[3H]-NFPS binding to the glycine transporter 1 in the hemi-parkinsonian rat brain.

L-3,4-dihydroxyphenylalanine (L-DOPA) is the main treatment for Parkinson's disease (PD) but with long term administration, motor complications such as dyskinesia are induced. Glycine transporter 1 (GlyT1) inhibition was shown to produce an anti-dyskinetic effect in parkinsonian rats and primates, coupled with an improvement in the anti-parkinsonian action of L-DOPA. The expression of GlyT1 in the brain in the dyskinetic state remains to be investigated. Here, we quantified the levels of GlyT1 across different brain regions using [3H]-NFPS in the presence of Org-25,935. Brain sections were chosen from sham-lesioned rats, L-DOPA-naïve 6-hydroxydopamine (6-OHDA)-lesioned rats and 6-OHDA-lesioned rats exhibiting mild or severe abnormal involuntary movements (AIMs). [3H]-NFPS binding decreased in the ipsilateral and contralateral thalamus, by 28% and 41%, in 6-OHDA-lesioned rats with severe AIMs compared to sham-lesioned animals (P < 0.01 and 0.001). [3H]-NFPS binding increased by 21% in the ipsilateral substantia nigra of 6-OHDA-lesioned rats with severe AIMs compared to 6-OHDA-lesioned rats with mild AIMs (P < 0.05). [3H]-NFPS binding was lower by 19% in the contralateral primary motor cortex and by 20% in the contralateral subthalamic nucleus of 6-OHDA-lesioned rats with mild AIMs animals compared to rats with severe AIMs (both P < 0.05). The severity of AIMs scores positively correlated with [3H]-NFPS binding in the ipsilateral substantia nigra (P < 0.05), ipsilateral entopeduncular nucleus (P < 0.05) and contralateral primary motor cortex (P < 0.05). These data provide an anatomical basis to explain the efficacy of GlyT1 inhibitors in dyskinesia in PD.

A biophysical model of the cortex-basal ganglia-thalamus network in the 6-OHDA lesioned rat model of Parkinson's disease.

Electrical stimulation of sub-cortical brain regions (the basal ganglia), known as deep brain stimulation (DBS), is an effective treatment for Parkinson's disease (PD). Chronic high frequency (HF) DBS in the subthalamic nucleus (STN) or globus pallidus interna (GPi) reduces motor symptoms including bradykinesia and tremor in patients with PD, but the therapeutic mechanisms of DBS are not fully understood. We developed a biophysical network model comprising of the closed loop cortical-basal ganglia-thalamus circuit representing the healthy and parkinsonian rat brain. The network properties of the model were validated by comparing responses evoked in basal ganglia (BG) nuclei by cortical (CTX) stimulation to published experimental results. A key emergent property of the model was generation of low-frequency network oscillations. Consistent with their putative pathological role, low-frequency oscillations in model BG neurons were exaggerated in the parkinsonian state compared to the healthy condition. We used the model to quantify the effectiveness of STN DBS at different frequencies in suppressing low-frequency oscillatory activity in GPi. Frequencies less than 40 Hz were ineffective, low-frequency oscillatory power decreased gradually for frequencies between 50 Hz and 130 Hz, and saturated at frequencies higher than 150 Hz. HF STN DBS suppressed pathological oscillations in GPe/GPi both by exciting and inhibiting the firing in GPe/GPi neurons, and the number of GPe/GPi neurons influenced was greater for HF stimulation than low-frequency stimulation. Similar to the frequency dependent suppression of pathological oscillations, STN DBS also normalized the abnormal GPi spiking activity evoked by CTX stimulation in a frequency dependent fashion with HF being the most effective. Therefore, therapeutic HF STN DBS effectively suppresses pathological activity by influencing the activity of a greater proportion of neurons in the output nucleus of the BG.

Autoradiographic labelling of metabotropic glutamate type 2/3 receptors in the hemi-parkinsonian rat brain.

L-3,4-dihydroxyphenylalanine (L-DOPA) is the treatment of choice for Parkinson's disease (PD) motor symptoms, but its chronic use is hindered by complications such as dyskinesia. Pre-clinical studies discovered that activation of metabotropic glutamate type 2 and 3 (mGlu2/3) receptors alleviates L-DOPA-induced dyskinesia. To gain mechanistic insight into the anti-dyskinetic activity of mGlu2/3 activation, we performed autoradiographic binding with [3H]-LY-341,495 in brain sections from L-DOPA-treated 6-hydroxydopamine (6-OHDA)-lesioned rats that developed mild or severe dyskinesia, as well as L-DOPA-untreated 6-OHDA-lesioned and sham-lesioned animals. In the ipsilateral hemisphere, mildly dyskinetic 6-OHDA-lesioned rats showed a decrease in [3H]-LY-341,495 binding in the entopeduncular nucleus (EPN, 30 % vs sham-lesioned rats, P<0.05), globus pallidus (GP, 28 % vs sham-lesioned rats, P<0.05; 23 % vs L-DOPA-untreated 6-OHDA-lesioned rats, P<0.001), and primary motor cortex (49 % vs sham-lesioned rats, P<0.05; 45 % vs L-DOPA-untreated 6-OHDA-lesioned rats, P<0.001). Severely dyskinetic 6-OHDA-lesioned rats exhibited an increase in binding in the primary motor cortex (43 % vs mildly dyskinetic 6-OHDA-lesioned rats, P<0.05). In the contralateral hemisphere, mildly dyskinetic 6-OHDA-lesioned rats harboured a decrease in binding in the EPN (30 % vs sham-lesioned rats; 24 % vs L-DOPA-untreated 6-OHDA-lesioned rats, both P<0.05), GP (34 % vs sham-lesioned rats, P<0.05; 23 % vs L-DOPA-untreated 6-OHDA-lesioned rats, P<0.001), and primary motor cortex (50 % vs sham-lesioned rats; 44 % vs L-DOPA-untreated 6-OHDA-lesioned rats, both P<0.05). Severely dyskinetic 6-OHDA-lesioned rats presented a decrease in binding in the GP (30 % vs sham-lesioned rats; 19 % vs L-DOPA-untreated 6-OHDA-lesioned rats, both P<0.05). Abnormal involuntary movements scores of 6-OHDA-lesioned animals were positively correlated with [3H]-LY-341,495 binding in the ipsilateral striatum, ipsilateral EPN, ipsilateral primary motor cortex and contralateral primary motor cortex (all P<0.05). These results suggest that alterations in mGlu2/3 receptor levels may be part of an endogenous compensatory mechanism to alleviate dyskinesia.

Design, synthesis and biological evaluation of peptide derivatives of L-dopa as anti-parkinsonian agents.

A series of dipeptide derivatives of L-dopa were synthesized and investigated for their pharmacological activity using the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat as an experimental model of Parkinson's disease. Among them, (S)-isopropyl 2-(2-amino-2-methylpropanamido)-3-(3,4-dihydroxyphenyl)propanoate (4 g) was found to be the most active compound, with 106% AUC activity and 149% peak activity of L-dopa after oral administration.

Effect of glycine transporter 1 inhibition with bitopertin on parkinsonism and L-DOPA induced dyskinesia in the 6-OHDA-lesioned rat.

Dyskinesia remains an unmet need in Parkinson's disease (PD). We have previously demonstrated that glycine transporter 1 (GlyT1) inhibition with ALX-5407 reduces dyskinesia and slightly improves parkinsonism in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned marmoset. Here, we sought to determine the effect of bitopertin, a clinically-tested GlyT1 inhibitor, on parkinsonism and dyskinesia in the 6-hydroxydopamine (6-OHDA)-lesioned rat. To do so, we assessed the effect of bitopertin on parkinsonism as monotherapy and as adjunct to a low dose of L-3,4-dihydroxyphenylalanine (L-DOPA). We then assessed the efficacy of bitopertin on dyskinesia in the context of acute challenge and chronic administration studies. Lastly, we evaluated whether de novo treatment with bitopertin, started concurrently with L-DOPA, would diminish the development of dyskinesia. We discovered that bitopertin (0.3 mg/kg), when administered alone, reduced the severity of parkinsonism by 35% (P < 0.01). As adjunct to a low dose of L-DOPA, bitopertin (3 mg/kg) enhanced the anti-parkinsonian effect of L-DOPA by 36% (P < 0.05). Moreover, the acute addition of bitopertin (0.03 mg/kg) to L-DOPA reduced dyskinesia by 27% (P < 0.001), and there was no tolerance to the anti-dyskinetic benefit after 4 weeks of daily administration. Lastly, bitopertin (0.03 mg/kg) started concurrently with L-DOPA, also attenuated the development of dyskinesia, by 33% (P < 0.01), when compared to L-DOPA alone. Our results suggest that GlyT1 inhibition may simultaneously reduce parkinsonism and L-DOPA-induced dyskinesia and represents a novel approach to treat, possibly prevent, motor complications in PD.

Human Embryonic Stem Cell-Derived Dopaminergic Grafts Alleviate L-DOPA Induced Dyskinesia.

BACKGROUND: First-in-human studies to test the efficacy and safety of human embryonic stem cells (hESC)-derived dopaminergic cells in the treatment of Parkinson's disease (PD) are imminent. Pre-clinical studies using hESC-derived dopamine neuron transplants in rat models have indicated that the benefits parallel those shown with fetal tissue but have thus far failed to consider how ongoing L-DOPA administration might impact on the graft. OBJECTIVE: To determine whether L-DOPA impacts on survival and functional recovery following grafting of hESC-derived dopaminergic neurons. METHODS: Unilateral 6-OHDA lesioned rats were administered with either saline or L-DOPA prior to, and for 18 weeks following surgical implantation of dopaminergic neural progenitors derived from RC17 hESCs according to two distinct protocols in independent laboratories. RESULTS: Grafts from both protocols elicited reduction in amphetamine-induced rotations. Reduced L-DOPA-induced dyskinesia preceded the improvement in amphetamine-induced rotations. Furthermore, L-DOPA had no effect on overall survival (HuNu) or dopaminergic neuron content of the graft (TH positive cells) but did lead to an increase in the number of GIRK2 positive neurons. CONCLUSION: Critically, we found that L-DOPA was not detrimental to graft function, potentially enhancing graft maturation and promoting an A9 phenotype. Early improvement of L-DOPA-induced dyskinesia suggests that grafts may support the handling of exogenously supplied dopamine earlier than improvements in amphetamine-induced behaviours indicate. Given that one of the protocols will be employed in the production of cells for the European STEM-PD clinical trial, this is vital information for the management of patients and achieving optimal outcomes following transplantation of hESC-derived grafts for PD.

Zonisamide attenuates the severity of levodopa-induced dyskinesia via modulation of the striatal serotonergic system in a rat model of Parkinson's disease.

Glutamate, GABA, acetylcholine, dopamine, and serotonin interact with each other to regulate the flow of neural information in the striatum. Serotonin type 1A receptor (5HT1A) is primarily expressed on glutamatergic nerve terminals, and 5HT1B is expressed on GABAergic medium spiny neurons (MSNs). Zonisamide (ZNS) reportedly improves the off period without worsening levodopa-induced dyskinesia (LID) in patients with advanced Parkinson's disease. In this study, LID model rats were prepared by administrating levodopa to unilaterally 6-OHDA-lesioned rats. We analyzed changes in serotonergic neurotransmission of LID model rats to elucidate the relationship between LID and the serotonergic system and pathomechanism of the anti-dyskinetic effects of ZNS. Abnormal involuntary movements (AIMs) were most severe in intermittently levodopa-treated rats but milder in rats intermittently medicated with levodopa and ZNS. Continuously levodopa-infused rats or intermittently ZNS-injected rats did not develop AIMs, and no differences in the expression of brain-derived neurotrophic factor, 5-HT transporter, 5HT1A, and 5HT1B mRNA between the lesioned striatum and normal side were observed. Expression of 5HT1B mRNA was elevated in the lesioned striatum of intermittently levodopa-treated rats, but this elevation was normalized by concomitant use of ZNS. The severity of AIMs was correlated with the ratio of 5HT1B to 5HT1A mRNA expression in the lesioned striatum, indicating that the anti-LID effect of ZNS is based on inhibition via 5HT1B receptors to direct pathway MSNs sensitized by intermittent levodopa treatment. Selectively acting serotonergic drugs, especially those that lower the 5HT1B to 5HT1A ratio, are promising new therapeutic agents to attenuate LID development.

Can adenosine A2A receptor antagonists modify motor behavior and dyskinesia in experimental models of Parkinson's disease?

Current treatment of the motor symptoms of Parkinson's disease (PD) focuses on dopamine replacement therapies. While these treatments are initially highly effective, with long-term use and disease progression, the therapeutic response is often limited by the development of motor complications, dopaminergic side effects, and residual unresponsive motor and non-motor symptoms. An alternative or additive treatment approach may be to target non-dopaminergic receptors within the motor control pathways, which function to modulate basal ganglia output. Adenosine A2A receptors are one potential non-dopaminergic target as they are selectively localized to the basal ganglia and to the indirect output pathway known to modulate the striato-thalamo-cortical loops critical to the expression of the motor symptoms of PD. This paper reviews the preclinical evidence base for the ability of adenosine A2A receptor blockade to influence motor function and modulate dyskinesia expression. There is consensus that adenosine A2A receptor antagonists - administered either as a monotherapy or in combination with l-DOPA or dopamine agonists - improve motor function in both rodent and primate models of PD, and should be effective for treating the motor symptoms of PD in humans. Importantly, the improvements in motor function were seen in the absence of dyskinesia. The introduction of a non-dopaminergic approach to modifying basal ganglia function provides a useful addition to the range of available therapies for treating PD, and there is a rational basis for a drug that focuses on modifying basal ganglia output.