Aristaless brain diseases: A class of pathologies associated with an altered GABAergic homeostasis ?

ARX is one of the most important X-linked brain disease genes encoding the homeo-Transcription Factor Aristaless with a crucial role in the development of cerebral cortex, thalamus, hippocampus, and striatum. Mutations in humans cause a large spectrum of neurological disorders including lissencephaly, severe epilepsy and mental retardation. Male embryonic Arx null mice shows aberrant migration and differentiation of gamma-aminobutyric acid (GABA)-ergic interneurons, characteristics recapitulating some of the clinical features of lissencephaly and epilepsy in humans. In this report, we present molecular and morphological signatures that appear altered during the transition from neuronal precursors to fully committed Arx deficient GABA cells. In ES and neurosphere cells from wt and Arx-/Y embryonic brains, we analysed specific proliferative and neuronal hallmarks, during in vitro GABAergic maturation. They were investigated in critical transition stages: proliferation, intermediate differentiation and full differentiation. In the absence of Aristaless, we established by real time PCR robust transcriptional changes of a number of critical determinants of proliferating precursor cells and of early neuronal fate. Subsequent analysis performed by immunofluorescence confirms a parallel deregulation of the corresponding protein. In addition, in full differentiation condition, confocal microscopic study puts in evidence abnormality in neuronal morphology and the high percentage of immature GABAergic neurons. In light of these results, we investigated whether impaired induction of neuronal differentiation was accompanied by generation of cells derived from the other germ layers. Transcript levels of mesoderm/endoderm specific genes revealed abnormal expression during neuronal differentiation. We are currently producing Arx deficient ES cell lines stably transfected with normal Arx to evaluate molecular and morphological rescue effects. In summary, we reveal the existence of changes in critical neuronal markers compatible with an impaired Arx-dependent synaptic function, which impacts on GABA homeostasis. One of the altered markers was suspected to be a direct transcriptional target of Aristaless, even if further work is required to define their functional connection. These findings emphasize the "interneuronopathy" hypothesis and will hopefully lead to the identification of a novel entry point to study ARX physiopathology in epilepsy and mental retardation.