Striatal Serotonin 4 Receptor is Increased in Experimental Parkinsonism and Dyskinesia

Alterations of serotonin type 4 receptor levels are linked to mood disorders and cognitive deficits in several conditions. However, few studies have investigated 5-HT4R alterations in movement disorders. We wondered whether striatal 5-HT4R expression is altered in experimental parkinsonism. We used a brain bank tissue from a rat and a macaque model of Parkinson’s disease (PD). We then investigated its in vivo PET imaging regulation in a cohort of macaques. Dopaminergic depletion increases striatal 5-HT4R in the two models, further augmented after dyskinesia-inducing L-Dopa. Pending confirmation in PD patients, the 5-HT4R might offer a therapeutic target for dampening PD’s symptoms.

Beyond the presynaptic 5-HT system, there is a growing interest towards the post-synaptic serotonin 4 receptor (5-HT 4 R) [5,6].This G-protein coupled receptor is widely distributed in the body and highly expressed in the brain, especially in the basal ganglia.Its activation modulates food intake [7] and supports pro-cognitive, anxiolytic and antidepressant effects [8,9].5-HT 4 R agonists treat chronic idiopathic constipation in humans [10] and improve memory [11].5-HT 4 R expression is knowingly altered in abnormal food intake, mood disorders and cognitive deficits [12][13][14].
Surprisingly few studies have focused on the 5-HT 4 R in PD, while the myriad of PD non-motor symptoms encompasses such manifestations [15,16].As a first step, we wondered whether the striatal 5-HT 4 R is increased after DA depletion and L-Dopa supplementation using an existing brain bank tissue from a rat and a non-human primate (NHP) model of PD.We then investigated its in vivo PET imaging regulation in a second cohort of NHPs.

Animals
Experiments were carried out in accordance with European Communities Council Directive of November 24, 1986 (86/609/EEC) revised in 2010 (2010/63/UE) and were approved by the local ethical committees.Following the three Rs (Reduction, Refinement, and Replacement) for animal experimentation, we first used existing well-validated brain collections featuring parkinsonian and dyskinetic rats [17] and Macaca mulatta NHPs [18].Briefly, rats were rendered hemiparkinsonian following unilateral injection of 6-hydroxydopamine (12 g) into the substantia nigra pars compacta, rendered dyskinetic by a 10 days-treatment with L-Dopa (25 mg/kg twice daily), and sacrificed 6 weeks after dopaminergic lesion [17].Macaca mulatta NHPs were rendered parkinsonian by daily injection of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP at 0.2 mg/kg/day) until stabilization of parkinsonian symptoms, dyskinetic by a 3-months oral L-Dopa treatment (20 mg/kg twice daily), and were sacrificed 6 months after onset of DA lesion [18].For both species, behavioral analysis and lesion characteristics were published in detail [17,18].However, parkinsonian and dyskinetic measures obtained for rats and macaques are indicated in Supplementary Table 1.These measures have been acquired through the use of specific, well-known tests and scales (the rotational behavior and the scoring of axial, orolingual and forelimb dyskinesia [19] for rats; the PD disability score [20], the monkey clinical assessment scale [21] and the NHP dyskinesia rating scale [22] for macaques).In vivo molecular imaging was conducted on a novel macaque cohort, the experimental details of which are given below.

Postmortem studies
Brain collections were issued from 15 rats (4 control, 5 hemiparkinsonian, 6 dyskinetic) and 11 macaques (4 controls, 4 parkinsonian, 3 dyskinetic).For each animal, four sections at the level of the posterior striatum were processed for immunohistochemistry (see detailed protocol in [23]) with the following antibodies: anti-5-HT 4 receptor (5-HT 4 R) 1/100 rabbit polyclonal from Merck (Merck, Molsheim, France) (catalog number S0195), anti-FosB/ FosB 1/200 rabbit polyclonal from Tebu-Bio (catalog number SC-7203; Tebu-Bio, Le Perray en Yvelines, France).The specificity of the immunostaining was assessed by omission of the primary antibodies from the protocol.At the end of the protocol, sections were examined with a light microscope using a computerized image analyzer (Mercator, ExploraNova, La Rochelle, France).Striatal 5-HT 4 R expression levels were analyzed under blinded conditions relative to the animal by optical density measurements using Image J software.

In vivo molecular imaging studies
Six adult male macaques (Macaca fascicularis) were used for in vivo molecular imaging.Monkeys weighed between 4 and 9 kg and were aged between 4 and 6 years.They were kept under standard conditions (12 h light cycles, 23 • C, and 50% humidity).They were rendered parkinsonian by systemic intoxication with MPTP (0.4 mg/kg) (MPTP from Sigma-Aldrich, Saint-Quentin-Fallavier, France).MPTP injections were stopped once Parkinsonian symptoms were established, as previously described [24].The six monkeys were scanned before (baseline) and two months after MPTP intoxication (post-MPTP) with [ 11 C]PE2I and [ 11 C]SB207145, which bind to the dopaminergic transporter [25] and the 5-HT 4 receptor [26,27], respectively.PET (positron emission tomography) and MRI (magnetic resonance imaging) acquisitions were performed at the imaging center (CERMEP, Lyon, France) under anesthesia (atropine 0.05 mg/kg intramuscularly followed 15 min later by zoletil 15 mg/kg intramuscularly).Anatomical MRI acquisition consisted of a 3D T1weighted sequence using a 1.5-T AvantoFit scanner (Siemens).The anatomical volume covered the whole The cerebellum (excluding the vermis) was considered as the reference region for the modelisation.Regional values of BP ND were extracted from parametric maps using MAXPROB atlas as described in [24].

Statistical analysis
All statistical analyses were performed using GraphPad Prism software.PET imaging and immunohistochemical data were analyzed using non-parametric Mann-Whitney tests with p < 0.05.Histograms represent mean ± SEM.
Such 5-HT 4 R upregulation was also observed in the gold-standard experimental model of PD, namely the MPTP-treated macaque NHP (Fig. 2A).Chronic (3 months) L-Dopa supplementation at therapeutic doses led to LID manifestations and to further upregulation of striatal 5-HT 4 R levels (Fig. 2A).
Given the translational value of Parkinsonian macaques, we then longitudinally investigated 5-HT 4 R in vivo binding by PET imaging before (control situation) and after MPTP intoxication (Parkinsonian situation).The extent of nigrostriatal lesion was documented using a clinical-grade radiotracer specific to the dopamine transporter, the [ 11 C]PE2I (Fig. 2B,  C).We then ran [ 11 C]SB207245, a highly specific radiotracer of 5-HT 4 R [30].[ 11 C]SB207145 BP ND was increased after parkinsonism induction in all stri-atal areas (Fig. 2D), notably in the posterior motor putamen (Fig. 2E).

DISCUSSION
This study shows that dopaminergic depletion is sufficient to induce a striatal upregulation of the 5-HT 4 R, and that this increase is potentiated, and concomitant with FosB/ FosB, following L-Dopa supplementation causing dyskinesias.
5-HT 4 R distribution in the brain is highly conserved across species [31,32].Very few studies investigated 5-HT 4 R expression regulation so far.Experimentally, they were performed only in rodents, i.e., rats and guinea pigs.In the 6-OHDA-injured rat, Compan and colleagues (1996) showed a 59% increase in 5-HT 4 R binding in the caudal part of the caudate-putamen [33].A recent in situ hybridization study, therefore measuring mRNA transcripts and not receptors themselves, did not detect changes in striatal 5-HT 4 R mRNA levels after dopaminergic lesion or after chronic L-Dopa treatment [34].These results suggest that the 5-HT 4 R expression must be functionally investigated with direct binding or immunohistochemistry, as 5-HT 4 R displays a commonly observed decoupling between the transcript abundance and the protein expression.
Only two studies, from the same lab in 1995, report tritiated radioligand binding studies of the 5-HT 4 R in postmortem human brain homogenates.Peculiarly, the authors did not report a difference in putaminal 5-HT 4 binding levels between control and Parkinsonian subjects although the mean binding values were increased [35,36].The low power of the studies associated to the lack of spatial resolution due to the homogenization of tissues as opposed to ligand binding or immunostaining of brain sections should account for this difference.The trend is however similar.
In conclusion, the 5-HT 4 R is over-expressed in the putamen both after DA depletion and DA dyskinesiogenic supplementation.This suggests that two distinct mechanisms are involved: firstly, a post-injury compensatory mechanism, and secondly, a LID-driven maladaptive plasticity mechanism involving FosB/ FosB, which may in turn regulate the transcription of 5-HT 4 R. Indeed, FosB likely binds to the 5-HT 4 R [37].Although we do not know, at this stage, whether 5-HT 4 R is involved in motor disorders or is due to compensatory mechanisms, this work raises the broader question of the role of the 5-HT 4 R in the pathophysiology of PD, with possible implications on the pathophysiology of cognitive deficits or mood disorders to which this receptor has been linked in other pathologies.Future PET imaging studies in humans urgently need to confirm (or infirm) these preclinical results since the 5-HT 4 R upregulation might offer a therapeutic target for dampening PD's motor symptomatology.However, the clinical use of 5-HT 4 antagonists could prove tricky given the lack of available pharmacological agents and the high risk of inducing non-targeted side effects, particularly due to the expression of these receptors outside the brain, such as in the gastrointestinal tract or the heart [5,38].Also, given that constipation [39,40] and nonmotor disorders [41,42] are reportedly improved by 5-HT 4 R agonists, the neurologists may have to opt for different pharmacological options to treat these non-motor symptoms alongside motor disorders.

Fig. 2 .
Fig. 2. Striatal upregulation of 5-HT 4 R after dopamine depletion and L-Dopa exposure in MPTP-treated macaques.A) Histogram represents optical density measurements of 5-HT 4 R (in arbitrary units) in the posterior putamen for the 3 experimental groups.*p < 0.05 versus control; & p < 0.05 versus parkinsonian.B) [ 11 C]PE2I PET averaged images (in color) on coronal planes at the level of the posterior caudate and putamen for each condition.C) Histogram represents 11 C-PE2I BP ND in the posterior putamen for each condition.D) [ 11 C]SB207145 PET averaged images (in color) on coronal planes at the level of the posterior caudate and putamen for each condition.E) Histogram represents 11 C-SB207145 BP ND in the posterior putamen for each condition.*p < 0.05, **p < 0.01 versus baseline.Color represents the level of BP ND using the cerebellum as the reference region (red indicates high whereas bleu indicates low BP ND on each scale).a.u., arbitrary units; BP ND , non-displaceable binding potential; Dysk, dyskinetic; OD, optical density; Park, parkinsonian.