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Degeneration of nigrostriatal dopaminergic (DA) neurons in the the substantia nigra pars compacta (SNc) leads an imbalance in the basal ganglia circuitry that results in Parkinson diseases (PD) motor abnormalities. Increased glutamatergic transmission in the basal ganglia circuits is a key pathophysiological feature of PD. The protection of midbrain DA neuron against N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity in mGluR5 knockout mice combined with the antiparkinsonian effects of mGluR5 antagonist in rodent models of PD suggest that mGluR5 antagonist may be an alternative therapeutic target for PD1,3. In the present study, we tested the neuroprotective effect of the mGluR5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), against MPTP toxicity of SNc neurons in the monkey model of PD. Four rhesus monkeys were divided into three groups (1 control, 1 MPTP-treated, and 2 MPTP/MTEP-treated). Monkeys received (0.2-0.5 mg/kg) MPTP once a week until they exhibited PD symptoms. MTEP-treated group received a daily dose of MTEP (10mg/kg), whereas the MPTP-treated group received vehicle. Postmortem midbrain tissue from the different groups was immunostained for tyrosine hydroxylase (TH) to quantify SNc DA neurons using unbiased stereological methods. Our results show a significant reduction in the number of TH-positive neurons in MPTP-treated monkeys [95,880] compared to controls [193,311] (P<0.05) and MTEP/MPTP-treated animals [136,272]. These observations were further correlated with the level of dopamine transporter binding (18F-FECNT) in the SN, as revealed with in vivo position emission tomography (PET). These preliminary data, therefore, indicate a strong potential for MTEP as non-dopaminergic neuroprotective therapy for PD.
Parkinson's disease (PD) is a chronic, progressive movement disorder that is the second most common neurodegenerative disease with a lifetime risk of 1 in 453.
Environmental factors, genetic factors or a combination of both may elevate the risk for PD. The majority of PD patients are thought to be idiopathic.
The pathological hallmark of PD is the loss of dopaminergic neurons in the substantia nigra pars compacta that eventually reduces dopaminergic output to the striatum.
Decreased striatal dopamine levels induce an imbalance in the basal ganglia circuitry that results in PD motor abnormalities.
STN output pathways become hyperactive as a consequence of striatal dopamine (DA) depletion. Hyperactivity of the STN neurons contributes to the progressive degeneration of nigral dopaminergic neurons via glutamate excitotoxicity.
Modulation of abnormal glutamate transmission provides opportunity for alternative neuroprotective therapies
Glutamate transmission is mediated via 2 types of receptors: the metabotropic glutamate receptors (mGluRs) play a modulatory role in shaping synaptic transmission and neuronal excitability as opposed to ionotropic glutamate receptors (iGluRs) which play the primary role in mediating fast synaptic transmission.
A promising drug target in PD is the mGluR5 receptor, which is distributed throughout the basal ganglia.
mGluR5 knockout mice are protected against MPTP-induced degeneration of midbrain dopaminergic neurons, the mouse model of PD.1
In addition, mGluR5 antagonist produce antiparkinsonian effects in rodent models of PD.3
Test the neuroprotective efficacy of the novel mGluR5 specific antagonist, MTEP in the gold standard MPTP monkey model of Parkinson’s disease.
Validate the [18] FECNT radioligand as a biomarker in PD.
In this study four female Rhesus monkeys (Macaca mulatta) (5.38–6.59 kg, 4 -5 years) were used. The four monkeys were divided into three groups:
i) one control (no MPTP)
ii) one MPTP treated
iii) two MTEP therapy (MTEP + MPTP).
In the MTEP therapy group monkeys received MTEP (10mg/Kg) 30 days prior to the beginning of the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment and continued up to the end the experiments. In contrast, MPTP alone treated group received vehicle. Besides the control monkey, all the three monkeys received i.m injection of 0.2-0.5 mg/kg, (MPTP) hydrochloride (Sigma-Aldrich, St Louis, MO, USA) once per week. After 24 weeks the MPTP alone monkey showed parkinsonian symptoms and experiments were ceased. The monkeys were then deeply anesthetized with an overdose of pentobarbital and perfused transcardially.
Tissue sections (60 um thick) obtained were from the monkey brains with a Vibratome. A systematic random series of every twelfth section was immunostained for tyrosine hydroxylase (TH) using Chemicon mouse antibody (1:1000). An adjacent series of every twelfth section was stained for calibindin D28K (CB). Two series (right and left hemispheres) were processed for each animal.
Corresponding adjacent TH and CB sections from each series were compared in order to delineate the regions of interest based on the differential expression of calbindin, a calcium buffering protein. The SNcV is characterized by the absence of CB-positive neurons.
Each of the 3 regions of interest were quantified for TH-stained dopaminergic neurons by an investigator blind to treatment using computer-assisted light microscopy and the StereoInvestigator program. An optical fractionator probe with a grid size of 249x413 um, a counting frame of 65x65 um, and an optical dissector height of 37 um with a guard of 2 um was run at 100x oil magnification.
The mounted tissue thickness was entered as 50um. The optical fractionator generated an estimated total number of DA neurons for each ROI. The total neuronal cell count data generated by the optical fractionator were then compared to the collected endpoint PET scan images. [18]FECNT, a DAT ligand, was used to monitor SNc dopaminergic loss during the course of the MPTP treatment.
In the SNcV we found a significant reduction in the number of TH-positive neurons in MPTP-treated monkeys [95,880] compared to controls [193,311] (P<0.05)
However, in the VTA and SNcD we did not find significant degeneration of DA neurons.
Our data indicates that by intervening before the onset of SNc DA degeneration, we were able to postpone the onset of parkinsonian symptoms in the MTEP therapy group.
The significant correlation between the postmortem DA neuron numbers and the in vivo PET data indicates that the [18]FECNT DAT-ligand could serve as a reliable biomarker for the progression of SNc DA degeneration in Parkinson’s disease.
Our preliminary data indicate a strong potential for MTEP as a non-dopaminergic neuroprotective therapy for PD.
More animals needs to be tested so that we can perform thorough statistical analysis to validate our preliminary findings.
In the future, we will test if MTEP intervention after the onset of DA degeneration confers neuroprotective properties.
We also will compare the quantitative data with the behavioral data to determine whether increased total SnCV DA neurons correlate with an improved PD score.
The authors would like to thank the Howard Hughes Medical Institute, the National Parkinson’s Foundation, and the Emory University SURE program. In addition we would like to thank Susan Maxson and Jean Francois Pare for their technical assistance.
-Battaglia G, Busceti LC, Molinaro G, Biagioni F, Storto M, Fornai F, Nicoletti F, Bruno V (2004) Endogenous activation of mGlu5 metabotropic glutamate receptors contributes to the development of nigro-striatal damage induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. J Neurosci 24:828–835.
-Shapira, A., & Olanow, C. (2004). Neuroprotection in Parkinson Disease: Mysteries, Myths, and Misconceptions. JAMA , 291 (3), 358-364.
-Spooren, W. & F. Gasparin (2004) mGlu5 receptor antagonists: a novel class of anxiolytics? Drug News Perspect., 17, 251–257.
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