Huntington disease (HD) is a genetic neurodegenerative disorder for which there is currently no cure and no way to stop or even slow the brain changes it causes. In the present study, we aimed to investigate whether FTY720, the first approved oral therapy for multiple sclerosis, may be effective in HD models and eventually constitute an alternative therapeutic approach for the treatment of the disease. Here, we utilized preclinical target validation paradigms and examined the in vivo efficacy of chronic administration of FTY720 in R6/2 HD mouse model. Our findings indicate that FTY720 improved motor function, prolonged survival and reduced brain atrophy in R6/2 mice. The beneficial effect of FTY720 administration was associated with a significant strengthening of neuronal activity and connectivity and, with reduction of mutant huntingtin aggregates, and it was also paralleled by increased phosphorylation of mutant huntingtin at serine 13/16 residues that are predicted to attenuate protein toxicity.
Huntington disease (HD) is an inherited brain disorder characterized by progressive degeneration of striatum and cortex and associated functional impairments in motor, cognitive and psychiatric domains (1). The disease is caused by abnormal expansion of CAG trinucleotide repeat encoding expanded polyglutamine (polyQ) stretch in the huntingtin (Htt) protein (2). Mutant Htt (mHtt) has detrimental effects on cell survival and neuronal cell functions such as synaptic transmission, gene transcription and production of a number of neurotrophins including the cortical brain-derived neurotrophic factor (BDNF) (3). The presence of the expanded polyQ stretch alters Htt structure and triggers the formation of mutant protein aggregates, thereby leading to cytotoxicity and interfering with overall cell functions (4). The expression of mutant protein has been also associated with deeply deregulated lipid metabolism, including sphingolipids breakdown (5).
FTY720, currently accepted for the treatment of relapsing remittent multiple sclerosis (6,7), is a synthetic analog of sphingosine, one of the major constituents of sphingolipids. Sphingosine is normally converted to sphingosine-1-phosphate (S1P) by sphingosine kinase-1 and -2 (SPHK1 and 2) (8). S1P is a potent signaling lipid that regulates a number of processes essential for cellular homeostasis, differentiation, motility and cell viability (8,9). Its action is mediated by the binding at S1P receptors (S1PRs1–5), normally expressed in neurons, astrocytes and oligodendrocytes (10). The pro-drug FTY720 is able to cross the blood–brain barrier (BBB) and distributes to the brain (11), where is reversibly phosphorylated to FTY720-phosphate (FTY720-P) by endogenous SPHK2 (12). FTY720-P acts as a potent agonist at four of the sphingosine-1-phosphate (S1P) receptors (S1P1, S1P3, S1P4 and S1P5) (10,13).
FTY720 normally acts as an immuno-modulator; however, in the central nervous system (CNS), it may have a number of additional effects (14). It has been previously described to induce the activation of mitogen-activated protein kinases (MAPK) in cultured astrocytes (15), to stimulate survival of oligodendrocyte progenitors through the activation of AKT and ERK pathways (16) and to protect cultured cortical neurons by increasing BDNF production (17) and reducing NMDA-mediated excitotoxicity (18). Administration of FTY720 has been recently reported to increase BDNF levels and significantly prolong survival in a mouse model of Rett syndrome (19), suggesting a potential therapeutic of FTY720 for the treatment of brain disorders, other than MS.
In the present study, we investigated whether FTY720 might be effective in HD models. Our findings indicate that chronic administration of FTY720 leads to improved motor function, prolonged survival and reduced brain atrophy in HD R6/2 mice. Remarkably, the benefits of the treatment were associated with increased BDNF levels, strengthening of neuronal activity and connectivity and reduction of mHtt aggregates. Interestingly, FTY720 treatment evoked phosphorylation of mutant huntingtin at serine 13/16 residues both in vitro and in vivo.
Administration of FTY720 protects HD cell model from apoptosis and promotes phosphorylation of pro-survival molecules, AKT and ERK
Striatal-derived cell line expressing endogenous levels of mHtt (STHdh111/111) showed higher susceptibility to apoptosis when compared with wild-type Htt-expressing cells (STHdh7/7) (5). First, to test the hypothesis that FTY720 was able to protect HD cells from apoptosis, STHdh7/7 and STHdh111/111 were grown in serum-free medium for 8 h in the presence of 1 μm FTY720. Administration of the pro-drug significantly reduced apoptosis in both cell lines (Fig. 1A) and rapidly induced phosphorylation of either AKT or ERK (Fig. 1B and C).
The neuroprotective effect of FTY720 is mimicked by FTY720-P and other S1P receptor agonists and is partially due to sphingosine kinase 2 activity
To explore additional molecular mechanism behind the neuroprotective effect of FTY720, we tested the involvement of S1PRs. A number of evidence demonstrates that stimulation of such receptors may exert anti-apoptotic effects in different cell death paradigm (20,21). First, to address the question whether a direct stimulation of S1PRs was neuroprotective also in HD models, STHdh111/111 cells were cultured in serum-free medium in the presence of different S1P receptor agonists: S1P, FTY720-P and SEW2871. Our results showed that all the S1PR agonists used efficiently reduced apoptosis in HD cell lines (Fig. 2A–C).
Next, in attempt to explore whether the neuroprotective effects of FTY720 were exclusively attributable to its active form FTY720-P, we co-treated HD cells with 1 μm FTY720 and 10 μm SPHKs inhibitor, d-erythro-N,N-dimethylsphingosine (22). SPHK2 mediates the conversion of the pro-drug FTY720 to the active form FTY720-P (12,13). DMS treatment failed to completely block the anti-apoptotic action of FTY720 (Fig. 2D), indicating that FTY720-mediated neuroprotection was partially dependent on the conversion of FTY720 to FTY720-P.
Infusion of FTY720 improves motor functions and prolongs lifespan in R6/2 mice
In order to test the hypothesis that FTY720 may exert beneficial effect in vivo in HD, R6/2 mice at different weeks of age and wild-type littermate controls were daily i.p.- injected with FTY720 or vehicle. Seven- and 10-week-old mice were treated in order to assess the effect of FTY720 on motor performance and lifespan (Figs 3 and 4). Motor function was analyzed before and at fixed time points (once a week) during the treatment. In line with previous studies, at the time when the treatment with FTY720 was initiated, 7- and 10-week-old R6/2 mice showed severe motor impairment compared with WT littermates (23), as assessed by open-field and horizontal ladder task (Fig. 3A–C). Remarkably, the beneficial effects of the compound on motor function and coordination were observed after 1 week of treatment (Fig. 3A–C). FTY720 chronically infused-R6/2 mice performed significantly better than vehicle-treated mice during the whole period of the treatment (Fig. 3A–C). No effects on motor behavior were observed in WT mice. According to the Kaplan–Meyer survival curve, both groups of FTY720-treated R6/2 mice (7- and 10-week-old mice) showed an extended survival compared with the vehicle groups (Fig. 4A and B). Furthermore, FTY720 treatment preserved HD mice from the weight loss, normally associated with the disease progression (Fig. 4C). Our results point out an overall good safety and tolerability profile of FTY720 with no evidence of adverse events throughout the period of the treatment.
FTY720 induces phosphorylation of AKT and ERK in vivo
Next, we explored the possibility that FTY720 was able to induce activation of pro-survival AKT and ERK also in mouse striatal tissues. To this purpose, 7-week-old vehicle- and FTY720-treated WT and R6/2 mice were sacrificed within 1 h from the last drug injection. In agreement with the results obtained in vitro, administration of FTY720 significantly incremented the phosphorylation levels of both AKT and ERK in either R6/2 (Fig. 5A and B) or WT mice (Fig. 5C and D), highlighting the ability of the drug to modulate the activity of ‘classical effectors’ of neuronal survival also in vivo. The increase of AKT and ERK phosphorylation, induced by FTY720 administration, appeared less pronounced in R6/2 with respect to WT brain tissues, corroborating the defects in the molecular pathways involving such kinases (24–26).
FTY720 ameliorates R6/2 mice neuropathology
Analysis of brain atrophy
The biological effect of FTY720 on neuropathology was assessed on serial brain coronal sections of whole brains from 11-week-old mice. R6/2 mice have been previously reported to progressively lose brain weight from ∼7 weeks of age (23). After the administration of FTY720, mice showed a marked reduction in brain weight loss when compared with the vehicle-treated mice (Fig. 6A). This result was associated with higher number of striatal cells as measured by hematoxylin/eosin staining (Fig. 6B) and with reduced extend of ventricle enlargement (Fig. 6C). Interestingly, semi-quantitative analyses of anatomical structure of corpus callosum (CC) revealed a significant reduction in its thickness in R6/2 mice when compared with WT mice. Importantly, chronic infusion of FTY720 partially prevented such reduction (Fig. 6C and D) in R6/2 mice, highlighting potential disease-modifying properties of the drug. Neuropathological changes in FTY720-treated mice were also associated with higher levels of striatal dopamine- and cAMP-regulated protein 32 (DARPP-32), a specific marker of medium spiny neurons (Fig. 6E), and increased levels of myelin–associated glycoprotein (MAG), a marker of myelin and white matter (WM) integrity (27), when compared with vehicle-treated mice (Fig. 6F). DARPP-32 protein levels were also increased in WT striatal tissues (Supplementary Material, Fig. S1), further confirming the potential ability of the drug to reinforce neuronal activity independently of the pathological condition.
Analysis of mutant huntingtin aggregates in R6/2 brain
To investigate the possibility that FTY720 may lower Htt toxicity, we evaluated its effect on mHtt aggregation. Formation of mHtt aggregates is a pathological hallmark that may conceivably cause neuronal dysfunction in HD (28). Immunohistochemical analysis using EM48 antibody revealed a dramatic reduction of either number or area of mHtt aggregates in the striatum of 11-week-old FTY720-treated R6/2 mice compared with controls (Fig. 6G–I).
FTY720 increases BDNF brain levels
In line with previous evidence highlighting the ability of FTY720 to promote elevation of BDNF both in vitro and in vivo (19), we found a significant increase of cortical BDNF mRNA levels both in R6/2 and WT treated mice (Fig. 7A). Consistently, BDNF protein levels were found elevated in the cortical tissues of FTY720-treated R6/2 mice (Fig. 7B and C) as well as in the striatum of the same mice, suggesting FTY720 to be potentially able to ameliorate the anterograde transport of the neurotrophin in R6/2 mice and supporting the hypothesis of a possible improvement of overall cortical-striatal transmission after the treatment.
FTY720 ameliorates cortical neuronal activity
BDNF has been reported to revert the increased GABAergic transmission in R6/2 mice (29), and its FTY720-mediated up-regulation may likely induce recovery of cortical-striatal transmission. Thus, we analyzed the GABAergic inputs to layer IV–V cortical pyramidal neurons. Vehicle- and FTY720-treated mice were sacrificed at 8 weeks of age, and frequency and amplitude of spontaneous inhibitory post-synaptic currents (sIPSCs) were measured. Similar to previous studies (29), the sIPSCs frequency was significantly higher in R6/2 than that in WT mice (Fig. 8A and B). The sIPSCs amplitude was also significantly increased, though to a lower extent (Fig. 8C). In FTY720-treated R6/2 mice, the observed reduction in sIPSCs frequency was not significant (Fig. 8B), whereas mean amplitude was significantly lowered to WT values (Fig. 8C). To deeper investigate the cortical GABAergic transmission, we analyzed miniature inhibitory post-synaptic currents (mIPSCs), owing to the spontaneous GABA release in the tetrodotoxin (TTX)-induced inhibition of action potential propagation. The mIPSCs frequency was obviously increased in R6/2 mice (Fig. 8D and E), whereas the amplitude was similar to WT values (Fig. 8F). Confirming the analysis of sIPSCs, the FTY720 treatment significantly reduced mIPSCs amplitude in R6/2 mice (Fig. 8F), leaving unaffected their frequency (Fig. 8E). These results indicated a clear FTY720-induced functional action on GABAergic synapses impinging cortical pyramidal neurons, with a partial but significant recovery of inhibitory transmission to physiological conditions.
FTY720 treatment increases endogenous GM1 levels in vitro and in vivo HD models
Evidence indicates that FTY720, but not FTY720-P, may increase levels of C18-ceramide, a natural precursor of brain sphingolipids including gangliosides (30). Defective ganglioside metabolism leads to increased susceptibility of HD cells to apoptosis, and administration of ganglioside GM1 is neuroprotective in HD models (5,31). Here, we observed that administration of FTY720 increased mRNA levels of GM1 synthase (β3galt4) and plasma membrane GM1 content in STHdh111/111 cells (Fig. 9A and B). Similarly, GM1 levels were restored in the striatum of FTY720-treated R6/2 mice compared with vehicle-treated mice (Fig. 9C). These results would suggest a potential ability of the drug to retrieve the altered ganglioside metabolism in HD models.
FTY720 treatment induces Htt phosphorylation at serine 13/16 residues both in vitro and in vivo.
To further investigate a possible molecular event by which the drug may specifically impact HD pathophysiology, we tested the potential of FTY720 to modulate mHtt phosphorylation at serine 13/16 residues, previously reported as critical determinants in the pathogenesis of the disease (32). Administration of 1 μm FTY720 increased levels of phospho-Ser13/16 Htt in HD STHdh111/111 cells as assessed by using the phospho-specific antibody (pN17) (31,33) (Fig. 10A–C). Phosphorylated huntingtin migrated at higher molecular weight than full-length Htt (FL-Htt), consistent with the formation of an SDS–insoluble protein complex which we have previously defined as high-molecular-weight huntingtin (HMW-Htt) (Fig. 10C) (31). Similar result was observed in R6/2 striatal lysates, where only the HMW-Htt complex was detected by pN17 (Supplementary Material, Fig. S2).
FTY720, a Food and Drug Administration (FDA)-approved oral agent for the treatment of MS, was first described as an immunosuppressive drug (34) and recently reported to have neuroprotective and neurorestorative properties (18,19,35) that make FTY720 a potential therapeutic drug for treatment of different neurodegenerative disorders (19). In this study, we evaluated whether FTY720 may represent a possible pharmacological intervention also in HD.
FTY720 significantly reduced apoptosis in STHdh cell models and evoked pro-survival pathways responses by inducing activation of AKT and ERK. This is particularly important in the context of HD where PI3 /AKT and ERK pathways are notoriously impaired (24–26) and stimulation of each of these molecules is reported to be beneficial (5,36–38). The efficacy of FTY720 may depend on the stimulation of S1PRs by its phosphorylated form (FTY720-P) (13). Coherently, stimulation of such receptors is neuroprotective in HD striatal cell lines and suggests that the direct stimulation of S1PRs may be considered as potential target for new therapeutic strategies for the disease. On the other hand, the pharmacological inhibition of the conversion of FTY720 to FTY720-P, by DMS, failed to completely block the anti-apoptotic properties of the drug in HD, indicating that FTY720-P may not be the only effective form.
Starting from the first week and throughout the duration of the treatment, FTY720-treated R6/2 mice performed significantly better than their controls. The progression of the disease was dramatically slowed down up to the animal death that was significantly delayed independently of the age at which animal underwent the treatment. Interestingly, the efficacy of FTY720 on motor symptoms, even at very late stages of the disease, pointed out an important therapeutic potential of the molecule in HD. Recovery of motor function and overall wellbeing of FTY720-treated R6/2 mice well correlated with reduced loss of brain weight limited ventricles enlargement and higher number of striatal cells probably including neuronal and non-neuronal cell populations (e.g. oligodendrocytes) (14,16). Neuropathological improvements were associated with increased levels of DARPP-32 protein, whose defective expression is an early sign of striatal dysfunction and neuronal loss in R6/2 (39). Increased levels of DARPP-32 in HD mice, after FTY720, may be due to reduced loss of striatal neurons. FTY720 may preserve medium spiny neuronal function and protect these neurons from degeneration. This effect may likely reinforce cell signaling and eventually correlate with an improved neuronal activity.
FTY720 mediates tissues remodeling processes and normally distributes in CNS WM preferentially along myelin sheaths (40), leading to attenuated injury to oligodendrocytes, myelin and axons in the CC of mice with chemical-induced WM damage (41). WM segmentation and myelin breakdown is described in different HD mouse models at different stages of the disease (42,43).
In this study, FTY720 significantly reduced the thinning of CC and restored levels of MAG protein, which normally localizes in periaxonal Schwann cells and oligodendroglial membranes of myelin sheaths, where it regulates signaling and stabilizes glia–axon interactions (27) as well as WM integrity (44). MAG-null phenotype results in loss of axonal neurons and increased susceptibility to excitotoxic stimuli and seizure activity (45,46). Restoring levels of MAG may likely stabilize axonal–glial interactions and preserve neuronal function and survival in HD.
The beneficial effects of FTY720 on HD striatum and CC neuropathology might be the result of a direct action of the drug on the two distinct regions or secondary to the effectiveness of the drug to specifically increase the levels of BDNF protein, as this neurotrophin has been reported to regulate the expression of DARPP-32 and MAG in medium spiny neurons and WM, respectively (47,48). BDNF is normally synthesized in the cortex and transported to the striatum via cortico-striatal projections and provides the main support for survival of striatal neurons in the adult brain (49). Reduced levels of BDNF, altered gene expression and/or abnormalities in its anterograde transport in cortical regions have been previously implicated in the pathogenesis of HD (3,50). Administration of FTY720 increased BDNF levels in both WT and HD mice. Although this effect was independent of the mouse genotype, increased levels of BDNF in HD mouse cortex and the restoration in the striatum of same mice suggest FTY720 as a modulator of BDNF expression and release in HD and rises the hypothesis that the neuroprotective effect of the drug may also be mediated by the neurotrophin, as BDNF prevents loss of striatal neurons and motor dysfunction in HD mouse models (51,52). HD mice, including R6/2, display a progressive impairment in both excitatory and inhibitory synaptic transmission, in both striatum and cortex (53). In particular, GABAergic inputs are functionally enhanced in the first stages of the pathology and then strongly decreased, likely due to interneuron death in the last period of R6/2 mice life (29). Here we found that FTY720 partially but significantly reduced GABAergic transmission in the cortices of 8-week-old R6/2 mice, making their fast inhibitory signaling similar to that of WT animals. This effect is likely to be mediated by FTY720-induced BDNF production, as this neurotrophin has been also shown to revert the increased GABAergic function in the striatum (29).
An important finding of our study is the dramatic reduction of mHtt aggregates of FTY720-treated R6/2 mice. Although it is still unclear whether aggregates are the toxic Htt species (54), their reduction is often associated with an improvement of disease phenotype in different HD models (55).
The pro-drug FTY720 has been previously demonstrated to have an intrinsic activity to influence lipids homeostasis (30,56). Disrupted metabolism of lipids, in particular gangliosides, has been described as critical determinant of HD pathogenesis (5). Administration of exogenous GM1 restored normal levels of ganglioside in HD cellular and animal models, and it resulted to be beneficial both in vitro and in vivo (5,31). Here, we reported that FTY720 modulated endogenous GM1 levels in STHdh111/111 cells and restored its normal content also in the striatum of R6/2 mice. Thus, the effect of FTY720 on gangliosides metabolism may reinforce its neuroprotective function in HD models and further contribute to the amelioration of the pathology.
Phosphorylation of Htt at serine 13/16 residues is reported to have several beneficial effects in HD models by changing mHtt conformation (33,57), reducing mutant Htt exon1 aggregation and toxicity (58) and by increasing mutant protein degradation (59). Importantly, in BACHD mice expressing Ser13 and Ser16 phosphomimic mHtt, behavioral features of the disease, including motor and psychiatric deficits, are significantly ameliorated and mHtt aggregates in the brain are not detected (32). In this study, we reported the evidence that FTY720 triggered Htt phosphorylation at serine 13/16 residues both in vitro and in vivo. In line with our previous findings (31), we were able to detect phosphorylation at serines 13 and 16 only in the HMW-Htt and not in the FL-Htt. These results corroborated the hypothesis that phosphorylation of Htt at such serine residues may be involved in the stabilization and/or formation of HMW-Htt complex (31) whose nature is not fully determined yet. HMW-Htt could represent mHtt aggregates species (60), and the increased phosphorylation state may appoint them to degradation (59), thus explaining the reduction of mHtt inclusions in R6/2 striatum.
Although in this study, there is no prove of a direct correlation between increased levels of phospho-mHtt and the neuroprotective effect of the drug, we believe that compounds like FTY720, which cross the BBB and promote all the beneficial effects we described, may likely result effective for treating the disease. To our best knowledge, this is the first evidence of a compound exerting neuroprotective action to such extent in HD models.
We believe that the pre-clinical efficacy of FTY720 reported in this study highlights the therapeutic potential of the drug to treat HD. What might make FTY720 superior to other drugs is the fact that, beside its ability to improve motor function and neuropathology in R6/2 mice, which would make it an effective disease-modifying drug in HD, it has the potential of impacting critical disease-related deficits. Neuroprotective and neurorestorative proprieties of the drug, as well as the safety profile observed in R6/2 mice and, importantly, also in clinical trials for other neurological disorders (61), indicate that FTY720 may represent a valuable and alternative therapeutic approach that can be transferred into clinical practice in HD in the next future.
MATERIALS AND METHODS
FTY720, FTY720-phosphate (FTY720-P), 1,2,4-oxadiazole, 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl] (SEW2871) were purchased from Santa Cruz. d-Erythro-N,N-dimethylsphingosine was purchased from ENZO Life Sciences. All chemicals were dissolved according to the manufacturer's instructions.
Conditionally immortalized mouse striatal knock-in cells expressing endogenous levels of wild-type (STHdh7/7) or mHtt (STHdh111/111) were purchased from the Coriell Cell Repositories (Coriell Institute for Medical Research, Camden, NJ, USA) and were maintained as previously described (5).
After pre-incubation for 5 h at 33°C in serum-free medium, cells were treated with 1 μm FTY720 for the indicated time. Cells were lysate in lysis buffer containing 20 mm Tris, pH 7.4, 1% Nonidet P-40, 1 mm EDTA, 20 mm NaF, 2 mm Na3VO4 and 1:1000 protease inhibitor mixture (Sigma–Aldrich), sonicated with 2 × 10 s pulses and then centrifuged for 10 min at 10 000g.
Analysis of apoptosis
The–anti-apoptotic effect of FTY720, FTY720-P, SEW2871 and S1P was tested on immortalized cells cultured in serum-free medium at 39°C at the indicated time in the presence of the specific compound. In FTY720 experiments, cells were pre-incubated with FTY720 for 2 h before they were placed at 39°C. In DMS experiments, cells were pre-incubated with DMS for 1 h before adding FTY720. At the end of each treatment, cells were collected and incubated with FITC-conjugated Annexin V (Southern Biotech) according to the manufacturer's instructions. Fluorescence-activated cell sorting (FACS) analysis was performed as previously described (5).
Breeding pairs of the R6/2 line of transgenic mice [strain name: B6CBA-tgN (HDexon1) 62Gpb/1J] with ∼160 ± 10 (CAG) repeat expansions were purchased from the Jackson Laboratories (Bar Harbor, ME, USA) and maintained in the animal facility at IRCCS Neuromed. In vivo experiments were approved by IRCCS Neuromed and carried out in both R6/2 mice and WT littermates, starting from 5 or 10 weeks of age. To ensure homogeneity of experimental cohorts, mice from the same F generation were assigned to experimental groups, such that age and weight were balanced. In total, 64 male R6/2 transgenic mice and 50 male WT littermates were used in this study. All animals used for biochemical and histological experiments were euthanized at fixed time points. R6/2 mice used for testing the effect of FTY720 on mouse survival died naturally.
Protein lysate preparation
Mice were sacrificed by cervical dislocation, brain regions were dissected out, snap-frozen in liquid N2 and pulverized in a mortar with a pestle. Pulverized tissue was homogenized in lysis buffer containing 20 mm Tris, pH 7.4, 1% Nonidet P-40, 1 mm EDTA, 20 mm NaF, 2 mm Na3VO4 and 1:1000 protease inhibitor mixture (Sigma–Aldrich), sonicated with 2 × 10 s pulses and then centrifuged for 10 min at 10 000g.
For analysis of phospho-AKT and phospho-ERK, 40 μg of total lysate was immunoblotted with the following antibodies: anti-phospho-AKT (1:1000), anti-AKT (1:1000), anti-phospho-ERK (1:1000) and anti-ERK (1:1000) (all from Cell Signaling). For DARPP-32, BDNF and MAG total lysate were immunoblotted with the following antibodies: anti-DARPP-32 (1:1000), anti-BDNF (1:500) and anti-MAG (L20) (1:1000) (all from Santa Cruz), respectively. For the analysis of huntingtin phosphorylation at serine 13/16 residues, 40 μg of striatal lysate was immunoblotted with the following antibodies: anti-phospho-Se13/16-specific antibody pN17 (1:6000) (31,33) and anti-Huntingtin N17 (1:6000) (31,33). For protein normalization, anti-α tubulin (1:5000) (Abcam) or anti-β Actin (1:3000) (Sigma) was used. HRP-conjugated secondary antibodies (GE Healthcare) were used at 1:5000 dilution. Protein bands were detected by ECL Prime (GE Healthcare) and quantitated with Quantity One (Bio-Rad Laboratories) and/or ImageJ software.
In vivo drug administration
FTY720 was dissolved in DMSO according to the manufactory instructions and daily administered by intraperitoneal (i.p.) injection at dose of 0.1 mg/kg of body weight according to Deogracias et al. (19). FTY720 solution was further dissolved in saline (vehicle). Control mice (WT and R6/2) were daily injected with the same volume of vehicle containing DMSO.
Motor behavior tests and survival study
Open-field test and horizontal ladder task were performed according to the standard recommendations. All tests took place during the light phase of the light–dark cycle. Male mice were first habituated to the test room and to the instrument and task to perform for 2 h before any test. Spontaneous horizontal locomotion and general motor activity were analyzed in the open field. Mice were placed in the center of the chamber, and their behavior was recorded for 5 min. Quantitative analyses were performed on the total distance traveled. Skilled walking, limb placement and limb coordination were all assessed by the ladder rung walking task as previously described (31). All tests were carried out blindly to the treatment. All mouse cages have been examined daily in order to determine lifespan.
Brain pathology and immunohistochemistry
WT and R6/2 mice were sacrificed by cervical dislocation. Brains were removed and trimmed by removing the olfactory bulbs and spinal cord. The remaining brain was weighed in milligrams, processed and embedded in paraffin wax, and 10 µm coronal sections were cut on an RM 2245 microtome (Leica Microsystems). Five mice/group (n = 5) were used, and four coronal sections spread over the anterior–posterior extent of the brain (200–300 μm inter-section distance) were scanned. For each coronal section, a total number of 10 fields at 63× magnification were analyzed. The number of striatal cells was determined by hematoxylin/eosin staining (62). Immunostaining for mutant Htt aggregates was carried out by using EM48 antibody (1:100) (Millipore) (63). The average number and area of striatal mHtt aggregates per brain section was quantiﬁed by ImageJ software. For the analysis of CC, five animals per group were analyzed. For each coronal section, a total number of 10 fields at 5× magnification were acquired. The thickness of CC was measured in correspondence of the middle line of the brain (Fig. 6C). All the assessments were performed blindly.
Mice were anesthetized with halothane and then decapitated, and the brain rapidly removed to ice-cold glycerol-based dissection artificial cerebrospinal fluid (ACSF) containing the following (in mm): glycerol 250, KCl 2.5, CaCl2 2.4, MgCl2 1.2, NaH2PO4 1.2, NaHCO3 26, glucose 11 and Na-pyruvate 0.1 (pH 7.35, aerated with 95% O2/5% CO2, 290–300 mOsm/l). The brain was glued to the stage of a vibrating microtome (model VT1000S; Leica), and 300 μm coronal slices were cut. Slices then were stored and allowed to recover at room temperature in a submerged chamber for at least 1 h before experimentation in oxygenated standard ACSF, containing the following (in mm): NaCl 125, KCl 2.5, CaCl2 2, NaH2PO4 1.25, MgCl2 1, NaHCO3 26, glucose 10, Na-pyruvate 0.1 (pH 7.35, aerated with 95% O2/5% CO2, 290–300 mOsm/l).
Whole-cell recordings in slices
Whole-cell patch-clamp recordings were obtained from layer IV–V pyramidal cells visualized in cortical slices with the aid of infrared-differential interference contrast microscopy. The patch pipette (3–4 MΩ) was filled with the following solution (in mm): KCl 140, HEPES 10, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid 5, MgCl2 2, MgATP 2 (pH 7.35, with KOH). Voltage-clamp recordings were performed using an Axon Multiclamp 700B patch-clamp amplifier. Spontaneous inhibitory post-synaptic currents (sIPSCs) were recorded filtering the membrane current at 1 kHz and digitizing at 100–200 μs, clamping cells at −70 mV, in the presence of 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 μmdl-2-amino-5-phosphonovaleric acid (AP-5). In these conditions, synaptic currents were GABAergic in nature, as they were all blocked by 20 μm bicuculline (not shown). To record miniature currents (mIPSCs), 1 μm TTX was bath-applied to slices. All drugs were obtained from Tocris. Spontaneous synaptic currents were analyzed offline using an automatic template-based detection protocol (Clampfit 10).
RNA extraction and analysis of gene expression by real-time PCR
Total RNA were extracted using RNeasy kit (Qiagen) according to the manufacturer's instructions. One microgram of total RNA was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo-dT primer, and the resulting cDNAs were amplified using Power SYBR Green PCR Master Mix (Applied Biosystems) following the manufacturers' instructions. Quantitative PCR analysis will be performed on a StepOne instrument (Applied Biosystems) using specific primers, as reported in Supplementary Material, Table S1, as previously described (5).
Analysis of GM1 content
Plasma membrane GM1 levels were measured by cholera toxin B subunit binding and FACS analysis. Cells were cultured seven days in regular medium supplemented with 1 μm FTY720. At the end of the treatment, cells were trypsinized, washed in ice-cold PBS (Invitrogen) and labeled with 2 mg/ml Alexa488-conjugated cholera toxin B (CTX-B) (Invitrogen) in PBS/0.1% BSA for 5 min on ice. After washing, cells were fixed with 2% paraformaldehyde and stored at 4°C until FACS analysis was performed. GM1 levels in dissected mouse brain tissues were analyzed by dot-blotting as previously described (31). Striatal tissues were lysed, spotted on nitrocellulose membrane and incubated with HRP-conjugated CTX-B. Total amount of GM1 was detected by ECL Prime (GE Healthcare) and quantitated with Quantity One software (Bio-Rad Laboratories).
Htt immunoprecipitation and immunoblotting
After pre-incubation for 3 h at 33°C in serum-free medium, cells were treated with 1 μm FTY720 for 30 min and lysed in lysis buffer containing 20 mm Tris, pH 7.4, 1% Nonidet P-40, 1 mm EDTA, 20 mm NaF, 2 mm Na3VO4 and 1:1000 protease inhibitor mixture (Sigma–Aldrich), sonicated with 2 × 10 s pulses and then centrifuged for 10 min at 10,000g. Immunoprecipitation (0.6 mg total lysate) was performed using polyclonal anti-Htt antibody N17 (31,33) complexed to protein G-Sepharose (Zymed, Invitrogen). The immunoprecipitated protein was resolved on 7% SDS–PAGE, transferred overnight on PVDF membrane (31) and detected by immunoblotting with mab2166 (1:1000) (Millipore) and anti-phospho-Ser13/16-Htt-specific antibody pN17 (1:6000) (31,33). HRP-conjugated secondary antibodies (GE Healthcare) were used at 1:5000 dilution. Protein bands were detected by ECL Prime (GE Healthcare) and visualized by Quantity One software (Bio-Rad Laboratories).
Cells were grown on glass coverslips coated with 50 μg/mL poly-l-lysine and treated with either vehicle or 1 μm FTY720 for 30 min. Cells were then washed in PBS, fixed in 4% paraformaldehyde for 15 min at room temperature (RT) and permeabilized with 0.5% Triton X-100 in PBS for 5 min. After blocking with 4% donkey serum in PBS for 1 h, cells were incubated with polyclonal pN17 antibody (1:1000) for 1 h at RT. Anti-rabbit Alexa 488-conjugated secondary antibody (Vector Laboratories) was used at 1:500 dilution for 1 h at RT. Cell nuclei were stained with 4′-6 diamidino-2-phenildindole (Vector Laboratories) for 10 min at RT. Coverslips were mounted using Prolong Gold antifade reagent (Invitrogen). Images of vehicle- and FTY720-treated cells were acquired at 63× magnification using the same microscope settings. The mean fluorescence intensity (MFI) per pixel in each cell was calculated with ImageJ software (National Institutes of Health) after manual selection of cell area.
Two-way ANOVA followed by Bonferroni post-test was used to compare treatment groups in the open field and horizontal ladder tests and for mouse body weight analysis. Non-parametric Mann–Whitey U-test was used to analyze cell survival, mouse brain weight, CC thickness, BDNF, DARPP-32 and MAG protein levels, mouse striatum GM1 content and cortical currents. Log-rank test was used to analyze mice survival. Two-tailed t-test was used to compare mean fluorescence intensity of vehicle- versus FTY720-treated samples in immunocytochemistry experiments and in all other experiments. All data are expressed as mean ± SD except in the electrophysiology studies in which data are expressed as mean ± SEM.
This research was supported by Marie Curie International Incoming Fellowship (PIIF-GA-2011-300197) granted to V.M. within the 7th European Community Framework Programme and by Telethon (Project GGP12218) to F.S.
We thank Professor Ray Truant (McMaster University, Hamiton, ON, Canada) for providing us with pN17 and N17 antibodies. We also thank N. Panebianco and S. Sciacca for technical assistance. We gratefully acknowledge the support of Lega Italiana Ricerca Huntington (www.lirh.it) e malattie correlate onlus.
Conflict of Interest statement: none declared.