Abstract
Despite the experimental evidence pointing to a significant role of the Wnt family of proteins in physiological and pathological rodent spinal cord functioning, its potential relevance in the healthy and traumatically injured human spinal cord as well as its therapeutic potential in spinal cord injury (SCI) are still poorly understood. To get further insight into these interesting issues, we first demonstrated by quantitative Real-Time PCR and simple immunohistochemistry that detectable mRNA expression of most Wnt components, as well as protein expression of all known Wnt receptors, can be found in the healthy human spinal cord, supporting its potential involvement in human spinal cord physiology. Moreover, evaluation of Frizzled (Fz) 1 expression by double immunohistochemistry showed that its spatio-temporal and cellular expression pattern in the traumatically injured human spinal cord is equivalent to that observed in a clinically relevant model of rat SCI and suggests its potential involvement in SCI progression/outcome. Accordingly, we found that long-term lentiviral-mediated overexpression of the Fz1 ligand Wnt1 after rat SCI improves motor functional recovery, increases myelin preservation and neuronal survival, and reduces early astroglial reactivity and NG2+ cell accumulation, highlighting the therapeutic potential of Wnt1 in this neuropathological situation.
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Acknowledgements
We would like to thank Virginia Pérez, Sandra Vázquez, and the technical staff from the Service of Microscopy and Image Analysis and the Service of Flow Cytometry at the National Hospital for Paraplegics for their outstanding technical help, especially to Javier Mazarío for his essential participation in the design of the automatized cell count method. We also like to thank Sam David for his help to obtain the lesioned human spinal cord samples. This work has been supported by the Fondo de Investigación Sanitaria (FIS) of Instituto de Salud Carlos III (Grant number PI12/2895; FEDER co-funded) and the Ministerio de Ciencia, Innovación y Universidades (Grant number RTI2018-097775-B-I00; FEDER co-funded).
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18_2019_3427_MOESM1_ESM.tif
Online Resource 1 Determination of the primary antibody specificity used to visualize Fz1. This figure shows representative images obtained from non-lesioned (NL) rat spinal cord sections (a1-6), lesioned rat spinal cord sections (7 days post-injury) (b1-6) and NL human spinal cord sections (c1-6) used to determine the specificity of the anti-Fz1 primary antibody. (a1-2), (b1-2) and (c1-2) correspond to images obtained from spinal cord sections processed for the immunohistochemical visualization of Fz1 and used as positive controls (C+); (a3-4), (b3-4) and (c3-4) correspond to images obtained from spinal cord sections processed for the immunohistochemical visualization of Fz1 without the primary antibody and used as negative controls (C-); (a5-6), (b5-6) and (c5-6) correspond to images obtained from spinal cord sections processed for the immunohistochemical visualization of Fz1 with the primary antibody pre-adsorbed with its corresponding blocking peptide (1:20 antibody/blocking peptide weight excess) (1/20). (a), (b) and (c) correspond to schematic drawings of prototypical NL rat, lesioned rat and and NL human spinal cord sections respectively, indicating the approximate areas shown in the corresponding images. Scale bars = 100 µm. (TIF 64125 kb)
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Online Resource 2 Determination of the proper functioning of the lentiviral vector generated to overexpress Wnt1. This figure shows the data and representative images obtained from the different studies performed in cultured B1a cells to evaluate the proper functioning of the lentiviral vector generated to overexpress Wnt1. Briefly, to determine the multiplicity of infection (MOI) that should be used to conduct this set of experiments we tested the effects of various MOI (MOI 5, 10, 20 and 40) on different aspects such as cell number (a), percentage of transduced cells (b) and GFP intensity (c) at 3 and 7 days post-transduction (dpt) (n = 3 per group) by flow cytometry. Based in the results obtained we selected MOI 10 to perform the subsequent experiments, since it induced a similar transduction efficiency to that observed with higher MOIs (MOI 20 and 40) (b), led to a high transgene expression (c) and did not affect cell viability and/or proliferation (a). The existence of significant differences between groups in a-c was assessed by Two-way ANOVA followed by Bonferroni post-hoc test (#, ## and ### = p < 0.05, p < 0.01 and p < 0.001 vs. 3 dpt; *, ** and *** = p < 0.05, p < 0.01 and p < 0.001 vs. non-transduced cells (NT); &, && and &&& = p < 0.05, p < 0.01 and p < 0.001 vs. the immediately lower MOI). We next determined by Real Time PCR and western blot that, in comparison with NT cells and those transduced with a lentiviral vector that only induce the expression of the green fluorescence protein (GFP) (LV-GFP), transduction with a lentiviral vector that induce the expression of both GFP and Wnt1 (LV-Wnt1) induced a robust increase in Wnt1 expression at both the mRNA (d, n = 3 per group) and protein levels (e, n = 3 per group) at 7 dpt, when we have previously observed the maximum transduction efficiency (b) and transgene expression (c). The existence of statistically significant differences between groups in e was assessed by One-way ANOVA followed by Bonferroni post-hoc test (*** represent p < 0.001 vs. NT; $$$ represent p < 0.001 vs. LV-GFP). It should be noted that Wnt1 is a soluble protein that acts in both autocrine and paracrine fashions and, thus, its correct secretion to the extracellular environment is a critical step to exert its multiple biological activities. In this regard, we found that the protein levels of Wnt1 were also strongly upregulated in the culture medium of cells transduced with LV-Wnt1 in comparison with those of NT and cells transduced with LV-GFP (f, n = 3 per group), demonstrating that Wnt1 was conveniently secreted to the extracellular space. We next aimed to evaluate whether overexpressed Wnt1 was able to activate the canonical Wnt/β-catenin signaling pathway which is the main Wnt signaling pathway that is activated by Wnt1. For this purpose, we determined that incubation of cell cultures with recombinant Wnt1 (100 and 200 ng/ml) (120-17, Peprotech) during 1 h led to the activation of the canonical Wnt/β-catenin signaling pathway (g) (n = 3 per group and time of incubation). The existence of significant differences between groups in g was assessed by Two-way ANOVA followed by Bonferroni post-hoc test (* = p < 0.05 vs. control). Finally, we found that, in comparison with cell cultures incubated with conditioned medium from cells transduced with LV-GFP (1:5) (CM LV-GFP), cell cultures incubated during 1 h with conditioned medium from cells cultures transduced with LV-Wnt1 (1:5) (CM LV-Wnt1) induced a similar activation of this signaling pathway than that observed in cell cultures incubated during 1 h with conditioned medium from cell cultures transduced with LV-GFP (1:5) and supplemented with 200 ng/ml of recombinant Wnt1 (CM LV-GFP+ Wnt1) (h, n = 3 per group), demonstrating that overexpressed Wnt1 is biologically active. The existence of significant differences between groups in h was assessed by One-way ANOVA followed by Bonferroni post-hoc test (* = p < 0.05 vs. CM LV-GFP). In all cases, data are presented as the mean + SEM. (TIF 4260 kb)
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Online Resource 3 Determination of the injection method of lentiviral vectors in vivo. This figure shows the results obtained from the experiments performed to establish the in vivo intraparenchymal injection method of the lentiviral vectors in the lesioned rat spinal cord. (a) and (b) shows schematic drawings from the two methods tested. In the first one (3ip), one injection per rostro-caudal level (stereotaxic coordinates: 0 mm lateral and 1 mm depth) was performed in three rostro-caudal levels corresponding to the lesion epicenter, 1.5 mm rostral and 1.5 mm caudal (a). In the second one (6ip), two injections per rostro-caudal level (stereotaxic coordinates: 0.6/-0.6 mm lateral and 1 mm depth) were performed in the same rostro-caudal levels detailed above (b). Evaluation of transduction at 7 days post-injury showed that both injection methods induced a similar transduction in longitudinal sections corresponding to the median line, where transduced green fluorescence protein (GFP)+ cells were observed surrounding the lesioned area, mainly rostral and caudal to the lesion epicenter, and to a lesser extent in the injury site (c-e) (non-injected (NT) group, n = 2; 3ip group, n = 3; 6ip group, n = 3) (arrows in d and e point the areas where GFP+ cells were observed). However, those animals that received 6 injections of lentiviral particles displayed a wider lateral distribution of transduced GFP+ cells than those that received 3 injections (f). As a consequence, we selected the second injection method for the subsequent experiments performed to evaluate the effects exerted by Wnt1 overexpression in the traumatically injured spinal cord. Scale bars in c-e represent 1 mm. (TIF 7420 kb)
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Online Resource 4 Evaluation of transduction at 7 days after SCI. This figure shows representative images from the evaluation of transduction at 7 days post-injury in those animals used to evaluate the effects of Wnt1 overexpression after SCI, which were injected with a lentiviral vector that only induce the expression of the green fluorescence protein (GFP) (LV-GFP control group) (n = 5) or with a lentiviral vector that induce the expression of both GFP and Wnt1 (LV-Wnt1 group) (n = 5). (a), shows a schematic drawing of a prototypical lesioned longitudinal spinal cord section and indicates the approximate rostro-caudal levels where the representative images were obtained. b, b1, c, c1, d, d1, e, e1, f and f1 shows representative images from the LV-GFP control group, while g, g1, h, h1, i, i1, j, j1, k and k1 show representative images from the LV-Wnt1 group. Squares in a-k indicate the areas shown in the corresponding higher magnification images. Arrows in a1-k1 point to the cells shown in the corresponding insets. Scale bars in a-k represent 500 µm, while those in a1-k1 and in their corresponding insets represent 100 and 20 µm, respectively. (TIF 17121 kb)
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Online Resource 5 Evaluation of transduction at 14 days after SCI. This figure shows representative images from the evaluation of transduction at 14 days post-injury in those animals used to evaluate the effects of Wnt1 overexpression after SCI, which were injected with a lentiviral vector that only induce the expression of the green fluorescence protein (GFP) (LV-GFP control group) (n = 5) or with a lentiviral vector that induce the expression of both GFP and Wnt1 (LV-Wnt1 group) (n = 5). (a), shows a schematic drawing of a prototypical lesioned longitudinal spinal cord section and indicates the approximate rostro-caudal levels where the representative images were obtained. b, b1, c, c1, d, d1, e, e1, f and f1 shows representative images from the LV-GFP control group, while g, g1, h, h1, i, i1, j, j1, k and k1 show representative images from the LV-Wnt1 group. Squares in a-k indicate the areas shown in the corresponding higher magnification images. Arrows in a1-k1 point to the cells shown in the corresponding insets. Scale bars in a-k represent 500 µm, while those in a1-k1 and in their corresponding insets represent 100 and 20 µm, respectively. (TIF 12872 kb)
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Online Resource 6 Evaluation of transduction at 126 days after SCI. This figure shows representative images from the evaluation of transduction at 126 days post-injury in those animals used to evaluate the effects of Wnt1 overexpression after SCI, which were injected with a lentiviral vector that only induce the expression of the green fluorescence protein (GFP) (LV-GFP control group) (n = 10) or with a lentiviral vector that induce the expression of both GFP and Wnt1 (LV-Wnt1 group) (n = 7). (a), shows a schematic drawing of a prototypical lesioned longitudinal spinal cord section and indicates the approximate rostro-caudal levels where the representative images were obtained. b, b1, c, c1, d, d1, e, e1, f and f1 shows representative images from the LV-GFP control group, while g, g1, h, h1, i, i1, j, j1, k and k1 show representative images from the LV-Wnt1 group. Squares in a-k indicate the areas shown in the corresponding higher magnification images. Arrows in a1-k1 point to the cells shown in the corresponding insets. Scale bars in a-k represent 500 µm, while those in a1-k1 and in their corresponding insets represent 100 and 20 µm, respectively. (TIF 17167 kb)
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Online Resource 7 Separate analysis of different individual aspects of locomotion analyzed in the 21-point BBB open-field test. This figure shows data obtained from the separate analysis of different individual aspects of locomotion such as coordination (a), paw positions (b), tail position (c), stepping (d), and toe clearance (e), which were evaluated using the 21-point BBB open-field test. The analysis was performed in lesioned animals injected with a lentiviral vector that only induce the expression of the green fluorescence protein (GFP) (LV-GFP control group) or with a lentiviral vector that induce the expression of both GFP and Wnt1 (LV-Wnt1 group) at 1, 3, 7, 14, 21, 35, 49, 63, 77, 91, 105 and 119 days post-injury (dpi). Data obtained from all animals used to evaluate the effects exerted by Wnt1 overexpression, which were sacrificed at 7 (LV-GFP group, n = 5; LV-Wnt1 group, n = 5), 14 (LV-GFP group, n = 5; LV-Wnt1 group, n = 5) and 126 dpi (LV-GFP group, n = 10; LV-Wnt1 group, n = 7), were included in the analysis. The existence of statistically significant differences between groups was assessed by Two-way ANOVA followed by Bonferroni post-hoc test. Data are presented as the mean + SEM. *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001 vs. LV-GFP. (TIF 4261 kb)
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González, P., González-Fernández, C., Campos-Martín, Y. et al. Frizzled 1 and Wnt1 as new potential therapeutic targets in the traumatically injured spinal cord. Cell. Mol. Life Sci. 77, 4631–4662 (2020). https://doi.org/10.1007/s00018-019-03427-4
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DOI: https://doi.org/10.1007/s00018-019-03427-4