Prenatal Exposure to Histone Deacetylase Inhibitors Affects Gene Expression of Autism-Related Molecules and Delays Neuronal Maturation

Valproic acid (VPA) is a multi-target drug and an inhibitor of histone deacetylase (HDAC). We have previously demonstrated that prenatal exposure to VPA at embryonic day 12.5 (E12.5), but not at E14.5, causes autism-like behavioral abnormalities in male mouse offspring. We have also found that prenatal VPA exposure causes transient histone hyperacetylation in the embryonic brain, followed by decreased neuronal cell numbers in the prefrontal and somatosensory cortices after birth. In the present study, we examined whether prenatal HDAC inhibition affects neuronal maturation in primary mouse cortical neurons. Pregnant mice were injected intraperitoneally with VPA (500 mg/kg) and the more selective HDAC inhibitor trichostatin A (TSA; 500 µg/kg) at E12.5 or E14.5, and primary neuronal cultures were prepared from the cerebral cortices of their embryos. Prenatal exposure to VPA at E12.5, but not at E14.5, decreased total number, total length, and complexity of neuronal dendrites at 14 days in vitro (DIV). The effects of VPA weakened at 21 DIV. Exposure to TSA at E12.5, but not at E14.5, also delayed maturation of cortical neurons. In addition, real-time quantitative PCR revealed that the prenatal exposure to TSA decreased neuroligin-1 (Nlgn1), Shank2, and Shank3 mRNA levels and increased contactin-associated protein-like 2 mRNA level. The delay in neuronal maturation was also observed in Nlgn1-knockdown cells, which were transfected with Nlgn1 siRNA. These findings suggest that prenatal HDAC inhibition causes changes in gene expression of autism-related molecules linked to a delay of neuronal maturation.     

The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells

Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Here, we show that Wnt signaling inhibits the self-renewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of beta-catenin in mouse cortical NPC cultures induced neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling led to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, the beta-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized beta-catenin did not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development.     

Gene expression profiling of primate neocortex: molecular neuroanatomy of cortical areas

One hundred years have passed since Brodmann's mapping of the mammalian neocortex. Solely on the basis of morphological observations, he envisaged the conservation and differentiation of cortical areal structures across various species. We now know that neurochemical, connectional and functional heterogeneity of the neocortex accompanies the morphological divergence observed in such cytoarchitectonic studies. Nevertheless, we are yet far from fully understanding the biological significance of this cortical heterogeneity. In this article, we review our past works on the gene expression profiling of the postnatal primate cortical areas, by quantitative real-time polymerase chain reaction (PCR), DNA array, differential display PCR and in situ hybridization analyses. These studies revealed both the overall homogeneity of gene expression across different cortical areas and the presence of a small number of genes that show markedly area-specific expression patterns. In situ hybridization showed that, among such genes, occ1 and retinol-binding protein (RBP) mRNAs are selectively expressed in the neuronal populations that seem to be involved in distinct neural processing such as sensory reception (for occ1 ) and associative function (for RBP). Such a molecular neuroanatomical approach has the promise to provide an important link between structure and function of the cerebral cortex.     

Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice.

Fibroblast growth factor-2 (FGF-2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF-2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF-2-deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin-positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin-positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF-2-deficient adult mice are hypotensive. They respond normally to angiotensin II-induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF-2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF-2-deficient adult mice uncovers more general roles in neural development and function.     

Comparative analysis of the neocortex during the ontogenesis of cetaceae and primates

Comparative ontogenetic investigation of cytoarchitectonics of the cerebral neocortex has been performed in Cetacea and Primates using paraffin frontal and sagittal cerebral sections stained after Nissl. Cerebral hemispheres of dolphins, whales, monkeys and human being have been studied at various periods of prenatal development and in mature individuals. The comparison has been made at similar stages of cytoarchitectonical differentiation of the cortical plate. At two first stages of the prenatal ontogenesis (formation of the cortical plate and its differentiation into layers) there is not any principle differences between the Cetacea and Primates. Peculiarities of the cerebral cortical plate differentiation in the Cetacea (absence of the internal granular layer IV) is determined at the stage of stratification. Similar agranular character of the cerebral cortex differentiation is maintained during the whole subsequent ontogenesis in the Cetacea (heterogenetic type of the neocortex after Brodman). Absence of the layer IV in the cerebral neocortex determines some other principles in the spatial organization of the cortical-subcortical and in the intracortical connections in the Cetacea brain. This is confirmed by modern data of morphological and electrophysiological investigations. Perhaps, a comparatively more simple initial architectonics of the Cetacea brain limited the level of their functional possibilities, the latter is comparable only with anthropoid apes.     

Timing of Expression of r and Its Encoding mRNAs in the Developing Cerebral Neocortex and Cerebellum of the Mouse

The expression of tau mRNA and of the corresponding encoded protein variants was studied during postnatal development in two brain regions differing in their timing of differentiation: the cerebral neocortex and the cerebellum. (a) The expression of tau mRNA was different in the two regions. Maximal contents were found at early stages in the cerebral neocortex, with a 10-fold decrease at later stages. In the cerebellum, two peaks of tau mRNA were observed soon after birth and in adulthood, with minimal values at postnatal day 6. (b) The expression of total tau proteins was similar to that of their encoding mRNAs in the cerebral neocortex, i.e., high concentrations after birth and low contents at later stages. In contrast, two peaks of tau proteins were observed in the cerebellum: the first perinatally and the second with a maximum at postnatal day 15. (c) Both in the cerebral neocortex and especially in the cerebellum, increasing concentrations of mature tau variants were expressed at late developmental stages, i.e., when total tau protein contents were decreased. In conclusion, the fluctuations in expression of tau and of its encoding mRNA seen in the cerebellum seem to reflect differences in the timing of differentiation of the various cell types, i.e., the macroneurons and the interneurons, present in this brain region. The adult tau variants appear in both the neocortex and the cerebellum only at late developmental stages, i.e., when most of the circuitry has been established, although these two regions markedly differ in their timing of differentiation.     

Morphological changes in ventricular germinal zone and neocortex of the cerebral hemispheres in human fetuses and newborns on weeks 22–40 of prenatal development

In this study, we investigated the morphology of the ventricular germinal zone and neocortex of the cerebral hemispheres in the projection field no. 4 of the motor area in human fetuses in dynamics from week 22 to 40 of fetal development. Morphological study allowed us to clarify the following patterns of prenatal ontogeny of the human CNS. On weeks 22–27, an intensive formation of the main sulci of the first order, differentiating the brain into lobes, is observed. By weeks 28–32, the formation of all sulci of the first order is completed; and on weeks 33–37, additional sulci characteristic of an individual are formed. The spurt of gyrification of the cortex (weeks 22–27) practically coincides with the completion of neuronal differentiation and formation of the motor neocortex. The structure of the latter is characterized by a clear stratification of cytoarchitectonic layers and modular organization of neurons with their vertical orientation in cell columns (weeks 25–27). In subsequent weeks of prenatal development until birth, no significant changes in the topography and structure of the neocortex are observed. Structural rearrangement of the ventricular germinal zone on weeks 22–40 of prenatal development consists in its gradual reduction and is completed on weeks 37–40. The criteria of physiological reduction of this area are the zonal location of glioblasts and a progressive decrease in its thickness on weeks 33–37 of prenatal development.     

Cell type-specific expression of the genes for the protein kinase C family: down regulation of mRNAs for PKC alpha and nPKC epsilon upon in vitro differentiation of a mouse neuroblastoma cell line neuro 2a

By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation.     

TrkB regulates neocortex formation through the Shc/PLCgamma-mediated control of neuronal migration

The generation of complex neuronal structures, such as the neocortex, requires accurate positioning of neurons and glia within the structure, followed by differentiation, formation of neuronal connections, and myelination. To understand the importance of TrkB signaling during these events, we have used conditional and knockin mutagenesis of the TrkB neurotrophin receptor, and we now show that this tyrosine kinase receptor, through docking sites for the Shc/FRS2 adaptors and phospholipase Cgamma (PLCgamma), coordinates these events in the cerebral cortex by (1) controlling cortical stratification through the timing of neuronal migration during cortex formation, and (2) regulating both neuronal and oligodendrocyte differentiation. These results provide genetic evidence that TrkB regulates important functions throughout the formation of the cerebral cortex via recruitment of the Shc/FRS2 adaptors and PLCgamma.     

Genetic mechanisms specifying cortical connectivity: let's make some projections together

Great neuroanatomists of the twentieth century recognized that the cerebral cortex of mammals is the single most complex structure of the central nervous system both in terms of neuronal diversity and connectivity. Understanding the cellular and molecular mechanisms specifying the afferent and efferent connectivity in the neocortex may seem like a daunting task. However, recent technical advances have greatly improved our ability to (1) profile gene expression of neuronal populations isolated based on their connectional properties, (2) manipulate gene expression in specific neuronal populations, and (3) visualize their axonal projections in vivo. These new tools are revolutionizing our ability to identify the molecular mechanisms patterning afferent and efferent cortical projections.