Amyloid beta peptide (Aβ) is the major protein component of the amyloid plaques that are present in the brains of Alzheimer's disease (AD) patients. Aβ42 peptide is a known neurotoxic agent that binds to neurons and, under specific aggregation conditions, triggers cell death. Aβ peptide can undergo specific amino acid posttranslational modifications, such as phosphorylation, that are important for modulating its proteolytic degradation, aggregation, binding to lipid membranes and neurotoxic functions. Peptides phosphorylated at serine 8 in full-length Aβ42 (pAβ42) were synthesised and compared to native Aβ42 peptide. Their secondary structures, aggregation properties and interactions with plasma membranes of primary cortical neurons were investigated. The results revealed that pAβ42 has increased β-sheet formation with rapid amyloid formation in a synthetic lipid environment, which was associated with increased cellular binding but concomitant diminished neurotoxicity. Our data support the notion that phosphorylation of Aβ42 promotes the formation of amyloid plaques in the brain, which lack the neurotoxic properties associated with oligomeric species causing pathogenesis in AD.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.