Navegando por Autor "Schroeder, Timm"

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Activity-Independent Effects of CREB on Neuronal Survival and Differentiation during Mouse Cerebral Cortex Development
(2016-12) Landeira, Bruna Soares; Santana, Themis Taynah da Silva; Araújo, Jéssica Alves de Medeiros; Tabet, Elie I.; Tannous, Bakhos A.; Schroeder, Timm; Costa, Marcos Romualdo
Neuronal survival and morphological maturation depends on the action of the transcription factor calcium responsive element binding protein (CREB), which regulates expression of several target genes in an activity-dependent manner. However, it remains largely unknown whether CREB-mediated transcription could play a role at early stages of neuronal differentiation, prior to the establishment of functional synaptic contacts. Here, we show that CREB is phosphorylated at very early stages of neuronal differentiation in vivo and in vitro, even in the absence of depolarizing agents. Using genetic tools, we also show that inhibition of CREB-signaling affects neuronal growth and survival in vitro without affecting cell proliferation and neurogenesis. Expression of A-CREB or M-CREB, 2 dominant-negative inhibitors of CREB, decreases cell survival and the complexity of neuronal arborization. Similar changes are observed in neurons treated with protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors, which also show decreased levels of pCREBSer133. Notably, expression of CREB-FY, a Tyr134Phe CREB mutant with a lower Km for phosphorylation, partly rescues the effects of PKA and CaMKII inhibition. Our data indicate that CREB-mediated signaling play important roles at early stages of cortical neuron differentiation, prior to the establishment of fully functional synaptic contacts.
Continuous live imaging of adult neural stem cell division and lineage progression in vitro
(2011) Costa, Marcos Romualdo; Ortega, Felipe; Brill, Monika S.; Beckervordersandforth, Ruth; Petrone, Ciro; Schroeder, Timm; Götz, Magdalena; Berninger, Benedikt
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.
Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations
(2017-12-16) Gómez-Villafuertes, Rosa; Paniagua-Herranz, Lucía; Gascon, Sergio; Agustín-Durán, David de; Ferreras, María de la O; Gil-Redondo, Juan Carlos; Queipo, María José; Menendez-Mendez, Aida; Pérez-Sen, Ráquel; G. Delicado, Esmerilda; Gualix, Javier; Costa, Marcos Romualdo; Schroeder, Timm; Miras-Portugal, María Teresa; Ortega, Felipe
Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.
Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System
(2017-08-09) Landeira, Bruna Soares; Araújo, Jéssica Alves de Medeiros; Schroeder, Timm; Müller, Ulrich; Costa, Marcos Romualdo
During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.
Specific phospholipids regulate the acquisition of neuronal and astroglial identities in post-mitotic cells
(2018-01-11) Montaner, Aneley; Santana, Themis Taynah da Silva; Schroeder, Timm; Einiker-Lamas, Marcelo; Girardini, Javier; Costa, Marcos Romualdo; Banchio, Claudia
Hitherto, the known mechanisms underpinning cell-fate specification act on neural progenitors, affecting their commitment to generate neuron or glial cells. Here, we show that particular phospholipids supplemented in the culture media modify the commitment of post-mitotic neural cells in vitro. Phosphatidylcholine (PtdCho)-enriched media enhances neuronal differentiation at the expense of astroglial and unspecified cells. Conversely, phosphatidylethanolamine (PtdEtn) enhances astroglial differentiation and accelerates astrocyte maturation. The ability of phospholipids to modify the fate of post-mitotic cells depends on its presence during a narrow time-window during cell differentiation and it is mediated by the selective activation of particular signaling pathways. While PtdCho-mediated effect on neuronal differentiation depends on cAMP-dependent kinase (PKA)/calcium responsive element binding protein (CREB), PtdEtn stimulates astrogliogenesis through the activation of the MEK/ERK signaling pathway. Collectively, our results provide an additional degree of plasticity in neural cell specification and further support the notion that cell differentiation is a reversible phenomenon. They also contribute to our understanding of neuronal and glial lineage specification in the central nervous system, opening up new avenues to retrieve neurogenic capacity in the brain.
Using an adherent cell culture of the mouse subependymal zone to study the behavior of adult neural stem cells on a single-cell level
(2011-11-03) Ortega, Felipe; Costa, Marcos Romualdo; Simon-Ebert, Tatiana; Schroeder, Timm; Götz, Magdalena
A comprehensive understanding of the cell biology of adult neural stem cells (aNSCNSCNSCs) requires direct observation of aNSCNSCNSC division and lineage progression in the absence of niche-dependent signals. Here we describe a culture preparation of the adult mouse subependymal zone (SESEZ), which allows for continuous single-cell tracking of aNSCNSCNSC behavior. The protocol involves the isolation (~3 h) and culture of cells from the adult SESEZ at low density in the absence of mitogenic growth factors in chemically defined medium and subsequent live imaging using time-lapse video microscopy (5–7 d); these steps are followed by postimaging immunocytochemistry to identify progeny (~7 h). This protocol enables the observation of the progression from slow-dividing aNSCNSCNSCs of radial/astroglial identity up to the neuroblast stage, involving asymmetric and symmetric cell divisions of distinct fast-dividing precursors. This culture provides an experimental system for studying instructive or permissive effects of signal molecules on aNSCNSCNSC modes of cell division and lineage progression.

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Navegando por Autor "Schroeder, Timm"