Marcos R. Costa

Functional properties of neurons derived from in vitro reprogrammed postnatal astroglia

With the exception of astroglia-like cells in the neurogenic niches of the telencephalic subependymal or hippocampal subgranular zone, astroglia in all other regions of the adult mouse brain do not normally generate neurons. Previous studies have shown, however, that early postnatal cortical astroglia in culture can be reprogrammed to adopt a neuronal fate after forced expression of Pax6, a transcription factor (TF) required for proper neuronal specification during embryonic corticogenesis. Here we show that also the proneural genes neurogenin-2 and Mash1 (mammalian achaete schute homolog 1) possess the ability to reprogram astroglial cells from early postnatal cerebral cortex. By means of time-lapse imaging of green fluorescent astroglia, we provide direct evidence that it is indeed cells with astroglial characteristics that give rise to neurons. Using patch-clamp recordings in culture, we show that astroglia-derived neurons acquire active conductances and are capable of firing action potentials, thus displaying hallmarks of true neurons. However, independent of the TF used for reprogramming, astroglia-derived neurons appear to mature more slowly compared with embryonic-born neurons and fail to generate a functional presynaptic output within the culturing period. However, when cocultured with embryonic cortical neurons, astroglia-derived neurons receive synaptic input, demonstrating that they are competent of establishing a functional postsynaptic compartment. Our data demonstrate that single TFs are capable of inducing a remarkable functional reprogramming of astroglia toward a truly neuronal identity.

Par-complex proteins promote proliferative progenitor divisions in the developing mouse cerebral cortex

The size of brain regions depends on the balance between proliferation and differentiation. During development of the mouse cerebral cortex, ventricular zone (VZ) progenitors, neuroepithelial and radial glial cells, enlarge the progenitor pool by proliferative divisions, while basal progenitors located in the subventricular zone (SVZ) mostly divide in a differentiative mode generating two neurons. These differences correlate to the existence of an apico-basal polarity in VZ, but not SVZ, progenitors. Only VZ progenitors possess an apical membrane domain at which proteins of the Par complex are strongly enriched. We describe a prominent decrease in the amount of Par-complex proteins at the apical surface during cortical development and examine the role of these proteins by gain- and loss-of-function experiments. Par3 (Pard3) loss-of-function led to premature cell cycle exit, reflected in reduced clone size in vitro and the restriction of the progeny to the lower cortical layers in vivo. By contrast, Par3 or Par6 (Pard6α) overexpression promoted the generation of Pax6+ self-renewing progenitors in vitro and in vivo and increased the clonal progeny of single progenitors in vitro. Time-lapse video microscopy revealed that a change in the mode of cell division, rather than an alteration of the cell cycle length, causes the Par-complex-mediated increase in progenitors. Taken together, our data demonstrate a key role for the apically located Par-complex proteins in promoting self-renewing progenitor cell divisions at the expense of neurogenic differentiation in the developing cerebral cortex.

Continuous live imaging of adult neural stem cell division and lineage progression in vitro.

Little is known about the intrinsic specification of adult neural stem cells (NSCs) and to what extent they depend on their local niche. To observe adult NSC division and lineage progression independent of their niche, we isolated cells from the adult mouse subependymal zone (SEZ) and cultured them at low density without growth factors. We demonstrate here that SEZ cells in this culture system are primarily neurogenic and that adult NSCs progress through stereotypic lineage trees consisting of asymmetric stem cell divisions, symmetric transit-amplifying divisions and final symmetric neurogenic divisions. Stem cells, identified by their astro/radial glial identity and their slow-dividing nature, were observed to generate asymmetrically and fast-dividing cells that maintained an astro/radial glia identity. These, in turn, gave rise to symmetrically and fast-dividing cells that lost glial hallmarks, but had not yet acquired neuronal features. The number of amplifying divisions was limited to a maximum of five in this system. Moreover, we found that cell growth correlated with the number of subsequent divisions of SEZ cells, with slow-dividing astro/radial glia exhibiting the most substantial growth prior to division. The fact that in the absence both of exogenously supplied growth factors and of signals provided by the local niche neurogenic lineage progression takes place in such stereotypic fashion, suggests that lineage progression is, to a significant degree, cell intrinsic or pre-programmed at the beginning of the lineage.

Late Origin of Glia-Restricted Progenitors in the Developing Mouse Cerebral Cortex

In order to unravel the molecular determinants of cell fate, it is important to understand when fate restriction occurs during brain development. Lineage analysis suggested that bi- or multipotent progenitors persist into late developmental stages in some central nervous system regions, whereas most progenitor cells in the cerebral cortex appeared to be restrained to generate only a single cell type already at early stages. Here we discuss this previous work and present new data demonstrating that cortical progenitors generating exclusively glial cells appear late in development. In utero transduction of cortical progenitors at early and mid-neurogenesis using a combination of replication-defective retroviral vectors encoding different fluorescent proteins indicated that the early developing cortex is devoid of glia-restricted progenitors, although these are frequent during mid- and late neurogenesis. Clonal analyses in vitro using retroviral vectors and live cell tracking by video time-lapse microscopy confirmed these findings, revealing that the early developing cortex harbors 2 main progenitor types: neuron-restricted and bipotent (neuron-glial) progenitors. The latter are responsible for the generation of glial-restricted progenitors at mid- and late neurogenesis.

Estresse: diagnóstico dos policiais militares em uma cidade brasileira

OBJETIVOS: Diagnosticar a ocorrencia e a fase de estresse em policiais militares da Cidade de Natal, Brasil, alem de determinar a prevalencia de sintomatologia fisica e mental. METODO: Estudo descritivo, com corte transversal. Foi investigada uma amostra de 264 individuos extraida de uma populacao de 3 193 militares do Comando de Policiamento da Capital. Os dados foram coletados entre junho de 2004 e janeiro de 2005 utilizando-se o Inventario de Sintomas de Stress para Adultos de Lipp. Foi determinada a presenca de estresse, a fase de estresse (alerta, resistencia, quase-exaustao, exaustao), a prevalencia de sintomas fisicos e mentais e a relacao entre estresse e unidade policial, posto policial, sexo, habito de beber, fumo, escolaridade, estado civil, idade, tempo de servico e faixa salarial. RESULTADOS: A proporcao de policiais sem sintomas de estresse foi de 52,6%, enquanto que 47,4% apresentaram sintomatologia. Dos 47,4% com estresse, 3,4% encontravam-se na fase de alerta, 39,8% na fase de resistencia, 3,8% na fase de quase-exaustao e 0,4% na fase de exaustao. Sintomas psicologicos foram registrados em 76,0% dos policiais com estresse, e sintomas fisicos, em 24,0%. Das variaveis investigadas, a unica que apresentou relacao com estresse foi o sexo (P = 0,0337), sendo as mulheres as mais afetadas. CONCLUSAO: Os niveis de estresse e de sintomas nao indicaram um quadro de fadiga critico. E recomendavel uma acao preventiva por parte da organizacao policial, que poderia incluir a aplicacao de um programa de diagnostico, orientacao e controle do estresse.

The Marginal Zone/Layer I as a Novel Niche for Neurogenesis and Gliogenesis in Developing Cerebral Cortex

The cellular diversity of the cerebral cortex is thought to arise from progenitors located in the ventricular zone and subventricular zone in the telencephalon. Here we describe a novel source of progenitors located outside these two major germinative zones of the mouse cerebral cortex that contributes to neurogenesis and gliogenesis. Proliferating cells first appear in the preplate of the embryonic cerebral cortex and further increase in the marginal zone during mid and late neurogenesis. The embryonic marginal zone progenitors differ in their molecular characteristics as well as the size and identity of their clonal progeny from progenitors isolated from the ventricular zone and subventricular zone. Time-lapse video microscopy and clonal analysis in vitro revealed that the marginal zone progenitor pool contains a large fraction of oligodendrocyte or astrocyte progenitors, as well as neuronal and bipotent progenitors. Thus, marginal zone progenitors are heterogenous in regard to their fate specification, as well as in regard to their region of origin (pallial and subpallial) as revealed by in vivo fate mapping. The local environment in the marginal zone tightly regulates the size of this novel progenitor pool, because both basement membrane defects in lamininγ1−/− mice or alterations in the cellular composition of the marginal zone in Pax6 Small Eye mutant mice lead to an increase in the marginal zone progenitor pool. In conclusion, we have identified a novel source of neuronal and glial progenitors in the marginal zone of the developing cerebral cortex with properties notably distinct from those of ventricular zone and subventricular zone progenitors.

Astrocyte heterogeneity in the brain: from development to disease

In the last decades, astrocytes have risen from passive supporters of neuronal activity to central players in brain function and cognition. Likewise, the heterogeneity of astrocytes starts to become recognized in contrast to the homogeneous population previously predicted. In this review, we focused on astrocyte heterogeneity in terms of their morphological, protein expression and functional aspects, and debate in a historical perspective the diversity encountered in glial progenitors and how they may reflect mature astrocyte heterogeneity. We discussed data that show that different progenitors may have unsuspected roles in developmental processes. We have approached the functions of astrocyte subpopulations on the onset of psychiatric and neurological diseases.

Expression and function of ganglioside 9-O-acetyl GD3 in postmitotic granule cell development.

We have shown previously that the Jones monoclonal antibody (Jones mAb) recognizes 9-O-acetyl GD3 expressed during periods of neuronal migration and neurite outgrowth in the developing rat nervous system. In the present study we investigated the expression of this ganglioside in the developing cerebellum and correlated this expression with granule cell migration. Electron microscopic immunocytochemistry revealed that around the peak of cerebellar neuronal migration (7-day-old rat), 9-O-acetyl GD3 was localized at the contact sites between migrating granule cells and radial glia in the external granular layer and prospective molecular layer. In addition, using microexplant and slice cultures of the postnatal rat cerebellum, we tested whether the ganglioside detected by our antibody contribute to the regulation of neuronal migration in the cerebellar cortex. We have shown that the Jones mAb blocks the migration of neurons in a dose-dependent manner. These findings suggest strongly that 9-O-acetyl GD3 is involved in granule cell migration in the developing cerebellum.

Gene Deletion Mutants Reveal a Role for Semaphorin Receptors of the Plexin-B Family in Mechanisms Underlying Corticogenesis

ABSTRACT Semaphorins and their receptors, plexins, are emerging as key regulators of various aspects of neural and nonneural development. Semaphorin 4D (Sema4D) and B-type plexins demonstrate distinct expression patterns over critical time windows during the development of the murine neocortex. Here, analysis of mice genetically lacking plexin-B1 or plexin-B2 revealed the significance of Sema4D-plexin-B signaling in cortical development. Deficiency of plexin-B2 resulted in abnormal cortical layering and defective migration and differentiation of several subtypes of cortical neurons, including Cajal-Retzius cells, GABAergic interneurons, and principal cells in vivo. In contrast, a lack of plexin-B1 did not impact on cortical development in vivo. In various ex vivo assays on embryonic forebrain, Sema4D enhanced the radial and tangential migration of developing neurons in a plexin-B2-dependent manner. These results suggest that Sema4D-plexin-B2 interactions regulate mechanisms underlying cell specification, differentiation, and migration during corticogenesis.

Proliferative Defects and Formation of a Double Cortex in Mice Lacking Mltt4 and Cdh2 in the Dorsal Telencephalon

Radial glial cells (RGCs) in the ventricular neuroepithelium of the dorsal telencephalon are the progenitor cells for neocortical projection neurons and astrocytes. Here we show that the adherens junction proteins afadin and CDH2 are critical for the control of cell proliferation in the dorsal telencephalon and for the formation of its normal laminar structure. Inactivation of afadin or CDH2 in the dorsal telencephalon leads to a phenotype resembling subcortical band heterotopia, also known as “double cortex,” a brain malformation in which heterotopic gray matter is interposed between zones of white matter. Adherens junctions between RGCs are disrupted in the mutants, progenitor cells are widely dispersed throughout the developing neocortex, and their proliferation is dramatically increased. Major subtypes of neocortical projection neurons are generated, but their integration into cell layers is disrupted. Our findings suggest that defects in adherens junctions components in mice massively affects progenitor cell proliferation and leads to a double cortex-like phenotype.