Alzheimer’s disease (AD) is the leading cause of dementia, and clinical symptoms develop due to neuronal death. This neurodegeneration is believed to be primarily initiated by the abnormal accumulation of Amyloid β (Aβ) peptide and tau protein aggregates. Among these, Aβ accumulation is considered the primary driver of AD and forms the main focus of this thesis. Different alloforms of Aβ (e.g. Aβ1-40 and Aβ1-42) are produced by differential cleavage of the Amyloid precursor protein (APP). The lifecycles of APP and Aβ depend on their localization to different cellular compartments such as endosomes, but they can also be trafficked out of the cells in exosomes, a type of extracellular vesicle (EV). It is hypothesized that halting the spread and accumulation of Aβ via EVs could slow neurodegeneration and disease progression. However, the mechanisms underlying Aβ packaging into these vesicles and how other AD-related cellular dysfunctions influence EV propagation, remain largely unclear. Understanding these mechanisms could be crucial for identifying new treatment targets. This thesis aims to elucidate some of these gaps.   

Differences in Aβ fibril conformations have been hypothesized to result in diverse clinical phenotypes of AD. Therefore, Paper I examined how different ratios of Aβ1-40 to Aβ1-42 affect fibril conformation. Given that there is no standard timeframe for fibril formation, we further investigated the effect of maturation time. Using electron microscopy, different dyes, and antibodies, we found that both Aβ1-40:42 ratio and maturation significantly influenced fibril conformation. Fibrils with higher Aβ1-42 ratio showed increased cellular association, although this did not substantially impact cytotoxicity and autophagy.

Mutations or dysfunctions related to endosomal trafficking (e.g. dysfunction of the retromer subunit Vps35), can cause Aβ accumulation but the impact on exosome biogenesis and EV release is lacking. Therefore, Paper II investigated the effect of Vps35 knockout (KO) on exosome biogenesis and EV cargo during Aβ challenge. We found that lack of Vps35 resulted in lower EV abundance, caused by a decrease in exosome biogenesis-related proteins, leading to increased Aβ cargo. Aβ challenge also led to increased EV release and affected EV cargo, revealing new mechanisms by which Vps35 dysfunction contributes to Aβ accumulation.  

Aβ is hypothesized to be released in exosomes via a neutral sphingomyelinase 2 (nSMase2)-dependent pathway. However, knowledge regarding how Aβ is packaged is still lacking. Therefore, Paper III aimed to confirm the nSmase2-dependency and to investigate whether the Aβ-interacting cellular prion protein (PrPC) could influence Aβ packaging into exosomes. Our findings confirmed that Aβ release occurs via an nSMase2-dependent pathway, independent of PrPC. Surprisingly, lack of PrPC caused cellular Aβ accumulation, changes to exosome biogenesis-related proteins, and an increase in EV production.   

Autophagy is heavily involved in both the accumulation and degradation of Aβ and has been connected to EV abundance. Therefore, Paper IV examined the role of autophagy in Aβ accumulation and EV dynamics. Using a cross between an Atg7 conditional KO mouse and a knock in APP mouse (with Swedish and Iberian mutation, APP^NL-F) we could demonstrate that plaques and intracellular Aβ was decreased while EV abundance was increased upon autophagy deficiency. However, the Aβ content of EVs was not altered. With additional mutations (Arctic mutation, APP^NL-G-F), leading to excessive Aβ production in autophagy deficient mice, similar decreases in Aβ plaque were seen but intracellular Aβ remained unchanged. This resulted in different cell death mechanisms, gamma oscillations, and behavioral outcomes between the autophagy deficient APP^NL-F and APP^NL-G-F mice.   In conclusion, this thesis expands our understanding of the impact of amyloid conformation, endosomal trafficking dysfunction, autophagy dysfunction, exosome biogenesis and EVs dynamics in the progression of AD.