Alzheimer’s disease (AD), the most common neurodegenerative disease, is a serious threat to the autonomy, mobility, and overall functionality of aging persons worldwide. Although the etiology of AD remains incompletely defined, over a decade of investigation increasingly implicates oligomers of the amyloid-β (Aβ) peptide as the primary toxic molecular species responsible for the disease. Several studies support a robust correlation between Aβ oligomer concentration and the extent of synaptic loss and severity of cognitive impairment, and Ab oligomers also trigger abnormalities of the microtubule-associated protein tau, which is associated with irreversible neuronal damage. The revised amyloid cascade hypothesis stipulates that when Aβ is generated, by β and g-secretase cleavage of amyloid precursor protein (APP), the non-toxic Aβ monomers rapidly form soluble, toxic Aβ oligomers, which are then converted to relatively non-toxic insoluble Aβ fibril plaques that are deposited extracellularly. However, the subcellular location, cellular basis of Aβ oligomerization and the mechanism of Ab release from cells remains largely unknown. These mechanisms may also explain the characteristic spreading of Ab pathology throughout the brain, and could lead to new therapeutic approaches. Recently we showed that exosomes, endocytically derived nanovesicles, carry Ab peptides out of the cell and to contain the enzymatic machinery required to generate these peptides from APP (1). The overarching goal of this proposal it to correlatively combine super-resolution light microscopy (SR-LM) with high-resolution transmission electron microscopy (EM), yielding Corrleative SR-LM/EM or short CSRLEM, and to apply this to demonstrate that exosomes are a major way to shuttle amyloids out of the cell, and that exosome-associated amyloids contribute to AD pathology.

By combining chemical biology tools, biophysical and structural biology, cell biology and neuroscience, we propose to explore the role of the exosomal nanovesicles in the formation of amyloid aggregates.

The subcellular mechanisms governing exosomal-Ab biogenesis and secretion will be addressed using immunofluorescence and electron microscopy in concert with a novel fluorescently labeled g-secretase inhibitor. The proteins involved in exosomal release will be investigated using an RNAi silencing screen and the application of various knock out cell lines. The role of exosomes in nucleating amyloid aggregation will be monitored using in vitro aggregation assays and the pathological consequences of exosomal-Ab will be examined in vivo by assaying plaque propagation after intracerebral injection of exosomal-Ab in AD mouse models, and by examining tau phosphorylation and synaptic functionality after exosomal-Ab treatment. The expected results will clarify the role of exosomes in amyloid pathology propagation and define the rules for exosomal-Ab biogenesis and release from neurons.