Pierre-Olivier Fernagut

Early and Progressive Sensorimotor Anomalies in Mice Overexpressing Wild-Type Human α-Synuclein

Accumulation of α-synuclein in brain is a hallmark of synucleinopathies, neurodegenerative diseases that include Parkinsons disease. Mice overexpressing α-synuclein under the Thy-1 promoter (ASO) show abnormal accumulation of α-synuclein in cortical and subcortical regions of the brain, including the substantia nigra. We examined the motor deficits in ASO mice with a battery of sensorimotor tests that are sensitive to alterations in the nigrostriatal dopaminergic system. Male wild-type and ASO mice were tested every 2 months for 8 months for motor performance and coordination on a challenging beam, inverted grid, and pole, sensorimotor deficits in an adhesive removal test, spontaneous activity in a cylinder, and gait. Fine motor skills were assessed by the ability to grasp cotton from a bin. ASO mice displayed significant impairments in motor performance and coordination and a reduction in spontaneous activity as early as 2 months of age. Motor performance and coordination impairments became progressively worse with age and sensorimotor deficits appeared at 6 months. Fine motor skills were altered at 4 months and worsened at 8 months. These data indicate that overexpression of α-synuclein induced an early and progressive behavioral phenotype that can be detected in multiple tests of sensorimotor function. These behavioral deficits provide a useful way to assess novel drug therapy in genetic models of synucleinopathies.

Direct regulation of adult brain function by the male-specific factor SRY.

The central dogma of mammalian brain sexual differentiation has contended that sex steroids of gonadal origin organize the neural circuits of the developing brain. Recent evidence has begun to challenge this idea and has suggested that, independent of the masculinizing effects of gonadal secretions, XY and XX brain cells have different patterns of gene expression that influence their differentiation and function. We have previously shown that specific differences in gene expression exist between male and female developing brains and that these differences precede the influences of gonadal hormones. Here we demonstrate that the Y chromosome-linked, male-determining gene Sry is specifically expressed in the substantia nigra of the adult male rodent in tyrosine hydroxylase-expressing neurons. Furthermore, using antisense oligodeoxynucleotides, we show that Sry downregulation in the substantia nigra causes a statistically significant decrease in tyrosine hydroxylase expression with no overall effect on neuronal numbers and that this decrease leads to motor deficits in male rats. Our studies suggest that Sry directly affects the biochemical properties of the dopaminergic neurons of the nigrostriatal system and the specific motor behaviors they control. These results demonstrate a direct male-specific effect on the brain by a gene encoded only in the male genome, without any mediation by gonadal hormones.

Alpha-synuclein and transgenic mouse models

Identified as the cause of some familial forms of Parkinson disease (PD) and as one of the major component of Lewy bodies, alpha-synuclein (alpha-syn) became the molecular hallmark of several neurodegenerative conditions now designated as synucleinopathies. Transgenic models have been generated to elucidate its physiological and pathologic roles. Although none of the lines created display dopaminergic neuronal death in the substantia nigra, the models recapitulate some features of synucleinopathies and are useful to study the potential pathogenic role of alpha-synuclein and its molecular partners. This review describes the different alpha-synuclein transgenic models, their clinical relevance to synucleinopathies, and their further utilization to understand the disease process.

Genetic Mouse Models of Parkinsonism: Strengths and Limitations

SummaryParkinson’s disease (PD) is a progressive neurodegenerative disorder. Patients with PD display a combination of motor symptoms including resting tremor, rigidity, bradykinesia, and postural instability that worsen over time. These motor symptoms are related to the progressive loss of dopamine neurons in the substantia nigra pars compacta. PD patients also suffer from nonmotor symptoms that may precede the cardinal motor symptoms and that are likely related to pathology in other brain regions. Traditional toxin models of PD have focused on the nigrostriatal pathway and the loss of dopamine neurons in this region, and these models have been important in our understanding of PD and in the development of symptomatic treatments for the disease. However, they are limited in that they do not reproduce the full pathology and progression seen in PD, thus creating a need for better models. The recent discovery of specific genes causing familial forms of PD has contributed to the development of novel genetic mouse models of PD. This review discusses the validity, benefits, and limitations of these new models.

Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone

Mitochondrial toxins such as the complex 1 inhibitor rotenone are widely used as pesticides and may be present in military environments. Administration of rotenone can induce biochemical and histological alterations similar to those of Parkinsons disease in rats. However, only a subset of animals show these effects and it is unclear whether more subtle alterations are caused by chronic administration of rotenone in those animals that appear resistant to its toxic effects on dopaminergic nerve terminals. To address this question, vehicle or rotenone (2.0, 2.5, or 3.5 mg/kg/day) was administered intravenously or subcutaneously for 21 days to adult rats, and rotenone effects on survival, motor behavior, and striatal tyrosine hydroxylase immunoreactivity (TH-IR) were examined. Both intravenous and subcutaneous rotenone induced a dose-dependent decrease in survival rates. Surviving animals showed a decrease in spontaneous rearing. Locomotor activity and movement initiation time were also altered in some of the experimental groups. Confirming previous results, TH-IR in the striatum was markedly decreased in rats that fell ill early in the study and in a few of the surviving rats with high rotenone doses. However, none of the surviving rats receiving 2.0 mg/kg/day showed TH-IR loss reminiscent of Parkinsons disease, and loss of striatal TH-IR across doses was not correlated with motor behavior in individual rats. Thus, chronic administration of low doses of rotenone induces motor anomalies even in animals that do not develop histological signs of Parkinsons disease, indicating a pervasive neurological effect of moderate mitochondrial dysfunction in vivo.

Deleterious effects of minocycline in animal models of Parkinson's disease and Huntington's disease.

Minocycline has been shown to exert anti‐inflammatory effects underlying its putative neuroprotective properties in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of Parkinsons disease and in the R6/2 mouse model of Huntingtons disease (HD). However, contradictory results have recently been reported. We report deleterious effects of minocycline in two phenotypic (toxic) models of Parkinsons disease and HD in monkey and mouse. Of seven MPTP‐intoxicated female cynomolgus monkeys (0.2 mg/kg, i.v. until day 15), three received minocycline (200 mg b.i.d.). While placebo‐MPTP‐treated animals displayed mild parkinsonism at day 15, the minocycline/MPTP‐treated animals tended to be more affected (P = 0.057) and showed a greater loss of putaminal dopaminergic nerve endings (P < 0.0001). In the 3‐nitropropionic acid (3‐NP) mouse model of HD, minocycline (45 mg/kg i.p.) was administered 30 min before each i.p. injection of 3‐NP (b.i.d., cumulated dose, 360 mg/kg in 5 days). Mice receiving minocycline exhibited a worsening of the mean motor score with a slower recovery slope, more impaired general activity and significantly deteriorated performances on the rotarod, pole test and beam‐traversing tasks. The histopathological outcome demonstrated that minocycline‐treated mice presented significantly more severe neuronal cell loss in the dorsal striatum. The effect of minocycline vs. 3‐NP was also investigated on hippocampal and cortical cell cultures. minocycline blocked 3‐NP‐induced neurotoxicity at certain doses (1 mm cortical neurons) but not at higher doses (10 mm). Thus, minocycline may have variable and even deleterious effects in different species and models according to the mode of administration and dose.

Variable effects of chronic subcutaneous administration of rotenone on striatal histology.

When infused in rats, rotenone, a mitochondrial complex I inhibitor, induces alterations that resemble the histological changes of Parkinsons disease, particularly degeneration of the nigrostriatal dopaminergic system. However, the specificity of rotenone effects has been challenged recently. We have re‐examined the alterations caused by rotenone in the substantia nigra and the striatum of rats after infusion of rotenone (2 mg/kg per day s.c.) for 21 days. Three patterns of striatal tyrosine‐hydroxylase immunoreactivity (TH‐IR) were observed: 46% of animals showed no reduction, and 46% of animals showed diffuse reduction in TH‐IR, whereas one animal presented a focal loss of TH‐IR in the striatum. Confocal microscopy analysis showed that the vesicular monoamine transporter (VMAT2) was decreased in parallel with TH‐IR, strongly suggesting a loss of striatal DA nerve terminals in animals with diffuse or central TH‐IR loss. However, no significant loss of TH‐IR neurons was observed in the substantia nigra. Analysis of NeuN and DARPP‐32 immunoreactivity, and Nissl staining, in the striatum showed no striatal neuronal loss in animals with either preserved TH‐IR or diffuse TH‐IR reduction. However, in the animal with focal TH‐IR loss, severe neuronal loss was evident in the center and the periphery of the striatum, together with microglial activation detected by OX‐6 and OX‐42 staining. Thus, in most cases, chronic subcutaneous infusion of low doses of rotenone does not induce significant striatal neuronal loss, despite TH‐IR and VMAT‐IR reduction in a subset of animals, supporting the use of rotenone as a model of Parkinsons disease under carefully controlled experimental conditions. J. Comp. Neurol. 478:418–426, 2004.

Ziram Causes Dopaminergic Cell Damage by Inhibiting E1 Ligase of the Proteasome

The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated α-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target.

Low dose rotenone treatment causes selective transcriptional activation of cell death related pathways in dopaminergic neurons in vivo.

Mitochondrial complex I inhibition has been implicated in the degeneration of midbrain dopaminergic (DA) neurons in Parkinsons disease. However, the mechanisms and pathways that determine the cellular fate of DA neurons downstream of the mitochondrial dysfunction have not been fully identified. We conducted cell-type specific gene array experiments with nigral DA neurons from rats treated with the complex I inhibitor, rotenone, at a dose that does not induce cell death. The genome wide screen identified transcriptional changes in multiple cell death related pathways that are indicative of a simultaneous activation of both degenerative and protective mechanisms. Quantitative PCR analyses of a subset of these genes in different neuronal populations of the basal ganglia revealed that some of the changes are specific for DA neurons, suggesting that these neurons are highly sensitive to rotenone. Our data provide insight into potentially defensive strategies of DA neurons against disease relevant insults.

In vivo models of multiple system atrophy

Multiple system atrophy (MSA) is a sporadic adult‐onset neurodegenerative disorder of unknown etiology clinically characterized by a combination of parkinsonian, pyramidal, and cerebellar signs. Levodopa‐unresponsive parkinsonism is present in 80% of MSA cases, and this dominant clinical presentation (MSA‐P) is associated with a combined degeneration of the substantia nigra pars compacta and the striatum in anatomically related areas. The limited knowledge of the pathophysiology of MSA and the lack of therapeutic strategies prompted the development of lesion models reproducing striatonigral degeneration, the substrate of levodopa‐unresponsive parkinsonism in MSA‐P. This method was carried out first in rats with two different stereotaxic strategies using either two neurotoxins (“double toxin–double lesion”) or a single neurotoxin (“single toxin–double lesion”). Double‐lesioned rat models showed severe motor impairment compared to those with a single nigral or striatal lesion and helped to mimic different stages of the disease. Systemic models were also developed in mice and primates using the nigral toxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and the striatal toxin 3‐nitropropionic (3‐NP). In mice, although MPTP reduced the subsequent sensitivity to 3‐NP in a sequential lesion, simultaneous nigral and striatal insults were shown to exacerbate striatal damage. MPTP‐treated monkeys displayed a significant worsening of parkinsonism and a loss of levodopa‐responsiveness after the appearance of hindlimb dystonia and striatal lesion formation induced by subsequent 3‐NP intoxication. The different species and intoxication paradigms used will be useful to investigate functional changes in substantia nigra and striatum and to define neuroprotective, neurorestorative, or symptomatic therapeutic strategies.