Anterior Pharynx Defective-1 Research Articles

Levels of nicastrin species are increased in Alzheimer’s disease cerebrospinal fluid

AbstractBackgroundPrevious studies demonstrated that presenilin‐1 (PS1), the active component of the intramembrane γ‐secretase complex, can be detected as soluble heteromeric aggregates in cerebrospinal fluid (CSF). The γ‐secretase components, APH‐1 (anterior pharynx‐defective 1) and PEN‐2 (presenilin enhancer 2), co‐exist within these CSF complexes, while nicastrin is not detected. Nicastrin plays a role in the complex regulation, and substrate recognition, being the unique γ‐secretase component that is a Type I, single‐pass transmembrane protein. Here, we aim to characterize and compare the levels of CSF nicastrin in Alzheimer’s disease (AD) and non‐AD control patients.MethodNicastrin levels were analyzed in the CSF by western blotting using antibodies against the ectodomain and the intracellular domain to identify the different species of the protein that are present in human CSF. Core AD CSF biomarkers (Aβ1‐42, T‐tau, and p‐Tau) were determined. We also assessed whether CSF nicastrin and PS1 are present in extracellular vesicles (EVs).ResultWe show that nicastrin is present in CSF as full‐length species of 130, 110, and 105 kDa. We demonstrated that nicastrin full‐length species are increased in AD patients (n = 21) in comparison with non‐AD controls (n = 21), and that the levels of the three species positively correlated with T‐tau and p‐Tau levels in AD patients. Nicastrin was also found to be present in EVs, different from PS1.ConclusionThe increased levels of nicastrin in the CSF of AD patients confirmed the potential alteration of γ‐secretase subunits during pathology progression. Further studies are required to evaluate whether these proteins could act as biomarkers to facilitate AD diagnosis and/or prognosis.

Effects of the Gamma Secretase Inhibitors (GSI) Dapt (GSI-IX) and LSN424354 on Gene Expression and Shedding of Bcma from Multiple Myeloma and Chronic Lymphocytic Leukemia Tumor Cells

Introduction: Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) patients have high levels of solubilized (s) BCMA in the blood, and this protein is responsible for the immune deficiency of MM. sBCMA may also interfere with the efficacy of anti-BCMA-CAR-transduced T cells or other anti-BCMA antibody-based immunotherapy for multiple myeloma patients. Recently, γ-secretase has been identified as the enzyme that leads to the shedding of BCMA from B-cells. Gamma secretase (GS) is a multi-subunit protease complex that includes four individual proteins: presenilin-1 (PSEN1), nicastrin, anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). CD147 acts as a non-essential regulator of the complex. We examined gene expression of PSEN1, APH-1, PEN-2, and CD147 among MM pts with progressive disease (PD) and complete remission (CR). We also determined the effects of the GS inhibitors (i) DAPT (GSI-IX) and LSN424354 (Eli Lilly, Indianapolis, IN) on solubilized BCMA levels from cultured human MM and CLL tumor cells.Study design: Bone marrow (BM) mononuclear cells (MCs) and serum were collected from monoclonal gammopathy of undetermined significance (MGUS) individuals, MM patients and healthy subjects after obtaining IRB approval. BMMCs from MM patients were treated with or without DAPT (GSI-IX) in a concentration dependent fashion for 24 hours. Following incubation, cells were washed three times using PBS. Flow cytometric analysis was performed using a Beckman Coulter Cytomics FC 500 with Cytomics CXP software. Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Louisville, KY) following the manufacturer's instructions. Quantitative PCR (qPCR) was applied to measure the relative abundance of human PSEN1, APH-1, PEN-2, and CD147 mRNA compared to that of the housekeeping gene HPRT mRNA. Single-cell suspensions were prepared from human MM LAGκ-1A xenografts which had been grown in the left superficial gluteal muscle of SCID mice. Cells were cultured and treated with the LSN424354 in a concentration dependent fashion. Supernatant BCMA levels were determined using an ELISA (R&D Systems, Minneapolis, MN) assay.Results and Discussion: qPCR showed PEN-2 gene expression was only slightly increased among MM patients with progressive disease (PD) compared to those in complete response or normal subjects whereas there was no change in expression of the PSEN1 or APH1 genes. CD147 gene expression was markedly increased in MM pts in PD (n=25) compared with those in CR (n=18; P=0.005) or MGUS (n=9; P=0.005). Next, we examined the effect of the DAPT (GSI-IX) on primary human MM tumor cells. Supernatants from primary human MM cells cultured for 24 hours in DAPT showed markedly reduced BCMA in a concentration dependent fashion (range,0.2 nM-2µM). Using qPCR, DAPT markedly reduced PEN-2 gene expression in a concentration dependent fashion. On the other hand, this GSI did not effect BCMA gene expression. DAPT reduces shedding of BCMA from B-cells but not its gene expression. We also determined BCMA levels in supernatants from cultured tumor cells derived from the human MM LAGκ-1A xenograft following 5 days of tissue culture with LSN424354 at concentrations ranging from 100 pM to 10 nM. Supernatant sBCMA levels were reduced > 90% at the highest concentrations (10nM). Similarly, freshly obtained CLL tumor cells cultured with LSN424354 at concentrations ranging from 100 pM to 10 nM also showed a marked reduction in supernatant BCMA levels at the higher concentrations.Conclusion: Gamma secretase sheds BCMA off B-cells. CD147, a regulator of gamma secretase activity, shows markedly higher gene expression among MM pts with PD compared with those in CR or MGUS individuals. The GSIs DAPT and LSN424354 reduce shedding of BCMA from malignant cells from MM and CLL patients. Since GSIs reduce solubilized BCMA levels, it is possible that treatment with these agents can reverse the immune dysfunction caused by this solubilized protein, and also improve the efficacy of anti-BCMA immunotherapies for treating patients with multiple myeloma and CLL. DisclosuresBerenson:Incyte: Consultancy, Research Funding; OncoTracker, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Mutations in γ-secretase subunit–encoding PSENEN gene alone may not be sufficient for the development of acne inversa

BackgroundThe 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. ObjectiveTo investigate the effects of PSENEN knockdown on γ-secretase subunits, biological behaviors, and related signaling pathways in keratinocytes. MethodsHaCaT 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. ResultsA 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. ConclusionsPSENEN mutations alone may be insufficient to cause the development of AI, or they may only induce a mild phenotype of AI.

Insight γ-Secretase: Structure, Function, and Role in Alzheimer's Disease.

In neurodegenerative disorders, there is a progressive degeneration of the body, leading to the death of nerve cells. In this state, a patient gets affected day by day with mental weakness, dementia, and ataxia. Alzheimer's disease (AD) is the most common irreversible neurodegenerative brain disorder mainly affecting people over the age of 65. Many types of research suggest that the main culprit for AD is the aggregated form of a (39-43) amino acid peptide called amyloid beta. Amyloid beta (Aβ) is generated by the action of beta-secretase and gamma-secretase on the larger glycoprotein. Gamma (γ) secretase is an intra-membrane protease complex that cleaves the single-- pass transmembrane protein, the amyloid precursor protein, and Notch. The γ-secretase complex contains presenilin, presenilin enhancer-2, anterior pharynx defective-1, and nicastrin. Any mutation in presenilin-1 or the cleavage of amyloid precursor protein by γ-secretase directly or indirectly is associated with AD. Therefore, the prevention of this enzyme is one of the solutions for AD. In this article, we discuss the γ-secretase complex and its inhibitors that can contribute to the prevention of AD.

High temperature promotes amyloid β-protein production and γ-secretase complex formation via Hsp90

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.

Membrane dynamics of γ-secretase with the anterior pharynx-defective 1B subunit.

The four-subunit protease complex γ-secretase cleaves many single-pass transmembrane (TM) substrates, including Notch and β-amyloid precursor protein to generate amyloid-β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx-defective 1 (APH-1) and presenilin (PS) exist in two homologous forms APH1-A and APH1-B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ-secretase medicine. Here, we developed the first complete structural model of the APH-1B subunit using the published cryo-electron microscopy (cryo-EM) structures of APH1-A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all-atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH-1B alone and in γ-secretase without and with substrate C83-bound. We show that APH-1B adopts a 7TM topology with a water channel topology similar to APH-1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo-EM structures with APH-1A, however with subtle differences: The substrate-bound APH-1B γ-secretase was quite stable, but some TM helices of PS1 and APH-1B rearranged in the membrane consistent with the disorder seen in the cryo-EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH-1B, that is, it represents a more closed state, due to interactions with the C-terminal fragment of PS1. Our structural-dynamic model of APH-1B alone and in γ-secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.

Isorhynchophylline ameliorates cognitive impairment via modulating amyloid pathology, tau hyperphosphorylation and neuroinflammation: Studies in a transgenic mouse model of Alzheimer’s disease

Isorhynchophylline (IRN) has been demonstrated to have distinct anti-Alzheimer’s disease (AD) activity in several animal models of AD. In this study, we aimed at evaluating the preventive effect of IRN on the cognitive deficits and amyloid pathology in TgCRND8 mice. Male TgCRND8 mice were administered with IRN (20 or 40 mg/kg) by oral gavage daily for 4 months, followed by assessing the spatial learning and memory functions with the Radial Arm Maze (RAM) test. Brain tissues were determined immunohistochemically or biochemically for changes in amyloid pathology, tau hyperphosphorylation and neuroinflammation. Our results revealed that IRN (40 mg/kg) significantly ameliorated cognitive deficits in TgCRND8 mice. In addition, IRN (40 mg/kg) markedly reduced the levels of Aβ40, Aβ42 and tumor necrosis factor (TNF-α), interleukin 6 (IL-6) and IL-1β, and modulated the amyloid precursor protein (APP) processing and phosphorylation by altering the protein expressions of β-site APP cleaving enzyme-1 (BACE-1), phosphorylated APP (Thr668), presenilin-1 (PS-1) and anterior pharynx-defective-1 (APH-1), as well as insulin degrading enzyme (IDE), a major Aβ-degrading enzyme. IRN was also found to inhibit the phosphorylation of tau at the sites of Thr205 and Ser396. Immunofluorescence showed that IRN reduced the Aβ deposition, and suppressed the activation of microglia (Iba-1) and astrocytes (GFAP) in the cerebral cortex and hippocampus of TgCRND8 mice. Furthermore, IRN was able to attenuate the ratios of p-c-Jun/c-Jun and p-JNK/JNK in the brains of TgCRND8 mice. IRN also showed marked inhibitory effect on JNK signaling pathway in the Aβ-treated rat primary hippocampus neurons. We conclude that IRN improves cognitive impairment in TgCRND8 transgenic mice via reducing Aβ generation and deposition, tau hyperphosphorylation and neuroinflammation through inhibiting the activation of JNK signaling pathway, and has good potential for further development into pharmacological treatment for AD.

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.

Impacts of Acute Hypoxia on Alzheimer's Disease-Like Pathologies in APPswe/PS1dE9 Mice and Their Wild Type Littermates.

Alzheimer's disease (AD) is the most common form of dementia and pathologically featured by β-amyloid (Aβ) plaque deposition and hyper-phosphorylated tau aggregation in the brain. Environmental factors are believed to contribute to the pathogenesis and progression of AD. In the present study, we investigated the impacts of acute hypoxia on Aβ and tau pathologies, neuroinflammation, mitochondrial function, and autophagy in APPswe/PS1dE9 AD mouse model. Male APPswe/PS1dE9 transgenic (Tg) mice and their age-matched wild type (Wt) littermates were exposed to one single acute hypoxic episode (oxygen 7%) for 24 h. We found that acute hypoxia exposure increased the expressions of amyloid precursor protein (APP), anterior pharynx-defective 1 (APH1) and cyclin-dependent kinase 5 (CDK5), and promoted tau phosphorylation at T181 and T231 residues in both Tg and Wt mice. In addition, acute hypoxia also induced autophagy through the mammalian target of rapamycin (mTOR) signaling, elicited abnormal mitochondrial function and neuroinflammation in both Tg and Wt mice. In summary, all these findings suggest that acute hypoxia could induce the AD-like pathological damages in the brain of APPswe/PS1dE9 mice and Wt mice to some extent.

Structural and Chemical Biology of Presenilin Complexes.

The presenilin proteins are the catalytic subunits of a tetrameric complex containing presenilin 1 or 2, anterior pharynx defective 1 (APH1), nicastrin, and PEN-2. Other components such as TMP21 may exist in a subset of specialized complexes. The presenilin complex is the founding member of a unique class of aspartyl proteases that catalyze the γ, ɛ, ζ site cleavage of the transmembrane domains of Type I membrane proteins including amyloid precursor protein (APP) and Notch. Here, we detail the structural and chemical biology of this unusual enzyme. Taken together, these studies suggest that the complex exists in several conformations, and subtle long-range (allosteric) shifts in the conformation of the complex underpin substrate access to the catalytic site and the mechanism of action for allosteric inhibitors and modulators. Understanding the mechanics of these shifts will facilitate the design of γ-secretase modulator (GSM) compounds that modulate the relative efficiency of γ, ɛ, ζ site cleavage and/or substrate specificity.

Huannao Yicong Formula () regulates γ-secretase activity through APH-1 and PEN-2 gene ragulation pathways in hippocampus of APP/PS1 double transgenic mice.

To observe the effects of Huannao Yicong Formula (, HYF) on learning and memory and it's regulating effect on γ-secretase related anterior pharynx defective 1 (APH-1), presenilin enhancer-2 (PEN-2) signaling pathway, so as to discuss and further clarify the mechanism of HYF on Alzheimer's disease. Sixty APP/PS1 transgenic mice, randomly allocated into 4 groups, the model group, the donepezil group (0.65 mg/kg), HYF low-dose group (HYF-L, 5.46 g/kg) and HYF high-dose group (HYF-H, 10.92 g/kg), 15 for each group. Another 15 C57BL/6J mice with the same age and same genetic background were allocated into the control group, proper dosage of drugs or distilled water were given by intragastric administration once daily for 12 weeks. After 12 weeks of administration, the learning and memory abilities of mice in each group was evaluated by the morris water maze test, amyloid precursor protein (APP), Aβ1-40 and Aβ1-42 levels in hippocampus were detected by enzyme-linked immunosorbent assay, γ-secretase was detected by dual luciferase assaying, the levels of APH-1a, hypoxia-inducible factor 1α (HIF-1α), cAMP response element-binding protein (CREB) and PEN-2 and their mRNA expression was measured by Western blot and real-time polymerase chain reaction. HYF can ameliorate learning and memory deficits in APP/PS1 transgenic mice by decreasing the escape latency, improving the number of platform crossing and swimming speed (P<0.01, P<0.05). HYF can decrease the levels of APP, Aβ1-40, Aβ1-42 and the activity of γ-secretase in hippocampus of Alzheimer's disease model mice. HYF can down-regulate the levels of CREB and PEN-2 and the expression of their mRNA. HYF can improve the learning and memory ability by inhibiting the activity of γ-secretase through the CREB/PEN-2 signaling pathway, and this may be one of the therapeutic mechanisms of HYF in Alzheimer's disease.

Gene Expression of Gamma Secretase (GS) Complex-Related Proteins, the Enzyme That Sheds B-Cell Maturation Antigen (BCMA), Among Patients with Multiple Myeloma (MM) and Effects of the GS Inhibitor LSN424354 on Solubilized Bcma in MM and Chronic Lymphocytic Leukemia

Introduction: B-cell maturation antigen (BCMA) has been shown to be highly expressed on the surface of tumor cells from patients (pts) with multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) and is the target for therapeutic approaches in these diseases. Our group has shown that serum and plasma levels of BCMA correlate with disease status and survival in MM and CLL patients. We have recently shown that solubilized BCMA prevents its ligand B cell activating factor (BAFF) from binding and stimulating B-cells to produce polyclonal antibody. Recently, γ-secretase has been identified as the enzyme that leads to shedding of BCMA from off of B-cells. Gamma secretase is a multi-subunit protease complex that includes four individual proteins: presenilin-1 (PSEN1), nicastrin, anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). CD147 is as a non-essential regulator of the complex. We examined gene expression of PSEN1, APH-1, PEN-2, and CD147 in MM pts with progressive (PD) and in complete remission (CR). We also determined the effects of the gamma secretase inhibitor (GSI) LSN424354 (Eli Lilly), a selective small-molecule inhibitor, on solubilized BCMA levels in MM and CLL tumor cells.Methods: Bone marrow (BM) mononuclear cells (MCs) were isolated from MM pts following Western Institutional Review Board (WIRB) approval and informed consent in accordance with the Declaration of Helsinki. Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Louisville, KY 40219) following the manufacturer’s instructions. Quantitative PCR (qPCR) was applied to measure the relative abundance of human PSEN1, APH-1, PEN-2, and CD147 mRNA compared to that of the housekeeping gene HPRT mRNA. qPCR was performed using TaqMan technology on an OneStepPlus instrument (Life Technology, Grand Island, NY 14072) and followed the standard procedure. Peripheral blood MCs were obtained from patients with CLL. The human MM LAGκ-1A xenograft was grown in the left superficial gluteal muscle of SCID mice for six weeks and removed. Single-cell suspensions were prepared. LAGκ-1A or CLL tumor cells were treated with the GSI LSN424354 in a concentration dependent fashion, and BCMA levels were determined using an ELISA (R&D Systems, Minneapolis, MN) assay. MM tumor cells were treated with the LSN424354 and performed non-radioactive MTS cell proliferation assay following a standard MTS protocol.Results: qPCR showed PEN-2 gene expression was slightly increased among MM patients in PD compared to those in CR or normal subjects whereas there was no change in expression of the PSEN1 or APH1 genes. CD147 gene expression was markedly increased in MM pts in PD (n=25) compared to those in CR (n=18; P=0.005) or MGUS (n=9; P=0.005). Next, we determined the effect of the GSI LSN424354 on cultured MM or CLL tumor cells. The GSI inhibitor LSN424354 markedly reduced BCMA levels (> 90%) in supernatants from human MM LAGκ-1A cells after 5 days of tissue culture in a dose dependent fashion at concentrations ranging from 100 pM to 10 nM. Similarly, freshly obtained CLL tumor cells exposed to LSN424354 at concentrations similarly from 100 pM to 10 nM also showed a marked reduction (also > 90%) in supernatant BCMA levels. Notably, MTS assay results showed LSN424354 did not inhibit cell proliferation in MM or CLL tumor cells at concentrations up to 100 nM.Conclusion: Gamma secretase sheds BCMA off of B-cells. CD147, a regulator of gamma secretase activity, shows markedly higher gene expression in MM pts with PD compared to those in CR or MGUS individuals. The GSI LSN424354 reduces solubilized BCMA from tumor cells from CLL patients and human MM xenografts. Since CD147 has been shown to be present in serum, we are currently evaluating CD147 in serum samples from pts with MM and other plasma cell dyscrasias. In addition, we are examining the expression of BCMA on the surface of tumor cells from MM and CLL pts following exposure to BCMA, whether this GSI by reducing solubilized BCMA levels can reverse the immune dysfunction in mice bearing MM, and improve the efficacy of anti-BCMA antibody therapies. DisclosuresBerenson:OncoTracker: Employment, Equity Ownership.

Mutation analysis of the nicastrin gene in a Chinese pedigree with acne inversa

Objective To identify mutations of the γ-secretase gene in two patients with acne inversa (AI)in a Chinese pedigree. Methods Blood samples were obtained from 5 family members (including 2 patients, the proband's father, 2 unaffected family members)and 100 unrelated healthy controls. Genomic DNA was extracted from these blood samples, and PCR was conducted to amplify all the exons and their flanking sequences of nicastrin (NCSTN), presenilin-1 (PSEN1), presenilin enhancer (PSENEN)and anterior pharynx-defective 1 (APH1)genes, followed by direct sequencing. Besides, the NCSTN mRNA expression in skin lesions of the proband was compared with that in the normal skin of 4 healthy controls. Results A heterozygous mutation (c.477C > A)at position 477 in the exon 5 of the NCSTN gene, which had not been described previously in the National Center for Biotechnology Information (NCBI)single nucleotide polymorphism database, was identified in the 2 patients, but not observed in the other 3 unaffected family members or the 100 unrelated controls. Additionally, the mRNA expression of NCSTN was evidently decreased in the lesions of the proband compared with the normal skin of healthy controls. Conclusion The novel nonsense mutation c.477C> A in the NCSTN gene is the causative mutation of AI in this family, and it may induce functional inactivation of NCSTN through the nonsense-mediated mRNA decay. Key words: Acne inversa; Genes; Mutation; Pedigree; Genes, nicastrin

Evidence That the “Lid” Domain of Nicastrin Is Not Essential for Regulating γ-Secretase Activity

Understanding of the structure of the γ-secretase complex consisting of presenilin (PS), anterior pharynx-defective 1 (APH-1), nicastrin (NCT), and presenilin enhancer 2 (PEN-2) is of significant therapeutic interest for the design of γ-secretase modulators for Alzheimer disease. The structure of γ-secretase revealed by cryo-EM approaches suggested a substrate binding mechanism for NCT, a bilobar structure that involved rotation of the two lobes around a central pivot and opening of a "lid" region that facilitates substrate recruitment. To validate this proposal, we expressed NCT that lacks the lid entirely, or a variety of NCT variants that harbor mutations at highly conserved residues in the lid region inNCT-deficient cells, and then assessed their impact on γ-secretase assembly, activity, and stability. In addition, we assessed the impact of mutating a critical residue proposed to be a pivot around which the two lobes of NCT rotate. Our results show that neither the mutations on the lid tested here nor the entire lid deletion has any significant impact on γ-secretase assembly, activity, and stability, and that NCT with the mutation of the proposed pivot rescues γ-secretase activity inNCT-deficient cells in a manner indistinguishable from WT NCT. These findings indicate that the NCT lid is not an essential element necessary for γ-secretase assembly, activity, and stability, and that rotation of the two lobes appears not to be a prerequisite for substrate binding and γ-secretase function.

Determination of the Interaction of γ-Secretase with its βCTF Substrate

A key step in the pathogenesis of Alzheimer's disease (AD) is γ-secretase's proteolysis of βCTF (C99) substrate, resulting in the formation of different sized Aβ peptides, incuding Aβ42, which is assumed to be the principale component of the characteristical cerebral plaques of the disease.It was proposed that the oligomeric state of C99 might play a role for its proteolytic processing by the γ-secretase enzyme. γ-Secretase is a multi-protein complex consisting of presenilin (PS), nicastrin (Nct), anterior pharynx defective-1 (Aph-1) and presenilin enhancer-2 (Pen-2). Of these, presenilin is the catalytic component and thus of special interest.Despite the importance of the presenilin subunit for the generation of Alzheimer's disease, there are still a number of important questions. The exact location of the C99 substrate binding site and the question if, or if not dimerization of the substrate plays a role for C99 substrate recognition and therefor intramembrane-cleavage is still under big debate.To answer the question which role dimerization plays, we designed four single-cysteine mutants of C99. These proteins were crosslinked to form covalent dimers, which were tested as substrates in an in-vitro γ-secretase assay. We show here that the covalently crosslinked C99 homodimer cannot be cleaved by γ-secretase, suggesting that dimerization may not be important for cleavage. To help identify the γ-secretase binding site, we used an in-vivo reporter assay to test for interaction between the transmembrane helix of C99 substrate and the isolated transmembrane helices of presenilin. The role of transmembrane helix interaction in the recognition of the C99 substrate is discussed.

Akt1 phosphorylates Nicastrin to regulate its protein stability and activity.

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.

The γ-secretase complex: from structure to function.

One of the most critical pathological features of Alzheimer’s disease (AD) is the accumulation of β-amyloid (Aβ) peptides that form extracellular senile plaques in the brain. Aβ is derived from β-amyloid precursor protein (APP) through sequential cleavage by β- and γ-secretases. γ-secretase is a high molecular weight complex minimally composed of four components: presenilins (PS), nicastrin, anterior pharynx defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). In addition to APP, γ-secretase also cleaves many other type I transmembrane (TM) protein substrates. As a crucial enzyme for Aβ production, γ-secretase is an appealing therapeutic target for AD. Here, we summarize current knowledge on the structure and function of γ-secretase, as well as recent progress in developing γ-secretase targeting drugs for AD treatment.

A Synthetic Antibody Fragment Targeting Nicastrin Affects Assembly and Trafficking of γ-Secretase

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.

CSF Presenilin-1 complexes are increased in Alzheimer’s disease

BackgroundPresenilin-1 (PS1) is the active component of the amyloid precursor protein cleaving γ-secretase complex. PS1 protein is a transmembrane protein containing multiple hydrophobic regions which presence in cerebrospinal fluid (CSF) has not been measured to date. This study assesses whether PS1 and other components of the γ-secretase complex are present in CSF.ResultsHere, we show that PS1 is present in ventricular post-mortem and lumbar ante-mortem CSF, and plasma as 100–150-kDa hetero-complexes containing both the N- and C-terminal fragments (NTF and CTF) of the protein. Immunoprecipitation and immunoblotting with different antibodies confirmed the identity of the PS1 species. The γ-secretase components, APH-1 (anterior pharynx-defective 1) and PEN-2 (presenilin enhancer 2), as well as presenilin-2 (PS2) fragments, co-exist within these CSF complexes, while nicastrin is not detected. These CSF-PS1 complexes differ from active γ-secretase membrane-complexes, and may represent nonspecific aggregation of the PS1 protein. Levels of PS1 complexes are increased in CSF samples from autopsy-confirmed Alzheimer’s disease (AD) cases and were found to be more stable than complexes in CSF from control subjects. Despite similar levels of total PS1 in CSF from probable AD patients and cognitively normal subjects, an increased proportion of highly stable PS1 complexes were observed in AD CSF.ConclusionsOur data suggest that fragments of the PS1 protein present in CSF as complexes may be useful as a biomarker for AD.

Chronic Administration of Anti-Stroke Herbal Medicine TongLuoJiuNao Reduces Amyloidogenic Processing of Amyloid Precursor Protein in a Mouse Model of Alzheimer’s Disease

Composed of Ginsenoside Rg1 and Geniposide, the herbal medicine TongLuoJiuNao (TLJN) injection liquid has anti-inflammatory properties and can improve learning and memory in mice. Recently, TLJN has been used to treat the patients with cerebral ischemic stroke and vascular dementia, which significantly increase the risk of developing Alzheimer’s disease (AD) in the early human beings. Although beneficial effects of TLJN have been reported in the vascular-associated brain disorders, the roles of TLJN in AD brains are still not clear. In this study, we chronically administered TLJN in amyloid precursor protein (APP) Swedish mutant transgenic mice (APP23) from 6 months old of age, which is at the onset of Aβ plaques, to 12 months old. We found that TLJN significantly decreased Aβ production and deposition in the brain of APP23 mice. Furthermore, we observed that TLJN down-regulated the levels and activity of β-secretase 1 (BACE1) protein as well as the expression levels of γ-secretase complex components: PS1, nicastrin and anterior pharynx-defective 1 (APH1) but not presenilin enhancer 2 (PEN2). The results suggest an inhibitory effect of TLJN on amyloidogenic APP processing by down-regulating the cleavage enzymes BACE1 and γ-secretase.