Development of monoclonal antibodies specific for the calpain‐generated ∆48 kDa calcineurin fragment, a marker of distressed astrocytes

Abstract

AbstractBackgroundCalcineurin (CN) is a Ca2+/calmodulin‐dependent protein phosphatase expressed at high levels in brain. In healthy tissue, CN exists mainly as a full‐length (∼60 kDa) highly‐regulated protein involved in essential cellular functions. However, in diseased or injured tissue, CN is proteolytically converted to a constitutively active fragment that has been causatively‐linked to numerous pathophysiologic processes. The 48 kDa CN fragment (DCN) appears at high levels in human brain at early stages of cognitive decline associated with Alzheimer’s disease. DCN tends to show‐up in regions of frank amyloid and cerebrovascular pathology, especially in select subsets of astrocytes, both in humans and in animal models. Our goal was to develop monoclonal antibodies to DCN for use in neuropathology research.MethodA peptide encompassing the calpain sensitive region of the CN carboxyl terminus was used for antibody generation. Antibodies were screened in ELISAs against the immunizing peptide, but decision‐making screens were carried out as a Western analysis of 5XFAD mouse brain extracts, which express high levels of the DCN fragment.ResultWe identified 2 monoclonals, one of which (17E1) was highly reactive to DCN in Western blots, but not in ELISAs, of 5xFAD brain extracts. In contrast, the second antibody (26A6) reacted well against the peptide in ELISAs, but worked only modestly in Westerns. Importantly, neither antibody reacted with full‐length (60 kDa) CN, nor did antibodies detect DCN in extracts from healthy WT mice. In immunostaining assays, both antibodies strongly labeled subsets of GFAP‐positive astrocytes in 5xFAD mice with a high signal‐to‐background. Some GFAP‐negative cells also labeled positively for DCN. Consistent with Westerns, there was no antibody reactivity to WT mouse brain sections.ConclusionNew monoclonals are highly selective for the 48 kDa CN proteolytic fragment and label subsets of astrocytes, and possibly other cell types, under pathological conditions. These antibodies could be a useful tool for marking insidious brain pathology and identifying novel astrocyte phenotypes.