Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

Investigation of a critical region for an X-linked mental retardation (XLMR) locus led us to identify a novel Aristaless related homeobox gene (ARX ). Inherited and de novo ARX mutations, including missense mutations and in frame duplications/insertions leading to expansions of polyalanine tracts in ARX, were found in nine familial and one sporadic case of MR. In contrast to other genes involved in XLMR, ARX expression is specific to the telencephalon and ventral thalamus. Notably there is an absence of expression in the cerebellum throughout development and also in adult. The absence of detectable brain malformations in patients suggests that ARX may have an essential role, in mature neurons, required for the development of cognitive abilities.

X-linked forms of mental retardation (MR) affect approximately 1 in 600 males and are likely to be highly heterogeneous. They can be categorized into syndromic (MRXS) and nonspecific (MRX) forms. In MRX forms, affected patients have no distinctive clinical or biochemical features. At least five MRX genes have been identified by positional cloning, but each accounts for only 0.5%-1.0% of MRX cases. Here we show that the gene TM4SF2 at Xp11.4 is inactivated by the X breakpoint of an X;2 balanced translocation in a patient with MR. Further investigation led to identification of TM4SF2 mutations in 2 of 33 other MRX families. RNA in situ hybridization showed that TM4SF2 is highly expressed in the central nervous system, including the cerebral cortex and hippocampus. TM4SF2 encodes a member of the tetraspanin family of proteins, which are known to contribute in molecular complexes including beta-1 integrins. We speculate that through this interaction, TM4SF2 might have a role in the control of neurite outgrowth.

Type I lissencephaly is a cortical malformation disorder characterized by disorganized cortical layers and gyral abnormalities and associated with severe cognitive impairment and epilepsy. The exact pathophysiological mechanisms underlying the epilepsy and mental retardation in this and related disorders remain unknown. Two genes, LIS1 and doublecortin, have both been shown to be mutated in a large proportion of cases of type I lissencephaly and a milder allelic disorder, subcortical laminar heterotopia (SCLH). Studying the protein products of these genes and the biochemical pathways in which they belong is likely to yield important information concerning both normal and abnormal cortical development. The relationships between the LIS1 and Doublecortin proteins are not yet well defined, but both are believed to play a critical role in cortical neuronal migration. Lis1 is expressed from very early development in the mouse and in both proliferating cells and post-mitotic neurons of the cortex. This protein is likely to have multiple functions since it is a subunit of the enzyme platelet-activating factor acetylhydrolase, which degrades platelet activating factor, and has also been shown to be involved in microtubule dynamics, potentially influencing nuclear migration through its interaction with the dynein motor protein complex. Doublecortin on the other hand is exclusively expressed in post-mitotic neurons and is developmentally regulated. In young developing neurons Doublecortin has a specific subcellular localization at the ends of neuritic and leading processes. This localization, combined with our previous data showing that it is a microtubule-associated protein and that it interacts with adapter complexes involved in vesicle trafficking, suggests a role in the growth of neuronal processes, downstream of directional or guidance signals. The observations summarized here favor the suggestion that whereas LIS1 may play a role in nuclear migration, Doublecortin is instead restricted to functions at the leading edge of the cell.

Type I lissencephaly, a genetic disease characterized by disorganized cortical layers and gyral abnormalities, is associated with severe cognitive impairment and epilepsy. Two genes, LIS1 and doublecortin (DCX), have been shown to be responsible for a large proportion of cases of type I lissencephaly. Both genes encode microtubule-associated proteins that have been shown to be important for radial migration of cortical pyramidal neurons. To investigate whether DCX also plays a role in cortical interneuron migration, we inactivated DCX in the ganglionic eminence of rat embryonic day 17 brain slices using short hairpin RNA. We found that, when DCX expression was blocked, the migration of interneurons from the ganglionic eminence to the cerebral cortex was slowed but not absent, similar to what had previously been reported for radial neuronal migration. In addition, the processes of DCX-deficient migrating interneurons were more branched than their counterparts in control experiments. These effects were rescued by DCX overexpression, confirming the specificity to DCX inactivation. A similar delay in interneuron migration was observed when Doublecortin-like kinase (DCLK), a microtubuleassociated protein related to DCX, was inactivated, although the morphology of the cells was not affected. The importance of these genes in interneuron migration was confirmed by our finding that the cortices of Dcx, Dclk, and Dcx/Dclk mutant mice contained a reduced number of such cells in the cortex and their distribution was different compared with wild-type controls. However, the defect was different for each group of mutant animals, suggesting that DCX and DCLK have distinct roles in cortical interneuron migration.

The aristaless-related homeobox (ARX) gene has been implicated in a wide spectrum of disorders ranging from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild forms of X-linked mental retardation without apparent brain abnormalities. To better understand its role in corticogenesis, we used in utero electroporation to knock down or overexpress ARX. We show here that targeted inhibition of ARX causes cortical progenitor cells to exit the cell cycle prematurely and impairs their migration toward the cortical plate. In contrast, ARX overexpression increases the length of the cell cycle. In addition, we report that RNA interferencemediated inactivation of ARX prevents cells from acquiring multipolar morphology in the subventricular and intermediate zones, resulting in decreased neuronal motility. In contrast, ARX overexpression appears to promote the development of tangentially oriented processes of cells in the subventricular and intermediate zones and affects radial migration of pyramidal neurons. We also demonstrate that the level of ARX expression is important for tangential migration of GABA-containing interneurons, because both inactivation and overexpression of the gene impair their migration from the ganglionic eminence. However, our data suggest that ARX is not directly involved in GABAergic cell fate specification. Overall, these results identify multiple and distinct cell-autonomous roles for ARX in corticogenesis.

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Previously, human genetics-based approaches allowed us to show that mutations in the IL-1 receptor accessory protein-like gene (IL1RAPL) are responsible for a non-specific form of X-linked mental retardation. This gene encodes a predicted protein of 696 amino acids that belongs to a novel class of the IL-1/Toll receptor family. In addition to the extracellular portion consisting of three Ig-like domains and the intracellular TIR domain characteristic of the IL-1/Toll receptor family, IL1RAPL contains a specific 150 amino acid carboxy terminus that has no significant homology with any protein of known function. In order to begin to elucidate the function of this IL-1/Toll receptor-like protein, we have assessed the effect of recombinant IL1RAPL on the binding affinity of type I IL-1R for its ligands IL-1alpha and beta and searched for proteins interacting with the specific carboxy terminus domain of IL1RAPL. Our results show that IL1RAPL is not a protein receptor for IL-1. In addition we present here the identification of Neuronal Calcium Sensor-1 (NCS-1) as an IL1RAPL interactor. Remarkably, although NCS-1 and its non-mammalian homologue, frequenin, are members of a highly conserved EF-hand Ca(2+) binding protein family, our data show that IL1RAPL interacts only with NCS-1 through its specific C-terminal domain. The functional relevance of IL1RAPL activity was further supported by the inhibitory effect on exocytosis in PC12 cells overexpressing IL1RAPL. Taken together, our data suggest that IL1RAPL may regulate calcium-dependent exocytosis and provide insight into the understanding of physiopathological mechanisms underlying cognitive impairment resulting from IL1RAPL dysfunction.