Research report
Neuroanatomical distribution of ARX in brain and its localisation in GABAergic neurons

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Abstract

Recent human genetics approaches identified the Aristaless-related homeobox (ARX) gene as the causative gene in X-linked infantile spasms, Partington syndrome, and non-syndromic mental retardation as well as in forms of lissencephaly with abnormal genitalia. The ARX predicted protein belongs to a large family of homeoproteins and is characterised by a C-terminal Aristaless domain and an octapeptide domain near the N-terminus. In order to learn more about ARX function, we have studied in detail Arx expression in the central nervous system during mouse embryonic development as well as in the adult. During early stages of development, Arx is expressed in a significant proportion of neurons in the cortex, the striatum, the ganglionic eminences and also in the spinal cord. In the adult, expression of Arx is still present and restricted to regions that are known to be rich in GABAergic neurons such as the amygdala and the olfactory bulb. A possible role for Arx in this type of neurons is further reinforced by the expression of Arx in a subset of GABAergic interneurons in young and mature primary cultures of cortical neuronal cells as well as in vivo. Moreover, these data could explain the occurrence of seizures in the great majority of patients with an ARX mutation, due to mislocalisation or dysfunction of GABAergic neurons. We also performed ARX wild-type and mutant over-expression experiments and found that the different ARX mutations tested did not modify the morphology of the cells. Moreover, no abnormal cell death or protein aggregation was observed, hence suggesting that more subtle pathogenic mechanisms are involved.

Introduction

The human Aristaless-related homeobox gene (ARX) was identified as the causative gene in X-linked infantile spasms (ISSX; West syndrome, MIM308350), in certain families with X-linked syndromic (Partington syndrome, MIM309510) and non-syndromic mental retardation, including patients with dystonia, and patients with myoclonic epilepsy with spasticity [5], [20]. Mutations identified in ARX in these patients included missense mutations, a recurrent in-frame 24-bp duplication predicted to cause an expansion of a polyalanine tract from 12 to 20 alanines and a small in-frame insertion leading to an increase of another polyalanine tract containing 16 residues. Notably, no nonsense mutations were identified in these cases. More recently, mutations in ARX have been identified in X-linked lissencephaly with abnormal genitalia (XLAG, MIM300215) [9]. However, these latter mutations in ARX result predominantly in premature termination signals because of frameshifts due to insertions or deletions, or nonsense mutations. In two cases of XLAG, missense mutations have been identified (R332H, L343Q) [9]. These two mutations are located in highly conserved regions of the homeodomain suggesting dysfunction of DNA binding. The striking differences in phenotype, which are observed (XLAG vs. X-linked mental retardation), are likely to be explained by the consequences of the different ARX mutations on the stability and/or the function of ARX protein.

The ARX predicted protein belongs to one of the three largest classes of homeoproteins, the paired (Prd) class. Within this class, it is member of a specific sub-class of proteins, which contain a glutamine residue at the critical position 50 of their homeodomain (Q50), a residue also found conserved in the Drosophila Aristaless protein (al). In addition to its paired/Q50 central homeodomain, ARX is characterised by a 14 amino acid C-terminal Aristaless domain and by an octapeptide domain located near the N-terminus designated as the Goosecoid Engrailed Homology (GEH) or the eh-1 domain in the Engrailed (En) homeoprotein [11].

In order to better understand the function of ARX and its involvement in X-linked mental retardation, we studied Arx expression in the central nervous system (CNS) during mouse embryonic development as well as in the adult. Our results suggest that Arx is likely to play an important role during embryonic CNS development, as well as in the adult were ARX expression is still present in the neocortex, hippocampus, hypothalamus, amygdala and especially in the olfactory bulb. High expression of Arx in regions which are known to be rich in GABAergic neurons, and the expression of Arx in a subset of GABAergic neurons suggest that mutations in ARX alter GABAergic neuronal development and/or function, which might contribute to the seizure phenotype observed in ARX-related disorders. Over-expression of wild-type and different ARX mutants involved in certain of these disorders, including a polyalanine tract expansion, revealed no dramatic changes in cell differentiation, morphology and cell death. Moreover, no abnormal protein aggregation was observed, suggesting that these mutations result in more subtle cellular changes.

Section snippets

Expression constructs

The QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) was used to introduce the L33P and the P353L mutations into the mouse Arx cDNA, subcloned in the pEGFP-C1 vector (Clontech, Palo Alto, CA, USA). All procedures were performed according to the manufacturer's instructions. In the case of the polyalanine stretch expansion construct (ARX-polyA), we amplified exon 2 of a patient known to carry the 7 alanine insertion within the first polyalanine stretch. This exon was

Characterisation of polyclonal antibodies specific to Arx

Two different polyclonal antibodies, one directed against the C-terminal part of the protein (anti-ARX-Cter) and the other against the homeodomain (anti-ARX-HD), were used to detect the Arx protein. As shown in Fig. 1A, anti-ARX-HD antibody revealed a band of approximately 70 kDa in rat neonatal and embryo (E17) brain extracts. In protein extracts from cerebellum, no protein was detected with these antibodies. This result is in line with the previously described in situ hybridisation and RT-PCR

Discussion

We analysed the expression of the Arx protein in the CNS during development and in the adult. Arx was found in a number of specific brain regions as well as in the developing spinal cord. During early development of the telencephalon, expression of Arx is observed in the ventricular zone and in post-mitotic neurons in the pre-plate. As development emerges, Arx expression is mainly observed in the marginal zone (MZ) as well as in post-mitotic neurons that are migrating through the intermediate

Acknowledgements

The authors are grateful to P. Billuart, J. Parnavelas, F. Fauchereau, M. Mildalgo-Sanchez and E. Bloch-Gallego for their helpful comments, M.C. Vinet for her technical assistance, A. Goffinet for providing the Reelin antibodies and A. Koulakoff for glial cell cultures. This work was supported by the European Community (QLG2-CT-1999-00791, QLG3-CT-2002-01810), CNRS, Fondation pour la recherche Médicale (FRM), Fondation Bettencourt Schuler, Fondation France Telecom, and Fondation Française pour

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