Biomedical subjects

Javier Diaz-Nido

Publications and source records attributed to Javier Diaz-Nido.

6 recordsLinked to original sources

PubMed2006

BDNF production by olfactory ensheathing cells contributes to axonal regeneration of cultured adult CNS neurons.

Olfactory ensheathing cells (OECs) are the main glial cell type that populates mammalian olfactory nerves. These cells have a great capacity to promote the regeneration of axons when transplanted into the injured adult mammalian CNS. However, little is still known about the molecular mechanisms they employ in mediating such a task. Brain-derived neurotrophic factor (BDNF) was identified as a candidate molecule in a genomic study that compared three functionally different OEC populations: Early passage OECs (OEC Ep), Late passage OECs (OEC Lp) and the OEC cell line TEG3 [Pastrana, E., Moreno-Flores, M.T., Gurzov, E.N., Avila, J., Wandosell, F., Diaz-Nido, J., 2006. Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2. J. Neurosci. 26, 5347-5359]. We have here set out to determine the role played by BDNF in the stimulation of axon outgrowth by OECs. We compared the extracellular BDNF levels in the three OEC populations and show that it is produced in significant amounts by the OECs that can stimulate axon regeneration in adult retinal neurons (OEC Ep and TEG3) but it is absent from the extracellular medium of OEC Lp cells which lack this capacity. Blocking BDNF signalling impaired axonal regeneration of adult retinal neurons co-cultured with TEG3 cells and adding BDNF increased the proportion of adult neurons that regenerate their axons on OEC Lp monolayers. Combining BDNF with other extracellular proteins such as Matrix Metalloproteinase 2 (MMP2) further augmented this effect. This study shows that BDNF production by OECs plays a direct role in the promotion of axon regeneration of adult CNS neurons.

Animals↗

PubMed2006

Gene transfer into Purkinje cells using herpesviral amplicon vectors in cerebellar cultures.

Purkinje cells play a crucial role in sensory motor coordination since they are the only output projection neurons in the cerebellar cortex and are affected in most spinocerebellar ataxias. They stand out in the central nervous system due to their large size and their profusely branched dendritic arbor. However, molecular and cellular studies on Purkinje cells are often hampered by the difficulty of maintaining these cells in culture. Here we report an easy, robust and reproducible method to obtain Purkinje-enriched mixed cerebellar cell cultures from day 16 mouse embryos using papain digestion and a semi-defined culture medium, being the composition of the culture approximately 20% Purkinje cells, 70% non-Purkinje neurons and 10% glial cells. We demonstrate that efficient gene transfer into Purkinje cells (as well as into other cerebellar populations) is possible using herpes simplex virus-1 (HSV-1)-derived vectors. Indeed, up to 50% of the Purkinje cells can be transduced and gene expression may persist for at least 14 days. As a result, this procedure permits functional gene expression studies to be carried out on cultured Purkinje neurons. To demonstrate this, we show that the expression of a dominant-negative form of glycogen synthase kinase-3 protects Purkinje neurons against cell death triggered by a chemical inhibitor of phosphatidylinositol-3 kinase. In summary, we have established reproducible and reliable cerebellar cell cultures enriched for Purkinje cells which enables gene transfer studies to be carried out using herpesviral vectors.

Animals↗

PubMed2006

Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2.

The molecular mechanisms used by olfactory ensheathing cells (OECs) to promote repair in the damaged adult mammalian CNS remain unknown. Thus, we used microarrays to analyze three OEC populations with different capacities to promote axonal regeneration in cultured rat retinal neurons. Gene expression in "long-term cultured OECs" that do not stimulate adult axonal outgrowth was compared with that of "primary olfactory ensheathing cells" and the immortalized OEC cell line TEG3. In this way, we identified a number of candidate genes that might play a role in promoting adult axonal regeneration. Among these genes, it was striking that both the matrix metalloproteinase 2 (MMP2) and an inhibitor of this protease were represented. The disruption of MMP2 activity in TEG3 cells impaired their capacity to trigger axon regeneration in cultured adult retinal neurons. Furthermore, the MMP2 protein was detected in grafts of OECs that elicited robust axonal regeneration in the injured spinal cord of adult rats in vivo. These data suggest that MMP2 does indeed participate in adult axonal regeneration induced by OECs.

Animals↗

PubMed2006

Differentiation of a human neuroblastoma into neuron-like cells increases their susceptibility to transduction by herpesviral vectors.

Gene transfer is a powerful tool for functional gene analysis in human cells. In this respect, there is a need to develop experimental models that involve homogeneous cultures of human neuron-like cells susceptible to gene transduction and that are easy to handle. Here we describe an optimized and reproducible procedure to differentiate human SH-SY5Y neuroblastoma cells into a homogeneous population of neuron-like cells. The fully differentiated cells are postmitotic and resemble primary cultured neurons in terms of their cytoskeletal polarity. Notably, differentiated SH-SY5Y cells are far more susceptible to transduction by herpes simplex virus (HSV-1)-based vectors than proliferating SH-SY5Y cells. This increase in transduction efficiency after neuronal differentiation may be due to the up-regulation of cell surface receptors for herpesvirus entry. In summary, we propose that fully differentiated human neuron-like cells obtained from the SH-SY5Y neuroblastoma may constitute an excellent and versatile experimental tool for gene transfer and functional genomic studies with HSV-1 vectors.

Blotting, Western↗

PubMed2004

Microtubule-associated protein 1B function during normal development, regeneration, and pathological conditions in the nervous system.

Microtubule-associated protein 1B is the first MAP to be expressed during the development of the nervous system. Several different approaches have revealed that MAP1B function is associated with microtubule and actin microfilament polymerization and dynamics. In recent years, the generation of molecular models to inactivate MAP1B function in invertebrates and mammals has sparked some controversy about the real role of MAP1B. Despite discrepancies between some studies, it is clear that MAP1B plays a principal role in the development of the nervous system. In this article, we summarize the evidence for MAP1B function in a wide variety of cellular processes implicated in the proper construction of the nervous system. We also discuss the role of MAP1B in pathological processes.

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PubMed2002

Highly efficient and specific gene transfer to Purkinje cells in vivo using a herpes simplex virus I amplicon.

The transduction of cerebellar neurons in vivo with herpes simplex virus 1 (HSV-1) amplicon carrying the lacZ gene has been investigated after injection of the vector in the cerebellar cortex, ventricles, and inferior olive of adult rats. Injection into the cerebellar cortex resulted in transduction of Purkinje cells near the needle tract and injection into the ventricles yielded no transduced neurons. In contrast, high transduction efficiency was achieved by vector injection into the inferior olive, resulting in one of three positive Purkinje cells all over the ipsilateral and contralateral cerebellar hemispheres. Because neurons in the deep cerebellar nuclei are also transduced, we suggest that the vector is delivered from the inferior olive to the cerebellar nuclei and then to Purkinje cells by retrograde axonal transport. Expression of the lacZ gene within Purkinje cells was surprisingly persistent and was maintained at the same level for at least 40 days. Importantly, no signs of either toxicity or inflammation were observed in the cerebellum after vector injection, except for the borders of the needle tract where some reactive astrocytes were detected. Indeed, motor coordination of treated animals was entirely normal, as assessed by the rota-rod test. These results demonstrate that HSV-1 amplicon vectors can effect safe and stable transgene expression in Purkinje cells in vivo, raising the possibility of using these vectors for long-term gene therapy of human cerebellar disorders.

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