Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons.
The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-9 toxins, an increase in free cytosolic Ca(2+) and a reduction in mitochondrial transmembrane potential (ΔΨm) became evident and occurred prior to the morphological changes and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed in the electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic Ca(2+) and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s.
Laboratory of Pharmacology, Butantan Institute, Av. Dr. Vital Brasil 1500, São Paulo, SP 05503 900, Brazil.
Nathalia Delazeri
ND
de Carvalho
Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP 05508 000, Brazil.
Raphael CaioTamborelli
RC
Garcia
Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, SP 05503 900, Brazil.
Adilson Kleber
AK
Ferreira
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.
Daniel Rodrigo
DR
Batista
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.
Antonio Carlos
AC
Cassola
Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, SP 05503 900, Brazil.
Durvanei
D
Maria
Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, SP 05503 900, Brazil.
Ivo
I
Lebrun
Laboratory of Cellular Biology, Butantan Institute, São Paulo, SP 05503 900, Brazil.
Sylvia Mendes
SM
Carneiro
Laboratory of Pharmacology, Butantan Institute, Av. Dr. Vital Brasil 1500, São Paulo, SP 05503 900, Brazil.
Solange Castro
SC
Afeche
Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP 05508 000, Brazil.
Tania
T
Marcourakis
Laboratory of Pharmacology, Butantan Institute, Av. Dr. Vital Brasil 1500, São Paulo, SP 05503 900, Brazil. Electronic address: mrlsando@usp.br.
Maria Regina Lopes
MR
Sandoval
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D004249:DNA Damage; D004546:Elapid Venoms / Q000201:enzymology*; D017815:Elapidae / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology* / Q000196:embryology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D009336:Necrosis; D009474:Neurons / Q000187:drug effects*; D009498:Neurotoxins / Q000302:isolation & purification / Q000633:toxicity*; D054467:Phospholipases A2 / Q000302:isolation & purification / Q000633:toxicity*; D051381:Rats; D017208:Rats, Wistar; D014617:Vacuoles / Q000187:drug effects / Q000648:ultrastructure
D004546:Elapid Venoms; D009498:Neurotoxins; C000592374:Mlx-8 phospholipase A2, Micrurus lemniscatus; C000592375:Mlx-9 phospholipase A2, Micrurus lemniscatus; D054467:Phospholipases A2
Calcium; Micrurus lemniscatus venom; Mitochondrial transmembrane potential; Neuronal cell death; Neurotoxic phospholipases A2; hippocampal cell culture