a b s t r a c tIncreasing amounts of tau protein were expressed in non-neuronal cells. When intracellular amounts reached a threshold level, tau protein was released to the extracellular culture medium in association with membrane vesicles. Hence, we propose that tau might be secreted through membrane vesicles as a cellular mechanism to eliminate the excess of tau protein, thereby avoiding its toxicity.

The mechanisms that underlie axon formation are still poorly understood. GSK3 has been recently implicated in establishing the axon and in its elongation. We have used four different GSK3 inhibitors to determine the role of GSK3 activity in hippocampal neurons at different periods of time. Inhibition of GSK3 activity impairs axon formation. The ''critical period'' of this activity of GSK3 is at least the first 24 h since afterwards the inhibition of GSK3 does not compromise the process of elongation, although it exacerbates axon branching. Moreover, interference RNAs impeding the expression of the GSK3 alpha or beta isoforms in hippocampal neurons prevents an axon from forming.

Background: Tau protein, the main component of neurofibrillary tangles, could be found in the extracellular space upon neuronal death or, as it has recently been suggested, could be secreted from cells through membrane vesicles. Objective: The purpose of this communication is to confirm that upon neuronal death, tau protein can be found, indeed, in the extracellular space and to analyze if tau could be secreted outside the cell in an alternative way. Methods: We have tested not only the extracellular release of tau, but also the toxicity of this extracellular tau. To do these studies, we have used neuronal cell cultures and tau-overexpressing non-neuronal cells. Membrane vesicles were isolated from culture medium from tau-overexpressing non-neuronal cells. Results: Our results indicate that extracellular tau, arising after neuron death, could be a toxic agent for neighboring neurons. On the other hand, we have found that an overexpression of tau protein could result in its secretion through membrane vesicles. However, the presence of this secreted tau does not result in cell death. Conclusion: We conclude that extracellular tau could arise by two different ways, by cell death or by secretion through membrane vesicles.

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Ensheathing glia have been demonstrated to have neuroregenerative properties but this cell type from human sources has not been extensively studied because tissue samples are not easily obtained, primary cultures are slow growing, and human cell lines are not available. We previously isolated immortalized ensheathing glia by gene transfer of BMI1 and telomerase catalytic subunit into primary cultures derived from olfactory bulbs of an elderly human cadaver donor. These cells escape the replicative senescence characteristic of primary human cells while conserving antigenic and neuroregenerative properties of ensheathing glia, but their low proliferative rate in culture complicates their utility as cell models and their application for preclinical cell therapy experiments. In this study we describe the use of a conditional SV40 T antigen (TAg) transgene to generate human ensheathing glia cell lines, which are easy to maintain due to their robust growth in culture. Although these fast growing clones exhibited polyploid karyotypes frequently observed in cells immortalized by TAg, they did not acquire a transformed phenotype, all of them maintaining neuroregenerative capacity and antigenic markers typical of ensheathing glia. These markers were also retained even after elimination of the TAg transgene using Cre/LoxP technology, although the cells died shortly after, confirming that their survival depended on the presence of the immortalizing genes. We have also demonstrated here the feasibility of using these human cell lines in animal models by genetically marking the cells with GFP and implanting them into the injured spinal cord of immunosuppressed rats. Our conditionally immortalized human ensheathing glia cell lines will thus serve as useful tools for advancing cell therapy approaches and understanding neuroregenerative mechanisms of this unique cell type.

The pathology associated with tau protein, tauopathy, has been recently analyzed in different disorders, leading to the suggestion that intracellular and extracellular tau may itself be the principal agent in the transmission and spreading of tauopathies. Tau pathology is based on an increase in the amount of tau, an increase in phosphorylated tau, and/or an increase in aggregated tau. Indeed, phosphorylated tau protein is the main component of tau aggregates, such as the neurofibrillary tangles present in the brain of Alzheimer's disease patients. It has been suggested that intracellular tau could be toxic to neurons in its phosphorylated and/or aggregated form. However, extracellular tau could also damage neurons and since neuronal death is widespread in Alzheimer's disease, mainly among cholinergic neurons, these cells may represent a possible source of extracellular tau. However, other sources of extracellular tau have been proposed that are independent of cell death. In addition, several ways have been proposed for cells to interact with, transmit, and spread extracellular tau, and to transduce signals mediated by this tau. In this work, we will discuss the role of extracellular tau in the spreading of the tau pathology.