α-Spectrins are major ubiquitinated proteins in rat hippocampal neurons and components of ubiquitinated inclusions in neurodegenerative disorders

We have demonstrated that rr-spectrins (alpha Spl Sigma* and alpha Spll Sigma1) are major ubiquitinated proteins in terminally differentiated hippocampal neurons in culture. Western blotting experiments, using alpha Spl Sigma1, alpha Spll Sigma1, and ubiquitin antibodies and lysates of 11-day-old cultured rat hippocampal neurons, have demonstrated that a single band comigrating with rYSplI* and alpha Spll Sigma1 in a 5% polyacrylamide sodium dodecyl sulfate gel is recognized by ubiquitin antibodies when I-125-protein A is used for detection, Immunofluorescence staining of the 7- and 12 -day-old rat hippocampal neuron cultures using ubiquitin, alpha Spl Sigma1, and alpha Spll Sigma1 antibodies demonstrated that all of these antibodies label neurons but not the astrocytes in the cultures. Immunoprecipitation of spectrin subunits in lysates of 12-day-old rat hippocampal neurons under stringent conditions (9.5 M urea) using alpha Spl Sigma1 and alpha Spll Sigma1 antibodies followed by Western blot experiments of the immunoprecipitated spectrin subunits using alpha Spl Sigma1, alpha Spll Sigma1 and ubiquitin antibodies confirmed that both alpha Spl Sigma* and alpha Spll Sigma1 are ubiquitinated in rat hippocampal neurons. Furthermore, we demonstrated by immunohistochemistry that cu-spectrins are components of the cytoplasmic ubiquitinated inclusions in hippocampal neurons in Alzheimers and Parkinson's disease patients. (C) 2001 Elsevier Science Inc.