Alpha adaptins show isoform-specific association with neurofibrillary tangles in Alzheimer's disease

Aims: The heterotetrameric assembly protein complex 2 (AP-2) is a central hub for clathrin-dependent endocytosis. The AP-2 α-adaptin subunit has two major isoforms, encoded by two separate genes: AP2A1 and AP2A2. Endocytosis has been implicated in the pathogenesis of neurodegenerative disease, and recent studies linked α-adaptins (gene variants, splicing defects and altered expression) with late-onset Alzheimer's disease (LOAD) risk. Here, we used multiple antibodies to investigate α-adaptin isoforms and their localization in human brains. Methods: The specificities of 10 different α-adaptin antibodies were evaluated using immunoblots after human AP2A1 and AP2A2 plasmid transfection in cultured cells. Additional immunoblot analyses were then performed on protein homogenates from control and LOAD subjects. Formalin-fixed, paraffin-embedded brain sections from control and LOAD subjects were immunohistochemically stained, and immunofluorescence experiments were performed for quantitation of colocalisation with digital image analysis. Results: Eight of the 10 evaluated antibodies recognised transfected α-adaptin proteins on immunoblots. The α-adaptin subspecies were relatively uniformly expressed in five different human brain regions. The α-adaptins were present in the detergent-insoluble fraction from cognitively impaired, but less so in control, brains. Immunohistochemical analyses showed colocalisation of AP2A1 with tau pathology in LOAD brains. By contrast, AP2A2 colocalised with microglial cells. Conclusions: These observations provide evidence of isoform-specific changes of α-adaptins in the brains of LOAD subjects. Antibodies that were verified to recognise AP2A1, but not AP2A2, labelled neurofibrillary tangles of LOAD patients. The findings extend our understanding of AP-2 proteins in the human brain in healthy and diseased states.