Joseph G. Gleeson

Doublecortin Is a Microtubule-Associated Protein and Is Expressed Widely by Migrating Neurons

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.

PAK3 mutation in nonsyndromic X-linked mental retardation

Nonsyndromic X-linked mental retardation (MRX) syndromes are clinically homogeneous but genetically heterogeneous disorders, whose genetic bases are largely unknown. Affected individuals in a multiplex pedigree with MRX (MRX30), previously mapped to Xq22, show a point mutation in the PAK3 (p21-activated kinase) gene, which encodes a serine-threonine kinase. PAK proteins are crucial effectors linking Rho GTPases to cytoskeletal reorganization and to nuclear signalling. The mutation produces premature termination, disrupting kinase function. MRI analysis showed no gross defects in brain development. Immunofluorescence analysis showed that PAK3 protein is highly expressed in postmitotic neurons of the developing and postnatal cerebral cortex and hippocampus. Signal transduction through Rho GTPases and PAK3 may be critical for human cognitive function.

De novo somatic mutations in components of the PI3K-AKT3-mTOR pathway cause hemimegalencephaly

De novo somatic mutations in focal areas are well documented in diseases such as neoplasia but are rarely reported in malformation of the developing brain. Hemimegalencephaly (HME) is characterized by overgrowth of either one of the two cerebral hemispheres. The molecular etiology of HME remains a mystery. The intractable epilepsy that is associated with HME can be relieved by the surgical treatment hemispherectomy, allowing sampling of diseased tissue. Exome sequencing and mass spectrometry analysis in paired brain-blood samples from individuals with HME (n = 20 cases) identified de novo somatic mutations in 30% of affected individuals in the PIK3CA, AKT3 and MTOR genes. A recurrent PIK3CA c.1633G>A mutation was found in four separate cases. Identified mutations were present in 8–40% of sequenced alleles in various brain regions and were associated with increased neuronal S6 protein phosphorylation in the brains of affected individuals, indicating aberrant activation of mammalian target of rapamycin (mTOR) signaling. Thus HME is probably a genetically mosaic disease caused by gain of function in phosphatidylinositol 3-kinase (PI3K)-AKT3-mTOR signaling.

Lis1 and doublecortin function with dynein to mediate coupling of the nucleus to the centrosome in neuronal migration

Humans with mutations in either DCX or LIS1 display nearly identical neuronal migration defects, known as lissencephaly. To define subcellular mechanisms, we have combined in vitro neuronal migration assays with retroviral transduction. Overexpression of wild-type Dcx or Lis1, but not patient-related mutant versions, increased migration rates. Dcx overexpression rescued the migration defect in Lis1 +/− neurons. Lis1 localized predominantly to the centrosome, and after disruption of microtubules, redistributed to the perinuclear region. Dcx outlined microtubules extending from the perinuclear “cage” to the centrosome. Lis1 +/− neurons displayed increased and more variable separation between the nucleus and the preceding centrosome during migration. Dynein inhibition resulted in similar defects in both nucleus–centrosome (N-C) coupling and neuronal migration. These N-C coupling defects were rescued by Dcx overexpression, and Dcx was found to complex with dynein. These data indicate Lis1 and Dcx function with dynein to mediate N-C coupling during migration, and suggest defects in this coupling may contribute to migration defects in lissencephaly.

Functional genomic screen for modulators of ciliogenesis and cilium length

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell–cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

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.

Neuronal migration disorders: from genetic diseases to developmental mechanisms

Neurons that constitute the cerebral cortex must migrate hundreds of cell-body distances from their place of birth, and through several anatomical boundaries, to reach their final position within the correct cortical layer. Human neurological conditions associated with abnormal neuronal migration, together with spontaneous and engineered mouse mutants, define at least four distinct steps in cortical neuronal migration. Many of the genes that control neuronal migration have strong genetic or biochemical links to the cytoskeleton, suggesting that the field of neuronal migration might be closing in on the underlying cytoskeletal events.

Nucleokinesis in neuronal migration.

Neuronal migration is a critical phase of nervous system development and can be divided into two distinct phases: extension of the leading process and movement of the cell body and nucleus (nucleokinesis). Nucleokinesis appears to require many of the same cytoskeletal and signaling molecules used in cell mitosis. Converging studies suggest it requires cytoplasmic dynein, cell polarity genes, and microtubule-associated proteins that coordinate microtubule remodeling. These coordinate first the positioning of the centrosome (microtubule organizing center) in the leading process in front of the nucleus and then the movement of the nucleus towards the centrosome. The positioning of the centrosome and the dynamic regulation that couples and uncouples the nucleus underlies directed migration of neurons.

Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders

Neurodegenerative Genetics The underlying genetics of neurodegenerative disorders tend not to be well understood. Novarino et al. (p. 506; see the Perspective by Singleton) investigated the underlying genetics of hereditary spastic paraplegia (HSP), a human neurodegenerative disease, by sequencing the exomes of individuals with recessive neurological disorders. Loss-of-function gene mutations in both novel genes and genes previously implicated for this condition were identified, and several were functionally validated. Analysis of hereditary spastic paraplegia genes identifies mutants involved in human neurodegenerative disease. [Also see Perspective by Singleton] Hereditary spastic paraplegias (HSPs) are neurodegenerative motor neuron diseases characterized by progressive age-dependent loss of corticospinal motor tract function. Although the genetic basis is partly understood, only a fraction of cases can receive a genetic diagnosis, and a global view of HSP is lacking. By using whole-exome sequencing in combination with network analysis, we identified 18 previously unknown putative HSP genes and validated nearly all of these genes functionally or genetically. The pathways highlighted by these mutations link HSP to cellular transport, nucleotide metabolism, and synapse and axon development. Network analysis revealed a host of further candidate genes, of which three were mutated in our cohort. Our analysis links HSP to other neurodegenerative disorders and can facilitate gene discovery and mechanistic understanding of disease.

TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum

Ciliary dysfunction leads to a broad range of overlapping phenotypes, collectively termed ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifier alleles to clinically distinct disorders. Here we show that mutations in TTC21B, which encodes the retrograde intraflagellar transport protein IFT139, cause both isolated nephronophthisis and syndromic Jeune asphyxiating thoracic dystrophy. Moreover, although resequencing of TTC21B in a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations showed a significant enrichment of pathogenic alleles in cases (P < 0.003), suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy cases. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies and interact in trans with other disease-causing genes and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of inherited disorders.