Culture and Phenotyping of Glial Cell Cultures, Gliospheres, and Neurospheres.

Cell cultures constitute an important tool for research as a way to reproduce pathological processes in a controlled system. However, the culture of brain-derived cells in monolayer presents significant challenges that obscure the fidelity of in vitro results. After a few number of passages, glial and neuronal cells begin to lose their morphological characteristics, and most importantly, their specific cellular markers and phenotype. In recent years, the discovery of neural progenitor cells, and the methodology to culture them in suspension maintaining their potentiality while still retaining the ability to differentiate into astrocytes, oligodendrocytes and neurons has been a significant contribution to the fields of neuroscience and neuropathology.In the brain, progenitor cells are located in the Germinal Matrix, the subventricular zone in what later would become the basal ganglia, and play an essential role in the homeostasis of the brain by providing the source to replace differentiated cells that have been lost or damaged by different pathological processes, such as senescence, injury, genetic conditions, or disease. The discovery of these neural stem cells in an organ traditionally thought to have limited or no regenerative capacity has opened the door to the development of novel treatments, which include cell replacement therapy. Here we describe the culture and differentiation of neural progenitor cells into neurospheres, and the phenotyping of the resulting cells using immunocytochemistry . The immunocytological methods outlined are not restricted to the analysis of neurosphere-derived cultures but are also applicable for cell typing of primary glial or cell line-derived samples.

Neurospheres and Glial Cell Cultures; from Plating to Cell Phenotyping.

Cell cultures constitute an important tool for research as a way to reproduce pathological processes in a controlled system. However, the culture of brain-derived cells in monolayer presents significant challenges that obscure the fidelity of in vitro results. This is because after a few number of passages, glial and neuronal cells begin to lose their morphological characteristics, and most importantly, their specific cellular markers and phenotype. In recent years, the discovery of neural progenitor cells, and the methodology to culture them in suspension maintaining their potentiality while still retaining the ability to differentiate into astrocytes, oligodendrocytes, and neurons has made significant contributions to the fields of neuroscience and neuropathology.In the brain, progenitor cells are located in the germinal matrix, in the subventricular zone and play an essential role in the homeostasis of the brain by providing the source to replace differentiated cells that have been lost or damaged by different pathological processes, such as injury, genetic conditions, or disease. The discovery of these Neural Stem Cells in an organ traditionally thought to have limited or no regenerative capacity has opened the door to the development of novel treatments, which include cell replacement therapy. Here we describe the culture and differentiation of neural progenitor cells from Neurospheres, and the phenotyping of the resulting cells using immunocytochemistry. The immunocytological methods outlined are not restricted to the analysis of neurosphere-derived cultures but are also applicable for cell typing of primary glial or cell line-derived samples.

JCPyV T-Antigen Activation of the Anti-Apoptotic Survivin Promoter-Its Role in the Development of Progressive Multifocal Leukoencephalopathy.

Progressive Multifocal Leukoencephalopathy (PML) is a fatal demyelinating disease of the CNS, resulting from the lytic infection of oligodendrocytes by the human neurotropic polyomavirus JC (JCPyV), typically associated with severe immunocompromised states and, in recent years, with the use of immunotherapies. Apoptosis is a homeostatic mechanism to dispose of senescent or damaged cells, including virally infected cells, triggered in the vast majority of viral infections of the brain. Previously, we showed upregulation of the normally dormant anti-apoptotic protein Survivin in cases of PML, which-in vitro-resulted in protection from apoptosis in JCPyV-infected primary cultures of astrocytes and oligodendrocytes. In the present study, we first demonstrate the absence of apoptotic DNA fragmentation and the lack of caspase activity in 16 cases of PML. We also identified the viral protein large T-Antigen as being responsible for the activation of the Survivin promoter. Chromatin Immunoprecipitation assay shows a direct binding between T-Antigen and the Survivin promoter DNA. Finally, we have identified the specific region of T-Antigen, spanning from amino acids 266 and 688, which binds to Survivin and translocates it to the nucleus, providing evidence of a mechanism that results in the efficient replication of JCPyV and a potential target for novel therapies.

HIV-1-Tat Protein Inhibits SC35-mediated Tau Exon 10 Inclusion through Up-regulation of DYRK1A Kinase.

The HIV-1 transactivator protein Tat is implicated in the neuronal damage that contributes to neurocognitive impairment affecting people living with HIV/AIDS. Aberrant splicing of TAU exon 10 results in tauopathies characterized by alterations in the proportion of TAU isoforms containing three (3R) or four (4R) microtubule-binding repeats. The splicing factor SC35/SRSF2 binds to nuclear RNA and facilitates the incorporation of exon 10 in the TAU molecule. Here, we utilized clinical samples, an animal model, and neuronal cell cultures and found that Tat promotes TAU 3R up-regulation through increased levels of phosphorylated SC35, which is retained in nuclear speckles. This mechanism involved Tat-mediated increased expression of DYRK1A and was prevented by DYRK1A silencing. In addition, we found that Tat associates with TAU RNA, further demonstrating that Tat interferes with host RNA metabolism in the absence of viral infection. Altogether, our data unravel a novel mechanism of Tat-mediated neuronal toxicity through dysregulation of the SC35-dependent alternative splicing of TAU exon 10. Furthermore, the increased immunostaining of DYRK1A in HIV+ brains without pathology points at dysregulation of DYRK1A as an early event in the neuronal complications of HIV infection.

Activation of c-Myc and Cyclin D1 by JCV T-Antigen and ß-catenin in colon cancer.

During the last decade, mounting evidence has implicated the human neurotropic virus JC virus in the pathology of colon cancer. However, the mechanisms of JC virus-mediated oncogenesis are still not fully determined. One candidate to mediate these effects is the viral early transcriptional product T-Antigen, which has the ability to inactivate cell cycle regulatory proteins such as p53. In medulloblastomas, T-Antigen has been shown to bind the Wnt signaling pathway protein ß-catenin; however, the effects of this interaction on downstream cell cycle regulatory proteins remain unknown. In light of these observations, we investigated the association of T-Antigen and nuclear ß-catenin in colon cancer cases and the effects of this complex in the activation of the transcription and cell cycle regulators c-Myc and Cyclin D1 in vitro. Gene amplification demonstrated the presence of viral sequences in 82.4% of cases and we detected expression of T-Antigen in 64.6% of cases by immunohistochemistry. Further, we found that T-Antigen and ß-catenin co-localized in the nuclei of tumor cells and we confirmed the physical binding between these two proteins in vitro. The nuclear presence of T-Antigen and ß-catenin resulted in the significant enhancement of TCF-dependent promoter activity and activation of the ß-catenin downstream targets, c-Myc and Cyclin D1. These observations provide further evidence for a role of JCV T-Antigen in the dysregulation of the Wnt signaling pathway and in the pathogenesis of colon cancer.