Structural characterization of APP family proteins

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Project Summary/Abstract Alzheimer disease (AD) is a neurodegenerative disease characterized by progressive decline of brain functions, progressive loss of synapses, neuronal death, brain atrophy and formation of extracellular amyloid plaques and intracellular neurofibrillary tangles. The amyloid precursor protein (APP) is a type I transmembrane glycoprotein which plays a central role in AD pathogenesis, as the precursor of the beta amyloid peptide (Aβ), the main component of amyloid plaques. In addition, several mutations on the APP gene are associated with the early-onset hereditary form of the disease (Familial Alzheimer disease or FAD). Apart from being the precursor of the toxic Aβ peptide and the carrier of FAD mutations, APP has been associated with a wide range of biological functions, including proliferation, neuronal development, intracellular transport, Ca2+ homeostasis, neurite outgrowth, shaping of dendritic complexity and, most importantly, synaptogenesis and synaptic function. Indeed, multiple reports link APP to the regulation of spine density, spine dynamics and synaptic plasticity. Reported synaptic roles of APP both in the presynaptic active zone and postsynaptic density include regulation of presynaptic nicotinic receptors, facilitation of neurotransmitter release, regulation of synaptic vesicle recycling and regulation of the cell surface expression and activity of NMDA receptors. Deregulation of one or more of the above functions is therefore likely to lead to synaptic dysfunction and neurodegeneration, also basic hallmarks of the AD. APP is part of a gene family, with two more mammalian members, the Amyloid Precursor Like Protein 1 (APLP1) and the Amyloid Precursor Like Protein 2 (APLP2). Both APLPs are highly homologous to APP, with similar molecular architecture and functions, especially related to synaptogenesis, spine dynamics and synaptic plasticity. The goal of this proposal is to develop suitable experimental resources for performing structural investigations of the physiological functions of APP and APLPs. To achieve this goal, we propose to establish an expression and purification pipeline for medium throughput screening of APP, APLP1 and APLP2 orthologs, investigate an alternative tagging procedure and compare extraction and reconstitution methods for a subset of orthologs. Finally, we propose to utilize single particle cryo-electron microscopy (cryoEM) to elucidate atomic-level details of the molecular architecture of APP family proteins. High-resolution structural information of a full- length APP family protein will enable us to better understand the structural basis of the physiological functions of APP and APP-like proteins and inform our understanding of how these functions are deregulated in AD.