A human dynein heavy chain mutation impacts cortical progenitor cells causing developmental defects, reduced brain size and altered brain architecture.

Dynein heavy chain (DYNC1H1) mutations can either lead to severe cerebral cortical malformations, or alternatively may be associated with the development of spinal muscular atrophy with lower extremity predominance (SMA-LED). To assess the origin of such differences, we studied a new Dync1h1 knock-in mouse carrying the cortical malformation p.Lys3334Asn mutation. Comparing with an existing neurodegenerative Dync1h1 mutant (Legs at odd angles, Loa, p.Phe580Tyr/+), we assessed Dync1h1's roles in cortical progenitor and especially radial glia functions during embryogenesis, and assessed neuronal differentiation. p.Lys3334Asn /+ mice exhibit reduced brain and body size. Embryonic brains show increased and disorganized radial glia: interkinetic nuclear migration occurs in mutants, however there are increased basally positioned cells and abventricular mitoses. The ventricular boundary is disorganized potentially contributing to progenitor mislocalization and death. Morphologies of mitochondria and Golgi apparatus are perturbed in vitro, with different effects also in Loa mice. Perturbations of neuronal migration and layering are also observed in p.Lys3334Asn /+ mutants. Overall, we identify specific developmental effects due to a severe cortical malformation mutation in Dync1h1, highlighting the differences with a mutation known instead to primarily affect motor function.

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.

Use of subcutaneous transponders to monitor body temperature in laboratory rats.

Implantable radiofrequency transponders may be adequate for the characterization of hazardous chemicals targeting body temperature control in experimental animals when colonic probes and automated monitoring systems based on intraperitoneal transmitters are not available, installable or applicable for any reason. In this work, we summarize a series of experiments showing the implantation protocol and utility of rice-grain size transponders to monitor subcutaneous temperature (Tsc) after exposure to pharmacological or toxicological treatments targeting body temperature control in laboratory rats. In addition, to explore the responsiveness of this thermometric system, the influence of physiological activity on Tsc readings was examined by monitoring Tsc after a motor exercise in a RotaRod system. Moreover, we characterized the effects of acute oral administration of the pyrethroid insecticide permethrin (PRM) in corn oil (1 mL/kg) on Tsc. PRM has been previously reported to cause dose-related increases in core temperature after administering oral doses ≥75 mg/kg, with peak effects at 2-4 h in adult rats. We monitored Tsc at 30 min intervals over a 4 h period after exposure to PRM (40-160 mg/kg). PRM caused a moderate increase in Tsc starting at ~3.5 h. Overall, Tsc assays showed minimal animal stress (if any) and rapid animal recovery from transponder implantation, simplicity to collect data, convenient testing room space requirements, and a competitive global cost per animal examined. However, various experimental factors may greatly influence the variability within and between individuals, some of which can be controlled by carefully setting up experimental conditions.

Mutations in the Heterotopia Gene Eml1/EML1 Severely Disrupt the Formation of Primary Cilia.

Apical radial glia (aRGs) are predominant progenitors during corticogenesis. Perturbing their function leads to cortical malformations, including subcortical heterotopia (SH), characterized by the presence of neurons below the cortex. EML1/Eml1 mutations lead to SH in patients, as well as to heterotopic cortex (HeCo) mutant mice. In HeCo mice, some aRGs are abnormally positioned away from the ventricular zone (VZ). Thus, unraveling EML1/Eml1 function will clarify mechanisms maintaining aRGs in the VZ. We pinpoint an unknown EML1/Eml1 function in primary cilium formation. In HeCo aRGs, cilia are shorter, less numerous, and often found aberrantly oriented within vesicles. Patient fibroblasts and human cortical progenitors show similar defects. EML1 interacts with RPGRIP1L, a ciliary protein, and RPGRIP1L mutations were revealed in a heterotopia patient. We also identify Golgi apparatus abnormalities in EML1/Eml1 mutant cells, potentially upstream of the cilia phenotype. We thus reveal primary cilia mechanisms impacting aRG dynamics in physiological and pathological conditions.

Genetics and mechanisms leading to human cortical malformations.

Cerebral cortical development involves a complex series of highly regulated steps to generate the laminated structure of the adult neocortex. Neuronal migration is a key part of this process. We provide here a detailed review of cortical malformations thought to be linked to abnormal neuronal migration. We have focused on providing updated views related to perturbed mechanisms based on the wealth of genetic information currently available, as well as the study of mutant genes in animal models. We discuss mainly type 1 lissencephaly, periventricular heterotopia, type II lissencephaly and polymicrogyria. We also discuss functional classifications such as the tubulinopathies, and emphasize how modern genetics is revealing genes mutated in atypical cases, as well as unexpected genes for classical cases. A role in neuronal migration is revealed for many mutant genes, although progenitor abnormalities also predominate, depending on the disorder. We finish by describing the advantages of human in vitro cell culture models, to examine human-specific cells and transcripts, and further mention non-genetic mechanisms leading to cortical malformations.

From the Cover: Vulnerability of C6 Astrocytoma Cells After Single-Compound and Joint Exposure to Type I and Type II Pyrethroid Insecticides.

A primary mode-of-action of all pyrethroid insecticides (PYRs) is the disruption of the voltage-gated sodium channel electrophysiology in neurons of target pests and nontarget species. The neurological actions of PYRs on non-neuronal cells of the nervous system remain poorly investigated. In the present work, we used C6 astrocytoma cells to study PYR actions (0.1-50 µM) under the hypothesis that glial cells may be targeted by and vulnerable to PYRs. To this end, we characterized the effects of bifenthrin (BF), tefluthrin (TF), α-cypermethrin (α-CYP), and deltamethrin (DM) on the integrity of nuclear, mitochondrial, and lysosomal compartments. In general, 24- to 48-h exposures produced concentration-related impairment of cell viability. In single-compound, 24-h exposure experiments, effective concentration (EC)15s 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT assay) were computed as follows (in µM): BF, 16.1; TF, 37.3; α-CYP, 7.8; DM, 5.0. We found concentration-related damage in several C6-cell subcellular compartments (mitochondria, nuclei, and lysosomes) at ≥ 10-1 µM levels. Last, we examined a mixture of all PYRs (ie, Σ individual EC15) using MTT assays and subcellular analyses. Our findings indicate that C6 cells are responsive to nM levels of PYRs, suggesting that astroglial susceptibility may contribute to the low-dose neurological effects caused by these insecticides. This research further suggests that C6 cells may provide relevant information as a screening platform for pesticide mixtures targeting nervous system cells by expected and unexpected toxicogenic pathways potentially contributing to clinical neurotoxicity.

Oxidative stress induced by a commercial glyphosate formulation in a tolerant strain of Chlorella kessleri.

We studied the toxicity of a glyphosate formulation and provide evidence of metabolic alterations due to oxidative stress caused in a Chlorella kessleri tolerant strain by exposure to the herbicide. After 96 h of exposure to increasing concentrations of the herbicide (0-70 mg L(-1)) with alkylaryl polyglycol ether surfactant, growth was inhibited (EC50-96 h 55.62 mg L(-1)). Glyphosate increased protein and malondialdehyde content which was significantly higher than in the control at 50-70 mg L(-1). Superoxide dismutase and catalase activities and reduced glutathione levels increased in a concentration-dependant manner. Morphological studies showed increases in vacuolisation and in cell and sporangia sizes. The glyphosate formulation studied has a cytotoxic effect on C. kessleri through a mechanism that would involve the induction of oxidative stress. Upon glyphosate exposure, oxidative stress parameters such as SOD and CAT activities and MDA level could be more sensitive biomarkers than usually tested growth parameters in C. kessleri.