Perturbation of serine enantiomers homeostasis in the striatum of MPTP-lesioned monkeys and mice reflects the extent of dopaminergic midbrain degeneration.

Loss of dopaminergic midbrain neurons perturbs l-serine and d-serine homeostasis in the post-mortem caudate putamen (CPu) of Parkinson's disease (PD) patients. However, it is unclear whether the severity of dopaminergic nigrostriatal degeneration plays a role in deregulating serine enantiomers' metabolism. Here, through high-performance liquid chromatography (HPLC), we measured the levels of these amino acids in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys and MPTP-plus-probenecid (MPTPp)-treated mice to determine whether and how dopaminergic midbrain degeneration affects the levels of serine enantiomers in various basal ganglia subregions. In addition, in the same brain regions, we measured the levels of key neuroactive amino acids modulating glutamatergic neurotransmission, including l-glutamate, glycine, l-aspartate, d-aspartate, and their precursors l-glutamine, l-asparagine. In monkeys, MPTP treatment produced severe denervation of nigrostriatal dopaminergic fibers (⁓75%) and increased the levels of serine enantiomers in the rostral putamen (rPut), but not in the subthalamic nucleus, and the lateral and medial portion of the globus pallidus. Moreover, this neurotoxin significantly reduced the protein expression of the astrocytic serine transporter ASCT1 and the glycolytic enzyme GAPDH in the rPut of monkeys. Conversely, concentrations of d-serine and l-serine, as well as ASCT1 and GAPDH expression were unaffected in the striatum of MPTPp-treated mice, which showed only mild dopaminergic degeneration (⁓30%). These findings unveil a link between the severity of dopaminergic nigrostriatal degeneration and striatal serine enantiomers concentration, ASCT1 and GAPDH expression. We hypothesize that the up-regulation of d-serine and l-serine levels occurs as a secondary response within a homeostatic loop to support the metabolic and neurotransmission demands imposed by the degeneration of dopaminergic neurons.

Homeostasis of serine enantiomers is disrupted in the post-mortem caudate putamen and cerebrospinal fluid of living Parkinson's disease patients.

L-serine generated in astrocytes plays a pivotal role in modulating essential neurometabolic processes, while its enantiomer, D-serine, specifically regulates NMDA receptor (NMDAR) signalling. Despite their physiological relevance in modulating cerebral activity, serine enantiomers metabolism in Parkinson's disease (PD) remains elusive. Using High-Performance Liquid Chromatography (HPLC), we measured D- and L-serine levels along with other amino acids known to modulate NMDAR function, such as L-glutamate, L-aspartate, D-aspartate, and glycine, in the post-mortem caudate putamen (CPu) and superior frontal gyrus (SFG) of PD patients. Moreover, we examined these amino acids in the cerebrospinal fluid (CSF) of de novo living PD, Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) patients versus subjects with other neurological disorders (OND), used as control. We found higher D-serine and L-serine levels in the CPu of PD patients but not in the SFG, a cerebral region that, in contrast to the CPu, is not innervated by nigral dopaminergic terminals. We also highlighted a significant elevation of both serine enantiomers in the CSF samples from PD but not in those of AD and ALS patients, compared with control subjects. By contrast, none or only minor changes were found in the amount of other NMDAR modulating amino acids. Our findings identify D-serine and L-serine level upregulation as a biochemical signature associated with nigrostriatal dopaminergic degeneration in PD.

Preliminary finding of a randomized, double-blind, placebo-controlled, crossover study to evaluate the safety and efficacy of 5-hydroxytryptophan on REM sleep behavior disorder in Parkinson's disease.

PURPOSE: Altered serotonergic neurotransmission may contribute to the non-motor features commonly associated with Parkinson's disease (PD) such as sleep disorders. The 5-hydroxytryptophan (5-HTP) is the intermediate metabolite of L-tryptophan in the production of serotonin and melatonin. The purpose of this study was to compare the effects of 5-HTP to placebo on REM sleep behavior disorder (RBD) status in patients with PD. METHODS: A single-center, randomized, double-blind placebo-controlled crossover trial was performed in a selected population of 18 patients with PD and RBD. The patients received a placebo and 50 mg of 5-HTP daily in a crossover design over a period of 4 weeks. RESULTS: 5-HTP produced an increase in the total percentage of stage REM sleep without a related increase of RBD episodes, as well as a marginal, non-significant reduction in both arousal index and wake after sleep onset. The self-reported RBD frequency and clinical global impression (CGI) were improved during 5-HTP and placebo treatment in comparison to baseline. 5-HTP significantly improved our patients' motor experiences of daily living as rated by the Unified Parkinson's Disease Rating Scale (UPDRS) part II. CONCLUSIONS: This study provides evidence that 5-HTP is safe and effective in improving sleep stability in PD, contributing to ameliorate patients' global sleep quality. Larger studies with higher doses and longer treatment duration are needed to corroborate these preliminary findings.

Preclinical Pharmacology of [2-(3-Fluoro-5-Methanesulfonyl-phenoxy)Ethyl](Propyl)amine (IRL790), a Novel Dopamine Transmission Modulator for the Treatment of Motor and Psychiatric Complications in Parkinson Disease.

IRL790 ([2-(3-fluoro-5-methanesulfonylphenoxy)ethyl](propyl)amine, mesdopetam) is a novel compound in development for the clinical management of motor and psychiatric disabilities in Parkinson disease. The discovery of IRL790 was made applying a systems pharmacology approach based on in vivo response profiling. The chemical design idea was to develop a new type of DA D3/D2 receptor type antagonist built on agonist rather than antagonist structural motifs. We hypothesized that such a dopamine antagonist with physicochemical properties similar to agonists would exert antidyskinetic and antipsychotic effects in states of dysregulated dopaminergic signaling while having little negative impact on physiologic dopamine transmission and, hence, minimal liability for side effects related to dopamine-dependent functions. At the level of in vivo pharmacology, IRL790 displays balancing effects on aberrant motor phenotypes, reducing l-DOPA-induced dyskinesias in the rodent 6-hydroxydopamine lesion model and reducing psychostimulant-induced locomotor hyperactivity elicited by pretreatment with either d-amphetamine or dizocilpine, without negatively impacting normal motor performance. Thus, IRL790 has the ability to normalize the behavioral phenotype in hyperdopaminergic as well as hypoglutamatergic states. Neurochemical and immediate early gene (IEG) response profiles suggest modulation of DA neurotransmission, with some features, such as increased DA metabolites and extracellular DA, shared by atypical antipsychotics and others, such as increased frontal cortex IEGs, unique to IRL790. IRL790 also increases extracellular levels of acetylcholine in the prefrontal cortex and ventral hippocampus. At the receptor level, IRL790 appears to act as a preferential DA D3 receptor antagonist. Computational docking studies support preferential affinity at D3 receptors with an agonist-like binding mode. SIGNIFICANCE STATEMENT: This paper reports preclinical pharmacology along with molecular modeling results on IRL790, a novel compound in clinical development for the treatment of motor and psychiatric complications in advanced Parkinson disease. IRL790 is active in models of perturbed dopaminergic and glutamatergic signaling, including rodent 6-hydroxydopamine l-DOPA-induced dyskinesias and psychostimulant-induced hyperactivity, in a dose range that does not impair normal behavior. This effect profile is attributed to interactions at dopamine D2/D3 receptors, with a 6- to 8-fold preference for the D3 subtype.

VGF peptides as novel biomarkers in Parkinson's disease.

Parkinson's disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra (SN). At disease onset, a diagnosis is often difficult. VGF peptides are abundant in the SN and peripheral circulation; hence, we investigate whether their plasma profile may reflect the brain dopamine reduction. Using antibodies against the VGF C-terminal portion, we analyzed the rat brain and human plasma, with immunohistochemistry and ELISA. Rats were unilaterally lesioned with 6-hyroxydopamine and sacrificed either 3 or 6 weeks later with or without levodopa treatment. Plasma samples were obtained from PD patients, either at the time of diagnosis (group 1, drug naïve, n = 23) or upon dopamine replacement (group 2, 1-6 years, n = 24; group 3, > 6 years, n = 16), compared with age-matched control subjects (group 4, n = 21). Assessment of the olfactory function was carried out in group 2 using the "Sniffin' Sticks" test. VGF immunoreactivity was present in GABAergic neurons and, on the lesioned side, it was reduced at 3 weeks and abolished at 6 weeks after lesion. Conversely, upon levopoda, VGF labeling was restored. In PD patients, VGF levels were reduced at the time of diagnosis (1504 ± 587 vs. 643 ± 348 pmol/mL, means ± S.E.M: control vs. naïve; p < 0.05) but were comparable with the controls after long-term drug treatment (> 6 years). A linear correlation was demonstrated between VGF immunoreactivity and disease duration, levodopa equivalent dose and olfactory dysfunction. Plasma VGF levels may represent a useful biomarker, especially in the early stages of PD.

5alpha-reductase inhibitors dampen L-DOPA-induced dyskinesia via normalization of dopamine D1-receptor signaling pathway and D1-D3 receptor interaction.

Although 1-3,4-dihydroxyphenylalanine (L-DOPA) is the mainstay therapy for treating Parkinson's disease (PD), its long-term administration is accompanied by the development of motor complications, particularly L-DOPA induced dyskinesia (LID), that dramatically affects patients' quality of life. LID has consistently been related to an excessive dopamine receptor transmission, particularly at the down-stream signaling of the striatal D1 receptors (D1R), resulting in an exaggerated stimulation of cAMP-dependent protein kinase and extracellular signal-regulated kinase (ERK) pathway. We previously reported that pharmacological blockade of 5alpha-reductase (5AR), the rate-limiting enzyme in neurosteroids synthesis, attenuates the severity of a broad set of behavioral alterations induced by D1R and D3R activation, without inducing extrapyramidal symptoms. In line with this evidence, in a recent study, we found that inhibition of 5AR by finasteride (FIN) produced a significant reduction of dyskinesia induced by L-DOPA and direct dopaminergic agonists in 6-OHDA-lesioned rats. In the attempt to further investigate the effect of 5AR inhibitors on dyskinesia and shed light on the mechanism of action, in the present study we compared the effect of FIN and dutasteride (DUTA), a potent dual 5AR inhibitor, on the development of LID, on the therapeutic efficacy of L-DOPA, on the molecular alterations downstream to the D1R, as well as on D1R-D3R interaction. The results indicated that both FIN and DUTA administration significantly reduced development and expression of LID; however, DUTA appeared more effective than FIN at a lower dose and produced its antidyskinetic effect without impacting the ability of L-DOPA to increase motor activation, or ameliorate forelimb use in parkinsonian rats. Moreover, this study demonstrates for the first time that 5AR inhibitors are able to prevent key events in the appearance of dyskinesia, such as L-DOPA-induced upregulation of striatal D1R-related cAMP/PKA/ERK signaling pathways and D1R-D3R coimmunoprecipitation, an index of heteromer formation. These findings are relevant as they confirm the 5AR enzyme as a potential therapeutic target for treatment of dyskinesia in PD, suggesting the first ever evidence that neurosteroidogenesis may affect functional interaction between dopamine D1R and D3R.

The serotonergic system in L-DOPA-induced dyskinesia: pre-clinical evidence and clinical perspective.

During the last decade, the serotonergic system has emerged as a key player in the appearance of L-DOPA-induced dyskinesia in animal models of Parkinson's disease. Clinical investigations, based on imaging and postmortem analyses, suggest that the serotonin neurons are also involved in the etiology of this complication of long-term L-DOPA treatment in parkinsonian patients. These findings have stimulated efforts to develop new therapies using drugs targeting the malfunctioning serotonin neurons. In this review, we summarize the experimental and clinical data obtained so far and discuss the prospects for further development of this therapeutic strategy.

Modulation of serotonergic transmission by eltoprazine in L-DOPA-induced dyskinesia: Behavioral, molecular, and synaptic mechanisms.

L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias (LIDs) represent the main side effect of Parkinson's Disease (PD) therapy. Among the various pharmacological targets for novel therapeutic approaches, the serotonergic system represents a promising one. In experimental models of PD and in PD patients the development of abnormal involuntary movements (AIMs) and LIDs, respectively, is accompanied by the impairment of bidirectional synaptic plasticity in key structures such as striatum. Recently, it has been shown that the 5-HT1A/1B receptor agonist, eltoprazine, significantly decreased LIDs in experimental PD and human patients. Despite the fact that several papers have tested this and other serotonergic drugs, nothing is known about the electrophysiological consequences on this combined serotonin receptors modulation at striatal neurons. The present study demonstrates that activation of 5-HT1A/1B receptors reduces AIMs via the restoration of Long-Term Potentiation (LTP) and synaptic depotentiation in a sub-set of striatal spiny projection neurons (SPNs). This recovery is associated with the normalization of D1 receptor-dependent cAMP/PKA and ERK/mTORC signaling pathways, and the recovery of NMDA receptor subunits balance, indicating these events as key elements in AIMs induction. Moreover, we analyzed whether the manipulation of the serotonergic system might affect motor behavior and cognitive performances. We found that a defect in locomotor activity in parkinsonian and L-DOPA-treated rats was reversed by eltoprazine treatment. Conversely, the impairment in the striatal-dependent learning was found exacerbated in L-DOPA-treated rats and eltoprazine failed to recover it.

Antidyskinetic effect of A2A and 5HT1A/1B receptor ligands in two animal models of Parkinson's disease.

BACKGROUND: The serotonin 5-HT1A/1B receptor agonist eltoprazine suppressed dyskinetic-like behavior in animal models of Parkinson's disease (PD) but simultaneously reduced levodopa (l-dopa)-induced motility. Moreover, adenosine A2A receptor antagonists, such as preladenant, significantly increased l-dopa efficacy in PD without exacerbating dyskinetic-like behavior. OBJECTIVES: We evaluated whether a combination of eltoprazine and preladenant may prevent or suppress l-dopa-induced dyskinesia, without impairing l-dopa's efficacy in relieving motor signs, in 2 PD models: unilateral 6-hydroxydopamine-lesioned rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. METHODS: Rotational behavior and abnormal involuntary movements, or disability and l-dopa-induced dyskinesia were evaluated in 6-hydroxydopamine-lesioned rats and MPTP-treated monkeys, respectively. Moreover, in the rodent striatum, induction of immediate-early gene zif-268, an index of long-term changes, was correlated with dyskinesia. RESULTS: In 6-hydroxydopamine-lesioned rats, combined administration of l-dopa (4 mg/kg) plus eltoprazine (0.6 mg/kg) plus preladenant (0.3 mg/kg) significantly prevented or reduced dyskinetic-like behavior without impairing motor activity. Zif-268 was increased in the striatum of rats treated with l-dopa and l-dopa plus preladenant compared with vehicle. In contrast, rats treated with eltoprazine (with or without preladenant) had lower zif-268 activation after chronic treatment in both the dyskinetic and l-dopa-non-primed groups. Moreover, acute l-dopa plus eltoprazine plus preladenant prevented worsening of motor performance (adjusting step) and sensorimotor integration deficit. Similar results were obtained in MPTP-treated monkeys, where a combination of preladenant with eltoprazine was found to counteract dyskinesia and maintain the full therapeutic effects of a low dose of l-dopa. CONCLUSIONS: Our results suggest a promising nondopaminergic pharmacological strategy for the treatment of dyskinesia in PD. © 2016 International Parkinson and Movement Disorder Society.

The anti-dyskinetic effect of dopamine receptor blockade is enhanced in parkinsonian rats following dopamine neuron transplantation.

Graft-induced dyskinesia (GID) is a serious complication induced by dopamine (DA) cell transplantation in parkinsonian patients. We have recently shown that DA D2 receptor blockade produces striking blockade of dyskinesia induced by amphetamine in grafted 6-OHDA-lesioned rats, a model of GID. This study was designed to investigate whether blockade of DA D1 receptors could produce similar outcome, and to see whether the effect of these treatments in grafted rats was specific for dyskinesia induced by amphetamine, or could also influence L-DOPA-induced dyskinesia (LID). L-DOPA-primed rats received transplants of fetal DA neurons into the DA-denervated striatum. Beginning at 20weeks after transplantation rats were subjected to pharmacological treatments with either L-DOPA (6mg/kg) or amphetamine (1.5mg/kg) alone, or in combination with the D1 receptor antagonist SCH23390, the D2 receptor antagonist eticlopride, and the 5-HT1A agonist/D2 receptor antagonist buspirone. Grafted rats developed severe GID, while LID was reduced. Both eticlopride and SCH23390 produced near-complete suppression of GID already at very low doses (0.015 and 0.1mg/kg, respectively). Buspirone induced similar suppression at a dose as low as 0.3mg/kg, which is far lower than the dose known to affect LID in non-grafted dyskinetic rats. In agreement with our previous results, the effect of buspirone was independent from 5-HT1A receptor activation, as it was not counteracted by the selective 5-HT1A antagonist WAY100635, but likely due to D2 receptor blockade. Most interestingly, the same doses of eticlopride, SCH23390 and buspirone were found to suppress LID in grafted but not in control dyskinetic rats. Taken together, these data demonstrate that the DA cell grafts strikingly exacerbate the effect of DA D1 and D2 receptor blockade against both GID and LID, and suggest that the anti-GID effect of buspirone seen in patients may also be due to blockade of DA D2 receptors.

The chronic use of serotonin norepinephrine reuptake inhibitors facilitates dyskinesia priming in early Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) patients are frequently exposed to antidepressant medications (ADMs). Norepinephrine (NE) and serotonin (5HT) systems have a role in levodopa-induced dyskinesias (LID) pathophysiology. METHODS: We performed a longitudinal analysis on the PPMI cohort including drug-naïve PD patients, who are progressively exposed to dopamine replacement therapies (DRTs) to test the effect of ADM exposure on LID development by the 4th year of follow-up. RESULTS: LID prevalence (according to MDS UPDRS score 4.1 ≥ 1) was 16% (42/251); these patients were more likely women (p = 0.01), had higher motor (p < 0.001) and depression scores (p = 0.01) and lower putaminal DAT binding ratio (p = 0.01). LID were associated with the exposure time to L-DOPA (2.2 ± 1.07 vs 2.6 ± 0.9, p = 0.02) and to the exposure to ADMs, in particular to SNRI (4.8% vs 21.4%, p < 0.001). The latter persisted after correcting for significant covariates (e.g., disease duration, cognitive status, motor impairment, depression, dopaminergic denervation). A similar difference in LID prevalence in PD patients exposed vs non-exposed to SNRI was observed on matched data by the real-world TriNetX repository (22% vs 13%, p < 0.001). DISCUSSION: This study supports the presence of an effect of SNRI on LID priming in patients with early PD. Independent prospective cohort studies are warranted to further verify such association.

5-Hydroxy-tryptophan for the treatment of L-DOPA-induced dyskinesia in the rat Parkinson's disease model.

The serotonin system has recently emerged as an important player in the appearance of L-DOPA-induced dyskinesia (LID) in experimental models of Parkinson's disease, as it provides an unregulated source of L-DOPA-derived dopamine release in the dopamine-depleted striatum. Accordingly, toxin lesion or pharmacological silencing of serotonin neurons suppressed LID in the rat and monkey models of Parkinson's disease. However, 5-HT1 receptor agonists were also found to partially reduce the therapeutic effect of L-DOPA. In this study, we evaluated whether enhancement of the serotonin tone induced by the administration of the serotonin precursor 5-hydroxy-tryptophan (5-HTP) could affect induction and expression of LID, as well as the therapeutic effect of L-DOPA, in 6-OHDA-lesioned rats. Drug naïve and L-DOPA-primed 6-OHDA-lesioned rats were chronically treated with a daily injection of L-DOPA (6 mg/kg plus benserazide, s.c.) alone, or in combination with 5-HTP (24-48 mg/kg, i.p.). The abnormal involuntary movements (AIMs) test, as well as the stepping and the motor activity tests, were performed during the chronic treatments. Results showed that 5-HTP reduced the appearance of LID of about 50% at both tested doses. A partial reduction of the therapeutic effect of L-DOPA was seen with the higher but not with the lower dose of 5-HTP. 5-HTP 24 mg/kg was also able to reduce the expression of dyskinesia in L-DOPA-primed dyskinetic rats, to a similar extent than in L-DOPA-primed rats. Importantly, the antidyskinetic effect of 5-HTP 24 mg/kg does not appear to be due to a competition with L-DOPA for crossing the blood-brain barrier; in fact, similar L-DOPA striatal levels were found in L-DOPA only and L-DOPA plus 5-HTP 24 mg/kg treated animals. These data further confirm the involvement of the serotonin system in the appearance of LID, and suggest that 5-HTP may be useful to counteract the appearance of dyskinesia in Parkinson's disease patients.

Study of the antidyskinetic effect of eltoprazine in animal models of levodopa-induced dyskinesia.

The serotonin (5-hydroxytryptamine [5HT]) system has recently emerged as an important player in the appearance of l-3,4-dihydroxyphenylalanine (levodopa [l-dopa])-induced dyskinesia in animal models of Parkinson's disease. In fact, dopamine released as a false transmitter from serotonin neurons appears to contribute to the pulsatile stimulation of dopamine receptors, leading to the appearance of the abnormal involuntary movements. Thus, drugs able to dampen the activity of serotonin neurons hold promise for the treatment of dyskinesia. The authors investigated the ability of the mixed 5-HT 1A/1B receptor agonist eltoprazine to counteract l-dopa-induced dyskinesia in 6-hydroxydopamine-lesioned rats and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated macaques. The data demonstrated that eltoprazine is extremely effective in suppressing dyskinesia in experimental models, although this effect was accompanied by a partial worsening of the therapeutic effect of l-dopa. Interestingly, eltoprazine was found to (synergistically) potentiate the antidyskinetic effect of amantadine. The current data indicated that eltoprazine is highly effective in counteracting dyskinesia in preclinical models. However, the partial worsening of the l-dopa effect observed after eltoprazine administration represents a concern; whether this side effect is due to a limitation of the animal models or to an intrinsic property of eltoprazine needs to be addressed in ongoing clinical trials. The data also suggest that the combination of low doses of eltoprazine with amantadine may represent a valid strategy to increase the antidyskinetic effect and reduce the eltoprazine-induced worsening of l-dopa therapeutic effects.

Pregnenolone for the treatment of L-DOPA-induced dyskinesia in Parkinson's disease.

Growing preclinical and clinical evidence highlights neurosteroid pathway imbalances in Parkinson's Disease (PD) and L-DOPA-induced dyskinesias (LIDs). We recently reported that 5α-reductase (5AR) inhibitors dampen dyskinesias in parkinsonian rats; however, unraveling which specific neurosteroid mediates this effect is critical to optimize a targeted therapy. Among the 5AR-related neurosteroids, striatal pregnenolone has been shown to be increased in response to 5AR blockade and decreased after 6-OHDA lesions in the rat PD model. Moreover, this neurosteroid rescued psychotic-like phenotypes by exerting marked antidopaminergic activity. In light of this evidence, we investigated whether pregnenolone might dampen the appearance of LIDs in parkinsonian drug-naïve rats. We tested 3 escalating doses of pregnenolone (6, 18, 36 mg/kg) in 6-OHDA-lesioned male rats and compared the behavioral, neurochemical, and molecular outcomes with those induced by the 5AR inhibitor dutasteride, as positive control. The results showed that pregnenolone dose-dependently countered LIDs without affecting L-DOPA-induced motor improvements. Post-mortem analyses revealed that pregnenolone significantly prevented the increase of validated striatal markers of dyskinesias, such as phospho-Thr-34 DARPP-32 and phospho-ERK1/2, as well as D1-D3 receptor co-immunoprecipitation in a fashion similar to dutasteride. Moreover, the antidyskinetic effect of pregnenolone was paralleled by reduced striatal levels of BDNF, a well-established factor associated with the development of LIDs. In support of a direct pregnenolone effect, LC/MS-MS analyses revealed that striatal pregnenolone levels strikingly increased after the exogenous administration, with no significant alterations in downstream metabolites. All these data suggest pregnenolone as a key player in the antidyskinetic properties of 5AR inhibitors and highlight this neurosteroid as an interesting novel tool to target LIDs in PD.

SMN deficiency perturbs monoamine neurotransmitter metabolism in spinal muscular atrophy.

Beyond motor neuron degeneration, homozygous mutations in the survival motor neuron 1 (SMN1) gene cause multiorgan and metabolic defects in patients with spinal muscular atrophy (SMA). However, the precise biochemical features of these alterations and the age of onset in the brain and peripheral organs remain unclear. Using untargeted NMR-based metabolomics in SMA mice, we identify cerebral and hepatic abnormalities related to energy homeostasis pathways and amino acid metabolism, emerging already at postnatal day 3 (P3) in the liver. Through HPLC, we find that SMN deficiency induces a drop in cerebral norepinephrine levels in overt symptomatic SMA mice at P11, affecting the mRNA and protein expression of key genes regulating monoamine metabolism, including aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DßH) and monoamine oxidase A (MAO-A). In support of the translational value of our preclinical observations, we also discovered that SMN upregulation increases cerebrospinal fluid norepinephrine concentration in Nusinersen-treated SMA1 patients. Our findings highlight a previously unrecognized harmful influence of low SMN levels on the expression of critical enzymes involved in monoamine metabolism, suggesting that SMN-inducing therapies may modulate catecholamine neurotransmission. These results may also be relevant for setting therapeutic approaches to counteract peripheral metabolic defects in SMA.

Serotonergic and dopaminergic mechanisms in graft-induced dyskinesia in a rat model of Parkinson's disease.

Dyskinesia seen in the off-state, referred as graft-induced dyskinesia (GID), has emerged as a serious complication induced by dopamine (DA) cell transplantation in parkinsonian patients. Although the mechanism underlying the appearance of GID is unknown, in a recent clinical study the partial 5-HT(1A) agonist buspirone was found to markedly reduce GID in three grafted patients, who showed significant serotonin (5-HT) hyperinnervation in the grafted striatum in positron emission tomography scanning (Politis et al., 2010, 2011). Prompted by these findings, this study was performed to investigate the involvement of serotonin neurons in the appearance of GID in the rat 6-hydroxydopamine model. L-DOPA-primed rats received transplants of DA neurons only, DA plus 5-HT neurons or 5-HT neurons only into the lesioned striatum. In DA cell-grafted rats, with or without 5-HT neurons, but not in 5-HT grafts, GID was observed consistently after administration of amphetamine (1.5mg/kg, i.p.) indicating that grafted DA neurons are required to induce GID. Strikingly, a low dose of buspirone produced a complete suppression of GID. In addition, activation of 5-HT(1A) and 5-HT(1B) receptors by 8-OH-DPAT and CP 94253, known to inhibit the activity of 5-HT neurons, significantly reduced GID, whereas induction of neurotransmitter release by fenfluramine administration significantly increased GID, indicating an involvement of the 5-HT system in the modulation of GID. To investigate the involvement of the host 5-HT system in GID, the endogenous 5-HT terminals were removed by intracerebral injection of 5,7-dihydroxytryptamine, but this treatment did not affect GID expression. However, 5-HT terminal destruction suppressed the anti-GID effect of 5-HT(1A) and 5-HT(1B) agonists, demonstrating that the 5-HT(1) agonist combination exerted its anti-GID effect through the activation of pre-synaptic host-derived receptors. By contrast, removal of the host 5-HT innervation or pre-treatment with a 5-HT(1A) antagonist did not abolish the anti-GID effect of buspirone, showing that its effect is independent from activation of either pre- or post-synaptic 5-HT(1A) receptors. Since buspirone is known to also act as a DA D(2) receptor antagonist, the selective D(2) receptor antagonist eticlopride was administered to test whether blockade of D(2) receptors could account for the anti-dyskinetic effect of buspirone. In fact, eticlopride produced complete suppression of GID in grafted animals already at very low dose. Together, these results point to a critical role of both 5-HT(1) and D(2) receptors in the modulation of GID, and suggest that 5-HT neurons exert a modulatory role in the development of this side effect of neuronal transplantation.

Nusinersen Induces Disease-Severity-Specific Neurometabolic Effects in Spinal Muscular Atrophy.

Intrathecal delivery of Nusinersen-an antisense oligonucleotide that promotes survival motor neuron (SMN) protein induction-is an approved therapy for spinal muscular atrophy (SMA). Here, we employed nuclear magnetic resonance (NMR) spectroscopy to longitudinally characterize the unknown metabolic effects of Nusinersen in the cerebrospinal fluid (CSF) of SMA patients across disease severity. Modulation of amino acid metabolism is a common denominator of biochemical changes induced by Nusinersen, with distinct downstream metabolic effects according to disease severity. In severe SMA1 patients, Nusinersen stimulates energy-related glucose metabolism. In intermediate SMA2 patients, Nusinersen effects are also related to energy homeostasis but involve ketone body and fatty acid biosynthesis. In milder SMA3 patients, Nusinersen mainly modulates amino acid metabolism. Moreover, Nusinersen modifies the CSF metabolome of a more severe clinical group towards the profile of untreated SMA patients with milder disease. These findings reveal disease severity-specific neurometabolic signatures of Nusinersen treatment, suggesting a selective modulation of peripheral organ metabolism by this CNS-directed therapy in severe SMA patients.

Role of aberrant striatal dopamine D1 receptor/cAMP/protein kinase A/DARPP32 signaling in the paradoxical calming effect of amphetamine.

Attention deficit/hyperactivity disorder (ADHD) is characterized by inattention, impulsivity, and motor hyperactivity. Several lines of research support a crucial role for the dopamine transporter (DAT) gene in this psychiatric disease. Consistently, the most commonly prescribed medications in ADHD treatment are stimulant drugs, known to preferentially act on DAT. Recently, a knock-in mouse [DAT-cocaine insensitive (DAT-CI)] has been generated carrying a cocaine-insensitive DAT that is functional but with reduced dopamine uptake function. DAT-CI mutants display enhanced striatal extracellular dopamine levels and basal motor hyperactivity. Herein, we showed that DAT-CI animals present higher striatal dopamine turnover, altered basal phosphorylation state of dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP32) at Thr75 residue, but preserved D(2) receptor (D(2)R) function. However, although we demonstrated that striatal D(1) receptor (D(1)R) is physiologically responsive under basal conditions, its stimulus-induced activation strikingly resulted in paradoxical electrophysiological, behavioral, and biochemical responses. Indeed, in DAT-CI animals, (1) striatal LTP was completely disrupted, (2) R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) treatment induced paradoxical motor calming effects, and (3) SKF 81297 administration failed to increase cAMP/protein kinase A (PKA)/DARPP32 signaling. Such biochemical alteration selectively affected dopamine D(1)Rs since haloperidol, by blocking the tonic inhibition of D(2)R, unmasked a normal activation of striatal adenosine A(2A) receptor-mediated cAMP/PKA/DARPP32 cascade in mutants. Most importantly, our studies highlighted that amphetamine, nomifensine, and bupropion, through increased striatal dopaminergic transmission, are able to revert motor hyperactivity of DAT-CI animals. Overall, our results suggest that the paradoxical motor calming effect induced by these drugs in DAT-CI mutants depends on selective aberrant phasic activation of D(1)R/cAMP/PKA/DARPP32 signaling in response to increased striatal extracellular dopamine levels.