Michael W. Jakowec

Time course and morphology of dopaminergic neuronal death caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Mechanisms responsible for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine (DA) neuronal death remain unknown and in mice it is even unclear whether neuronal death does occur. In vitro studies suggest that 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP, kills neurons by apoptosis. Herein, we investigated whether MPTP induces DA neuronal death in vivo in mice and whether the mechanism is that of apoptosis. C57/bl Mice received different doses of MPTP administered in four intraperitoneal injections every 2 hours and were sacrificed at different time points for analyses of tyrosine hydroxylase (TH) immunohistochemistry, silver staining, and Nissl staining within the mesencephalon. We found that MPTP induces neuronal destruction in the substantia nigra pars compacta (SNpc) and the ventral tegmental area (VTA). The active phase of degeneration began at 12 h postinjection and continued up to 4 days. During this period, there was a greater decrease in TH-defined neurons than in Nissl-stained neurons suggesting that MPTP can cause a loss in TH without necessarily destroying the neuron. Thereafter, neuronal counts by both techniques equalized and there was no further loss of DA neurons. Dying neurons showed shrunken eosinophilic cytoplasm and shrunken darkly stained nuclei. Double staining revealed degenerating neurons solely among TH positive neurons of SNpc and VTA. At no time point and at no dose of MPTP was apoptosis observed. In addition, in situ labelling revealed no evidence of DNA fragmentation. This study demonstrates that the MPTP mouse model replicates several key features of neurodegeneration of DA neurons in PD and provides no in vivo evidence that, using this specific paradigm of injection, MPTP kills DA neurons by apoptosis.

The parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) : A technical review of its utility and safety

Parkinsons disease (PD) is a common disabling neurodegenerative disorder the cardinal clinical features of which include tremor, rigidity and slowness of movement (Fahn and Przedborski 2000). These symptoms are attributed mainly to a profound reduction of dopamine in the striatum due to a dramatic loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (Fahn and Przedborski 2000). Thus far, both the cause and the mechanisms of PD remain unknown. Over the years, investigators have used experimental models of PD produced by several compounds such as reserpine, 6-hydroxydopamine, methamphetamine, and 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) to provide insights into the mechanisms responsible for the demise of dopaminergic neurons in PD. To this end, MPTP has emerged unquestionably as a popular tool for inducing a model of PD in a variety of animal species including monkeys, rodents, cats, and pigs (Kopin and Markey 1988). The sensitivity to MPTP and therefore its ability to induce parkinsonism closely follows the phylogenetic tree where the species most closely related to humans are the most vulnerable to this neurotoxin. Due to the signi®cant neurotoxicity of MPTP, it is important that researchers appreciate the potential hazards of this toxin. Given this, the purpose of this review is to inform the researcher of the hazardous nature of MPTP and to provide guidance for its safe handling and use.

α-Synuclein Up-Regulation in Substantia Nigra Dopaminergic Neurons Following Administration of the Parkinsonian Toxin MPTP

Abstract: Mutations in α‐synuclein cause a form of familial Parkinson’s disease (PD), and wild‐type α‐synuclein is a major component of the intraneuronal inclusions called Lewy bodies, a pathological hallmark of PD. These observations suggest a pathogenic role for α‐synuclein in PD. Thus far, however, little is known about the importance of α‐synuclein in the nigral dopaminergic pathway in either normal or pathological situations. Herein, we studied this question by assessing the expression of synuclein‐1, the rodent homologue of human α‐synuclein, in both normal and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐intoxicated mice. In normal mice, detectable levels of synuclein mRNA and protein were seen in all brain regions studied and especially in ventral midbrain. In the latter, there was a dense synuclein‐positive nerve fiber network, which predominated over the substantia nigra, and only few scattered synuclein‐positive neurons. After a regimen of MPTP that kills dopaminergic neurons by apoptosis, synuclein mRNA and protein levels were increased significantly in midbrain extracts; the time course of these changes paralleled that of MPTP‐induced dopaminergic neurodegeneration. In these MPTP‐injected mice, there was also a dramatic increase in the number of synuclein‐immunoreactive neurons exclusively in the substantia nigra pars compacta; all synuclein‐positive neurons were tyrosine hydroxylase‐positive, but none coexpressed apoptotic features. These data indicate that synuclein is highly expressed in the nigrostriatal pathway of normal mice and that it is up‐regulated following MPTP‐induced injury. In light of the synuclein alterations, it can be suggested that, by targeting this protein, one may modulate MPTP neurotoxicity and, consequently, open new therapeutic avenues for PD.

Effects of Treadmill Exercise on Dopaminergic Transmission in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Lesioned Mouse Model of Basal Ganglia Injury

Studies have suggested that there are beneficial effects of exercise in patients with Parkinsons disease, but the underlying molecular mechanisms responsible for these effects are poorly understood. Studies in rodent models provide a means to examine the effects of exercise on dopaminergic neurotransmission. Using intensive treadmill exercise, we determined changes in striatal dopamine in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse. C57BL/6J mice were divided into four groups: (1) saline, (2) saline plus exercise, (3) MPTP, and (4) MPTP plus exercise. Exercise was started 5 d after MPTP lesioning and continued for 28 d. Treadmill running improved motor velocity in both exercise groups. All exercised animals also showed increased latency to fall (improved balance) using the accelerating rotarod compared with nonexercised mice. Using HPLC, we found no difference in striatal dopamine tissue levels between MPTP plus exercise compared with MPTP mice. There was an increase detected in saline plus exercise mice. Analyses using fast-scan cyclic voltammetry showed increased stimulus-evoked release and a decrease in decay of dopamine in the dorsal striatum of MPTP plus exercise mice only. Immunohistochemical staining analysis of striatal tyrosine hydroxylase and dopamine transporter proteins showed decreased expression in MPTP plus exercise mice compared with MPTP mice. There were no differences in mRNA transcript expression in midbrain dopaminergic neurons between these two groups. However, there was diminished transcript expression in saline plus exercise compared with saline mice. Our findings suggest that the benefits of treadmill exercise on motor performance may be accompanied by changes in dopaminergic neurotransmission that are different in the injured (MPTP-lesioned) compared with the noninjured (saline) nigrostriatal system.

Exercise-induced behavioral recovery and neuroplasticity in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse basal ganglia.

Physical activity has been shown to be neuroprotective in lesions affecting the basal ganglia. Using a treadmill exercise paradigm, we investigated the effect of exercise on neurorestoration. The 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐lesioned mouse model provides a means to investigate the effect of exercise on neurorestoration because 30–40% of nigrostriatal dopaminergic neurons survive MPTP lesioning and may provide a template for neurorestoration to occur. MPTP‐lesioned C57 BL/6J mice were administered MPTP (four injections of 20 mg/kg free‐base, 2 hr apart) or saline and divided into the following groups: (1) saline; (2) saline + exercise; (3) MPTP; and (4) MPTP + exercise. Mice in exercise groups were run on a motorized treadmill for 30 days starting 4 days after MPTP lesioning (a period after which MPTP‐induced cell death is complete). Initially, MPTP‐lesioned + exercise mice ran at slower speeds for a shorter amount of time compared to saline + exercise mice. Both velocity and endurance improved in the MPTP + exercise group to near normal levels over the 30‐day exercise period. The expression of proteins and genes involved in basal ganglia function including the dopamine transporter (DAT), tyrosine hydroxylase (TH), and the dopamine D1 and D2 receptors, as well as alterations on glutamate immunolabeling were determined. Exercise resulted in a significant downregulation of striatal DAT in the MPTP + exercise compared to MPTP nonexercised mice and to a lesser extent in the saline + exercised mice compared to their no‐exercise counterparts. There was no significant difference in TH protein levels between MPTP and MPTP + exercise groups at the end of the study. The expression of striatal dopamine D1 and D2 receptor mRNA transcript was suppressed in the saline + exercise group; however, dopamine D2 transcript expression was increased in the MPTP + exercise mice. Immunoelectron microscopy indicated that treadmill exercise reversed the lesioned‐induced increase in nerve terminal glutamate immunolabeling seen after MPTP administration. Our data demonstrates that exercise promotes behavioral recovery in the injured brain by modulating genes and proteins important to basal ganglia function.

Exercise-enhanced neuroplasticity targeting motor and cognitive circuitry in Parkinson's disease

Exercise interventions in individuals with Parkinsons disease incorporate goal-based motor skill training to engage cognitive circuitry important in motor learning. With this exercise approach, physical therapy helps with learning through instruction and feedback (reinforcement) and encouragement to perform beyond self-perceived capability. Individuals with Parkinsons disease become more cognitively engaged with the practice and learning of movements and skills that were previously automatic and unconscious. Aerobic exercise, regarded as important for improvement of blood flow and facilitation of neuroplasticity in elderly people, might also have a role in improvement of behavioural function in individuals with Parkinsons disease. Exercises that incorporate goal-based training and aerobic activity have the potential to improve both cognitive and automatic components of motor control in individuals with mild to moderate disease through experience-dependent neuroplasticity. Basic research in animal models of Parkinsons disease is beginning to show exercise-induced neuroplastic effects at the level of synaptic connections and circuits.

Localization of nicotinic receptor subunit mRNAs in monkey brain by in situ hybridization

Nicotinic receptors are implicated in memory, learning, locomotor activity, and addiction. Identification of the specific receptor subtypes that mediate these behaviors is essential for understanding their role in central nervous system (CNS) function. Although expression of nicotinic receptor transcript has been studied in rodent brain, their localization in the monkey CNS, which may be a better model for the human brain, is not yet known. We therefore investigated the distribution of α4, α6, α7, β2, β3, and β4 receptors subunit mRNAs in the monkey brain by using in situ hybridization. α4 and α7 mRNAs were very widely expressed, with a substantial degree of overlap in their distribution, except for the reticular nucleus of the thalamus in which α7 mRNA was much more prominently expressed. β2 and β4 mRNA were also widely distributed, although β4 was more prominently localized in thalamic nuclei than β2. The distribution of α6 and β3 mRNA was very distinct from that of the other transcripts, being restricted to catecholaminergic nuclei, the cerebellum, and a few other areas. Although there were similarities in distribution of the nicotinic receptor subunit mRNAs in monkey and rodent brain, there were prominent differences in areas such as the caudate, putamen, locus coeruleus, medial habenula, and cerebellum. In fact, the distribution of α4 and α7 mRNAs in the monkey caudate and putamen was more similar to that reported in the human than rodent brain. These findings have implications for the development of drug therapies for neurological disorders, such as Alzheimers and Parkinsons disease, in which nicotinic receptors are decreased. J. Comp. Neurol. 425:58–69, 2000.

Enhancing neuroplasticity in the basal ganglia: the role of exercise in Parkinson's disease.

Epidemiological and clinical trials have suggested that exercise is beneficial for patients with Parkinsons disease (PD). However, the underlying mechanisms and potential for disease modification are currently unknown. This review presents current findings from our laboratories in patients with PD and animal models. The data indicate that alterations in both dopaminergic and glutamatergic neurotransmission, induced by activity‐dependent (exercise) processes, may mitigate the cortically driven hyper‐excitability in the basal ganglia normally observed in the parkinsonian state. These insights have potential to identify novel therapeutic treatments capable of reversing or delaying disease progression in PD.

Combined assessment of tau and neuronal thread protein in Alzheimer’s disease CSF

Objective: Comparative study of CSF levels of tau and AD7C-neuronal thread protein (NTP) in patients with AD and control subjects. Background: AD is characterized by neurofibrillary tangles composed of the abnormally hyperphosphorylated microtubule-associated protein tau. AD7C-NTP is a proposed AD marker expressed at early stages of neurofibrillary degeneration. Methods: Enzyme-linked immunosorbent assays specific for tau and AD7C-NTP. CSF samples were obtained from 35 demented patients (25 with antemortem clinical diagnosis of probable AD, 5 with neuropathologic diagnosis of definite AD, 5 with Lewy body pathology), 29 nondemented patients with PD, and 16 elderly healthy control subjects. Receiver operating characteristics (ROC) and multivariate discriminant analysis for AD versus controls. Correlational analysis of CSF tau and AD7C-NTP and of each marker with Mini-Mental State Examination (MMSE) scores was performed. Results: Levels of both tau and AD7C-NTP were significantly elevated in the AD patients compared with control subjects. ROC analysis showed that CSF tau distinguished between patients with AD and nondemented control subjects with 63% sensitivity and 89% specificity, AD7C-NTP with 70% sensitivity and 87% specificity. Combined evaluation of both markers with discriminant analysis raised the specificity to 93% at a 63% sensitivity level. Both markers positively correlated with each other within the AD group, but not among control subjects. CSF levels of AD7C-NTP, but not of tau, showed a small but significant inverse correlation ( r = −0.43) with MMSE scores of AD patients. Conclusions: CSF levels of tau and AD7C-NTP may be useful biomarkers for AD.

Exercise elevates dopamine D2 receptor in a mouse model of Parkinson's disease: In vivo imaging with [18F]fallypride

The purpose of the current study was to examine changes in dopamine D2 receptor (DA‐D2R) expression within the basal ganglia of MPTP mice subjected to intensive treadmill exercise. Using Western immunoblotting analysis of synaptoneurosomes and in vivo positron emission tomography (PET) imaging employing the DA‐D2R specific ligand [18F]fallypride, we found that high intensity treadmill exercise led to an increase in striatal DA‐D2R expression that was most pronounced in MPTP compared to saline treated mice. Exercise‐induced changes in the DA‐D2R in the dopamine‐depleted basal ganglia are consistent with the potential role of this receptor in modulating medium spiny neurons (MSNs) function and behavioral recovery. Importantly, findings from this study support the rationale for using PET imaging with [18F]fallypride to examine DA‐D2R changes in individuals with Parkinsons Disease (PD) undergoing high‐intensity treadmill training.