Effects of phospholipases isolated from Micrurus lemniscatus snake venom on cell proliferation and apoptosis of cultured astrocytes and their mechanisms

Abstract

Endogenous phospholipases A2 have a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity (beta-neurotoxins) are found in snake venoms from Elapidae and Viperidae families and these induce neuronal death. Neuronal cultures have been used as a model in order to characterize the mechanisms of action of these neurotoxins. However, the use of glial cells as a model is scarce. Glial cells, in particular astrocytes, are proliferative cells that participate in homeostasis and defense responses against pathological events in the central nervous system. Moreover, astrocytes are partners at the synapses, composing the tripartite synapse, where pre-synaptic neurons release neurotransmitters that change pos-synaptic neurons and astrocytes activities. Astrocytes in its turn release gliotransmitters that modulate the synaptic transmission by acting on pre- and post-synaptic neurons. The mechanisms of PLA2 neurotoxicity are still in debate and they could be attributed to the enzymatic activity or to the interaction with its own receptor and cellular internalization. The aim of this study is to characterize the effects of two PLA2 (Mlx-8 and Mlx-9) isolated from Micrurus lemniscatus venom on cell viability, cell proliferation, cell cycle phases, apoptosis, and the intracellular pathways involved, in cultured astrocytes. Cell proliferation, cell cycle phases and cell viability will be analyzed by flow citometry and MTT assay. TNF-alpha release and NFkB activation will be verified by immunoassays. Apoptosis will be evaluated by DNA fragmentation, annexin-V assay and mRNA and protein expression of Bcl2, Bax, caspases 3 and 8 and Hsp-70. mRNA and protein expression for the cell proliferation markers like p53, p21, p27, Ki67, A and D cyclins, and for PLA2 receptor and MAPK will be also analyzed. Mitochondrial viability and integrity will be evaluated by membrane potential measurement using rhodamine-123. The reactive oxigen species formation will be evaluated by fluorimetry. Toxin internalization will be verified by its labeling with the Alexa-488 or Alexa- 568 fluorofores and confocal microscopy. (AU)