Mutations in ARX Result in Several Defects Involving GABAergic Neurons

Abstract

Genetic investigations of X-linked mental retardation have demonstrated the implication of ARX in a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild or moderate forms of mental retardation without apparent brain abnormalities, but with associated features of dystonia and epilepsy. These investigations have in recent years directed attention to the role of this gene in brain development. Analysis of its spatio-temporal localization profile revealed expression in telencephalic structures at all stages of development, mainly restricted to populations of GABA-containing neurons. Furthermore, studies of the effects of ARX loss of function either in humans or in lines of mutant mice revealed varying defects, suggesting multiple roles of this gene during development. In particular, Arx has been shown to contribute to almost all fundamental processes of brain development: patterning, neuronal proliferation and migration, cell maturation and differentiation, as well as axonal outgrowth and connectivity. In this review, we will present and discuss recent findings concerning the role of ARX in brain development and how this information will be useful to better understand the pathophysiological mechanisms of mental retardation and epilepsy associated with ARX mutations.

Keywords: ARX; GABA; basal ganglia; epilepsy; interneurons; lissencephaly; neuronal migration.

Figure 1

While non-malformation phenotypes [non-specific X-linked mental retardation (MRX), Partington (PRTS) and West syndromes, XMESID, IEDE and Ohtahara syndrome] tend to be caused by pathogenic variations outside the homeodomain or inside the first two polyalanines tracts of ARX, brain and genital malformation phenotypes [XLAG, hydranencephaly with abnormal genitalia (HYD-AG) and Proud syndrome] are associated with pathogenic variations that truncate the ARX protein or alter residues in the highly conserved homeodomain. Interestingly, a non-conservative missense mutation near the C-terminal aristaless domain (p.A521T) causes unusually severe XLAG with microcephaly and mild cerebellar hypoplasia.

Figure 2

Tangential migration of a few ARX-overexpressing cells in the cortex. (A,B) Examination of coronal sections of E16.5 mouse brains electroporated at E13.5 with an ARX-overexpressing construct. Tangentially orientated cells migrating away from the site of electroporation are detectable in the IZ (see arrows). Some of these cells have long and complex processes, orientated tangentially. (C,D) Examination of E18.5 coronal sections of mouse brains electroporated at E13.5 with an ARX-overexpressing construct. Five days after electroporation, the number of tangentially orientated cells observed was reduced, but some had migrated long distances (see arrows). Scale bars: (A,B) 100 μm, (C,D) 200 μm.

Figure 3

Measure of the transcriptional repression capacity of different ARX mutants corresponding to identified mutations in human. The capacity of transcriptional repression of mutant forms of ARX was tested on Lmo1 enhancer, which was cloned upstream TK-luciferase in a similar design as Fulp et al. (2008). ARX wild-type or mutant constructs were transfected in Neuro2a cells and the luciferase activity was measured. Luciferase data were normalized to Renilla expression and data are presented as the percentage of transcriptional activity compared to an empty vector control. Although P353L mutation does not have a detectable effect on ARX transcriptional repression, (GCG) + 7 and P353R both decrease ARX capacity to repress expression of the reporter gene.