Yue-Ming Li

Development and Mechanism of γ-Secretase Modulators for Alzheimer’s Disease

γ-Secretase is an aspartyl intramembranal protease composed of presenilin, Nicastrin, Aph1, and Pen2 with 19 transmembrane domains. γ-Secretase cleaves the amyloid precursor proteins (APP) to release Aβ peptides that likely play a causative role in the pathogenesis of Alzheimers disease (AD). In addition, γ-secretase cleaves Notch and other type I membrane proteins. γ-Secretase inhibitors (GSIs) have been developed and used for clinical studies. However, clinical trials have shown adverse effects of GSIs that are potentially linked with nondiscriminatory inhibition of Notch signaling, overall APP processing, and other substrate cleavages. Therefore, these findings call for the development of disease-modifying agents that target γ-secretase activity to lower levels of Aβ42 production without blocking the overall processing of γ-secretase substrates. γ-Secretase modulators (GSMs) originally derived from nonsteroidal anti-inflammatory drugs (NSAIDs) display such characteristics and are the focus of this review. However, first-generation GSMs have limited potential because of the low potency and undesired neuropharmacokinetic properties. This generation of GSMs has been suggested to interact with the APP substrate, γ-secretase, or both. To improve the potency and brain availability, second-generation GSMs, including NSAID-derived carboxylic acid and non-NSAID-derived heterocyclic chemotypes, as well as natural product-derived GSMs have been developed. Animal studies of this generation of GSMs have shown encouraging preclinical profiles. Moreover, using potent GSM photoaffinity probes, multiple studies unambiguously have showed that both carboxylic acid and heterocyclic GSMs specifically target presenilin, the catalytic subunit of γ-secretase. In addition, two types of GSMs have distinct binding sites within the γ-secretase complex and exhibit different Aβ profiles. GSMs induce a conformational change of γ-secretase to achieve modulation. Various models are proposed and discussed. Despite the progress of GSM research, many outstanding issues remain to be investigated to achieve the ultimate goal of developing GSMs as effective AD therapies.

Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 γ-Secretase Complexes

γ-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Aβ42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of γ-secretase, invariably increase the Aβ42:Aβ40 ratio. However, the mechanism by which these mutations affect γ-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active γ-secretase complexes. Furthermore we reveal that the equilibrium of PS1- and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased Aβ42:Aβ40 ratios. These data suggest that perturbations to γ-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased Aβ42:Aβ40 ratio.

Familial Alzheimer Disease Presenilin-1 Mutations Alter the Active Site Conformation of γ-secretase

Background: The effect of PS1 mutations on the active site of γ-secretase is unknown. Results: PS1 mutations reduce photoprobe interaction with the S2 subsite of γ-secretase and Notch1 cleavage. Conclusion: Certain PS mutations specifically alter the S2 subsite of γ-secretase active site that leads to changes in APP and Notch1 cleavage. Significance: It provides structural insights into the mechanism of PS1 mutations in regards to γ-secretase regulation. Presenilin-1 (PS1) is the catalytic subunit of γ-secretase, and mutations in this protein cause familial Alzheimer Disease (FAD). However, little is known about how these mutations affect the active site of γ-secretase. Here, we show that PS1 mutations alter the S2 subsite within the active site of γ-secretase using a multiple photoaffinity probe approach called “photophore walking.“ Moreover, we developed a unique in vitro assay with a biotinylated recombinant Notch1 substrate and demonstrated that PS1 FAD mutations directly and significantly reduced γ-secretase activity for Notch1 cleavage. Substitution of the Notch Cys-1752 residue, which interacts with the S2 subsite, with Val, Met, or Ile has little effect on wild-type PS1 but leads to more efficient substrates for mutant PS1s. This study indicates that alteration of this S2 subsite plays an important role in determining the activity and specificity of γ-secretase for APP and Notch1 processing, which provides structural basis and insights on how certain PS1 FAD mutations lead to AD pathogenesis.

Structure of γ-Secretase and Its Trimeric Pre-activation Intermediate by Single-particle Electron Microscopy

The γ-secretase membrane protein complex is responsible for proteolytic maturation of signaling precursors and catalyzes the final step in the production of the amyloid β-peptides implicated in the pathogenesis of Alzheimer disease. The incorporation of PEN-2 (presenilin enhancer 2) into a pre-activation intermediate, composed of the catalytic subunit presenilin and the accessory proteins APH-1 (anterior pharynx-defective 1) and nicastrin, triggers the endoproteolysis of presenilin and results in an active tetrameric γ-secretase. We have determined the three-dimensional reconstruction of a mature and catalytically active γ-secretase using single-particle cryo-electron microscopy. γ-Secretase has a cup-like shape with a lateral belt of ∼40–50 Å in height that encloses a water-accessible internal chamber. Active site labeling with a gold-coupled transition state analog inhibitor suggested that the γ-secretase active site faces this chamber. Comparison with the structure of a trimeric pre-activation intermediate suggested that the incorporation of PEN-2 might contribute to the maturation of the active site architecture.

Dual Role of α-Secretase Cleavage in the Regulation of γ-Secretase Activity for Amyloid Production

Processing of the amyloid precursor protein (APP) by β- and γ-secretases generates pathogenic β-amyloid (Aβ) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by α-secretases precludes Aβ formation. Little is known about the role of α-secretase cleavage in γ-secretase regulation. Here, we show that α-secretase-cleaved APP C-terminal product (αCTF) functions as an inhibitor of γ-secretase. We demonstrate that the substrate inhibitory domain (ASID) within αCTF, which is bisected by the α-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns αCTF into a better substrate for γ-secretase. Moreover, α-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of γ-secretase activity by αCTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for α-secretase in generating an endogenous γ-secretase inhibitor that down-regulates the production of Aβ. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.

Identification of a tetratricopeptide repeat-like domain in the nicastrin subunit of γ-secretase using synthetic antibodies

The γ-secretase complex, composed of presenilin, anterior-pharynx-defective 1, nicastrin, and presenilin enhancer 2, catalyzes the intramembranous processing of a wide variety of type I membrane proteins, including amyloid precursor protein (APP) and Notch. Earlier studies have revealed that nicastrin, a type I membrane-anchored glycoprotein, plays a role in γ-secretase assembly and trafficking and has been proposed to bind substrates. To gain more insights regarding nicastrin structure and function, we generated a conformation-specific synthetic antibody and used it as a molecular probe to map functional domains within nicastrin ectodomain. The antibody bound to a conformational epitope within a nicastrin segment encompassing residues 245–630 and inhibited the processing of APP and Notch substrates in in vitro γ-secretase activity assays, suggesting that a functional domain pertinent to γ-secretase activity resides within this region. Epitope mapping and database searches revealed the presence of a structured segment, located downstream of the previously identified DAP domain (DYIGS and peptidase; residues 261–502), that is homologous to a tetratricopeptide repeat (TPR) domain commonly involved in peptide recognition. Mutagenesis analyses within the predicted TPR-like domain showed that disruption of the signature helical structure resulted in the loss of γ-secretase activity but not the assembly of the γ-secretase and that Leu571 within the TPR-like domain plays an important role in mediating substrate binding. Taken together, these studies offer provocative insights pertaining to the structural basis for nicastrin function as a “substrate receptor” within the γ-secretase complex.

Intron 2 (T/C) CYP46 Polymorphism Is Associated with Alzheimer’s Disease in Chinese Patients

Background: Cholesterol metabolism has been implicated in the pathophysiology of Alzheimer’s disease (AD), and cholesterol-related genes are plausible candidate genes for AD. Genetic association of CYP46A1 polymorphisms with AD had been under extensive investigations; however, observations on intron 2 T→C (rs754203) generated inconclusive results. Objective: To analyse an independent data set in a Chinese population to see whether the polymorphic site rs754203 of the CYP46A1 gene is associated with AD. Methods: We analysed 130 sporadic AD patients and 110 healthy controls of the Southern Chinese origin. Results: An association between the genotype frequency and AD was suggested in the general population (p = 0.047, odds ratio, OR = 1. 61, 95% confidence interval, CI = 0.96–2.70), while the association was most significant in the apolipoprotein E (ApoE) Ε4-negative group (p = 0.004, OR = 2.54, 95% CI =1.31–4.95). Linkage disequilibrium block prediction results also favoured this association. Consistent with previous reports, intron 3 C→T (rs4900442) polymorphism did not show any evidence of association; in our data set ApoEΕ4 was confirmed to be a genetic risk factor for AD (p = 0.0016, OR = 2.76, 95% CI = 1.50–5.11).

Soluble γ-Secretase Modulators Selectively Inhibit the Production of the 42-Amino Acid Amyloid β Peptide Variant and Augment the Production of Multiple Carboxy-Truncated Amyloid β Species

Alzheimer’s disease (AD) is characterized pathologically by an abundance of extracellular neuritic plaques composed primarily of the 42-amino acid amyloid β peptide variant (Aβ42). In the majority of familial AD (FAD) cases, e.g., those harboring mutations in presenilin 1 (PS1), there is a relative increase in the levels of Aβ42 compared to the levels of Aβ40. We previously reported the characterization of a series of aminothiazole-bridged aromates termed aryl aminothiazole γ-secretase modulators or AGSMs [Kounnas, M. Z., et al. (2010) Neuron 67, 769–780] and showed their potential for use in the treatment of FAD [Wagner, S. L., et al. (2012) Arch. Neurol. 69, 1255–1258]. Here we describe a series of GSMs with physicochemical properties improved compared to those of AGSMs. Specific heterocycle replacements of the phenyl rings in AGSMs provided potent molecules with improved aqueous solubilities. A number of these soluble γ-secretase modulators (SGSMs) potently lowered Aβ42 levels without inhibiting proteolysis of Notch or causing accumulation of amyloid precursor protein carboxy-terminal fragments, even at concentrations approximately 1000-fold greater than their IC50 values for reducing Aβ42 levels. The effects of one potent SGSM on Aβ peptide production were verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, showing enhanced production of a number of carboxy-truncated Aβ species. This SGSM also inhibited Aβ42 peptide production in a highly purified reconstituted γ-secretase in vitro assay system and retained the ability to modulate γ-secretase-mediated proteolysis in a stably transfected cell culture model overexpressing a human PS1 mutation validating the potential for use in FAD.

Characterization of an Atypical γ-Secretase Complex from Hematopoietic Origin

Gamma-secretase is a widely expressed multisubunit enzyme complex which is involved in the pathogenesis of Alzheimer disease and hematopoietic malignancies through its aberrant processing of the amyloid precursor protein (APP) and Notch1, respectively. While gamma-secretase has been extensively studied, there is a dearth of information surrounding the activity, composition, and function of gamma-secretase expressed in distinct cellular populations. Here we show that endogenous gamma-secretase complexes of hematopoietic origin are distinct from epithelial derived gamma-secretase complexes. Hematopoietic gamma-secretase has reduced activity for APP and Notch1 processing compared to epithelial gamma-secretase. Characterization of the active complexes with small molecule affinity probes reveals that hematopoietic gamma-secretase has an atypical subunit composition with significantly altered subunit stoichiometry. Furthermore, we demonstrate that these discrete complexes exhibit cell-line specific substrate selectivity suggesting a possible mechanism of substrate regulation. These data underscore the need for studying endogenous gamma-secretase to fully understand of the biology of gamma-secretase and its complexity as a molecular target for the development of disease therapeutics.

γ-Secretase Inhibitors and Modulators Induce Distinct Conformational Changes in the Active Sites of γ-Secretase and Signal Peptide Peptidase

γ-Secretase inhibitors (GSIs) and modulators (GSMs) are at the frontline of cancer and Alzheimers disease research, respectively. While both are therapeutically promising, not much is known about their interactions with proteins other than γ-secretase. Signal peptide peptidase (SPP), like γ-secretase, is a multispan transmembrane aspartyl protease that catalyzes regulated intramembrane proteolysis. We used active site-directed photophore walking probes to study the effects of different GSIs and GSMs on the active sites of γ-secretase and SPP and found that nontransition state GSIs inhibit labeling of γ-secretase by activity-based probes but enhance labeling of SPP. The opposite is true of GSMs, which have little effect on the labeling of γ-secretase but diminish labeling of SPP. These results demonstrate that GSIs and GSMs are altering the structure of not only γ-secretase but also SPP, leading to potential changes in enzyme activity and specificity that may impact the clinical outcomes of these molecules.