Neurabin II mediates doublecortin-dephosphorylation on actin filaments

Mutations in the human Doublecortin (DCX) gene cause X-linked lissencephaly, a neuronal migration disorder. DCX binds to microtubules and actin filaments. Association of Dcx with F-actin is regulated by site-specific phosphorylation and by neurabin II, an F-actin binding protein that also binds to Dcx. We show here that neurabin II mediates dephosphorylation of Dcx by protein phosphatase 1 (PP1). Furthermore, overexpression of PP1 reduces Dcx phosphorylation and decreases Dcx binding to F-actin. By contrast, abolishing PP1 binding to neurabin II maintains phosphorylation levels of Dcx, leading to a retention of Dcx at F-actin. We suggest that a dynamic regulation of Dcx mediated by neurabin II regulates the translocation of Dcx from F-actin to microtubules and vice versa.     

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.     

Doublecortin microtubule affinity is regulated by a balance of kinase and phosphatase activity at the leading edge of migrating neurons

Doublecortin (Dcx) is a microtubule-associated protein that is mutated in X-linked lissencephaly (X-LIS), a neuronal migration disorder associated with epilepsy and mental retardation. Although Dcx can bind ubiquitously to microtubules in nonneuronal cells, Dcx is highly enriched in the leading processes of migrating neurons and the growth cone region of differentiating neurons. We present evidence that Dcx/microtubule interactions are negatively controlled by Protein Kinase A (PKA) and the MARK/PAR-1 family of protein kinases. In addition to a consensus MARK site, we identified a serine within a novel sequence that is crucial for the PKA- and MARK-dependent regulation of Dcx's microtubule binding activity in vitro. This serine is mutated in two families affected by X-LIS. Immunostaining neurons with an antibody that recognizes phosphorylated substrates of MARK supports the conclusion that Dcx localization and function are regulated at the leading edge of migrating cells by a balance of kinase and phosphatase activity.     

A LIS1/NUDEL/cytoplasmic dynein heavy chain complex in the developing and adult nervous system

Mutations in mammalian Lis1 (Pafah1b1) result in neuronal migration defects. Several lines of evidence suggest that LIS1 participates in pathways regulating microtubule function, but the molecular mechanisms are unknown. Here, we demonstrate that LIS1 directly interacts with the cytoplasmic dynein heavy chain (CDHC) and NUDEL, a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE. LIS1 and NUDEL colocalize predominantly at the centrosome in early neuroblasts but redistribute to axons in association with retrograde dynein motor proteins. NUDEL is phosphorylated by Cdk5/p35, a complex essential for neuronal migration. NUDEL and LIS1 regulate the distribution of CDHC along microtubules, and establish a direct functional link between LIS1, NUDEL, and microtubule motors. These results suggest that LIS1 and NUDEL regulate CDHC activity during neuronal migration and axonal retrograde transport in a Cdk5/p35-dependent fashion.     

Regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian Lis1

Whereas total loss of Lis1 is lethal, disruption of one allele of the Lis1 gene results in brain abnormalities, indicating that developing neurons are particularly sensitive to a reduction in Lis1 dosage. Here we show that Lis1 is enriched in neurons relative to levels in other cell types, and that Lis1 interacts with the microtubule motor cytoplasmic dynein. Production of more Lis1 in non-neuronal cells increases retrograde movement of cytoplasmic dynein and leads to peripheral accumulation of microtubules. These changes may reflect neuron-like dynein behaviours induced by abundant Lis1. Lis1 deficiency produces the opposite phenotype. Our results indicate that abundance of Lis1 in neurons may stimulate specific dynein functions that function in neuronal migration and axon growth.     

Drosophila Lis1 is required for neuroblast proliferation, dendritic elaboration and axonal transport

Haplo-insufficiency of human Lis1 causes lissencephaly. Reduced Lis1 activity in both humans and mice results in a neuronal migration defect. Here we show that Drosophila Lis1 is highly expressed in the nervous system. Lis1 is essential for neuroblast proliferation and axonal transport, as shown by a mosaic analysis using a Lis1 null mutation. Moreover, it is cell-autonomously required for dendritic growth, branching and maturation. Analogous mosaic analysis shows that neurons containing a mutated cytoplasmic-dynein heavy chain (Dhc64C) exhibit phenotypes similar to Lis1 mutants. These results implicate Lis1 as a regulator of the microtubule cytoskeleton and show that it is important for diverse physiological functions in the nervous system.     

Complete loss of Ndel1 results in neuronal migration defects and early embryonic lethality

Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1(-/-) mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1(-/-) blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1(+/-) mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1(cko/-)) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1(cko/+)-Ndel1(+/-) mice or Lis1(+/-)-Ndel1(+/-) mice displayed more severe neuronal migration defects than Lis1(cko/+)-Ndel1(+/)(+) mice or Lis1(+/-)-Ndel1(+/+) mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of beta-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.     

Cdk5 phosphorylation of doublecortin ser297 regulates its effect on neuronal migration

Mutations in the doublecortin (DCX) gene in human or targeted disruption of the cdk5 gene in mouse lead to similar cortical lamination defects in the developing brain. Here we show that Dcx is phosphorylated by Cdk5. Dcx phosphorylation is developmentally regulated and corresponds to the timing of expression of p35, the major activating subunit for Cdk5. Mass spectrometry and Western blot analysis indicate phosphorylation at Dcx residue Ser297. Phosphorylation of Dcx lowers its affinity to microtubules in vitro, reduces its effect on polymerization, and displaces it from microtubules in cultured neurons. Mutation of Ser297 blocks the effect of Dcx on migration in a fashion similar to pharmacological inhibition of Cdk5 activity. These results suggest that Dcx phosphorylation by Cdk5 regulates its actions on migration through an effect on microtubules.     

Doublecortin association with actin filaments is regulated by neurabin II

Mutations in the human Doublecortin (DCX) gene cause X-linked lissencephaly, a neuronal migration disorder affecting the neocortex and characterized by mental retardation and epilepsy. Because dynamic cellular asymmetries such as those seen in cell migration critically depend on a cooperation between the microtubule and actin cytoskeletal filament systems, we investigated whether Dcx, a microtubule-associated protein, is engaged in cytoskeletal cross-talk. We now demonstrate that Dcx co-sediments with actin filaments (F-actin), and using light and electron microscopy and spin down assays, we show that Dcx induces bundling and cross-linking of microtubules and F-actin in vitro. It has recently been shown that binding of Dcx to microtubules is negatively regulated by phosphorylation of the Dcx at Ser-47 or Ser-297. Although the phosphomimetic green fluorescent protein (GFP)-Dcx(S47E) transfected into COS-7 cells had a reduced affinity for microtubules, we found that pseudophosphorylation was not sufficient to cause Dcx to bind to F-actin. When cells were co-transfected with neurabin II, a protein that binds F-actin as well as Dcx, GFP-Dcx and to an even greater extent GFP-Dcx(S47E) became predominantly associated with filamentous actin. Thus Dcx phosphorylation and neurabin II combinatorially enhance Dcx binding to F-actin. Our findings raise the possibility that Dcx acts as a molecular link between microtubule and actin cytoskeletal filaments that is regulated by phosphorylation and neurabin II.     

Ndel1 controls the dynein-mediated transport of vimentin during neurite outgrowth

Ndel1, the mammalian homologue of the Aspergillus nidulans NudE, is emergently viewed as an integrator of the cytoskeleton. By regulating the dynamics of microtubules and assembly of neuronal intermediate filaments (IFs), Ndel1 promotes neurite outgrowth, neuronal migration, and cell integrity (1-6). To further understand the roles of Ndel1 in cytoskeletal dynamics, we performed a tandem affinity purification of Ndel1-interacting proteins. We isolated a novel Ndel1 molecular complex composed of the IF vimentin, the molecular motor dynein, the lissencephaly protein Lis1, and the cis-Golgi-associated protein alphaCOP. Ndel1 promotes the interaction between Lis1, alphaCOP, and the vimentin-dynein complex. The functional result of this complex is activation of dynein-mediated transport of vimentin. A loss of Ndel1 functions by RNA interference fails to incorporate Lis1/alphaCOP in the complex, reduces the transport of vimentin, and culminates in IF accumulations and altered neuritogenesis. Our findings reveal a novel regulatory mechanism of vimentin transport during neurite extension that may have implications in diseases featuring transport/trafficking defects and impaired regeneration.     

Dictyostelium LIS1 is a centrosomal protein required for microtubule/cell cortex interactions, nucleus/centrosome linkage, and actin dynamics

The widespread LIS1-proteins were originally identified as the target for sporadic mutations causing lissencephaly in humans. Dictyostelium LIS1 (DdLIS1) is a microtubule-associated protein exhibiting 53% identity to human LIS1. It colocalizes with dynein at isolated, microtubule-free centrosomes, suggesting that both are integral centrosomal components. Replacement of the DdLIS1 gene by the hypomorphic D327H allele or overexpression of an MBP-DdLIS1 fusion disrupted various dynein-associated functions. Microtubules lost contact with the cell cortex and were dragged behind an unusually motile centrosome. Previously, this phenotype was observed in cells overexpressing fragments of dynein or the XMAP215-homologue DdCP224. DdLIS1 was coprecipitated with DdCP224, suggesting that both act together in dynein-mediated cortical attachment of microtubules. Furthermore, DdLIS1-D327H mutants showed Golgi dispersal and reduced centrosome/nucleus association. Defects in DdLIS1 function also altered actin dynamics characterized by traveling waves of actin polymerization correlated with a reduced F-actin content. DdLIS1 could be involved in actin dynamics through Rho-GTPases, because DdLIS1 interacted directly with Rac1A in vitro. Our results show that DdLIS1 is required for maintenance of the microtubule cytoskeleton, Golgi apparatus and nucleus/centrosome association, and they suggest that LIS1-dependent alterations of actin dynamics could also contribute to defects in neuronal migration in lissencephaly patients.     

Doublecortin-like kinase functions with doublecortin to mediate fiber tract decussation and neuronal migration

The potential role of doublecortin (Dcx), encoding a microtubule-associated protein, in brain development has remained controversial. Humans with mutations show profound alterations in cortical lamination, whereas in mouse, RNAi-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. Here, we report that the doublecortin-like kinase (Dclk) gene functions in a partially redundant pathway with Dcx in the formation of axonal projections across the midline and migration of cortical neurons. Dosage-dependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. Surprisingly, RNAi-mediated knockdown of either gene results in similar migration defects. These results indicate the Dcx microtubule-associated protein family is required for proper neuronal migration and axonal wiring.     

Genetic interactions between doublecortin and doublecortin-like kinase in neuronal migration and axon outgrowth

Although mutations in the human doublecortin gene (DCX) cause profound defects in cortical neuronal migration, a genetic deletion of Dcx in mice produces a milder defect. A second locus, doublecortin-like kinase (Dclk), encodes a protein with similar "doublecortin domains" and microtubule stabilization properties that may compensate for Dcx. Here, we generate a mouse with a Dclk mutation that causes no obvious migrational abnormalities but show that mice mutant for both Dcx and Dclk demonstrate perinatal lethality, disorganized neocortical layering, and profound hippocampal cytoarchitectural disorganization. Surprisingly, Dcx(-/y);Dclk(-/-) mutants have widespread axonal defects, affecting the corpus callosum, anterior commissure, subcortical fiber tracts, and internal capsule. Dcx/Dclk-deficient dissociated neurons show abnormal axon outgrowth and dendritic structure, with defects in axonal transport of synaptic vesicle proteins. Dcx and Dclk may directly or indirectly regulate microtubule-based vesicle transport, a process critical to both neuronal migration and axon outgrowth.     

Ndel1 operates in a common pathway with LIS1 and cytoplasmic dynein to regulate cortical neuronal positioning

Correct neuronal migration and positioning during cortical development are essential for proper brain function. Mutations of the LIS1 gene result in human lissencephaly (smooth brain), which features misplaced cortical neurons and disarrayed cerebral lamination. However, the mechanism by which LIS1 regulates neuronal migration remains unknown. Using RNA interference (RNAi), we found that the binding partner of LIS1, NudE-like protein (Ndel1, formerly known as NUDEL), positively regulates dynein activity by facilitating the interaction between LIS1 and dynein. Loss of function of Ndel1, LIS1, or dynein in developing neocortex impairs neuronal positioning and causes the uncoupling of the centrosome and nucleus. Overexpression of LIS1 partially rescues the positioning defect caused by Ndel1 RNAi but not dynein RNAi, whereas overexpression of Ndel1 does not rescue the phenotype induced by LIS1 RNAi. These results provide strong evidence that Ndel1 interacts with LIS1 to sustain the function of dynein, which in turn impacts microtubule organization, nuclear translocation, and neuronal positioning.     

The structure of the coiled-coil domain of Ndel1 and the basis of its interaction with Lis1, the causal protein of Miller-Dieker lissencephaly

Ndel1 and Nde1 are homologous and evolutionarily conserved proteins, with critical roles in cell division, neuronal migration, and other physiological phenomena. These functions are dependent on their interactions with the retrograde microtubule motor dynein and with its regulator Lis1--a product of the causal gene for isolated lissencephaly sequence (ILS) and Miller-Dieker lissencephaly. The molecular basis of the interactions of Ndel1 and Nde1 with Lis1 is not known. Here, we present a crystallographic study of two fragments of the coiled-coil domain of Ndel1, one of which reveals contiguous high-quality electron density for residues 10-166, the longest such structure reported by X-ray diffraction at high resolution. Together with complementary solution studies, our structures reveal how the Ndel1 coiled coil forms a stable parallel homodimer and suggest mechanisms by which the Lis1-interacting domain can be regulated to maintain a conformation in which two supercoiled alpha helices cooperatively bind to a Lis1 homodimer.     

Three-dimensional regulation of radial glial functions by Lis1-Nde1 and dystrophin glycoprotein complexes

Radial glial cells (RGCs) are distinctive neural stem cells with an extraordinary slender bipolar morphology and dual functions as precursors and migration scaffolds for cortical neurons. Here we show a novel mechanism by which the Lis1-Nde1 complex maintains RGC functions through stabilizing the dystrophin/dystroglycan glycoprotein complex (DGC). A direct interaction between Nde1 and utrophin/dystrophin allows for the assembly of a multi-protein complex that links the cytoskeleton to the extracellular matrix of RGCs to stabilize their lateral membrane, cell-cell adhesion, and radial morphology. Lis1-Nde1 mutations destabilized the DGC and resulted in deformed, disjointed RGCs and disrupted basal lamina. Besides impaired RGC self-renewal and neuronal migration arrests, Lis1-Nde1 deficiencies also led to neuronal over-migration. Additional to phenotypic resemblances of Lis1-Nde1 with DGC, strong synergistic interactions were found between Nde1 and dystroglycan in RGCs. As functional insufficiencies of LIS1, NDE1, and dystroglycan all cause lissencephaly syndromes, our data demonstrated that a three-dimensional regulation of RGC's cytoarchitecture by the Lis1-Nde1-DGC complex determines the number and spatial organization of cortical neurons as well as the size and shape of the cerebral cortex.     

Inactivation of a testis-specific Lis1 transcript in mice prevents spermatid differentiation and causes male infertility

Lis1 protein is the non-catalytic component of platelet-activating factor acetylhydrolase 1b (PAF-AH 1B) and associated with microtubular structures. Hemizygous mutations of the LIS1 gene cause type I lissencephaly, a brain abnormality with developmental defects of neuronal migration. Lis1 is also expressed in testis, but its function there has not been determined. We have generated a mouse mutant (LIS1GT/GT) by gene trap integration leading to selective disruption of a Lis1 splicing variant in testis. Homozygous mutant males are infertile with no other apparent phenotype. We demonstrate that Lis1 is predominantly expressed in spermatids, and spermiogenesis is blocked when Lis1 is absent. Mutant spermatids fail to form correct acrosomes and nuclei appear distorted in size and shape. The tissue architecture in mutant testis appears severely disturbed displaying collapsed seminiferous tubules, mislocated germ cells, and increased apoptosis. These results provide evidence for an essential and hitherto uncharacterized role of the Lis1 protein in spermatogenesis, particularly in the differentiation of spermatids into spermatozoa.     

Neuronal migration.

Like other motile cells, neurons migrate in three schematic steps, namely leading edge extension, nuclear translocation or nucleokinesis, and retraction of the trailing process. In addition, neurons are ordered into architectonic patterns at the end of migration. Leading edge extension can proceed at the extremity of the axon, by growth cone formation, or from the dendrites, by formation of dendritic tips. Among both categories of leading edges, variation seems to be related to the rate of extension of the leading process. Leading edge extension is directed by microfilament polymerization following integration of extracellular cues and is regulated by Rho-type small GTPases. In humans, mutations of filamin, an actin-associated protein, result in heterotopic neurons, probably due to defective leading edge extension. The second event in neuron migration is nucleokinesis, a process which is critically dependent on the microtubule network, as shown in many cell types, from slime molds to vertebrates. In humans, mutations in the PAFAH1B1 gene (more commonly called LIS1) or in the doublecortin (DCX) gene result in type 1 lissencephalies that are most probably due to defective nucleokinesis. Both the Lis1 and doublecortin proteins interact with microtubules, and two Lis1-interacting proteins, Nudel and mammalian NudE, are components of the dynein motor complex and of microtubule organizing centers. In mice, mutations of Cdk5 or of its activators p35 and p39 result in a migration phenotype compatible with defective nucleokinesis, although an effect on leading edge formation is also likely. The formation of architectonic patterns at the end of migration requires the integrity of the Reelin signalling pathway. Other known components of the pathway include members of the lipoprotein receptor family, the intracellular adaptor Dab1, and possibly integrin alpha 3 beta 1. Defective Reelin leads to poor lamination and, in humans, to a lissencephaly phenotype different from type 1 lissencephaly. Although the action of Reelin is unknown, it may trigger some recognition-adhesion among target neurons. Finally, pattern formation requires the integrity of the external limiting membrane, defects of which lead to overmigration of neurons in meninges and to human type 2 lissencephaly.     

Neurons acetylate their way to migration

The centrosome is crucial for neuronal migration and polarisation, processes that are disrupted in a number of neurodevelopmental disorders including schizophrenia. Mutation of DISC1, associated with increased risk of schizophrenia and psychiatric illness, has been shown to affect the centrosome, but the mechanisms involved have not been elucidated. In this issue of EMBO Reports, Fukuda and colleagues demonstrate that a DISC1‐interacting protein, CAMDI, suppresses the activity of the histone deacetylase HDAC6, thereby promoting centrosome stability and consequently neuronal migration 1 . Loss of CAMDI leads to cortical migration defects and behavioural phenotypes that model autism spectrum disorders and which can be rescued by inhibition of HDAC6. The study provides novel mechanistic insight into centrosome regulation in neurodevelopment.