Toxic Effects And The Underlving Mechanisms Of Microcystin-lr On Neuroendocrine(PC12)Cells

Contamination of freshwater and marine environments by cyanobacterial blooms has been a worldwide ecological issue, triggering both water eutrophication and health threats to livestock and humans. Among the various cyanobacteria (blue-green algae), Microcystis aeruginosa is the most common genera producing microcystins (MCs), a family of over80structurally monocyclic heptapeptide toxins, with microcystin-LR (MC-LR) considered to be the most toxic and most commonly encountered variant. MC-LR is thought to act as a hepatotoxin, because it preferentially accumulates in the vertebrate liver, and its presence in contaminated drinking water has been frequently reported to cause acute liver hemorrhage, liver necrosis and primary liver cancer. Recent evidence has demonstrated that MC-LR is also brain-permeable as MC-LR is present in the brain, raising the possibility that MC-LR could exert neurotoxicity. Although the potential neurotoxicity of MC-LR has been proposed, little is known about its molecular basis.The neuroendocrine PC12cell line has been widely utilized as a model system for studying various aspects of neuronal function and neurotoxins. The current study was designed to elucidate the neurological toxicity of the cyanobacterial toxin MC-LR on the PC12cell line and to determine its underlying mechanisms. Studies were undertaken with the objective of evaluating (a) the cellular effects including cell viability, apoptotic rate, cytoskeleton morphology including MT and F-actin architectures, and the level of ROS,(b) protein phosphatase2A activity, the protein profile of PP2A composition subunits including A subunit, C subunit, as well as the regulatory B family subunits, Bpand By, and methylation and phosphorylation modification of PP2A-C,(c) the phosphorylation status of cytoskeletal-associated proteins including tau and HSP27, and the roles of PP2A and p38MAPK signaling in MC-LR-induced cytoskeletal structure remodeling. Our work may further our knowledge of MC-LR-induced neurotoxicity and provide some new theoretical bases and ways for the inhibition of MC-LR-induced neurotoxicity.Results:1.In the present study, MC-LR had no effect on the cell viability of PC12cells.2. In the present study, MC-LR had no effect on neither the apoptotic rate of PC12cells, nor the activation of caspase-33. In the present study, MC-LR had no effect on the cell morphology of PC12cells.4. MC-LR triggered microtubule (MT) and actin cytoskeleton rearrangement, as well as MT destabilization. Additionally, MC-LR induced disruption of PP2A Bcc-MT interaction.5. MC-LR induced the elevation of the ROS level.6. MC-LR directly targeted the PP2A catalytic subunit which was followed by a strongly dose-dependent inhibition of PP2A activity, as well as altered composition subunits levels and post-translational modifications of PP2A.7. MC-LR induced hyperphosphorylation of the neural microtubule-associated protein tau, which correlated with a decrease in cytoskeleton-associated tau. Besides, the phosphorylation of the actin-associated protein HSP27was also increased in MC-LR-treated cells.8. Direct inhibition of protein phosphatase2A (PP2A) activity and indirect activation of the p38mitogen-activated protein kinase (MAPK) could be the mechanisms underlying MC-LR induced hyperphosphorylation of tau and HSP27.9. Pretreatment with the p38MAPK inhibitor SB203580effectively abolished hyperphosphorylation of tau and HSP27, and blocked MC-LR-triggered cytoskeletal alterations.Conclusions:1. PP2A inhibition induced by MC-LR may be the result of the combined effects of alterations of various composition subunits levels that suggesting changes in holoenzyme assembly and PP2A-C post-translational modifications,which provides a new insight into the effect pattern of MC-LR on PP2A.2. Our data showed that the level of ROS as well as the cytoskeleton structure responded relatively sensitively to MC-LR in the neuroendocrine PC12cell line.3. MC-LR induced hyperphosphorylation of the neural microtubule-associated protein tau and the actin-associated protein HSP27, which may be a likely mechanistic link to MC-LR-induced cytoskeletal alterations.4. MC-LR-caused hyperphosphorylation of HSP27and tau may be mediated directly by reduced dephosphorylation from PP2A activity impairment and indirectly by elevated phosphorylation via subsequent p38MAPK activation.5. The protective effect of SB203580on MC-LR-induced cytoskeleton remodeling might be via the conversion of the phosphorylation levels of HSP27and tau, which are involved in cytoskeletal structure maintenance. Thus, a therapeutic strategy based on p38MAPK inhibitors may be effective in counteracting the neurotoxicity of MC-LR.