Altered striatal endocannabinoid signaling in a transgenic mouse model of spinocerebellar ataxia type-3

Spinocerebellar ataxia type-3 (SCA-3) is the most prevalent autosomal dominant inherited ataxia. We recently found that the endocannabinoid system is altered in the post-mortem cerebellum of SCA-3 patients, and similar results were also found in the cerebellar and brainstem nuclei of a SCA-3 transgenic mouse model. Given that the neuropathology of SCA-3 is not restricted to these two brain regions but rather, it is also evident in other structures (e.g., the basal ganglia), we studied the possible changes to endocannabinoid signaling in the striatum of these transgenic mice. SCA-3 mutant mice suffer defects in motor coordination, balance and they have an abnormal gait, reflecting a cerebellar/brainstem neuropathology. However, they also show dystonia-like behavior (limb clasping) that may be related to the malfunction/deterioration of specific neurons in the striatum. Indeed, we found a loss of striatal projecting neurons in SCA-3 mutant mice, accompanied by a reduction in glial glutamate transporters that could potentially aggravate excitotoxic damage. In terms of endocannabinoid signaling, no changes in CB2 receptors were evident, yet an important reduction in CB1 receptors was detected by qPCR and immunostaining. The reduction in CB1 receptors was presumed to occur in striatal afferent and efferent neurons, also potentially aggravating excitotoxicity. We also measured the endocannabinoid lipids in the striatum and despite a marked increase in the FAAH enzyme in this area, no overall changes in these lipids were found. Collectively, these studies confirm that the striatal endocannabinoid system is altered in SCA-3 mutant mice, adding to the equivalent changes found in other strongly affected CNS structures in this type of ataxia (i.e.: the cerebellum and brainstem). These data open the way to search for drugs that might correct these changes.

Introduction Important changes in the endocannabinoid signaling system have been found in the CNS structures affected in most acute and chronic neurodegenerative disorders ( [1,2] for review). Such changes have been interpreted in two ways. On the one hand, dysregulation of cannabinoid receptor type-1 (CB 1 ) signaling has been associated with maladaptive responses that eventually contribute to disease symptoms (e.g., bradykinesia, ataxia, choreic movements) and/or to enhanced neuronal deterioration (e.g., heightened glutamate toxicity: [1,2]). Indeed, the elevated activity of endocannabinoid hydrolyzing enzymes like fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) has been associated with such maladaptive responses, provoking excessive degradation of endocannabinoids that dampens their prosurvival properties ( [2,3] for review). On the other hand, the up-regulation of cannabinoid receptor type-2 (CB 2 ), particularly in activated glial elements, produces an endogenous protective response that helps limit the consequences of neurotoxic insults, mainly inflammatory events but also oxidative stress and excitotoxicity, all of which operate in most neurodegenerative disorders ( [2,4,5] for review). A similar interpretation has been employed when considering the massive generation of endocannabinoids under neurotoxic conditions in vitro and in vivo ( [2] for review).
We recently found that endocannabinoid signaling appears to be dysregulated in the cerebellum SCA patients (including SCA-3), with important alterations to CB 1 and CB 2 receptors [15], and to FAAH and MAGL enzymes in post-mortem tissue [16]. Such alterations were also found recently in a transgenic mouse model of SCA-3 [17], which reproduces many of the neurological and neuropathological signs of the disease [28]. This earlier study [17] concentrated on two key CNS structures (cerebellum and brainstem) closely related to the classic symptoms (motor incoordination, imbalance, gait anomalies) and neuropathological lesions (the loss of specific neuronal subpopulations in the dentate nucleus, Purkinje layer and pontine nuclei) observed in SCA-3 [25,26,29,30]. Hence, the pharmacological correction of such changes in endocannabinoid signaling may represent a promising therapeutic option for this disease. However, the neuropathology of SCA-3 is not restricted to these two CNS areas and it is also affects other important structures like the basal ganglia, which explains the occurrence of extrapyramidal defects in SCA-3 patients [31] and in our SCA-3 transgenic mouse model (e.g., dystonia, rigidity: [17]). Therefore, the goal of this follow-up study was to investigate the changes to the endocannabinoid signaling system in the striatum of these transgenic mice using different biochemical and histological approaches.

Animals and sampling
All experiments were carried out on pCMVMJD135 transgenic mice and their wild-type littermates [28]. Offspring were genotyped to detect those that carried the transgene containing the ataxin-3 mutation (around 135Q), as described previously [28]. All animals were housed under a controlled photoperiod (08:00-20:00 lights on) and temperature (22 ± 1˚C), with free access to standard food and water. All the experiments were conducted in accordance with local and European legislation (directive 2010/63/EU), and following the principles of the ARRIVE guidelines. The protocols were also approved by the animal experimentation committee at our university (ref. CEA-UCM 56/2012). We used non-transgenic and pCMVMJD135Q transgenic male mice for longitudinal studies to initially characterize the appearance of striatal malfunctioning/degeneration at the biochemical and histological levels. Subsequently possible alterations in the endocannabinoid signaling were evaluated in this structure. These animals were characterized 7-55 weeks after birth [17], detecting dystonic-like behavior (limb clasping) and establishing three different disease states: (i) an early symptomatic stage, up to 16 weeks after birth, characterized by a loss of muscle strength in the hanging wire test [28]; (ii) a stable symptomatic stage, up to 32 weeks after birth, involving impaired performance on the balance beam and rotarod indicative of impaired balance and motor coordination [28]; and (iii) an advanced stage, up to 56 weeks after birth, in which marked deficiencies in gait and dystonia were reflected by the animal performance in footprinting tests and through their clasping behavior, respectively [17].
Groups of SCA-3 transgenic and wild-type mice of these three ages were perfused transcardially with saline followed by fresh 4% paraformaldehyde in 0.1M phosphate buffered saline (PBS, pH 7.4), and their brain was collected and post-fixed for one day at 4˚C. The tissue was 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 striatum was dissected out of brain of other mice and rapidly frozen on dry ice for subsequent biochemical analysis (qRT-PCR and western blotting), as well as to analyze the endocannabinoid concentrations. In all of the experiments, 6-8 male mice were used per experimental group.

Biochemical analyses
Real time qRT-PCR analysis. Total RNA was extracted from the striatum using the Sure-Prep™ RNA/Protein Purification kit (Fisher BioReagents, Fair Lawn, NJ, USA) and it was quantified by spectrometry at 260 nm. The RNA purity was evaluated through the absorbance ratio at 260 and 280 nm, whereas its integrity was confirmed in agarose gels. Genomic DNA was removed enzymatically and single-stranded complementary DNA was synthesized from 1 μg of total RNA using a commercial kit (Rneasy Mini Quantitect Reverse Transcription, Qiagen, Izasa, Madrid, Spain). The cDNAs were amplified by quantitative real-time PCR using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA: see Table 1), using GADPH as an endogenous control gene for normalization. The PCR assays were performed using the 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the threshold cycle (Ct) was calculated using the instrument's software (7300 Fast System, Applied Biosystems, Foster City, CA, USA).
Western blotting. Purified protein fractions were isolated using the SurePrep™ RNA/Protein Purification kit (Fisher BioReagents, Fair Lawn, NJ, USA) and aliquots of protein (20 μg) were boiled for 5 min in Laemmli SDS loading buffer (10% glycerol, 5% SDS, 5% β-mercaptoethanol, 0.01% bromophenol blue and 125 mM TRIS-HCl [pH 6.8]) prior to loading onto a 12% acrylamide gel (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were transferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA, USA) using a mini Trans-Blot Electrophoretic transfer cell (Bio-Rad Laboratories, Hercules, CA, USA), which were then blocked with 5% non-fat milk. The membranes were probed overnight at 4˚C with the anti-FAAH antibody (see Table 2) and then for 2 hours at room temperature with an ECL™ Horseradish Peroxidase-linked whole secondary antibody (GE Healthcare UK Limited, Buckinghamshire, UK) diluted 1:5000. Reactive bands were detected by chemiluminescence with the Amersham™ ECL™ Prime Western Blotting Detection Reagent (GE Healthcare UK Limited, Buckinghamshire, UK) and the membranes were analyzed on a ChemiDoc station with Quantity one software (Bio-Rad Laboratories, Madrid, Spain). The optical density of the specific protein bands were assessed relative to the housekeeping protein GAPDH, and they were normalized to the controls.
LC/MS analysis for endocannabinoid levels. The amounts of endocannabinoids, and their related N-acylethanolamines and 2-acylglycerols, were determined by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC/MS; 1100 LC-MSD, SL model; Agilent Technologies Inc., Wilmington, DE, USA), incorporating slight modifications to a previously described protocol [32]. Tissue samples were weighed and placed into borosilicate glass tubes containing 2 ml of acetonitrile with 84 pmol of [ 2 H 8 ]AEA and 186 pmol of [ 2 H 8 ]2-AG (Cayman Chemical, Ann Arbor, MI, USA). The tissues were homogenized with a glass rod and sonicated for 60 min. The samples were incubated overnight at -20˚C to precipitate the proteins, they were centrifuged at 1,500g for 3 min, and the supernatant was removed to a new glass tube and evaporated to dryness under nitrogen. The samples were then resuspended in methanol (300 μl) to recapture any lipids adhering to the glass tube and dried again. Finally, the lipid extracts were suspended in 30 μl of methanol, 5 μl of which was used for the LC/MS analysis. Each sample was separated on a reverse-phase C18 column (Kromasil, 250 × 2 mm, 5-μm diameter) using mobile phase A (deionized water with 1 mM ammonium acetate and 0.005% acetic acid) and mobile phase B (methanol with 1 mM ammonium acetate and 0.005% acetic acid). The samples were eluted at a flow rate of 300 μl/min under a linear gradient, with the percentage of solvent B increasing linearly from 85% to 100% over 25 min and maintaining 100% solvent B for 10 min. Over the next 10 min, solvent B decreased linearly from 100 to 85% and it was held at 85% for an additional 10 min.
MS detection was carried out in the selected ion monitoring mode using m/z values to detect

Histological procedures
Tissue slices. Sagittal cryostat sections of fixed brains (30 μm thick) were collected on gelatin-coated slides, and either Nissl-stained or used for immunohistochemistry.
Nissl staining. Frozen sagittal brain sections were incubated with 0.1% methylene blue for 5 min and washed with distilled water. The sections were then dehydrated, sealed and coverslipped with non-aqueous mounting medium. 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 cells, high resolution photomicrographs were taken with a 20x objective under the same conditions of light, brightness and contrast. Four images from at least 3 sections per animal were analyzed to establish the mean of all the animals studied from each group. The data are expressed relative to the wild-type animals.
Immunohistochemistry. Sections were preincubated for 20 min in 0.1M PBS with 0.1% Triton X-100 [pH 7.4], and endogenous peroxidase activity was blocked by incubating them for 1 hour at room temperature in blocking solution (Dako Cytomation, Glostrup, Denmark). After incubation overnight at 4˚C with the corresponding primary antibody (see Table 2) diluted in 0.1M PBS with 0.01% Triton X-100 [pH 7.4], the sections were washed in 0.1M PBS and incubated for 2 hours at room temperature with the appropriate biotin-conjugated antigoat or anti-rabbit secondary antibodies (1:200; Vector Laboratories, Burlingame, CA, USA). An avidin-biotin complex (Vectastain 1 Elite ABC kit; Vector Laboratories, Burlingame, CA, USA) and a DAB 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 the omission of the primary antibody. All sections for each immunohistochemical procedure were processed at the same time and under the same conditions. The sections were observed and photographed on a Leica DMRB microscope and with a Leica DFC300FX camera (Leica, Wetzlar, Germany). To quantify the immunoreactivity, the NIH Image Processing and Analysis software was used (ImageJ; NIH, Bethesda, MD, USA) on 4-5 randomly selected sections. The tissue was visualized with 20x objective by researchers blind to the experimental treatments and the data were expressed relative to the wild-type tissue.

Statistics
The data were assessed using the GraphPad software (version 5.0), applying an unpaired Student's t-test or two-way ANOVA for repeated measures followed by the Bonferroni test, as required.

Results
Characterization of the striatal damage in SCA-3 transgenic mice We recently described the progressive motor deterioration experienced by SCA-3 transgenic mice, as evident in the hanging wire, balance beam and rotarod tests [17,28]. In these studies, we also detected the presence of hindlimb clasping, a common marker of dystonia in mouse models of neurological disorders (including certain cerebellar ataxias [21]) and an extremely frequent response in SCA-3 mouse models [17,33]. Dystonia is an extrapyramidal sign often related to basal ganglia malfunction [34,35]. Thus, we have attempted to find a neuropathological basis for this response by analyzing the striatum of SCA-3 transgenic mice, a brain region significantly altered in SCA-3 patients [21]. We first assessed different biochemical markers that reflect either neuronal malfunction or neurodegeneration, combining qPCR with histological procedures to define neuronal loss or glial activation in this structure. Our qPCR studies revealed a marked reduction in the expression of the calbindin gene that appeared to commence at 16 weeks but that was significantly weaker at 32 and 56 weeks (Fig 1A). A decrease in DARPP32 expression was also seen at 56 weeks, a selective marker for striatal projection neurons, whereas other less-selective neuronal markers like NSE and BDNF did not appear to be affected (Fig 1A). The number of Nissl-stained cells in the striatal parenchyma of 56 week-old SCA-3 transgenic mice was also lower than in wild-type animals, and the surviving Nissl-stained cells in these mutant mice generally had a smaller cell body (see arrows in Fig  1B). Collectively, these data confirmed the deterioration of striatal neurons, in particular projection neurons, in SCA-3 transgenic mice, as also described in other SCA-3 models [33]. These data were consistent with the atrophy of the striatum and its output nuclei (globus pallidus, substantia nigra) described in SCA-3 patients [31].
We also investigated the possible glial activation in this structure using Iba-1 immunostaining to label microglial cells (Fig 2A). However, there were no significant differences in the number of Iba-1 positive cells or the staining intensity between 56 week-old (Fig 2B) or younger (data not shown) SCA-3 transgenic and wild-type mice. Similarly no such changes were evident in the expression of the proinflammatory cytokines IL-1β and TNF-α (Fig 2C). By contrast, the expression of glial glutamate transporters was significantly lower in the striatum of SCA-3 transgenic mice, both that of GLT-1 (16, 32 and 56 weeks) and GLAST (32 and 56 weeks: Fig 2C). However, the reduction in glutamate transporters was not associated with changes in GFAP immunostaining in the striatal parenchyma of SCA-3 transgenic mice relative to the wild-type animals (data not shown), a marker of astrocytes.

Analysis of the endocannabinoid signaling system in the striatum of SCA-3 transgenic mice
We initially explored the changes in endocannabinoid signaling by studying CB 1 receptor expression by qPCR, detecting a significant reduction in 32 and 56 week-old SCA-3 transgenic  (Fig 3A). This reduction might be related to the loss of CB 1 receptor-containing striatal projection neurons in SCA-3 transgenic mice, the cell bodies of which are located in the striatum (see above). Indeed, this response was similar to that described in Huntington's disease, another polyQ disorder in which the striatum is preferentially affected [36]. Accordingly, we expected to find less CB 1 receptor immunoreactivity in the cell bodies of striatal projection neurons in SCA-3 transgenic mice. However, we were unable to label these cell bodies in either SCA-3 transgenic or wild-type mice (Fig 3B), possibly because CB 1 receptors localize predominantly to the terminals of neurons that arise in the globus pallidus and the substantia nigra, and not significantly to their cell bodies [37]. Conversely, we did detect CB 1 receptors in terminals projecting to the striatum (see arrows in Fig 3B), almost certainly belonging to corticostriatal neurons [38]. Quantification of this immunolabeling indicated there were no changes in young, 16 week-old SCA-3 transgenic and wild-type mice, although there was notable reduction of immunoreactivity in SCA-3 transgenic mice at 32 and 56 weeks of age (Fig 3C). This reduction was not correlated with the reduction found in CB 1 receptor mRNA in the striatum (Fig 3A), as the cell bodies for these neurons are located in cortical areas.
We detected an increase in FAAH mRNA expression in the whole striatum at 32 and 56 weeks (Fig 4A), a change also seen in patients and animal models of Huntington's disease [36]. However, no such increase was seen in western blots (Fig 4B). FAAH immunostaining demonstrated an intense signal in the cell bodies of striatal neurons (see arrows in Fig 4C), although there were no apparent differences in the cellular and subcellular distribution between SCA-3 transgenic and wild-type mice at 16, 32 and 56 weeks of age (Fig 4C). However, quantification of the immunostaining revealed elevated immunolabeling in SCA-3 transgenic mice at 32 weeks of age (Fig 4D), despite the deterioration of the striatal projection neurons described above (Fig 1A). Thus, as surviving neurons may express more FAAH (Fig  4A), this change may only be evident at local levels as it was not seen in western blots of the whole striatum (Fig 4B).
Lastly, no differences were detected between SCA-3 transgenic and wild-type mice when other endocannabinoid genes were assessed at the three ages studied, including the CB 2 receptor, NAPE-PLD, DAGL and MAGL (Fig 5A-5D). We also measured anandamide and 2-AG, as well as their related signaling lipids PEA, OEA and 2-OG, again failing to detect differences between SCA-3 transgenic mice and wild-type animals at the ages studied (Fig 5E-5I).

Discussion
This study demonstrates that certain elements of the endocannabinoid signaling system (CB 1 receptors, the FAAH enzyme) are altered in the striatum of SCA-3 transgenic mice, as previously found in other important brain structures affected in this type of ataxia (cerebellum and brainstem [17]). The SCA-3 transgenic mice used in this study exhibited a broad-spectrum of motor anomalies that are comparable to the clinical symptoms found in patients. These included basal ganglia-related extrapyramidal signs like dystonia [17,28], which should a priori be the consequence of the selective deterioration of striatal projecting neurons that are also affected in the human pathology [24,25]. As in humans, neurodegeneration does not appear to be associated with marked glial activation or inflammatory events [21]. Rather, early alterations to glial glutamate transporters associate the degenerative process with elevated glutamate toxicity, consistent with our earlier observations in the brainstem nuclei [17]. Such toxicity was also found in other SCA-3 models [39] and it has been proposed to be the basis for the extension of neurodegeneration to extracerebellar structures in SCA-3 [20]. As such, the CMVMJD135 SCA-3 mouse model would appear to be useful for the study of extrapyramidal signs/basal ganglia deterioration in the progression of SCA-3, including the contribution of the endocannabinoid signaling system to this progression, the objective pursued herein.
Our goal was to determine whether endocannabinoid signaling is altered in the striatum of CMVMJD135 mice, possibly contributing to disease progression. This is indeed the case, as our findings are consistent with a possible reduction in CB 1 receptor signaling with elevated FAAH levels in this structure, both events that are compatible with enhanced excitotoxic damage. The nature of such altered endocannabinoid signals is clearly dependent on the cellular context, as the changes were not exactly the same in the striatum as in the cerebellum and brainstem [17]. In addition to contributing to disease progression, the changes in endocannabinoid signaling in SCA-3 transgenic mice may be associated with the appearance of specific neurological symptoms like the loss of motor coordination observed in these mice. Such symptoms were previously related to the elevated CB 1 receptor-mediated signaling in the Purkinje cell layer, predominantly in terminals of the basket cells [17]. Along similar lines, the reduced CB 1 receptor signaling found in the striatum may be related to the dystonia (clasping behavior) described in these mice [17], particularly given the relevance of the basal ganglia circuits in controlling dystonic responses [34,35]. However, recent data related the onset of dystonia to the malfunction of other CNS structures, including the cerebellum [40], making it possible if this is also related to the altered endocannabinoid signals in the cerebellum.
Such alterations to CB 1 receptor signaling could be pharmacologically corrected using direct agonists of this receptor or even by elevating the endocannabinoid tone by inhibiting endocannabinoid inactivation (e.g. FAAH/MAGL inhibition). Such an approach should attenuate dystonia, as documented in a few pharmacological studies on dystonic patients [41] or experimental models [42]. While this may relieve these symptoms, our aim was to obtain a more effective and sustained alleviation of these symptoms by controlling disease progression through cannabinoid-based therapies [3]. In this sense, our data indicate that the activation of CB 1 receptors may also be beneficial to avoid the loss of striatal projection neurons, as described previously in murine models of Huntington's disease [36]. In both cases, alleviating dystonia or preserving striatal neurons, the therapy aims to correct the changes in endocannabinoid signaling by enhancing CB 1 receptor function, assuming that these changes represent a maladaptive response that contributes to disease progression. This appears to be the case for the elevated FAAH levels found in the striatum, which would also be a maladaptive response possibly contributing to disease progression through excessive endocannabinoid degradation. In this sense, it may be better to selectively inhibit the FAAH enzyme in order to enhance the levels of endocannabinoids and their protective function. In both cases, enhancing CB 1 receptor signaling or inhibiting FAAH enzyme would address the changes in both targets that may contribute to this pathology.
The neuroprotection elicited by cannabinoids may also involve an enhancement of specific responses that represent adaptive mechanisms aimed at restoring neuronal integrity and homeostasis in the face of neurotoxic insults [43]. We expect this may be the case of CB 2 receptor-mediated signaling, given that it is up-regulated in an attempt to preserve neurons against inflammatory events in most neurodegenerative disorders [2,4], including SCAs when postmortem tissue from patients is studied [15]. However, we were unable to find any evidence of such events in the striatum of these SCA-3 transgenic mice, as occurs in the cerebellum and brainstem [17] where CB 2 receptor levels also remain unaffected. We assume that the lack of a CB 2 receptor response may reflect the relatively limited glial reactivity found in the affected CNS areas in this model [28].
Another adaptive response frequently elicited by endocannabinoid signaling in response to damage is an elevation in the generation of endocannabinoids. This has been described for different chronic neurodegenerative disorders [1], yet as in the other CNS structures investigated previously [17], we did not find elevated endocannabinoid levels in the striatum of SCA-3 transgenic mice. In fact, given the elevated FAAH immunostaining in specific striatal neurons the expected local reduction of these signaling lipids was not evident in Western blots, most likely due to the use of the whole striatum. Nevertheless, such a response, elevated FAAH activity/local reduction of endocannabinoids, is likely to contribute to disease progression rather than being an adaptive protective response.

Conclusions
Our results from SCA-3 transgenic mice confirm that certain elements of the endocannabinoid signaling system are altered in the striatum, as previously found in other important brain structures affected in this type of ataxia (cerebellum, brainstem [17]). Such changes may contribute to the pathogenesis of this condition by aggravating the influence of different neurotoxic stimuli in these mice (e.g. excitotoxicity). Hence, a pharmacological manipulation that corrects the changes in endocannabinoid signaling could be an interesting objective for future research.