DU145 cells expressing endogenous GPR55 were stimulated with either VCE-006.1 (5 µM), LPI (2 µM), or a combination of both for 30 min. Then, cells were washed with phosphate-buffered saline (PBS) and proteins extracted in lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% glycerol, and 1% NP-40) supplemented with 10 mM NaF, 1 mM Na₃VO₄, 10 μg/mL leupeptin, 1 μg/mL pepstatin and aprotinin, and 1 μL/mL saturated PMSF. Thirty μg of proteins were boiled at 95 °C in Laemmli buffer and electrophoresed in 10% SDS/PAGE gels. Total ERK was used as a loading control. Separated proteins were transferred to PVDF membranes, and after blocking with non-fat milk in TBST buffer, primary antibodies were added. The washed membranes were incubated with appropriate secondary antibodies coupled to horseradish peroxidase that were detected by an enhanced chemiluminescence system (USB). Antibodies against total and phospho-ERK1/2 were purchased from Sigma-Aldrich (Madrid, Spain).

U937 cells expressing endogenous GPR55 receptor were incubated for 1 h at 37 °C in Tyrode’s salt solution (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 0.4 mM NaH₂PO₄, 12.0 mM NaHCO₃, and 5.6 mM D-glucose) containing 5 µM Indo1-AM (Invitrogen, Waltham, MA, USA) for 30 min at 37 °C in the dark. Cells were then harvested, washed three times with buffer to remove extracellular Indo1 dye, readjusted to 10⁶ cells/mL in the appropriate buffer, and analyzed in a spectrofluorometer operated in the ratio mode (model F-2500; Hitachi Ltd., Tokyo, Japan) under continuous stirring and at a constant temperature of 37 °C using a water-jacketed device. After a 5-min accommodation to equilibrate temperatures, samples were excited at 338 nm, and emission was collected at 405 and 485 nm, corresponding to the fluorescence emitted by Ca²⁺ bound and -free Indo1, respectively. The cells were stimulated with either LPI or VCE-006.1, and maximal ratio values for calculations were determined by the addition at the end of the measurements of 10 µM ionomycin. [Ca²⁺]i changes are presented as changes in the ratio of bound to free calcium (340 nm/380 nm).

The determination of GPR55 activity was carried out using the HEK293T-GPR55 cells stably transfected with the human GPR55 cDNA. Briefly, HEK293T-GPR55 cells were transiently transfected with 0.2 µg of the reporter plasmid CRE-Luc that contains six consensus cAMP-responsive elements (CRE) linked to the firefly luciferase reporter gene using Roti©-Fect (Carl Roth, Karlsruhe, Germany). Transfected cells were treated with either VCE-006.1, LPI, or a combination of both. After 6 h of stimulation, cells were washed twice with PBS 1× and lysed in 100 µL lysis buffer containing 25 mM Tris-phosphate (pH 7.8), 8 mM MgCl₂, 1 mM DTT, 1% Triton X-100, and 7% glycerol for 15 min at room temperature in a horizontal shaker. Luciferase activity was measured using a TriStar2 Berthold/LB942 multimode reader (Berthold Technologies, Bad Wildbad, Germany) following the instructions of the luciferase assay kit (Promega, Madison, WI, USA). The RLUs (relative light units) were calculated, and the results were expressed as fold induction over unstimulated cells. The experiment was performed 5–6 times.

Male C57BL/6 mice were housed in a room with a controlled photoperiod (08:00–20:00 light) and temperature (22 ± 1 °C). They had free access to standard food and water and were used at adult age (3–4 month-old; 25–30 g weight). All experiments were conducted according to national and European guidelines (directive 2010/63/EU), as well as conformed to ARRIVE guidelines and approved by the “Comité de Experimentación Animal” of our university (PROEX: 056/19).

In a first experiment, male C57BL/6 mice were subjected to stereotaxic unilateral application of 6-hydroxydopamine (6-OHDA) or saline [24,75]. To do that, mice were anesthetized (ketamine 40 mg/kg + xylazine 4 mg/kg, i.p.) 30 min after pretreatment with desipramine (25 mg/kg, i.p.), and then 6-OHDA free base (2 μL at a concentration of 2 μg/μL saline in 0.2% ascorbate to avoid oxidation) or saline (for control mice) were injected stereotaxically into the right striatum at a rate of 0.5 μL/min, using the following coordinates: + 0.4 mm AP, −1.8 mm ML and −3.5 mm DV, as described in [75]. Once injected, the needle was left in place for 5 min before being slowly withdrawn, thus avoiding reflux and a rapid increase in intracranial pressure. Control animals were sham-operated and injected with 2 μL of saline using the same coordinates. The lesions were generated using unilateral injection, the advantage of which is that contralateral structures serve as controls for the different analyses. After the application of 6-OHDA or saline, animals were subjected to a daily treatment with VCE-006.1 (20 mg/kg, i.p.) or vehicle (cremophor-saline, 1:18) for two weeks, at the end of which (24 h after the last injection), they were analyzed in the pole test and the cylinder rearing test just before being killed by rapid and careful decapitation and their brains rapidly removed. Brains were divided coronally into two parts, following the procedure described by Palkovits and Brownstein [76]. The anterior halves were used to dissect the striatum (both ipsilateral and contralateral sides separately), and tissues were rapidly frozen by immersion in cold 2-methylbutane and stored at −80 °C for qPCR analysis. The posterior halves containing the midbrains were fixed for one day at 4 °C in fresh 4% paraformaldehyde prepared in 0.1 M PBS, pH 7.4. Samples were cryoprotected by immersion in a 30% sucrose solution for a further day, and finally stored at −80 °C for immunohistochemical analysis in the substantia nigra.

In a second experiment, mice were anesthetized (ketamine 40 mg/kg + xylazine 4 mg/kg, i.p.) and subjected to unilateral injections of S. Minnesota LPS (Sigma-Aldrich, Madrid, Spain) into two points of the right striatum following the procedure developed by Hunter et al. [77]. We used the following stereotaxic coordinates from bregma: + 1.1 mm AP, −1.8 mm ML, and −3.5 mm DV, as well as −0.3 mm AP, −2.5 mm ML, and −3.2 mm DV (see details in [77]). At each intrastriatal coordinate, 5 μg of LPS in a volume of 1 μL of saline was injected slowly (0.5 μL/30 s), and the needle was again left in place for 5 min before being slowly withdrawn. This avoids generating reflux and a rapid increase in intracranial pressure. Control animals were sham-operated and injected with 1 μL of saline using the same coordinates. Again, the lesions were generated using unilateral administration, the advantage of which is that contralateral structures serve as controls for the different analyses. After the application of LPS or saline, animals were subjected to a daily treatment with VCE-006.1 (20 mg/kg, i.p.) or vehicle (cremophor-saline, 1:18) for two weeks, at the end of which (24 h after the last injection), they were analyzed in the pole test and the cylinder rearing test just before being killed by rapid and careful decapitation and their brains rapidly removed and processed as described before 6-OHDA-lesioned mice.

In a third experiment, cultures of SH-SY5Y neuronal cell line (kindly provided by Dr. Ana Martínez, CIB-CSIC, Madrid, Spain) were used to induce cell death with 6-OHDA and to investigate in vitro the possible cytoprotective effects of VCE-006.1, following a procedure described previously [78]. To this end, SH-SY5Y cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS), 2 mM Ultraglutamine, and 1% antibiotics (Lonza, Verviers, Belgium) under a humidified 5% CO₂ atmosphere at 37 °C. For cytotoxicity experiments, cells were seeded at 60,000 cells/well in 96-well plates and maintained under a humidified atmosphere (5% CO₂) at 37 °C overnight. For experiments, 24 h after seeding, cells were treated with the vehicle (DMEM + 0.1% DMSO) or with five different concentrations of VCE-006.1 (0.5, 1, 2, 5, 10, and 20 μM; selected according to [65]), 60 min before being exposed to 200 µM 6-OHDA (or saline) following our previously published studies with different concentrations of 6-OHDA in these cells [43,44]. Cells were incubated 24 h before the neuronal death was analyzed with the MTT assay (Panreac AppliChem., Barcelona, Spain). Data of cell viability were normalized in relation to the corresponding control group (cells exposed to vehicles for 6-OHDA and VCE-006.1).

In a fourth experiment, cultured BV-2 cells were maintained in DMEM (Lonza, Verviers, Belgium) supplemented with 10% FBS (Sigma-Aldrich, Madrid, Spain), 2 mM Ultraglutamine, and antibiotics (Lonza, Verviers, Belgium) in a humidified atmosphere of 5% CO₂ at 37 °C. Cells were plated at a density of 45 × 10⁴ cells per well in 12-well culture plates and incubated in DMEM with a reduction of FBS to 1%. Three hours later, cells were treated with 0.5 μg/mL LPS (from Escherichia coli 055:B5, Sigma-Aldrich, Madrid, Spain), alone or in combination with VCE-006.1, used at a concentration of 1 μM, and added 1 h before LPS. Twenty hours after the addition of LPS, media were removed, and cell pellets were collected for analyzing mRNA levels of GPR55, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) using qPCR analysis.

Experiments were conducted with two mouse colonies: (i) B6SJL-Tg(SOD1*G93A)1Gur/J transgenic (mSOD1 mice) and non-transgenic littermate sibling mice bred in our animal facilities from initial breeders provided by Dr. Rosario Osta (LagenBio-Ingen, University of Zaragoza, Spain), and (ii) Prp-hTDP-43(A315T) transgenic and non-transgenic littermate sibling mice bred in our animal facilities from initial breeders purchased from Jackson Laboratories (Bar Harbor, ME, USA). In both cases, animals were subjected to genotyping for identifying the presence or absence of the transgene containing the SOD-1 or the TDP-43 mutation (see details in [30,45], respectively). As in PD experiments, all animals were housed in a room with controlled photoperiod (08:00–20:00 light) and temperature (22 ± 1 °C) with free access to standard food or, in the case of TDP-43 transgenic mice, to a high-fat jelly diet (DietGel Boost, ClearH20, Portland, ME, USA) [79], and water. All experiments were conducted according to local and European rules (directive 2010/63/EU), as well as conformed to ARRIVE guidelines. They were approved by the ethical committees of our university and the regulatory institution (PROEX: 056/19).

In a first experiment, wild-type and mSOD-1 transgenic mice were identified by numbered ear marks, and prior to the start of the different experiments, they were randomly allocated to the different treatment groups. We treated B6SJL-Tg(SOD-1*G93A)1Gur/J transgenic male mice with VCE-006.1, synthesized as previously described [65], and administered i.p. to mice at the dose of 20 mg/kg. Additional transgenic mice, as well as wild-type animals, were treated with vehicle (cremophor-saline, 1:18). The treatment was initiated when animals were 63 days old and prolonged daily up to the age of 18 weeks (125 days of age). During this period, animals were weighed every day and subjected to several neurological analyses and behavioral tests at specific time points. Twenty-four hours after the last injection, animals were euthanized by rapid decapitation, and their spinal cords were dissected and removed. The spinal cords (lumbar level) to be used for histology were fixed for one day at 4 °C in 4% formaldehyde solution in PBS. Samples were then cryoprotected by immersion in a 30% sucrose solution for a further day, and finally stored at −80 °C for Nissl staining and immunohistochemical analysis. The spinal samples (also lumbar area) to be used for qPCR analyses were collected and rapidly frozen by immersion in cold 2-methylbutane and stored at −80 °C for qPCR analysis.

In a second experiment, we treated non-transgenic and Prp-hTDP-43(A315T) transgenic male mice with VCE-006.1, again synthesized as previously described [65] and administered i.p. to mice at the dose of 20 mg/kg. Additional transgenic mice, as well as wild-type animals, were treated with vehicle (cremophor-saline, 1:18). The treatment was initiated when animals were 65 days old and prolonged daily up to the age of 95 days, the same treatment window used in our previous study [30], which extends from early symptomatic phases (around the 9th week of age) up to an advanced stage (around the 13th week of age). Animal weight was logged daily. Weight loss of 20% was established as the human end-point. Rotarod performance and clasping reflex to detect dystonia were recorded weekly during the 4 weeks of the treatment period (including a recording just before the first injection). All animals were euthanized by rapid decapitation at the age of 95 days, at least 24 h after the last administration. Their spinal cords were rapidly removed and processed as described for mSOD-1 mice.

Mice were placed head-upward on the top of a vertical rough-surfaced pole (diameter 8 mm; height 55 cm), and the time until animals descended to the floor was recorded with a maximum duration of 120 s. When the mouse was not able to turn downward and instead dropped from the pole, the time was taken as 120 s (default value) (see details in [44]).

Given that the lesion was unilateral in the experiment with 6-OHDA or LPS, this test attempted to quantify the degree of forepaw (ipsilateral, contralateral, or both) preference for wall contacts after placing the mouse in a methacrylate transparent cylinder (diameter: 15.5 cm; height: 12.7 cm [80]). Each score was made out of a 3 min trial with a minimum of 4 wall contacts.

Mice were evaluated for neurological decline using a numerical scale published previously [45]. The scale ranged from 0 to 15 distributed in three sub-scales (0–5) concentrated on ambulation, strength analysis, and hind-foot reflex test. A final score of 0 corresponds to animals that are not symptomatic, whereas a score of 15 reflects a state of total functional loss in hindlimbs and postural control. The assessment of ambulation was carried out by placing the animal inside a corridor (10 × 10 × 80 cm) while evaluating postural control and the way in which hindlimbs were leaned during motion. The strength test evaluated the animal’s ability to drag and offer resistance when the tail was pulled softly to the opposite direction in which the animal moves. Lastly, the hind-foot reflex test evaluated the stiffness of the limbs and their coordination when the mouse was suspended by the tail 10 cm over the surface. The final score was calculated from the sum of values reached in each sub-scale.

Mice were evaluated for possible motor weakness using the rotarod test, using an LE8200 device (Panlab, Barcelona, Spain). Mice were exposed to a period of acclimation and training (first session: 0 r.p.m. for 30 s; second and third sessions: 4 r.p.m. for 60 s, with periods of 10 min between sessions), followed 30 min later by the assay. Mice were placed into the apparatus, and the rotational speed was increased from 4 to 40 r.p.m. over a period of 300 s to measure the time to fall off. Mice were tested for 3 consecutive trials with a rest period of approximately 15 min between trials, and the mean of the 3 trials was calculated.

Dystonia was evaluated by picking up the mouse by the base of the tail for 30 s so that the mouse was facing downwards away from any object. The position of the hindlimbs was observed and scored following the scale reported by Guyenet et al. [81]. Animals were scored as follows: 0 if the hindlimbs were consistently extended away from the abdomen; 1 if one hindlimb was retracted toward the abdomen; 2 if both hindlimbs were partially retracted toward the abdomen; 3 if both hindlimbs were entirely retracted and touching the abdomen. Mice were tested for three consecutive trials, and the mean clasping score of the three trials was calculated.

The latency of mice to fall from a wire cage top, which was slowly inverted and suspended at approximately 30 cm to the floor, was also used as an index of motor weakness. The test was repeated three times to obtain the mean value of the three trials.

In the PD experiment, brains were sliced in coronal sections (containing the substantia nigra) in a cryostat (30 µm thick) and collected on antifreeze solution (glycerol/ethylene glycol/PBS; 2:3:5) and stored at −20 °C until used for immunostaining. In the ALS experiment, fixed spinal cords were sliced with a cryostat at the lumbar level (L4-L6) to obtain coronal sections (20 μm thick) that were collected on gelatin-coated slides. Sections were used for procedures of Nissl-staining and immunostaining.

Brain sections containing the substantia nigra were mounted on gelatin-coated slides and, once adhered, washed in 0.1 M potassium PBS (KPBS) at pH 7.4. Endogenous peroxidase was blocked by 30 min incubation at room temperature in peroxidase blocking solution (Dako Cytomation, Glostrup, Denmark). After several washes with KPBS, sections were incubated overnight at room temperature with the following polyclonal antibodies: (i) rabbit anti-tyrosine hydroxylase (TH) (Chemicon-Millipore, Temecula, CA, USA) used at 1/200; (ii) rat anti-mouse Cd68 antibody (AbD Serotec, Oxford, UK) used at 1/200; or (iii) rabbit anti-mouse GFAP antibody (Dako Cytomation, Glostrup, Denmark) used at 1/200. In the case of LAMP-1 immunostaining, we used the hybridoma monoclonal rat anti-mouse LAMP-1 antibody 1D4B, which was deposited by Dr. J. Thomas in the Developmental Studies Hybridoma Bank (DSHB; Hybridoma Product 1D4B), created by the NICHD (NIH, Bethesda, MD, USA) and maintained at The University of Iowa, Department of Biology, Iowa City, IA, USA. Dilutions were carried out in KPBS containing 2% bovine serum albumin and 0.1% Triton X-100 (Sigma Chem., Madrid, Spain). After incubation, sections were washed in KPBS, followed by incubation with the corresponding biotinylated secondary antibody (1/200) (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine substrate–chromogen system (Dako Cytomation, Glostrup, Denmark) were used to obtain a visible reaction product. Negative control sections were obtained using the same protocol with omission of the primary antibody. A Leica DMRB microscope and a DFC300FX camera (Leica, Wetzlar, Germany) were used for the observation and photography of the slides, respectively. For quantification of TH, LAMP-1, GFAP, or Cd68 immunostaining in the substantia nigra, we used the NIH Image Processing and Analysis software (ImageJ; NIH, Bethesda, MD, USA) using 4–5 sections, separated approximately by 200 µm, and observed with 5x-20x objectives depending on the method and the brain area under quantification. In all sections, the same area of the substantia nigra pars compacta was analyzed. Analyses were always conducted by experimenters who were blinded to all animal characteristics. Data were expressed as a percentage of immunostaining intensity in the ipsilateral (lesioned) side over the contralateral (non-lesioned) side.

Slices were used for Nissl staining using cresyl violet, as previously described [82], which permitted us to determine the effects of particular treatments on cell numbers. A Leica DMRB microscope (Leica, Wetzlar, Germany) and a DFC300Fx camera (Leica) were used to study and photograph the tissue, respectively. To count the number of Nissl-stained motor neurons (>400 μm²) in the ventral horn, high-resolution photomicrographs were taken with a 10× objective under the same conditions of light, brightness, and contrast. Counting was carried out with ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/, 1997–2012). At least 6 images per animal were analyzed to establish the mean of all animals studied in each group. Analyses were always conducted by experimenters who were blinded to all animal characteristics. In all analyses, data were transformed to the percentage over the mean obtained in the wild-type group for each parameter.

Spinal slices were used for the detection and quantification of GFAP or Iba-1 immunofluorescence. After preincubation for 1 h with Tris-buffered saline with 0.1% Triton X-100 (pH 7.5), sections were sequentially incubated overnight at 4 °C with the following polyclonal antibodies: (i) anti-Iba-1 (Wako Chemicals, Richmond, VI, USA) used at 1:500; or (ii) anti-GFAP (Dako Cytomation, Glostrup, Denmark) used at 1:200, followed by washing in Tris-buffered saline and a new incubation (at 37 °C for 2 h) with an anti-rabbit secondary antibody conjugated with Alexa 488 (Invitrogen, Carlsbad, CA, USA). A DMRB microscope and a DFC300Fx camera (Leica, Wetzlar, Germany) were used for slide observation and photography. The mean density of immunolabelling was measured in the selected areas. Again, all data were transformed to the percentage over the mean obtained in the wild-type group for each parameter.