Alzheimer's disease : uncovering mechanisms for amyloid precursor protein processing and trafficking

Abstract

Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder characterized by impairment of memory and, eventually, by disturbances in cognitive abilities. Brain regions crucial for learning and memory exhibit atrophy, but the underlying mechanisms for neurodegeneration continues to be point of debate. One fundamental abnormality that certainly plays a pivotal role is altered proteolytic processing of the amyloid precursor protein (APP) such that toxic amyloid-β (Aβ) peptides are formed. To prevent aggregation, enhance clearance or preclude formation of Aβ represent notable attempted strategies in the fight against AD. Because none of these strategies have - as of yet - proved triumphant, there is a persistent demand for understanding basic biological features of cells in an AD state. In this regard, this thesis collectively aims to uncover mechanisms which regulate processing and trafficking of APP and APP-relevant molecules.

Paper 1 aims to expand our understanding of endogenous APP transport to synapses of hippocampal neurons. Using immunocytochemical approaches, we find that under normal physiological conditions, APP primarily exists as cleaved fragments at nerve terminals. Perturbation of BACE1 activity, either through genetic manipulation or pharmacological inhibition, enhanced accumulation of APP at presynaptic terminals. Together with biochemical observations, this finding suggests the existence of a full-length APP trafficking pathway in neurons. Moreover, it raises questions on whether strong perturbation of BACE1 activity may be deleterious for synaptic function.

Paper 2 aims to elucidate how a protein involved in protein sorting and membrane trafficking, SNX3, may be involved in APP processing and Aβ generation. An in vitro cell culture model involving genetically manipulated expression of SNX3 was used in conjunction with a number of immunocytochemical techniques, flow cytometry and biochemical approaches to fulfill this aim. We found that SNX3 overexpression can perturb physical interaction of APP and BACE1 such that it results in decreased Aβ levels. This was likely the result of reduced APP internalization from the surface of cells. As such, SNX3 regulates Aβ production by influencing APP endocytosis.

Paper 3 aims to understand how exercise can lessen Aβ accumulation and how BDNF may be involved in associated regulation of APP processing. Using a transgenic mouse model of AD and cultured human neural cells, we demonstrate that exercise and BDNF can reduce production of toxic Aβ peptides through a mechanism involving enhanced α-secretase activity. Flow cytometry, biochemical techniques and immunocytochemistry enabled us to determine that this anti-amyloidogenic APP processing involves subcellular redistribution of α-secretase and an increase in intracellular neuroprotective APP peptides capable of binding and inhibiting BACE1. Exercise, and other factors which enhance BDNF signaling, may - therefore - have both therapeutic and prophylactic potential in AD.

Paper 4 aims to determine the contribution of extracellular vesicles (EVs) to Aβ production and pathogenesis of AD. Using EVs isolated from cerebrospinal fluid and plasma of AD patients, plasma of AD mouse models and media of cultured neural cells expressing AD mutations, we determined that EVs have the capacity to destabilize neuronal Ca2+ homeostasis, impair mitochondrial function and sensitize neurons to excitotoxicity. Though it was found that EVs contain relatively low levels of Aβ species, the ratio between more toxic Aβ42 isoforms and Aβ40 was enhanced. The majority of this Aβ appeared to be on the surface of EVs and this appeared to be an important feature in the transcellular spread of Aβ and associated toxicity.

In summary, this thesis expands our understanding of mechanisms which regulate processing and trafficking of APP and APP-relevant molecules. In doing so, the work presented here collectively advocates for novel strategies in the fight against AD.