γ-Secretase in Alzheimer’s disease

Alzheimer’s disease (AD) is caused by synaptic and neuronal loss in the brain. One of the characteristic hallmarks of AD is senile plaques containing amyloid β-peptide (Aβ). Aβ is produced from amyloid precursor protein (APP) by sequential proteolytic cleavages by β-secretase and γ-secretase, and the polymerization of Aβ into amyloid plaques is thought to be a key pathogenic event in AD. Since γ-secretase mediates the final cleavage that liberates Aβ, γ-secretase has been widely studied as a potential drug target for the treatment of AD. γ-Secretase is a transmembrane protein complex containing presenilin, nicastrin, Aph-1, and Pen-2, which are sufficient for γ-secretase activity. γ-Secretase cleaves >140 substrates, including APP and Notch. Previously, γ-secretase inhibitors (GSIs) were shown to cause side effects in clinical trials due to the inhibition of Notch signaling. Therefore, more specific regulation or modulation of γ-secretase is needed. In recent years, γ-secretase modulators (GSMs) have been developed. To modulate γ-secretase and to understand its complex biology, finding the binding sites of GSIs and GSMs on γ-secretase as well as identifying transiently binding γ-secretase modulatory proteins have been of great interest. In this review, decades of findings on γ-secretase in AD are discussed.


INTRODUCTION
Alzheimer's disease (AD) is the most common form of dementia 1 . Two major pathological hallmarks of AD are senile plaques, which result from extracellular accumulation and deposition of amyloid β-peptide (Aβ), and neurofibrillary tangles containing the hyperphosphorylated tau protein in neurons 2,3 . AD progresses slowly, and the progression is estimated to occur 25 years prior to the onset of symptoms 4 . The current treatment for AD is to use acetylcholinesterase inhibitors and the N-methyl-D-aspartate receptor antagonist memantine for the symptomatic improvement of AD 5 , and there is no cure available. Recently, aducanumab targeting Aβ aggregates in the brain 6 was approved with some controversy.
According to the amyloid cascade hypothesis, the accumulation of Aβ in the brain is the primary cause of AD 7 . The chronic imbalance between the production and clearance rate of Aβ may lead to increased Aβ42 levels, followed by Aβ oligomerization, fibril formation, and accumulation in plaques 7 . Both Aβ oligomers and plaques damage neurons by astrocytic activation, oxidative injury, and altered kinase/phosphatase activities, followed by the formation of neurofibrillary tangles 7 . Therefore, therapeutics aimed at lowering Aβ levels could be clinically useful for the treatment of AD 7 . Genetically inherited familial Alzheimer's disease (FAD) genes also support Aβ as the key driver in the amyloid cascade hypothesis. In most cases, APP mutations increase the ratio of Aβ42/Aβ40 or total Aβ production 8 . Missense mutations, insertions, or deletions in PSEN are mostly located in the transmembrane regions or hydrophilic loops in the cytosol, and they result in an increased ratio of Aβ42/Aβ40 8 .
It has been commonly referred to as the γ-secretase cleavage to release Aβ40 or Aβ42, and AICD. The γ-secretase cleavage site can be further separated into γ-, ζ-, and ε-cleavage sites (Fig. 2) 10 . The γ-site ends at Aβ40 or Aβ42, and AICD starts at Aβ49 or Aβ50. This discrepancy with missing amino acid residues led to the new identification of the ε-cleavage site at Aβ49 [11][12][13][14] . The question of whether the γand ε-cleavages occur sequentially or independently from each other was answered by a new identification of the ζ-cleavage site at Aβ46 15,16 . Aβ peptides are cleaved mainly by tripeptide trimming via the Aβ40 product line (Aβ49→46→43→40→37) or the Aβ42 product line with the last cleavage step by tetrapeptide trimming (Aβ48→45→42→38) (Fig.  2) 17 . In addition, other Aβ peptides found in varying lengths support the link between two major Aβ40 and Aβ42 product lines and multiple interactive pathways releasing tri-, tetra-, penta-, and hexapeptides 18,19 .
The physiological role of Aβ is not yet clear. The length of Aβ found in CSF or brain varies from 37 to 43 amino acids [20][21][22] . Aβ42 is more prone to aggregate and more toxic than Aβ40, even though the ratio of production for Aβ42 and Aβ40 is approximately one to nine 23 . Aβ42 is the major component of amyloid plaques [23][24][25] , and Aβ43 was reported to exist in amyloid deposition of the human AD brain 20 .
γ-Secretase γ-Secretase carries out a sequential cleavage of the substrate C99 to generate Aβ peptides 9 . As such, γ-secretase has been an attractive target for the potential treatment of AD. However, it has been found to be challenging, and more studies are needed to fully understand γ-secretase. γ-Secretase is a transmembrane protein complex containing presenilin (PS), nicastrin, anterior pharynx defective-1 (Aph-1), and presenilin enhancer-2 (Pen-2) (Fig. 3a, b). γ-Secretase belongs to a new class of proteases, intramembrane-cleaving proteases (I-CliPs), and its unusual cleavage processes substrates in the lipid bilayer of membranes 26 .
Nicastrin was discovered by its association with PS after immunoaffinity purification using an anti-PS antibody 36 . Two additional cofactors, Aph-1 and Pen-2, were discovered by genetic screening in Caenorhabditis elegans 37,38 . Nicastrin is a single-pass transmembrane protein with a large extracellular domain. Immature nicastrin is~110 kDa, and the apparent molecular weight is increased to~130 kDa after N-glycosylation in the Golgi/ TGN compartments 39 . This mature form of nicastrin is associated with the active γ-secretase complex [39][40][41][42] . Aph-1 is required for the cell-surface localization of nicastrin 38 , and Pen-2 is required for both the expression of PS and the maturation of nicastrin 43 .
The mRNA and protein expression of the γ-secretase complex subunits is ubiquitously expressed in the body 44 . The physiological functions of γ-secretase complex subunits were studied by using KO mice. PS1 KO mice are lethal, resulting Notch signaling deficiency, while the phenotype of PS2 KO mice is normal, and double KO of PS1 and PS2 is embryonic lethal, showing a severe Notch deficiency 45,46 . Nicastrin KO mice showed a Notch phenotype with embryonic lethality 47 . Aph-1a KO mice showed embryonic lethality, and Aph-1b/c KO mice (equivalent to human Aph-1b loss) showed reduced APP processing in several regions in the adult brain 48 . A KO study in zebrafish showed that Pen-2 is important for neuronal cell survival and protects cells from apoptosis 49 .
γ-secretase activity 50 . This was also shown in Drosophila and mammalian cells [50][51][52][53][54] . Co-expression of all four components also increased the PS heterodimeric form, fully glycosylated nicastrin, and γ-secretase activity in mammalian cells 52 . In a postmortem human brain study, it was shown that human brain-derived γsecretase is present as a high molecular weight protein complex containing PS, nicastrin, Aph-1, and Pen-2 and that these are associated with γ-secretase activity 55 . The activity of the γ-secretase complex was inhibited by the specific GSI L-685,458, suggesting that this γ-secretase complex isolated from the human brain is functional 55 .
The assembly of the γ-secretase complex is initiated in the endoplasmatic reticulum (ER) 56 , where Aph-1 and nicastrin interact, followed by the binding of PS 53 . Thereafter, Pen-2 binds to the complex and facilitates the endoproteolysis of PS to PS-NTF and PS-CTF, resulting in an active γ-secretase complex 53 . In a γsecretase activity study using a biotinylated affinity ligand, it was confirmed that PS heterodimers and mature nicastrin exist in the active enzyme complex 57 . It was also reported that bacterially synthesized recombinant proteins in liposomes such as PS1-ΔE9 (FAD mutation with PS1 exon 9 deletion) alone or PS1-full-length (FL)/Pen-2 have active γ-secretase activity 58 .
Trafficking and localization of γ-secretase How APP, BACE1, and γ-secretase are trafficked and processed through subcellular compartments has been studied to identify Fig. 3 The γ-secretase complex. a γ-Secretase complexes require at least four essential components: presenilin (PS), nicastrin (Nct), Aph-1, and Pen-2. The two catalytic aspartyl residues in PS are indicated by 'D' (Asp257 in TM6 and Asp385 in TM7). PS undergoes endoproteolysis (indicated by arrow) and becomes a PS-NTF/PS-CTF heterodimer. b The γ-secretase complex structure is shown in the surface view. Presenilin (blue), nicastrin (magenta), Aph-1 (green), and Pen-2 (yellow). Rendered from Protein Data Bank entry 7D8X. The structural figure was prepared with UCSF ChimeraX 1.2.5.
the sites for Aβ production in cells. Aβ is found in the TGN 65 and endosomes 66 . The subcellular localization of Aβ in brain tissue is mainly endosomal as well 67,68 . APP is cleaved by α-secretase at the cell surface 69 , while BACE1 cleavage occurs mostly in the late Golgi/TGN and endosomes 70 . γ-Secretase components have been found in many subcellular compartments, such as the ER, ER-Golgi intermediate compartment, Golgi, TGN, endosomes, and plasma membrane [71][72][73][74] . Interestingly, PS was also found in synaptic compartments [75][76][77][78] . In addition, all four γ-secretase components were found in phagosomes 79 . PS1, nicastrin, and APP are localized in the outer membranes of lysosomes 80 .
Importantly, the sites for γ-secretase activity have been investigated. A biotinylated active site probe labeled γ-secretase in the plasma membrane of cells 73,81 . Additionally, a small fraction of active γ-secretase was found in mitochondria 82 . γ-Secretase enriched in endosomes, in the plasma membranes, and at synapses is active to produce Aβ or AICD, and active γ-secretase was labeled by a GSI in the brain or primary cortical neurons 74,83 .
The lipid membrane environment can also affect the activity of proteins. Since γ-secretase is a transmembrane-bound protein, different detergents have been used to extract proteins from membranes and study the complex. However, γ-secretase can also be studied in a membrane environment, preserving some of its natural interactions with lipids. Cholesterol and sphingolipids are the major lipid constituents of ordered microdomains called lipid rafts in cell membranes 84 . Lipid rafts are considered to be dynamic platforms for cell signaling, membrane protein sorting, and transport 84 . Several findings suggest that the trafficking and processing of APP are regulated in lipid rafts [85][86][87][88][89] . APP, BACE, and γ-secretase have been shown to localize to lipid rafts. APP and BACE residing in separate lipid rafts can merge in endosomes, where amyloidogenic processing occurs 85 . Active γ-secretase was found in lipid rafts 86 and brain lipid rafts 90 , and γ-secretase was active in lipid rafts from post-Golgi compartments and endosomes 87 . The reconstitution study of γ-secretase with different lipid mixtures showed that a lipid raft-like condition gave the highest γ-secretase activity 91 .
A recent high-throughput functional genomics screen using the FLeXSelect human FL cDNA library identified orphan G proteincoupled receptor 3 (GPR3) 92 . GPR3 appears to promote complex assembly of γ-secretase, resulting in increased trafficking of the γsecretase components and the mature γ-secretase complex to the cell surface and increased localization in lipid rafts, which eventually leads to an increase in Aβ generation 92 . Therefore, specific inhibition of γ-secretase in certain organelles or microdomains could be an attractive approach 91,93,94 , and a membraneanchored version of a β-secretase transition state inhibitor reduced enzyme activity 95 .
γ-Secretase structure The catalytic residues of I-CliPs are located within transmembrane regions, and they hydrolyze the peptide bonds of their substrates in the transmembrane regions 96 . The I-CliP family can be categorized into aspartyl proteases (including γ-secretase and signal peptide peptidase), metalloproteases (site-2 protease, Eep), and serine proteases (Rhomboid, AarA) 97 . It had been challenging for γ-secretase structure studies due to its many subunits and transmembrane domains.
An electron microscopy study on the 3D structure of γ-secretase revealed that there is a low-density interior chamber and two pores (apical and basal pores), which allow for water molecule entry into the structure 98 . These pores for water molecules could explain this unusual intramembrane cleavage (peptide bond hydrolysis) by γ-secretase 98 . Aβ and AICD could be released through two pores into outer spaces (extracellular and cytosolic spaces, respectively) 98 . In 2015, single-particle cryoelectron microscopy (cryo-EM) revealed an atomic structure of γ-secretase in a substrate-free state with a 3.4 Å resolution 99 . In recent years, cryo-EM structures of the γ-secretase complex either bound to APP (C83) at a 2.6 Å resolution or Notch (Notch-100) at a 2.7 Å resolution have shown that PS1 undergoes conformational changes upon substrate binding [100][101][102] . Substrate-bound γ-secretase showed that the β-strand from the C-terminal of APP together with two APP-induced β-strands of PS1 form a hybrid β sheet, which guides γ-secretase cleavage for substrates 101 .
Nicastrin acts as a gatekeeper for the entry of γ-secretase substrates to block substrates with long extracellular domains 103 . APP can enter, either in whole or in part, at the substrate docking site between PS-NTF and PS-CTF to access the internal active site 104 . In other words, after a substrate binds to the docking site on PS, the substrate is moved into the S1′, S2′, and S3′ sites (three substrate binding pockets) in the active site of PS by lateral gating, and long Aβ peptides are produced 105 . Then, long Aβ peptides were cleaved by tripeptide trimming (Aβ49→46→43→40→37 or Aβ48→45→42→38) to release Aβ peptides (Fig. 2) 17 .
A well-known γ-secretase substrate, Notch, undergoes ectodomain shedding by metalloprotease at the S2 site, which is further cleaved by γ-secretase at the S3 site and releases the Notch intracellular domain (NICD) (Fig. 4) 97 . Rare genetic variants of TREM2 (ex. R47H) are associated with AD 109 . The microglial surface receptor TREM2 and its adaptor protein DAP12 (TYROBP) cascade TREM2 signaling, which promotes phagocytosis 109 . After TREM2 undergoes ectodomain shedding by ADAM10, it was reported that TREM2-CTF can be cleaved by γ-secretase in cells 110 . The processing of several substrates by γ-secretase was investigated by ICD formation and the accumulation of substrate-C-terminal fragments (CTFs) by western blotting 111 . Ideally, an in vitro assay can confirm substrate cleavages 106 . Structurally, the β-strand region of several substrates (CD43, CD44, N-cadherin, ErbB4, and CD33) was aligned with the β-strand sequences of APP and Notch 112 . CD43 and CD44 closely resemble Notch 1, whereas Ncadherin, ErbB4, and CD33 share similar features with APP (C99) 112 . There is still much to be investigated for these substrate cleavages by γ-secretase and their physiological functions.

Gain or loss of function of PS
The common feature of PSEN1 or PSEN2 FAD mutations is the increased Aβ42/40 ratio. However, it has been debated whether it is due to a gain or loss of PS function that results in an increased Aβ42/40 ratio 113 . An Aβ42/40 ratio increase could be due to increased Aβ42 production, decreased Aβ40 production, or a combination of both 111 . Analysis of the formation of substrate CTFs, ICDs, and Aβ species as the effect of FAD mutations of PSEN1 or PSEN2 on the cleavage of various γ-secretase substrates, such as APP, Notch, syndecan-3, N-cadherin, and β1-integrin, showed that different mutations had a varying effect on substrate processing, indicating "variable" or "partial" loss of PS protein function, and PS2 was less efficient than PS1 111 . Reconstitution of the PS protein from 138 PS1 FAD mutations with Aph-1aL containing γ-secretase mostly decreased the production of Aβ42 and Aβ40, increased the Aβ42/40 ratio, and suggested the loss of PS1 function 114 . However, these 138 PS1 FAD mutations also showed that different mutations displayed variations in Aβ42 or Aβ40 production (increase or decrease) 114 . In addition, further studies addressing the effect of PS FAD mutations on the structure of γ-secretase and how those conformational changes could affect the cleavage of different substrates by γ-secretase remain to be investigated. For instance, E280 in PS1 forms hydrogen bonds with Y159 and Y154 102 . PS1 E280A (the Columbian mutation) disrupts hydrogen bonds and causes a local conformational change 102 .
Small molecules targeting γ-secretase Over the years, small molecule inhibitors and modulators targeting γ-secretase have been developed as potential diseasemodifying agents in AD. The main goal is to target γ-secretase and reduce toxic Aβ42 species while sparing other substrate cleavage processing by γ-secretase.

γ-Secretase inhibitors
GSIs bind to the active site of PS and inhibit γ-secretase cleavage, thereby reducing total Aβ production. GSIs such as L-685,458 35,115 , BrA-1-Bt 34 , III-31C 116 , DAPT 117 , and Merck C 57 as well as GSI-based chemical probes have been widely used to study γ-secretase. A GSIbased photoaffinity probe showed that <14% of PS1 is incorporated into active γ-secretase complexes and catalytically active while leaving the rest of PS1 in inactive γ-secretase complexes 118 . Thus, GSI-based chemical probes are critical to differentiating enzymatically active γ-secretase complexes from inactive complexes 119 . On the other hand, a co-immunoprecipitation study against γ-secretase complex components pulled down both active and inactive γsecretase complexes.
In clinical trials, GSIs such as semagacestat (LY-450,139, Eli Lilly) and avagacestat (BMS-708,163, Bristol-Myers Squibb) reduced Aβ production in AD patients 124,125 . However, the multitude of γsecretase substrates has made the development of clinically useful inhibitors difficult. Due to the decreased Notch signaling and the accumulation of APP-CTFs 122 , side effects such as the risk of skin cancer and infection, gastrointestinal bleeding, and worsening cognition led to the pause of clinical trials 5,124,125 . Therefore, these GSIs are nonselective and inhibit both APP and Notch 121,124,126 . Avagacestat was reported as a "Notch-sparing" GSI and was shown to have a higher selectivity for APP over Notch cleavage 127 . However, avagacestat was suggested to be nonselective later based on poor Notch-sparing activity 122,128 and its binding site as PS1-NTF 128 . Another "Notch-sparing" GSI, begacestat (GSI-953, Wyeth/Pfizer), was also discontinued in phase I clinical trial, and the reasons are unclear 129 . Another concern regarding GSI treatment is the Aβ rebound effect. GSIs at lower doses increased Aβ levels, and discontinuation of GSI treatment was observed with a rebound of Aβ levels 130,131 . These GSIs target PS1-NTF 128 . GSIs have been repurposed in the cancer field for Notch signaling inhibition and are currently in clinical trials.
Nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, indomethacin, and sulindac sulfide, were found to modulate γ-secretase and represent first-generation GSMs (NSAID-derived carboxylic acid GSMs) 132 . These NSAIDs lowered Aβ42 and increased Aβ38 without affecting Notch cleavage 133 . This Aβ modulation was not due to the inhibition of cyclooxygenase activity, the pharmacological target of NSAIDs 133 . Sulindac sulfide treatment showed a varying degree of Aβ42 reduction levels while increasing high Aβ38 levels in cells overexpressing PS1 FAD mutants 134 .
Second-generation GSMs were developed to improve in vivo potency and blood-brain penetrance, including NSAID-derived carboxylic acid GSMs, non-NSAID-derived imidazole GSMs, and natural product-derived GSMs 132 . Acid GSMs decrease Aβ42, increase Aβ38, and have little effect on Aβ40 levels, total Aβ levels, and NICD production 132 . GSM-1 (acid GSM, GSM-2, and GSM-10 h as close analogs) reduced Aβ42 in many PS mutants but did not reduce Aβ42 levels in cells overexpressing PS1 L166P or PS2 N141I mutants 134,135 . GSM-2 improved memory in Tg2576 mice and did not affect cognition in wild-type mice 122 . Acute and subchronic administration of GSM-10 h decreased Aβ42 with no effect on Notch signaling, and there was no Aβ rebound effect and no accumulation of APP-CTFs (C83 and C99) 136,137 . E2012 (imidazole GSM) decreased Aβ42, Aβ40, and Aβ39 and increased Aβ37 and slightly Aβ38 without affecting Notch processing 138 .
Several GSMs have entered AD clinical trials. Tarenflurbil ((R)flurbiprofen, NSAID GSM, Myriad Genetics & Laboratories) failed in phase III clinical trial due to lack of efficacy 139 . However, there were no Notch inhibition-related adverse effects 140 . A safety study in rats indicated that E2012 (Eisai) induced cataracts by inhibiting the final step in cholesterol biosynthesis 141 . Therefore, E2012 was withdrawn from the phase I clinical trial, and Eisai pursued E2212 119 . E2212 has a better safety profile than E2012 and was evaluated for safety, tolerability, pharmacokinetics, and pharmacodynamics in healthy subjects in phase I clinical trial 140 . The most common adverse effect was diarrhea 140 . PF-06648671 (Pfizer) was well tolerated at single doses in healthy subjects, lowered plasma Aβ40 and Aβ42, and increased Aβ37 and Aβ38 142 . However, this small molecule was discontinued due to Pfizer's discontinuation of R&D in neurology in 2018. EVP-0962 (NSAID GSM, Forum Pharmaceuticals, Inc.) was discontinued after the phase II clinical trial, and the results were not reported 129 . CHF5074 (acid GSM, CereSpir Incorporated, Chiesi Pharmaceuticals, Inc.) was first thought of as a GSM and improved memory and reduced microglial activation in Tg2576 mice 143 . CHF5074 lowered soluble CD40 ligand levels (microglia activation marker) 144 and is considered a microglia modulator 145 . The natural product GSM NIC5-15 (Humanetics Pharmaceuticals Corporation), which is found in soy, plants, and fruits, reduced Aβ production without affecting Notch processing (ALZFORUM, http://www.alzforum. org) 129 . Amyloid PET in APP-Swe transgenic mice revealed that chronic treatment with RO5506284 reduced de novo amyloid plaque formation 146 . The GSM-based brain imaging agent [ 11 C] SGSM-15606 was also developed and showed γ-secretase imaging in the brains of mice and macaques 147 . Recently, chronic treatment with GSM UCSD-776890 in PSAPP mice reduced amyloid deposition and microgliosis 148 .
GSI and GSM-binding sites GSIs bind to the active site of PS and inhibit the γ-secretase cleavage of APP and Notch. The mechanism of how GSMs shift Aβ cleavages from longer Aβ peptides to shorter Aβ peptides and where GSMs bind in γ-secretase complexes are still poorly understood 129 . Moreover, the physiological role of shorter Aβ peptides (Aβ38 or Aβ37) is not well understood. To understand the mechanism of action of GSMs in modifying γ-secretase activity, researchers have investigated GSM target proteins. Studies have shown that NSAID GSMs bind to APP or γsecretase 132 . GSM-1-based photoaffinity probes label PS1 or PS1-NTF, and E2012 targets PS1-NTF in γ-secretase complexes [149][150][151] . Further identification of potential GSM-binding proteins is important to understand the modulation of γ-secretase and to avoid possible side effects of GSMs in clinical trials.
Various chemical probes have been developed to identify the multiple binding sites within the active γ-secretase complex. The "photophore walking" approach to modifying GSI-based photoaffinity probes with a benzophenone group on P2, P1, P1′, and P3′ of L-685,458 (GSI compounds: L646, GY4, JC8, and L505, respectively) can label subsites such as S2, S1, S1′, and S3′ in the active site of the γ-secretase complex 149,152 . GSI GY4 photolabeling in the presence of GSM-1 altered the S1 subsite of PS1 and increased GY4 labeling 149 . These results indicated that GSM-1 (acid GSM) caused an active site shape change (conformational change) in the γ-secretase complex and that there is a GSM-1 allosteric binding site on PS1 apart from the GSI L-685,458 binding site (Fig. 5) 149 . Photolabeling by E2012-Bpyne (imidazole GSM E2012-based photoaffinity probe) also revealed PS1-NTF as the imidazole GSM-binding site within γ-secretase complexes (Fig.  5) 150 .
In summary, biochemical studies suggest that distinct GSI and GSM modulation sites exist in PS of the γ-secretase complex: binding sites for TSA GSI and allosteric GSMs (acid GSM and imidazole GSM, respectively) (Fig. 5) 132 . These different classes of small molecules occupy different distinctive sites within the γsecretase complex. Therefore, they interact and induce conformational changes in γ-secretase complexes, which lead to different Aβ cleavages 132 . For example, E2012-BPyne shows enhanced labeling of PS1-NTF in the presence of GSI L-685,458 150 . Recently, cryo-EM structure studies confirmed the different binding sites for TSA GSI (L-685,458) and imidazole GSM (E2012) found in the γsecretase complex (Fig. 5) 112 . L-685,458 binds the active site of PS1, while the imidazole GSM E2012 binds to the allosteric binding site in PS1 112 . For a non-TSA GSI, semagacestat occupies the same location as APP (C99) and Notch (N100) 112 . It suggests that semagacestat could block hybrid β sheet formation between substrates and PS1, therefore inhibiting substrate cleavages 112 . Another non-TSA GSI (avagacestat) also occupies a similar binding site as semagacestat except with some variations 112 . L-685,458, semagacestat, and avagacestat share the same binding pocket in PS1, whereas L-685,458 has an additional unique binding pocket 112 . Co-incubation with L-685,458 and E2012 also showed that E2012 binds to the interface between Nct and PS1 (Fig. 5) 112 . Yang et al. 112 suggested that GSIs and GSMs could be used in combination for a synergistic effect, and this structural information could also improve the design of substrate-selective small molecules for AD.

γ-Secretase modulatory proteins (GSMPs)
Bateman et al. 153 reported that Aβ production and clearance rates per hour in human CSF are 7.6% and 8.3%, respectively. Sporadic AD (SAD) human brain gray matter has 4.8 mg more total Aβ than healthy controls 154 . This gives the estimated Aβ accumulation rate in the brain~28 ng/hour, and a 2-5% slight increase in Aβ deposition could lead to AD in~20 years 154 . For FAD, PS FAD mutations increase the ratio of Aβ42/40, which leads to AD over several decades 155 . Therefore, reducing Aβ production by a few percent 154 by modulating γ-secretase activity with GSMs and/or other means could be therapeutic for AD patients.
Although PS, nicastrin, Aph-1, and Pen-2 are essential subunits for γ-secretase activity 50 , it is plausible that other transiently binding proteins could regulate γ-secretase activity and/or substrate specificity in different types of tissues, cells, or subcellular organelles. In addition, the reported size of the γsecretase complex varies between 200 and 2000 kDa 50,52,55 , indicating that there is a possibility of unknown components being present in the γ-secretase complex. Moreover, most of the γ-secretase complexes are inactive, while <14% are enzymatically active 118 . How the activation of inactive γ-secretase complexes occurs is still unclear 119 . It is possible that the binding of GSMPs could shift γ-secretase complexes from inactive to active. Thus, these GSMPs could be new targets to modulate γ-secretase activity in AD. Over the years, several GSMPs have been identified by using different methods: multiple purification columns followed by amino acid sequencing 156 , IP followed by mass spectrometry (MS) 62 , and tandem affinity purification 157 . To capture GSMPs in enzymatically active γ-secretase complexes, researchers used GSI pulldown or GSM photolabeling followed by MS 158,159 . The following are some of the GSMPs reported.

CD147
CD147 (also known as basigin, extracellular matrix metalloproteinase inducer (EMMPRIN)) is a transmembrane glycoprotein with two Ig-like domains, and CD147 was initially identified as a regulatory subunit of γ-secretase 156 . CD147 is ubiquitously expressed in various cells and tissues 160 and is suggested to be involved in many biological functions, such as neural-glial cell interactions, reproduction, neural function, inflammation, protein trafficking, and tumor invasion 156 . The deletion of CD147 in mice has resulted in severe defects in nervous system development, spatial learning deficits, and working memory deficits 161 . Coimmunoprecipitation with anti-PS1-CTF and nicastrin antibodies indicated that CD147 is present in the γ-secretase complex 156 . Suppression of CD147 expression by siRNA resulted in dosagedependent increases in the Aβ40 and Aβ42 levels without changes in the expression levels of the other γ-secretase components or APP substrates 156 . However, it was later questioned whether CD147 is associated with the γ-secretase complex. GSI pulldown using Merck C (biotinylated GSI L-685,458) did not co-purify CD147, indicating that CD147 is not engaged in active γsecretase complexes 162 . Vetrivel et al. 163 also reported that CD147 degrades Aβ and that it is independent of γ-secretase activity.
TMP21 TMP21 (also known as p23) was identified by immunoprecipitation with an anti-PS antibody followed by MS 62 . TMP21 is a type I transmembrane protein 164 , is a member of the p24 cargo-protein family 164 and is involved in protein transport and quality control in the ER and Golgi 165 . When TMP21 was suppressed by siRNA, Aβ production (Aβ40 and Aβ42) was increased 62,166 . TMP21 might have two pools. The major pool joins the p23 cargo family without affecting Aβ production, and the minor makes a complex with PS1 62 . Since TMP21 siRNA affects Aβ production (γ-cleavage site) but not AICD or NICD production (ε-cleavage site), TMP21 in the minor pool regulates only γ-cleavage 62 . The transmembrane domain of TMP21 interacts with γ-secretase to alter γ-cleavage 167 . Later, it was questioned whether TMP21 is truly a GSMP. It was reported that TMP21 also plays a role in APP trafficking, which affects Aβ production 168 . In addition, GSI Merck C (L-685,458 derivative) using detergent-solubilized human cell line membranes did not pulldown TMP21, suggesting that TMP21 might not be a part of active γ-secretase complexes 162,168 . However, TMP21 was found to be associated with endogenous active γsecretase complexes using GSI GCB (L-685,458 derivative) in the brain 159 and brain lipid rafts 169 . This discrepancy between cell lines and brain studies might be because TMP21 in the minor pool is transiently associated with γ-secretase, and different materials and methods were used 62,159,162,169 . Interestingly, it was easier to detect TMP21 in lipid raft-associated γ-secretase than in detergent-solubilized membranes from the brain 159,169 . For AD, TMP21 protein expression levels (normalized to nicastrin protein levels) were decreased in the brains of SAD and FAD patients 170 , and the TMP21 SNP rs12435391 was associated with SAD 171 .

GSMPs in membrane microdomains
Previously, it was reported that γ-secretase is localized at membrane microdomains called lipid rafts 86,89,90,172 . Several GSMPs in lipid raft-like microdomains or lipid rafts were identified. Proteins in the tetraspanin web were identified as GSMPs: EWI-F, CD81, CD98hc, and CD9 157 . Members of this family form lipid raftlike microdomains in cellular membranes 157 and have a role in intracellular and intercellular processes, cell fusion, cell proliferation, adhesion, and migration 173 . EWI proteins (EWI-F) form a primary interaction with tetraspanins (CD81), followed by a secondary interaction with integrins (β1-integrin), and the tetraspanin web makes a tertiary interaction with the γ-secretase complex 157 . Suppressing CD81, EWI-F, or CD98hc by siRNA decreased Aβ production 157 . CD81, CD9, and EWI-F are in γsecretase complexes, as shown by Aβ production in coimmunoprecipitates 157 . The α-secretase ADAM10 was also associated with tetraspanins for the cleavage of TNF-α and epidermal growth factor (EGF) 174 , and a new APP processing model by αand γ-secretases in the tetraspanin web was proposed 175,176 .
GSI pulldown using lipid rafts from the brain identified several endogenous GSMPs that regulate active γ-secretase. Voltagedependent anion channel 1 (VDAC1, also known as porin) and contactin-associated protein 1 (CNTNAP1, also known as Caspr) are associated with active γ-secretase complexes in brain lipid rafts, and silencing those genes in HEK293-APP695 cells decreased Aβ production 169 . VDAC is a major protein at the outer mitochondrial membrane 177 . A new member of the VDAC family, B-36 VDAC at 36 kDa, was found during the purification of the GABA A receptor and was localized at the membrane of nonpyramidal neurons in the human prefrontal cortex 178 . VDAC at the plasma membrane of neurons was also reported 179 . VDAC accumulates around amyloid plaques in APP/PS1 Tg mice 177 . Nitrated VDAC1 protein was increased in the hippocampus of AD brains 180 , and VDAC and estrogen receptor alpha in caveolae are highly expressed in AD human brains 179 . CNTNAP1 and contactin are essential for forming the paranodal junction in myelinated axons 181 . Contactin interacts with APP in neurons and brains 182,183 . CNTNAP1 interacted with APP, and overexpression of CNTNAP1 decreased Aβ production in HEK293 cells overexpressing APP V717F (Indiana mutant) 184 . Erlin-2, which is known to be located at lipid rafts from the ER, is also engaged in active γsecretase from lipid rafts to regulate Aβ levels 185 . VDAC1, Erlin-1, and Erlin-2 were previously shown to interact with PS 157 . Flotillin-2, syntaxin-binding protein 1, solute carrier family 2 member 3, and growth-associated protein 43 were also found as γ-secretaseassociated proteins in lipid rafts 186 .
Endogenous GSMPs in the brain Using a biotinylated GSI, GCB (GSI coupled to biotin via a cleavable linker, L-685,458 derivative), in the brain instead of using overexpressed cell lines identified several endogenous GSMPs. Previously, a yeast two-hybrid screening identified a synaptic plasma membrane protein, syntaxin 1A, which binds to PS1 187 . Less than 1% of syntaxin 1 associates with endogenous active γsecretase complexes in brain membranes 159 and brain lipid rafts 169 . Proton myoinositol cotransporter (SLC2A13) is another endogenous brain GSMP that regulates Aβ production without affecting Notch processing 188 .
GSI GCB also pulled down several endogenous GSMPs from synapses in the brain, such as NADH dehydrogenase iron-sulfur protein 7 (NDUFS7) from synaptic vesicles and tubulin polymerization promoting protein (TPPP) from synaptic membranes 189 . Silencing NDUFS7 decreased Aβ levels, while TPPP increased Aβ production, and both proteins were co-immunoprecipitated with Nct and PS1-CTF in the human brain 189 . Previously, it was shown that monoamine oxidase B (MAO-B) is increased in AD brains and platelets 190 . Overexpression of MAO-B increased Aβ production, and MAO-B was associated with active γ-secretase 191 . MAO-B levels were increased in neurons of AD human brains 191 .
γ-Secretase activating protein The treatment for chronic myeloid leukemia, Gleevec (an anticancer drug, imatinib mesylate, STI571), was shown to reduce Aβ production but spare Notch cleavage 192 . A biotinylated derivative of imatinib identified GSAP (γ-secretase activating proteins)-16 kDa, and GSAP is the C-terminal region of an uncharacterized protein, pigeon homologue protein (PION) 63 . GSAP-16 kDa, γsecretase, and APP-CTF form a tertiary complex 63 . Knockdown (KD) of GSAP by siRNA reduced Aβ production and did not change NICD production, and recombinant GSAP-16 kDa increased Aβ production 63 . KD of GSAP by crossing AD X 2 mice with doxycycline-inducible GSAP RNAi mice resulted in the reduction of Aβ and amyloid plaques in the brain 63 . Knockout (KO) of GSAP also decreased Aβ production while sparing Notch cleavage 193 . Overexpression of FL GSAP in GSAP KO cells increased Aβ generation 193 . Treatment with imatinib in 3XTg mice (mutant APP, mutant PS1, and mutant MAPT) decreased GSAP-16 kDa protein, Aβ production, brain Aβ deposits, and phosphorylated tau 194 .
However, the relationship between GSAP and γ-secretase for Aβ generation was later questioned by several groups 195 . Hussain et al. 195 reported that KD of GSAP decreased Aβ levels, but overexpression of GSAP-16 kDa did not increase Aβ production, and APP-CTF/PS1-CTF complexes were immunoprecipitated without GSAP. Hussain et al. 195 suggested that Aβ reduction by KD of GSAP might be due to some effects on the trafficking or assembly of γsecretase but not a direct effect of GSAP on γ-secretase. In addition, imatinib did not decrease Aβ generation in cell lines and in vivo while sparing Notch processing in cell lines. Another study also showed that, unlike GSI L-685,458, imatinib treatment did not inhibit Aβ production in cell lines, mouse primary neurons, and differentiated human embryonic stem cells 196 . In humans, imatinib treatment in chronic myeloid leukemia patients for up to 12 months also did not result in an Aβ decrease in plasma 196 . In contrast, overexpression of GSAP-FL in GSAP KO cells rescued γ-secretase activity, and the dual GSI photoprobe L631 for PS1-NTF and PS1-CTF labeled PS1-NTF, PS1-CTF, and FL PS1 when GSAP-FL was overexpressed in GSAP KO cells compared to KO cells 193 . This result suggested that the presence of GSAP aligned PS1-NTF and PS1-CTF in a specific confirmation with higher γ-secretase activity for Aβ cleavage 193 . In human brains, an immunohistochemistry study showed that GSAP-positive deposits are present both in control and AD brains, while the quantification of GSAP-positive deposits is higher in AD brains, and these GSAP-positive deposits are closely localized to PS1 and Aβ deposits in AD brains 197 . It was also reported that the GSAP SNP rs4727380 was associated with APOE4 noncarriers of AD patients from Han Chinese in a small sample size 198 .

GSMPs induced by other factors
Hif-1α was identified as a GSMP for Notch processing. Hif-1 expression is upregulated by aging in the frontal cortex of the human brain 199 , and stroke increases the risk for dementia 200 . Brain ischemia/hypoxia-induced Aβ deposits in the human brain 201 . The BACE1 gene contains a hypoxia response element (HRE) in the promoter region, and hypoxia increases BACE1 protein expression as well as β-secretase cleavage for APP 202 . A transcription factor, Hif-1α (hypoxia-inducible factor-1α), works as an oxygen sensor, and Hif-1α is degraded by the ubiquitinproteasome system under normoxia 203 . Under hypoxia, the canonical hypoxic response leads to the binding of Hif-1α/Hif-1β to HRE elements in the promoter regions of several genes, such as vascular endothelial growth factor, erythropoietin (Epo), and glucose transporters 1, for angiogenesis, erythropoiesis, and energy metabolism 203 . Gustafsson et al. 204 discovered crosstalk between the noncanonical pathway of Hif-1α and Notch signaling. Under hypoxia, Hif-1α binds to the NICD and induces Notch downstream genes such as Hes and Hey for the undifferentiated cell state in the stem cell population 204 . Villa et al. 64 found that hypoxia also increases active γ-secretase complex formation and upregulates γ-secretase activity to cleave Notch. Nontranscriptional Hif-1α converts the pool of inactive γ-secretase to active γsecretase, and GSI-34 decreases hypoxia-induced cell invasion and metastatic progression in cells and animal models of breast cancer 64 .
GPCRs require adaptor proteins such as arrestins to prevent further G protein-mediated signaling 206 . β-Arrestin1 is highly expressed in the brain 206 , and β-arrestin1 KO mice exhibit reduced Aβ production and spared Notch cleavage 207 . β-arrestin1 interacts only with Aph-1 in γ-secretase, and overexpression of β-arrestin1 enhanced mature γ-secretase complex formation at 440 kDa 207 . KO of β-arrestin1 in APP/PS1 mice decreased Aβ production and improved memory deficits 207 . Stress-associated endoplasmic reticulum protein 1 (SERP1) was also reported to regulate the assembly of γ-secretase complexes and contribute to Aβ pathogenesis 208 . SERP1 interacts with the Aph-1a/Nct subcomplex of γ-secretase and increases γ-secretase activity for Aβ generation but reduces Notch processing 208 .

GSMP in neuroinflammation
Recently, IFITM3 (interferon-induced transmembrane protein 3, also known as fragilis) was identified as an imidazole GSM, E2012, Fig. 6 Aβ production by IFITM3-γ-secretase complexes. Normally, active γ-secretase cleaves its substrate to release Aβ. Under inflammatory conditions such as aging and infection, proinflammatory cytokines are induced by microglia and astrocytes. These cytokines upregulate IFITM3 protein expression in astrocytes and neurons, which in turn increases the processing of APP-CTF (C99) by active IFITM3-γ-secretase complexes to produce Aβ40 and Aβ42. The accumulation of amyloid leads to amyloid build-ups in the brain. Note that less than 14% of γsecretase complexes are enzymatically active, while the rest are inactive.
binding protein 158 . Photolabeling with E2012-BPyne (an E2012based photoaffinity probe) followed by LC-MS/MS identified IFITM3 at 15 kDa as a GSMP 158 . IFITM3 plays a role in innate immunity as an antiviral protein that restricts viral protein entry into host cell membranes by inhibiting membrane fusion 209 . IFITM3 KO mice are susceptible to viral infections 210 . Previously, microarray analysis and RT-PCR showed 19.9-and 3.4-fold increases in IFITM3 in SAD brains 211 . Hur et al. 158 showed that IFITM3 binds to PS1-NTF in active γ-secretase complexes and regulates γ-secretase activity for Aβ production (Aβ40 and Aβ42) (Fig. 6). KD or KO of IFITM3 decreased Aβ production, and overexpression of IFITM3 increased Aβ levels in IFITM3 KO cells 158 . Moreover, crossing IFITM3 KO mice with 5XFAD Tg mice decreased Aβ production and amyloid plaque formation in the cortex and hippocampus 158 . Aging mouse models also showed increased IFITM3 levels, γ-secretase activity, and active IFITM3-γsecretase complex formation levels by aging 158 . A positive correlation between the amount of active IFITM3-γ-secretase complexes and the high γ-secretase activity resulting in high Aβ production was shown in the subsets of SAD patient brains expressing high IFITM3 protein levels 158 . Proinflammatory cytokines such as Type I IFN or Type II IFN can induce IFITM3 protein expression, increase the engagement of IFITM3 in active γsecretase complexes, and increase Aβ production in mouse primary cortical neurons 158 . This result shows the direct link between inflammation and Aβ production via IFITM3-γ-secretase in neurons 158,212,213 . IFITM3 modulates γ-secretase under inflammation in neurons and astrocytes and may contribute to aging and the pathogenesis of AD 158 . The "antimicrobial protection hypothesis of AD" proposes that Aβ is beneficial as an antimicrobial peptide and that Aβ fibrilization entraps bacteria and viruses as an innate immune response to pathogens 214 . The involvement of IFITM3 in Aβ production might suggest the role of the "neuronal innate immune response" against pathogens, and Aβ has resulted as a protective pathway against infection 212 . At the same time, the accumulation of Aβ poses a risk of developing AD 158 . Further studies on regulating other γ-secretase substrates by IFITM3-γ-secretase complexes are needed to understand possible adverse effects when targeting IFITM3 in AD.

CONCLUSION
Aβ is the key driver in AD according to the amyloid cascade hypothesis. Since γ-secretase cleaves its immediate substrate APP-CTF to release Aβ, which causes AD, and its unique biology as a transmembrane protein complex enzyme is still much to be learned, γ-secretase is still interesting to study. How γ-secretase cleaves over 100 substrates and how those signaling cascades could result in different physiological functions remain to be determined in the future. To validate γ-secretase as an Aβ modifying drug, further studies on the regulation/modulation of γsecretase by GSMs and transiently binding GSMPs are needed. In addition, the effects of GSMs and GSMPs on different substrate processing need to be elucidated. This knowledge could advance the development of AD-modifying drugs by selectively inhibiting APP processing by γ-secretase.