Generation of an induced pluripotent stem cell line (SDQLCHi067-A) from a patient with subcortical band heterotopia harboring a heterozygous mutation in DCX gene.

Subcortical band heterotopia (SHB) is a rare severe brain developmental malformation caused by deficient neuronal migration during the development of cerebral cortex. Here, a human induced pluripotent stem cell (iPSCs) line was established from a 4-year-1-month-old girl with SHB carrying a heterozygous mutation (c.568A > G, p.K190E) in DCX. The generated iPSC line showed the ability to differentiate into three lineages in vitro and was confirmed by pluripotency markers and the original gene mutation.

Novel role of the synaptic scaffold protein Dlgap4 in ventricular surface integrity and neuronal migration during cortical development.

Subcortical heterotopias are malformations associated with epilepsy and intellectual disability, characterized by the presence of ectopic neurons in the white matter. Mouse and human heterotopia mutations were identified in the microtubule-binding protein Echinoderm microtubule-associated protein-like 1, EML1. Further exploring pathological mechanisms, we identified a patient with an EML1-like phenotype and a novel genetic variation in DLGAP4. The protein belongs to a membrane-associated guanylate kinase family known to function in glutamate synapses. We showed that DLGAP4 is strongly expressed in the mouse ventricular zone (VZ) from early corticogenesis, and interacts with key VZ proteins including EML1. In utero electroporation of Dlgap4 knockdown (KD) and overexpression constructs revealed a ventricular surface phenotype including changes in progenitor cell dynamics, morphology, proliferation and neuronal migration defects. The Dlgap4 KD phenotype was rescued by wild-type but not mutant DLGAP4. Dlgap4 is required for the organization of radial glial cell adherens junction components and actin cytoskeleton dynamics at the apical domain, as well as during neuronal migration. Finally, Dlgap4 heterozygous knockout (KO) mice also show developmental defects in the dorsal telencephalon. We hence identify a synapse-related scaffold protein with pleiotropic functions, influencing the integrity of the developing cerebral cortex.

Hepatic loss of Lissencephaly 1 (Lis1) induces fatty liver and accelerates liver tumorigenesis in mice.

The liver is a major organ in lipid metabolism, and its malfunction leads to various diseases. Nonalcoholic fatty liver disease, the most common chronic liver disorder in developed countries, is characterized by the abnormal retention of excess lipid within hepatocytes and predisposes individuals to liver cancer. We previously reported that the levels of Lissencephaly 1 (LIS1, also known as PAFAH1B1) are down-regulated in human hepatocellular carcinoma. Following up on this observation, we found that genetic deletion of Lis1 in the mouse liver increases lipid accumulation and inflammation in this organ. Further analysis revealed that loss of Lis1 triggers endoplasmic reticulum (ER) stress and reduces triglyceride secretion. Attenuation of ER stress by addition of tauroursodeoxycholic acid (TUDCA) diminished lipid accumulation in the Lis1-deficient hepatocytes. Moreover, the Golgi stacks were disorganized in Lis1-deficient liver cells. Of note, the Lis1 liver-knockout mice exhibited increased hepatocyte ploidy and accelerated development of liver cancer after exposure to the liver carcinogen diethylnitrosamine (DEN). Taken together, these findings suggest that reduced Lis1 levels can spur the development of liver diseases from steatosis to liver cancer and provide a useful model for delineating the molecular pathways that lead to these diseases.

Eml1 loss impairs apical progenitor spindle length and soma shape in the developing cerebral cortex.

The ventricular zone (VZ) of the developing cerebral cortex is a pseudostratified epithelium that contains progenitors undergoing precisely regulated divisions at its most apical side, the ventricular lining (VL). Mitotic perturbations can contribute to pathological mechanisms leading to cortical malformations. The HeCo mutant mouse exhibits subcortical band heterotopia (SBH), likely to be initiated by progenitor delamination from the VZ early during corticogenesis. The causes for this are however, currently unknown. Eml1, a microtubule (MT)-associated protein of the EMAP family, is impaired in these mice. We first show that MT dynamics are perturbed in mutant progenitor cells in vitro. These may influence interphase and mitotic MT mechanisms and indeed, centrosome and primary cilia were altered and spindles were found to be abnormally long in HeCo progenitors. Consistently, MT and spindle length regulators were identified in EML1 pulldowns from embryonic brain extracts. Finally, we found that mitotic cell shape is also abnormal in the mutant VZ. These previously unidentified VZ characteristics suggest altered cell constraints which may contribute to cell delamination.

An Organoid-Based Model of Cortical Development Identifies Non-Cell-Autonomous Defects in Wnt Signaling Contributing to Miller-Dieker Syndrome.

Miller-Dieker syndrome (MDS) is caused by a heterozygous deletion of chromosome 17p13.3 involving the genes LIS1 and YWHAE (coding for 14.3.3ε) and leads to malformations during cortical development. Here, we used patient-specific forebrain-type organoids to investigate pathological changes associated with MDS. Patient-derived organoids are significantly reduced in size, a change accompanied by a switch from symmetric to asymmetric cell division of ventricular zone radial glia cells (vRGCs). Alterations in microtubule network organization in vRGCs and a disruption of cortical niche architecture, including altered expression of cell adhesion molecules, are also observed. These phenotypic changes lead to a non-cell-autonomous disturbance of the N-cadherin/ß-catenin signaling axis. Reinstalling active ß-catenin signaling rescues division modes and ameliorates growth defects. Our data define the role of LIS1 and 14.3.3ε in maintaining the cortical niche and highlight the utility of organoid-based systems for modeling complex cell-cell interactions in vitro.

Neuroanatomical characterization of the cellular and axonal architecture of subcortical band heterotopia in the BXD29-Tlr4lps-2J/J mouse cortex.

Subcortical band heterotopia (SBH) are malformations of the human cerebral cortex typically associated with epilepsy and cognitive delay/disability. Rodent models of SBH have demonstrated strong face validity as they are accompanied by both cognitive deficits and spontaneous seizures or reduced seizure threshold. BXD29-Tlr4lps-2J/J recombinant inbred mice display striking bilateral SBH, partial callosal agenesis, morphological changes in subcortical structures of the auditory pathway, and display sensory deficits in behavioral tests (Rosen et al., 2013; Truong et al., 2013, 2015). Surprisingly, these mice show no cognitive deficits and have a higher seizure threshold to chemi-convulsive treatment (Gabel et al., 2013) making them different than other rodent SBH models described previously. In the present report, we perform a detailed characterization of the cellular and axonal constituents of SBH in BXD29-Tlr4lps-2J/J mice and demonstrate that various types of interneurons and glia as well as cortical and subcortical projections are found in SBH. In addition, the length of neuronal cilia was reduced in SBH compared to neurons in the overlying and adjacent normotopic cortex. Finally, we describe additional and novel malformations of the hippocampus and neocortex present in BXD29-Tlr4lps-2J/J mice. Together, our findings in BXD29-Tlr4lps-2J/J mice are discussed in the context of the known neuroanatomy and phenotype of other SBH rodent models.

Thyroid hormone-dependent formation of a subcortical band heterotopia (SBH) in the neonatal brain is not exacerbated under conditions of low dietary iron (FeD).

Thyroid hormones (TH) are critical for brain development and insufficiencies can lead to structural abnormalities in specific brain regions. Administration of the goitrogen propylthiouracil (PTU) reduces TH production by inhibiting thyroperoxidase (TPO), an enzyme that oxidizes iodide for the synthesis of TH. TPO activity is iron (Fe)-dependent and dietary iron deficiency (FeD) also reduces circulating levels of TH. We have previously shown that modest degrees of TH insufficiency induced in pregnant rat dams alters the expression of TH-responsive genes in the cortex and hippocampus of the neonate, and results in the formation of a subcortical band heterotopia (SBH) in the corpus callosum (Royland et al., 2008, Bastian et al., 2014, Gilbert et al., 2014). The present experiment investigated if FeD alone was sufficient to induce a SBH or if FeD would augment SBH formation at lower doses of PTU. One set of pregnant rats was administered 0, 1, 3, or 10ppm of PTU via drinking water starting on gestational day (GD) 6. FeD was induced in a 2nd set of dams beginning on GD2. A third set of dams received the FeD diet from GD2 paired with either 1ppm or 3ppm PTU beginning on GD6. All treatments continued until the time of sacrifice. On PN18, one female pup from each litter was sacrificed and the brain examined for SBH. We observed lower maternal, PN2 and PN18 pup serum T4 in response to PTU. FeD reduced serum T4 in pups on PN16, but did not affect serum T4 in dams or PN2 pups. Neither did FeD in combination with PTU alter T4 levels in dams on PN18 or pups on PN2 compared to PTU treatment alone. By PN16, however more severe T4 reductions were observed in pups when FeD was combined with PTU. SBH increased with increasing dosage of PTU, but counter to our hypothesis, no SBH was detected in the offspring of FeD dams. As such, T4 levels in dams and newborn pups rather than older neonates appear to be a better predictor SBH associated with TH insufficiency. These data indirectly support previous work indicating prenatal TH insufficiency but not postnatal TH insufficiency in offspring is required for SBH formation.

Recurrent Guillain-Barré syndrome, Miller Fisher syndrome and Bickerstaff brainstem encephalitis.

OBJECTIVE: Guillain-Barré syndrome (GBS), Miller Fisher syndrome (MFS), and Bickerstaff brainstem encephalitis (BBE) are usually monophasic, but some patients experience recurrences after long asymptomatic intervals. We aimed to investigate clinical features of recurrent GBS, MFS, and BBE at a single hospital. METHODS: Records from 97 consecutive patients with GBS, MFS or BBE who were admitted to a tertiary hospital between 2001 and 2013 were reviewed. Clinical and laboratory features of patients with recurrent GBS, MFS, or BBE were investigated. RESULTS: Patients included 55 (32 males) with GBS, 34 (22 males) with MFS, and 8 (6 males) with BBE. Recurrent cases occurred in 2 (4%) of the 55 patients with GBS, 4 (12%) of the 34 patients with MFS, and 2 (25%) of the 8 patients with BBE. Patients with recurrent MFS had a tendency to be younger at the first episode than patients with non-recurrent MFS (median, 22 versus 37years old). Symptoms and signs were less severe during relapses than during the initial episode in recurrent patients. CONCLUSIONS: Recurrences occurred more frequently in patients with MFS or BBE compared with those with GBS. Patients with recurrent MFS might be younger than those with non-recurrent MFS.

Ccdc85C, a causative protein for hydrocephalus and subcortical heterotopia, is expressed in the systemic epithelia with proliferative activity in rats.

Coiled-coil domain containing 85c (Ccdc85c) is a causative gene for spontaneous mutant mouse with non-obstructive hydrocephalus and subcortical heterotopia. Detailed functions of Ccdc85C protein have not been clarified. To reveal roles of Ccdc85C, we examined the distribution and expression pattern of Ccdc85C in the systemic developing organs in rats. Ccdc85C was expressed in various simple epithelia but not stratified epithelia. In the various epithelia, Ccdc85C was localized at cell-cell junctions and its expression was strong at apical junctions. Furthermore, intense expression was seen at developing period and gradually decreased with advancing development. Distribution of Ccdc85C coincides with that of proliferating epithelial cells. These results suggest that Ccdc85C plays an important role in the proliferative property of simple epithelia.

Small Rho-GTPases and cortical malformations: fine-tuning the cytoskeleton stability.

Rho-GTPases have been found to be crucial for cytoskeleton remodelling and cell polarity, as well as key players in directed cell migration in various tissues and organs, therefore becoming good candidates for involvement in neuronal migration disorders. We recently found that genetic deletion of the small GTPase RhoA in the developing mouse cerebral cortex results in three distinct cortical malformations: a defect in the proliferation of progenitor cells during development that leads to a bigger cerebral cortex in the adult mouse, a change in the morphology of radial glial cells that results in the formation of a subcortical band heterotopia (SBH, also called Double Cortex) and an increase in the speed of migrating newborn neurons. The latter, together with the aberrant radial glial shape, is likely to be the cause of cobblestone lissencephaly, where neurons protrude beyond layer I at the pial surface of the brain.

A radial glia-specific role of RhoA in double cortex formation.

The positioning of neurons in the cerebral cortex is of crucial importance for its function as highlighted by the severe consequences of migrational disorders in patients. Here we show that genetic deletion of the small GTPase RhoA in the developing cerebral cortex results in two migrational disorders: subcortical band heterotopia (SBH), a heterotopic cortex underlying the normotopic cortex, and cobblestone lissencephaly, in which neurons protrude beyond layer I at the pial surface of the brain. Surprisingly, RhoA(-/-) neurons migrated normally when transplanted into wild-type cerebral cortex, whereas the converse was not the case. Alterations in the radial glia scaffold are demonstrated to cause these migrational defects through destabilization of both the actin and the microtubules cytoskeleton. These data not only demonstrate that RhoA is largely dispensable for migration in neurons but also showed that defects in radial glial cells, rather than neurons, can be sufficient to produce SBH.

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.

Inhibition of histone deacetylases in rats self-administering cocaine regulates lissencephaly gene-1 and reelin gene expression, as revealed by microarray technique.

Injection of the histone deacetylase inhibitor trichostatin A (TsA) to rats has been shown to decrease their motivation to self-administer cocaine. In the present study, we investigated alterations in gene expression patterns in the anterior cingulate cortex and nucleus accumbens of rats self-administering cocaine and treated with TsA. Using oligonucleotide microarrays, we identified 722 probe sets in the cortex and 136 probe sets in the nucleus accumbens that were differentially expressed between vehicle and TsA-treated rats that self-administered cocaine. Microarray data were validated by real-time PCR for seven genes. Using immunohistochemistry, we further investigated the expression of Lis1 and reelin genes, because (i) they were similarly regulated by TsA at the mRNA level; (ii) they belong to the same signal transduction pathway; (iii) mutations within both genes cause lissencephaly. Cocaine self-injection was sufficient to activate the two genes at both the mRNA and protein levels. TsA treatment was found to up-regulate both Lis1 and reelin protein expression in the cortex and to down-regulate it in the nucleus accumbens of rats self-administering cocaine. The data suggest that the two proteins contribute to establish neurobiological mechanisms underlying brain plasticity whereby TsA lowers the motivation for cocaine.

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.

Lissencephaly 1 linking to multiple diseases: mental retardation, neurodegeneration, schizophrenia, male sterility, and more.

Lissencephaly 1 (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.