alpha-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 Sig ma1) are major ubiquitinated proteins in terminally differentiated hippocam pal neurons in culture. Western blotting experiments, using alpha Spl Sigma 1, alpha Spll Sigma1, and ubiquitin antibodies and lysates of 11-day-old cu ltured rat hippocampal neurons, have demonstrated that a single band comigr ating with rYSplI* and alpha Spll Sigma1 in a 5% polyacrylamide sodium dode cyl sulfate gel is recognized by ubiquitin antibodies when I-125-protein A is used for detection, Immunofluorescence staining of the 7- and 12 -day-ol d rat hippocampal neuron cultures using ubiquitin, alpha Spl Sigma1, and al pha Spll Sigma1 antibodies demonstrated that all of these antibodies label neurons but not the astrocytes in the cultures. Immunoprecipitation of spec trin subunits in lysates of 12-day-old rat hippocampal neurons under string ent conditions (9.5 M urea) using alpha Spl Sigma1 and alpha Spll Sigma1 an tibodies followed by Western blot experiments of the immunoprecipitated spe ctrin subunits using alpha Spl Sigma1, alpha Spll Sigma1 and ubiquitin anti bodies confirmed that both alpha Spl Sigma* and alpha Spll Sigma1 are ubiqu itinated in rat hippocampal neurons. Furthermore, we demonstrated by immuno histochemistry that cu-spectrins are components of the cytoplasmic ubiquiti nated inclusions in hippocampal neurons in Alzheimers and Parkinson's disea se patients. (C) 2001 Elsevier Science Inc.