the accumulation of β-Catenin. A, B) Representative western blot analysis (A) and quantification of the intensity of the band (B) of β-Catenin expression in wild-type (Wt) cerebral cortical neurons incubated 30 min with 5 nM of uPA or a comparable volume of vehicle (control). n = 6 observations per experimental group. Statistical analysis: two-tailed student’s t-test. C, D) Representative western blot analysis (C) and quantification of the intensity of the band (D) of β-Catenin abundance in Wt cerebral cortical neurons incubated 24 h with 100 nM of soluble Aβ oligomers or a comparable volume of vehicle (control), alone or in the presence of 5 nM of uPA. n = 6 observations per experimental group. Statistical analysis: one-way ANOVA with Holm-Sidak’s multiple comparisons test. E, F) Representative western blot analysis (E) and quantification of the intensity of the band (F) of β-Catenin abundance in N2asw cells incubated 15 min with 5 nM of uPA or a comparable volume of vehicle (control). n = 4 per experimental condition. Statistical analysis: two-tailed student’s t-test. G) Representative confocal micrographs at 60 X magnification of bassoon (green)- and PSD-95 (red)- immunoreactive puncta in extensions of Wt neurons treated during 24 h with 1μM of XAV-939 or vehicle (control), and then incubated for 24 h with 100 nM of soluble Aβ oligomers or vehicle (control), alone or in the presence of 5 nM of uPA. Scale bar: 10μm. H) Number of bassoon/PSD-95-positive puncta in extensions of 40 Wt neurons exposed to the experimental conditions described in G. Statistical analysis: one-way ANOVA with Holm-Sidak’s multiple comparisons test.

β-Catenin plays a pivotal role in the formation of synaptic contacts and our previous studies indicate that uPA protects the synapse from the harmful effects soluble Aβ [19]. Thus, to determine if β-Catenin mediates the protective effect of uPA on the synapse, we used confocal microscopy to quantify the number of intact synaptic contacts (bassoon/PSD-95-positive puncta) in extensions of Wt cerebral cortical neurons incubated during 24 h with soluble Aβ oligomers in the presence of 5 nM of uPA, alone or in combination with 1μM of XAV-939 (promotes β-Catenin degradation [32, 33]). We found that the effect of soluble Aβ on the number of intact synaptic contacts (a decrease from 26.41±7.8 in control cells to 9±7.46 in neurons incubated with soluble Aβ; p < 0.0001) was attenuated by treatment with uPA (22.28±8.2; p < 0.0001). However, the protective effect of uPA on the synapse was abrogated when the accumulation of β-Catenin was prevented with XAV-939 (10.85±7.83; p = 0.13 compared to control cells and p < 0.0001 compared to cells treated with uPA alone. n = 40 per experimental group; Fig. 1 G, H).

UPA/uPAR signaling antagonizes the inhibitory effect of Aβ on LRP6 by providing a Wnt-independent alternative pathway for LRP6 phosphorylation

When the Wnt β-Catenin pathway is inactive, GSK3β targets β-Catenin to proteasomal degradation by triggering its phosphorylation at Ser33/Ser37/Thr41 [10]. Thus, to investigate if uPA promotes the accumulation of β-Catenin by preventing its phosphorylation by GSK3β, we used an antibody that does not recognize phosphorylated β-Catenin, to study the expression of β-Catenin non-phosphorylated at Ser33/Ser37/Thr41 (also known as active β-Catenin, is not degraded by the proteasome) in Wt cerebral cortical neurons treated during 30 min with 5 nM of uPA or vehicle (control). These experiments revealed that uPA prevents GSK3β-induced β-Catenin phosphorylation, as denoted by an increase in the abundance of active β-Catenin in uPA-treated neurons (Fig. 2A, B).

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Fig. 2

UPA binding to uPAR induces LRP6 phosphorylation via a Wnt-independent mechanism. A, B) Representative western blot analysis (A) and quantification of the intensity of the band (B) of the abundance of active β-Catenin in Wt cerebral cortical neurons incubated 30 min with 5 nM of uPA or vehicle (control). n = 6 per experimental condition. Statistical analysis: two-tailed student’s t-test. C, D) Representative western blot analysis (C) and quantification of the intensity of the band (D) of the abundance LRP6 phosphorylated at S1490 (pLRP6) in Wt cerebral cortical neurons incubated 15 min with 5 nM of uPA or a comparable volume of vehicle (control). n = 4 per experimental condition. Statistical analysis: two-tailed student’s t-test. E) Representative confocal micrographs at 60X magnification of pLRP6 (red) and phalloidin (green) staining in extensions of Wt cerebral cortical neurons incubated 15 min with 5 nM of uPA or a comparable volume of vehicle (control). Scale bar: 10μm. F) Number of pLRP6-positive puncta in extensions of 52 Wt cerebral cortical neurons exposed to the experimental conditions described in E. Statistical analysis: two-tailed student’s t-test. G) Representative confocal micrographs at 60X magnification of puncta immunoreactive to anti-pLRP6 antibodies (red) in extensions of Wt cerebral cortical neurons treated during 30 min with 10μg/ml of WIF-1 or vehicle (control), and then incubated 15 min with 5 nM of uPA or 80μg/ml of Wnt3a. Green is phalloidin. Scale bar: 10μm. H) Number of pLRP6-positive puncta in extensions of Wt cerebral cortical neurons exposed to the experimental conditions described in G. n = 52 - 58 cells per experimental condition. Statistical analysis: one-way ANOVA with Holm-Sidak’s multiple comparison’s test. I) Representative confocal micrographs at 60X magnification of puncta immunoreactive to pLRP6 antibodies (red) in extensions of Wt cerebral cortical neurons incubated during 15 min with vehicle (control; a), or 5 nM of uPA (b), or 6μg/ml of anti-uPAR antibodies (c), or a combination of uPA and anti-uPAR antibodies (d). Scale bars: 50μm. Lower panels correspond to a 1.5 magnification of the area depicted by the corresponding white squares. Green corresponds to phalloidin. J) Number of pLRP6-positive puncta in extensions of Wt cerebral cortical neurons exposed to the experimental conditions described in I. n = 57 - 60 cells per experimental condition. Lower panels correspond to a 1.5 magnification of the area depicted by the corresponding white squares. Statistical analysis: two-way ANOVA with Holm-Sidak’s multiple comparisons test. K) Representative confocal micrographs at 60X magnification of pLRP6-positive puncta (red) in extensions of Wt cerebral cortical neurons incubated during 15 min with vehicle (control; a), or 80μg/ml of Wnt3a (b), or 6μg/ml of anti-uPAR antibodies (c), or a combination of Wnt3a and anti-uPAR antibodies (d). Lower panels correspond to a 1.5 magnification of the area depicted by the corresponding white squares. Green corresponds to phalloidin. Scale bars: 50μm. L) Number of pLRP6-positive puncta in extensions of Wt cerebral cortical neurons exposed to the experimental conditions described in K. n = 49 –52 cells per experimental condition. Statistical analysis: two-way ANOVA with Holm-Sidak’s multiple comparisons test.

Because LRP6 phosphorylation at Ser1490 (pLRP6) triggered by binding of Wnts to LRP6 on the cell membrane prompts the recruitment and inactivation GSK3β, we then decided to investigate the effect of uPA on the abundance of pLRP6. We found that 15 min of incubation with 5 nM of uPA induces the phosphorylation of LRP6 in Wt cerebral cortical neurons (Fig. 2 C, D), and in line with these observations, our confocal microscopy studies show that the number of pLRP6-positive puncta in extensions of Wt cerebral cortical neurons increases from 13±1.81 in control cells to 24.63±1.2 in uPA-treated neurons (p < 0.0001; n = 48; Fig. 2E, F).

To determine if uPA increases the abundance of pLRP6 by promoting the binding of Wnts to LRP6 on the cell membrane, we studied the abundance of pLRP6 in extensions of Wt cerebral cortical neurons treated with either vehicle (control), or Wnt3a (Wnt3a binding to LRP6 triggers its phosphorylation in cerebral cortical neurons [34]), or uPA, or with the Wnt inhibitor WIF-1 in combination with either Wnt3a or uPA. We found that the number of pLRP6-positive puncta increases from 12.32±1.9 in control cells (n = 57) to 22.84±1.27 (n = 57) and 22.96±1.57 (n = 52), in Wnt3a- and uPA-treated cells, respectively (p < 0.0001 when control cells are compared to either Wnt3a- or uPA-treated neurons; Fig. 2 G, H). However, while WIF-1 abrogates the effect of Wnt3a (10.7±0.9; p < 0.0001 compared to cells treated with Wnt3a alone), it does not have an effect on uPA-treated neurons (22±2.3; p = 0.96 compared to cells treated with uPA alone, n = 51). To determine if uPAR mediates the effect of uPA and Wnt3a, we performed similar observations in the presence of anti-uPAR neutralizing antibodies. Our data indicate that uPAR mediates the effect of uPA (Fig. 2I, J) but not Wnt3a (Fig. 2K, L) on the abundance of pLRP6.

Because soluble Aβ inhibits Wnt-β-catenin pathway signaling by preventing LRP6 phosphorylation [35], we decided to investigate if uPA is capable to antagonize this effect. We used confocal microscopy to quantify the abundance of pLRP6 in extensions of Wt cerebral cortical neurons incubated during 24 h with 100 nM of soluble Aβ oligomers, and then treated during 15 min with 5 nM of uPA or 80μg/ml of Wnt3a. We found that the number of pLRP6-positive puncta decreases from 20.11±10.07 in control cells (n = 80) to 10.68±4.68 in neurons incubated with soluble Aβ (p < 0.0001). Significantly, this effect was antagonized by treatment with uPA (20.31±10.88; p < 0.0001, n = 74) but not Wnt3a (12.09±9.49; p = 0.6, n = 65; Fig. 3A, B).

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Fig. 3

UPA reverses the inhibitory effect of soluble Aβ on LRP6 phosphorylation. A) Representative confocal micrographs at 60X magnification of Wt cerebral cortical neurons incubated during 24 h with vehicle (control; panel a), or 100 nM of soluble Aβ oligomers (b, c, and d), followed by 15 min of treatment with vehicle (control; b), or 80μg/ml of Wnt3a (c), or 5 nM of uPA (d). Lower panels correspond to a 1.5 magnification of the area depicted by the corresponding white squares. Scale bars: 50μm. B) Number of pLRP6-positive puncta in extensions of Wt cerebral cortical neurons exposed to the experimental conditions described in A. n = 80 (control cells), 78 (cells treated with Aβ and vehicle-control), 65 (cells treated with Aβ and Wnt3a), and 74 (cells treated with Aβ and uPA). Statistical analysis: one-way ANOVA with Holm-Sidak’s multiple comparison’s test.

UPA induces the nuclear translocation of β-Catenin via a plasminogen-independent mechanism

Because the cytoplasmic accumulation of β-Catenin is followed by its nuclear translocation [10], we used confocal microscopy to quantify the area of the nucleus immunoreactive to anti-β-Catenin antibodies in Wt cerebral cortical neurons treated during 60 min with 5 nM of uPA or a comparable volume of vehicle (control). Our data indicate that compared to control-treated cells, uPA induces a 23.71±5.1% increase in the area of the nucleus immunoreactive to anti-β-Catenin antibodies (Fig. 4A, B). To further characterize these observations, we studied the abundance of β-Catenin in nuclear extracts prepared from Wt cerebral cortical neurons treated during 60 min with 5 nM of either uPA, or its amino terminal fragment [ATF (Ser1 -Lys135): this fragment of uPA binds to uPAR as its contains its epidermal growth factor-like (EGF) domain, but is devoid of proteolytic activity because lacks the serine protease domain], or vehicle (control). We found that the cytoplasmic accumulation of β-Catenin induced by uPA treatment is followed by its nuclear translocation (Fig. 4 C, D) via a mechanism that does not require plasmin generation (Fig. 4E, F).

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Fig. 4

UPA induces the nuclear translocation of β-Catenin via a plasminogen-independent mechanism. A) Representative confocal micrographs of cerebral cortical neurons stained with anti-β-Catenin antibodies (red) and Hoechst (blue) following 60 min of treatment with vehicle (control: C; panels a-c) or 5 nM of uPA (panels d-f). Magnification in panels a, b, d, and e: 60 X. Panels c and f correspond to a 1.5 X magnification of the areas denoted by the white squares in b and e. Arrows in f denote examples of β-Catenin immunoreactivity in the nucleus of uPA-treated neurons. Scale bars: 20μm. B) β-Catenin-immunoreactive area in the nucleus of Wt neurons exposed to the experimental conditions described in A. n = 14 neurons from three different cultures per experimental group. Statistical analysis: two-tailed student’s t-test. C-F) Representative western blot analyses (C, E) and quantification of the intensity of the band (D, F) of β-Catenin expression in nuclear extracts prepared from Wt cerebral cortical neurons following 60 min of treatment with 5 nM of either uPA (C, D) or its amino terminal fragment (ATF: E, F), or with a comparable volume of vehicle (control). n = 5 in D and 6 in F. Statistical analysis in D and F: two-tailed student’s t-test.

UPA-triggered β-Catenin signaling inhibits the amyloidogenic processing of the AβPP

Because the nuclear translocation of β-Catenin represses BACE1 transcription by promoting binding of TCF-4 to its promoter [31], we studied the expression of BACE1 mRNA in Wt cerebral cortical neurons incubated during 24 h with 5 nM of uPA, alone or in the presence of XAV-939, which inhibits Wnt-β-Catenin signaling by promoting the degradation of β-Catenin. We found that uPA effectively decreases the abundance of BACE1 mRNA and that this effect is abrogated by XAV-939 (Fig. 5A, B). In line with these observations, our confocal microscopy studies show that the number of BACE-1-positive puncta in extensions from Wt cerebral cortical neurons decreases from 23.98±7.93 in control cells to 13.25±2.5 in neurons incubated with uPA (n = 48 per experimental group. p < 0.0001; Fig. 5 C, D). To study if the effect of uPA requires its proteolytic activity, we studied the expression of BACE1 mRNA in cerebral cortical neurons treated with 5 nM of uPA’s ATF (binds to uPAR but is devoid or proteolytic activity as it lacks uPA’s serine protease domain). These experiments revealed that the effect of uPA on BACE1 mRNA does not require the conversion of plasminogen to plasmin (Fig. 5E, F). Because BACE1 plays a pivotal role in the amyloidogenic processing of AβPP [36], we quantified BACE1 mRNA and the concentration of Aβ₄₀ and Aβ₄₂ in N2asw cells and their culture medium, respectively, following 24 h of incubation with 5 nM of uPA or vehicle (control). These experiments revealed that uPA effectively decreases the abundance of BACE1 mRNA (Fig. 5 G, H) and the generation of Aβ₄₀ and Aβ₄₂ (Fig. 5I) in cells with enhanced amyloidogenic processing of AβPP [31]. To determine the translational relevance of these observations, we quantified the concentrations of Aβ₄₀ and Aβ₄₂ in the cerebral cortex of 6-month-old Wt, uPA^-/- and Plg^-/- mice. In line with our previous observations, we found that genetic deficiency of uPA is associated with increased generation of Aβ₄₀ and Aβ₄₂ in the cerebral cortex via a plasminogen-independent mechanism (Fig. 5J).

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Fig. 5

UPA-induced activation of the Wnt-β-Catenin pathway inhibits the expression of BACE-1 and the amyloidogenic processing of AβPP. A, B) Representative PCR analysis (A) and quantification of BACE1 mRNA normalized to actin and to BACE1 mRNA in control cells (B) in Wt cerebral cortical neurons incubated during 24 h with vehicle (control) or 1μM of XAV-939, followed by 24 h of treatment with either 5 nM of uPA or vehicle (control). n = 5 per experimental condition. Statistical analysis: two-way ANOVA with Holm-Sidak’s multiple comparison’s test. C) Representative confocal micrographs at 60X magnification of BACE 1 (red) and phalloidin (green) expression in Wt cerebral cortical neurons treated 24 h with 5 nM of uPA or vehicle (control). Lower panels correspond to 1.5 magnifications of the areas depicted by the white squares. Scale bar: 50μm. D) Number of BACE1-positive puncta in extensions of 48 Wt cerebral cortical neurons exposed to the experimental conditions described in C. Statistical analysis: two-tailed student’s t-test. E, F) Representative PCR analysis (E) and quantification of BACE1 mRNA normalized to actin and to BACE1 mRNA in control cells (F) in Wt cerebral cortical neurons incubated during 24 h with vehicle (control) or 5 nM of uPA’s ATF. n = 6 per experimental condition. Statistical analysis: two-tailed student’s t-test. G, H) Representative PCR analysis of BACE1 mRNA (G) and quantification of the intensity of the band (H) in N2asw cells treated during 24 h with 5 nM of uPA or a comparable volume of vehicle (control). n = 4 per experimental group. Statistical analysis: two-tailed student’s t-test. I) Aβ₄₀ and Aβ₄₂ in the culture medium of N2asw cells treated during 24 h with 5 nM of uPA or a comparable volume of vehicle (control). n = 4 per experimental group. Statistical analysis: two-tailed student’s t-test. J) Aβ₄₀ and Aβ₄₂ in the cerebral cortex of 6-month-old Wt (n = 7), uPA ^-/- (n = 6) and Plg^-/- mice (n = 3). Statistical analyses: one-way ANOVA with Holm-Sidak’s multiple comparisons test.