Oral administration of the cannabigerol derivative VCE-003.2 promotes subventricular zone neurogenesis and protects against mutant huntingtin-induced neurodegeneration.

Experimental design for pharmacological manipulation and characterization of in vivo model of Huntington’s disease based on adeno-associated viral expression of mutant huntingtin exon1

VCE-003.2 exerts a pro-neurogenic effect in vitro. a-b Mouse embryonic stem cells (R1 line) were treated with Vehicle, VCE-003.2 (500 nM) during neural differentiation for 21 days. Representative immunofluorescence images and quantification of CTIP2- and doublecortin-positive cells is shown (n = 3). c Luciferase reporter assay of the A4-MARS sequence of the CTIP2 locus was performed 24 h after P19 cell transfection under neuronal differentiation conditions and subject to pharmacological regulation as above. d P19 neurosphere formation assay was performed by culturing the cells for 24 h in the presence of Vehicle or VCE-003.2. Statistics: Unpaired t-test vs Vehicle a * p < 0.05; t = 2.82; df = 4; 95% Confidence interval (CI) = 0.3149 to 36.85; R2 = 0.66. b * p < 0.05; t = 2.91; df = 4; 95% CI = 0.02 to 1.03; R2 = 0.67. c * p < 0.05; t = 2.87; df = 5; 95% CI = 0.09 to 1.72; R2 = 0.62. d ** < 0.01 t = 5.91; df = 362; 95% CI = 66.20 to 132.20; R2 = 0.08. Scale bar, 50 μm

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Oral administration of VCE-003.2 attenuates neuroinflammation and is neuroprotective in a viral model of mutant huntingtin expression

To evaluate the neuroprotective profile of oral VCE-003.2 administration, a viral model of HD was employed [20] in which wild-type mice were subjected to bilateral intrastriatal injection of adeno-associated virus (AAV) expressing either exon 1 of human pathogenic huntingtin with a polyQ tract of 94 CAG repeats, or a normal, non-pathogenic huntingtin with a polyQ tract of 16 CAG repeats (AAV-htt16Q and AAV-htt94Q, respectively), and treated with VCE-003.2 (10 mg/kg/day). Whereas expression of (exon1)-huntingtin-16Q during 14 days did not affect motor coordination, mutant huntingtin-94Q decreased RotaRod latency to fall (Fig. 3a). Motor impairment in htt94Q mice was accompanied by profound activation of microglial cells, as evidenced by Iba1 confocal immunofluorescence, in the area surrounding the infection site (Fig.

(Fig.3b).

3b). Mice treated with VCE-003.2 performed better in the RotaRod test than their vehicle-treated AAV-htt94Q counterparts, and showed reduced microglial activation (Fig.

(Fig.3a-b).

3a-b). Furthermore, VCE-003.2 administration prevented htt94Q-induced MSN degeneration as evidenced by DARPP-32 and NeuN immunofluorescence (Fig. 4a-b). These results are in line with previous findings on the neuroprotective ability of VCE-003.2 in toxin-based models of neurodegeneration [14, 21] and expand its potential clinical profile since oral administration was as effective as i.p. delivery. Likewise, oral VCE-003.2 administration was also neuroprotective and anti-inflammatory in the 3-NP model of striatal neurodegeneration (Additional file 6).

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Oral administration of VCE-003.2 promotes striatal neurogenesis

The neuroprotective action of oral VCE-003.2 and its pro-neurogenic impact in vitro prompted us to investigate if this compound could contribute to striatal neurorepair in vivo by promoting SVZ-derived neurogenesis. We analyzed the SVZ neural stem cell population, identified as double-labelled GFAP/Ki-67 cells, which represents the radial glia type B cell compartment. VCE-003.2 increased the number of proliferating GFAP-positive cells in AAV-htt94Q-injected mice, indicating its positive impact on NSC mobilization (Fig. 5). Next, SVZ-derived neural progenitors were identified by immunofluorescence against Ascl1 (a transcription factor characteristic of the transit amplifying progenitor subpopulation). Mutant huntingtin expression induced an increase in Ascl1+ cell number, and VCE-003.2 administration further promoted Ascl1-positive cell expansion in AAV-htt94Q-mice (Fig. 6). To determine the impact of VCE-003.2 on striatal neurogenesis we next evaluated doublecortin-positive neuroblasts after 30 days of AAV-mediated huntingtin expression and daily VCE-003.2 administration. AAV-htt94Q-induced injury resulted in a slight increase in neuroblast formation (Fig. 7a) and effective neurogenesis (BrdU+NeuN+ cells) (Fig.

(Fig.7b)

7b) as compared to AAV-htt16Q. Oral administration of VCE-003.2 was effective in promoting neurogenesis in AAV-htt16Q- and AAV-htt94Q-treated mice, both determined as increased doublecortin-expressing cells and double-positive NeuN/BrdU neurons (Fig.

(Fig.7a-b).

7a-b). Hence, oral VCE-003.2 administration is able to restore striatal neurogenesis affected by mutant huntingtin expression.

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Subventricular zone neural progenitor mobilization is increased by oral administration of VCE-003.2. Mice were analyzed 4 weeks after lesion induced by AAV-htt16Q and AAV-htt94Q bilateral injection and daily treatment with vehicle or VCE-003.2 (10 mg/kg). Confocal microscopy characterization of the SVZ was performed with GFAP, htt and Ki-67-specific antibodies in the indicated experimental groups. Proliferating SVZ-neural stem cells were quantified based on GFAP and Ki-67 immunofluorescence colocalization. AAV-htt16Q (Veh and VCE-003.2, n = 7 and 6, respectively) and AAV-htt94Q (Veh and VCE-003.2, n = 7 and 8, respectively). Statistics: One-way ANOVA. F = 5.78; R2 = 0.42. ##p < 0.01; q = 5.29 AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh. Scale bar, 100 μm

Subventricular zone neural progenitor mobilization is increased by oral administration of VCE-003.2. Mice were analyzed 4 weeks after lesion induced by AAV-htt16Q and AAV-htt94Q bilateral injection and daily treatment with vehicle or VCE-003.2 (10 mg/kg). Confocal microscopy characterization of the SVZ was performed with Ascl1-specific antibody in the indicated experimental groups. Quantification of transit amplifying progenitors labelled with Ascl1. AAV-htt-16Q (Vehicle and VCE-003.2, n = 5 each) and AAV-htt94Q (Vehicle and VCE-003.2, n = 5 each). Statistics: One-way ANOVA. F = 10.12; R2 = 0.65. ##p < 0.01; q = 4.25 AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh and **p < 0.01; q = 7.12 AAV-htt94Q VCE-003.2 vs. AAV-htt16Q Veh. Scale bar, 100 μm

Oral administration of VCE-003.2 exerts a pro-neurogenic action. Mice were analyzed 4 weeks after lesion induced by AAV-htt16Q and AAV-htt94Q bilateral injection and administered daily with vehicle or VCE-003.2 (10 mg/kg). a Confocal microscopy characterization of migrating neuroblasts identified with doublecortin antibody and quantification in the striatum of the indicated mice groups. b Effective neurogenesis was determined by quantification of double positive cells labelled with BrdU and NeuN antibodies. AAV-htt16Q Vehicle and VCE-003.2 (n = 7 and 5, respectively); AAV-htt94Q Vehicle and VCE-003.2 (n = 9 and 11, respectively). Statistics: One-way ANOVA. a F = 14.43; R2 = 0.69. ##p < 0.01; q = 6.95 AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh and **p < 0.01; q = 7.59 AAV-htt94Q VCE-003.2 vs. AAV-htt16Q Veh. b F = 13.99; R2 = 0.59. ##p < 0.01; q = 6.50 AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh and **p < 0.01; q = 7.59 AAV-htt94Q VCE-003.2 vs. AAV-htt16Q Veh. Scale bar, 100 μmGo to:

Discussion

VCE-003.2 is an aminoquinone derivative of CBG that has been designated by the FDA as an orphan drug for HD treatment (www.accessdata.fda.gov/scripts/opdlisting/oopd/detailedIndex.cfm?cfgridkey=623717). In the present study, we demonstrate the pro-neurogenic activity of this cannabinoid derivative in a preclinical model of HD that adds to its neuroprotective activity. Oral administration of VCE-003.2 exerted an anti-inflammatory and neuroprotective action in a HD experimental model. In addition, VCE-003.2 was able to augment mutant huntingtin-induced SVZ-derived neurogenesis, thus favoring neural stem cell mobilization (double-labelled GFAP+/Ki-67+ and Ascl1+ cells), neuronal differentiation (doublecortin+ cells) and effective neurogenesis (double-labelled Brdu+/NeuN+ cells). In agreement with previous studies, mutant huntingtin expression induced a pro-neurogenic response that was evident by the trend towards increased Ascl1+, Dcx+ and double-labelled BrdU+/NeuN+ cells. The molecular signals and mechanisms underlying injury-induced neurogenesis are diverse, and numerous therapeutic approaches aim to promote this endogenous process strategy [22]. Hence, the results obtained in this study support that striatal neurogenesis is improved by oral VCE-003.2 administration. This new mechanism of action of VCE-003.2 is relevant when considering its potential clinical applications, as it suggests that this molecule could constitute a disease-modifying drug rather than merely symptom-palliative. It is worth noting that the preclinical model of HD employed in this study is based on the expression of exon 1 of either mutant huntingtin or normal, non-pathogenic huntingtin. The validity of this approach for mechanistic and pharmacological investigations is demonstrated by the high number of studies using it [23]. However, improved animal models, for example based on the expression of full-length huntingtin, are likely to provide novel insights of additional aspects of HD.

The use of plant derived-cannabinoid ligands has been proposed to exert symptomatic relief of motor symptoms in neurodegenerative diseases, including HD-associated dystonia and chorea, levodopa-induced dyskinesia and tremor in Parkinson’s disease, and multiple sclerosis-associated spasticity [6, 24]. Despite the great interest in the therapeutic potential of cannabinoids in neurodegenerative diseases, the undesired side-effects of CB1 receptor agonists significantly limit their use in clinical practice. In addition, during HD progression presynaptic CB1 receptor levels decline very early, even at presymptomatic stages [7, 9, 10]. Hence, despite the fact that corticostriatal projections preserve CB1 receptor expression [17], the use of CB1 agonists alone may have a limited therapeutic window, and the development of new cannabinoid-derived molecules acting via different signaling mechanisms and with reduced undesired side-effects constitutes an attractive strategy to solve these limitations.

PPARγ receptors are required for appropriate neurogenesis as it controls neural progenitor cell proliferation and differentiation [25]. Noteworthy, the positive effect of PPARγ in neurogenesis, requires fine-tune regulation, as either its ablation or overactivation may hamper neuronal differentiation [26]. Under pathological conditions, for instance under an inflammatory insult, PPARγ activation can restore neurogenesis and cognitive impairment [27]. Similarly, PPARγ activation can restore neurogenesis in the OH-DA model of Parkinson’s disease [28] and prevent amyloid-beta-induced cell death of human neural stem cells [29]. In HD models, the administration of PPARγ agonists protects from striatal neurodegeneration, attenuates neuroinflammation and decreases oxidative damage [30–32], supporting PPARγ as a valid target for the management of HD.

CBD is the most widely investigated phytocannabinoid devoid of psychotomimetic actions, and whereas its precise molecular mechanism of action remains the subject of research, some CBD actions, including its protective effect in counteracting amyloid-induced inflammation and neurogenesis deficits, are mediated by PPARγ receptors [33]. Hence, CBD administration can restore hippocampal neurogenesis deficits induced by different nervous system insults such as amyloid-beta pathology, chronic unpredictable stress and aging [34]. Other plant-derived cannabinoids such as CBG and tetrahydrocannabinolic acid regulate PPARγ activity [15, 35] and, hence, constitute interesting template structures for the development of new molecules with improved selectivity, reduced side-effects and better pharmacokinetic profile. Different strategies have been applied to improve CBD pharmacological properties. Fluorinated derivatives or quinone modifications have successfully generated new cannabinoid molecules with better translational perspectives (e.g., HUF-101, VCE-004). Thus, HUF-101 possesses improved anxiolytic, antidepressant and antipsychotic properties compared to the original CBD molecule [36]. Regarding CBG, its aminoquinone derivative VCE-003.2 has demonstrated efficacy as a neuroprotective molecule in HD [14], Parkinson’s disease [21] and amyotrophic lateral sclerosis models [37]. Noteworthy, the VCE-003.2 compound is devoid of the side effects seen with full PPARγ-agonists and, hence, does not interfere with osteoblast differentiation and is less adipogenic [14].

Cell replacement therapy has demonstrated efficacy in HD experimental models and in preliminary studies in patients [38]. Exogenous cell grafts of either fetal tissue or stem-cell derived neurons are able to survive in the striatum, as well as integrate and ameliorate motor symptoms and survival, however they face various challenges that impede their development for disease-modifying strategy. Considering that in HD postmortem tissue an increased SVZ cell proliferation and neurogenesis is evident [39], promoting endogenous SVZ-derived striatal neurogenesis constitutes an alternative approach of interest. Promoting SVZ neurogenesis by gene therapy-mediated delivery of BDNF and noggin successfully delayed progression of the disease in the R6/2 mouse model [40, 41], and the same approach induced neurogenesis in adult squirrel monkeys [40]. These findings evidence that, even if endogenous newly-born neurons express mutant huntingtin, neurogenesis is effective and able to counteract, at least in part, neurodegeneration-evoked symptoms. In this scenario, pharmacological promotion of adult neurogenesis by cannabinoids [42, 43] may constitute a plausible alternative to gene therapy. In addition, cannabinoids could be used to promote ES-derived striatal neurogenesis ex vivo for mechanistic studies or cell replacement therapies.

In summary, our findings demonstrate that oral administration of VCE-003.2 exerts a neuroprotective action in a striatal neurodegeneration model that is accompanied by an improved endogenous neurogenic response. The capability of VCE-003.2 to increase effective neurogenesis suggests that this CBG derivative may possess the ability to act as a disease-modifying drug rather than only a symptomatic reliever.

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Conclusions

Oral administration of the cannabigerol derivative VCE-003.2 is neuroprotective and improves subventricular zone-derived neurogenesis in response to mutant huntingtin-induced neurodegeneration. These findings are relevant in the search for novel therapeutic strategies against Huntington’s disease progression, considering the lack of undesired side actions of this novel cannabinoid-derived molecule and good bioavailability upon oral administration.

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Additional files

Additional file 1:(373K, pdf)

Microsomal Metabolic Stability. Pooled human liver microsomes (final protein concentration 0.5 mg/mL), 0.1 M phosphate buffer pH 7.4 and the test compounds (VCE-003.2, CBG, verapamil and dextromethorphan) were pre-incubated at 37 °C prior to the addition of 1 mM NADPH to initiate the reaction. The final incubation volume was 25 μL. Each compound was incubated for 0, 5, 15, 30 and 45 min. The control (minus NADPH) was incubated for 45 min only. The reactions were stopped by the addition of 50 μL methanol containing internal standard at the appropriate time points. The incubation plates were centrifuged at 2500 rpm for 20 min at 4 °C to precipitate the protein. Following protein precipitation, the sample supernatants were analyzed using LC-MS/MS. From a plot of ln peak area ratio (compound peak area/internal standard peak area) against time, the gradient of the line was determined. Subsequently, half-life and intrinsic clearance was calculated using the equations below: Elimination rate constant (k) = (− gradient). Half-life (t1/2) (min) =

0.693

k

. Intrinsic Clearance (CLint) (μL/min/mg protein) =

V×0.693

t

1/2

. where V=Incubation volume mL/mg microsomal protein. (PDF 373 kb)

Additional file 2:(324K, pdf)

Cytochrome P450 Inhibition (IC50 Determination). CYP1A Inhibition. VCE-003.2 (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration = 0.3%) was incubated with human liver microsomes (0.25 mg/mL) and NADPH (1 mM) in the presence of the probe substrate ethoxyresorufin (0.5 μM) for 5 min at 37 °C. The selective CYP1A inhibitor, alpha-naphthoflavone, was screened alongside the test compounds as a positive control. CYP2B6 Inhibition. VCE-003.2 (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration = 0.3%) was incubated with human liver microsomes (0.1 mg/mL) and NADPH (1 mM) in the presence of the probe substrate bupropion (110 μM) for 5 min at 37 °C. The selective CYP2B6 inhibitor, ticlopidine, was screened alongside the test compounds as a positive control. CYP2C8 Inhibition. VCE-003.2 (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration = 0.3%) was incubated with human liver microsomes (0.25 mg/mL) and NADPH (1 mM) in the presence of the probe substrate paclitaxel (7.5 μM) for 30 min at 37 °C. The selective CYP2C8 inhibitor, montelukast, was screened alongside the test compounds as a positive control. CYP2C9 Inhibition. VCE-003.2 (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration = 0.3%) was incubated with human liver microsomes (1 mg/mL) and NADPH (1 mM) in the presence of the probe substrate tolbutamide (120 μM) for 60 min at 37 °C. The selective CYP2C9 inhibitor, sulphaphenazole, was screened alongside the test compounds as a positive control. CYP2C19 Inhibition. VCE-003.2 (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration = 0.3%) was incubated with human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence of the probe substrate mephenytoin (25 μM) for 60 min at 37 °C. The selective CYP2C19 inhibitor, tranylcypromine, was screened alongside the test compounds as a positive control. CYP2D6 Inhibition. VCE-003.2 (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration = 0.3%) was incubated with human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence of the probe substrate dextromethorphan (5 μM) for 5 min at 37 °C. The selective CYP2D6 inhibitor, quinidine, was screened alongside the test compounds as a positive control. CYP3A4 Inhibition. VCE-003.2 (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration = 0.3%) was incubated with human liver microsomes (0.1 mg/mL) and NADPH (1 mM) in the presence of the probe substrate midazolam (2.5 μM) for 5 min at 37 °C. The selective CYP3A4 inhibitor, ketoconazole, was screened alongside the test compounds as a positive control. CYP3A4 Inhibition. VCE-003.2 (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration = 0.3%) was incubated with human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence of the probe substrate testosterone (50 μM) for 5 min at 37 °C. The selective CYP3A4 inhibitor, ketoconazole, was screened alongside the test compounds as a positive control. For the CYP1A incubations, the reactions were terminated by methanol, and the formation of the metabolite, resorufin, was monitored by fluorescence (excitation wavelength = 535 nm, emission wavelength = 595 nm). For the CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 incubations, the reactions were terminated by methanol. The samples were centrifuged, and the supernatants combined for the simultaneous analysis of 4-hydroxytolbutamide, 4-hydroxymephenytoin, dextrorphan, and 1-hydroxymidazolam by LC-MS/MS. Hydroxybupropion, 6α-hydroxypaclitaxel and 6ß-hydroxytestosterone were analyzed separately by LC-MS/MS. A decrease in the formation of the metabolites compared to vehicle control was used to calculate IC50 values. (PDF 323 kb)

Additional file 3:(322K, pdf)

hERG Channel Inhibition (IC50 Determination). The experiments were performed on an IonWorks™ HT instrument (Molecular Devices Corporation), which automatically performs electrophysiology measurements in 48 single cells simultaneously in a specialized 384-well plate (PatchPlate™). The cells used were Chinese hamster ovary (CHO) cells stably transfected with hERG (cell-line obtained from Cytomyx, UK). A single-cell suspension was prepared in extracellular solution (Dulbecco’s phosphate buffered saline with calcium and magnesium pH 7–7.2) and aliquots added to each well of a PatchPlate™. Cells were positioned over a small hole at the bottom of each well by applying a vacuum beneath the plate to form an electrical seal. The resistance of each seal was measured via a common ground-electrode in the intracellular compartment and individual electrodes placed into each of the upper wells. Electrical access to the cell was achieved by circulating a perforating agent, amphotericin, underneath the PatchPlate™ and then measuring the pre-compound hERG current. An electrode is positioned in the extracellular compartment and a holding potential of − 80 mV applied for 15 s. The hERG channels were then activated by applying a depolarizing step to + 40 mV for 5 s and then clamped at − 50 mV for 4 s to elicit the hERG tail current, before returning to − 80 mV for 0.3 s. VCE-003.2 were added to the upper wells of the PatchPlate™. Solutions were prepared by diluting 10 mM DMSO solutions of the test compound into extracellular buffer such that the final concentrations tested are 0.008, 0.04, 0.2, 1, 5 and 25 μM (final DMSO concentration 0.25%). Quinidine, an established hERG inhibitor, was included as a positive control and buffer containing 0.25% DMSO was included as a negative control. Post-compound currents were then expressed as a percentage of pre-compound currents and plotted against concentration for each compound. Where concentration-dependent inhibition is observed, the data are fitted and IC50 values were calculated. (PDF 322 kb)

Additional file 4:(568K, pdf)

AMES Data Summary. AMES Experimental Procedure. Approximately ten million bacteria are exposed in triplicate to VCE-003-.2 (7.8, 15.6, 31.3, 62.5, 125 and 250 μg/ml), a negative control (vehicle) and a positive control for 90 min in medium containing a low concentration of histidine (sufficient for about 2 doublings). The cultures were then diluted into indicator medium lacking histidine and dispensed into 48 wells of a 384 well plate (micro-plate format, MPF). The plate was incubated for 48 h at 37 °C, and the number of wells showing growth were counted and compared to the vehicle control. An increase in the number of colonies of at least two-fold over baseline (mean + SD of the vehicle control) and a dose response indicates a positive response. An unpaired, one-sided Student’s T-test was used to identify conditions that are significantly different from the vehicle control. Where indicated, S9 fraction from the livers of Aroclor 1254-treated rats was included in the incubation at a final concentration of 4.5%. An NADPH-regenerating system is also included to ensure a steady supply of reducing equivalents. The strains used in this study were S. typhimurium TA98 (hisD3052, rfa, uvrB/pKM10 for detection of frame-shift mutations) and S. typhimurium TA100 (hisG45, rfa, uvrB/pKM101 for detection of base-pair substitutions). VCE-003.2 was assessed for its mutagenic potential in the AMES reverse mutation assay. This test was performed in the absence and presence of S9 metabolic activation. VCE-003.2 was found to be negative for genotoxicity in this AMES study. The positive controls all behaved as expected. (PDF 568 kb)

Additional file 5:(338K, pdf)

Effect of oral VCE-003.2 on plasmatic biomarkers. Plasma samples from the indicated groups of animals (n = 6) were pooled and subjected to mouse cytokine array (ARY028; R&D Systems) and mouse adipokine array (ARY013; R&D Systems) analysis. The relative expression of the indicated biomarkers is shown. (PDF 338 kb)

Additional file 6:(325K, pdf)

3-Nitroproprionic acid model of striatal neurodegeneration. 16-week- old C57BL/6 N male mice (Harlan Ibérica, Barcelona, Spain) were subjected to seven intraperitoneal (i.p.) injections of 50 mg/kg 3-NP (one injection each every 12 h prepared in phosphate-buffered saline (PBS)]. 3-NP-treated animals and their respective non-lesioned controls (injected with PBS) were used for pharmacological studies with VCE-003.2. Treatments consisted of oral gavage every 24 h with VCE-003.2 at a dose of 10 mg/kg using sesame oil as vehicle. 12 h after the last injection of 3-NP motor activity, hindlimb clasping and kyphosis were evaluated as previously described [12]. Animals were euthanized at the indicated time after huntingtin-AAV infection or 12 h after the last injection of 3NP and their brains removed. Statistical analysis: One-way ANOVA followed by the Tuckey’s post hoc test was used to determine the statistical significance. All the in vivo data were expressed as mean ± SEM. Kruskal-Wallis test was used to determine the statistical in the case of non-parametric analysis. a) Hindlimb Clasping (F = 8.069; p = 0.0047) post hoc test: p = 0.0036 3NP vs Veh; Locomotor activity (F = 18.62; p = 0.0001) post hoc test p < 0.0001 3NP vs Veh, p = 0.0027 3NP + VCE-003.2 vs 3NP; Kyphosis (F = 28.24 p < 0.0001) post hoc test: p < 0.0001 3NP vs Veh, p < 0.0001 3NP + VCE-003.2 vs 3NP. b) TNF-α (F = 18.17 p = 0.0028) post hoc test: p = 0.0027 3NP vs Veh, p = 0.0138 3NP + VCE-003.2 vs 3NP. IL-6 (Kruskal-Wallis statistic = 6.880 p = 0.0071) post hoc test: p = 0.0265 3NP vs Veh. c) Average number of neurons per field (F = 15.69 p = 0.0012) post hoc test: p = 0.0011 3NP vs Veh, p = 0.0086 3NP + VCE-003.2 vs 3NP; Number of Iba1+ cells (F = 10.82 p = 0.0040) post hoc test: p = 0.0101 3NP vs Veh, p = 0.0059 3NP + VCE-003.2 vs 3NP. (PDF 325 kb)

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Acknowledgements

We are thankful to J. Díaz-Alonso and L. Bellochio for intellectual input to the project, assistance designing and preparing AAV constructs and particles; E. Resel for grant administration and the rest of Galve-Roperh, Guzmán and Muñoz laboratories for enriching scientific environment.

Funding

This work was supported by the MINECO grant RTC-2015-3364 to EM and IGR cofounded by the European Development Regional Fund in the Framework of the Operative Program “Reinforcement of research, technological development and innovation”. IGR was also supported by grant PI15–00310 and PI18–00941 cofinanced by the European Development Regional Fund “A way to achieve Europe” and EM by the MINECO grant SAF2017–87701-R. JA and JPL were supported by FPI and FPU program fellowship (Ministerio de Educación, Cultura y Deporte) and DGR by Fundación Tatiana de Guzmán el Bueno. BP is a predoctoral fellow supported by the i-PFIS program, Instituto de Salud Carlos III (IFI15/00022; European Social Fund “Investing in your future”).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Abbreviations