Extracellular Phosphorylation of the Amyloid β-Peptide Promotes Aggregation

Alzheimer’s disease (AD) is the most prevalent, progressive neurodegenerative disorder and its incidence is increasing with the aging population worldwide. The presence of extracelluar neuritic plaques containing amyloid beta (Aβ) and intracellular neurofibrillary tangles (NFT) in the brain are pathological hallmarks of the disease. Aβ is generated by proteolytic processing, involving sequential cleavages of the amyloid precursor protein (APP) by beta (β) and gamma (γ) secretase. Aggregation of Aβ is believed to be critical for its neurotoxicity and pathogenesis of AD. Mutations that lead to amino acid substitutions within the Aβ can cause early onset AD and promote the formation of neurotoxic Aβ assemblies. However, such mutations are very rare and account for only a very small number of cases. Mechanisms that increase the aggregation of wild-type Aβ and cause the more common sporadic forms of AD are largely unknown. It is plausible that the aggregation of Aβ in AD might be induced by unknown post-translational modification. The role of post-translational modification of Aβ in its aggregation and the pathogenesis of AD are unclear. The amino acid sequence of Aβ contains three potential phosphorylation sites at serine residue 8, tyrosine residue 10 and serine residue 26. In silico and in vitro studies indicated that Aβ can undergo phosphorylation by different protein kinases. In vivo and ex vivo phosphorylation experiments using cultured cells, mouse cerebellar neurons and in cerebrospinal fluid (CSF) of AD patients showed the presence of extracellular PKs activity. Extracellular Aβ is phosphorylated by protein kinase A (PKA) that is secreted by or localized at the cell surface of culture neurons. In addition, the presence of PKA-like kinase activity was identified in CSF of AD patients. Different biophysical methods such as Circular dichroism, NMR, Thioflavin T, Congo red binding, Dynamic light scattering and Electron microscope using synthetic phosphorylated and non-phosphorylated variants of Aβ elucidated the role of phosphorylation on Aβ conformation, oligomerization and aggregation. Polyclonal phosphorylation-state Aβ specific antibody was generated to study the occurrence of phosphorylated Aβ in vivo. Biochemical and immunohistological stainings using brains of APP transgenic mice and human AD patients employing phosphorylation-state Aβ specific antibodyshowed the specific detection of phosphorylated and non-phosphorylated Aβ species. Importantly, phosphorylated Aβ was detected in small deposits in the brains of young transgenic mice and showed age-dependent accumulation in plaques. Phosphorylated Aβ was also detected in the core of neuritic plaques and associated with dystrophic neurites in human AD brains. In summary, the undertaken study shows that extracellular Aβ is phosphorylated by PKs present at the cell surface and in the CSF of the human brain. The phosphorylation at serine residue 8 increases the propensity of Aβ to adopt b-sheet conformation and promotes the formation of small oligomeric aggregates that could seed aggregation into larger oligomeric and fibrillar assemblies. The specific detection of phosphorylated and non-phosphorylated Aβ species in APP transgenicmice and human AD brain indicates the preferential aggregation of phosphorylated Aβ in vivo. The combined data demonstrate that extracellular phosphorylation of Aβ strongly promotes its assembly into neurotoxic species and thus might represent an important molecular mechanism in the pathogenesis of the most common sporadic AD. Thus, targeting extracellular phosphorylation of Aβ could be explored for therapeutic or preventive strategies to decrease Aβ aggregation in sporadic AD