Differential Activation States of Direct Pathway Striatal Output Neurons during l-DOPA-Induced Dyskinesia Development.

l-DOPA-induced dyskinesia (LID) is a debilitating motor side effect arising from chronic dopamine (DA) replacement therapy with l-DOPA for the treatment of Parkinson's disease. LID is associated with supersensitivity of striatal dopaminergic signaling and fluctuations in synaptic DA following each l-DOPA dose, shrinking the therapeutic window. The heterogeneous composition of the striatum, including subpopulations of medium spiny output neurons (MSNs), interneurons, and supporting cells, complicates the identification of cell(s) underlying LID. We used single-nucleus RNA sequencing (snRNA-seq) to establish a comprehensive striatal transcriptional profile during LID development. Male hemiparkinsonian mice were treated with vehicle or l-DOPA for 1, 5, or 10 d, and striatal nuclei were processed for snRNA-seq. Analyses indicated a limited population of DA D1 receptor-expressing MSNs (D1-MSNs) formed three subclusters in response to l-DOPA treatment and expressed cellular markers of activation. These activated D1-MSNs display similar transcriptional changes previously associated with LID; however, their prevalence and transcriptional behavior were differentially influenced by l-DOPA experience. Differentially expressed genes indicated acute upregulation of plasticity-related transcription factors and mitogen-activated protein kinase signaling, while repeated l-DOPA-induced synaptic remodeling, learning and memory, and transforming growth factor-ß (TGF-ß) signaling genes. Notably, repeated l-DOPA sensitized Inhba, an activin subunit of the TGF-ß superfamily, in activated D1-MSNs, and its pharmacological inhibition impaired LID development, suggesting that activin signaling may play an essential role in LID. These data suggest distinct subsets of D1-MSNs become differentially l-DOPA-responsive due to aberrant induction of molecular mechanisms necessary for neuronal entrainment, similar to processes underlying hippocampal learning and memory.

5-HT4R agonism reduces L-DOPA-induced dyskinesia via striatopallidal neurons in unilaterally 6-OHDA lesioned mice.

Parkinson's disease is caused by a selective vulnerability and cell loss of dopaminergic neurons of the Substantia Nigra pars compacta and, consequently, striatal dopamine depletion. In Parkinson's disease therapy, dopamine loss is counteracted by the administration of L-DOPA, which is initially effective in ameliorating motor symptoms, but over time leads to a burdening side effect of uncontrollable jerky movements, termed L-DOPA-induced dyskinesia. To date, no efficient treatment for dyskinesia exists. The dopaminergic and serotonergic systems are intrinsically linked, and in recent years, a role has been established for pre-synaptic 5-HT1a/b receptors in L-DOPA-induced dyskinesia. We hypothesized that post-synaptic serotonin receptors may have a role and investigated the effect of modulation of 5-HT4 receptor on motor symptoms and L-DOPA-induced dyskinesia in the unilateral 6-OHDA mouse model of Parkinson's disease. Administration of RS 67333, a 5-HT4 receptor partial agonist, reduces L-DOPA-induced dyskinesia without altering L-DOPA's pro-kinetic effect. In the dorsolateral striatum, we find 5-HT4 receptor to be predominantly expressed in D2R-containing medium spiny neurons, and its expression is altered by dopamine depletion and L-DOPA treatment. We further show that 5-HT4 receptor agonism not only reduces L-DOPA-induced dyskinesia, but also enhances the activation of the cAMP-PKA pathway in striatopallidal medium spiny neurons. Taken together, our findings suggest that agonism of the post-synaptic serotonin receptor 5-HT4 may be a novel therapeutic approach to reduce L-DOPA-induced dyskinesia.

Antagonism of metabotropic glutamate receptor type 5 prevents levodopa-induced dyskinesia development in a male rat model of Parkinson's disease: Electrophysiological evidence.

Levodopa-induced dyskinesia (LID) is a common complication in patients with advanced Parkinson's disease (PD) undergoing treatment with levodopa. Glutamate receptor antagonists can suppress LID; however, the underlying mechanisms remain unclear. Here, we aimed to evaluate the effect of 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP), a metabotropic glutamate receptor 5 (mGluR5) antagonist, on dyskinesia. We recorded the neuronal activity of the entopeduncular nucleus and examined responses to cortical electric stimulation in the control group (n = 6) and three groups of rats (male PD model). Saline was intraperitoneally administered to dopamine lesioned (DL) rats (n = 6), levodopa/benserazide (L/B) was administered to LID rats (n = 8), and L/B combined with MTEP was administered to MTEP rats (n = 6) twice daily for 14 days. We administered L/B to LID and MTEP rats 48 h after the final administration of MTEP to examine the chronic effect of MTEP. The control and DL groups did not have LID. The MTEP group had less LID than the LID group (p < .01) on day 1 and day 18. The control group had a typical triphasic pattern consisting of early excitation (early-Ex), inhibition, and late excitation (late-Ex). However, the inhibition phase disappeared, was partially observed, and was fully suppressed in the DL, LID, and MTEP groups, respectively. The cortico-striato-entopeduncular pathway is important in the pathophysiology of LID. mGluR5 antagonism suppresses LID progression by preventing physiological changes in the cortico-striato-entopeduncular pathway. Future studies are required to validate these results.

Vitexin Mitigates Haloperidol-Induced Orofacial Dyskinesia in Rats through Activation of the Nrf2 Pathway.

Vitexin (VTX), a C-glycosylated flavone found in various medicinal herbs, is known for its antioxidant, anti-inflammatory, and neuroprotective properties. This study investigated the protective effects of VTX against orofacial dyskinesia (OD) in rats, induced by haloperidol (HPD), along with the neuroprotective mechanisms underlying these effects. OD was induced by administering HPD (1 mg/kg i.p.) to rats for 21 days, which led to an increase in the frequency of vacuous chewing movements (VCMs) and tongue protrusion (TP). VTX (10 and 30 mg/kg) was given intraperitoneally 60 min after each HPD injection during the same period. On the 21st day, following assessments of OD, the rats were sacrificed, and nitrosative and oxidative stress, antioxidant capacity, mitochondrial function, neuroinflammation, and apoptosis markers in the striatum were measured. HPD effectively induced OD, while VTX significantly reduced HPD-induced OD, decreased oxidative stress, enhanced antioxidant capacity, prevented mitochondrial dysfunction, and reduced neuroinflammatory and apoptotic markers in the striatum, and the protective effects of VTX on both behavioral and biochemical aspects of HPD-induced OD were significantly reduced when trigonelline (TGN), an inhibitor of the nuclear factor erythroid-2-related factor 2 (Nrf2)-mediated pathway, was administered. These findings suggest that VTX provides neuroprotection against HPD-induced OD, potentially through the Nrf2 pathway, indicating its potential as a therapeutic candidate for the prevention or treatment of tardive dyskinesia (TD) in clinical settings. However, further detailed research is required to confirm these preclinical findings and fully elucidate VTX's therapeutic potential in human studies.

Downregulation of striatal CaV1.3 inhibits the escalation of levodopa-induced dyskinesia in male and female parkinsonian rats of advanced age.

In the past 25 years, the prevalence of Parkinson's disease (PD) has nearly doubled. Age remains the primary risk factor for PD and as the global aging population increases this trend is predicted to continue. Even when treated with levodopa, the gold standard dopamine (DA) replacement therapy, individuals with PD frequently develop therapeutic side effects. Levodopa-induced dyskinesia (LID), a common side effect of long-term levodopa use, represents a significant unmet clinical need in the treatment of PD. Previously, in young adult (3-month-old) male parkinsonian rats, we demonstrated that the silencing of CaV1.3 (Cacan1d) L-type voltage-gated calcium channels via striatal delivery of rAAV-CaV1.3-shRNA provides uniform protection against the induction of LID, and significant reduction of established severe LID. With the goal of more closely replicating a clinical demographic, the current study examined the effects of CaV1.3-targeted gene therapy on LID escalation in male and female parkinsonian rats of advanced age (18-month-old at study completion). We tested the hypothesis that silencing aberrant CaV1.3 channel activity in the parkinsonian striatum would prevent moderate to severe dyskinesia with levodopa dose escalation. To test this hypothesis, 15-month-old male and female F344 rats were rendered unilaterally parkinsonian and primed with low-dose (3-4 mg/kg) levodopa. Following the establishment of stable, mild dyskinesias, rats received an intrastriatal injection of either the Cacna1d-specific rAAV-CaV1.3-shRNA vector (CAV-shRNA), or the scramble control rAAV-SCR-shRNA vector (SCR-shRNA). Daily (M-Fr) low-dose levodopa was maintained for 4 weeks during the vector transduction and gene silencing window followed by escalation to 6 mg/kg, then to 12 mg/kg levodopa. SCR-shRNA-shRNA rats showed stable LID expression with low-dose levodopa and the predicted escalation of LID severity with increased levodopa doses. Conversely, complex behavioral responses were observed in aged rats receiving CAV-shRNA, with approximately half of the male and female subjects-therapeutic 'Responders'-demonstrating protection against LID escalation, while the remaining half-therapeutic 'Non-Responders'-showed LID escalation similar to SCR-shRNA rats. Post-mortem histological analyses revealed individual variability in the detection of Cacna1d regulation in the DA-depleted striatum of aged rats. However, taken together, male and female therapeutic 'Responder' rats receiving CAV-shRNA had significantly less striatal Cacna1d in their vector-injected striatum relative to contralateral striatum than those with SCR-shRNA. The current data suggest that mRNA-level silencing of striatal CaV1.3 channels maintains potency in a clinically relevant in vivo scenario by preventing dose-dependent dyskinesia escalation in rats of advanced age. As compared to the uniform response previously reported in young male rats, there was notable variability between individual aged rats, particularly females, in the current study. Future investigations are needed to derive the sex-specific and age-related mechanisms which underlie variable responses to gene therapy and to elucidate factors which determine the therapeutic efficacy of treatment for PD.

BDNF/TrkB pathway activation in D1 receptor-expressing striatal projection neurons plays a protective role against L-DOPA-induced dyskinesia.

L-DOPA-induced dyskinesia (LID) is a frequent adverse side effect of L-DOPA treatment in Parkinson's disease (PD). Understanding the mechanisms underlying the development of these motor disorders is needed to reduce or prevent them. We investigated the role of TrkB receptor in LID, in hemiparkinsonian mice treated by chronic L-DOPA administration. Repeated L-DOPA treatment for 10 days specifically increased full-length TrkB receptor mRNA and protein levels in the dopamine-depleted dorsal striatum (DS) compared to the contralateral non-lesioned DS or to the DS of sham-operated animals. Dopamine depletion alone or acute L-DOPA treatment did not significantly increase TrkB protein levels. In addition to increasing TrkB protein levels, chronic L-DOPA treatment activated the TrkB receptor as evidenced by its increased tyrosine phosphorylation. Using specific agonists for the D1 or D2 receptors, we found that TrkB increase is D1 receptor-dependent. To determine the consequences of these effects, the TrkB gene was selectively deleted in striatal neurons expressing the D1 receptor. Mice with TrkB floxed gene were injected with Cre-expressing adeno-associated viruses or crossed with Drd1-Cre transgenic mice. After unilateral lesion of dopamine neurons in these mice, we found an aggravation of axial LID compared to the control groups. In contrast, no change was found when TrkB deletion was induced in the indirect pathway D2 receptor-expressing neurons. Our study suggests that BDNF/TrkB signaling plays a protective role against the development of LID and that agonists specifically activating TrkB could reduce the severity of LID.

Role of P11 through serotonergic and glutamatergic pathways in LID.

Parkinson's disease is a progressive neurodegenerative disorder caused by the degeneration of dopaminergic neurons. This leads to the pathogenesis of multiple basal ganglia-thalamomotor loops and diverse neurotransmission alterations. Dopamine replacement therapy, and on top of that, levodopa and l-3,4-dihydroxyphenylalanine (L-DOPA), is the gold standard treatment, while it develops numerous complications. Levodopa-induced dyskinesia (LID) is well-known as the most prominent side effect. Several studies have been devoted to tackling this problem. Studies showed that metabotropic glutamate receptor 5 (mGluR5) antagonists and 5-hydroxytryptamine receptor 1B (5HT1B) agonists significantly reduced LID when considering the glutamatergic overactivity and compensatory mechanisms of serotonergic neurons after L-DOPA therapy. Moreover, it is documented that these receptors act through an adaptor protein called P11 (S100A10). This protein has been thought to play a crucial role in LID due to its interactions with numerous ion channels and receptors. Lately, experiments have shown successful evidence of the effects of P11 blockade on alleviating LID greater than 5HT1B and mGluR5 manipulations. In contrast, there is a trace of ambiguity in the exact mechanism of action. P11 has shown the potential to be a promising target to diminish LID and prolong L-DOPA therapy in parkinsonian patients owing to further studies and experiments.

Brainstem Modulates Parkinsonism-Induced Orofacial Sensorimotor Dysfunctions.

Parkinson's Disease (PD), treated with the dopamine precursor l-3,4-dihydroxyphenylalanine (L-DOPA), displays motor and non-motor orofacial manifestations. We investigated the pathophysiologic mechanisms of the lateral pterygoid muscles (LPMs) and the trigeminal system related to PD-induced orofacial manifestations. A PD rat model was produced by unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. Abnormal involuntary movements (dyskinesia) and nociceptive responses were determined. We analyzed the immunodetection of Fos-B and microglia/astrocytes in trigeminal and facial nuclei and morphological markers in the LPMs. Hyperalgesia response was increased in hemiparkinsonian and dyskinetic rats. Hemiparkinsonism increased slow skeletal myosin fibers in the LPMs, while in the dyskinetic ones, these fibers decreased in the contralateral side of the lesion. Bilateral increased glycolytic metabolism and an inflammatory muscle profile were detected in dyskinetic rats. There was increased Fos-B expression in the spinal nucleus of lesioned rats and in the motor and facial nucleus in L-DOPA-induced dyskinetic rats in the contralateral side of the lesion. Glial cells were increased in the facial nucleus on the contralateral side of the lesion. Overall, spinal trigeminal nucleus activation may be associated with orofacial sensorial impairment in Parkinsonian rats, while a fatigue profile on LPMs is suggested in L-DOPA-induced dyskinesia when the motor and facial nucleus are activated.

Subregion-Specific Regulation of Dopamine D1 Receptor Signaling in the Striatum: Implication for L-DOPA-Induced Dyskinesia.

The striatum is the main structure of the basal ganglia. The striatum receives inputs from various cortical areas, and its subregions play distinct roles in motor and emotional functions. Recently, striatal maps based on corticostriatal connectivity and striosome-matrix compartmentalization were developed, and we were able to subdivide the striatum into seven subregions. Dopaminergic modulation of the excitability of medium spiny neurons (MSNs) is critical for striatal function. In this study, we investigated the functional properties of dopamine signaling in seven subregions of the striatum from male mice. By monitoring the phosphorylation of PKA substrates including DARPP-32 in mouse striatal slices, we identified two subregions with low D1 receptor signaling: the dorsolateral portion of the intermediate/rostral part (DL-IR) and the intermediate/caudal part (IC). Low D1 receptor signaling in the two subregions was maintained by phosphodiesterase (PDE)10A and muscarinic M4 receptors. In an animal model of 6-hydroxydopamine (6-OHDA)-induced hemi-parkinsonism, D1 receptor signaling was upregulated in almost all subregions including the DL-IR, but not in the IC. When L-DOPA-induced dyskinesia (LID) was developed, D1 receptor signaling in the IC was upregulated and correlated with the severity of LID. Our results suggest that the function of the striatum is maintained through the subregion-specific regulation of dopamine D1 receptor signaling and that the aberrant activation of D1 receptor signaling in the IC is involved in LID. Future studies focusing on D1 receptor signaling in the IC of the striatum will facilitate the development of novel therapeutics for LID.SIGNIFICANCE STATEMENT Recent progress in striatal mapping based on corticostriatal connectivity and striosome-matrix compartmentalization allowed us to subdivide the striatum into seven subregions. Analyses of D1 receptor signaling in the seven subregions identified two unique subregions with low D1 receptor signaling: the dorsolateral portion of the intermediate/rostral part (DL-IR) and the intermediate/caudal part (IC). Aberrant activation of D1 receptor signaling in the IC is involved in L-DOPA-induced dyskinesia (LID). Previous studies of LID have mainly focused on the DL-IR, but not on the IC of the striatum. Future studies to clarify aberrant D1 receptor signaling in the IC are required to develop novel therapeutics for LID.

Striatal Nurr1 Facilitates the Dyskinetic State and Exacerbates Levodopa-Induced Dyskinesia in a Rat Model of Parkinson's Disease.

The transcription factor Nurr1 has been identified to be ectopically induced in the striatum of rodents expressing l-DOPA-induced dyskinesia (LID). In the present study, we sought to characterize Nurr1 as a causative factor in LID expression. We used rAAV2/5 to overexpress Nurr1 or GFP in the parkinsonian striatum of LID-resistant Lewis or LID-prone Fischer-344 (F344) male rats. In a second cohort, rats received the Nurr1 agonist amodiaquine (AQ) together with l-DOPA or ropinirole. All rats received a chronic DA agonist and were evaluated for LID severity. Finally, we performed single-unit recordings and dendritic spine analyses on striatal medium spiny neurons (MSNs) in drug-naïve rAAV-injected male parkinsonian rats. rAAV-GFP injected LID-resistant hemi-parkinsonian Lewis rats displayed mild LID and no induction of striatal Nurr1 despite receiving a high dose of l-DOPA. However, Lewis rats overexpressing Nurr1 developed severe LID. Nurr1 agonism with AQ exacerbated LID in F344 rats. We additionally determined that in l-DOPA-naïve rats striatal rAAV-Nurr1 overexpression (1) increased cortically-evoked firing in a subpopulation of identified striatonigral MSNs, and (2) altered spine density and thin-spine morphology on striatal MSNs; both phenomena mimicking changes seen in dyskinetic rats. Finally, we provide postmortem evidence of Nurr1 expression in striatal neurons of l-DOPA-treated PD patients. Our data demonstrate that ectopic induction of striatal Nurr1 is capable of inducing LID behavior and associated neuropathology, even in resistant subjects. These data support a direct role of Nurr1 in aberrant neuronal plasticity and LID induction, providing a potential novel target for therapeutic development.SIGNIFICANCE STATEMENT The transcription factor Nurr1 is ectopically induced in striatal neurons of rats exhibiting levodopa-induced dyskinesia [LID; a side-effect to dopamine replacement strategies in Parkinson's disease (PD)]. Here we asked whether Nurr1 is causing LID. Indeed, rAAV-mediated expression of Nurr1 in striatal neurons was sufficient to overcome LID-resistance, and Nurr1 agonism exacerbated LID severity in dyskinetic rats. Moreover, we found that expression of Nurr1 in l-DOPA naïve hemi-parkinsonian rats resulted in the formation of morphologic and electrophysiological signatures of maladaptive neuronal plasticity; a phenomenon associated with LID. Finally, we determined that ectopic Nurr1 expression can be found in the putamen of l-DOPA-treated PD patients. These data suggest that striatal Nurr1 is an important mediator of the formation of LID.

Distinct patterns of dyskinetic and dystonic features following D1 or D2 receptor stimulation in a mouse model of parkinsonism.

L-DOPA-induced dyskinesia (LID) is a significant complication of dopamine replacement therapy in Parkinson's disease (PD), and the specific role of different dopamine receptors in this disorder is poorly understood. We set out to compare patterns of dyskinetic behaviours induced by the systemic administration of L-DOPA and D1 or D2 receptor (D1R, D2R) agonists in mice with unilateral 6-hydroxydopamine lesions. Mice were divided in four groups to receive increasing doses of L-DOPA, a D1R agonist (SKF38393), a D2/3 agonist (quinpirole), or a selective D2R agonist (sumanirole). Axial, limb and orofacial abnormal involuntary movements (AIMs) were rated using a well-established method, while dystonic features were quantified in different body segments using a new rating scale. Measures of abnormal limb and trunk posturing were extracted from high-speed videos using a software for markerless pose estimation (DeepLabCut). While L-DOPA induced the full spectrum of dyskinesias already described in this mouse model, SKF38393 induced mostly orofacial and limb AIMs. By contrast, both of the D2-class agonists (quinpirole, sumanirole) induced predominantly axial AIMs. Dystonia ratings revealed that these agonists elicited marked dystonic features in trunk/neck, forelimbs, and hindlimbs, which were overall more severe in sumanirole-treated mice. Accordingly, sumanirole induced pronounced axial bending and hindlimb divergence in the automated video analysis. In animals treated with SKF38393, the only appreciable dystonic-like reaction consisted in sustained tail dorsiflexion and stiffness. We next compared the effects of D1R or D2R selective antagonists in L-DOPA-treated mice, where only the D2R antagonist had a significant effect on dystonic features. Taken together these results indicate that the dystonic components of LID are predominantly mediated by the D2R.

Role of 5-HT1A Receptor in Vilazodone-Mediated Suppression of L-DOPA-Induced Dyskinesia and Increased Responsiveness to Cortical Input in Striatal Medium Spiny Neurons in an Animal Model of Parkinson's Disease.

L-DOPA therapy in Parkinson's disease (PD) is limited due to emerging L-DOPA-induced dyskinesia. Research has identified abnormal dopamine release from serotonergic (5-HT) terminals contributing to this dyskinesia. Selective serotonin reuptake inhibitors (SSRIs) or 5-HT receptor (5-HTr) agonists can regulate 5-HT activity and attenuate dyskinesia, but they often also produce a loss of the antiparkinsonian efficacy of L-DOPA. We investigated vilazodone, a novel multimodal 5-HT agent with SSRI and 5-HTr1A partial agonist properties, for its potential to reduce dyskinesia without interfering with the prokinetic effects of L-DOPA, and underlying mechanisms. We assessed vilazodone effects on L-DOPA-induced dyskinesia (abnormal involuntary movements, AIMs) and aberrant responsiveness to corticostriatal drive in striatal medium spiny neurons (MSNs) measured with in vivo single-unit extracellular recordings, in the 6-OHDA rat model of PD. Vilazodone (10 mg/kg) suppressed all subtypes (axial, limb, orolingual) of AIMs induced by L-DOPA (5 mg/kg) and the increase in MSN responsiveness to cortical stimulation (shorter spike onset latency). Both the antidyskinetic effects and reversal in MSN excitability by vilazodone were inhibited by the 5-HTr1A antagonist WAY-100635, demonstrating a critical role for 5-HTr1A in these vilazodone actions. Our results indicate that vilazodone may serve as an adjunct therapeutic for reducing dyskinesia in patients with PD.

Targeting the cannabinoid receptor CB2 in a mouse model of l-dopa induced dyskinesia.

L-dopa induced dyskinesia (LID) is a debilitating side-effect of the primary treatment used in Parkinson's disease (PD), l-dopa. Here we investigate the effect of HU-308, a cannabinoid CB2 receptor agonist, on LIDs. Utilizing a mouse model of PD and LIDs, induced by 6-OHDA and subsequent l-dopa treatment, we show that HU-308 reduced LIDs as effectively as amantadine, the current frontline treatment. Furthermore, treatment with HU-308 plus amantadine resulted in a greater anti-dyskinetic effect than maximally achieved with HU-308 alone, potentially suggesting a synergistic effect of these two treatments. Lastly, we demonstrated that treatment with HU-308 and amantadine either alone, or in combination, decreased striatal neuroinflammation, a mechanism which has been suggested to contribute to LIDs. Taken together, our results suggest pharmacological treatments with CB2 agonists merit further investigation as therapies for LIDs in PD patients. Furthermore, since CB2 receptors are thought to be primarily expressed on, and signal through, glia, our data provide weight to suggestion that neuroinflammation, or more specifically, altered glial function, plays a role in development of LIDs.

GABA storage and release in the medial globus pallidus in L-DOPA-induced dyskinesia priming.

Levo-dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinson's disease; however, most patients develop uncontrollable abnormal involuntary movements known as L-DOPA-induced dyskinesia. L-DOPA-induced dyskinesia can be reduced by pallidotomy of the medial globus pallidus or pallidal deep brain stimulation, suggesting that the medial globus pallidus plays a significant role in the development of L-DOPA-induced dyskinesia. In the present study, the pathological changes of the medial globus pallidus in L-DOPA-induced dyskinesia were studied in rat models of Parkinson's disease (unilateral 6-hydroxydopamine lesioning) and L-DOPA-induced dyskinesia (L-DOPA injection in Parkinson's disease-model rats twice daily for 2 weeks, confirmed by display of dyskinesia-like abnormal involuntary movements). L-DOPA-induced dyskinesia-model rats displayed medial globus pallidus hypertrophy, enlarged axon terminals surrounding the dendrites of medial globus pallidus neurons, and increased density of synaptic vesicles in enlarged axon terminals on the lesioned side. Synaptic terminal enlargement reversed after discontinuation of L-DOPA. Histological studies revealed the enlarged synaptic terminals were those of GABAergic striatal (direct pathway) neurons. A single injection of L-DOPA enhanced GABA release in the medial globus pallidus on the lesioned side in L-DOPA-induced dyskinesia-model rats compared to Parkinson's disease-model rats. In addition, microinjection of muscimol, a GABAA receptor agonist, into the medial globus pallidus on the lesioned side of Parkinson's disease-model rats induced dyskinesia-like abnormal involuntary movements. Microinjection of bicuculline, a GABAA receptor antagonist, into the medial globus pallidus on the lesioned side alleviated L-DOPA-induced dyskinesia in Parkinson's disease-model rats that had received L-DOPA prior to the microinjection. These results indicate that priming for L-DOPA-induced dyskinesia comprises excessive GABA storage in axon terminals of the direct pathway and that expression of L-DOPA-induced dyskinesia is associated with enhanced GABA release into the medial globus pallidus after L-DOPA dosing and the resultant excessive stimulation of GABAA receptors.

Simultaneous mass spectrometry imaging of multiple neuropeptides in the brain and alterations induced by experimental parkinsonism and L-DOPA therapy.

Neuropeptides are important signalling molecules in the brain and alterations in their expression levels have been linked to neurological disorders such as Parkinson's disease. It is challenging to map neuropeptide changes across and within brain regions because of their low in vivo concentrations and complex post-translational processing. Consequently, the role of neuropeptides in Parkinson's disease is not well understood. Thus, we have developed and evaluated a method to image multiple neuropeptides simultaneously in both rat and primate brain tissue sections by matrix-assisted laser desorption/ionisation mass spectrometry imaging at high lateral resolution. Using a unilateral 6-hydroxydopamine rat model of Parkinson's disease, we imaged changes in enkephalins, dynorphins, tachykinins and neurotensin associated with the dopaminergic denervation and L-DOPA treatment in multiple brain regions. L-DOPA administration significantly affected neuropeptides in the globus pallidus, while neuropeptides in the caudate-putamen were mostly affected by dopamine depletion. Using high lateral resolution imaging, we observed an increase of neurotensin in the dorsal sub-region of the globus pallidus after dopamine depletion. This study highlights the capacity of mass spectrometry imaging to elucidate the dynamics of neuropeptide signalling during Parkinson's disease and its treatment.

Striatal and nigral muscarinic type 1 and type 4 receptors modulate levodopa-induced dyskinesia and striato-nigral pathway activation in 6-hydroxydopamine hemilesioned rats.

Acetylcholine muscarinic receptors (mAChRs) contribute to both the facilitation and inhibition of levodopa-induced dyskinesia operated by striatal cholinergic interneurons, although the receptor subtypes involved remain elusive. Cholinergic afferents from the midbrain also innervate the substantia nigra reticulata, although the role of nigral mAChRs in levodopa-induced dyskinesia is unknown. Here, we investigate whether striatal and nigral M1 and/or M4 mAChRs modulate dyskinesia and the underlying striato-nigral GABAergic pathway activation in 6-hydroxydopamine hemilesioned rats. Reverse microdialysis allowed to deliver the mAChR antagonists telenzepine (M1 subtype preferring), PD-102807 and tropicamide (M4 subtype preferring), as well as the selective M4 mAChR positive allosteric modulator VU0152100 in striatum or substantia nigra, while levodopa was administered systemically. Dyskinetic movements were monitored along with nigral GABA (and glutamate) and striatal glutamate dialysate levels, taken as neurochemical correlates of striato-nigral pathway and cortico-basal ganglia-thalamo-cortical loop activation. We observed that intrastriatal telenzepine, PD-102807 and tropicamide alleviated dyskinesia and inhibited nigral GABA and striatal glutamate release. This was partially replicated by intrastriatal VU0152100. The M2 subtype preferring antagonist AFDX-116, used to elevate striatal acetylcholine levels, blocked the behavioral and neurochemical effects of PD-102807. Intranigral VU0152100 prevented levodopa-induced dyskinesia and its neurochemical correlates whereas PD-102807 was ineffective. These results suggest that striatal, likely postsynaptic, M1 mAChRs facilitate dyskinesia and striato-nigral pathway activation in vivo. Conversely, striatal M4 mAChRs can both facilitate and inhibit dyskinesia, possibly depending on their localization. Potentiation of striatal and nigral M4 mAChR transmission leads to powerful multilevel inhibition of striato-nigral pathway and attenuation of dyskinesia.

Angiotensin AT1 and AT2 receptor heteromer expression in the hemilesioned rat model of Parkinson's disease that increases with levodopa-induced dyskinesia.

BACKGROUND/AIMS: The renin-angiotensin system (RAS) is altered in Parkinson's disease (PD), a disease due to substantia nigra neurodegeneration and whose dopamine-replacement therapy, using the precursor levodopa, leads to dyskinesias as the main side effect. Angiotensin AT1 and AT2 receptors, mainly known for their role in regulating water homeostasis and blood pressure and able to form heterodimers (AT1/2Hets), are present in the central nervous system. We assessed the functionality and expression of AT1/2Hets in Parkinson disease (PD). METHODS: Immunocytochemistry was used to analyze the colocalization between angiotensin receptors; bioluminescence resonance energy transfer was used to detect AT1/2Hets. Calcium and cAMP determination, MAPK activation, and label-free assays were performed to characterize signaling in homologous and heterologous systems. Proximity ligation assays were used to quantify receptor expression in mouse primary cultures and in rat striatal sections. RESULTS: We confirmed that AT1 and AT2 receptors form AT1/2Hets that are expressed in cells of the central nervous system. AT1/2Hets are novel functional units with particular signaling properties. Importantly, the coactivation of the two receptors in the heteromer reduces the signaling output of angiotensin. Remarkably, AT1/2Hets that are expressed in both striatal neurons and microglia make possible that candesartan, the antagonist of AT1, increases the effect of AT2 receptor agonists. In addition, the level of striatal expression increased in the unilateral 6-OH-dopamine lesioned rat PD model and was markedly higher in parkinsonian-like animals that did not become dyskinetic upon levodopa chronic administration if compared with expression in those that became dyskinetic. CONCLUSION: The results indicate that boosting the action of neuroprotective AT2 receptors using an AT1 receptor antagonist constitutes a promising therapeutic strategy in PD.

The Delta-Specific Opioid Glycopeptide BBI-11008: CNS Penetration and Behavioral Analysis in a Preclinical Model of Levodopa-Induced Dyskinesia.

In previous work we evaluated an opioid glycopeptide with mixed µ/δ-opioid receptor agonism that was a congener of leu-enkephalin, MMP-2200. The glycopeptide analogue showed penetration of the blood-brain barrier (BBB) after systemic administration to rats, as well as profound central effects in models of Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesia (LID). In the present study, we tested the glycopeptide BBI-11008 with selective δ-opioid receptor agonism, an analogue of deltorphin, a peptide secreted from the skin of frogs (genus Phyllomedusa). We tested BBI-11008 for BBB-penetration after intraperitoneal (i.p.) injection and evaluated effects in LID rats. BBI-11008 (10 mg/kg) demonstrated good CNS-penetrance as shown by microdialysis and mass spectrometric analysis, with peak concentration levels of 150 pM in the striatum. While BBI-11008 at both 10 and 20 mg/kg produced no effect on levodopa-induced limb, axial and oral (LAO) abnormal involuntary movements (AIMs), it reduced the levodopa-induced locomotor AIMs by 50% after systemic injection. The N-methyl-D-aspartate receptor antagonist MK-801 reduced levodopa-induced LAO AIMs, but worsened PD symptoms in this model. Co-administration of MMP-2200 had been shown prior to block the MK-801-induced pro-Parkinsonian activity. Interestingly, BBI-11008 was not able to block the pro-Parkinsonian effect of MK-801 in the LID model, further indicating that a balance of mu- and delta-opioid agonism is required for this modulation. In summary, this study illustrates another example of meaningful BBB-penetration of a glycopeptide analogue of a peptide to achieve a central behavioral effect, providing additional evidence for the glycosylation technique as a method to harness therapeutic potential of peptides.

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.

NMDA receptor GluN2D subunit participates to levodopa-induced dyskinesia pathophysiology.

In the striatum, specific N-methyl-d-aspartate receptor (NMDAR) subtypes are found in different neuronal cells. Spiny projection neurons (SPNs) are characterized by NMDARs expressing GluN2A and GluN2B subunits, while GluN2D is exclusively detected in striatal cholinergic interneurons (ChIs). In Parkinson's disease (PD), dopamine depletion and prolonged treatment with levodopa (L-DOPA) trigger adaptive changes in the glutamatergic transmission from the cortex to the striatum, also resulting in the aberrant function of striatal NMDARs. While modifications of GluN2A- and GluN2B-NMDARs in SPNs have been extensively documented, only few studies report GluN2D dysfunction in PD and no data are available in L-DOPA-induced dyskinesia (LID). Here we investigate the contribution of a specific NMDAR subtype (GluN2D-NMDAR) to PD and LID, and whether this receptor could represent a candidate for future pharmacological interventions. Our results show that GluN2D synaptic abundance is selectively augmented in the striatum of L-DOPA-treated male parkinsonian rats displaying a dyskinetic phenotype. This event is associated to a dramatic increase in GluN2D binding to the postsynaptic protein scaffold PSD-95. Moreover, immunohistochemistry and electrophysiology experiments reveal that GluN2D-NMDARs are expressed not only by striatal ChIs but also by SPNs in dyskinetic rats. Notably, in vivo treatment with a well-characterized GluN2D antagonist ameliorates the severity of established dyskinesia in L-DOPA-treated animals. Our findings support a role for GluN2D-NMDARs in LID, and they confirm that cell-type and subunit specific modifications of NMDARs underlie the pathophysiology of LID.