The goal of this proposal is to test a new paradigm for the causative mechanisms of Alzheimer’s disease (AD): The accumulation of amyloid aggregates inside neurons leading to their degeneration and initiating plaque formation. The hypothesis we will test is the inverse or mirror image of the commonly held view of the amyloid hypothesis of AD, which proposes that soluble Abeta is secreted from neurons, aggregates in the extracellular space and causes neuronal dysfunction from the outside. Based on new preliminary data, we propose that amyloid aggregation begins inside neurons, leading to neuronal dysfunction and death, which then releases the amyloid to the extracellular environment as a neuritic plaque. Recent reports of clinical trials of drugs that inhibit secretion of Abeta and decrease the concentration of Abeta in the interstitial fluid indicate that inhibition of Abeta secretion may exacerbate cognitive dysfunction in humans and have no effect on plaque accumulation. These results support the alternative amyloid hypothesis and suggest that the intracellular retention and accumulation of insoluble long Abetas may be an initial pathological event leading to intraneuronal accumulation of amyloid and ultimately neuronal death and neuritic plaque formation. We propose two specific aims that are pilot studies to generate reagents and models to test key predictions of this hypothesis. The first aim is to develop end-specific antibodies that specifically recognize long Abeta peptides ending at residues 45, 46, 47, 48 and 49. The second aim is to develop organotypic slice models of transgenic mouse brain to test the pathological significance of intraneuronal amyloid and examine whether it is the precursor to amyloid deposited in neuritic plaques. The successful completion of this project may have an important impact on the road map plan of Cure Alzheimer’s Fund and research in Alzheimer’s disease by initiating a paradigm shift and providing a clearer understanding of the mechanisms of neuronal dysfunction and degeneration underlying cognitive dysfunction. An accurate understanding of the disease mechanisms will provide a focus for the development of drugs that prevent or reverse cognitive decline rather than making drugs that make patients cognitively worse.