Extracellular α-synuclein alters synaptic transmission in brain neurons by perforating the neuronal plasma membrane.

It has been postulated that the accumulation of extracellular α-synuclein (α-syn) might alter the neuronal membrane by formation of 'pore-like structures' that will lead to alterations in ionic homeostasis. However, this has never been demonstrated to occur in brain neuronal plasma membranes. In this study, we show that α-syn oligomers rapidly associate with hippocampal membranes in a punctate fashion, resulting in increased membrane conductance (5 fold over control) and the influx of both calcium and a fluorescent glucose analogue. The enhancement in intracellular calcium (1.7 fold over control) caused a large increase in the frequency of synaptic transmission (2.5 fold over control), calcium transients (3 fold over control), and synaptic vesicle release. Both primary hippocampal and dissociated nigral neurons showed rapid increases in membrane conductance by α-syn oligomers. In addition, we show here that α-syn caused synaptotoxic failure associated with a decrease in SV2, a membrane protein of synaptic vesicles associated with neurotransmitter release. In conclusion, extracellular α-syn oligomers facilitate the perforation of the neuronal plasma membrane, thus explaining, in part, the synaptotoxicity observed in neurodegenerative diseases characterized by its extracellular accumulation. We propose that α-synuclein (α-syn) oligomers form pore-like structures in the plasma membrane of neurons from central nervous system (CNS). We believe that extracellular α-syn oligomers facilitate the formation of α-syn membrane pore-like structures, thus explaining, in part, the synaptotoxicity observed in neurodegenerative diseases characterized by its extracellular accumulation. We think that alterations in ionic homeostasis and synaptic vesicular depletion are key steps that lead to synaptotoxicity promoted by α -syn membrane pore-like structures.

Cell lines as in vitro models for drug screening and toxicity studies.

Cell culture is highly desirable, as it provides systems for ready, direct access and evaluation of tissues. The use of tissue culture is a valuable tool to study problems of clinical relevance, especially those related to diseases, screening, and studies of cell toxicity mechanisms. Ready access to the cells provides the possibility for easy studies of cellular mechanisms that may suggest new potential drug targets and, in the case of pathological-derived tissue, it has an interesting application in the evaluation of therapeutic agents that potentially may treat the dysfunction. However, special considerations must be addressed to establish stable in vitro function. In primary culture, these factors are primarily linked to greater demands of tissue to adequately survive and develop differentiated conditions in vitro. Additional requirements include the use of special substrates (collagen, laminin, extracellular matrix preparations, etc.), growth factors and soluble media supplements, some of which can be quite complex in their composition. These demands, along with difficulties in obtaining adequate tissue amounts, have prompted interest in developing immortalized cell lines which can provide unlimited tissue amounts. However, cell lines tend to exhibit problems in stability and/or viability, though they serve as a feasible alternative, especially regarding new potential applications in cell transplant therapy. In this regard, stem cells may also be a source for the generation of various cell types in vitro. This review will address aspects of cell culture system application, with focus on immortalized cell lines, in studying cell function and dysfunction with the primary aim being to identify cell targets for drug screening.