Publications

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) is a nuclear protein that plays a crucial role in various biological functions, such as RNA splicing and chromatin organization. HNRNPU/scaffold attachment factor A (SAF-A) activities are essential for regulating gene expression, DNA replication, genome integrity, and mitotic fidelity. These functions are critical to ensure the robustness of developmental processes, particularly those involved in shaping the human brain. As a result, HNRNPU is associated with various neurodevelopmental disorders (HNRNPU-related neurodevelopmental disorder, HNRNPU-NDD) characterized by developmental delay and intellectual disability. Our research demonstrates that the loss of HNRNPU function results in the death of both neural progenitor cells and post-mitotic neurons, with a higher sensitivity observed in the former. We reported that HNRNPU truncation leads to the dysregulation of gene expression and alternative splicing of genes that converge on several signaling pathways, some of which are likely to be involved in the pathology of HNRNPU-related NDD.

HNRNPU encodes the heterogeneous nuclear ribonucleoprotein U, which participates in RNA splicing and chromatin organization. Microdeletions in the 1q44 locus encompassing HNRNPU and other genes and point mutations in HNRNPU cause brain disorders, including early-onset seizures and severe intellectual disability. We aimed to understand HNRNPUs roles in the developing brain. Our work revealed that HNRNPU loss of function leads to rapid cell death of both postmitotic neurons and neural progenitors, with an apparent higher sensitivity of the latter. Further, expression and alternative splicing of multiple genes involved in cell survival, cell motility, and synapse formation are affected following Hnrnpus conditional truncation. Finally, we identified pharmaceutical and genetic agents that can partially reverse the loss of cortical structures in Hnrnpu mutated embryonic brains, ameliorate radial neuronal migration defects and rescue cultured neural progenitors cell death.

The cortex is a highly organized structure that develops from the caudal regions of the segmented neural tube. Its spatial organization sets the stage for future functional arealization. Here, we suggest using a developmental perspective to describe and understand the etiology of common cortical malformations and their manifestation in the human brain.

During the last few decades we have witnessed an impressive gain in the knowledge regarding the basic mechanisms underlying human neuronal migration disorders by the usage of mouse models. Nevertheless, despite the remarkable conservation both in the genetic encoded information and the developmental processes, there are still numerous important differences between human and mouse. This may explain the vast excitement following the realization that technological breakthroughs enabled generating tissue-like human-based organoids for modeling human neuronal migration diseases. This review will provide a short introduction on human and mouse neuronal migration processes, and highlight human brain organoid models of neuronal migration diseases.

In recent years, it has been revealed that Parkinsons disease pathology may begin to manifest in the gastrointestinal track at a much earlier time point than in the brain. This paradigm shift has been suggested following evidence in humans that has been reproduced in animal models. Since rodent models cannot recapitulate many of the human disease features, human induced pluripotent stem cells derived from Parkinsons patients have been used to generate brain organoids, greatly contributing to our understanding of the disease pathophysiology. To understand the multifaced aspects of Parkinsons disease, it may be desirable to expand the complexity of these models, to include different brain regions, vasculature, immune cells as well as additional diverse organ-specific organoids such as gut and intestine. Furthermore, the contribution of gut microbiota to disease progression cannot be underestimated. Recent biotechnological advances propose that such combinations may be feasible. Here we discuss how this need can be met and propose that additional brain diseases can benefit from this approach.

LifeTime aims to track, understand and target human cells during the onset and progression of complex diseases and their response to therapy at single-cell resolution. This mission will be implemented through the development and integration of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during progression from health to disease. Analysis of such large molecular and clinical datasets will discover molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. Timely detection and interception of disease embedded in an ethical and patient-centered vision will be achieved through interactions across academia, hospitals, patient-associations, health data management systems and industry. Applying this strategy to key medical challenges in cancer, neurological, infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

Nucleokinesis, literally the movement of the nucleus, may or may not be coupled with cellular motility. In this chapter, we present our point of view on the moving nucleus. We depict the structure of the nucleus, discuss how the chromatin is organized within the nucleus, highlight nuclear substructure, and describe the molecules that anchor it to the cytoskeleton. We further discuss diseases that arise when these molecules malfunction. How the nucleus connects with the cytoskeleton is key in understanding the relationships between these two different domains. The structure and the role of the bridging complex, the LINC complex, are presented as well. As an example for nuclear motility, we focus on dynamic nucleokinesis during interkinetic nuclear movement. Collectively, we aim to introduce the concept that the migrating nucleus is more than just a passive cargo.

In the middle of March 2019, a group of scientists and clinicians (as well as those who wear both hats) gathered in the green campus of the Weizmann Institute of Science to share recent scientific findings, to establish collaborations, and to discuss future directions for better diagnosis, etiology modeling and treatment of brain malformations. One hundred fifty scientists from twenty-two countries took part in this meeting. Thirty-eight talks were presented and as many as twenty-five posters were displayed. This review is aimed at presenting some of the highlights that the audience was exposed to during the three-day meeting.

Palmitoyl-protein thioesterase 1 (PPT1) is a depalmitoylation enzyme that is mutated in cases of neuronal ceroid lipofuscinosis (NCL). The hallmarks of the disease include progressive neurodegeneration and blindness, as well as seizures. In the current study, we identified 62 high-confident PPT1-binding proteins. These proteins included a self-interaction of PPT1, two V-type ATPases, calcium voltage-gated channels, cytoskeletal proteins and others. Pathway analysis suggested their involvement in seizures and neuronal morphology. We then proceeded to demonstrate that hippocampal neurons from Ppt1-/- mice exhibit structural deficits, and further investigated electrophysiology parameters in the hippocampi of mutant mice, both in brain slices and dissociated postnatal primary cultures. Our studies reveal new mechanistic features involved in the pathophysiology of this devastating neurodegenerative disease.

The convoluted human cerebral cortex is one of the key features that allows for an increased neuronal density packing essential for the complex cognitive and socioemotional behaviours man possesses. Nevertheless, the underlying mechanisms involved in cortical folding remained a both intriguing and functionally important enigma. A crucial component known to be involved in the formation and maintenance of all tissues is the extracellular matrix (ECM), providing scaffolds which tie tissues and organs in place. The composition of the ECM in both developing and mature structures is constantly remodelled, degraded and secreted by numerous types of cells, and its role as a source of growth factors and signalling in morphogenesis, migration, and proliferation is increasingly appreciated. Evidence for the differential expression of ECM during gyrification pinpoints its potentially fundamental role in shaping the folds of the cerebral cortex through both mechanical and molecular configurations. This review aims at addressing key ideas, potential directions and discoveries that highlight biomechanics of the ECM during the construction of the cortex cerebral gyrification.

The cerebral cortex is a highly organized structure whose development depends on diverse progenitor cell types, namely apical radial glia, intermediate progenitors, and basal radial glia cells, which are responsible for the production of the correct neuronal output. In recent years, these progenitor cell types have been deeply studied, particularly basal radial glia and their role in cortical expansion and gyrification. We review here a broad series of factors that regulate progenitor behavior and daughter cell fate. We first describe the different neuronal progenitor types, emphasizing the differences between lissencephalic and gyrencephalic species. We then review key factors shown to influence progenitor proliferation versus differentiation, discussing their roles in progenitor dynamics, neuronal production, and potentially brain size and complexity. Although spindle orientation has been considered a critical factor for mode of division and daughter cell output, we discuss other features that are emerging as crucial for these processes such as organelle and cell cycle dynamics. Additionally, we highlight the importance of adhesion molecules and the polarity complex for correct cortical development. Finally, we briefly discuss studies assessing progenitor multipotency and its possible contribution to the production of specific neuronal populations. This review hence summarizes recent aspects of cortical progenitor cell biology, and pinpoints emerging features critical for their behavior.

The complement system, which is part of the innate immune response system, has been recently shown to participate in multiple key processes in the developing brain. Here we aimed to elucidate downstream signaling responses linking complement C3, a key molecule of the pathway, to small GTPases, known to affect the cytoskeleton. The expression pattern of the activated small GTPase Rac1 resembled that of complement C3. C3-deficient mice exhibited reduced Rac1 and elevated RhoA activity in comparison with control mice. The most pronounced reduction of Rac1 activity occurred at embryonic day 14. Rac1 has been implicated in neuronal migration as well as neuronal stem cell proliferation and differentiation. Consistent with the reduction in Rac1 activity, the expression of phospho-cofilin, decreased in migrating neurons. Reduced Rac1-GTP was also correlated with a decrease in the expression of progenitor markers ( Nestin, Pax6 and Tbr2) and conversely the expression of neuronal markers (Dcx and NeuN) increased in C3 knockout (KO) cortices in comparison with wild-type (WT) cortices. More specifically, C3 deficiency resulted in a reduction in the number of the cells in S-phase and an elevation in the number of cells that precociously exited the cell cycle. Collectively, our findings suggest that C3 impacts the activity of small GTPases resulting in cell cycle defects and premature neuronal differentiation.

Autism spectrum disorders (ASD) encompass a group of neurodevelopmental diseases that demonstrate strong heritability, however, the inheritance is not simple and many genes have been associated with these disorders. ASD is regarded as a neurodevelopmental disorder, and abnormalities at different developmental stages are part of the disease etiology. This review provides a general background on neuronal migration during brain development and discusses recent advancements in the field connecting ASD and aberrant neuronal migration. We propose that neuronal migration impairment may be an important common pathophysiology in autism spectrum disorders (ASD). This review provides a general background on neuronal migration during brain development and discusses recent advancements in the field connecting ASD and aberrant neuronal migration. We propose that neuronal migration impairment may be an important common pathophysiology in autism spectrum disorders (ASD). This review provides a general background on neuronal migration during brain development and discusses recent advancements in the field connecting ASD and aberrant neuronal migration.

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at ≥6 months after birth. We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Keywords: Immunology

The nuclei of neuroepithelial cells move along the apicobasal axis in synchronization with their cell cycle status. This motility is known as interkinetic nuclear movement. We discuss here the importance of cytoskeleton organization, the centrosome, molecular motors, cell polarity proteins, and their regulators in controlling and maintaining this typical behavior. Furthermore, due to the tight linkage between cell proliferation, cell cycle, and nuclear motility, we speculate that interkinetic nuclear movement is likely to be affected in the pathophysiology of microcephaly, where the brain size is markedly reduced.

Platelet-activating factor acetylhydrolase 1B (PAF-AH) inactivates the potent phospholipid platelet-activating factor (PAF) and is composed of two catalytic subunits (α1 and α2) and a dimeric regulatory subunit, LIS1. The function of the catalytic subunits in brain development remains unknown. Here we examined their effects on proliferation in the ganglionic eminences and tangential migration. In α1 and α2 catalytic subunits knockout mice we noticed an increase in the size of the ganglionic eminences resulting from increased proliferation of GABAergic neurons. Our results indicate that the catalytic subunits act as negative regulators of the Wnt signaling pathway. Overexpression of each of the PAF-AH catalytic subunits reduced the amount of nuclear beta-catenin and provoked a shift of this protein from the nucleus to the cytoplasm. In the double mutant mice, Wnt signaling increased in the ganglionic eminences and in the dorsal part of the cerebral cortex. In situ hybridization revealed increased and expanded expression of a downstream target of the Wnt pathway (Cyclin D1), and of upstream Wnt components (Tcf4, Tcf3 and Wnt7B). Furthermore, the interneurons in the cerebral cortex were more numerous and in a more advanced position. Transplantation assays revealed a non-cell autonomous component to this phenotype, which may be explained in part by increased and expanded expression of Sdf1 and Netrin-1. Our findings strongly suggest that PAF-AH catalytic subunits modulate the Wnt pathway in restricted areas of the developing cerebral cortex. We hypothesize that modulation of the Wnt pathway is the evolutionary conserved activity of the PAF-AH catalytic subunits.

Abnormal neuronal migration is manifested in brain malformations such as lissencephaly. The impairment in coordinated cell motility likely reflects a faulty mechanism of cell polarization or coupling between polarization and movement. Here we report on the relationship between the polarity kinase MARK2/Par-1 and its substrate, the well-known lissencephaly-associated gene doublecortin (DCX), during cortical radial migration. We have previously shown using in utero electroporation that reduced MARK2 levels resulted in multipolar neurons stalled at the intermediate zone border, similar to the phenotype observed in the case of DCX silencing. However, whereas reduced MARK2 stabilized microtubules, we show here that knock-down of DCX increased microtubule dynamics. This led to the hypothesis that simultaneous reduction may alleviate the phenotype. Coreduction ofMARK2and DCX resulted in a partial restoration of the normal neuronal migration phenotype in vivo. The kinetic behavior of the centrosomes reflected the different molecular mechanisms activated when either protein was reduced. In the case of reducing MARK2 processive motility of the centrosome was hindered, whereas when DCX was reduced, centrosomes moved quickly but bidirectionally. Our results stress the necessity for successful coupling between the polarity pathway and cytoplasmic dynein-dependent activities for proper neuronal migration.

Directed cell migration is a property central to multiple basic biological processes. Here, we show that directed cell migration is associated with global changes in the chromatin fiber. Polarized posttranslational changes in histone H1 along with a transient decrease in H1 mobility were detected in cells facing the scratch in a wound healing assay. In parallel to the changes in H1, the levels of the heterochromatin marker histone H3 lysine 9 tri-methylation were elevated. Interestingly, reduction of the chromatin-binding affinity of H1 altered the cell migration rates. Moreover, migration-associated changes in histone H1 were observed during nuclear motility in the simple multicellular organism Neurospora crassa . Our studies suggest that dynamic reorganization of the chromatin fiber is an early event in the cellular response to migration cues.

The doublecortin-like (DCX) domains serve as protein-interaction platforms. DCX tandem domains appear in the product of the X-linked doublecortin (DCX) gene, in retinitis pigmentosa-1 (RP1), as well as in other gene products. Mutations in the human DCX gene are associated with abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, while DCDC2 has been associated with dyslectic reading disabilities. Motivated by the possible importance of this gene family, a thorough analysis to detect all family members in the mouse was conducted. The DCX-repeat gene superfamily is composed of eleven paralogs, and we cloned the DCX domains from nine different genes. Our study questioned which functions attributed to the DCX domain, are conserved among the different members. Our results suggest that the proteins with the DCX-domain have conserved and unique roles in microtubule regulation and signal transduction. All the tested proteins stimulated microtubule assembly in vitro. Proteins with tandem repeats stabilized the microtubule cytoskeleton in transfected cells, while those with single repeats localized to actin-rich subcellular structures, or the nucleus. All tested proteins interacted with components of the JNK/MAP-kinase pathway, while only a subset interacted with Neurabin 2, and a nonoverlapping group demonstrated actin association. The sub-specialization of some members due to confined intracellular localization, and protein interactions may explain the success of this superfamily.

Lissencephaly (LIS1) was the first gene implicated in the pathogenesis of type-1 lissencephaly. More than a decade of research by multiple laboratories has revealed that LIS1 is a key node protein, which participates in several pathways, including association with the molecular motor cytoplasmic dynein, the reelin signaling pathway, and the platelet-activating factor pathway. Mutations in LIS1-interacting proteins, either in human, or in mouse models has suggested that LIS1 might play a role in the pathogenesis of numerous diseases such as male sterility, schizophrenia, neuronal degeneration, and viral infections.

For neuronal migration to occur, the cell must undergo morphological changes that require modifications of the cytoskeleton. Several different MAPs (microtubule-associated proteins) or actin-binding proteins are proposed to be involved in the migration of neurons. Therefore we have specifically analysed how two members of the MAP family, MAP1B and LIS1 (lissencephaly-related protein 1), interact with one another and participate in neuronal migration. Our results indicate that, in hippocampal neurons, MAP1B and LIS1 co-localize, associate and interact with each another. The interaction between these two MAPs is regulated by the phosphorylation of MAP1B. Furthermore, this interaction interferes with the association between LIS1 and the microtubule-dependent molecular motor, dynein. Clearly, the differential binding of these cytoskeletal proteins could regulate the functions attributed to the LIS1-dynein complex, including those related to extension of the neural processes necessary for neuronal migration.

Proper human brain formation is dependent upon the integrated activity of multiple genes. Malfunctioning of key proteins results in brain developmental abnormalities. Mutation(s) in the LIS1 gene or the X-linked gene doublecortin (DCX) results in a spectrum of disorders including lissencephaly, or 'smooth brain', and subcortical band heterotopia, or 'doublecortex'. Here, we will focus on a particular subset of missense mutations in these two genes and their effect on protein structure and function.

Mutations in the Lis1 gene result in lissencephaly (smooth brain), a debilitating developmental syndrome caused by the impaired ability of postmitotic neurons to migrate to their correct destination in the cerebral cortex. Sequence similarities suggest that the LIS1 protein contains a C-terminal seven-blade β-propeller domain, while the structure of the N-terminal fragment includes the LisH (Lis-homology) motif, a pattern found in over 100 eukaryotic proteins with a hitherto unknown function. We present the 1.75 Å resolution crystal structure of the N-terminal domain of mouse LIS1, and we show that the LisH motif is a novel, thermodynamically very stable dimerization domain. The structure explains the molecular basis of a low severity form of lissencephaly.

Alternative splicing of mRNA transcripts expands the range of protein products from a single gene locus. Several splice variants of DCLK (doublecortin-like kinase) have previously been reported. Here, we report the genomic organization underlying the splice variants of DCLK and examine the expression profile of two splice variants affecting the kinase domain of DCLK and CPG16 (candidate plasticity gene 16), one containing an Arg-rich domain and the other affecting the C terminus of the protein. These splice alternatives were differentially expressed in embryonic and adult brain. Both splice variants disrupted DCLK PEST domains; however, all splice variants remained sensitive to proteolysis by calpain. The adult-specific C-terminal splice variant of DCLK had reduced autophosphorylation activity, but similar kinase activity for myelin basic protein relative to the embryonic splice variant. The splice variant adding an Arg-rich domain gained an autophosphorylation site at Ser-382. Although this protein isoform was expressed mainly in the adult brain, the phosphorylated form was strongly enriched in embryonic brain and adult olfactory bulb, suggesting a possible role in migrating neurons.

LIS1 is one of the genes that has a principle role in brain development since hemizygote mutations in LIS1 result in a severe brain malformation known as lissencephaly ('smooth brain'). LIS1 is a WD repeat protein and is known to be involved in several protein complexes that are likely to play a functional role in brain development. We discuss here the brain developmental phenotype observed in mice heterozygote for an N-terminal truncated LIS1 protein in view of known LIS1 protein interactions.

Lissencephaly is a severe brain malformation in humans. To study the function of the gene mutated in lissencephaly (LIS1), we deleted the first coding exon from the mouse Lis1 gene. The deletion resulted in a shorter protein (sLIS1) that initiates from the second methionine, a unique situation because most LIS1 mutations result in a null allele. This mutation mimics a mutation described in one lissencephaly patient with a milder phenotype. Homozygotes are early lethal, although heterozygotes are viable and fertile. Most strikingly, the morphology of cortical neurons and radial glia is aberrant in the developing cortex, and the neurons migrate more slowly. This is the first demonstration, to our knowledge, of a cellular abnormality in the migrating neurons after Lis1 mutation. Moreover, cortical plate splitting and thalamocortical innervation are also abnormal. Biochemically, the mutant protein is not capable of dimerization, and enzymatic activity is elevated in the embryos, thus a demonstration of the in vivo role of LIS1 as a subunit of PAF-AH. This mutation allows us to determine a hierarchy of functions that are sensitive to LIS1 dosage, thus promoting our understanding of the role of LIS1 in the developing cortex.

Mutations in either LIS1 or DCX are the most common cause for type I lissencephaly. Here we report that LIS1 and DCX interact physically both in vitro and in vivo. Epitope-tagged DCX transiently expressed in COS cells can be co-immunoprecipitated with endogenous LIS1. Furthermore, endogenous DCX could be co-immunoprecipitated with endogenous LIS1 in embryonic brain extracts, demonstrating an in vivo association. The two protein products also co-localize in transfected cells and in primary neuronal cells. In addition, we demonstrate homodimerization of DCX in vitro. Using fragments of both LIS1 and DCX, the domains of interaction were mapped. LIS1 and DCX interact with tubulin and microtubules. Our results suggest that addition of DCX and LIS1 to tubulin enhances polymerization in an additive fashion. In in vitro competition assays, when LIS1 is added first, DCX competes with LIS1 in its binding to microtubules, but when DCX is added prior to the addition of LIS1 it enhances the binding of LIS1 to microtubules. We conclude that LIS1 and DCX cross-talk is important to microtubule function in the developing cerebral cortex.

Mutations in the X-linked gene doublecortin (DCX) result in lissencephaly in males or subcortical laminar heterotopia ('double cortex') in females. Various types of mutation were identified and the sequence differences included nonsense, splice site and missense mutations throughout the gene. Recently, we and others have demonstrated that DCX interacts and stabilizes microtubules. Here, we performed a detailed sequence analysis of DCX and DCX-like proteins from various organisms and defined an evolutionarily conserved Doublecortin (DC) domain. The domain typically appears in the N-terminus of proteins and consists of two tandemly repeated 80 amino acid regions. In the large majority of patients, missense mutations in DCX fall within the conserved regions. We hypothesized that these repeats may be important for microtubule binding. We expressed DCX or DCLK (KIAA0369) repeats in vitro and in vivo. Our results suggest that the first repeat binds tubulin but not microtubules and enhances microtubule polymerization. To study the functional consequences of DCX mutations, we overexpressed seven of the reported mutations in COS7 cells and examined their effect on the microtubule cytoskeleton. The results demonstrate that some of the mutations disrupt microtubules. The most severe effect was observed with a tyrosine to histidine mutation at amino acid 125 (Y125H). Produced as a recombinant protein, this mutation disrupts microtubules in vitro at high molar concentration. The positions of the different mutations are discussed according to the evolutionarily defined DC-repeat motif. The results from this study emphasize the importance of DCX-microtubule interaction during normal and abnormal brain development.

Doublecortin-like kinase (DCLK) shares sequence similarity to Doublecortin (DCX) in its N-terminal region. It contains the evolutionary conserved DC repeat motif as well a C-terminal kinase domain. Ectopic expression of DCLK in COS cells results in colocalization with microtubules, and phosphorylated DCLK copurifies with microtubules during assembly from embryonic brain extract. During brain development DCLK is expressed mainly in postmigratory neurons in a similar pattern to DCX. We demonstrate that DCLK is a microtubule-associated active protein kinase expressed in growth cones of postmitotic neurons.

During embryonic development, the cerebral cortex attains its characteristic adult laminated structure. The finding that X-linked lissencephaly patients harbor mutations in the doublecortin gene implicated this gene product in the process of corticogenesis. An autosomal human gene, KIAA0369, with a high level of similarity to doublecortin, has been cloned from human adult brain. This gene product contains a kinase domain in addition to a doublecortin-like domain. In order to evaluate whether this doublecortin-like kinase also plays a role during brain development, we cloned and studied the expression pattern of the mouse homolog. Three cDNA products of this gene were cloned: one, doublecortin-like kinase, the second containing only the doublecortin-like region, and the third containing only the kinase domain, a homolog of the previously cloned rat CPG16 gene. We studied doublecortin-like kinase expression in mouse using Northern blot analysis, in situ hybridization, and Western blot analysis, and conclude that doublecortin-like kinase is expressed in multiple regions of embryonic brain including the developing cerebral cortex.

Lissencephaly patients are born with severe brain malformations and suffer from recurrent seizures. LIS1, the gene mutated in isolated lissencephaly patients, is a subunit of the heterotrimeric cytosolic enzyme platelet-activating factor acetylhydrolase (PAF-AH), interacts with tubulin, and affects microtubule dynamics. In order to gain molecular insights into the possible involvement of LIS1 in seizures in lissencephaly patients, we induced seizures in rats by injection of kainate. PAF-AH activity was markedly reduced as early as 30 min following initiation of seizures, making this parameter a sensitive indicator of seizure events. PAF-AH activity returned to and surpassed control values I week following initiation of seizures. Expression of LIS1 in the dentate gyrus changed significantly in a manner similar to that of PAF-AH enzymatic activity. This is the first correlation found between LIS1 expression and PAF-AH activity. Furthermore, the expression of the α2 catalytic subunit, which is the major PAF-AH catalytic subunit in rat adult brain, changed in a dramatic fashion. An additional higher-mobility LIS1 crossreactive band was detected in samples isolated a week following seizure occurrence. This LIS1 isoform was enriched in the microtubule-associated fraction. We propose that LIS1 expression is an important factor in regulation of PAF-AH activity. We postulate that reductions in LIS1 protein levels found in lissencephaly patients may render them more susceptible to seizures.

Platelet-activating factor is a phospholipid with several documented roles in the pre-implantation embryo. Enzymes that belong to the platelet-activating factor acetylhydrolases family inactivate platelet-activating factor. Cytosolic platelet-activating factor acetylhydrolase (Ib) is a heterotetramer composed of two catalytic subunits (α1/α2) and two regulatory LIS1 subunits. The expression of these components was monitored in the mouse oocytes and zygotes using reverse-transcribed PCR and Western blot analysis. Interestingly, these proteins are expressed in the oocyte and zygote and their expression increases after fertilization, probably due to stabilization of maternal RNA. Lis1 mRNA transcription also increases after fertilization. However, assaying for expression of a specific paternal LIS1 isoform detected no zygotic translation in the one cell stage. These findings suggest a potential role for platelet-activating factor acetylhydrolase (Ib) components in the early mouse embryo. Copyright (C) 1999 Federation of European Biochemical Societies.

Lissencephaly is a relatively common brain malformation. Lissencephaly type 1 is characterized by the smooth appearance of the cortex and the presence of four abnormally positioned layers instead of the normal six. Lissencephaly is considered to be an abnormality in neuronal migration. The gene mutated in type 1 lissencephaly was cloned by us and designated LIS1. Recently, several genes involved in cortical development have been cloned in the mouse. In human an additional X-linked lissencephaly gene has been identified. We summarize here our current knowledge on the LIS1 gene and its function. It has been identified as a non-catalytic subunit of PAF-acetylhydrolase, a heterotrimeric enzyme which inactivates the platelet-activating factor (PAF). In addition, we have demonstrated that LIS1 interacts with tubulin, and affects the dynamics properties of microtubules. LIS1 contains seven WD repeats and may structurally resemble the beta-subunit of heterotrimeric G proteins. Interestingly, the catalytic subunit of PAF-acetylhydrolase was found to resemble the alpha-subunit of heterotrimeric G proteins. We raise the possibility that LIS1 is part of an intracellular signaling pathway involved in neuronal migration.

Familial juvenile nephronophthisis (NPH) is an autosomal recessive cystic disease of the kidney that leads to end-stage renal failure in adolescence. NPH is the most common genetic cause of end-stage renal disease in children. A gene locus for nephronophthisis (NPH1) has been mapped by linkage analysis to chromosome 2q13. We report here the construction of a complete YAC contig in the minimum genetic region for NPH1 by STS content mapping using clones of the CEPH YAC libraries. A physical map of maximum distances between 32 STS markers was constructed, thereby defining the order of a total of 27 STS markers. Since D2S340 and D2S121 have previously been identified as flanking markers to the NPH1 gene, the new contig defines on a physical map the NPH1 minimum genetic region to a 6.4-Mb interval. As a novel assignment, expressed genes, some of which may be candidates for the disease, were localized to the NPH1 region. In addition, the known interstitial telomeric repeat on chromosome 2 was physically mapped to this region. This contig assembly provides the basis for closer definition of the NPH1 critical region through identification of more narrow flanking markers and for the construction of a transcriptional map of the region towards isolation of the NPH1 gene.

Mutated cDNA clones containing the entire coding sequence of human glucocerebrosidase were isolated from libraries originated from Gaucher patients. Sequence analysis of a mutated cDNA derived from a type II Gaucher patient revealed a C-to-G transversion causing a substitution of an arginine for a proline at residue 415. This change creates a new cleavage site for the enzyme HhaI in the mutated cDNA. Allele-specific oligonucleotide hybridization made it possible to show that this mutation exists in the genomic DNA of the patient. From a cDNA library originated from a type I Gaucher patient, a mutated allele was cloned that contains a T-to-C transition causing a substitution of proline for leucine at residue 444 and creating a new NciI site. This mutation is identical to that described by S. Tsuji and colleagues in genomic DNA from type I, type II, and type III patients. Since the new NciI site generates RFLP, it was used to test the existence of this mutated allele in several Gaucher patients by Southern blot analysis. This allele was found in type I (Jewish and non-Jewish), type II, and type III Gaucher patients. These findings led us to conclude that the patient suffering from type II disease (denoted GM1260) carried both mutations described above. Any one of the amino acid changes described reduces the glucocerebrosidase activity as tested by transfection of COS cells with expression vectors harboring the mutated cDNAs. The base changes in the two mutated cDNAs do not affect the electrophoretic mobility of the corresponding polypeptides on an SDS polyacrylamide gel.