Mutations in ARX Result in Several Defects Involving GABAergic Neurons

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

Identification of Arx targets unveils new candidates for controlling cortical interneuron migration and differentiation

Mutations in the homeobox transcription factor ARX have been found to be responsible for a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild forms of intellectual disabilities without apparent brain abnormalities, but with associated features of dystonia and epilepsy. Arx expression is mainly restricted to populations of GABA-containing neurons. Studies of the effects of ARX loss of function, either in humans or mutant mice, revealed varying defects, suggesting multiple roles of this gene in brain patterning, neuronal proliferation and migration, cell maturation and differentiation, as well as axonal outgrowth and connectivity. However, to date, little is known about how Arx functions as a transcription factor or which genes it binds and regulates. Recently, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified approximately 1000 gene promoters bound by Arx in transfected neuroblastoma N2a cells and mouse embryonic brain. To narrow the analysis of Arx targets to those most likely to control cortical interneuron migration and/or differentiation, we compare here our data to previously published studies searching for genes enriched or down-regulated in cortical interneurons between E13.5 and E15.5. We thus identified 14 Arx-target genes enriched (Cxcr7, Meis1, Ppap2a, Slc 12a5, Ets2, Phlda1, Egr1, Igf1, Lmo3, Sema6, Lgi1, Alk, Tgfb3, and Napb) and 5 genes specifically down-regulated (Hmgn3, Lmo1, Ebf3, Rasgef1b, and Slit2) in cortical migrating neurons. In this review, we present these genes and discuss how their possible regulation by Arx may lead to the dysfunction of GABAergic neurons, resulting in mental retardation and epilepsy

Both doublecortin and doublecortin-like kinase play a role in cortical interneuron migration

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 microtubule-associated 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

High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

Genetic investigations of X-linked intellectual disabilities have implicated the ARX (Aristaless-related homeobox) gene in a wide spectrum of disorders extending from phenotypes characterised by 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. Analysis of Arx spatio-temporal localisation profile in mouse revealed expression in telencephalic structures, mainly restricted to populations of GABAergic neurons at all stages of development. Furthermore, studies of the effects of ARX loss of function in humans and animal models revealed varying defects, suggesting multiple roles of this gene during brain development. However, to date, little is known about how ARX functions as a transcription factor and the nature of its targets. To better understand its role, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified a total of 1006 gene promoters bound by Arx in transfected neuroblastoma (N2a) cells and in mouse embryonic brain. Approximately 24% of Arx-bound genes were found to show expression changes following Arx overexpression or knock-down. Several of the Arx target genes we identified are known to be important for a variety of functions in brain development and some of them suggest new functions for Arx. Overall, these results identified multiple new candidate targets for Arx and should help to better understand the pathophysiological mechanisms of intellectual disability and epilepsy associated with ARX mutations

Role of cytoskeletal abnormalities in the neuropathology and pathophysiology of type I lissencephaly

Type I lissencephaly or agyria-pachygyria is a rare developmental disorder which results from a defect of neuronal migration. It is characterized by the absence of gyri and a thickening of the cerebral cortex and can be associated with other brain and visceral anomalies. Since the discovery of the first genetic cause (deletion of chromosome 17p13.3), six additional genes have been found to be responsible for agyria–pachygyria. In this review, we summarize the current knowledge concerning these genetic disorders including clinical, neuropathological and molecular results. Genetic alterations of LIS1, DCX, ARX, TUBA1A, VLDLR, RELN and more recently WDR62 genes cause migrational abnormalities along with more complex and subtle anomalies affecting cell proliferation and differentiation, i.e., neurite outgrowth, axonal pathfinding, axonal transport, connectivity and even myelination. The number and heterogeneity of clinical, neuropathological and radiological defects suggest that type I lissencephaly now includes several forms of cerebral malformations. In vitro experiments and mutant animal studies, along with neuropathological abnormalities in humans are of invaluable interest for the understanding of pathophysiological mechanisms, highlighting the central role of cytoskeletal dynamics required for a proper achievement of cell proliferation, neuronal migration and differentiation

Uncovering memory-related gene expression in contextual fear conditioning using ribosome profiling

Contextual fear conditioning (CFC) in rodents is the most widely used behavioural paradigm in neuroscience research to elucidate the neurobiological mechanisms underlying learning and memory. It is based on the pairing of an aversive unconditioned stimulus (US; e.g. mild footshock) with a neutral conditioned stimulus (CS; e.g. context of the test chamber) in order to acquire associative long-term memory (LTM), which persists for days and even months. Using genome-wide analysis, several studies have generated lists of genes modulated in response to CFC in an attempt to identify the "memory genes", which orchestrate memory formation. Yet, most studies use naïve animals as a baseline for assessing gene-expression changes, while only few studies have examined the effect of the US alone, without pairing to context, using genome-wide analysis of gene-expression. Herein, using the ribosome profiling methodology, we show that in male mice an immediate shock, which does not lead to LTM formation, elicits pervasive translational and transcriptional changes in the expression of Immediate Early Genes (IEGs) in dorsal hippocampus (such as Fos and Arc), a fact which has been disregarded by the majority of CFC studies. By removing the effect of the immediate shock, we identify and validate a new set of genes, which are translationally and transcriptionally responsive to the association of context-to-footshock in CFC, and thus constitute salient "memory genes"

Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression

<p>Abstract</p> <p>Background</p> <p>Neuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem.</p> <p>Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment.</p> <p>Methods</p> <p>We analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration) and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively.</p> <p>Results</p> <p>Doublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness.</p> <p>Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin.</p> <p>Conclusion</p> <p>a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness;</p> <p>b) Retinoic acid treatment reduces migration and invasiveness of the more aggressive cell components of SK-N-SH cells;</p> <p>c) The cells that after retinoic acid exposure show migration and invasive capability may be identified on the basis of doublecortin expression.</p

Global Developmental Gene Expression and Pathway Analysis of Normal Brain Development and Mouse Models of Human Neuronal Migration Defects

Heterozygous LIS1 mutations are the most common cause of human lissencephaly, a human neuronal migration defect, and DCX mutations are the most common cause of X-linked lissencephaly. LIS1 is part of a protein complex including NDEL1 and 14-3-3ε that regulates dynein motor function and microtubule dynamics, while DCX stabilizes microtubules and cooperates with LIS1 during neuronal migration and neurogenesis. Targeted gene mutations of Lis1, Dcx, Ywhae (coding for 14-3-3ε), and Ndel1 lead to neuronal migration defects in mouse and provide models of human lissencephaly, as well as aid the study of related neuro-developmental diseases. Here we investigated the developing brain of these four mutants and wild-type mice using expression microarrays, bioinformatic analyses, and in vivo/in vitro experiments to address whether mutations in different members of the LIS1 neuronal migration complex lead to similar and/or distinct global gene expression alterations. Consistent with the overall successful development of the mutant brains, unsupervised clustering and co-expression analysis suggested that cell cycle and synaptogenesis genes are similarly expressed and co-regulated in WT and mutant brains in a time-dependent fashion. By contrast, focused co-expression analysis in the Lis1 and Ndel1 mutants uncovered substantial differences in the correlation among pathways. Differential expression analysis revealed that cell cycle, cell adhesion, and cytoskeleton organization pathways are commonly altered in all mutants, while synaptogenesis, cell morphology, and inflammation/immune response are specifically altered in one or more mutants. We found several commonly dysregulated genes located within pathogenic deletion/duplication regions, which represent novel candidates of human mental retardation and neurocognitive disabilities. Our analysis suggests that gene expression and pathway analysis in mouse models of a similar disorder or within a common pathway can be used to define novel candidates for related human diseases

Loss of STOP Protein Impairs Peripheral Olfactory Neurogenesis

International audienceIn conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia

Epilepsy in Dcx Knockout Mice Associated with Discrete Lamination Defects and Enhanced Excitability in the Hippocampus

Patients with Doublecortin (DCX) mutations have severe cortical malformations associated with mental retardation and epilepsy. Dcx knockout (KO) mice show no major isocortical abnormalities, but have discrete hippocampal defects. We questioned the functional consequences of these defects and report here that Dcx KO mice are hyperactive and exhibit spontaneous convulsive seizures. Changes in neuropeptide Y and calbindin expression, consistent with seizure occurrence, were detected in a large proportion of KO animals, and convulsants, including kainate and pentylenetetrazole, also induced seizures more readily in KO mice. We show that the dysplastic CA3 region in KO hippocampal slices generates sharp wave-like activities and possesses a lower threshold for epileptiform events. Video-EEG monitoring also demonstrated that spontaneous seizures were initiated in the hippocampus. Similarly, seizures in human patients mutated for DCX can show a primary involvement of the temporal lobe. In conclusion, seizures in Dcx KO mice are likely to be due to abnormal synaptic transmission involving heterotopic cells in the hippocampus and these mice may therefore provide a useful model to further study how lamination defects underlie the genesis of epileptiform activities