Tese de Doutoramento Biomedicina 2023 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

One of the significant misfortunes of the elderly is dementia, and one of its leading causes is late-onset Alzheimer’s disease (AD), whose major risk factor is aging. Our brain is particularly susceptible to aging because neurons are mostly embryonically born and not replaced during our lifetime. As we age, our neurons age, and some loose synapses likely contribute to age-dependent cognitive decline. In AD, the loss of synapses is accelerated, and it may start during aging, in an asymptomatic phase, since the main AD biomarker, β-amyloid (Aβ), is detected as early as 25 years before the disease onset. Importantly, there is still no cure for AD. Therefore, with an increasingly aging population worldwide, there is an urgent need to understand the biological mechanisms of neuronal aging to achieve healthy brain aging. During neuronal aging, Aβ accumulates, and synapses decline. However, little is known about the underlying cell biological mechanisms that cause age-dependent synapse decline. Here, we decided to study neuronal aging in vivo and in vitro using physiologically aged mouse brains and aged mouse primary neurons. Aged primary neurons accumulate lysosomal lipofuscin, an aging marker, and show synapse loss. First, we identified the up-regulation of amyloid precursor protein (APP) endocytosis as a neuronal aging mechanism that potentiates APP processing in vitro and in vivo, increasing Aβ production and intraneuronal accumulation. Also, we observed the enlargement of early endosomes with aging in vitro and in vivo, in agreement with the endocytosis up-regulation. Mechanistically, we showed that clathrin-dependent APP endocytosis requires F-actin and that clathrin and endocytic F-actin increased with neuronal aging. Moreover, we established causality between Aβ accumulation intraneuronally and synapse decline by inhibiting Aβ production and partially reverting synaptic decline in aged neurons. Additionally, by genetically inducing endocytosis up-regulation in younger neurons, we were able to promote Aβ accumulation and recapitulate synapse decline similarly to aged neurons, supporting a role of increased endocytosis in Aβ production and synapse decline with aging. Since inhibition of Aβ production in aged neurons was not enough to protect neurons from synapse loss, we decided to investigate which other mechanisms would be altered with neuronal aging and contribute to age-dependent synapse decline. We decided to focus our research on lysosomes since correct lysosome function is essential for synapses. Additionally, lysosome dysfunction is considered a hallmark of cellular aging, and in this study, we observed that lysosomes accumulate lipofuscin in aged neurons. Therefore, we set out to investigate lysosome dysfunction with neuronal aging and whether it contributes to driving synapse dysfunction and loss. We started by characterizing endo-lysosomes (late-endosomes and lysosomes) in mature and aged neurons and physiologically aged brains. Mature neurons showed a higher endo-lysosomal area in neurites than soma and had lysosomal activity in distal neurites. Strikingly, lysosomes accumulated cathepsin D in aged neurons but were less degradative in distal neurites, most likely due to reduced acidification. Moreover, we observed enlarged endo-lysosomes present distally, close to synapses, in aged neurons and aged brain synaptosomes, possibly related to lysosome degradative capacity defects. Interestingly, restoring aged lysosomes function by using the mucolipin transient receptor potential channel 1 (TRPML1) agonist, mucolipin synthetic agonist 1 (ML-SA1), reverted synaptic decline while inducing lysosome dysfunction in mature neurons through increased alkalinization by chloroquine (CQN) treatment mimicked age-dependent synapse decline. Overall, we identified APP endocytosis up-regulation, as well as reduced degradative capacity and acidification of distal lysosomes, as mechanisms of neuronal aging contributing to age-dependent synapse loss. We identified endocytosis and lysosomes as potential druggable targets to therapeutically contribute to preventing or delaying synapse decline with aging, thus reducing the contribution of aging to the development of neurodegenerative disorders such as late-onset AD