Structural and Functional Determinants of γ-Secretase, an Intramembrane Protease Implicated in Alzheimer’s Disease

Alzheimer’s disease is the most common form of neurodegenerative diseases in humans, characterized by the progressive accumulation and aggregation of amyloid-β peptides (Aβ) in brain regions subserving memory and cognition. These 39-43 amino acids long peptides are generated by the sequential proteolytic cleavages of the amyloid-β precursor protein (APP) by β- and γ-secretases, with the latter being the founding member of a new class of intramembrane-cleaving proteases (I-CliPs) characterized by their intramembranous catalytic residues hydrolyzing the peptide bonds within the transmembrane regions of their respective substrates. These proteases include the S2P family of metalloproteases, the Rhomboid family of serine proteases, and two aspartyl proteases: the signal peptide peptidase (SPP) and γ-secretase. In sharp contrast to Rhomboid and SPP that function as a single component, γ-secretase is a multi-component protease with complex assembly, maturation and activation processes. Recently, two low-resolution three-dimensional structures of γ-secretase and three high-resolution structures of the GlpG rhomboid protease have been obtained almost simultaneously by different laboratories. Although these proteases are unrelated by sequence or evolution, they seem to share common functional and structural mechanisms explaining how they catalyze intramembrane proteolysis. Indeed, a water-containing chamber in the catalytic cores of both γ-secretase and GlpG rhomboid provides the hydrophilic environment required for proteolysis and a lateral gating mechanism controls substrate access to the active site. The studies that have identified and characterized the structural determinants critical for the assembly and activity of the γ-secretase complex are reviewed here

The Alzheimer’s disease γ-secretase generates higher 42:40 ratios for β-amyloid than for p3 peptides

Alzheimer’s disease is characterized by intracerebral deposition of β-amyloid (Aβ). While Aβ40 is the most abundant form, neurotoxicity is mainly mediated by Aβ42. Sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases gives rise to full-length Aβ (Aβ1-x) and N-terminally truncated Aβ′ (Aβ11-x) whereas cleavage by α- and γ-secretases leads to the shorter p3 peptides (Aβ17-x). We uncovered significantly higher ratios of 42- versus 40-ending variants for Aβ and Aβ′ than for p3 secreted by mouse neurons and human induced pluripotent stem cell (iPSC)-derived neurons or produced in a cell-free γ-secretase assay with recombinant APP-CTFs. The 42:40 ratio was highest for Aβ′, followed by Aβ and then p3. Mass spectrometry analysis of APP intracellular domains revealed differential processing of APP-C83, APP-C89, and APP-C99 by γ-secretase already at the ε-cleavage stage. This mechanistic insight could aid in developing substrate-targeted modulators of APP-C99 processing to specifically lower the Aβ42:Aβ40 ratio without compromising γ-secretase function

The adipocyte differentiation protein APMAP is an endogenous suppressor of Aβ production in the brain

The deposition of amyloid-beta (Aβ) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aβ is generated from the cleavage of C-terminal fragments of the amyloid precursor protein (APP-CTFs) by γ-secretase, an intramembrane-cleaving protease with multiple substrates, including the Notch receptors. Endogenous modulation of γ-secretase is pointed to be implicated in the sporadic, age-dependent form of AD. Moreover, specifically modulating Aβ production has become a priority for the safe treatment of AD because the inhibition of γ-secretase results in adverse effects that are related to impaired Notch cleavage. Here, we report the identification of the adipocyte differentiation protein APMAP as a novel endogenous suppressor of Aβ generation. We found that APMAP interacts physically with γ-secretase and its substrate APP. In cells, the partial depletion of APMAP drastically increased the levels of APP-CTFs, as well as uniquely affecting their stability, with the consequence being increased secretion of Aβ. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aβ production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aβ production through its interaction with APP and γ-secretase. All observed APMAP phenotypes can be explained by an impaired degradation of APP-CTFs, likely caused by an altered substrate transport capacity to the lysosomal/autophagic syste

The FDA-approved natural product dihydroergocristine reduces the production of the Alzheimer's disease amyloid-beta peptides

Known gamma-secretase inhibitors or modulators display an undesirable pharmacokinetic profile and toxicity and have therefore not been successful in clinical trials for Alzheimer's disease (AD). So far, no compounds from natural products have been identified as direct inhibitors of gamma-secretase. To search for bioactive molecules that can reduce the amount of amyloid-beta peptides (A beta) and that have better pharmacokinetics and an improved safety profile, we completed a screen of similar to 400 natural products by using cell-based and cell-free gamma-secretase activity assays. We identified dihydroergocristine (DHEC), a component of an FDA-(Food and Drug Administration)-approved drug, to be a direct inhibitor of gamma-secretase. Micromolar concentrations of DHEC substantially reduced A beta levels in different cell types, including a cell line derived from an AD patient. Structure-activity relationship studies implied that the key moiety for inhibiting gamma-secretase is the cyclized tripeptide moiety of DHEC. A Surface Plasmon Resonance assay showed that DHEC binds directly to gamma-secretase and Nicastrin, with equilibrium dissociation constants (K-d) of 25.7 nM and 9.8 mu M, respectively. This study offers DHEC not only as a new chemical moiety for selectively modulating the activity of gamma-secretase but also a candidate for drug repositioning in Alzheimer's disease

Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified γ-Secretase Complexes

BACKGROUND: Mutations linked to early onset, familial forms of Alzheimer's disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1-42/Aβ1-40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1-42 production, decreasing Aβ1-40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q. METHODOLOGY/PRINCIPAL FINDINGS: We took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1-40, Aβ1-42 and APP intracellular domain productions in vitro. CONCLUSION/SIGNIFICANCE: Our data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1-42/Aβ1-40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer's disease

The metalloprotease ADAMTS4 generates N-truncated Aβ4–x species and marks oligodendrocytes as a source of amyloidogenic peptides in Alzheimer’s disease

Brain accumulation and aggregation of amyloid-β (Aβ) peptides is a critical step in the pathogenesis of Alzheimer’s disease (AD). Full-length Aβ peptides (mainly Aβ1–40 and Aβ1–42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble Aβ peptides are truncated at the N-terminus, with Aβ4–x peptides being particularly abundant. Aβ4–x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the Aβ peptide sequence, which facilitates Aβ4–x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of Aβ4–40 but unchanged levels of Aβ1–x peptides. In the 5xFAD mouse model of amyloidosis, Aβ4–x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4−/− knockout background, Aβ4–40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of Aβ species, but Aβ4–40 peptides were absent in cultures derived from ADAMTS4−/− mice indicating that the enzyme was essential for Aβ4–x production in this cell type. These findings establish an enzymatic mechanism for the generation of Aβ4–x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic Aβ peptides

Gene Expression Profiling in Cells with Enhanced γ-Secretase Activity

BACKGROUND: Processing by gamma-secretase of many type-I membrane protein substrates triggers signaling cascades by releasing intracellular domains (ICDs) that, following nuclear translocation, modulate the transcription of different genes regulating a diverse array of cellular and biological processes. Because the list of gamma-secretase substrates is growing quickly and this enzyme is a cancer and Alzheimer's disease therapeutic target, the mapping of gamma-secretase activity susceptible gene transcription is important for sharpening our view of specific affected genes, molecular functions and biological pathways. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes and molecular functions transcriptionally affected by gamma-secretase activity, the cellular transcriptomes of Chinese hamster ovary (CHO) cells with enhanced and inhibited gamma-secretase activity were analyzed and compared by cDNA microarray. The functional clustering by FatiGO of the 1,981 identified genes revealed over- and under-represented groups with multiple activities and functions. Single genes with the most pronounced transcriptional susceptibility to gamma-secretase activity were evaluated by real-time PCR. Among the 21 validated genes, the strikingly decreased transcription of PTPRG and AMN1 and increased transcription of UPP1 potentially support data on cell cycle disturbances relevant to cancer, stem cell and neurodegenerative diseases' research. The mapping of interactions of proteins encoded by the validated genes exclusively relied on evidence-based data and revealed broad effects on Wnt pathway members, including WNT3A and DVL3. Intriguingly, the transcription of TERA, a gene of unknown function, is affected by gamma-secretase activity and was significantly altered in the analyzed human Alzheimer's disease brain cortices. CONCLUSIONS/SIGNIFICANCE: Investigating the effects of gamma-secretase activity on gene transcription has revealed several affected clusters of molecular functions and, more specifically, 21 genes that hold significant potential for a better understanding of the biology of gamma-secretase and its roles in cancer and Alzheimer's disease pathology

Ferritin H gene deletion in the choroid plexus and forebrain results in hydrocephalus

Ferritin H, the major iron storage protein, has essential functions in early embryonic development as well as in adult liver and intestine. To address the question whether ferritin H has similarly essential functions in the brain we used the Cre/loxP system to generate mice with a forebrain-specific inactivation of the ferritin H gene. Ferritin H deficiency in most cells of the forebrain including cells of the choroid plexus caused accumulation of cerebrospinal fluid in the lateral ventricles and the subarachnoid space. Brain tissue iron content was unchanged. (C) 2014 Elsevier Ltd. All rights reserved