Zebrafish lacking Alzheimer presenilin enhancer 2 (Pen-2) demonstrate excessive p53-dependent apoptosis and neuronal loss

Gamma-secretase cleavage, mediated by a complex of presenilin, presenilin enhancer (Pen-2), nicastrin, and Aph-1, is the final proteolytic step in generating amyloid beta protein found in brains of Alzheimer's disease patients and Notch intracellular domain critical for proper neuronal development. Here, we employ the zebrafish model to study the role of Pen-2 in neuronal survival. We found that (i) knockdown of Pen-2 using antisense morpholino led to a reduction of islet-1 positive neurons, (ii) Notch signaling was reduced in embryos lacking Pen-2 or other gamma-secretase components, (iii) neuronal loss in Pen-2 knockdown embryos is not as a result of a lack of neuronal precursor cells or cell proliferation, (iv) absence of Pen-2 caused massive apoptosis in the whole animal, which could be suppressed by simultaneous knockdown of the tumor suppressor p53, (v) loss of islet-1 or acetylated tubulin positive neurons in Pen-2 knockdown embryos could be partially rescued by knockdown of p53. Our results demonstrate that knockdown of Pen-2 directly induces a p53-dependent apoptotic pathway that contributes to neuronal loss and suggest that Pen-2 plays an important role in promoting neuronal cell survival and protecting from apoptosis in vivo.     

Active gamma-secretase complexes contain only one of each component

Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.     

Abeta42 overproduction associated with structural changes in the catalytic pore of gamma-secretase: common effects of Pen-2 N-terminal elongation and fenofibrate

gamma-Secretase is an atypical aspartyl protease that cleaves amyloid beta-precursor protein to generate Abeta peptides that are causative for Alzheimer disease. gamma-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2. Pen-2 directly binds to transmembrane domain 4 of PS and confers proteolytic activity on gamma-secretase, although the mechanism of activation and its role in catalysis remain unknown. Here we show that an addition of amino acid residues to the N terminus of Pen-2 specifically increases the generation of Abeta42, the longer and more aggregable species of Abeta. The effect of the N-terminal elongation of Pen-2 on Abeta42 generation was independent of the amino acid sequences, the expression system and the presenilin species. In vitro gamma-secretase assay revealed that Pen-2 directly affects the Abeta42-generating activity of gamma-secretase. The elongation of Pen-2 N terminus caused a reduction in the water accessibility of the luminal side of the catalytic pore of PS1 in a similar manner to that caused by an Abeta42-raising gamma-secretase modulator, fenofibrate, as determined by substituted cysteine accessibility method. These data suggest a unique mechanism of Abeta42 overproduction associated with structural changes in the catalytic pore of presenilins caused commonly by the N-terminal elongation of Pen-2 and fenofibrate.     

Functional and topological analysis of Pen-2, the fourth subunit of the gamma-secretase complex

The γ-secretase complex is a member of the family of intramembrane cleaving proteases, involved in the generation of the Aβ peptides in Alzheimer disease. One of the four subunits of the complex, presenilin, harbors the catalytic site, although the role of the other three subunits is less well understood. Here, we studied the role of the smallest subunit, Pen-2, in vivo and in vitro. We found a profound Notch-deficiency phenotype in Pen-2-/- embryos confirming the essential role of Pen-2 in the γ-secretase complex. We used Pen-2-/- fibroblasts to investigate the structure-function relation of Pen-2 by the scanning cysteine accessibility method. We showed that glycine 22 and proline 27 in hydrophobic domain 1 of Pen-2 are essential for complex formation and stability of γ-secretase. We also demonstrated that hydrophobic domain 1 and the loop domain of Pen-2 are located in a water-containing cavity and are in short proximity to the presenilin C-terminal fragment. We finally demonstrated the essential role of Pen-2 for the proteolytic activity of the complex. Our study supports the hypothesis that Pen-2 is more than a structural component of the γ-secretase complex and may contribute to the catalytic mechanism of the enzyme.     

Partial purification and characterization of gamma-secretase from post-mortem human brain

One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.     

Pen-2 is sequestered in the endoplasmic reticulum and subjected to ubiquitylation and proteasome-mediated degradation in the absence of presenilin

The gamma-secretase complex catalyzes intramembrane proteolysis of a number of transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and E-cadherin. gamma-Secretase is known to contain four major protein constituents: presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane proteins. There is increasing evidence that the formation of the complex and the stability of the individual components are tightly controlled in the cell, assuring correct composition of functional complexes. In this report, we investigate the topology, localization, and mechanism for destabilization of Pen-2 in relation to PS function. We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature gamma-secretase complexes reside. PS deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2, which adopts a hairpin structure with the N and C termini facing the luminal space, is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the gamma-secretase complex leads to degradation of Pen-2 through the ER-associated degradation-proteasome pathway.     

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.     

In vitro reconstitution of gamma-secretase activity using yeast microsomes

γ-Secretase is composed of at least four transmembrane proteins, presenilin (PS) 1/2, nicastrin, anterior pharynx-1 (Aph-1) and presenilin enhancer-2 (Pen-2), and cleaves amyloid precursor protein (APP) to produce amyloid β peptides (Aβ) that is deposited in the brains of Alzheimer disease. However, the mechanism of γ-secretase-mediated cleavage remains unclear. To examine the enzymatic properties of γ-secretase, we established an in vitro assay system using Saccharomyces cerevisiae, which does not possess homologs of human PS1/2, nicastrin, Aph-1, or Pen-2. We transformed these subunits and the substrate in pep4Δ cells with vacuole proteases inactivated, and microsome was isolated for in vitro assay. In the assay, Aβ40, Aβ42, and Aβ43 were produced with an optimal pH of ∼7.0. We also detected Aβ-production by yeast endogenous protease(s), which was abolished by the addition of phosphatidyl choline. This novel system will facilitate the analysis of substrate recognition by γ-secretase.     

Analysis of Notch function in presomitic mesoderm suggests a gamma-secretase-independent role for presenilins in somite differentiation

The role of Notch signaling in general and presenilin in particular was analyzed during mouse somitogenesis. We visualize cyclical production of activated Notch (NICD) and establish that somitogenesis requires less NICD than any other tissue in early mouse embryos. Indeed, formation of cervical somites proceeds in Notch1; Notch2-deficient embryos. This is in contrast to mice lacking all presenilin alleles, which have no somites. Since Nicastrin-, Pen-2-, and APH-1a-deficient embryos have anterior somites without gamma-secretase, presenilin may have a gamma-secretase-independent role in somitogenesis. Embryos triple homozygous for both presenilin null alleles and a Notch allele that is a poor substrate for presenilin (N1(V-->G)) experience fortuitous cleavage of N1(V-->G) by another protease. This restores NICD, anterior segmentation, and bilateral symmetry but does not rescue rostral/caudal identities. These data clarify multiple roles for Notch signaling during segmentation and suggest that the earliest stages of somitogenesis are regulated by both Notch-dependent and Notch-independent functions of presenilin.     

Pen-2 is incorporated into the gamma-secretase complex through binding to transmembrane domain 4 of presenilin 1

gamma-Secretase is a multimeric membrane protein complex comprised of presenilin (PS), nicastrin (Nct), Aph-1, and Pen-2. It is a member of an atypical class of aspartic proteases that hydrolyzes peptide bonds within the membrane. During the biosynthetic process of the gamma-secretase complex, Nct and Aph-1 form a heterodimeric intermediate complex and bind to the C-terminal region of PS, serving as a stabilizing scaffold for the complex. Pen-2 is then recruited into this trimeric complex and triggers endoproteolysis of PS, conferring gamma-secretase activity. Although the Pen-2 accumulation depends on PS, the binding partner of Pen-2 within the gamma-secretase complex remains unknown. We reconstituted PS1 in Psen1/Psen2 deficient cells by expressing a series of PS1 mutants in which one of the N-terminal six transmembrane domains (TMDs) was swapped with those of CD4 (a type I transmembrane protein) or CLAC-P (a type II transmembrane protein). We report that the proximal two-thirds of TMD4 of PS1, including the conserved Trp-Asn-Phe sequence, are required for its interaction with Pen-2. Using a chimeric CD4 molecule harboring PS1 TMD4, we further demonstrate that the PS1 TMD4 bears a direct binding motif to Pen-2. Pen-2 may contribute to the activation of the gamma-secretase complex by directly binding to the TMD4 of PS1.     

The gamma-secretase complex: machinery for intramembrane proteolysis

Gamma-secretase is a membrane protease complex that possesses presenilin as a catalytic subunit. Presenilin generates amyloid beta peptides in the brains of Alzheimer's patients and is indispensable to Notch signaling in tissue development and renewal. Recent studies have revealed how presenilin is assembled with its cofactor proteins and acquires the gamma-secretase activity: Aph-1 and nicastrin initially form a subcomplex to bind and stabilize presenilin, and then Pen-2 confers the gamma-secretase activity and facilitates endoproteolysis of presenilin. Understanding the mechanism of gamma-secretase cleavage will help to clarify how intercellular cell signaling through transmembrane proteins is regulated by intramembrane proteolysis, and will hopefully eventually lead to a cure for Alzheimer's disease.     

Pro-apoptotic function of calsenilin/DREAM/KChIP3.

Apoptotic cell death and increased production of amyloid b peptide (Ab) are pathological features of Alzheimer's disease (AD), although the exact contribution of apoptosis to the pathogenesis of the disease remains unclear. Here we describe a novel pro-apoptotic function of calsenilin/DREAM/KChIP3. By antisense oligonucleotide-induced inhibition of calsenilin/DREAM/KChIP3 synthesis, apoptosis induced by Fas, Ca2+-ionophore, or thapsigargin is attenuated. Conversely, calsenilin/DREAM/KChIP3 expression induced the morphological and biochemical features of apoptosis, including cell shrinkage, DNA laddering, and caspase activation. Calsenilin/DREAM/KChIP3-induced apoptosis was suppressed by caspase inhibitor Z-VAD and by Bcl-XL, and was potentiated by increasing cytosolic Ca2+, expression of Swedish amyloid precursor protein mutant (APPSW) or presenilin 2 (PS2), but not by a PS2 deletion lacking its C-terminus (PS2/411stop). In addition, calsenilin/DREAM/KChIP3 expression increased Ab42 production in cells expressing APPsw, which was potentiated by PS2, but not by PS2/411stop, which suggests a role for apoptosis-associated Ab42 production of calsenilin/DREAM/KChIP3.     

Assembly of the gamma-secretase complex involves early formation of an intermediate subcomplex of Aph-1 and nicastrin

The gamma-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the beta-amyloid precursor protein and Notch. Genetic and biochemical data have revealed that this protease consists of the presenilin heterodimer, a highly glycosylated form of nicastrin, and the recently identified gene products, Aph-1 and Pen-2. Whereas current evidence supports the notion that presenilin comprises the active site of the protease and that the other three components are members of the active complex required for proteolytic activity, the individual roles of the three co-factors remain unclear. Here, we demonstrate that endogenous Aph-1 interacts with an immature species of nicastrin, forming a stable intermediate early in the assembly of the gamma-secretase complex, prior to the addition of presenilin and Pen-2. Our data suggest 1) that Aph-1 is involved in the early stages of gamma-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature gamma-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease.     

The overexpression of presenilin2 and Alzheimer's-disease-linked presenilin2 variants influences TRPC6-enhanced Ca2+ entry into HEK293 cells

Mutations in the presenilin (PS) genes are linked to the development of early-onset Alzheimer's disease by a gain-of-function mechanism that alters proteolytic processing of the amyloid precursor protein (APP). Recent work indicates that Alzheimer's-disease-linked mutations in presenilin1 and presenilin2 attenuate calcium entry and augment calcium release from the endoplasmic reticulum (ER) in different cell types. However, the regulatory mechanisms underlying the altered profile of Ca(2+) signaling are unknown. The present study investigated the influence of two familial Alzheimer's-disease-linked presenilin2 variants (N141I and M239V) and a loss-of-function presenilin2 mutant (D263A) on the activity of the transient receptor potential canonical (TRPC)6 Ca(2+) entry channel. We show that transient coexpression of Alzheimer's-disease-linked presenilin2 mutants and TRPC6 in human embryonic kidney (HEK) 293T cells abolished agonist-induced TRPC6 activation without affecting agonist-induced endogenous Ca(2+) entry. The inhibitory effect of presenilin2 and the Alzheimer's-disease-linked presenilin2 variants was not due to an increase in amyloid beta-peptides in the medium. Despite the strong negative effect of the presenilin2 and Alzheimer's-disease-linked presenilin2 variants on agonist-induced TRPC6 activation, conformational coupling between inositol 1,4,5-trisphosphate receptor type 3 (IP(3)R3) and TRPC6 was unaffected. In cells coexpressing presenilin2 or the FAD-linked presenilin2 variants, Ca(2+) entry through TRPC6 could still be induced by direct activation of TRPC6 with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Furthermore, transient coexpression of a loss-of-function PS2 mutant and TRPC6 in HEK293T cells enhanced angiotensin II (AngII)- and OAG-induced Ca(2+) entry. These results clearly indicate that presenilin2 influences TRPC6-mediated Ca(2+) entry into HEK293 cells.     

Pen‐2 is dispensable for endoproteolysis of presenilin 1, and nicastrin‐Aph subcomplex is important for both γ‐secretase assembly and substrate recruitment

γ‐secretase is a protease complex with at least four components: presenilin, nicastrin (NCT), anterior pharynx‐defective 1 (Aph‐1), and presenilin enhancer 2 (Pen‐2). In this study, using knockout cell lines and small interfering RNA technology, our data demonstrated that the disappeared presenilin 1 C‐terminal fragment (PS1C) caused by knockdown of pen‐2 or knockout of NCT or Aph‐1 was recovered by the addition of proteasome inhibitors, indicating that Pen‐2, as well as NCT and Aph‐1α, is dispensable for presenilin endoproteolysis. Our data also demonstrate that the formation of the nicastrin‐Aph‐1 subcomplex plays not only an important role in γ‐secretase complex assembly but also in recruiting substrate C‐terminal fragment of amyloid precursor protein generated by β‐cleavage. Ablating any one component resulted in the instability of other components of the γ‐secretase complex, and the presence of all three of the other components is required for full maturation of NCT.     

Membrane topology and nicastrin-enhanced endoproteolysis of APH-1, a component of the gamma-secretase complex

APH-1, presenilin, nicastrin, and Pen-2 are proteins with varying membrane topologies that compose the gamma-secretase complex, which is responsible for the intramembrane proteolysis of several substrates including the amyloid precursor protein. APH-1 is known to be necessary for gamma-secretase activity, but its precise function in the complex is not fully understood, and its membrane topology has not been described, although it is predicted to traverse the membrane seven times. To investigate this, we used selective permeabilization of the plasma membrane and immunofluorescence microscopy to show that the C terminus of the APH-1 resides in the cytosolic space. Insertion of N-linked glycosylation sites into each of the hydrophilic loop domains and the N terminus of APH-1 showed that the N-terminal domain as well as loops 2, 4, and 6 could be glycosylated, whereas loops 1, 3, and 5 were not. Thus, APH-1 topologically resembles a seven-transmembrane domain receptor with the N terminus and even-numbered loops facing the endoplasmic reticulum lumen, and the C terminus and odd-numbered loops reside in the cytosolic space. By using these glycosylation mutants, we provide evidence that the association between nicastrin and APH-1 may occur very soon after APH-1 synthesis and that the interaction between these two proteins may rely more heavily on the transmembrane domains of APH-1 than on the loop domains. Furthermore, we found that APH-1 can be processed by several endoproteolytic events. One of these cleavages is strongly up-regulated by co-expression of nicastrin and generates a stable C-terminal fragment that associates with nicastrin.     

Fas-associated factor-1 inhibits nuclear factor-kappaB (NF-kappaB) activity by interfering with nuclear translocation of the RelA (p65) subunit of NF-kappaB

Fas-associated factor-1 (FAF1) is a Fas-binding pro-apoptotic protein that is a component of the death-inducing signaling complex in Fas-mediated apoptosis. Here, we show that FAF1 is involved in negative regulation of NF-kappaB activation. Overexpression of FAF1 decreased the basal level of NF-kappaB activity in 293 cells. NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha, interleukin-1beta, and lipopolysaccharide was also inhibited by FAF1 overexpression. Moreover, FAF1 suppressed NF-kappaB activation induced by transducers of diverse NF-kappaB-activating signals such as TNF receptor-associated factor-2 and -6, MEKK1, and IkappaB kinase-beta as well as NF-kappaB p65, one of the end point molecules in the NF-kappaB activation pathway, suggesting that NF-kappaB p65 might be a target molecule upon which FAF1 acts. Subsequent study disclosed that FAF1 physically interacts with NF-kappaB p65 and that the binding domain of FAF1 is the death effector domain (DED)-interacting domain (amino acids 181-381), where DEDs of the Fas-associated death domain protein and caspase-8 interact. The NF-kappaB activity-modulating potential of FAF1 was also mapped to the DED-interacting domain. Finally, overexpression of FAF1 prevented translocation of NF-kappaB p65 into the nucleus and decreased its DNA-binding activity upon TNFalpha treatment. This study presents a novel function of FAF1, in addition to the previously known function as a component of the Fas death-inducing signaling complex, i.e. NF-kappaB activity suppressor by cytoplasmic retention of NF-kappaB p65 via physical interaction.     

Sh3rf2/POSHER protein promotes cell survival by ring-mediated proteasomal degradation of the c-Jun N-terminal kinase scaffold POSH (Plenty of SH3s) protein

We report that Sh3rf2, a homologue of the pro-apoptotic scaffold POSH (Plenty of SH3s), acts as an anti-apoptotic regulator for the c-Jun N-terminal kinase (JNK) pathway. siRNA-mediated knockdown of Sh3rf2 promotes apoptosis of neuronal PC12 cells, cultured cortical neurons, and C6 glioma cells. This death appears to result from activation of JNK signaling. Loss of Sh3rf2 triggers activation of JNK and its target c-Jun. Also, apoptosis promoted by Sh3rf2 knockdown is inhibited by dominant-negative c-Jun as well as by a JNK inhibitor. Investigation of the mechanism by which Sh3rf2 regulates cell survival implicates POSH, a scaffold required for activation of pro-apoptotic JNK/c-Jun signaling. In cells lacking POSH, Sh3rf2 knockdown is unable to activate JNK. We further find that Sh3rf2 binds POSH to reduce its levels by a mechanism that requires the RING domains of both proteins and that appears to involve proteasomal POSH degradation. Conversely, knockdown of Sh3rf2 promotes the stabilization of POSH protein and activation of JNK signaling. Finally, we show that endogenous Sh3rf2 protein rapidly decreases following several different apoptotic stimuli and that knockdown of Sh3rf2 activates the pro-apoptotic JNK pathway in neuronal cells. These findings support a model in which Sh3rf2 promotes proteasomal degradation of pro-apoptotic POSH in healthy cells and in which apoptotic stimuli lead to rapid loss of Sh3rf2 expression, and consequently to stabilization of POSH and JNK activation and cell death. On the basis of these observations, we propose the alternative name POSHER (POSH-eliminating RING protein) for the Sh3rf2 protein.