# Specific Mutations in Aph1 Cause \(\gamma\)-Secretase Activation
## 1 Introduction  
\(\gamma\)-Secretase is an intramembrane-cleaving protease responsible for generating the amyloid beta peptide (A\(\beta\)), the accumulation and deposition of which is a major pathological hallmark of Alzheimer's disease (AD) [1]. A\(\beta\) is generated from amyloid precursor protein (APP) by sequential cleavages of \(\beta\)- and \(\gamma\)-secretase [2, 3]. In the first step, the extracellular portion of APP is cleaved by \(\beta\)-secretase, known as BACE1, to generate a \(\beta\)-carboxyl terminal fragment (\(\beta\)-CTF). \(\gamma\)-Secretase cleaves \(\beta\)-CTF in its transmembrane domain through two different protease activities [3]. An initial endopeptidase-like cleavage of \(\beta\)-CTF releases the APP intracellular domain (AICD), giving rise to A\(\beta\)48 or A\(\beta\)49. Further stepwise carboxypeptidase-like trimming occurs to produce major A\(\beta\) species composed of 38-43 amino acids [4, 5]. The longer A\(\beta\)42 and A\(\beta\)43 species are highly aggregation-prone and neurotoxic, compared with the shorter A\(\beta\)s (A\(\beta\)38 and A\(\beta\)40) [1, 6]. An increase in the ratio of A\(\beta\)42/43 species leads to the accumulation of A\(\beta\) fibrils and the deposition of senile plaques in the brain, resulting in AD [7, 8]. Recently, \(\gamma\)-secretase modulators (GSM) were found to alter the ratios of A\(\beta\) species, particularly decreasing the ratios of longer A\(\beta\)s [3]. Small molecules reducing toxic A\(\beta\)42/43 have been proposed as a potential cure, and GSM is a potential therapeutic agent for the treatment of AD. \(\gamma\)-Secretase is a protein complex composed of four membrane proteins: presenilin (PS1 or PS2), nicastrin (NCT), anterior pharynx-1 (Aph1), and presenilin enhancer 2 (Pen2) [9]. PS1 possesses nine transmembrane domains (TMD1-9); TMD6 and TMD7 contain two catalytic aspartic acids [10; 11]. Upon assembly and maturation of the complex, PSI is cleaved in the loop between TMD6 and TMD7, thereby generating N-terminal fragments (NTFs) composed of TMD1-6 and C-terminal fragments (CTFs) composed of TMD7-9 [3]. More than 150 pathological mutations in PS1 are associated with familial Alzheimer's disease (FAD) [1]. These clinical PS1 mutations negatively affect \(\gamma\)-secretase activity, resulting in a decrease in A\(\beta\) generation. Decreased trimming activity leads to increases in the ratios of A\(\beta\)42 or A\(\beta\)43, which are responsible for AD [12; 13]. The four transmembrane domains (TMD1-4) of Aph1 interact with PS1 and NCT [14]. To form the \(\gamma\)-secretase complex, Aph1 first binds NCT; it then binds PS1 and Pen2 [9]. Thus, Aph1 has an important role as a scaffold protein and stabilizes PS1 holoprotein in the complex, whereas Pen2 is required for endoproteolytic processing of PS1 and conferring \(\gamma\)-secretase activity of the complex [9]. Two Aph1 homologs, Aph1a and Aph1b, have been identified in humans. The \(\gamma\)-secretase complex containing Aph1b has been shown to produce more A\(\beta\)42 than the complex containing Aph1a, the major homolog; different conformations and functions have been proposed for the two Aph1 homologs [15]. Structural analyses of \(\gamma\)-secretase have provided insight into its cleavage mechanism. Cryo-electron microscopy (EM) of \(\gamma\)-secretase revealed that the transmembrane domains of each subunit are arranged in a horseshoe shape and the extracellular domain of NCT rests on the transmembrane helices [16]. PS1 exhibits a hydrophilic catalytic pore structure, which is water-accessible (according to the findings in biochemical studies) [17; 18; 19]. Aph1 transmembrane domains are in contact with PS1 transmembrane domains and the C-terminal loop, as well as NCT extracellular and transmembrane domains [20]. Pen2 are in contact with PS1 transmembrane and NCT extracellular domains on the opposite side of the horseshoe. Lateral movement of \(\beta\)-CTF into the pore structure has been proposed as a cleavage mechanism [21].