Presenilin Enhancer Research Articles

Specific combinations of presenilins and Aph1s affect the substrate specificity and activity of γ-secretase

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.

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

Identification of the novel activity-driven interaction between synaptotagmin 1 and presenilin 1 links calcium, synapse, and amyloid beta.

BackgroundSynaptic loss strongly correlates with memory deterioration. Local accumulation of amyloid β (Aβ) peptide, and neurotoxic Aβ42 in particular, due to abnormal neuronal activity may underlie synaptic dysfunction, neurodegeneration, and memory impairments. To gain an insight into molecular events underlying neuronal activity-regulated Aβ production at the synapse, we explored functional outcomes of the newly discovered calcium-dependent interaction between Alzheimer’s disease-associated presenilin 1 (PS1)/γ-secretase and synaptic vesicle proteins.ResultsMass spectrometry screen of mouse brain lysates identified synaptotagmin 1 (Syt1) as a novel synapse-specific PS1-binding partner that shows Ca2+-dependent PS1 binding profiles in vitro and in vivo. We found that Aβ level, and more critically, conformation of the PS1 and the Aβ42/40 ratio, are affected by Syt1 overexpression or knockdown, indicating that Syt1 and its interaction with PS1 might regulate Aβ production at the synapse. Moreover, β-secretase 1 (BACE1) stability, β- and γ-secretase activity, as well as intracellular compartmentalization of PS1 and BACE1, but not of amyloid precursor protein (APP), nicastrin (Nct), presenilin enhancer 2 (Pen-2), or synaptophysin (Syp) were altered in the absence of Syt1, suggesting a selective effect of Syt1 on PS1 and BACE1 trafficking.ConclusionsOur findings identify Syt1 as a novel Ca2+-sensitive PS1 modulator that could regulate synaptic Aβ, opening avenues for novel and selective synapse targeting therapeutic strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0248-3) contains supplementary material, which is available to authorized users.

Identification of candidate genes in atopic dermatitis based on bioinformatic methods.

Atopic dermatitis (AD) is a chronic or relapsing inflammatory disorder of the skin that frequently precedes asthma and allergic disorders. This study aimed to identify candidate genes related to AD using bioinformatic methods. The microarray data of GSE32924, including 12 nonlesional AD (ANL) and 13 lesional AD (AL) skin samples obtained from 14 patients with AD as well as eight other normal human skin samples, was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were screened in ANL and AL skin samples compared with normal controls, followed by gene ontology (GO) and pathway enrichment analyses. Furthermore, the selected overlapping DEGs were analyzed to identify co-expressed genes, and a co-expression network was established. GeneCodis database was selected for functional annotation of the differentially co-expressed genes and a regulatory network was constructed. Compared with normal controls, 438 DEGs were identified in ANL skin samples and were mainly enriched in two pathways and the GO terms associated with epidermis development. Besides, 779 DEGs were identified in AL skin samples and were mainly enriched in four pathways and GO terms associated with immune response. Stimulated by retinoic acid 13 (STRA13), presenilin enhancer gamma secretase subunit (PSENEN), and nucleosome assembly protein 1-like 2 (NAP1L2) were significantly enriched by integration analysis of the co-expression and regulatory network. Genes STRA13, PSENEN, and NAP1L2 were presumed to play critical roles in AD and they may serve as potential targets for the treatment of AD.

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.

Nicastrin is required for amyloid precursor protein (APP) but not Notch processing, while anterior pharynx-defective 1 is dispensable for processing of both APP and Notch.

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.

Propylthiouracil Attenuates Experimental Pulmonary Hypertension via Suppression of Pen-2, a Key Component of Gamma-Secretase.

Gamma-secretase-mediated Notch3 signaling is involved in smooth muscle cell (SMC) hyper-activity and proliferation leading to pulmonary arterial hypertension (PAH). In addition, Propylthiouracil (PTU), beyond its anti-thyroid action, has suppressive effects on atherosclerosis and PAH. Here, we investigated the possible involvement of gamma-secretase-mediated Notch3 signaling in PTU-inhibited PAH. In rats with monocrotaline-induced PAH, PTU therapy improved pulmonary arterial hypertrophy and hemodynamics. In vitro, treatment of PASMCs from monocrotaline-treated rats with PTU inhibited their proliferation and migration. Immunocyto, histochemistry, and western blot showed that PTU treatment attenuated the activation of Notch3 signaling in PASMCs from monocrotaline-treated rats, which was mediated via inhibition of gamma-secretase expression especially its presenilin enhancer 2 (Pen-2) subunit. Furthermore, over-expression of Pen-2 in PASMCs from control rats increased the capacity of migration, whereas knockdown of Pen-2 with its respective siRNA in PASMCs from monocrotaline-treated rats had an opposite effect. Transfection of PASMCs from monocrotaline-treated rats with Pen-2 siRNA blocked the inhibitory effect of PTU on PASMC proliferation and migration, reflecting the crucial role of Pen-2 in PTU effect. We present a novel cell-signaling paradigm in which overexpression of Pen-2 is essential for experimental pulmonary arterial hypertension to promote motility and growth of smooth muscle cells. Propylthiouracil attenuates experimental PAH via suppression of the gamma-secretase-mediated Notch3 signaling especially its presenilin enhancer 2 (Pen-2) subunit. These findings provide a deep insight into the pathogenesis of PAH and a novel therapeutic strategy.

The topology of pen-2, a γ-secretase subunit, revisited: evidence for a reentrant loop and a single pass transmembrane domain.

BackgroundThe γ-secretase complex, composed of transmembrane proteins termed presenilin (PS), anterior pharynx defective (APH), nicastrin (NCT), and presenilin enhancer-2 (Pen-2) catalyzes intramembranous hydrolysis of a variety of Type I membrane protein substrates. In order to understand aspects of subunit assembly, interactions, dynamics and catalysis, it is essential to clarify the membrane topology of each polypeptide. Hydophathicity plots predict that the 101 amino acid Pen-2 molecule has two hydrophobic domains (HP1 and HP2) that may serve as transmembrane spanning domains. Earlier reports indicated that transiently overexpressed Pen-2 uses these two hydrophobic domains as transmembrane helices that generates a “U-shaped” hairpin topology with both amino- (N-) and carboxyl-(C-) termini facing the lumen. In this report, we have reexamined the topology of endogenous Pen-2 and Pen-2 chimeras that are stably expressed in mammalian cells, and have assessed the function of these molecules in rescuing γ-secretase activity in Pen-2-deficient fibroblasts.ResultsWe confirm that the Pen-2 C-terminus is lumenal, but the N-terminus of Pen-2 is exposed to the cytoplasm, thus indicating that HP1 does not traverse the lipid bilayer as a transmembrane domain. Domain swapping studies reveal the importance of specific regions within the first hydrophobic domain of Pen-2 that are critical for generating the topology that is a prerequisite for mediating PS1 endoproteolysis and γ-secretase activity. Finally, we report that the first fourteen amino acids of the Pen-2 HP1 are required for γ-secretase activity.ConclusionsWe propose that the first hydrophobic domain of Pen-2 forms a structure similar to a reentrant loop while the second hydrophobic domain spans the lipid bilayer.

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.

γ-Secretase Mutation and Consequently Immune Reaction Involved in Pathogenesis of Acne Inversa.

Acne inversa (AI) is a chronic recurrent inflammatory skin condition, and gamma secretase gene mutation was found related to familial AI. However, other scholars found that only a few familial AI patients had pathogenic gamma secretase mutation. Here, we collected another seven Chinese AI families including 32 patients and 48 normal individuals, and detected gene mutations of gamma secretase. Clinical features of patients with mutations of nicastrin or presenilin enhancer were summarized, and serum levels of different cytokines including IL-2, IL-4, IL-5, IL-10, IL-17A, TNF-a, and IFN-g were investigated. We revealed seven novo mutations of gamma secretase in these families. Young male patients were found more frequently affected and posterior neck was the most easily affected area in patients with nicastrin or presenilin enhancer mutation. Comparing with normal controls, we also discovered that TNF-a and IL-10 levels were elevated in AI patients with nicastrin or presenilin enhancer mutation. It was concluded that gamma secretase mutation had a close relationship with Chinese familial AI, and Chinese AI patients had specific clinical characteristics. In patients with both nicastrin and presenilin enhancer mutation, TNF-a and IL-10 may play important roles in AI pathogenesis. MEETING REPORT

Function of Integrin-Linked Kinase in Modulating the Stemness of IL-6–Abundant Breast Cancer Cells by Regulating γ-Secretase–Mediated Notch1 Activation in Caveolae

Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6–driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6–driven Notch1 activation by ILK in IL-6–producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7IL-6 cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase–mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6–induced breast CSCs.

Distribution and expression of Pen-2 in the central nervous system of APP/PS1 double transgenic mice.

The γ-secretase complex catalyzes the final cleavage step of amyloid β-protein precursor (APP) to generate amyloid β (Aβ) peptide, a pathogenic component of senile plaques in the brain of Alzheimer's disease (AD) patients. Recent studies have shown that presenilin enhancer-2 (Pen-2), presenilin (PS, including PS1 and PS2), nicastrin, and anterior pharynx-defective 1 are essential components of the γ-secretase. The structure and function of Pen-2 in vitro have been well defined. However, little is known about the neuroanatomical distribution and expression of Pen-2 in the central nervous system (CNS) of AD model mice. We report here, using various methods such as immunohistochemical staining and immunoblotting, that Pen-2 is widely expressed at specific neuronal cells of major areas in AD model mice, including the olfactory bulb, basal forebrain, striatum, cortex, hippocampus, amygdala, thalamus, hypothalamus, cerebellum, brainstem, and spinal cord. It is co-expressed with PS1 in specific neuronal cells in mouse brain. Pen-2 is distributed much more extensively than extracellular amyloid deposits, suggesting the importance of other factors in localized amyloid deposition. Pen-2 is localized predominantly in cell membrane and cytoplasma in adult AD mice, but only distributed at cell membrane in controls. At the early stages of postnatal development, the expression level of Pen-2 is relatively high in CNS, but declines, gradually in adult mice. The present study provides an anatomical basis for Pen-2 as a key component of γ-secretase complex in the brain of developing and adult mice, and Pen-2 might be closely related to Aβ burden in aging nervous system.

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.

Reduction of β-amyloid and γ-secretase by calorie restriction in female Tg2576 mice

Research indicates that female risk of developing Alzheimer's disease (AD) is greater than that of males. Moderate reduction of calorie intake, known as calorie restriction (CR), reduces pathology in AD mouse models and is a potentially translatable prevention measure for individuals at-risk for AD, as well as an important tool for understanding how the brain endogenously attenuates age-related pathology. Whether sex influences the response to CR remains unknown. In this study, we assessed the effect of CR on beta-amyloid peptide (Aβ) pathology and hippocampal CA1 neuron specific gene expression in the Tg2576 mouse model of cerebral amyloidosis. Relative to ad libitum (AL) feeding, CR feeding significantly reduced hippocampal Aβ burden in 15-month-old female, but not age-matched male, Tg2576 mice. Sustained CR also significantly reduced expression of presenilin enhancer 2 (Psenen) and presenilin 1, components of the γ-secretase complex, in Tg2576 females. These results indicate that long-term CR significantly reduces age-dependent female Tg2576 Aβ pathology, which is likely to involve CR-mediated reductions in γ-secretase–dependent amyloid precursor protein (APP) metabolism.

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.

G206D Mutation of Presenilin-1 Reduces Pen2 Interaction, Increases Aβ42/Aβ40 Ratio and Elevates ER Ca(2+) Accumulation.

Early-onset familial Alzheimer's disease (AD) is most commonly associated with the mutations in presenilin-1 (PS1). PS1 is the catalytic component of the γ-secretase complex, which cleaves amyloid precursor protein to produce amyloid-β (Aβ), the major cause of AD. Presenilin enhancer 2 (Pen2) is critical for activating γ-secretase and exporting PS1 from endoplasmic reticulum (ER). Among all the familial AD-linked PS1 mutations, mutations at the G206 amino acid are the most adjacent position to the Pen2 binding site. Here, we characterized the effect of a familial AD-linked PS1 G206D mutation on the PS1-Pen2 interaction and the accompanied alteration in γ-secretase-dependent and -independent functions. We found that the G206D mutation reduced PS1-Pen2 interaction, but did not abolish γ-secretase formation and PS1 endoproteolysis. For γ-secretase-dependent function, the G206D mutation increased Aβ42 production but not Notch cleavage. For γ-secretase-independent function, this mutation disrupted the ER calcium homeostasis but not lysosomal calcium homeostasis and autophagosome maturation. Impaired ER calcium homeostasis may due to the reduced mutant PS1 level in the ER. Although this mutation did not alter the cell survival under stress, both increased Aβ42 ratio and disturbed ER calcium regulation could be the mechanisms underlying the pathogenesis of the familial AD-linked PS1 G206D mutation.

Functional analysis and purification of a Pen-2 fusion protein for γ-secretase structural studies.

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.

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.