Functional characterization of AP-1 and Arc/Arg3.1 in the transport and processing of APP at the late secretory pathway

Author(s)

Yunan Costa Januário

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2021-11-04

Examining board members

Luis Lamberti Pinto da Silva; Enilza Maria Espreafico; Merari de Fatima Ramires Ferrari; Sergio Teixeira Ferreira

• Advisor: Luis Lamberti Pinto da Silva

Abstract

Alzheimer\'s disease is a common type of dementia caused by neuronal degeneration and death. A hallmark of this disease in the brain is the accumulation of toxic amyloid-β (Aβ) peptides in the extracellular space, contributing to the formation of senile plaques. The direct precursor of Aβ is the carboxy-terminal fragment (CTFβ or C99) of the amyloid-beta precursor protein (APPβ). C99 is detected at high levels in the brain with Alzheimer\'s disease and its intracellular accumulation has been linked to early neurotoxicity, independently of Aβ. The proteolytic cleavage of APP at C99 occurs during its transport in the secretory pathway. However, the causes for the increased cellular levels of C99 are poorly understood. This work aimed to study the participation of adaptor protein 1 (AP-1) and Arc/Arg3.1 in the transport and processing of APP at the late secretory pathway. Immunoprecipitation and yeast two-hybrid assays showed that AP-1 interacts with APP. This interaction requires the conserved domain 682YENPTY687 in the cytosolic tail of APP and two binding sites in the µ1A subunit of AP-1. To understand the AP-1 importance in the APP transport, immunofluorescence and confocal microscopy assays were performed in human cell lines (HeLa and H4) or primary rat neurons. Analyzes were performed using RNAi silencing techniques, CRISPR/Cas9 gene deleted cells combined with the rescue of expression, dominant-negative expression and the expression of an APP mutant deficient in the interaction with AP-1. Observation of the data revealed that AP-1 is essential for transferring APP from the Golgi complex to endosomes. Interference with AP-1-mediated transport leads to APP retention in the trans-Golgi network. To analyze APP processing and C99 fragment generation in cells, we used western blot assays using the synchronized transport tool in the secretory pathway with Retention Using Selective Hooks (RUSH). Delaying APP transport in AP-1 deleted cells reduces C99 generation kinetics. Interestingly, at the stead state of APP subcellular distribution, the intracellular levels of C99 were increased upon AP-1 depletion. Our results indicate that AP-1 directly controls the subcellular distribution of APP in a way that regulates the generation and intracellular accumulation of the pathogenic C99 fragment. This indicates that defects in AP-1-mediated transport of APP/C99 may be a potential contributing factor to Alzheimer\'s disease. Together with these results, the participation of the Arc/Arg3.1 protein in the transport and processing of APP was also studied. Arc is a protein that controls neuronal plasticity and whose activity was previously related to Aβ accumulation. Western blot assays in H4 cells show that Arc expression increases APP levels and all of its carboxy-terminal fragments (CTFs), including the C99 fragment. The use of a drug that inhibits lysosomal activity showed that Arc favors the generation of the C99 fragment. Immunofluorescence and confocal microscopy assays indicated that Arc favors the accumulation of APP in the endosomal system. In an attempt to elucidate the possible mechanism by which Arc controls the intracellular transport of APP in the late secretory pathway, was analyzed whether Arc interacts with vesicle adapter proteins (APs). It was observed by immunoprecipitation and yeast two-hybrid assay that Arc interacts with the mediu µ subunit of AP1, AP-2, AP-3 and AP-4. These data indicated that Arc alters APP endolysosomal transit, leading to an increase in the C99 fragment and interacts with different APs as a possible mechanism by which Arc regulates APP transport. (AU)

• FAPESP's process: 17/12022-0 - Characterization of the molecular mechanisms used by Arc protein to modify the intracellular traffic and processing of AMPA receptors and beta-amyloid precursor protein (APP)
• Grantee: Yunan Costa Januário
• Support Opportunities: Scholarships in Brazil - Doctorate