Françoise Condé

Replicating Huntington's disease phenotype in experimental animals

Huntingtons disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder characterized by involuntary choreiform movements, cognitive decline and a progressive neuronal degeneration primarily affecting the striatum. There is at present no effective therapy against this disorder. The gene responsible for the disease (IT15) has been cloned and the molecular defect identified as an expanded polyglutamine tract in the N-terminal region of a protein of unknown function, named huntingtin (The Huntingtons Disease Collaborative Research Group, 1993. Cell 72, 971-983). An intense, search for the cell pathology attached to this molecular defect is currently under way [see Sharp and Ross (1996, Neurobiol. Dis. 3, 3-15) for review]. Huntingtin interacts with a number of proteins, some of which have well identified functions, and it has thus been suggested that alterations in glycolysis, vesicle trafficking or apoptosis play a role in the physiopathology of HD. On the other hand data derived from positron emission tomography (PET), magnetic resonance spectroscopy and post-mortem biochemical evidence for a defect in succinate oxidation have suggested the implication of a primary impairment of mitochondrial energy metabolism. All these hypotheses are not necessarily to be opposed and recent findings indicate that the HD mutation could possibly directly alter mitochondrial functions which would in turn activate apoptotic pathways. To test this mitochondrial hypothesis, we studied the effects in rodents and non-human primates of a chronic blockade of succinate oxidation by systemic administration of the mitochondrial toxin 3-nitropropionic acid (3NP). Extensive behavioural and neuropathological evaluations showed that a partial but prolonged energy impairment induced by 3NP is sufficient to replicate most of the clinical and pathophysiological hallmarks of HD, including spontaneous choreiform and dystonic movements, frontal-type cognitive deficits, and progressive heterogeneous striatal degeneration at least partially by apoptosis. 3NP produces the preferential degeneration of the medium-sized spiny GABAergic neurons with a relative sparing of interneurons and afferents, as was observed in HD striatum. The present manuscript reviews the different aspects of this neurotoxic treatment in rodents and non-human primates, and its interest as a phenotypic model of HD to understand the degenerative process of HD and test new therapeutic strategies.

Cellular distribution of the calcium-binding proteins parvalbumin, calbindin, and calretinin in the neocortex of mammals: phylogenetic and developmental patterns

The three calcium-binding proteins parvalbumin, calbindin, and calretinin are found in morphologically distinct classes of inhibitory interneurons as well as in some pyramidal neurons in the mammalian neocortex. Although there is a wide variability in the qualitative and quantitative characteristics of the neocortical subpopulations of calcium-binding protein-immunoreactive neurons in mammals, most of the available data show that there is a fundamental similarity among the mammalian species investigated so far, in terms of the distribution of parvalbumin, calbindin, and calretinin across the depth of the neocortex. Thus, calbindin- and calretinin-immunoreactive neurons are predominant in layers II and III, but are present across all cortical layers, whereas parvalbumin-immunoreactive neurons are more prevalent in the middle and lower cortical layers. These different neuronal populations have well defined regional and laminar distribution, neurochemical characteristics and synaptic connections, and each of these cell types displays a particular developmental sequence. Most of the available data on the development, distribution and morphological characteristics of these calcium-binding proteins are from studies in common laboratory animals such as the rat, mouse, cat, macaque monkey, as well as from postmortem analyses in humans, but there are virtually no data on other species aside of a few incidental reports. In the context of the evolution of mammalian neocortex, the distribution and morphological characteristics of calcium-binding protein-immunoreactive neurons may help defining taxon-specific patterns that may be used as reliable phylogenetic traits. It would be interesting to extend such neurochemical analyses of neuronal subpopulations to other species to assess the degree to which neurochemical specialization of particular neuronal subtypes, as well as their regional and laminar distribution in the cerebral cortex, may represent sets of derived features in any given mammalian order. This could be particularly interesting in view of the consistent differences in neurochemical typology observed in considerably divergent orders such as cetaceans and certain families of insectivores and metatherians, as well as in monotremes. The present article provides an overview of calcium-binding protein distribution across a large number of representative mammalian species and a review of their developmental patterns in the species where data are available. This analysis demonstrates that while it is likely that the developmental patterns are quite consistent across species, at least based on the limited number of species for which ontogenetic data exist, the distribution and morphology of calcium-binding protein-containingneurons varies substantially among mammalian orders and that certain species show highly divergent patterns compared to closely related taxa. Interestingly, primates, carnivores, rodents and tree shrews appear closely related on the basis of the observed patterns, marsupials show some affinities with that group, whereas prototherians have unique patterns. Our findings also support the relationships of cetaceans and ungulates, and demonstrates possible affinities between carnivores and ungulates, as well as the existence of common, probably primitive, traits in cetaceans and insectivores.

Lesion to the Nigrostriatal Dopamine System Disrupts Stimulus-Response Habit Formation

Acquisition and performance of instrumental actions are assumed to require both action-outcome and stimulus-response (S-R) habit processes. Over the course of extended training, control over instrumental performance shifts from goal-directed action-outcome associations to S-R associations that progressively gain domination over behavior. Lesions of the lateral part of the dorsal striatum disrupt this process, and rats with lesions to the lateral striatum showed selective sensitivity to devaluation of the instrumental outcome (Yin et al., 2004), indicating that this area is necessary for habit formation. The present experiment further explored the basis of this dysfunction by examining the ability of rats subjected to bilateral 6-hydroxydopamine lesions of the nigrostriatal dopaminergic pathway to develop behavioral autonomy with overtraining. Rats were given extended training on two cued instrumental tasks associating a stimulus (a tone or a light) with an instrumental action (lever press or chain pull) and a food reward (pellets or sucrose). Both tasks were run daily in separate sessions. Overtraining was followed by a test of goal sensitivity by satiety-specific devaluation of the reward. In control animals, one action (lever press) was insensitive to reward devaluation, indicating that it became a habit, whereas the second action (chain pull) was still sensitive to goal devaluation. This result provides evidence that the development of habit learning may depend on the characteristics of the response. In dopamine-depleted rats, lever press and chain pull remained sensitive to reward devaluation, evidencing a role of striatal dopamine transmission in habit formation.

Functional Recovery in a Primate Model of Parkinson's Disease following Motor Cortex Stimulation

A concept in Parkinsons disease postulates that motor cortex may pattern abnormal rhythmic activities in the basal ganglia, underlying the genesis of observed motor symptoms. We conducted a preclinical study of electrical interference in the primary motor cortex using a chronic MPTP primate model in which dopamine depletion was progressive and regularly documented using 18F-DOPA positron tomography. High-frequency motor cortex stimulation significantly reduced akinesia and bradykinesia. This behavioral benefit was associated with an increased metabolic activity in the supplementary motor area as assessed with 18-F-deoxyglucose PET, a normalization of mean firing rate in the internal globus pallidus (GPi) and the subthalamic nucleus (STN), and a reduction of synchronized oscillatory neuronal activities in these two structures. Motor cortex stimulation is a simple and safe procedure to modulate subthalamo-pallido-cortical loop and alleviate parkinsonian symptoms without requiring deep brain stereotactic surgery.

Early N-Acetylaspartate Depletion Is a Marker of Neuronal Dysfunction in Rats and Primates Chronically Treated with the Mitochondrial Toxin 3-Nitropropionic Acid:

N-acetylaspartate (NAA) quantification by 1H-magnetic resonance spectroscopy has been commonly used to assess in vivo neuronal loss in neurodegenerative disorders. Here, the authors used ex vivo and in vivo1H-magnetic resonance spectroscopy in rat and primate models of progressive striatal degeneration induced by the mitochondrial toxin 3-nitropropionate (3NP) to determine whether early NAA depletions could also be associated with neuronal dysfunction. In rats that were treated for 3 days with 3NP and had motor symptoms, the authors found a significant decrease in NAA concentrations, specifically restricted to the striatum. No cell loss or dying cells were found at this stage in these animals. After 5 days of 3NP treatment, a further decrease in striatal NAA concentrations was observed in association with the occurrence of dying neurons in the dorsolateral striatum. In 3NP-treated primates, a similar striatal-selective and early decrease in NAA concentrations was observed after only a few weeks of neurotoxic treatment, without any sign of ongoing cell death. This early decrease in striatal NAA was partially reversed after 4 weeks of 3NP withdrawal. These results demonstrate that early NAA depletions reflect a reversible state of neuronal dysfunction preceding cell degeneration and suggest that in vivo quantification of NAA 1H-magnetic resonance spectroscopy may become a valuable tool for assessing early neuronal dysfunction and the effects of potential neuroprotective therapies in neurodegenerative disorders.

Encapsulated GDNF-producing C2C12 cells for Parkinson's disease: a pre-clinical study in chronic MPTP-treated baboons

Glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophic factor with restorative effects in a variety of rodent and primate models of Parkinsons disease (PD), could be of therapeutic value to PD. In this study, we show that intraventricular chronic infusion of low doses of GDNF using encapsulated genetically engineered C2C12 cells can exert: (1) transient recovery of motor deficits (hypokinesia); (2) significant protection of intrinsic striatal dopaminergic function in the immediate vicinity of the site of implantation of the capsule in the caudate nucleus, and (3) significant-long-lasting-neurotrophic properties at the nigral level with an increase volume of the cell bodies. These observations confirm the potent neurorestorative potential of GDNF in PD and the safety/efficacy of the encapsulation technology as a means to deliver in situ this neurotrophic cytokine even using an intraventricular approach.

Transgenic expression of CTLA4-Ig by fetal pig neurons for xenotransplantation.

The transplantation of fetal porcine neurons is a potential therapeutic strategy for the treatment of human neurodegenerative disorders. A major obstacle to xenotransplantation, however, is the immune-mediated rejection that is resistant to conventional immunosuppression. To determine whether genetically modified donor pig neurons could be used to deliver immunosuppressive proteins locally in the brain, transgenic pigs were developed that express the human T cell inhibitory molecule hCTLA4-Ig under the control of the neuron-specific enolase promoter. Expression was found in various areas of the brain of transgenic pigs, including the mesencephalon, hippocampus and cortex. Neurons from 28-day old embryos secreted hCTLA4-Ig in vitro and this resulted in a 50% reduction of the proliferative response of human T lymphocytes in xenogenic proliferation assays. Transgenic embryonic neurons also secreted hCTLA4-Ig and had developed normally in vivo several weeks after transplantation into the striatum of immunosuppressed rats that were used here to study the engraftment in the absence of immunity. In conclusion, these data show that neurons from our transgenic pigs express hCTLA4-Ig in situ and support the use of this material in future pre-clinical trials in neuron xenotransplantation.

Learning and memory dissociation in rats with lesions to the subthalamic nucleus or to the dorsal striatum

The striatum and the subthalamic nucleus (STN) are the two main cortical inputs to the basal ganglia. Both structures are involved in motor and cognitive functions, particularly executive functions, known to rely mainly on fronto-basal ganglia circuits. The present work investigated the respective role of the dorsal part of the striatum (dST) and the STN by studying their involvement in learning and memory processes in two separate experiments. In a first experiment, rats with lesions to the STN or to the dST were trained in a light-tone discrimination task. When the learning criterion was reached, rats were then trained to the reversed discrimination. In a second experiment, surgery was done when the learning criterion had been reached. Three weeks after surgery, animals were then subjected to two relearning sessions and then to either a reversal learning or a working memory task. When surgery was done before learning, dysfunction of the dorsal striatum induced slight difficulties in acquisition, whereas dysfunction of the STN induced no difficulties during the initial learning but induced a more rapid inhibition of responses to the first lever press following the presentation of the tone during the reversed discrimination. In the second experiment, dST-lesioned rats showed long-term memory deficit in contrast to STN-lesioned rats which showed no difficulties during relearning but deficits in working memory. These results indicate a clear dissociation in cognitive functions in which STN and dorsal striatum are involved, suggesting that the fronto-striatal circuit and the fronto-STN circuit support, at least in part, different cognitive functions.

Up-regulation of glutamate concentration in the putamen and in the prefrontal cortex of asymptomatic SIVmac251-infected macaques without major brain involvement

We quantified putamen and prefrontal cortex metabolites in macaques with simian immunodeficiency virus infection and searched for virological and histological correlates. Fourteen asymptomatic macaques infected since 8–78 months (median: 38) were compared with eight uninfected ones. Absolute concentrations of acetate, alanine, aspartate, choline, creatine, GABA, glutamate, glutamine, lactate, myo‐inositol, N‐acetylaspartate, taurine and valine were determined by ex vivo proton magnetic resonance spectroscopy. Glutamate concentration in the CSF was determined by HPLC. Gliosis was assessed by glial fibrillary acidic protein and CD68 immunohistochemistry. Glutamate concentration was slightly increased in the prefrontal cortex (19%, p = 0.0152, t‐test) and putamen (13%, p = 0.0354, t‐test) of the infected macaques, and was unaffected in the CSF. Myo‐inositol concentration was increased in the prefrontal cortex only (27%, p = 0.0136). The concentrations of glutamate and myo‐inositol in the prefrontal cortex were higher in the animals with marked or intense microgliosis (p = 0.0114). The other studied metabolites, including N‐acetylaspartate, were not altered. Glutamate concentration may thus increase in the cerebral parenchyma in asymptomatic animals, but is not accompanied by a detectable decrease in N‐acetylaspartate concentration (neuronal dysfunction). Thus, there are probably compensatory mechanisms that may limit glutamate increase and/or counterbalance its effects.

Learning-dependent activation of Fra-1: involvement of ventral hippocampus and SNc/VTA complex in learning and habit formation.

Although the effect of overtraining on learning processes in rats has long been studied, only few studies have specifically assessed the differential involvement of brain areas in habit formation. We used the analysis of expression of the immediate early gene Fra-1 as a tool to differentiate the areas involved in training and overtraining. Behavioural experiments showed that instrumental performance (signalled and non-signalled instrumental tasks), but not pavlovian conditioned responses, were no longer under the control of the incentive value of the reward after overtraining. The number of Fra-1 expressing neurons was increased in SNc/VTA and ventral hippocampus after training in all groups independently of behavioural performance. After overtraining, the number of learning-induced Fra-1 immunoreactive neurons remained increased in the SNc/VTA. However, in CA1, it significantly decreased in the signalled instrumental group, whereas it further increased in the pavlovian group, with no modulation in non-signalled instrumental animals. The increase in the number of Fra-1 neurons observed after training in SNc/VTA and ventral hippocampus suggests that a general underlying incentive process regulates Fra-1. Moreover, the sustained increased expression of Fra-1 in the SNc/VTA after instrumental overtraining could reflect a possible role of dopaminergic neurons in habit formation.