LIS1: cellular function of a disease-causing gene

Brain development is severely defective in children with lissencephaly. The highly organized distribution of neurons within the cerebral cortex is disrupted, a condition that might arise from improper migration of neuronal progenitors to their cortical destinations. Type I lissencephaly results from mutations in the LIS1 gene, which has been implicated in the cytoplasmic dynein and platelet-activating factor pathways. Recent studies have identified roles for the product of LIS1 in nuclear migration, mitotic spindle orientation and chromosome alignment, where it appears to act in concert with cytoplasmic dynein. A unifying hypothesis for the subcellular function of LIS1 is presented.     

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.     

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.     

Direct association of LIS1, the lissencephaly gene product, with a mammalian homologue of a fungal nuclear distribution protein, rNUDE

LIS1 is a product of the causative gene for type I lissencephaly characterized by a smooth brain surface due to a defect in neuronal migration during brain development and a regulatory subunit of platelet-activating factor acetylhydrolase (PAF-AH). It is also a mammalian homologue of the fungal nuclear distribution (nud) gene, nudF, which controls the migration of fungal nuclei. Using the two-hybrid system, we identified a novel LIS1-interacting protein, rat NUDE (rNUDE), and found that it is a mammalian homologue of another fungal nud gene product, NUDE, and Xenopus mitotic phosphoprotein 43 which is phosphorylated in a cell cycle-dependent manner. rNUDE and the catalytic subunits of PAF-AH interact with the N- and C-termini of LIS1, respectively. However, these proteins, instead of simultaneously binding to LIS1, appeared to bind to LIS1 in a competitive manner. These results suggest that LIS1 functions in nuclear migration by interacting with multiple intracellular proteins in mammals.     

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.     

Lissencephaly associated mutations suggest a requirement for the PAFAH1B heterotrimeric complex in brain development

Human brain malformations, such as Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) may result from abnormal neuronal migration during brain development. MDS and ILS patients have a hemizygous deletion or mutation in the LIS1 gene (PAFAH1B1), therefore, the LIS1 encoded protein (Lis1) may play a role in neuronal migration. Lis1 is a subunit of a brain platelet-activating factor acetylhydrolase (PAFAH1B) where it forms a heterotrimeric complex with two hydrolase subunits, referred to as 29 kDa (PAFAH1B3) and 30 kDa (PAFAH1B2). In order to determine whether this heterotrimer is required for the developmental functions of PAFAH1B, we examined the binding properties of 29 and 30 kDa subunits to mutant Lis1 proteins. The results defined the critical regions of Lis1 for PAFAH1B complex formation and demonstrated that all human LIS1 mutations examined resulted in abolished or reduced capacity of Lis1 to interact with the 29 and 30 kDa subunits, suggesting that the PAFAH1B complex participates in the process of neuronal migration.     

mNUDC is required for plus‐end‐directed transport of cytoplasmic dynein and dynactins by kinesin‐1

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. The LIS1 (or PAFAH1B1) gene was identified as the gene mutated in lissencephaly patients, and was found to regulate cytoplasmic dynein function and localization. In particular, LIS1 is essential for anterograde transport of cytoplasmic dynein as a part of the cytoplasmic dynein–LIS1–microtubule complex in a kinesin‐1‐dependent manner. However, the underlying mechanism by which a cytoplasmic dynein–LIS1–microtubule complex binds kinesin‐1 is unknown. Here, we report that mNUDC (mammalian NUDC) interacts with kinesin‐1 and is required for the anterograde transport of a cytoplasmic dynein complex by kinesin‐1. mNUDC is also required for anterograde transport of a dynactin‐containing complex. Inhibition of mNUDC severely suppressed anterograde transport of distinct cytoplasmic dynein and dynactin complexes, whereas motility of kinesin‐1 remained intact. Reconstruction experiments clearly demonstrated that mNUDC mediates the interaction of the dynein or dynactin complex with kinesin‐1 and supports their transport by kinesin‐1. Our findings have uncovered an essential role of mNUDC for anterograde transport of dynein and dynactin by kinesin‐1.     

Phosphorylation of doublecortin by protein kinase A orchestrates microtubule and actin dynamics to promote neuronal progenitor cell migration

Doublecortin (DCX) is a microtubule-associated protein that is specifically expressed in neuronal cells. Genetic mutation of DCX causes lissencephaly disease. Although the abnormal cortical lamination in lissencephaly is thought to be attributable to neuronal cell migration defects, the regulatory mechanisms governing interactions between DCX and cytoskeleton in the migration of neuronal progenitor cells remain obscure. In this study we found that the G(s) and protein kinase A (PKA) signal elicited by pituitary adenylate cyclase-activating polypeptide promotes neuronal progenitor cells migration. Stimulation of G(s)-PKA signaling prevented microtubule bundling and induced the dissociation of DCX from microtubules in cells. PKA phosphorylated DCX at Ser-47, and the phospho-mimicking mutant DCX-S47E promoted cell migration. Activation of PKA and DCX-S47E induced lamellipodium formation. Pituitary adenylate cyclase-activating polypeptide and DCX-S47E stimulated the activation of Rac1, and DCX-S47E interacted with Asef2, a guanine nucleotide exchange factor for Rac1. Our data reveal a dual reciprocal role for DCX phosphorylation in the regulation of microtubule and actin dynamics that is indispensable for proper brain lamination.     

A novel strategy for therapeutic intervention for the genetic disease: Preventing proteolytic cleavage using small chemical compound

Haploinsufficiency is a state of genetic disease, which is caused by hemizygous mutations of functional alleles. Lissencephaly is a typical example of haploinsufficiency disorders characterized by a smooth cerebral surface, thick cortex and dilated lateral ventricules associated with mental retardation and seizures due to defective neuronal migration. LIS1 was the first gene cloned in an organism, which was deleted or mutated in patients with lissencephaly in a heterozygous fashion. Series of studies uncovered that LIS1 is an essential regulator of cytoplasmic dynein. In particular, we reported that LIS1 is essential for dynein transport to the plus-end of microtubules by kinesin, which is essential for maintaining proper distribution of cytoplasmic dynein within the cell. Fortuitously, we found that a substantial fraction of LIS1 is degraded by the cystein protease, calpain after reaching the plus-end of microtubules. We further demonstrated that inhibition of calpain-mediated LIS1 degradation increased LIS1 level at the cortex of the cell, resulting in therapeutic benefit using genetic mouse models with reduced levels of LIS1. Our work might provide a potential therapeutic approach for the treatment of a fraction of haploinsufficiency disorders through augmenting reduced proteins by the targeting inhibition of degradation machinery.     

The essential role of LIS1, NDEL1 and Aurora-A in polarity formation and microtubule organization during neurogensis

Lissencephaly is a devastating neurological disorder due to defective neuronal migration. LIS1 (or PAFAH1B1), the gene mutated in lissencephaly patients and its binding protein NDEL1 were found to regulate cytoplasmic dynein function and localization. LIS1 and NDEL1 also play a pivotal role on a microtubule regulation and determination of cell polarity. For example, LIS1 is required for the precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. On the other hand, NDEL1 is essential for mitotic entry as an effector molecule of Aurora-A kinase. In addition, an atypical protein kinase C (aPKC)-Aurora-A-NDEL1 pathway is critical for the regulation of microtubule organization during neurite extension. These findings suggest that physiological functions of LIS1 and NDEL1 in neurons have been ascribed for proteins fundamentally required for cell cycle progression and control. In turn, cell cycle regulators may exert other functions during neurogenesis in a direct or an indirect fashion. Thus far, only a handful of cell cycle regulators have been shown to play physiological cell-cycle-independent roles in neurons. Further identification of such proteins and elucidation of their underlying mechanisms of action will likely reveal novel concepts and/or patterns that provide a clear link between their seemingly distinct cell cycle and neuronal functions.     

Patient mutations in doublecortin define a repeated tubulin-binding domain

Doublecortin (DCX) missense mutations are found in two clusters in patients with defective cortical neuronal migration. Although DCX can function as a microtubule-associated protein (MAP), the potential relationship between its MAP activity and neuronal migration is not understood. Here we show that the two clusters of patient mutations precisely define an internal tandem repeat. Each repeat alone binds tubulin, whereas neither repeat is sufficient for co-assembly with microtubules. The two tandem repeats are sufficient to mediate microtubule polymerization, and representative patient missense mutations lead to impaired polymerization both in vitro and in vivo as well as impaired microtubule stabilization. Furthermore, each repeat is predicted to have the secondary structure of a beta-grasp superfold motif, a motif not found in other MAPs. The patient mutations are predicted to disrupt the structure of the motif, suggesting that the motif may be critical for the DCX-tubulin interaction. These data provide both genetic and biochemical evidence that the interaction of DCX with microtubules is dependent upon this novel repeated tubulin-binding motif.     

Microtubule association of the neuronal p35 activator of Cdk5

Cdk5 and its neuronal activator p35 play an important role in neuronal migration and proper development of the brain cortex. We show that p35 binds directly to alpha/beta-tubulin and microtubules. Microtubule polymers but not the alpha/beta-tubulin heterodimer block p35 interaction with Cdk5 and therefore inhibit Cdk5-p35 activity. p25, a neurotoxin-induced and truncated form of p35, does not have tubulin and microtubule binding activities, and Cdk5-p25 is inert to the inhibitory effect of microtubules. p35 displays strong activity in promoting microtubule assembly and inducing formation of microtubule bundles. Furthermore, microtubules stabilized by p35 are resistant to cold-induced disassembly. In cultured cortical neurons, a significant proportion of p35 localizes to microtubules. When microtubules were isolated from rat brain extracts, p35 co-assembled with microtubules, including cold-stable microtubules. Together, these findings suggest that p35 is a microtubule-associated protein that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation.     

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.     

Smooth,rough and upside-down neocortical development

Lissencephaly, which means 'smooth cortex', is caused by defective neuronal migration during development of the cerebral cortex and has devastating clinical consequences. 'Classical' lissencephaly seems to reflect mutations in regulators of the microtubule cytoskeleton, whereas 'cobblestone' lissencephaly is caused by mutations in genes needed for the integrity of the basal lamina of the central nervous system. Reelin, which is mutated in a third type of lissencephaly, may represent a unifying link because it encodes an extracellular protein that regulates neuronal migration and may also regulate the microtubule cytoskeleton.     

The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast

During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother-bud neck via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway. Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Third, Pac1 localized to the plus ends (distal tips) of cytoplasmic microtubules in the bud. This localization did not depend on the dynein heavy chain Dyn1. Moreover, the Pac1 fluorescence intensity at the microtubule end was enhanced in cells lacking dynactin or the cortical attachment molecule Num1. Fourth, dynein heavy chain Dyn1 also localized to the tips of cytoplasmic microtubules in wild-type cells. Dynein localization required Pac1 and, like Pac1, was enhanced in cells lacking the dynactin component Arp1 or the cortical attachment molecule Num1. Our results suggest that Pac1 targets dynein to microtubule tips, which is necessary for sliding of microtubules along the bud cortex. Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.     

Coupling PAF signaling to dynein regulation: structure of LIS1 in complex with PAF-acetylhydrolase

Mutations in the LIS1 gene cause lissencephaly, a human neuronal migration disorder. LIS1 binds dynein and the dynein-associated proteins Nde1 (formerly known as NudE), Ndel1 (formerly known as NUDEL), and CLIP-170, as well as the catalytic alpha dimers of brain cytosolic platelet activating factor acetylhydrolase (PAF-AH). The mechanism coupling the two diverse regulatory pathways remains unknown. We report the structure of LIS1 in complex with the alpha2/alpha2 PAF-AH homodimer. One LIS1 homodimer binds symmetrically to one alpha2/alpha2 homodimer via the highly conserved top faces of the LIS1 beta propellers. The same surface of LIS1 contains sites of mutations causing lissencephaly and overlaps with a putative dynein binding surface. Ndel1 competes with the alpha2/alpha2 homodimer for LIS1, but the interaction is complex and requires both the N- and C-terminal domains of LIS1. Our data suggest that the LIS1 molecule undergoes major conformational rearrangement when switching from a complex with the acetylhydrolase to the one with Ndel1.     

Dynein binds and stimulates axonal motility of the endosome adaptor and NEEP21 family member,calcyon

The neuron-enriched, endosomal protein Calcyon (Caly) regulates endocytosis and vesicle sorting, and is important for synaptic plasticity and brain development. In the current investigation of Caly interacting proteins in brain, the microtubule retrograde motor subunit, cytoplasmic dynein 1 heavy chain (DYNC1H), and microtubule structural proteins, α and β tubulin, were identified as Caly associated proteins by MALDI-ToF/ToF. Direct interaction of the Caly-C terminus with dynein and tubulin was further confirmed in in vitro studies. In Cos-7 cells, mCherry-Caly moved along the microtubule network in organelles largely labeled by the late endosome marker Rab7. Expression of the dynein inhibitor CC1, produced striking alterations in Caly distribution, consistent with retrograde motors playing a prominent role in Caly localization and movement. In axons of cultured adult rat sensory neurons, Caly-positive organelles co-localized with dynein intermediate chain (DYNC1I1-isoform IC-1B) and the dynein regulator, lissencephaly 1 (LIS1), both of which co-precipitated from brain with the Caly C-terminus. Manipulation of dynein function in axons altered the motile properties of Caly indicating that Caly vesicles utilize the retrograde motor. Altogether, the current evidence for association with dynein motors raises the possibility that the endocytic and cargo sorting functions of Caly in neurons could be regulated by interaction with the microtubule transport system.     

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.     

Lissencephaly: Mechanistic insights from animal models and potential therapeutic strategies

Lissencephaly is a severe human neuronal migration defect characterized by a smooth cerebral surface, mental retardation and seizures. The two most common genes mutated in patients with lissencephaly are LIS1 and DCX. LIS1 was the first gene cloned that was important for neuronal migration in any organism, and heterozygous mutations or deletions of LIS1 are found in the majority of patients with lissencephaly, while DCX mutations were found in males with X-linked lissencephaly. In this review, we will discuss how an understanding of the molecular and cellular pathways disrupted in model organisms with Lis1 and Dcx mutations or knock-down not only provide insights into the normal processes of neuronal migration, including neurogenesis, but they also may lead to potential novel therapeutic strategies for these severe cortical malformations.