# Specific Mutations in Aph1 Cause \(\gamma\)-Secretase Activation
## 3 Discussion  
In this study, we identified Aph1 mutations that increase \(\gamma\)-secretase activity using random mutagenesis by error-prone PCR and a yeast reconstitution system. Analysis using a yeast reporter system, an in vitro assay using microsomes, and MEF cells showed that the Aph1 aL L30F/T164A double mutation increased the \(\varepsilon\)-cleavage activity of \(\gamma\)-secretase and A\(\beta\) production. Previous studies analyzing mutations in Aph1 showed that conserved amino acids in the transmembrane region--Gln83, Glu84, Arg87 (TMD3), Gly122, Gly126 (GxxxG motif, TMD4), His171 (TMD5), and His197 (TMD6)--are important for \(\gamma\)-secretase complex formation and activity [33]. However, these analyses only introduced mutations in targeted regions and amino acids; they did not assess random mutations along the entire length of the protein. To our knowledge, this is the first report of Aph1a mutations that increase \(\gamma\)-secretase activity. The role of Aph1 as a scaffold in complex formation is well-known [9; 34; 35]. Because the mutation found in this study may have also affected complex formation, we examined the amount of complex by immunoprecipitation. The amounts of Aph1 aL and PS1 fragments (NTF or CTF) in the complex were identical in the Aph1 aL WT and mutant in yeast and MEF cells. Since the endoproteolytic fragments of PS1 reflect the formation of the full complement active \(\gamma\)-secretase complex with NCT and Pen2, these results suggest that the mutation directly affected activity, rather than complex-forming ability. Cryo-EM analysis of the \(\gamma\)-secretase structure revealed that the Leu30 of Aph1 is located on the amino-terminal side of TMD1 of PS1, while Thr164 is close to the carboxy-terminus of PS1 (Figure 7). The conformation of TMD1 of PS1 changes upon binding E2012, a phenylimidazole-type GSM; it is predicted to undergo piston movement [36]. Therefore, we predict that the L30F mutation in Aph1 aL affected the conformation of TMD1 in PS1, resulting in altered activity. The carboxy terminus of PS1 forms substrate-binding sites in cooperation with the hydrophilic loop 1 (HL1) [24]. The distance between the oxygen atom of Thr164 of Aph1 and the oxygen atom of Tyr466 of PS1 is predicted to be 3.5 A, suggesting that a hydrogen bond (H...O distance, approximately 1.8 A) is formed in these side chains (Figure 7b). The T164A mutation may eliminate the hydrogen bond with PS1 Y466, resulting in flexible movement of the carboxy terminus of PS1. We propose that Aph1 mutations regulate protease activity through conformational changes in PS1. This study revealed that Aph1 aL may be actively involved in \(\gamma\)-secretase catalytic activity. We analyzed the production of different A\(\beta\) species and found that the ratio of long A\(\beta\) species (A\(\beta\)42 and A\(\beta\)43) with A\(\beta\)40 was similar between the Aph1 aL WT and L30F/T164A double mutant. This finding will lead to a detailed understanding of the generation of A\(\beta\) molecular species (A\(\beta\)42 and A\(\beta\)43) in AD, as well as new \(\gamma\)-secretase inhibitors or modulators targeting Aph1. In a future study, it will be interesting to measure ROS or other biomarkers in apoptotic pathways to analyze the neurotoxicity caused by the Aph1 aL mutant. As for the \(\gamma\)-secretase mutations, more than 150 mutations in PS1 are associated with familial Alzheimer's disease (FAD) [1]. Recent genome-wide association studies identified Aph1b as an Alzheimer risk gene, and Aph1b T27I is likely the single causal variant [37]. In addition, SNPs in a promoter region of the Aph1a gene was associated with risk for developing sporadic Alzheimer's disease in a Chinese population [38]. In acne inversa, also known as hidradenitis suppurativa, loss of function mutations were identified in PS1, NCT, and Pen2, but not in Aph1 [39].