Altered structure of cortical sulci in gilles de la Tourette syndrome: Further support for abnormal brain development.

Gilles de la Tourette syndrome is a neurodevelopmental disorder characterized by the presence of motor and vocal tics. We hypothesized that patients with this syndrome would present an aberrant pattern of cortical formation, which could potentially reflect global alterations of brain development. Using 3 Tesla structural neuroimaging, we compared sulcal depth, opening, and length and thickness of sulcal gray matter in 52 adult patients and 52 matched controls. Cortical sulci were automatically reconstructed and identified over the whole brain, using BrainVisa software. We focused on frontal, parietal, and temporal cortical regions, in which abnormal structure and functional activity were identified in previous neuroimaging studies. Partial correlation analysis with age, sex, and treatment as covariables of noninterest was performed amongst relevant clinical and neuroimaging variables in patients. Patients with Gilles de la Tourette syndrome showed lower depth and reduced thickness of gray matter in the pre- and post-central as well as superior, inferior, and internal frontal sulci. In patients with associated obsessive-compulsive disorder, additional structural changes were found in temporal, insular, and olfactory sulci. Crucially, severity of tics and of obsessive-compulsive disorder measured by Yale Global Tic severity scale and Yale-Brown Obsessive-Compulsive scale, respectively, correlated with structural sulcal changes in sensorimotor, temporal, dorsolateral prefrontal, and middle cingulate cortical areas. Patients with Gilles de la Tourette syndrome displayed an abnormal structural pattern of cortical sulci, which correlated with severity of clinical symptoms. Our results provide further evidence of abnormal brain development in GTS. © 2015 International Parkinson and Movement Disorder Society.

The AMPA receptor subunit GluR-B in its Q/R site-unedited form is not essential for brain development and function

Calcium permeability of l-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in excitatory neurons of the mammalian brain is prevented by coassembly of the GluR-B subunit, which carries an arginine (R) residue at a critical site of the channel pore. The codon for this arginine is created by site-selective adenosine deamination of an exonic glutamine (Q) codon at the pre-mRNA level. Thus, central neurons can potentially control the calcium permeability of AMPARs by the level of GluR-B gene expression as well as by the extent of Q/R-site editing, which in postnatal brain, positions the R codon into >99% of GluR-B mRNA. To study whether the small amount of unedited GluR-B is of functional relevance, we have generated mice carrying GluR-B alleles with an exonic arginine codon. We report that these mutants manifest no obvious deficiencies, indicating that AMPAR-mediated calcium influx into central neurons can be solely regulated by the levels of Q/R site-edited GluR-B relative to other AMPAR subunits. Notably, a targeted GluR-B gene mutant with 30% reduced GluR-B levels had 2-fold higher AMPAR-mediated calcium permeability in hippocampal pyramidal cells with no sign of cytotoxicity. This constitutes proof in vivo that elevated calcium influx through AMPARs need not generate pathophysiological consequences.

In vitro-generated neural precursors participate in mammalian brain development

During embryogenesis, pluripotent stem cells segregate into daughter lineages of progressively restricted developmental potential. In vitro, this process has been mimicked by the controlled differentiation of embryonic stem cells into neural precursors. To explore the developmental potential of these cell-culture-derived precursors in vivo, we have implanted them into the ventricles of embryonic rats. The transplanted cells formed intraventricular neuroepithelial structures and migrated in large numbers into the brain tissue. Embryonic-stem-cell-derived neurons, astrocytes, and oligodendrocytes incorporated into telencephalic, diencephalic, and mesencephalic regions and assumed phenotypes indistinguishable from neighboring host cells. These observations indicate that entirely in vitro-generated neural precursors are able to respond to environmental signals guiding cell migration and differentiation and have the potential to reconstitute neuronal and glial lineages in the central nervous system.

Neural tube defects and abnormal brain development in F52-deficient mice.

F52 is a myristoylated, alanine-rich substrate for protein kinase C. We have generated F52-deficient mice by the gene targeting technique. These mutant mice manifest severe neural tube defects that are not associated with other complex malformations, a phenotype reminiscent of common human neural tube defects. The neural tube defects observed include both exencephaly and spina bifida, and the phenotype exhibits partial penetrance with about 60% of homozygous embryos developing neural tube defects. Exencephaly is the prominent type of defect and leads to high prenatal lethality. Neural tube defects are observed in a smaller percentage of heterozygous embryos (about 10%). Abnormal brain development and tail formation occur in homozygous mutants and are likely to be secondary to the neural tube defects. Disruption of F52 in mice therefore identifies a gene whose mutation results in isolated neural tube defects and may provide an animal model for common human neural tube defects.

Distribution of parthenogenetic cells in the mouse brain and their influence on brain development and behavior.

A systematic analysis of parthenogenetic (PG) cell fate within the central nervous system (CNS) was made throughout fetal development and neonatal and adult life. Chimeras were made between PG embryos carrying a ubiquitously expressed lacZ transgene and normal fertilized embryos. After detailed histological analysis, we find that the developmental potential of PG cells is spatially restricted to certain parts of the brain. PG cells are prevalent in telencephalic structures and are largely excluded from diencephalic structures, especially the hypothalamus. These spatial restrictions are established early in development. Behavioral studies with chimeras identified an increase in male aggression when the proportion of PG cells in the brain was high. These studies demonstrate that imprinted genes play key roles in development of the CNS and may be involved in behavior.

Vitamin D3 and brain development

Evidence for the presence of the vitamin D receptor in brain implies this vitamin may have some function in this organ. This study investigates whether vitamin D-3 acts during brain development. We demonstrate that rats born to vitamin D-3-deficient mothers had profound alterations in the brain at birth. The cortex was longer but not wider, the lateral ventricles were enlarged, the cortex was proportionally thinner and there was more cell proliferation throughout the brain. There were reductions in brain content of nerve growth factor and glial cell line-derived neurotrophic factor and reduced expression of p75(NTR), the low-affinity neurotrophin receptor. Our findings would suggest that low maternal vitamin D3 has important ramifications for the developing brain. (C) 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Bridging the link between trauma, brain development and depression: epigenetic mechanisms

Despite its large impact on the individual and society, we currently have only a rudimentary understanding of the biological basis of Major Depressive Disorder, even less so in adolescent populations. This thesis focuses on two research questions. First, how do adolescents with depression differ from adolescents who have never been depressed on (1a) brain morphology and (1b) DNA methylation? We studied differences in the fronto-limbic system (a collection of areas responsible for emotion regulation) and methylation at the serotonin transporter (SLC6A4) and FK506 binding protein gene (FKBP5) genes (two genes strongly linked to stress regulation and depression). Second, how does childhood trauma, which is known to increase risk for depression, affect (2a) brain development and (2b) SLC6A4 and FKBP5 methylation? Further, (2c) how might DNA methylation explain how trauma affects brain development in depression? We studied these questions in 24 adolescent depressed patients and 21 controls. We found that (1a) depressed adolescents had decreased left precuneus volume and greater volume of the left precentral gyrus compared to controls; however, no differences in fronto-limbic morphology were identified. Moreover, (1b) individuals with depression had lower levels of FKBP5 methylation than controls. In line with our second hypothesis (2a) greater levels of trauma were associated with decreased volume of a number of fronto-limbic regions. Further, we found that (2b) greater trauma was associated with decreased SLC6A4, but not FKBP5, methylation. Finally, (2c) greater FKBP5, but not SLC6A4, methylation was associated with decreased volume of a number of fronto-limbic regions. The results of this study suggest an association among trauma, DNA methylation and brain development in youth, but the direction of these relationships appears to be inconsistent. Future studies using a longitudinal design will be necessary to clarify these results and help us understand how the brain and epigenome change over time in depressed youth.

Imaging of activated complement using ultrasmall superparamagnetic iron oxide particles (USPIO) - conjugated vectors: an in vivo in utero non-invasive method to predict placental insufficiency and abnormal fetal brain development.

In the current study, we have developed a magnetic resonance imaging-based method for non-invasive detection of complement activation in placenta and foetal brain in vivo in utero. Using this method, we found that anti-complement C3-targeted ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles bind within the inflamed placenta and foetal brain cortical tissue, causing a shortening of the T2* relaxation time. We used two mouse models of pregnancy complications: a mouse model of obstetrics antiphospholipid syndrome (APS) and a mouse model of preterm birth (PTB). We found that detection of C3 deposition in the placenta in the APS model was associated with placental insufficiency characterised by increased oxidative stress, decreased vascular endothelial growth factor and placental growth factor levels and intrauterine growth restriction. We also found that foetal brain C3 deposition was associated with cortical axonal cytoarchitecture disruption and increased neurodegeneration in the mouse model of APS and in the PTB model. In the APS model, foetuses that showed increased C3 in their brains additionally expressed anxiety-related behaviour after birth. Importantly, USPIO did not affect pregnancy outcomes and liver function in the mother and the offspring, suggesting that this method may be useful for detecting complement activation in vivo in utero and predicting placental insufficiency and abnormal foetal neurodevelopment that leads to neuropsychiatric disorders.

Brain spectrin isoforms during development of chick dorsal root ganglion cells in vivo and in vitro

Brain spectrin is one of the major cytoskeletal proteins associated with the plasma membrane. In many tissues this protein occurs in a variety of isoforms, for which at least three have been described in the brain: i) brain spectrin 240/235 is localized in neurons most prominently in axons and is present early during brain development. ii) Brain spectrin 240/235E is immunologicaly related to erythrocyte spectrin and restricted to somato-dendritic regions in neurons and to glia. It appears late in brain development. iii) A third form, brain spectrin 240/ 235A, is found exclusively in astrocytes. In this study we have investigated the appearance and distribution of brain spectrins 240/235 and 240/235E during embryonic chick dorsal root ganglia development in vivo and in vitro. This system provides a unique model due to the lack of dendrites on developing sensory neurons. Both isoforms first appeared at embryonic day 6. Brain spectrin 240/235 increased transiently around embryonic day 10 and 14, and was first expressed in ventrolateral neurons. It was localized abundantly in perikarya and their axons. This somato-axonal distribution pattern found in situ was also observed in vitro. In contrast, brain spectrin 240/235E only slightly increased between E6 and E15 and remained unchanged thereafter. It was localized mainly in small neurons of the mediodorsal area, where it was found as punctate staining in the cytoplasm, forming first a nuclear cap and in subsequent stages becoming distributed evenly throughout cytoplasm. This brain spectrin isoform was absent from axons, both in situ and in vitro. In conclusion, this study suggests i) that brain spectrin 240/235 may contribute towards the outgrowth, elongation and possibly maintenance of axonal processes, ii) that brain spcctrin 240/235E could be involved in the stablization of the cytoarchilecture of cell bodies in a sclected population of ganglion cells, and iii) that isoform expression of brain spectrin 240/235E in DRG cells may depend on environmental factors.

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.

Widespread pH abnormalities in patients with malformations of cortical development and epilepsy: a phosphorus-31 brain MR spectroscopy study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ethanol inhibition of brain ornithine decarboxylase activity in the postnatal rat

The purpose of this study was to determine the relationship between ornithine decarboxylase activity (ODC; a marker for perturbed cell development), the blood alcohol level, and alcohol-induced microencephaly in the developing rat brain after binge treatment with ethanol vapour. By manipulating ethanol flow we were able to adjust vapour concentrations (24-65 mg ethanol/l air) such that an acute exposure of ethanol vapour for 3 h resulted in a range of blood alcohol levels (2.3-5.5 mg/ml). Acute studies showed that ethanol dose-dependently inhibited rat hippocampal and cerebellar ODC activity at PND4-PND10. There was a significant correlation between the blood alcohol level and degree of inhibition at all ages tested. Chronic treatment from PND4 to PND9 caused a significant decrease in both brain to body weight ratio and in hippocampal and cerebellar ODC activities at PND10. These results indicate that ethanol-induced disruption in ODC could play a significant role in ethanol's teratogenic effects during early postnatal development. (C) 1998 Elsevier Science Inc.