Identification of distinct pathological signatures induced by patient-derived α-synuclein structures in nonhuman primates.

Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)-rich protein aggregates [termed "Lewy bodies" (LBs)], is a well-established characteristic of Parkinson's disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning-based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration.

In utero delivery of rAAV2/9 induces neuronal expression of the transgene in the brain: towards new models of Parkinson's disease.

Animal models are essential tools for basic pathophysiological research as well as validation of therapeutic strategies for curing human diseases. However, technical difficulties associated with classical transgenesis approaches in rodent species higher than Mus musculus have prevented this long-awaited development. The availability of viral-mediated gene delivery systems in the past few years has stimulated the production of viruses with unique characteristics. For example, the recombinant adeno-associated virus serotype 9 (rAAV2/9) crosses the blood-brain barrier, is capable of transducing developing cells and neurons after intravenous injection and mediates long-term transduction. Whilst post-natal delivery is technically straightforward, in utero delivery bears the potential of achieving gene transduction in neurons at embryonic stages during which the target area is undergoing development. To test this possibility, we injected rAAV2/9 carrying either A53T mutant human α-synuclein or green fluorescent protein, intracerebroventricularly in rats at embryonic day 16.5. We observed neuronal transgene expression in most regions of the brain at 1 and 3 months after birth. This proof-of-concept experiment introduces a new opportunity to model brain diseases in rats.

The associative and limbic thalamus in the pathophysiology of obsessive-compulsive disorder: an experimental study in the monkey.

Obsessive-compulsive disorder (OCD) is a frequent psychiatric disorder characterized by repetitive intrusive thoughts and severe anxiety, leading to compulsive behaviors. Although medical treatment is effective in most cases, resistance is observed in about 30% of patients. In this context, deep brain stimulation (DBS) of the caudate or subthalamic nuclei has been recently proposed with encouraging results. However, some patients were unimproved or exhibited awkward side effects. Therefore, exploration of new targets for DBS remains critical in OCD. In the latter, functional imaging studies revealed overactivity in the limbic and associative cortico-subcortical loops encompassing the thalamus. However, the role of the thalamus in the genesis of repetitive behaviors and related anxiety is unknown. Here, we tested the hypothesis that pharmacological-induced overactivity of the medial thalamus could give rise to abnormal behaviors close to that observed in OCD. We modulated the ventral anterior (VA) and medial dorsal (MD) nuclei activity by in situ bicuculline (GABA(A) antagonist) microinjections in subhuman primates and assessed their pharmacological-induced behavior. Bicuculline injections within the VA caused significant repetitive and time-consuming motor acts whereas those performed within the MD induced symptoms of dysautonomic dysregulation along with abnormal vocalizations and marked motor hypoactivity. These findings suggest that overactivation of the VA and MD nuclei of the thalamus provokes compulsive-like behaviors and neurovegetative manifestations usually associated with the feeling of anxiety in OCD patients. In further research, this translational approach should allow us to test the effectiveness and side effects of these thalamic nuclei DBS in monkey and perhaps, in a second step, to propose a transfer of this technique to severely disabled OCD patients.

Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson's disease.

The concept of a threshold of dopamine (DA) depletion for onset of Parkinson's disease symptoms, although widely accepted, has, to date, not been determined experimentally in nonhuman primates in which a more rigorous definition of the mechanisms responsible for the threshold effect might be obtained. The present study was thus designed to determine (1) the relationship between Parkinsonian symptom appearance and level of degeneration of the nigrostriatal pathway and (2) the concomitant presynaptic and postsynaptic striatal response to the denervation, in monkeys treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine according to a regimen that produces a progressive Parkinsonian state. The kinetics of the nigrostriatal degeneration described allow the determination of the critical thresholds associated to symptom appearance, these were a loss of 43.2% of tyrosine hydroxylase-immunopositive neurons at the nigral level and losses of 80.3 and 81.6% DA transporter binding and DA content, respectively, at the striatal level. Our data argue against the concept that an increase in DA metabolism could act as an efficient adaptive mechanism early in the disease progress. Surprisingly, the D(2)-like DA receptor binding showed a biphasic regulation in relation to the level of striatal dopaminergic denervation, i.e., an initial decrease in the presymptomatic period was followed by an upregulation of postsynaptic receptors commencing when striatal dopaminergic homeostasis is broken. Further in vivo follow-up of the kinetics of striatal denervation in this, and similar, experimental models is now needed with a view to developing early diagnosis tools and symptomatic therapies that might enhance endogenous compensatory mechanisms.

Activation of GABA(A) receptors in subthalamic neurons in vitro: properties of native receptors and inhibition mechanisms.

The subthalamic nucleus (STN) influences the output of the basal ganglia, thereby interfering with motor behavior. The main inputs to the STN are GABAergic. We characterized the GABA(A) receptors expressed in the STN and investigated the response of subthalamic neurons to the activation of GABA(A) receptors. Cell-attached and whole cell recordings were made from rat brain slices using the patch-clamp technique. The newly identified epsilon subunit confers atypical pharmacological properties on recombinant receptors, which are insensitive to barbiturates and benzodiazepines. We tested the hypothesis that native subthalamic GABA(A) receptors contain epsilon proteins. Applications of increasing concentrations of muscimol, a selective GABA(A) agonist, induced Cl(-) and HCO currents with an EC(50) of 5 microM. Currents induced by muscimol were fully blocked by the GABA(A) receptor antagonists, bicuculline and picrotoxin. They were strongly potentiated by the barbiturate, pentobarbital (+190%), and by the benzodiazepines, diazepam (+197%) and flunitrazepam (+199%). Spontaneous inhibitory postsynaptic currents were also significantly enhanced by flunitrazepam. Furthermore, immunohistological experiments with an epsilon subunit-specific antibody showed that the epsilon protein was not expressed within the STN. Native subthalamic GABA(A) receptors did not, therefore, display pharmacological or structural properties consistent with receptors comprising epsilon. Burst firing is a hallmark of Parkinson's disease. Half of the subthalamic neurons have the intrinsic capacity of switching from regular-firing to burst-firing mode when hyperpolarized by current injection. This raises the possibility that activation of GABA(A) receptors might trigger the switch. Statistical analysis of spiking activity established that 90% of intact neurons in vitro were in single-spike firing mode, whereas 10% were in burst-firing mode. Muscimol reversibly stopped recurrent electrical activity in all intact neurons. In neurons held in whole cell configuration, membrane potential hyperpolarized by -10 mV whilst input resistance decreased by 50%, indicating powerful membrane shunting. Muscimol never induced burst firing, even in neurons that exhibited the capacity of switching from regular- to burst-firing mode. These molecular and functional data indicate that native subthalamic GABA(A) receptors do not contain the epsilon protein and activation of GABA(A) receptors induces membrane shunting, which is essential for firing inhibition but prevents switching to burst-firing. They suggest that the STN, like many other parts of the brain, has the physiological and structural features of the widely expressed GABA(A) receptors consisting of alphabetagamma subunits.

Spontaneous long-term compensatory dopaminergic sprouting in MPTP-treated mice.

The present study sought to determine whether severe 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication elicits spontaneous long-term compensatory sprouting in mice. Animals, once treated, were kept without further treatment for 0.5, 1, 5, or 7 months. The stability of the nigral degeneration was checked by evaluation of the number of tyrosine hydroxylase immunoreactive (TH-IR) neurons, whereas sprouting was assessed using both [(3)H]-dopamine (DA) uptake by striatal synaptosomes and optical density of TH-immunolabeled fibers in the striatum as markers. At 0.5 month after MPTP intoxication (80 mg/kg, i.p.), we observed comparable decreases of 83% in DA uptake, 83.3% in TH fiber density, and 74% in the number of TH-IR neurons compared to age-matched saline-treated animals. From 5 months onwards, both DA uptake and striatal TH fiber density increased significantly (50% and 34.9% at 5 months, 65% and 67.4% at 7 months, respectively) in comparison with age-matched saline-treated animals, although the number of TH-IR neurons remained stable (73% of degeneration at 7 months). These results indicate clearly that spontaneous long-term compensatory dopaminergic sprouting is a phenomenon that is not restricted to situations of partial nigral degeneration but can, on the contrary, constitute a response even to severe stable MPTP-induced nigral degeneration.

Trichloroethylene and parkinsonism: a human and experimental observation.

This report describes the case of a 47-year-old woman who developed Parkinson's disease after seven years of professional exposure to trichloroethylene. In the light of this clinical report, mice were intoxicated with trichloroethylene and tyrosine hydroxylase immunoreactivity was used to measure neuronal death in the substantia nigra pars compacta. Treated mice presented significant dopaminergic neuronal death in comparison with control mice (50%). The environmental trichlorethylene pollution, as well as other unspecific neurotoxic solvents, could potentially contribute to the genesis of some cases of Parkinson's disease.

Absence of MPTP-induced neuronal death in mice lacking the dopamine transporter.

MPTP has been shown to induce parkinsonism both in human and in nonhuman primates. The precise mechanism of dopaminergic cell death induced following MPTP treatment is still subject to intense debate. MPP+, which is the oxidation product of MPTP, is actively transported into presynaptic dopaminergic nerve terminals through the plasma membrane dopamine transporter (DAT). In this study, we used mice lacking the DAT by homologous recombination and demonstrated that the MPTP-induced dopaminergic cell loss is dependent on the presence of the DAT. For this we have used tyrosine hydroxylase immunoreactivity (TH-IR) labeling of dopamine cells of the substantia nigra compacta in wild-type, heterozygote, and homozygote mice that were given either saline or MPTP treatments (two ip injections of 30 mg/kg, 10 h apart). Our results show a significant loss of TH-IR in wild type (34.4%), less loss in heterozygotes (22.5%), and no loss in homozygote animals. Thus dopamine cell loss is related to levels of the DAT. These results shed light on the degenerative process of dopamine neurons and suggest that individual differences in developing Parkinson's disease in human may be related to differences of uptake through the DAT of a yet unidentified neurotoxin.

Effects of different schedules of MPTP administration on dopaminergic neurodegeneration in mice.

Although a valuable 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of human Parkinson's disease has been developed, our knowledge of the course of nigral degeneration remains fragmentary. Experimental factors which could possibly influence the destructive process must be taken into account. To evaluate the impact of experimental design, we compared the effects of different schedules of injection of the same cumulative dose of MPTP, in mice, by measuring tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta. Massive injection of the total dose over 1 day (4 injections of 20 mg/kg) destroyed more dopaminergic neurons than did the long-term daily injections of a lower dose of MPTP (20 injections of 4 mg/kg). This suggests that different schedules of administration of MPTP might induce different mechanisms of neuronal death. These mechanisms need to be better understood if chronic models of intoxication that replicate the evolution of human Parkinson's disease more precisely are to be developed.

Kinetics of nigral degeneration in a chronic model of MPTP-treated mice.

The chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model we have developed in monkey reproduces all the cardinal features of Parkinson's disease, and, in particular the characteristic slow evolution of clinical signs. We still know little, however, of the kinetics of the nigral degeneration induced. This present study charts the progressive destruction of tyrosine hydroxylase-immunoreactive neurones in mice treated daily with low doses of MPTP for 20 days. Our results show that the neuronal death rate is initially high, subsequently decreases, and stabilizes. This new protocol thus mirrors closely the pattern of evolution assumed to be that of Parkinson's disease and should prove useful for studies on neuroprotection and compensatory mechanisms.