Biochemical and Histochemical Evidence of Nonspecific Binding of α7nAChR Antibodies to Mouse Brain Tissue

Alpha 7 nicotinic acetylcholine receptors are involved in learning and memory, and are implicated in the pathology of Alzheimer's disease and schizophrenia. Detection of α7 subunits can be accomplished via immunodetection or α-bungarotoxin-binding techniques. Standard protocols for immunohistochemistry and Western blotting were followed using several commercially available antibodies. Various mice were evaluated, including non-transgenics, APP, PS1, APP + PS1, and α7 knockouts. Initial results with amyloid-depositing mice revealed α7 immunolabeled astrocytes, in addition to expected neuronal staining. Subsequent studies with intrahippocampal injections of lipopolysaccharide (LPS) into α7 knockout mice showed that both neuronal and astrocytic labeling by α7 antibodies was nonspecific. On Western blots of mouse brain proteins, none of the bands detected with antibodies directed against α7 subunits diminished in the α7 knockout mice. Although LPS-related changes in the expression of some bands were found, these also were unaffected by the α7 genotype of the mice. In general, the Western staining patterns for these antibodies revealed few overlapping bands. These immunodetection data are in contrast to genotyping results and mRNA analyses that confirmed the disruption of the α7 allele and lack of α7 message in the knockouts. These findings suggest caution in interpreting results when using several commercially available α7 nicotinic receptor antibodies.