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

Abstract

We have demonstrated that α-spectrins (αSpIΣ∗ and αSpIIΣ1) are major ubiquitinated proteins in terminally differentiated hippocampal neurons in culture. Western blotting experiments, using αSpIΣ1, αSpIIΣ1, and ubiquitin antibodies and lysates of 11-day-old cultured rat hippocampal neurons, have demonstrated that a single band comigrating with αSpIΣ∗ and αSpIIΣ1 in a 5% polyacrylamide sodium dodecyl sulfate gel is recognized by ubiquitin antibodies when 125I-protein A is used for detection. Immunofluorescence staining of the 7- and 12 -day-old rat hippocampal neuron cultures using ubiquitin, αSpIΣ1, and αSpIIΣ1 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 αSpIΣ1 and αSpIIΣ1 antibodies followed by Western blot experiments of the immunoprecipitated spectrin subunits using αSpIΣ1, αSpIIΣ1 and ubiquitin antibodies confirmed that both αSpIΣ∗ and αSpIIΣ1 are ubiquitinated in rat hippocampal neurons. Furthermore, we demonstrated by immunohistochemistry that α-spectrins are components of the cytoplasmic ubiquitinated inclusions in hippocampal neurons in Alzheimer’s and Parkinson’s disease patients.