Interictal hyperemia correlates with epileptogenicity in polymicrogyric cortex

Objective: To investigate pathophysiological factors underlying the presence of interictal hyper-perfusion within the limits of the polymicrogyric (PMG) cortex in epileptic patients. Methods: Retrospective observational study on interictal perfusion by Single Photon Emission Computed Tomography (SPECT) in 16 patients with PMG and its correlations with a number of clinical and neurophysiological variables. Patients underwent video-EEG monitoring, neurological and psychiatric assessments, invasive EEG, and the interictal SPECT coregistered to Magnetic Resonance Imaging (MRI). Results: Patients with interictal hyperperfusion within the PMG cortex had a significantly higher spike rate on interictal EEG than patients with normal perfusion. Interictal hyperperfusion was not correlated to sex, age at epilepsy onset, age at evaluation, number of seizures per month, presence of initial precipitating insult (IPI), abnormal neurological examination, EEG findings, ictal serniology, and seizure outcome. The high interictal spike rate did not correlate to a high frequency of seizures per month. Conclusions: Our work provides further evidences for an intrinsic epileptogenesis of the PMG cortex during the interictal state, which accounts for the major rote of PMG tissue in seizure generation. These results might help to increase our understanding about epileptogenesis related to the PMG cortex, providing new toots for more tailored epilepsy surgery in PMG patients. (c) 2008 Elsevier B.V. All rights reserved.

Mild cognitive deficits associated to neocortical microgyria in mice with genetic deletion of cellular prion protein

The cellular prion protein (PrP(c)) has been implicated with the modulation of neuronal apoptosis, adhesion, neurite outgrowth and maintenance which are processes involved in the neocortical development. Malformations of cortical development (MCD) are frequently associated with neurological conditions including mental retardation, autism, and epilepsy. Here we investigated the behavioral performance of female adult PrP(c)-null mice (Prnp(%)) and their wild-type controls (Prnp(+/+)) presenting unilateral polymicrogyria, a MCD experimentally induced by neonatal freeze-lesion in the right hemisphere. injured mice from both genotypes presented similar locomotor activity but Prnp(%) mice showed a tendency to increase anxiety-related responses when compared to Prnp(+/+) animals. Additionally, injured Prnp(%) mice have a poorer performance in the social recognition task than sham-operated and Prnp(%) injured ones. Moreover the step-down inhibitory avoidance task was not affected by the procedure or the genotype of the animals. These data suggest that the genetic deletion of PrP(c) confers increased susceptibility to short-term social memory deficits induced by neonatal freezing model of polymicrogyria in mice. (C) 2008 Published by Elsevier B.V.

Overexpression of a Slit homologue impairs convergent extension of the mesoderm and causes cyclopia in embryonic zebrafish

Slit is expressed in the midline of the central nervous system both in vertebrates and invertebrates. In Drosophila, it is the midline repellent acting as a ligand for the Roundabout (Robo) protein, the repulsive receptor which is expressed on the growth cones of the commissural neurons. We have isolated cDNA fragments of the zebrafish slit2 and slit3 homologues and found that both genes start to be expressed by the midgastrula stage well before the axonogenesis begins in the nervous system, both in the axial mesoderm, and slit2 in the anterior margin of the neural plate and slit3 in the polster at the anterior end of the prechordal mesoderm. Later, expression of slit2 mRNA is detected mainly in midline structures such as the floor plate cells and the hypochord, and in the anterior margins of the neural plates in the zebrafish embryo, while slit3 expression is observed in the anterior margin of the prechordal plate, the floorplate cells in the hindbrain, and the motor neurons both in the hindbrain and the spinal cord. To study the role of Slit in early embryos, we overexpressed Slit2 in the whole embryos either by injection of its mRNA into one-cell stage embryos or by heat-shock treatment of the transgenic embryos which carries the slit2 gene under control of the heat-shock promoter. Overexpression of Slit2 in such ways impaired the convergent extension movement of the mesoderm and the rostral migration of the cells in the dorsal diencephalon and resulted in cyclopia. Our results shed light on a novel aspect of Slit function as a regulatory factor of mesodermal cell movement during gastrulation. (C) 2001 Academic Press.

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.

Laminar disorganisation of mitral cells in the olfactory bulb does not affect topographic targeting of primary olfactory axons

Primary olfactory neurons expressing the same odorant receptor protein typically project to topographically fixed olfactory bulb sites. While cell adhesion molecules and odorant receptors have been implicated in guidance of primary olfactory axons. the postsynaptic mitral cells may also have a role in final target selection. We have examined the effect of disorganisation of the mitral cell soma layer in mutant mice heterozygous for the beta-subunit of platelet activating factor acetylhydrolase (Lis1(-/+)) on the targeting of primary olfactory axons. Lis1(-/+) mice display abnormal lamination of neurons in the olfactory bulb. Lis1(-/+) mice were crossed with the P2-IRES-tau:LacZ line of transgenic mice that selectively expresses beta-galactosidase in primary olfactory neurons expressing the P2 odorant receptor. LacZ histochemistry revealed blue-stained P2 axons that targeted topographically fixed glomeruli in these mice in a manner similar to that observed in the parent P2-IRES-tau:LacZ line. Thus, despite the aberrant organisation of postsynaptic mitral cells in Lis1(-/+) mice, primary olfactory axons continued to converge and form glomeruli at correct sites in the olfactory bulb. Next we examined whether challenging primary olfactory axons in adult Lis(-/+) mice with regeneration would affect their ability to converge and form glomeruli. Following partial chemical ablation of the olfactory neuroepithelium with dichlobenil, primary olfactory neurons die and are replaced by newly differentiating neurons that project axons to the olfactory bulb where they converge and form glomeruli. Despite the aberrant mitral cell layer in Lis(-/+) mice. primary olfactory axons continued to converge and form glomeruli during regeneration. Together these results demonstrate that the convergence of primary olfactory axons during development and regeneration is not affected by gross perturbations to the lamination of the mitral cell layer. Thus, these results support evidence from other studies indicating that mitral cells do not play a major role in the convergence and targeting of primary olfactory axons in the olfactory bulb. (C) 2002 Elsevier Science B.V. All rights reserved.

Exogenous Slit2 does not affect ureteric branching or nephron formation during kidney development

In an attempt to elucidate the role of Slit2 invertebrate kidney development, the effect of adding exogenous human Slit2 protein (hSlit2) to developing murine metanephric kidney explants was examined. To confirm the activity of the recombinant Slit2 protein, neurons from 8 day old chick sympathetic nerve chain dorsal root ganglia were cultured with hSlit2 protein, which induced significant neurite branching and outgrowth. Using kidney explants as a model system, metanephric development in the presence of hSlit2 protein was examined. Addition of hSlit2 up to a final concentration of 1 mug/ml had no detectable effect on the formation of nephrons or on branching morphogenesis of the ureteric tree after 2 or 4 days in culture, as assessed via immunofluorescence for the markers WT1 and calbindin 28K respectively. Similarly, maturation of the nephrogenic mesenchyme occurred in a phenotypically normal fashion. In situ analysis of the Slit receptors, Robot and Robot, the vasculogenic markers VEGFA and Flk-1, and the stromal cell marker BF2 displayed no difference in comparison to controls.

The role of the prokineticin 2 pathway in human reproduction: evidence from the study of human and murine gene mutations.

A widely dispersed network of hypothalamic GnRH neurons controls the reproductive axis in mammals. Genetic investigation of the human disease model of isolated GnRH deficiency has revealed several key genes crucial for GnRH neuronal ontogeny and GnRH secretion. Among these genes, prokineticin 2 (PROK2), and PROK2 receptor (PROKR2) have recently emerged as critical regulators of reproduction in both mice and humans. Both prok2- and prokr2-deficient mice recapitulate the human Kallmann syndrome phenotype. Additionally, PROK2 and PROKR2 mutations are seen in humans with Kallmann syndrome, thus implicating this pathway in GnRH neuronal migration. However, PROK2/PROKR2 mutations are also seen in normosmic GnRH deficiency, suggesting a role for the prokineticin signaling system in GnRH biology that is beyond neuronal migration. This observation is particularly surprising because mature GnRH neurons do not express PROKR2. Moreover, mutations in both PROK2 and PROKR2 are predominantly detected in the heterozygous state with incomplete penetrance or variable expressivity frequently seen within and across pedigrees. In some of these pedigrees, a "second hit" or oligogenicity has been documented. Besides reproduction, a pleiotropic physiological role for PROK2 is now recognized, including regulation of pain perception, circadian rhythms, hematopoiesis, and immune response. Therefore, further detailed clinical studies of patients with PROK2/PROKR2 mutations will help to map the broader biological role of the PROK2/PROKR2 pathway and identify other interacting genes/proteins that mediate its molecular effects in humans.

Characterization of the HeCo mutant mouse: a new model of subcortical band heterotopia associated with seizures and behavioral deficits.

In human, neuronal migration disorders are commonly associated with developmental delay, mental retardation, and epilepsy. We describe here a new mouse mutant that develops a heterotopic cortex (HeCo) lying in the dorsolateral hemispheric region, between the homotopic cortex (HoCo) and subcortical white matter. Cross-breeding demonstrated an autosomal recessive transmission. Birthdating studies and immunochemistry for layer-specific markers revealed that HeCo formation was due to a transit problem in the intermediate zone affecting both radially and tangentially migrating neurons. The scaffold of radial glial fibers, as well as the expression of doublecortin is not altered in the mutant. Neurons within the HeCo are generated at a late embryonic age (E18) and the superficial layers of the HoCo have a correspondingly lower cell density and layer thickness. Parvalbumin immunohistochemistry showed the presence of gamma-aminobutyric acidergic cells in the HeCo and the mutant mice have a lowered threshold for the induction of epileptic seizures. The mutant showed a developmental delay but, in contrast, memory function was relatively spared. Therefore, this unique mouse model resembles subcortical band heterotopia observed in human. This model represents a new and rare tool to better understand cortical development and to investigate future therapeutic strategies for refractory epilepsy.

Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse

The reelin gene encodes an extracellular protein that is crucial for neuronal migration in laminated brain regions. To gain insights into the functions of Reelin, we performed high-resolution in situ hybridization analyses to determine the pattern of reelin expression in the developing forebrain of the mouse. We also performed double-labeling studies with several markers, including calcium-binding proteins, GAD65/67, and neuropeptides, to characterize the neuronal subsets that express reelin transcripts. reelinexpression was detected at embryonic day 10 and later in the forebrain, with a distribution that is consistent with the prosomeric model of forebrain regionalization. In the diencephalon, expression was restricted to transverse and longitudinal domains that delineated boundaries between neuromeres. During embryogenesis,reelin was detected in the cerebral cortex in Cajal-Retzius cells but not in the GABAergic neurons of layer I. At prenatal stages, reelin was also expressed in the olfactory bulb, and striatum and in restricted nuclei in the ventral telencephalon, hypothalamus, thalamus, and pretectum. At postnatal stages, reelin transcripts gradually disappeared from Cajal-Retzius cells, at the same time as they appeared in subsets of GABAergic neurons distributed throughout neocortical and hippocampal layers. In other telencephalic and diencephalic regions,reelin expression decreased steadily during the postnatal period. In the adult, there was prominent expression in the olfactory bulb and cerebral cortex, where it was restricted to subsets of GABAergic interneurons that co-expressed calbindin, calretinin, neuropeptide Y, and somatostatin. This complex pattern of cellular and regional expression is consistent with Reelin having multiple roles in brain development and adult brain function.

Reelin regulates the development and synaptogenesis of the layer-specific entorhino-hippocampal connections

Here we examine the role of Reelin, an extracellular protein involved in neuronal migration, in the formation of hippocampal connections. Both at prenatal and postnatal stages, the general laminar and topographic distribution of entorhinal projections is preserved in the hippocampus of reeler mutant mice, in the absence of Reelin. However, developing and adult entorhinal afferents show severe alterations, including increased numbers of misrouted fibers and the formation of abnormal patches of termination from the medial and lateral entorhinal cortices. At perinatal stages, single entorhinal axons in reeler mice are grouped into thick bundles, and they have decreased axonal branching and decreased extension of axon collaterals. We also show that the number of entorhino-hippocampal synapses is lower in reeler mice than in control animals during development. Studies performed in mixed entorhino-hippocampal co-cultures combining slices from reeler and wild-type mice indicate that these abnormalities are caused by the lack of Reelin in the target hippocampus. These findings imply that Reelin fulfills a modulatory role during the formation of layer-specific and topographic connections in the hippocampus. They also suggest that Reelin promotes maturation of single fibers and synaptogenesis by entorhinal afferents.

CELLULAR AND MOLECULAR STUDY OF SUBCORTICAL BAND HETEROTOPIA IN THE MOUSE MUTANT HECO

The HeCo mouse model is characterized by a subcortical heterotopia formed by misplaced neurons normally migrating into the superficial cortical layers. The mutant mouse has a tendency to epileptic seizures. In my thesis project we discovered the mutated Eml1 gene, a member of the echinoderm microtubule-associated protein (EMAP) family, in HeCo as well as in a family of three children showing complex malformation of cortical development. This discovery formed an important step in exploring the pathogenic mechanisms underlying the HeCo phenotype. In vitro results showed that during cell division the EML1 protein is associated with the midbody and a mutated version of Eml1 highlighted an important role of the protein in the astral MT array during cell cycle. In vivo, we found that already at an early age of cortical development (E13), ectopic progenitors such as RGs (PAX6) and IPCs (TBR2) accumulate in the IZ along the entire neocortex. We demonstrated that in the VZ of the HeCo mouse, spindle orientation and cell cycle exit are perturbed. In later stages (E17), RG fibers are strongly disorganized with deep layer (TBR1) and upper layer (CUX1) neurons trapped within an ectopic mass. At P3, columns of upper layer neurons were present between the heterotopia and the developing cortex; these columns were also present at P7 but at lesser extent. Time lapse video recording (E15.5) revealed that the parameters characterizing the migration of individual neurons are not disturbed in HeCo; however, this analysis showed that the density of migrating neuron was smaller in HeCo. In conclusion, truncated EML1 is likely to play a prominent role during cell cycle but also acts on the cytoskeletal architecture altering the shape of RG fibers thus influencing the pattern of neuronal migration. The signal transduction between external cues and intracellular effector pathways through MTs may be secondary but sustains the heterotopia development and further studies are needed to clarify the impact of EML1 in progenitors versus post-mitotic cells.

The interplay of JNK and SCG10 during cortical development and in excitotoxic responses in brain

Initially identified as stress activated protein kinases (SAPKs), the c-Jun Nterminal kinases (JNKs) are currently accepted as potent regulators of various physiologically important cellular events. Named after their competence to phosphorylate transcription factor c-Jun in response to UVtreatment, JNKs play a key role in cell proliferation, cell death or cell migration. Interestingly, these functions are crucial for proper brain formation. The family consists of three JNK isoforms, JNK1, JNK2 and JNK3. Unlike brain specific JNK3 isoform, JNK1 and JNK2 are ubiquitously expressed. It is estimated that ten splice variants exist. However, the detailed cellular functions of these remain undetermined. In addition, physiological conditions keep the activities of JNK2 and JNK3 low in comparison with JNK1, whereas cellular stress raises the activity of these isoforms dramatically. Importantly, JNK1 activity is constitutively high in neurons, yet it does not stimulate cell death. This suggests a valuable role for JNK1 in brain development, but also as an important mediator of cell wellbeing. The aim of this thesis was to characterize the functional relationship between JNK1 and SCG10. We found that SCG10 is a bona fide target for JNK. By employing differential centrifugation we showed that SCG10 co-localized with active JNK, MKK7 and JIP1 in a fraction containing endosomes and Golgi vesicles. Investigation of JNK knockout tissues using phosphospecific antibodies recognizing JNK-specific phosphorylation sites on SCG10 (Ser 62/Ser 73) showed that phosphorylation of endogenous SCG10 was dramatically decreased in Jnk1-/- brains. Moreover, we found that JNK and SCG10 co-express during early embryonic days in brain regions that undergo extensive neuronal migration. Our study revealed that selective inhibition of JNK in the cytoplasm significantly increased both the frequency of exit from the multipolar stage and radial migration rate. However, as a consequence, it led to ill-defined cellular organization. Furthermore, we found that multipolar exit and radial migration in Jnk1 deficient mice can be connected to changes in phosphorylation state of SCG10. Also, the expression of a pseudo-phosphorylated mutant form of SCG10, mimicking the JNK1- phopshorylated form, brings migration rate back to normal in Jnk1 knockout mouse embryos. Furthermore, we investigated the role of SCG10 and JNK in regulation of Golgi apparatus (GA) biogenesis and whether pathological JNK action could be discernible by its deregulation. We found that SCG10 maintains GA integrity as with the absence of SCG10 neurons present more compact fragmented GA structure, as shown by the knockdown approach. Interestingly, neurons isolated from Jnk1-/- mice show similar characteristics. Block of ER to GA is believed to be involved in development of Parkinson's disease. Hence, by using a pharmacological approach (Brefeldin A treatment), we showed that GA recovery is delayed upon removal of the drug in Jnk1-/- neurons to an extent similar to the shRNA SCG10-treated cells. Finally, we investigated the role of the JNK1-SCG10 duo in the maintenance of GA biogenesis following excitotoxic insult. Although the GA underwent fragmentation in response to NMDA treatment, we observed a substantial delay in GA disintegration in neurons lacking either JNK1 or SCG10.

Transient outward potassium current and Ca2+ homeostasis in the heart: beyond the action potential

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

Expression Profile and Distribution of Efhc1 Gene Transcript During Rodent Brain Development

One of the putative causative genes for juvenile myoclonic epilepsy (JME) is EFHC1. We report here the expression profile and distribution of Efhc1 messenger RNA (mRNA) during mouse and rat brain development. Real-time polymerase chain reaction revealed that there is no difference in the expression of Efhc1 mRNA between right and left hemispheres in both species. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-day-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME.

Evidence of the Importance of the First Intracellular Loop of Prokineticin Receptor 2 in Receptor Function

Prokineticin receptors (PROKR) are G protein-coupled receptors (GPCR) that regulate diverse biological processes, including olfactory bulb neurogenesis and GnRH neuronal migration. Mutations in PROKR2 have been described in patients with varying degrees of GnRH deficiency and are located in diverse functional domains of the receptor. Our goal was to determine whether variants in the first intracellular loop (ICL1) of PROKR2 (R80C, R85C, and R85H) identified in patients with hypogonadotropic hypogonadism interfere with receptor function and to elucidate the mechanisms of these effects. Because of structural homology among GPCR, clarification of the role of ICL1 in PROKR2 activity may contribute to a better understanding of this domain across other GPCR. The effects of the ICL1 PROKR2 mutations on activation of signal transduction pathways, ligand binding, and receptor expression were evaluated. Our results indicated that the R85C and R85H PROKR2 mutations interfere only modestly with receptor function, whereas the R80C PROKR2 mutation leads to a marked reduction in receptor activity. Cotransfection of wild-type (WT) and R80C PROKR2 showed that the R80C mutant could exert a dominant negative effect on WT PROKR2 in vitro by interfering with WT receptor expression. In summary, we have shown the importance of Arg80 in ICL1 for PROKR2 expression and demonstrate that R80C PROKR2 exerts a dominant negative effect on WT PROKR2. (Molecular Endocrinology 26: 1417-1427, 2012)