Retrograde transport of γ-secretase from endosomes to the trans-Golgi network regulates Aß42 production.

The aberrant metabolism of amyloid-ß protein (Aß) in the human brain has been implicated in the etiology of Alzheimer disease (AD). γ-Secretase is the enzyme that generates various forms of Aß, such as Aß40 and Aß42, the latter being an aggregation-prone toxic peptide that is involved in the pathogenesis of AD. Recently, we found that clathrin-mediated endocytosis of γ-secretase affects the production and deposition of Aß42 in vivo, suggesting that the membrane trafficking of γ-secretase affects its enzymatic activity. However, the detailed intracellular trafficking pathway of γ-secretase and its contribution to Aß42 generation remain unclear. Here, we show that Retro-2, which inhibits the retrograde transport, elevated the Aß42-generating activity both in cultured cells and mice brain. However, the result of in vitro γ-secretase assay using a recombinant substrate suggested that Retro-2 did not elevate the intrinsic Aß42-production activity of γ-secretase. Immunocytochemistry and cell-surface biotinylation experiments revealed that γ-secretase is recycled via the endosome-to-trans-Golgi network transport. In addition, γ-secretase is retrogradely transported by syntaxin 5/6, known as targets of Retro-2, independent pathway. Conversely, TPT-260, which enhances the trafficking function of retromers, lowered Aß42 levels and the Aß42/(Aß40 + Aß42) ratio in secreted Aß from cultured cells. Our results strongly suggest that the endosome-to-trans-Golgi network trafficking of γ-secretase regulates its Aß42 production activity. Modulation of this trafficking pathway might be a potential target for the development of Aß42-lowering AD therapeutics. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Partial loss of CALM function reduces Aß42 production and amyloid deposition in vivo.

Aberrant production, clearance and deposition of amyloid-ß protein (Aß) in the human brain have been implicated in the aetiology of Alzheimer disease (AD). γ-Secretase is the enzyme responsible for generating various Aß species, such as Aß40 and toxic Aß42. Recently, genome-wide association studies in late-onset AD patients have identified the endocytosis-related phosphatidylinositol-binding clathrin assembly protein (PICALM) gene as a genetic risk factor for AD. We previously found that the loss of expression of CALM protein encoded by PICALM affects the ratio of production of Aß42, through the regulation of the clathrin-mediated endocytosis of γ-secretase. Here, we show that the binding capacity of the assembly protein 180 N-terminal homology (ANTH) domain of CALM to phosphatidylinositol-4,5-biphosphate, as well as to nicastrin, is critical to the modulation of the internalization of γ-secretase and to the Aß42 production ratio. Moreover, reduction of CALM decreases Aß deposition as well as brain levels of insoluble Aß42 in vivo These results suggest that CALM expression modifies AD risk by regulating Aß pathology.

Membrane trafficking and proteolytic activity of γ-secretase in Alzheimer's disease.

γ-Secretase is an intramembrane-cleaving protease that generates various forms of amyloid-ß peptides (Aß) that accumulate in the brains of Alzheimer's disease (AD) patients. The intracellular trafficking and subcellular localization of γ-secretase are linked to both qualitative and quantitative changes in Aß production. However, the precise intracellular localization of γ-secretase as well as its detailed regulatory mechanisms have remained elusive. Recent genetic studies on AD provide ample evidence that alteration of the subcellular localization of γ-secretase contributes to the pathogenesis of AD. Here we review our current understanding of the intracellular membrane trafficking of γ-secretase, the association between its localization and proteolytic activity, and the possibility of γ-secretase as a therapeutic target against AD.

Histidine 131 in presenilin 1 is the pH-sensitive residue that causes the increase in Aß42 level in acidic pH.

Alzheimer disease (AD) is the most common neurodegenerative disease worldwide. The pathological hallmark of AD is the presence of senile plaques in the brain, which are accumulations of amyloid-ß peptide (Aß) ending at the 42nd residue (i.e. Aß42), which is produced through multistep cleavage by γ-secretase. Thus, methods to regulate γ-secretase activity to attenuate the production of Aß42 are in urgent demand towards the development of treatments for AD. We and others have demonstrated that γ-secretase activity is affected by its localization and ambient environment. In particular, an increase in Aß42 production is correlated with the intracellular transport of γ-secretase and endosomal maturation-dependent luminal acidification. In this study, we focused on the mechanism by which γ-secretase affects Aß42 production together with alterations in pH. Histidine is known to function as a pH sensor in many proteins, to regulate their activities through the protonation state of the imidazole side chain. Among the histidines facing the luminal side of presenilin (PS) 1, which is the catalytic subunit of γ-secretase, point mutations at H131 had no effect on the Aß42 production ratio in an acidic environment. We also observed an increase in Aß42 ratio when histidine was introduced into N137 of PS2, which is the corresponding residue of H131 in PS1. These results indicated that H131 serves as the pH sensor in PS1, which contains γ-secretase, to regulate Aß42 production depending on the luminal pH. Our findings provide new insights into therapeutic strategies for AD targeting endosomes or the intracellular transport of γ-secretase.

Molecular mechanisms of the genetic risk factors in pathogenesis of Alzheimer disease.

Alzheimer disease (AD) is a neurodegenerative disease characterized by the extensive deposition of senile plaques and neurofibrillary tangles. Until recently, only the APOE gene had been known as a genetic risk factor for late-onset AD (LOAD), which accounts for more than 95% of all AD cases. However, in addition to this well-established genetic risk factor, genome-wide association studies have identified several single nucleotide polymorphisms as genetic risk factors of LOAD, such as PICALM and BIN1. In addition, whole genome sequencing and exome sequencing have identified rare variants associated with LOAD, including TREM2. We review the recent findings related to the molecular mechanisms by which these genetic risk factors contribute to AD, and our perspectives regarding the etiology of AD for the development of therapeutic agents.

Decreased CALM expression reduces Aß42 to total Aß ratio through clathrin-mediated endocytosis of γ-secretase.

A body of evidence suggests that aberrant metabolism of amyloid-ß peptide (Aß) underlies the aetiology of Alzheimer disease (AD). Recently, a single-nucleotide polymorphism in phosphatidylinositol binding clathrin assembly protein (PICALM/CALM) gene, which encodes a protein implicated in the clathrin-mediated endocytosis, was identified as a genetic protective factor for AD, although its mechanistic details have little been explored. Here we show that loss of CALM leads to the selective decrease in the production ratio of the pathogenic Aß species, Aß42. Active form of γ-secretase is constitutively endocytosed via the clathrin-mediated pathway in a CALM dependent manner. Alteration in the rate of clathrin-mediated endocytosis of γ-secretase causes a shift in its steady-state localization, which consequently impacts on the production ratio of Aß42. Our study identifies CALM as an endogenous modulator of γ-secretase activity by regulating its endocytosis and also as an excellent target for Aß42-lowering AD therapeutics.