THE Neurotoxic Effects of Manganese on Dopamine Post-Synaptic Receptors ARE REVERSED BY p-Aminosalicylic Acid (PAS)

Manganese is a neurotoxin that causes the human disease Manganism.  It disrupts dopamine neurotransmission in the brain, however the neurotoxic mechanism of action is not fully resolved and there is no effective clinical treatment for it.  Reports in the literature postulate that manganese toxicity is more related to dysfunction of the post-synaptic dopamine D2 receptors (D2DR) rather than degeneration of dopamine neurons.  Our previous work showed that gill lateral ciliated cells of Crassostrea virginica have post-synaptic dopamine receptors that are D2 type and are innervated by cilio-inhibitory dopamine fibers via the branchial nerve.  We also found that manganese treatments block the cilio-inhibitory effects of dopamine on gill lateral cells and that treatments with manganese in the presence of the drug p-aminosalicylic acid (PAS) prevented this toxic effect.  Our previous immunofluorescence work with 1̊ antibodies against D2DR and FITC-linked 2̊ antibodies showed that treatments with manganese caused reduced fluorescent intensity in the gill lateral cells, suggesting that manganese caused a loss of D2DR number or disruption of their structural integrity.  In this study we hypothesized that PAS would reverse the neurotoxic effects of manganese on D2DR when applied after manganese exposure.  To test this C. virginia were treated for up to 3 days with manganese (500 µM) followed by 5 more days with PAS (500 µM).  Gills were excised, fixed, exposed to 1̊ antibodies against D2DR and FITC-linked 2̊ antibodies, paraffin embedded, sectioned at 10 µm and then visualized on a fluorescence microscope.  Fluorescence intensity was quantified using ImageJ software from NSF.  The results showed that the fluorescence intensity in animals treated with manganese had a progressive decrease in fluorescence of up to 40% less than non-manganese treated controls.  Animals treated with PAS after manganese and animals treated only with PAS did not show reduced fluorescence, indicating PAS reversed the manganese induced loss of post-synaptic D2DR fluorescence.  This immunohistological study shows a positive correlation between the losses of D2DR fluorescence in manganese treated animals vs controls. It also shows PAS effectively reversed the toxic effects of manganese on D2DR after 3 days of manganese treatments.  The actions of PAS are likely due to its ability to chelate manganese.  The results suggest that manganese chelating agents like PAS that can penetrate the human blood-brain barrier might be effective therapeutic agents for Manganism.  This work was supported in part by 2R25GM06003 of the Bridge Program of NIGMS and 0516041071 of NYSDOE.