P3-188: Immunohistochemical localization of iron regulatory protein-2 in Alzheimer’s disease hippocampus

The link between perturbed brain iron metabolism and Alzheimer's Disease (AD) warrants study. Iron regulatory protein–2 (IRP–2) plays a role in brain iron metabolism by serving as a cytosolic iron sensor and post transcriptional control point for iron uptake or storage. Studies of IRP–2 in the AD brain with polyclonal antibodies report abnormal IRP–2 localization and interactions with iron responsive elements of RNA transcripts. This is the first study of IRP–2 localization in the AD and elderly control hippocampus using monoclonal antibodies (mAbs) against specific epitopes. Three mAbs developed in our laboratory are directed to two different epitopes in the 73 amino acid (aa) iron degradation domain (IDD, aa position 138–200) of IRP–2 and to one of the regions flanking the IDD, either aa100–128 or aa209–262. Specificity of antibodies has been established by immunoblots. Formalin fixed hippocampi from age–matched elderly control (n=8), AD (n=9) (documented clinically and pathologically by CERAD/NIH criteria) and Lewy Body Disease (LBD; n=4) have been studied. Formalin fixed brains of wild–type and IRP–2 knockout mice were used for positive and negative controls, respectively. A standard immunohistochemistry protocol with AEC as the chromogen was followed. IRP–2 expression was found to be primarily intraneuronal with sparing of oligodendrocytes, astrocytes, and white matter tracts. Hippocampal pyramidal neurons demonstrated remarkable perinuclear IRP–2 buildup, with colocalization in dystrophic neurites within the background neuropil. The dentate gyrus also expressed IRP–2 in some cases. The exact relationship of IRP–2 reactivity to tangles and plaques could not be adequately evaluated in the pilot samples examined, but additional studies are under way. IRP–2 expression appeared to be more prominent in the hippocampal pyramidal cells from AD and LBD, compared to control brains in the few cases studied to date, but more quantitative analysis in additional brain regions is being planned. Further characterization of the pathogenic role of the identified IRP–2 immunoreactive material in multiple brain regions relevant to neurodegenerative disease processes remains an important continuing objective. More sophisticated approaches will include laser–capture microdissection and application of techniques developed for proteomic analysis. This research was funded by NIH Grant #AG20948.