Selective disappearance of an axonal protein, 440-kDa ankyrinB, associated with neuronal degeneration induced by methylmercury

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Abstract
The 440‐kDa isoform of brain ankyrin, 440‐kDa ankyrinB, is a neuron‐specific protein and is confined to axons. Cerebellum is one of the areas characteristically altered by methylmercury intoxication both in the adult and during development. When rat cerebellar neurons matured for 7 days in vitro were exposed to methylmercury at 0.03 μM for 48 hr, viability of the cells was unaffected. However, the immunocytochemical staining of 440‐kDa ankyrinB diminished drastically, whereas that of microtubule‐associated protein‐2, which is localized in dendrites and cell bodies, and of glial fibrillary acidic protein (GFAP), a marker for astroglial cells coexisting in the culture, remained unchanged. To confirm these observations, a simplified dot blot assay was established to determine 440‐kDa ankyrinB and several other marker proteins in cultured cell samples. With this assay, we found that methylmercury at a submicromolar range induced a decrease of 440‐kDa ankyrinB and an increase of GFAP in a dose‐dependent manner in cerebellar cells in primary culture. Surprisingly, another axonal protein, tau, remained mostly in its intact molecular sizes even in the presence of 0.3–1.0 μM methylmercury, though its immunocytochemical localization was substantially altered. These results indicate that selective loss of the axonal protein 440‐kDa ankyrinB is associated with the early stage of degeneration of cerebellar neurons induced by methylmercury. Therefore, 440‐kDa ankyrinB should be useful as a specific and sensitive marker for the neurotoxicity of methylmercury.