The mutations that cause familial Alzheimer's disease (FAD) are found in amyloid precursor protein (APP) and presenilin (a catalytic component of γ-secretase), which together produce amyloid β peptide (Aβ). However, whether Aβ is a major driver of disease remains controversial. On February 12, 2024, Michael S. Wolfe from the University of Kansas and Yigong Shi from Tsinghua University/Westlake University published an online paper titled "Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes" in Cell Reports The study found that FAD mutations disrupt the initial proteolytic event of the γ-secretase in the multi-step processing of APP substrate C99. Cryo-electron microscopy showed that in the transition state, the substrate mimics captured γ-secretase, a structure consistent with the activated enzyme-substrate complex captured by molecular dynamics simulations. FAD mutations in silicon mimicry and in cellulose fluorescence microscopy are supported to stabilize enzyme-substrate complexes. Neuronal expression of C99 and/or presenilin-1 in Caenorhabditis elegans results in synaptic loss only in FAD-mutated transgenes. Mutations designed to stabilize the enzyme-substrate complex and block Aβ production likewise lead to synaptic loss. Overall, these findings imply that in the pathogenesis of FAD, there is a arrest process of γ-secretase cleavage substrates, rather than a product.
The discovery of dominant missense mutations associated with familial Alzheimer's disease (FAD) in amyloid precursor protein (APP) led to the initial formation of the 1991 amyloid hypothesis for the pathogenesis of Alzheimer's disease, which postulated the aggregation of secreted amyloid β (Aβ) peptides, particularly Aβ42, leading to a cascade of events that would eventually lead to neurodegeneration and dementia. Subsequent studies have found that progerin is the site of FAD mutations, alters the production of Aβ, is essential for the processing of APP's γ-secretase into Aβ, and contains the catalytic component of the γ-secretase complex, providing strong support for the amyloid hypothesis.
However, the assembly status of neurotoxic Aβ and related signaling pathways remain unclear, and clinical candidates targeting Aβ or its aggregates have shown little or no benefit in the prevention or treatment of Alzheimer's disease, raising doubts about Aβ as a major driver of the disease process, despite the recent approval of anti-Aβ monoclonal antibodies.
γ-分泌酶对APP底物的蛋白水解过程示意图（图源自Cell Reports ）
The pathology, presentation, and progression of FAD are very similar to those of the more common sporadic late-onset Alzheimer's disease, and the monogenic nature of the predominantly inherited nature of FAD suggests that it should be easier to elucidate the pathogenic mechanism. Since dominant missense FAD mutations are found only in substrates and Aβ-producing enzymes, these mutations may result in alterations in the proteolytic process of APP substrates by γ-secretase. However, this process is complex, with the APP transmembrane domain (TMD) being cleaved multiple times by the γ-secretase complex embedded in the membrane, producing Aβ peptides along the Aβ49→ Aβ46→ Aβ43→ Aβ40 and Aβ48→Aβ45→Aβ42→ Aβ38 pathways. The researchers' comprehensive analysis of the effects of the 14 FAD mutations in APP TMD on these proteolytic events found that each mutation was defective in either the first or second carboxypeptidase pruning step, elevating Aβ peptide levels of Aβ45 or longer. This complete and quantitative analysis has not reported any presenilin FAD mutations.
文章模式图（图源自Cell Reports ）
The study expands on the analysis of each proteolytic processing step of C99 by γ-secretase to elucidate the effect of FAD mutations on progerin-1 (PSEN1), revealing defects that are consistent in the initial processing step, rather than in the later steps that produce the secreted form of Aβ (e.g., Aβ42). The atomic-resolution structure of the active γ-secretase complex binds non-covalently to the full transmembrane substrate mimic, providing a snapshot of the enzyme in or near a transitional state, just as it is preparing for intramembrane proteolysis. This new structure, in turn, validates a molecular dynamics model system that captures the activating enzyme-substrate (E-S) complex.
The silicon model showed poor conformational flexibility of the FAD-mutant E-S complex, indicating that the complex was stable, which was supported by fluorescence lifetime imaging microscopy (FLIM) of intact cells. A Caenorhabditis elegans FAD model system used to test the mechanisms of neurodegeneration has shown that the stabilization of the E-S complex alone, without the production of Aβ-peptide products, is sufficient to lead to age-dependent synaptic loss and shortened lifespan.
来源：iNature