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Background The effects of PSENEN mutations in patients with acne inversa (AI) are poorly understood. Hyperproliferation of follicular keratinocytes and resulting occlusion may constitute the initial pathophysiology. Objective To investigate the effects of PSENEN knockdown on γ-secretase subunits, biological behaviors, and related signaling pathways in keratinocytes. Methods HaCaT cells were divided into an experimental group (PSENEN knock down), a negative control group, and a blank control group. Whole transcriptome sequencing was used to measure differences in mRNA expression of the whole genome; real-time PCR and Western blotting were performed to determine the interference efficiency and the effects of interference on the components of γ-secretase and related molecules. CCK-8 was used to measure cell proliferation, and flow cytometry was used to measure apoptosis and the cell cycle. Results A comparison of five healthy controls with three patients with PSENEN mutation (c.66delG, c.279delC, c.229_230insCACC) revealed decreased expression of mRNA and protein in skin lesions of the experimental group. In this group, expression of the other components of γ-secretase presenilin C-terminal fragment decreased, expression of immature nicastrin increased, expression of mature nicastrin decreased, and expression of anterior pharynx defective-1 remained unchanged. KEGG analysis revealed that differentially expressed molecules were enriched in m-TOR signaling pathways. Subsequent verification confirmed that differences in PI3K-AKT-mTOR signaling pathway molecules, cell proliferation, apoptosis, cell cycle and the expression levels of Ki-67, KRT1, and IVL between the groups were not statistically significant. Conclusions PSENEN mutations alone may be insufficient to cause the development of AI, or they may only induce a mild phenotype of AI.

Alzheimer's disease (AD) is characterized by neuronal loss and accumulation of β-amyloid-protein (Aβ) in the brain parenchyma. Sleep impairment is associated with AD and affects about 25–40% of patients in the mild-to-moderate stages of the disease. Sleep deprivation leads to increased Aβ production; however, its mechanism remains largely unknown. We hypothesized that the increase in core body temperature induced by sleep deprivation may promote Aβ production. Here, we report temperature-dependent regulation of Aβ production. We found that an increase in temperature, from 37 °C to 39 °C, significantly increased Aβ production in amyloid precursor protein-overexpressing cells. We also found that high temperature (39 °C) significantly increased the expression levels of heat shock protein 90 (Hsp90) and the C-terminal fragment of presenilin 1 (PS1-CTF) and promoted γ-secretase complex formation. Interestingly, Hsp90 was associated with the components of the premature γ-secretase complex, anterior pharynx-defective-1 (APH-1), and nicastrin (NCT) but was not associated with PS1-CTF or presenilin enhancer-2. Hsp90 knockdown abolished the increased level of Aβ production and the increased formation of the γ-secretase complex at high temperature in culture. Furthermore, with in vivo experiments, we observed increases in the levels of Hsp90, PS1-CTF, NCT, and the γ-secretase complex in the cortex of mice housed at higher room temperature (30 °C) compared with those housed at standard room temperature (23 °C). Our results suggest that high temperature regulates Aβ production by modulating γ-secretase complex formation through the binding of Hsp90 to NCT/APH-1.

The aberrant metabolism of amyloid β peptide (Aβ) has been implicated in the etiology of Alzheimer disease (AD). Aβ is produced via the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. However, the precise regulatory mechanism of Aβ generation still remains unclear. To gain a better understanding of the molecular mechanism of Aβ production, we established a genetic screening method based on the CRISPR/Cas9 system to identify novel regulators of Aβ production. We successfully identified calcium and integrin-binding protein 1 (CIB1) as a potential negative regulator of Aβ production. The disruption of Cib1 significantly upregulated Aβ levels. In addition, immunoprecipitation experiments demonstrated that CIB1 interacts with the γ-secretase complex. Moreover, the disruption of Cib1 specifically reduced the cell-surface localization of mature Nicastrin (Nct), which is a component of the γ-secretase complex, without changing the intrinsic activity of γ-secretase. Finally, we confirmed using the single-cell RNA-seq data in human that CIB1 mRNA level in neuron was decreased in the early stage of AD. Taken together, our results indicate that CIB1 regulates Aβ production via controlling the subcellular localization of γ-secretase, suggesting CIB1 is involved in the development of AD.

Nicastrin (NICT) is a transmembrane protein physically associated with the polytypical aspartyl protease presenilin that plays a vital role in the correct localization and stabilization of presenilin to the membrane-bound γ-secretase complex. This complex is involved in the regulation of a wide range of cellular events, including cell signaling and the regulation of endocytosed membrane proteins for their trafficking and protein processing. Methods: In Trypanosoma cruzi, the causal agent of the Chagas disease, a NICT-like protein (Tc/NICT) was identified with a short C-terminus orthologous to the human protein, a large ectodomain (ECD) with numerous glycosylation sites and a single-core transmembrane domain containing a putative TM-domain (457GSVGA461) important for the γ-secretase complex activity. Results: Using the Spot-synthesis strategy with Chagasic patient sera, five extracellular epitopes were identified and synthetic forms were used to generate rabbit anti-Tc/NICT polyclonal serum that recognized a ~72-kDa molecule in immunoblots of T. cruzi epimastigote extracts. Confocal microscopy suggests that Tc/NICT is localized in the flagellar pocket, which is consistent with data from our previous studies with a T. cruzi presenilin-like protein. Phylogenetically, Tc/NICT was localized within a subgroup with the T. rangeli protein that is clearly detached from the other Trypanosomatidae, such as T. brucei. These results, together with a comparative analysis of the selected peptide sequence regions between the T. cruzi and mammalian proteins, suggest a divergence from the human NICT that might be relevant to Chagas disease pathology. As a whole, our data show that a NICT-like protein is expressed in the infective and replicative stages of T. cruzi and may be considered further evidence for a γ-secretase complex in trypanosomatids.

Biological sex exerts distinct influences on brain levels of the β-amyloid (Aβ) peptide in both clinical depression and Alzheimer disease (AD), yet studies in animal models focus primarily on males. We examined behavioral 'despair'/depression (using the tail-suspension test) and memory (using the novel object recognition task) in J20 (hAPPSwe/Ind) mice. Three month-old male (but not female) J20 mice exhibited less despair-like behavior, but more evidence of cognitive deficits. In young J20 mice, only soluble Aβ peptides -primarily Aβ(1-40)- were detected. There was no evidence of an effect on despair-like behavior in the six month-old J20 mice, although cognitive deficits were now evident in both sexes, and coincided with a greater proportion of the neurotoxic Aβ(1-42) species (in soluble as well as insoluble fractions). This age-dependent shift in Aβ peptide profile coincided with reduced expression of glycosylated species of ADAM-10 (α-secretase) and BACE1 (β-secretase), and an increased co-immunoprecipitation of presenilin-1 with nicastrin (components of the γ-secretase complex). Sex-dependent changes in depression-related monoaminergic, e.g. serotonin and dopamine (but not noradrenaline), systems were evident already in young J20 mice. It is critical to acknowledge that sex-dependent APP-related phenotypes might differentially influence modifiable depression-related monoaminergic signalling at some of the earliest pathological stages of clinical AD.

Nicastrin (NCSTN) mutations are associated with familial acne inversa (AI), and emerging evidence suggests that microRNAs (miRNAs) are involved in various skin diseases. However, whether NCSTN mutations affect miRNA levels and their subsequent signaling pathways in familial AI patients has not been studied. We aimed to elucidate the relationship between NCSTN mutations and familial AI pathogenesis by investigating differential miRNA expression and their related pathways. Combined with miRNA microarray data from familial AI patients, Ncstn keratinocyte-specific–knockout (NcstnΔKC) mice and bioinformatics predictions showed that NCSTN mutations led to decreased miR-30a-3p levels, which negatively regulated RAB31 expression. Moreover, enhanced RAB31 levels accelerated degradation of activated EGFR, leading to abnormal differentiation in keratinocytes. The impaired EGFR signaling and its effects on epidermal differentiation were also observed in familial AI patients and NcstnΔKC mice. Thus, our study showed that miR-30a-3p/RAB31/EGFR signaling pathway may play a key role in the pathogenesis of familial AI with NCSTN mutations.

Genetic, biochemical, and anatomical grounds led to the proposal of the amyloid cascade hypothesis centered on the accumulation of amyloid beta peptides (Aβ) to explain Alzheimer's disease (AD) etiology. In this context, a bulk of efforts have aimed at developing therapeutic strategies seeking to reduce Aβ levels, either by blocking its production (γ- and β-secretase inhibitors) or by neutralizing it once formed (Aβ-directed immunotherapies). However, so far the vast majority of, if not all, clinical trials based on these strategies have failed, since they have not been able to restore cognitive function in AD patients, and even in many cases, they have worsened the clinical picture. We here propose that AD could be more complex than a simple Aβ-linked pathology and discuss the possibility that a way to reconcile undoubted genetic evidences linking processing of APP to AD and a consistent failure of Aβ-based clinical trials could be to envision the pathological contribution of the direct precursor of Aβ, the β-secretase-derived C-terminal fragment of APP, βCTF, also referred to as C99. In this review, we summarize scientific evidences pointing to C99 as an early contributor to AD and postulate that γ-secretase should be considered as not only an Aβ-generating protease, but also a beneficial C99-inactivating enzyme. In that sense, we discuss the limitations of molecules targeting γ-secretase and propose alternative strategies seeking to reduce C99 levels by other means and notably by enhancing its lysosomal degradation.

Prolonged neuroinflammation is a driving force for neurodegenerative disease, and agents against inflammatory responses are regarded as potential treatment strategies. Here we aimed to evaluate the prevention effects on gliosis by dexamethasone (DEX), an anti-inflammation drug. We used DEX to treat the nicastrin conditional knockout (cKO) mouse, a neurodegenerative mouse model. DEX (10 mg/kg) was given to 2.5-month-old nicastrin cKO mice, which have not started to display neurodegeneration and gliosis, for 2 months. Immunohistochemistry (IHC) and Western blotting techniques were used to detect changes in neuroinflammatory responses. We found that activation of glial fibrillary acidic protein (GFAP) positive or ionized calcium binding adapter molecule1 (Iba1) positive cells was not inhibited in nicastrin cKO mice treated with DEX as compared to those treated with saline. These data suggest that DEX does not prevent or ameliorate gliosis in a neurodegenerative mouse model when given prior to neuronal or synaptic loss.

γ-Secretase complex, the assembly of nicastrin (NCT), Presenilin (PS), Presenilin Enhancer-2 (PEN-2) and Anterior pharynx defective 1 (Aph-1), catalyzes the cleavage of amyloid precursor protein to generate amyloid-β protein (Aβ), the main culprit of Alzheimer's disease. NCT becomes matured through complex glycosylation and play important role in γ-secretase activity by interacting with catalytic subunit PS. However, the role of NCT glycosylation on γ-secretase activity and substrate specificity is still unknown. The purpose of this study is to investigate the effect of NCT glycosylation on γ-secretase activity and substrate specificity in a group of glycosylation mutant lectin resistant CHO (Lec) cells. CHO Lec-1 cells lack glycosyltransferase-I, GnT-I, thus N-glycan on NCT are all oligomannose type, whereas CHO Lec-2 cells synthesize NCT containing sialic acid deficient oligosaccharides due to the impairment of cytidine 5′-monophosphate-sialic acid transporter. Here, we reported that mutant CHO Lec-1 and Lec-2 reduced γ-secretase activity in both cell-based and biochemical assays, and that CHO Lec-1 preferentially reduced Aβ generation. Endogenous level of γ-secretase complex, subcellular distribution of γ-secretase subunits and the level of functional γ-secretase complex remained unchanged in mutants. Interestingly, Coimmunoprecipitation study revealed that mutant γ-secretase could recognize substrate as well as parental γ-secretase. Our data suggests that thorough glycosylation of NCT is critical for enzymatic activity and substrate preference of γ-secretase.

Γ-secretase is a membrane-embedded protease that cleaves single transmembrane helical domains of various integral membrane proteins. The amyloid precursor protein (APP) is an important substrate due to its pathological relevance to Alzheimer's disease. The mechanism of the cleavage of APP by γ-secretase that leads to accumulation of Alzheimer's disease causing amyloid-β (Aβ) is still unknown. Coarse-grained molecular dynamics simulations in this study reveal initial lipids raft formation near the catalytic site of γ-secretase as well as changes in dynamic behavior of γ-secretase once interacting with APP. The results suggest a precursor of the APP binding mode and hint at conformational changes of γ-secretase in the nicastrin (NCT) domain upon APP binding.

γ-secretase is an intramembrane protease complex consisting of nicastrin, presenilin-1/2, APH-1a/b, and Pen-2. Hydrolysis of the 99-residue transmembrane fragment of amyloid precursor protein (APP-C99) by γ-secretase produces β-amyloid (Aβ) peptides. Pathogenic mutations in PSEN1 and PSEN2 , which encode the catalytic subunit presenilin-1/2 of γ-secretase, lead to familial Alzheimer’s disease in an autosomal dominant manner. However, the underlying mechanism of how the mutant PSEN gene may affect the function of the WT allele remains to be elucidated. Here we report that each of the loss-of-function γ-secretase variants that carries a PSEN1 mutation suppresses the protease activity of the WT γ-secretase on Aβ production. Each of these γ-secretase variants forms a stable oligomer with the WT γ-secretase in vitro in the presence of the detergent CHAPSO {3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate}, but not digitonin. Importantly, robust protease activity of γ-secretase is detectable in the presence of CHAPSO, but not digitonin. These experimental observations suggest a dominant negative effect of the γ-secretase, in which the protease activity of WT γ-secretase is suppressed by the loss-of-function γ-secretase variants through hetero-oligomerization. The relevance of this finding to the genesis of Alzheimer’s disease is critically evaluated.

Mitochondrial dysfunction is implicated in the pathological mechanism of Alzheimer's disease (AD). Amyloid β-protein (Aβ), which plays a central role in AD pathogenesis, is reported to accumulate within mitochondria. However, a question remains as to whether Aβ is generated locally from amyloid precursor protein (APP) within mitochondria. We investigated this issue by analyzing the expression patterns of APP, APP-processing secretases, and APP metabolites in mitochondria separated from human neuroblastoma SH-SY5Y cells and those expressing Swedish mutant APP. APP, BACE1, and PEN-2 protein levels were significantly lower in crude mitochondria than microsome fractions while those of ADAM10 and the other γ-secretase complex components (presenilin 1, nicastrin, and APH-1) were comparable between fractions. The crude mitochondrial fraction containing substantial levels of cathepsin D, a lysosomal marker, was further separated via iodixanol gradient centrifugation to obtain mitochondria- and lysosome-enriched fractions. Mature APP, BACE1, and all γ-secretase complex components (in particular, presenilin 1 and PEN-2) were scarcely present in the mitochondria-enriched fraction, compared to the lysosome-enriched fraction. Moreover, expression of the β-C-terminal fragment (β-CTF) of APP was markedly low in the mitochondria-enriched fraction. Additionally, immunocytochemical analysis showed very little co-localization between presenilin 1 and Tom20, a marker protein of mitochondria. In view of the particularly low expression levels of BACE1, γ-secretase complex proteins, and β-CTF in mitochondria, we propose that it is unlikely that Aβ generation from APP occurs locally within this organelle.

Skin depigmentation diseases, such as vitiligo, are pigmentation disorders that often destroy melanocytes. However, their pathological mechanisms remain unclear, and therefore, promising treatments or prevention has been lacking. Here, we demonstrate that a zebrafish insertional mutant showing a significant reduction of nicastrin transcript possesses melanosome maturation defect, Tyrosinase-dependent mitochondrial swelling, and melanophore cell death. The depigmentation phenotypes are proven to be a result of γ-secretase inactivation. Furthermore, live imaging demonstrates that macrophages are recruited to and can phagocytose melanophore debris. Thus, we characterize a potential zebrafish depigmentation disease model, a nicastrinhi1384 mutant, which can be used for further treatment or drug development of diseases related to skin depigmentation and/or inflammation.

Gamma-secretase mediates the final proteolytic cleavage, which liberates amyloid beta-peptide (Abeta), the major component of senile plaques in the brains of Alzheimer disease patients. Therefore, gamma-secretase is a prime target for Abeta-lowering therapeutic strategies. gamma-Secretase is a protein complex composed of four different subunits, presenilin (PS), APH-1, nicastrin, and PEN-2, which are most likely present in a 1:1:1:1 stoichiometry. PS harbors the catalytically active site, which is critically required for the aspartyl protease activity of gamma-secretase. Moreover, numerous familial Alzheimer disease-associated mutations within the PSs increase the production of the aggregation-prone and neurotoxic 42-amino acid Abeta. Nicastrin may serve as a substrate receptor, although this has recently been challenged. PEN-2 is required to stabilize PS within the gamma-secretase complex. No particular function has so far been assigned to APH-1. The four components are sufficient and required for gamma-secretase activity. At least six different gamma-secretase complexes exist that are composed of different variants of PS and APH-1. All gamma-secretase complexes can exert pathological Abeta production. Assembly of the gamma-secretase complex occurs within the endoplasmic reticulum, and only fully assembled and functional gamma-secretase complexes are transported to the plasma membrane. Structural analysis by electron microscopy and chemical cross-linking reveals a water-containing cavity, which allows intramembrane proteolysis. Specific and highly sensitive gamma-secretase inhibitors have been developed; however, they interfere with the physiological function of gamma-secretase in Notch signaling and thus cause rather significant side effects in human trials. Modulators of gamma-secretase, which selectively affect the production of the pathological 42-amino acid Abeta, do not inhibit Notch signaling.

γ-Secretase is a multisubunit membrane protein complex containing catalytic presenilin (PS1 or PS2) and cofactors such as nicastrin, Aph-1, and Pen2. γ-Secretase hydrolyzes the transmembrane domains of type-I membrane proteins, which include the amyloid precursor protein (APP). APP is cleaved by γ-secretase to produce amyloid β peptide (Aβ), which is deposited in the brains of Alzheimer disease patients. However, the mechanism of this unusual proteolytic process within the lipid bilayer remains unknown. We have established a yeast transcriptional activator Gal4p system with artificial γ-secretase substrates containing APP or Notch fragments to examine the enzymatic properties of γ-secretase. The γ-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4 release from the membrane bound substrates as assessed by growth of yeast or β-galactosidase assay. We also established an in vitro yeast microsome assay system which identified different Aβ species produced by trimming. The yeast system allows for the screening of mutations and chemicals that inhibit or modulate γ-secretase activity. Herein we describe the genetic and biochemical methods used to analyze γ-secretase activity using the yeast reconstitution system. By studying the loss-of-function properties of PS1 mutants, it is possible to successfully screen FAD suppressor mutations and identify γ-secretase modulators (GSMs), which are promising Alzheimer disease therapeutic agents.

Aberrant production, clearance and deposition of amyloid-β protein (Aβ) in the human brain have been implicated in the aetiology of Alzheimer disease (AD). γ-Secretase is the enzyme responsible for generating various Aβ species, such as Aβ40 and toxic Aβ42. Recently, genome-wide association studies in late-onset AD patients have identified the endocytosis-related phosphatidylinositol-binding clathrin assembly protein (PICALM) gene as a genetic risk factor for AD. We previously found that the loss of expression of CALM protein encoded by PICALM affects the ratio of production of Aβ42, through the regulation of the clathrin-mediated endocytosis of γ-secretase. Here, we show that the binding capacity of the assembly protein 180 N-terminal homology (ANTH) domain of CALM to phosphatidylinositol-4,5-biphosphate, as well as to nicastrin, is critical to the modulation of the internalization of γ-secretase and to the Aβ42 production ratio. Moreover, reduction of CALM decreases Aβ deposition as well as brain levels of insoluble Aβ42 in vivo These results suggest that CALM expression modifies AD risk by regulating Aβ pathology.

The γ-secretase complex is composed of at least four components: presenilin 1 or presenilin-2, nicastrin (NCT), anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2. In this study, using knockout cell lines, our data demonstrated that knockout of NCT, as well as knockout of presenilin enhancer 2, completely blocked γ-secretase-catalyzed processing of C-terminal fragment (CTF)α and CTFβ, the C-terminal fragments of β-amyloid precursor protein (APP) produced by α-secretase and β-secretase cleavages, respectively. Interestingly, in Aph-1-knockout cells, CTFα and CTFβ were still processed by γ-secretase, indicating Aph-1 is dispensable for APP processing. Furthermore, our results indicate that Aph-1 as well as NCT is not absolutely required for Notch processing, suggesting that NCT is differentially required for APP and Notch processing. In addition, our data revealed that components of the γ-secretase complex are also important for proteasome- and lysosome-dependent degradation of APP and that endogenous APP is mostly degraded by lysosome while exogenous APP is mainly degraded by proteasome. There are unanswered questions regarding the roles of each component of the γ-secretase complex in amyloid precursor protein (APP) and Notch processing. The most relevant, novel finding of this study is that nicastrin (NCT) is required for APP but not Notch processing, while Aph-1 is not essential for processing of both APP and Notch, suggesting NCT as a therapeutic target to restrict Aβ formation without impairing Notch signaling.

Oxidative stress is a common feature of the aging process and of many neurodegenerative disorders, including Alzheimer's disease. Understanding the direct causative relationship between oxidative stress and amyloid pathology, and determining the underlying molecular mechanisms is crucial for the development of more effective therapeutics for the disease. By employing microdialysis technique, we report local increase in the amyloid-β42 levels and elevated amyloid-β42/40 ratio in the interstitial fluid within 6 h of direct infusion of oxidizing agents into the hippocampus of living and awake wild type mice. The increase in the amyloid-β42/40 ratio correlated with the pathogenic conformational change of the amyloid precursor protein-cleaving enzyme, presenilin1/γ-secretase. Furthermore, we found that the product of lipid peroxidation 4-hydroxynonenal, binds to both nicastrin and BACE, differentially affecting γ- and β-secretase activity, respectively. The present study demonstrates a direct cause-and-effect correlation between oxidative stress and altered amyloid-β production, and provides a molecular mechanism by which naturally occurring product of lipid peroxidation may trigger generation of toxic amyloid-β42 species.

The γ-secretase complex is composed of four membrane protein subunits, including presenilin as the catalytic component with aspartyl protease activity. The enzyme cleaves within the transmembrane domain of >70 different type I integral membrane proteins and has been dubbed "the proteasome of the membrane". The most studied substrates include the Notch family of receptors, involved in cell differentiation, and the amyloid precursor protein (APP), involved in the pathogenesis of Alzheimer's disease. A central mechanistic question is how γ-secretase recognizes helical transmembrane substrates and carries out processive proteolysis. Recent findings addressing substrate recognition and processing will be discussed, including the role of protease subunit nicastrin as a gatekeeper, the effects of Alzheimer-causing mutations in presenilin on processive proteolysis of APP, and evidence that three pockets in the active site (S1', S2', and S3') determine carboxypeptidase cleavage of substrate in intervals of three residues. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

The 99 amino acid C-terminal fragment of Amyloid Precursor Protein APP-C99 (C99) is cleaved by γ-secretase to form Aβ peptide, which plays a critical role in the etiology of Alzheimer's Disease (AD). The structure of C99 consists of a single transmembrane domain flanked by intra and intercellular domains. While the structure of the transmembrane domain has been well characterized, little is known about the structure of the flanking domains and their role in C99 processing by γ-secretase. To gain insight into the structure of full-length C99, REMD simulations were performed for monomeric C99 in model membranes of varying thickness. We find equilibrium ensembles of C99 from simulation agree with experimentally-inferred residue insertion depths and protein backbone chemical shifts. In thin membranes, the transmembrane domain structure is correlated with extra-membrane structural states and the extra-membrane domain structural states become less correlated to each other. Mean and variance of the transmembrane and G37G38 hinge angles are found to increase with thinning membrane. The N-terminus of C99 forms β-strands that may seed aggregation of Aβ on the membrane surface, promoting amyloid formation. In thicker membranes the N-terminus forms α-helices that interact with the nicastrin domain of γ-secretase. The C-terminus of C99 becomes more α-helical as the membrane thickens, forming structures that may be suitable for binding by cytoplasmic proteins, while C-terminal residues essential to cytotoxic function become α-helical as the membrane thins. The heterogeneous but discrete extra-membrane domain states analyzed here open the path to new investigations of the role of C99 structure and membrane in amyloidogenesis. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.

The gamma-secretase is a multiprotein complex that cleaves many type-I membrane proteins, such as the Notch receptor and the amyloid precursor protein. Nicastrin (NCT) is an essential component of the multimeric gamma-secretase complex and functions as a receptor for gamma-secretase substrates. In this study, we found that Akt1 markedly regulated the protein stability of NCT. Importantly, the kinase activity of Akt1 was essential for the inhibition of gamma-secretase activity through degradation of NCT. Notably, the protein level of endogenous NCT was higher in shAkt1-expressing cells than in shCon-expressing cells. Akt1 physically interacted with NCT and mediated its degradation through proteasomal and lysosomal pathways. We also found that Akt1 phosphorylates NCT at Ser437, resulting in a significant reduction in NCT protein stability. Importantly, a phospho-deficient mutation in NCT at Ser437 stabilized its protein levels. Collectively, our results reveal that Akt1 functions as a negative regulator of the gamma-secretase activity through phosphorylation and degradation of NCT. Generation of the amyloid peptide (A-beta) and the amyloid precursor protein (APP) intracellular domain (AICD) can happen by sequential proteolysis of APP by beta and gamma-secretase. The gamma-secretase complex consists of four essential proteins: presenilin (PS1 or PS2), presenilin enhancer 2 (PEN-2), anterior pharynx-defective 1 (APH-1), and the Nicastrin (NCT). NCT can interact and be phosphorylated by Akt1, and phosphorylated NCT promotes its proteasomal and lysosomal degradation. As a result, Akt1 plays role in reducing gamma-secretase activity through phosphorylation-dependent regulation of NCT protein degradation.

Alzheimer's disease (AD)-associated gamma-secretase is a ubiquitously expressed multi-subunit protease complex embedded in the lipid bilayer of cellular compartments including endosomes and the plasma membrane. Although gamma-secretase is of crucial interest for AD drug discovery, its atomic structure remains unresolved. Gamma-secretase assembly and maturation is a multistep process, which includes extensive glycosylation on nicastrin (NCT), the only g-secretase subunit having a large extracellular domain. These posttranslational modifications lead to protein heterogeneity that likely prevents the three-dimensional (3D) crystallization of the protease complex. To overcome this issue, we have engineered a Chinese hamster ovary (CHO) cell line deficient in complex sugar modifications (CHO lec1) to overexpress the four subunits of g-secretase as a functional complex. We purified glycosylation-deficient g-secretase from this recombinant cell line (CL1-9) and fully glycosylated g-secretase from a recombinant CHO DG44-derived cell line (SS20). We characterized both complexes biochemically and pharmacologically in vitro. Interestingly, we found that the complex oligosaccharides, which largely decorate the extracellular domain of fully glycosylated NCT, are not involved in the proper assembly and maturation of the complex, and are dispensable for the specific generation, in physiological ratios, of the amyloid precursor protein (APP) cleavage products. In conclusion, we propose a novel bioengineering approach for the production of functional glycosylation-deficient g-secretase, which may be suitable for crystallization studies. We expect that these findings will contribute both to solving the high-resolution 3D structure of g-secretase and to structure-based drug discovery for AD. Biotechnol. Bioeng. 2015;112: 2516-2526. (c) 2015 Wiley Periodicals, Inc.

The structure and function of the γ-secretase proteases are of vast interest because of their critical roles in cellular and disease processes. We established a novel purification protocol for γ-secretase complex that involves a conformation and complex-specific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the γ-secretase structure and its conformational variability. Under steady state conditions the complex adopts three major conformations, which are different in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate binding sites by inhibitors differentially stabilize sub-populations of particles with compact conformations, whereas a Familial Alzheimer Disease-linked mutation results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the γ-secretase complex as a dynamic population of inter-converting conformations, involving rearrangements at the nanometer scale and high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect Aβ generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely instrumental for highly allosteric behavior of the enzyme. ispartof: Journal of Cell Science vol:128 issue:3 pages:589-98 ispartof: location:England status: published

The γ-secretase complex, composed of presenilin, nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), is assembled in a highly regulated manner and catalyzes the intramembranous proteolysis of many type I membrane proteins, including Notch and amyloid precursor protein. The Notch family of receptors plays important roles in cell fate specification during development and in adult tissues, and aberrant hyperactive Notch signaling causes some forms of cancer. γ-Secretase-mediated processing of Notch at the cell surface results in the generation of the Notch intracellular domain, which associates with several transcriptional coactivators involved in nuclear signaling events. On the other hand, γ-secretase-mediated processing of amyloid precursor protein leads to the production of amyloid β (Aβ) peptides that play an important role in the pathogenesis of Alzheimer disease. We used a phage display approach to identify synthetic antibodies that specifically target NCT and expressed them in the single-chain variable fragment (scFv) format in mammalian cells. We show that expression of a NCT-specific scFv clone, G9, in HEK293 cells decreased the production of the Notch intracellular domain but not the production of amyloid β peptides that occurs in endosomal and recycling compartments. Biochemical studies revealed that scFvG9 impairs the maturation of NCT by associating with immature forms of NCT and, consequently, prevents its association with the other components of the γ-secretase complex, leading to degradation of these molecules. The reduced cell surface levels of mature γ-secretase complexes, in turn, compromise the intramembranous processing of Notch.

γ-Secretase generates amyloid beta peptides (Aβ) from amyloid precursor protein through multistep cleavages, such as endoproteolysis (e-cleavage) and trimming (γ-cleavage). Familial Alzheimer's disease (FAD) mutations within the catalytic subunit protein of presenilin 1 (PS1) decrease γ-cleavage, resulting in the generation of toxic, long Aβs. Reducing long Aβ levels has been proposed as an AD therapeutic strategy. Previously, we identified PS1 mutations that are active in the absence of nicastrin (NCT) using a yeast γ-secretase assay. Here, we analysed these PS1 mutations in the presence of NCT, and found that they were constitutively active in yeast. One triple, 13 double, and 5 single mutants enhanced e-cleavage activity up to 2.7-fold. Furthermore, L241I, F411Y, S438P and F441L mutations modulated trimming activities to produce more short-Aβ in yeast microsomes. When introduced in mouse embryonic fibroblasts, these mutations possessed similar or reduced e-cleavage activity. However, two mutations, L241I and S438P, modulated trimming activities and changed the conformation of transmembrane domain 1, the substrate recognition site. These mutants had the opposite modulatory effects of FAD mutations and produced more short Aβs and fewer long Aβs. Our results provide insights into the relationship between PS1 conformational changes and γ-secretase activities.

Alzheimer's disease (AD) is characterized by accumulation of the β-amyloid peptide (Aβ), which is generated through sequential proteolysis of the amyloid precursor protein (APP), first by the action of β-secretase, generating the β-C-terminal fragment (βCTF), and then by the Presenilin 1 (PS1) enzyme in the γ-secretase complex, generating Aβ. γ-Secretase is an intramembranous protein complex composed of Aph1, Pen2, Nicastrin, and Presenilin 1. Although it has a central role in the pathogenesis of AD, knowledge of the mechanisms that regulate PS1 function is limited. Here, we show that phosphorylation of PS1 at Ser367 does not affect γ-secretase activity, but has a dramatic effect on Aβ levels in vivo. We identified CK1γ2 as the endogenous kinase responsible for the phosphorylation of PS1 at Ser367. Inhibition of CK1γ leads to a decrease in PS1 Ser367 phosphorylation and an increase in Aβ levels in cultured cells. Transgenic mice in which Ser367 of PS1 was mutated to Ala, show dramatic increases in Aβ peptide and in βCTF levels in vivo. Finally, we show that this mutation impairs the autophagic degradation of βCTF, resulting in its accumulation and increased levels of Aβ peptide and plaque load in the brain. Our results demonstrate that PS1 regulates Aβ levels by a unique bifunctional mechanism. In addition to its known role as the catalytic subunit of the γ-secretase complex, selective phosphorylation of PS1 on Ser367 also decreases Aβ levels by increasing βCTF degradation through autophagy. Elucidation of the mechanism by which PS1 regulates βCTF degradation may aid in the development of potential therapies for Alzheimer's disease.

Chronic cerebral hypoperfusion (CCH) contributes to the Alzheimer’s-like pathogenesis, but the relationship between CCH and the occurrence of Alzheimer’s disease (AD) remains obscure. The aim is to elucidate the potential pathophysiological mechanism in the field of amyloid-beta (Aβ) pathology induced by CCH. A rat model of CCH has been developed with permanent bilateral occlusion of common carotid arteries (BCCAO). The cognitive function of rats was tested by the Morris water maze. The levels of Aβ (Aβ40 and Aβ42) and soluble amyloid precursor protein (sAPP: sAPPα and sAPPβ) were determined by enzyme linked immunosorbent assay. The expression of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), presenilin1 (PS1), nicastrin (NCT), anterior pharynx-defective 1alpha (Aph-1α) and presenilin enhancer 2 (Pen-2), sAPPα and sAPPβ were detected by Western blotting. Morris water maze test showed that CCH induced decline in learning and memory related to Aβ levels in the hippocampus. The levels of sAPPα, ADAM10 and ADAM17 in the hippocampus of CCH rats were higher than the control ones (P

Recent evidence suggests involvement of biometal homeostasis in the pathological mechanisms in Alzheimer's disease (AD). For example, increased intracellular copper or zinc has been linked to a reduction in secreted levels of the AD-causing amyloid-β peptide (Aβ). However, little is known about whether these biometals modulate the generation of Aβ. In the present study we demonstrate in both cell-free and cell-based assays that zinc and copper regulate Aβ production by distinct molecular mechanisms affecting the processing by γ-secretase of its Aβ precursor protein substrate APP-C99. We found that Zn2+ induces APP-C99 dimerization, which prevents its cleavage by γ-secretase and Aβ production, with an IC50 value of 15 μm. Importantly, at this concentration, Zn2+ also drastically raised the production of the aggregation-prone Aβ43 found in the senile plaques of AD brains and elevated the Aβ43:Aβ40 ratio, a promising biomarker for neurotoxicity and AD. We further demonstrate that the APP-C99 histidine residues His-6, His-13, and His-14 control the Zn2+-dependent APP-C99 dimerization and inhibition of Aβ production, whereas the increased Aβ43:Aβ40 ratio is substrate dimerization-independent and involves the known Zn2+ binding lysine Lys-28 residue that orientates the APP-C99 transmembrane domain within the lipid bilayer. Unlike zinc, copper inhibited Aβ production by directly targeting the subunits presenilin and nicastrin in the γ-secretase complex. Altogether, our data demonstrate that zinc and copper differentially modulate Aβ production. They further suggest that dimerization of APP-C99 or the specific targeting of individual residues regulating the production of the long, toxic Aβ species, may offer two therapeutic strategies for preventing AD.

Presenilin is a catalytic subunit of γ-secretase, which hydrolyzes several transmembrane proteins within the lipid bilayer, together with binding cofactors such as nicastrin, Aph-1, and Pen-2. However, the structural basis as well as molecular mechanism of this unusual proteolytic process remains unknown. We have analyzed the structure and function relationships of presenilin using the substituted-cysteine accessibility method (SCAM), which enables identification of the hydrophilic environment by the accessibility of sulfhydryl reagents to cysteine residues introduced at a desired position. In combination with small molecule inhibitors/modulators and cross-linking experiments, we were able to identify certain residues and regions of presenilin that contribute to its intramembrane-cleaving activity. In addition, we revealed the structural dynamics of the transmembrane domains of presenilin during the formation of the complex and its proteolytic process. The SCAM provides new insights into the relationship between the structure and activity of presenilin, and is useful for probing the protein dynamics of the membrane-embedded enzymes.

Presenilin is a membrane-embedded intramembrane-cleaving protease with a conserved catalytic G×GD motif. It is the catalytic subunit of γ-secretase, which plays critical roles in developmental biology and the molecular etiology of Alzheimer disease, together with three membrane protein cofactors, nicastrin, Aph-1 and Pen-2. Biochemical and enzymatic analyses have revealed that γ-secretase executes two types of proteolytic activities on a single substrate; an endopeptidase-like cleavage followed by carboxypeptidase-like processive cleavage. Utilizing small molecule inhibitors/modulators together with the substituted cysteine accessibility method, we identified certain residues and regions of presenilin that contribute to the formation of a catalytic pore structure within the lipid bilayer required for its intramembrane-cleaving activity. Recently, determination of the crystal structure of the archaeal presenilin homologue has confirmed the intramembranous location of the two conserved and essential aspartates. In this review, I will introduce the recent progresses in the understanding of the molecular mechanisms of action of this atypical protease.

The γ-secretase complex, comprising presenilin 1 (PS1), PEN-2, APH-1 and nicastrin, is a membrane-embedded protease that controls a number of important cellular functions through substrate cleavage. Aberrant cleavage of the amyloid precursor protein (APP) results in aggregation of amyloid-β, which accumulates in the brain and consequently causes Alzheimer’s disease. Here we report the three-dimensional structure of an intact human γ-secretase complex at 4.5 A resolution, determined by cryo-electron-microscopy single-particle analysis. The γ-secretase complex comprises a horseshoe-shaped transmembrane domain, which contains 19 transmembrane segments (TMs), and a large extracellular domain (ECD) from nicastrin, which sits immediately above the hollow space formed by the TM horseshoe. Intriguingly, nicastrin ECD is structurally similar to a large family of peptidases exemplified by the glutamate carboxypeptidase PSMA. This structure serves as an important basis for understanding the functional mechanisms of the γ-secretase complex. The three-dimensional structure of intact human γ-secretase complex at 4.5 A resolution is revealed by cryo-electron-microscopy single-particle analysis; the complex comprises a horseshoe-shaped transmembrane domain containing 19 transmembrane segments, and a large extracellular domain from nicastrin, which sits immediately above the hollow space formed by the horseshoe. This paper reports a high-resolution cryo-electron-microscopy structure of the human γ-secretase complex, a membrane-embedded protease that controls important cellular functions and is linked to the aberrant cleavage of the amyloid precursor protein seen in Alzheimer's disease. The complex, comprising presenilin 1, PEN-2, APH-1 and nicastrin, is horseshoe-shaped, with 19 transmembrane segments. The nicastrin extracellular domain, thought to be responsible for substrate recruitment, sits immediately above the hollow space formed by the transmembrane horseshoe.

The 19-transmembrane, multisubunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β-amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen-2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high-resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen-2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N-terminal maltose binding protein tag on Pen-2 still permits incorporation into the complex and subsequent presenilin-1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen-2 and the γ-secretase complex.

We have developed a suite of clickable γ-secretase active site-directed dual-photoaffinity probes possessing photoactivatable benzophenones that are located within the inhibitor scaffold as well as spaced away from the core by a linker. Through photoactivation of these dual photoprobes and subsequent click chemistry mediated conjugation of a reporter group, we have been able to specifically label PS1-N-terminal fragment (PS1-NTF), PS1-C-terminal fragment (PS1-CTF) and nicastrin and form a pseudo-full length PS1 through crosslinks between PS1-NTF and PS1-CTF. Probe-mediated protein–protein crosslinks were confirmed by in-gel fluorescence, western blotting and LC-MS/MS detection of tryptic peptides of the electrophoretically separated proteins.

The gamma (γ)-secretase holoenzyme is composed of four core proteins and cleaves APP to generate amyloid beta (Aβ), a key molecule that causes major neurotoxicity during the early stage of Alzheimer's disease (AD). However, despite its important role in Aβ production, little is known about the regulation of γ-secretase. OCIAD2, a novel modulator of γ-secretase that stimulates Aβ production, and which was isolated from a genome-wide functional screen using cell-based assays and a cDNA library comprising 6,178 genes. Ectopic expression of OCIAD2 enhanced Aβ production, while reduction of OCIAD2 expression suppressed it. OCIAD2 expression facilitated the formation of an active γ-secretase complex and enhanced subcellular localization of the enzyme components to lipid rafts. OCIAD2 interacted with nicastrin to stimulate γ-secretase activity. OCIAD2 also increased the interaction of nicastrin with C99 and stimulated APP processing via γ-secretase activation, but did not affect Notch processing. In addition, a cell-permeable Tat-OCIAD2 peptide that interfered with the interaction of OCIAD2 with nicastrin interrupted the γ-secretase-mediated AICD production. Finally, OCIAD2 expression was significantly elevated in the brain of AD patients and PDAPP mice. This study identifies OCIAD2 as a selective activator of γ-secretase to increase Aβ generation.

Alzheimer’s disease (AD) is primarily caused by overproduction/deposition of β-amyloid (Aβ) in the brain. Dysregulation of iron in the brain also contributes to AD. Although iron affects β-amyloid precursor protein (APP) expression and Aβ deposition, detailed role of iron in AD requires further elucidation. Aβ is produced by sequential proteolytic cleavages of APP by β-secretase and γ-secretase. The γ-secretase complex comprises presenilins (PS1 or PS2), Nicastrin, APH-1, and PEN-2. Herein, we find that PEN-2 can interact with ferritin light chain (FTL), an important component of the iron storage protein ferritin. In addition, we show that overexpression of FTL increases the protein levels of PEN-2 and PS1 amino-terminal fragment (NTF) and promotes γ-secretase activity for more production of Aβ and Notch intracellular domain (NICD). Furthermore, iron treatments increase the levels of FTL, PEN-2 and PS1 NTF and promote γ-secretase-mediated NICD production. Moreover, downregulation of FTL decreases the levels of PEN-2 and PS1 NTF. Together, our results suggest that iron can increase γ-secretase activity through promoting the level of FTL that interacts with and stabilizes PEN-2, providing a new molecular link between iron, PEN-2/γ-secretase and Aβ generation in AD.

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 Alzheimer's 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.

gamma-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel gamma-secretase activating protein (GSAP) was identified that interacts with gamma-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-beta production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of gamma-secretase activity and amyloid-beta production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-beta levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-beta generation. Likewise, purified recombinant GSAP had no effect on amyloid-beta generation in two distinct in vitro gamma-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-beta levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-beta levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of gamma-secretase activity and amyloid-beta generation are uncertain.

Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been reported to attenuate amyloid-β peptide (Aβ) production in various cellular models. However, the mechanisms by which statins affect neuronal Aβ production have not yet been clarified. Here, we investigated this issue in rat primary cortical neurons using two statins, pitavastatin (PV) and atorvastatin (AV). Treatment of neurons with 0.2-2.5 μM PV or AV for 4 days induced a concentration- and time-dependent reduction in the secretion of both Aβ40 and Aβ42. Moreover, Western blot analyses of cell lysates showed that treatment with PV or AV significantly reduced expression levels of the mature form of amyloid precursor protein (APP) and Thr668-phosphorylated APP (P-APP), but not immature form of APP; the decreases in P-APP levels were more notable than those of mature APP levels. The statin treatment did not alter expression of BACE1 (β-site APP-cleaving enzyme 1) or γ-secretase complex proteins (presenilin 1, nicastrin, APH-1, and PEN-2). In neurons overexpressing APP via recombinant adenoviruses, PV or AV similarly reduced Aβ secretion and the levels of mature APP and P-APP. Statins also markedly reduced cellular cholesterol content in neurons in a concentration-dependent manner. Co-treatment with mevalonate reversed the statin-induced decreases in Aβ secretion and mature APP and P-APP levels, whereas co-treatment with cholesterol did not, despite recovery of cellular cholesterol levels. Finally, cell-surface biotinylation experiments revealed that both statins significantly reduced the levels of cell-surface P-APP without changing those of cell surface mature APP. These results suggest that statins reduce Aβ production by selectively modulating APP maturation and phosphorylation through a mechanism independent of cholesterol reduction in cultured neurons.

The γ-secretase complex comprises presenilin (PS), nicastrin (NCT), anterior pharynx-defective 1 (Aph1), and presenilin enhancer 2 (Pen2). PS has two homologues, PS1 and PS2. Aph1 has two isoforms, Aph1a and Aph1b, with the former existing as two splice variants Aph1aL and Aph1aS. Each complex consists of one subunit each, resulting in six different γ-secretases. To better understand the functional differences among the γ-secretases, we reconstituted them using a yeast system and compared Notch1-cleavage and amyloid precursor protein (APP)-cleavage activities. Intriguingly, PS2/Aph1b had a clear substrate specificity: APP-Gal4, but not Notch-Gal4, was cleaved. In HEK cell lines expressing defined γ-secretase subunits, we showed that PS1/Aph1b, PS2/Aph1aL, PS2/Aph1aS and PS2/Aph1b γ-secretase produced amyloid β peptide (Aβ) with a higher Aβ42+Aβ43-to-Aβ40 (Aβ42(43)/Aβ40) ratio than the other γ-secretases. In addition, PS2/Aph1aS γ-secretase produced less Notch intracellular domain (NICD) than did the other 5 γ-secretases. Considering that the Aβ42(43)/Aβ40 ratio is relevant in the pathogenesis of Alzheimer's disease (AD), and that inhibition of Notch cleavage causes severe side effect, these results suggest that the PS2/Aph1aS γ-secretase complex is a potential therapeutic target in AD.

Intramembrane proteases execute fundamental biological processes ranging from crucial signaling events to general membrane proteostasis. Despite the availability of structural information on these proteases, it remains unclear how these enzymes bind and recruit substrates, particularly for the Alzheimer's disease-associated γ-secretase. Systematically scanning amyloid precursor protein substrates containing a genetically inserted photocrosslinkable amino acid for binding to γ-secretase allowed us to identify residues contacting the protease. These were primarily found in the transmembrane cleavage domain of the substrate and were also present in the extramembranous domains. The N-terminal fragment of the catalytic subunit presenilin was determined as principal substrate-binding site. Clinical presenilin mutations altered substrate binding in the active site region, implying a pathogenic mechanism for familial Alzheimer's disease. Remarkably, PEN-2 was identified besides nicastrin as additional substrate-binding subunit. Probing proteolysis of crosslinked substrates revealed a mechanistic model of how these subunits interact to mediate a stepwise transfer of bound substrate to the catalytic site. We propose that sequential binding steps might be common for intramembrane proteases to sample and select cognate substrates for catalysis.

Nicastrin is the largest component of γ-secretase that is an intramembrane protease important in the development of Alzheimer’s disease. Nicastrin contains a large extracellular domain, a single transmembrane (TM) domain and a short C-terminus. Its TM domain is important for the γ-secretase complex formation. Here we report nuclear magnetic resonance (NMR) studies of the TM and C-terminal regions of human nicastrin in both sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelles. Structural study and dynamic analysis reveal that the TM domain is largely helical and stable under both SDS and DPC micelles with its N-terminal region undergoing intermediate time scale motion. The TM helix contains a hydrophilic patch that is important for TM-TM interactions. The short C-terminus is not structured in solution and a region formed by residues V697-A702 interacts with the membrane, suggesting that these residues may play a role in the γ-secretase complex formation. Our study provides structural insight into the function of the nicastrin TM domain and the C-terminus in γ-secretase complex.

The biological underpinnings and the pathological lesions of psychiatric disorders are centuries-old questions that have yet to be understood. Recent studies suggest that schizophrenia and related disorders likely have their origins in perturbed neurodevelopment and can result from a large number of common genetic variants or multiple, individually rare genetic alterations. It is thus conceivable that key neurodevelopmental pathways underline the various genetic changes and the still unknown pathological lesions in schizophrenia. Here, we report that mice defective of the nicastrin subunit of γ-secretase in oligodendrocytes have hypomyelination in the central nervous system. These mice have altered dopamine signaling and display profound abnormal phenotypes reminiscent of schizophrenia. In addition, we identify an association of the nicastrin gene with a human schizophrenia cohort. These observations implicate γ-secretase and its mediated neurodevelopmental pathways in schizophrenia and provide support for the "myelination hypothesis" of the disease. Moreover, by showing that schizophrenia and obsessive-compulsive symptoms could be modeled in animals wherein a single genetic factor is altered, our work provides a biological basis that schizophrenia with obsessive-compulsive disorder is a distinct subtype of schizophrenia. ispartof: Journal of Biological Chemistry vol:291 issue:22 pages:11647-56 ispartof: location:United States status: published