ACTIVATION OF THE SIGMA-1 RECEPTOR BY SPECIFIC LIGANDS INHIBITS HUMAN INFLAMMATORY DENDRITIC CELL FUNCTIONS AND EFFECTOR T-LYMPHOCYTE RESPONSES

Abstract

CN autoinhibitory domain located near the C terminus of the CN catalytic subunit. Commercially available antibodies that target the N terminus of the CN catalytic subunit reveal the presence of a highly-active 48 kDa proteolytic fragment in human brain tissue during early stages of cognitive decline and also in a variety of experimental models of neurodegeneration. While useful for determining the extent of CN proteolysis in Western blot applications, N terminus antibodies do not reveal the cellular location of the proteolysis. Knowing where CN is proteolyzed in nervous tissue seems critical to understanding the mechanistic basis of its many deleterious actions, particularly because CN is found in both neurons and glial cells where it is involved in different cellular functions.Methods: To address this gap in our understanding of CN regulation, we generated custom rabbit polyclonal antibodies to CNA based on previously identified calpain-dependent cleavage sites. One of these antibodies (referred to as “DCN48”) detects a 48 kDa fragment in Western blot assays, but does not detect full-length (60 kDa) CN. TheDCN48 antibody was then used for DAB immunohistochemical labeling of human brain sections characterized by AD andmixed AD/vascular pathologies. Results: The anatomical features labeled by the DCN48 antibody included astrocyte clusters and vascular-associated elements and/or processes. We also observed numerous DCN48-positive astrocytes associated with microinfarcts. Surprisingly, we have seen very little neuronal labeling with this antibody. Conclusions: The results suggest that astrocytes and, perhaps astrocyte end-feet, are a primary locus for CPdependent CN proteolysis in injured or diseased nervous tissue. CN proteolysis appears to be especially pronounced in astrocytes associated with vascular pathology. This work may provide new mechanistic insights into the impact of Ca 2+ dysregulation on neurodegenerative diseases.