Aberrant sorting of hippocampal complex pyramidal cells in type I lissencephaly alters topological innervation.

Layering has been a long-appreciated feature of higher order mammalian brain structures but the extent to which it plays an instructive role in synaptic specification remains unknown. Here we examine the formation of synaptic circuitry under cellular heterotopia in hippocampal CA1, using a mouse model of the human neurodevelopmental disorder Type I Lissencephaly. We identify calbindin-expressing principal cells which are mispositioned under cellular heterotopia. Ectopic calbindin-expressing principal cells develop relatively normal morphological features and stunted intrinsic physiological features. Regarding network development, a connectivity preference for cholecystokinin-expressing interneurons to target calbindin-expressing principal cells is diminished. Moreover, in vitro gamma oscillatory activity is less synchronous across heterotopic bands and mutants are less responsive to pharmacological inhibition of cholecystokinin-containing interneurons. This study will aid not only in our understanding of how cellular networks form but highlight vulnerable cellular circuit motifs that might be generalized across disease states.

Neuronal migration genes and a familial translocation t (3;17): candidate genes implicated in the phenotype.

BACKGROUND: While Miller-Dieker syndrome critical region deletions are well known delineated anomalies, submicroscopic duplications in this region have recently emerged as a new distinctive syndrome. So far, only few cases have been described overlapping 17p13.3 duplications. METHODS: In this study, we report on clinical and cytogenetic characterization of two new cases involving 17p13.3 and 3p26 chromosomal regions in two sisters with familial history of lissencephaly. Fluorescent In Situ Hybridization and array Comparative Genomic Hybridization were performed. RESULTS: A deletion including the critical region of the Miller-Dieker syndrome of at least 2,9 Mb and a duplication of at least 3,6 Mb on the short arm of chromosome 3 were highlighted in one case. The opposite rearrangements, 17p13.3 duplication and 3p deletion, were observed in the second case. This double chromosomal aberration is the result of an adjacent 1:1 meiotic segregation of a maternal reciprocal translocation t(3,17)(p26.2;p13.3). CONCLUSIONS: 17p13.3 and 3p26 deletions have a clear range of phenotypic features while duplications still have an uncertain clinical significance. However, we could suggest that regardless of the type of the rearrangement, the gene dosage and interactions of CNTN4, CNTN6 and CHL1 in the 3p26 and PAFAH1B1, YWHAE in 17p13.3 could result in different clinical spectrums.

Spontaneous epileptiform activity in a rat model of bilateral subcortical band heterotopia.

OBJECTIVE: Malformations of cortical development are common causes of intellectual disability and epilepsy, yet there is a crucial lack of relevant preclinical models associating seizures and cortical malformations. Here, we describe a novel rat model with bilateral subcortical band heterotopia (SBH) and examine whether this model develops spontaneous epileptic seizures. METHODS: To generate bilateral SBH in rats, we combined RNAi-mediated knockdown of Dcx and in utero electroporation with a tripolar electrode configuration enabling simultaneous transfection of the two brain hemispheres. To determine whether bilateral SBH leads to epileptiform activity, rats of various ages were implanted for telemetric electrocorticographic recordings and histopathological examination was carried out at the end of the recording sessions. RESULTS: By 2 months, rats with bilateral SBH showed nonconvulsive spontaneous seizures consisting of spike-and-wave discharges (SWDs) with dominant frequencies in the alpha and theta bands and secondarily in higher-frequency bands. SWDs occurred during both the dark and the light period, but were more frequent during quiet awake state than during sleep. Also, SWDs were more frequent and lasted longer at older ages. No sex differences were found. Although frequencies and durations of SWDs were found to be uncorrelated with the size of SBH, SWDs were initiated in some occasions from brain hemispheres comprising a larger SBH. Lastly, SWDs exhibited absence-like pharmacological properties, being temporarily alleviated by ethosuximide administration. SIGNIFICANCE: This novel model of bilateral SBH with spontaneous epilepsy may potentially provide valuable new insights into causality between cortical malformations and seizures, and help translational research aiming at designing novel treatment strategies for epilepsy.

Loss of Lgl1 Disrupts the Radial Glial Fiber-guided Cortical Neuronal Migration and Causes Subcortical Band Heterotopia in Mice.

Radial glial cells (RGCs) are neuronal progenitors and function as scaffolds for neuronal radial migration in the developing cerebral cortex. These functions depend on a polarized radial glial scaffold, which is of fundamental importance for brain development. Lethal giant larvae 1 (Lgl1), a key regulator for cell polarity from Drosophila to mammals, plays a key role in tumorigenesis and brain development. To overcome neonatal lethality in Lgl1-null mice and clarify the role of Lgl1 in mouse cerebral cortex development and function, we created Lgl1 dorsal telencephalon-specific knockout mice mediated by Emx1-Cre. Lgl1Emx1 conditional knockout (CKO) mice had normal life spans and could be used for function research. Histology results revealed that the mutant mice displayed an ectopic cortical mass in the dorsolateral hemispheric region between the normotopic cortex and the subcortical white matter, resembling human subcortical band heterotopia (SBH). The Lgl1Emx1 CKO cortex showed disrupted adherens junctions (AJs), which were accompanied by ectopic RGCs and intermediate progenitors, and disorganization of the radial glial fiber system. The early- and late-born neurons failed to reach the destined position along the disrupted radial glial fiber scaffold and instead accumulated in ectopic positions and formed SBH. Additionally, the absence of Lgl1 led to severe abnormalities in RGCs, including hyperproliferation, impaired differentiation, and increased apoptosis. Lgl1Emx1 CKO mice also displayed deficiencies in anxiety-related behaviors. We concluded that Lgl1 is essential for RGC development and neural migration during cerebral cortex development.

Direct and Collateral Alterations of Functional Cortical Circuits in a Rat Model of Subcortical Band Heterotopia.

Subcortical band heterotopia (SBH), also known as double-cortex syndrome, is a neuronal migration disorder characterized by an accumulation of neurons in a heterotopic band below the normotopic cortex. The majority of patients with SBH have mild to moderate intellectual disability and intractable epilepsy. However, it is still not clear how cortical networks are organized in SBH patients and how this abnormal organization contributes to improper brain function. In this study, cortical networks were investigated in the barrel cortex in an animal model of SBH induced by in utero knockdown of Dcx, main causative gene of this condition in human patients. When the SBH was localized below the Barrel Field (BF), layer (L) four projection to correctly positioned L2/3 pyramidal cells was weakened due to lower connectivity. Conversely, when the SBH was below an adjacent cortical region, the excitatory L4 to L2/3 projection was stronger due to increased L4 neuron excitability, synaptic strength and excitation/inhibition ratio of L4 to L2/3 connection. We propose that these developmental alterations contribute to the spectrum of clinical dysfunctions reported in patients with SBH.

Quadruple Semitendinosus Graft Construct With Double Cortical Suspensory Fixation for Anterior Cruciate Ligament Reconstruction: A Biomechanical Study.

The purpose of this study was to evaluate the biomechanical properties of a graft construct with quadrupled Semitendinosus and two cortical buttons with adjustable loops concerning elongation, stiffness and resistance. A total of 15 fresh human cadaveric semitendinosus tendons were quadrupled over the two adjustable loops and stitched at the tibial tip with a cerclage type suture. They underwent pre-tensioning at 300 N for 2 minutes followed by cyclic loading (1000 cycles between 50-250 N) and finally a load-to-failure test. Statistical analysis was performed using SPSS Statistics software and groups were compared using a paired t-test, with a significance level set at α = 0.05. Graft construct elongation after pre-tensioning at 300 N was 12.8 mm (9.3 mm-16.5 mm) and mean cyclic elongation 0.4 mm (0.2 mm-0.9 mm), considered significant (p < 0,001). The resistance and stiffness values were respectively 849.46 N (649.30 N-1027.90 N) and 221.49 N (178,30 N - 276.10 N). Quadruple ST graft construct using two cortical buttons and adjustable loops showed a high stiffness and resistance with a very low elongation after cycling.

Analysis of 17 genes detects mutations in 81% of 811 patients with lissencephaly.

PURPOSE: To estimate diagnostic yield and genotype-phenotype correlations in a cohort of 811 patients with lissencephaly or subcortical band heterotopia. METHODS: We collected DNA from 756 children with lissencephaly over 30 years. Many were tested for deletion 17p13.3 and mutations of LIS1, DCX, and ARX, but few other genes. Among those tested, 216 remained unsolved and were tested by a targeted panel of 17 genes (ACTB, ACTG1, ARX, CRADD, DCX, LIS1, TUBA1A, TUBA8, TUBB2B, TUBB, TUBB3, TUBG1, KIF2A, KIF5C, DYNC1H1, RELN, and VLDLR) or by whole-exome sequencing. Fifty-five patients studied at another institution were added as a validation cohort. RESULTS: The overall mutation frequency in the entire cohort was 81%. LIS1 accounted for 40% of patients, followed by DCX (23%), TUBA1A (5%), and DYNC1H1 (3%). Other genes accounted for 1% or less of patients. Nineteen percent remained unsolved, which suggests that several additional genes remain to be discovered. The majority of unsolved patients had posterior pachygyria, subcortical band heterotopia, or mild frontal pachygyria. CONCLUSION: The brain-imaging pattern correlates with mutations in single lissencephaly-associated genes, as well as in biological pathways. We propose the first LIS classification system based on the underlying molecular mechanisms.

Disruption of YWHAE gene at 17p13.3 causes learning disabilities and brain abnormalities.

There is a broad phenotypic spectrum of patients with 17p13.3 deletions. One of the most prominent feature is lissencephaly caused by haploinsufficiency of the gene PAFAH1B1. The deletion of this gene and those distal to it, results in Miller-Dieker syndrome, however there have been many reports of patients with haploinsufficiency of the distal genes alone. The deletions of these genes including YWHAE CRK and TUSC5 have been studied extensively and YWHAE has been postulated to be the cause of neurological abnormalities. The patients with deletions of the Miller-Dieker syndrome distal region present with variable clinical features including brain abnormalities, growth retardation, developmental delay, facial dysmorphisms and seizures. While there have been many patients reported to have deletions involving the YWHAE gene along with other genes, here we present the first detailed clinical description of a patient with deletion of YWHAE alone, allowing a more accurate characterization of the pathogenicity of YWHAE haploinsufficiency. The patient reported here demonstrated brain abnormalities, learning disabilities, and seizures supporting the role of YWHAE in these features. We review the literature and use this case report to better characterize and further confirm the genotype-phenotype relationship of the genes within the critical region of Miller-Dieker Syndrome.

Lissencephaly: Expanded imaging and clinical classification.

Lissencephaly ("smooth brain," LIS) is a malformation of cortical development associated with deficient neuronal migration and abnormal formation of cerebral convolutions or gyri. The LIS spectrum includes agyria, pachygyria, and subcortical band heterotopia. Our first classification of LIS and subcortical band heterotopia (SBH) was developed to distinguish between the first two genetic causes of LIS-LIS1 (PAFAH1B1) and DCX. However, progress in molecular genetics has led to identification of 19 LIS-associated genes, leaving the existing classification system insufficient to distinguish the increasingly diverse patterns of LIS. To address this challenge, we reviewed clinical, imaging and molecular data on 188 patients with LIS-SBH ascertained during the last 5 years, and reviewed selected archival data on another ∼1,400 patients. Using these data plus published reports, we constructed a new imaging based classification system with 21 recognizable patterns that reliably predict the most likely causative genes. These patterns do not correlate consistently with the clinical outcome, leading us to also develop a new scale useful for predicting clinical severity and outcome. Taken together, our work provides new tools that should prove useful for clinical management and genetic counselling of patients with LIS-SBH (imaging and severity based classifications), and guidance for prioritizing and interpreting genetic testing results (imaging based- classification).

17p13.3 microduplication including CRK leads to overgrowth and elevated growth factors: A case report.

17p13.3 microduplications classified as class I duplications involving YWHAE but not PAFAH1B1 (formerly LIS1) and class II duplications which extend to involve PAFAH1B1, are associated with diverse phenotypes including intellectual disability and structural brain malformations. We report a girl with an approximately 1.58 Mb apparently terminal gain of 17p13.3, which contains more than 20 genes including the YWHAE and CRK genes (OMIM: 164762). She had increased growth factors accompanied by pathologic tall stature. In addition to these, she developed central precocious puberty at 7 years old. In individuals with class I 17p13.3 microduplications including CRK, we recommend biochemical evaluation of the growth hormone axis. Providers caring for these patients should be aware of their possible risk for the development of central precocious puberty.

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.

Subcortical heterotopia appearing as huge midline mass in the newborn brain.

INTRODUCTION: We report the case of a 2-year-old boy who showed a huge midline mass in the brain at prenatal assessment. CASE REPORT: After birth, magnetic resonance imaging (MRI) revealed a conglomerate mass with an infolded microgyrus at the midline, which was suspected as a midline brain-in-brain malformation. MRI also showed incomplete cleavage of his frontal cortex and thalamus, consistent with lobar holoprosencephaly. The patient underwent an incisional biopsy of the mass on the second day of life. The mass consisted of normal central nervous tissue with gray and white matter, representing a heterotopic brain. The malformation was considered to be a subcortical heterotopia. With maturity, focal signal changes and decreased cerebral perfusion became clear on brain imaging, suggesting secondary glial degeneration. Coincident with these MRI abnormalities, the child developed psychomotor retardation and severe epilepsy focused on the side of the intracranial mass.

Impairment of radial glial scaffold-dependent neuronal migration and formation of double cortex by genetic ablation of afadin.

Studies of human brain malformations, such as lissencephaly and double cortex, have revealed the importance of neuronal migration during cortical development. Afadin, a membrane scaffolding protein, regulates the formation of adherens junctions (AJs) and cell migration to form and maintain tissue structures. Here, we report that mice with dorsal telencephalon-specific ablation of afadin gene exhibited defects similar to human double cortex, in which the heterotopic cortex was located underneath the normotopic cortex. The normotopic cortex of the mutant mice was arranged in the pattern similar to the cortex of the control mice, while the heterotopic cortex was disorganized. As seen in human patients, double cortex in the mutant mice was formed by impaired neuronal migration during cortical development. Genetic ablation of afadin in the embryonic cerebral cortex disrupted AJs of radial glial cells, likely resulting in the retraction of the apical endfeet from the ventricular surface and the dispersion of radial glial cells from the ventricular zone to the subventricular and intermediate zones. These results indicate that afadin is required for the maintenance of AJs of radial glial cells and that the disruption of AJs might cause an abnormal radial scaffold for neuronal migration. In contrast, the proliferation or differentiation of radial glial cells was not significantly affected. Taken together, these findings indicate that afadin is required for the maintenance of the radial glial scaffold for neuronal migration and that the genetic ablation of afadin leads to the formation of double cortex.

Mimics and chameleons in Guillain-Barré and Miller Fisher syndromes.

Guillain-Barré syndrome (GBS) and its variant, Miller Fisher syndrome (MFS) have several subtypes, together forming a continuous spectrum of discrete and overlapping syndromes. Such is the heterogeneity within this spectrum that many physicians may be surprised to learn that these disorders are related pathophysiologically, and therefore share certain clinical features. These include history of antecedent infection, monophasic disease course and symmetrical cranial or limb weakness. The presence of cerebrospinal fluid albuminocytological dissociation (raised protein, normal cell count), antiganglioside antibodies and neurophysiological evidence of axonal or demyelinating neuropathy also support a diagnosis in many cases, but should not be relied upon. Mimics of GBS and MFS can broadly be divided into those presenting with symmetrical limb weakness and those presenting with brainstem signs. MFS and the pharyngeal-cervical-brachial variant of GBS are frequently mistaken for brainstem stroke, botulism or myasthenia gravis, whereas Bickerstaff's brainstem encephalitis is often diagnosed as Wernicke's encephalopathy. Chameleons or atypical presentations of GBS-related disorders include: paraparetic GBS, bifacial weakness with paraesthesias, acute ataxic neuropathy, acute ophthalmoparesis, acute ptosis and acute mydriasis. Many neurologists may also not be aware that deep tendon reflexes remain present or may even appear brisk in up to 10% of patients with GBS. Correct diagnosis of GBS-related disorders helps to avoid unnecessary investigations and allows early immunotherapy if appropriate.

Normotopic cortex is the major contributor to epilepsy in experimental double cortex.

OBJECTIVE: Subcortical band heterotopia (SBH) is a cortical malformation formed when neocortical neurons prematurely stop their migration in the white matter, forming a heterotopic band below the normotopic cortex, and is generally associated with intractable epilepsy. Although it is clear that the band heterotopia and the overlying cortex both contribute to creating an abnormal circuit prone to generate epileptic discharges, it is less understood which part of this circuitry is the most critical. Here, we sought to identify the origin of epileptiform activity in a targeted genetic model of SBH in rats. METHODS: Rats with SBH (Dcx-KD rats) were generated by knocking down the Dcx gene using shRNA vectors transfected into neocortical progenitors of rat embryos. Origin, spatial extent, and laminar profile of bicuculline-induced interictal-like activity on neocortical slices were analyzed by using extracellular recordings from 60-channel microelectrode arrays. Susceptibility to pentylenetetrazole-induced seizures was assessed by electrocorticography in head-restrained nonanesthetized rats. RESULTS: We show that the band heterotopia does not constitute a primary origin for interictal-like epileptiform activity in vitro and is dispensable for generating induced seizures in vivo. Furthermore, we report that most interictal-like discharges originating in the overlying cortex secondarily propagate to the band heterotopia. Importantly, we found that in vivo suppression of neuronal excitability in SBH does not alter the higher propensity of Dcx-KD rats to display seizures. INTERPRETATION: These results suggest a major role of the normotopic cortex over the band heterotopia in generating interictal epileptiform activity and seizures in brains with SBH.

Subcortical band heterotopia in rat offspring following maternal hypothyroxinaemia: structural and functional characteristics.

Thyroid hormones (TH) play crucial roles in brain maturation and are important for neuronal migration and neocortical lamination. Subcortical band heterotopia (SBH) represent a class of neuronal migration errors in humans that are often associated with childhood epilepsy. We have previously reported the presence of SBH in a rodent model of low level hypothyroidism induced by maternal exposure to the goitrogen, propylthiouracil (PTU). In the present study, we report the dose-response characteristics of this developmental malformation and the connectivity of heterotopic neurones with other brain regions, as well as their functionality. Pregnant rats were exposed to varying concentrations of PTU through the drinking water (0-10 p.p.m.) beginning on gestational day 6 to produce graded levels of TH insufficiency. Dose-dependent increases in the volume of the SBH present in the corpus callosum were documented in the adult offspring, with a clear presence at concentrations of PTU that resulted in minor (< 15%) reductions in maternal serum thyroxine as measured when pups were weaned. SBH contain neurones, oligodendrocytes, astrocytes and microglia. Monoaminergic and cholinergic processes were prevalent and many of the axons were myelinated. Anatomical connectivity of SBH neurones to cortical neurones and the synaptic functionality of these anatomical connections was verified by ex vivo field potential recordings. SBH persisted in adult offspring despite a return to euthyroid status on termination of exposure and these offspring displayed an increased sensitivity to seizures. Features of this model are attractive with respect to the investigation of the molecular mechanisms of cortical development, the effectiveness of therapeutic intervention in hypothyroxinaemia during pregnancy and the impact of the very modest TH imbalance that accompanies exposure to environmental contaminants.