p73 gene in dopaminergic neurons is highly susceptible to manganese neurotoxicity

Abstract

Chronic exposure to elevated levels of manganese (Mn) has been linked to a Parkinsonian-like movement disorder, resulting from dysfunction of the extrapyramidal motor system within the basal ganglia. However, the exact cellular and molecular mechanisms of Mn-induced neurotoxicity remain elusive. In this study, we treated C57/BL6J mice with 30 mg/kg Mn via oral gavage for 30 days. Interestingly, in nigral tissues of Mn-exposed mice, we found a significant downregulation of the truncated isoform of p73 protein at the N-terminus (ΔNp73). To further determine the functional role of Mn-induced p73 downregulation in Mn neurotoxicity, we examined the interrelationship between the effect of Mn on p73 gene expression and apoptotic cell death in an N27 dopaminergic neuronal model. Consistent with our animal study, a 300 μM Mn treatment significantly suppressed p73 mRNA expression in N27 dopaminergic cells. We further determined that protein levels of the ΔNp73 isoform was also reduced in Mn-treated N27 cells and primary striatal cultures. Furthermore, overexpression of ΔNp73 conferred modest cellular protection against Mn-induced neurotoxicity. Taken together, our results demonstrate that Mn exposure downregulates p73 gene expression resulting in enhanced susceptibility to apoptotic cell death. Thus, further characterization of the cellular mechanism underlying p73 gene downregulation will improve our understanding of the molecular underpinnings of Mn neurotoxicity.

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“…Microdissected brain tissues were lysed in modified RIPA lysis buffer (1% Triton X‐100, 1 mM EDTA, 100 mM NaCl, 1 mM EGTA, 1 mM NaF, 20 mM Na 4 P 2 O 7 , 2 mM Na 3 VO 4 , 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 50 mM Tris‐HCl, and pH 7.4) via sonication. Brain homogenates were prepared as described previously . Protein concentration of samples was measured using the Bradford assay.…”

“…Protein concentration of samples was measured using the Bradford assay. Western blotting was performed as described previously . Briefly, samples containing equal amounts of proteins were loaded and fractionated in a 10–12% SDS‐PAGE gel and transferred onto a nitrocellulose membrane.…”

Cobinamide is effective for treatment of hydrogen sulfide–induced neurological sequelae in a mouse model

“…With the aim of studying protein changes via Western blot, the inferior colliculus samples were lysed in modified RIPA lysis buffer (1% Triton X‐100, 1 mM EDTA, 100 mM NaCl, 1 mM EGTA, 1 mM NaF, 20 mM Na 4 P 2 O 7 , 2 mM Na 3 VO 4 , 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 50 mM Tris–Cl, pH7.4) via sonication. Brain homogenates were prepared as described previously . Protein concentration of samples was measured using the Bradford assay.…”

“…Protein concentration of samples was measured using the Bradford assay. Western blotting was performed as described previously . Briefly, the samples, containing equal amounts of proteins, were loaded and fractionated in a 10–12% SDS‐PAGE gel and transferred onto a nitrocellulose membrane.…”

“…Brain homogenates were prepared as described previously. 35 Protein concentration of samples was measured using the Bradford assay. Western blotting was performed as described previously.…”