Implication of APP phosphorylation at threonina 668 induced by Aß oligomers in amyloidogenic processing of APP: Role of GßƔ/p38MAPK signaling

ALMIRON, ROMINA; ANTONINO, MAGDALENA; ALFREDO, LORENZO; BIGNANTE, ELENA ANAHI

Rosario

Congreso; Congreso Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2023

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Protein phosphorylation is a fundamental post-translational modification, with serine, threonine and tyrosine residues generally being covalently linked to phosphate groups. Protein phosphorylation is closely involved in a variety of cellular events, indicating their crucial role in physiological and pathological conditions. Pathologic hallmarks of AD include amyloid-beta (AB) plaques, neurofibrillary tangles mainly composed of abnormally phosphorylated tau, dystrophic neurites surrounding Ab plaques, and overt loss of synapses and neurons. The study of phosphorylation signaling pathways in AD focuses mainly on tau protein, due to their abundant phosphorylation sites and kinases, while much less is known about APP phosphorylation and its impact on thisdisease. The phosphorylation of APP at threonine 668 (APP-P-T668) of the cytoplasmic domain has been correlated with an increase in the proteolysis of APP by beta-secretase, due to the activation of kinases such as GSK-3, JNK. It has been shown that APP-P-T668 is prevalent in AD and is increased in the hippocampus of AD patients. Furthermore, APP-P-T668 colocalizes withBACE1 in enlarged endosomes in hippocampal neurons. Previous studies from our laboratory demonstrated that p38MAPK kinase is a downstream effector of the APP/Go/Gβγ pathway, mediating tau phosphorylation and Aβ fiber-induced dendritic dystrophy. Based on this evidence, we propose to study the implication of APP phosphorylation at T668 induced by the Go/Gβγ/p38MAPK pathway as a possible triggering event for the amyloidogenic processing of APP. Our experiments were performed in cultures of cortical neurons from 10 DIV rat embryos, which were treated with preparations enriched in oligomeric AB (ABo,1uM), gallein, a beta-gamma subunit blocker (5uM),SB203580, inhibitor of p38MAPK,(20uM) or vehicle.Subsequently, western blot assays were performed to quantify the levels of endogenous phosphorylated APP in the different conditions, observing a significant increase in the same in neurons treated with ABo, which was prevented in those neurons that previously received gallein. Immunofluorescence studies show an increase in the APP-P-T668 label in both soma and dendritesof neurons treated with ABo and a decrease in the synaptophysin label, the latter being an indicator of synapse loss. These effects were avoided with pretreatment of the cultures with gallein or SB203580. In conclusion, ABo induces an increase in APP-P-T668 which correlates with an increase in its amyloidogenic processing. This effect is mediated by the Go/GβƔ/ p38MAPK pathway since it was inhibited by treatments with gallein and SB203580. Clarifying the mechanisms by which the cycle of toxicity induced by AB is generated and its consequences on neurons is important to be able to find early alterations of the pathology as well as to develop therapeutic strategies that allow a more selective intervention in the treatment of AD.