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FP6

AGLAEA Sintesi della relazione

Project ID: 37554
Finanziato nell'ambito di: FP6-LIFESCIHEALTH
Paese: France

Final Report Summary - AGLAEA (Development of novel animal models of glutamatergic central nervous system disorders using in vivo sirna and transgenic approaches)

Glutamate is the most abundant excitatory transmitter in the brain. However, few animal models exist to explore the potential of drugs that modulate glutamate transmission. Altered glutamate transmission is involved in numerous psychiatric diseases, resulting in the need for models to characterise the effects of hypo- and hyper-glutamatergic states involved in the onset and development of psychiatric diseases. AGLAEA aimed to develop and characterise models of selective, partial knockdown of specific components of the brain glutamatergic system in mice: glutamate membrane transporter (EAAT2 / GLT-1) and the vesicular membrane transporter (VGLUT1). These models are believed to provide better comprehension of the implication of glutamate signalling in diseases such as schizophrenia, anxiety and cognitive disorders. In order to selectively turn off specific components of the glutamatergic pathways, both siRNA and shRNA approaches were attempted for both molecular targets.

In vitro studies identified suitable siRNA and shRNA constructs for the glutamate membrane transporter (GLT-1 / EAAT2) and the vesicular membrane transporter (VGLUT1) respectively. Beyond the initial plan and foreseen deliverables, in vitro system was developed for assessing the functional consequence on proteins knock-down. It was possible to show the consequence of altering the gene expression and its repercussions on protein level and function of one glutamate transporter.

Initial in vivo studies showed poor brain penetration following ICV administration of TurboGFPencoding lentiviral particles, indicating the shRNA approach required localised administration (in contrast to siRNA that will be applied by ICV infusion). In general both siRNA and shRNA were tolerated and had no effect on gross behavioural measures.

Behavioural studies indicated that disruption of GLT1 transporter resulted in an anxiogenic behavioural profile, as expected given the role of this transporter in maintaining extracellular glutamate at physiologic levels. Interestingly, benzodiazepine was able to reverse the anxiogenic phenotype. At this stage, the conclusion is that a model exists although there is some work still to do to guarantee robustness and reproducibility for its use in routine for the testing of new compounds.

Also behavioural studies indicated that shRNA directed at VGLUT1 impaired specific aspects of cognition (after bilateral intra-hippocampal injections), as expected from the role of this transporter in the control of glutamate release. Interestingly, it was shown that one antagonist of 5-HT6 receptor was able to reverse cognitive deficits. Results gained with shRNA-VGLUT1 demonstrated that the initial plan to produce an inducible transgenic mouse showing partial loss of VGLUT1 expression as a model of the cognitive deficits was a valid goal. Despite a dedicated strategy and efforts, the consortium did not deliver the transgenic mice but did progress till the stage of the constructions of some lentiviruses encoding shRNA.

The consortium managed to produce germline transmitting C57 / Bl6 mES cell line for generation of animal models using in behavioural studies. Mice with C57 / BL6 background are much more amenable to behavioural studies than the 129 SV line, for which ES cell lines are already available. Mycoplasma free cell clones were characterised in vitro and tested in vivo for germline transmission potential.

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Reported by

ADDEX PHARMACEUTICALS FRANCE SAS
ARCHAMPS
France