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Abstract: Immunolocalization and expression of endogenous nicastrin (NCT) and presenilin 1 (PS1) fragments during postnatal development of rat cerebellum were investigated with fragment-specific antibodies. Immunoblotting for NCT revealed the expected mature and immature species, which gradually declined during development. In contrast, the expression of PS1 N-terminal fragment exhibited a peak at postnatal day 14 (P14) and declined thereafter. This chronological change was similarly observed with PS1 C-terminal fragment. Immunoprecipitation of NCT indicated its physical association with PS1 fragments. Colocalization of these molecules to the endoplasmic reticulum in cerebellar Purkinje cells indicates that they are organized into a complex in developing neurons. In addition, active sites of synaptogenesis, the base of the external granular layer and glomeruli, contained PS1 fragments and smaller amount of NCT. Isolated synaptic fraction contained both PS1 and NCT, suggesting their functional association within synapses. Transient abundance of NCT and PS1 fragments as a complex, when (P14) and where synaptogenesis is active, is consistent with intracellular trafficking of this complex in developing neurons and suggests its role as γ-secretase in synaptogenesis. [Copyright &y& Elsevier]

Abstract: The γ-secretase complex, a membrane-bound aspartyl protease, hydrolyzes the transmembrane domains of several integral membrane proteins including the key signaling molecules amyloid precursor protein (APP), Notch, deleted in colorectal cancer (DCC), and N- and E-cadherins. The proteolysis processing of these proteins is critical for generation of signaling molecules that may participate in neuronal communication and plasticity. Using a potent γ-secretase inhibitor, L-685,458, we examined if blockade of its activity in the hippocampus can influence contextual and spatial memory in rats. Surprisingly, we observed that post-training blockade of γ-secretase activity leads to enhanced long-term memory in two hippocampus-dependent tasks. This suggests that a signaling molecule(s) generated by γ-secretase activity may have a negative influence on long-term memory formation. [Copyright &y& Elsevier]

The γ-secretase complex processes substrate proteins within membranes and consists of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. PS harbours the enzymatic activity of the complex, and there are two mammalian PS homologues: PS1 and PS2. PS undergoes endoproteolysis, generating the N- and C-terminal fragments, NTF and CTF, which represent the active species of PS. To characterize the functional similarity between complexes of various PS composition, we analysed PS1, PS2, and chimeric PS composed of the NTF from PS1 and CTF from PS2, or vice versa, in assembly and function of the γ-secretase complex. Chimeric PSs, like PS1 and PS2, undergo normal endoproteolysis when introduced into cells devoid of endogenous PS. Furthermore, PS2 CTF can, at least partially, restore processing in a truncated PS1, which cannot undergo endoproteolysis. All PS forms enable maturation of nicastrin and cleave full length Notch receptors, indicating that both PS1 and PS2 are present at the cell surface. Finally, when co-introduced as separate molecules, NTF and CTF of different PS origin reconstitute γ-secretase activity. In conclusion, these data show that endoproteolysis, NTF–CTF interactions, and the assembly and activity of γ-secretase complexes are very conserved between PS1 and PS2. [ABSTRACT FROM AUTHOR]

Presenilins (PS) are thought to contain the active site for presenilinase endoproteolysis of PS and γ-secretase cleavage of substrates. The structural requirements for PS incorporation into the γ-secretase enzyme complex, complex stability and maturation, and appropriate presenilinase and γ-secretase activity are poorly understood. We used rescue assays to identify sequences in transmembrane domain one (TM1) of PS1 required to support presenilinase and γ-secretase activities. Swap mutations identified an N-terminal TM1 domain that is important for γ-secretase activity only and a C-terminal TM1 domain that is essential for both presenilinase and γ-secretase activities. Exchange of residues 95–98 of PS1 (sw95–98) completely abolishes both activities while the familial Alzheimer's disease mutation V96F significantly inhibits both activities. Reversion of residue 96 back to valine in the sw95–98 mutant rescues PS function, identifying V96 as the critical residue in this region. The TM1 mutants do not bind to an aspartyl protease transition state analog γ-secretase inhibitor, indicating a conformational change induced by the mutations that abrogates catalytic activity. TM1 mutant PS1 molecules retain the ability to interact with γ-secretase substrates and γ-secretase complex members, although Nicastrin stability is decreased by the presence of these mutants. γ-Secretase complexes that contain V96F mutant PS1 molecules display a partial loss of function for γ-secretase that alters the ratio of amyloid-β peptide species produced, leading to the amyloid-β peptide aggregation that causes familial Alzheimer's disease. [ABSTRACT FROM AUTHOR]

γ-Secretase is an aspartyl protease complex that catalyzes the intramembrane cleavage of a subset of type I transmembrane proteins including theβ-amyloid precursor protein (APP) implicated in Alzheimer's disease. Presenilin (PS), nicastrin (NCT), anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2) constitute the activeγ-secretase complex. PEN-2, the smallest subunit, is required for triggering PS endoproteolysis. Stabilization of the resultant N- and C-terminal fragments, which carry the catalytically active site aspartates, but not endoproteolysis itself, requires the C-terminal domain of PEN-2. To functionally dissect the C-terminal domain we created C-terminal deletion mutants and mutagenized several evolutionary highly conserved residues. The PEN-2 mutants were then probed for functional complementation of a PEN-2 knockdown, which displays deficient PS1 endoproteolysis and impaired NCT maturation. Progressive truncation of the C-terminus caused increasing loss of function. This was also observed for an internal deletion mutant as well as for C-terminally tagged PEN-2 with a twofold elongated C-terminal domain. Interestingly, only simultaneous, but not individual substitution of the highly conserved D90, F94, P97 and G99 residues with alanine interfered with PEN-2 function. All loss of function mutants identified allowed PS1 endoproteolysis, but failed to stably associate with the resultant PS1 fragments, which like the PEN-2 loss of function mutants underwent proteasomal degradation. However, complex formation of the PEN-2 mutants with PS1 fragments could be recovered when proteasomal degradation was blocked. Taken together, our data suggest that the PS-subunit stabilizing function of PEN-2 depends on length and overall sequence of its C-terminal domain. [ABSTRACT FROM AUTHOR]

Abstract: APH-1 is one of the four essential components of the presenilin-γ-secretase complex and has two human homologs, APH-1a, and APH-1b, both of which are seven-pass membrane proteins. Here, we identified a novel splice variant of human APH-1b. This variant lacks exon 4, which encodes the entire fourth transmembrane domain. The mRNA expression of this variant was detected in most tissues at low levels. In transiently transfected cells, protein expression of the APH-1b variant was much lower than that of the wild-type. Furthermore, exogenous expression of the APH-1-interacting protein, nicastrin, significantly increased the variant protein levels. These data suggest that the APH-1b variant protein is destabilized, and implies that the fourth transmembrane domain plays an important role in the protein stability and function of APH-1. [Copyright &y& Elsevier]

Abstract: Nicastrin is a type 1 transmembrane glycoprotein that interacts with presenilin, Aph-1, and Pen-2 proteins to form a high molecular complex with gamma secretase activity. Then, nicastrin has a central role in presenilin-mediated processing of beta-amyloid precursor protein and in some aspects of Notch/glp-1 signaling in vivo. Here, we isolated a rat nicastrin cDNA and investigated gene expression in embryonic and adult rat tissues. The predicted amino acid sequence is comprised of 708 residues and showed a high degree of identity with other vertebrate orthologs. Besides full-length nicastrin mRNA, we identified an alternative spliced variant lacking the whole exon 3 and predicted to encode a 62-residue-long truncated protein. Full-length nicastrin mRNA was observed to be ubiquitously expressed, while the spliced variant was preferentially transcribed in the nervous system, whether in embryonic or adult neural tissues. Studies performed on primary cell cultures demonstrated that the short isoform was expressed in neurons, but not in astrocyte and microglial cells. Further experiments performed to verify the presence of the variant in neuroblastoma culture failed to show any truncated protein. Treatments by cyclohexamide showed the involvement of a quality control-based surveillance mechanism, which selectively degrades the exon 3-skipped isoform. In summary, this is the first report describing a novel skipped isoform of nicastrin which may suggest a new possible control mechanism based on the alternative splicing and nonsense-mediated mRNA decay to regulate brain protein expression and provide newer insights into potential implication in Alzheimer''s disease. [Copyright &y& Elsevier]

The activity of theγ-secretase complex is critical for the processing of a number of transmembrane proteins, including Notch. Functionalγ-secretase activity can be reconstituted from four proteins– presenilin, nicastrin, Pen-2 and Aph-1– but the role of the individual proteins remains unclear. In this report we describe the cellular localization and protein interactions of Aph-1, with particular regard to Notch receptor processing. We found that Aph-1 is present at the cell surface, where it interacts with Pen-2, the mature forms of presenilin and nicastrin, and full-length Notch. Aph-1 also interacts with a truncated form of Notch, which is a direct substrate forγ-secretase, but not with the Notch intracellular domain. Immunoprecipitation data for Notch and Aph-1 showed that the Notch-containingγ-secretase complexes most likely form a small subset of the total number ofγ-secretase complexes. In conclusion, these data demonstrate that Aph-1 is present at the cell surface, presumably in activeγ-secretase complexes, and interacts with the Notch receptor, both before and after ligand activation. [ABSTRACT FROM AUTHOR]

The structural requirements for presenilin (PS) to produce active presenilinase andγ-secretase enzymes are poorly understood. Here we investigate the role the cytoplasmic C-terminal region of PS1 plays in PS1 activity. Deletion or addition of residues at the PS C-terminus has been reported to inhibit presenilinase endoproteolysis of PS and alterγ-secretase activity. In this study, we use a sensitive assay in PS1/2KO MEFs to define a domain at the extreme C-terminus of PS1 that is essential for both presenilinase andγ-secretase activities. Progressive deletion of the C-terminus demonstrated that removal of nine residues produces a PS1 molecule (458ST) that lacks both presenilinase processing andγ-secretase cleavage of Notch and APP substrates. In contrast, removal of four or five residues had no effect (462ST, 463ST), while intermediate truncations partially inhibited PS1 activity. The 458ST mutant was unable to replace endogenous wtPS1 in HEK293 cells. Although 458ST was able to form aγ-secretase complex, this complex was not matured, illustrated by mutant PS1 instability, lack of endoproteolysis, and little production of mature Nicastrin. These data indicate that the C-terminal end of PS1 is essential for Nicastrin trafficking and modification as well as the replacement of endogenous PS1 by PS1 transgenes. [ABSTRACT FROM AUTHOR]

Abstract: APH-1 is a polytopic membrane protein that functions as a component of presenilin–γ-secretase complexes. Two homologous genes of APH-1 exist in humans, APH-1a and APH-1b, and alternative splicing of the former generates two isoforms, APH-1aS and APH-1aL. We performed semi-quantitative reverse transcription-PCR analysis to investigate mRNA expression of these three APH-1 forms in human cell lines and tissues. We found that both APH-1a and APH-1b were expressed in almost all tissues, and that APH-1aS was 1.5–3 times more abundantly expressed than APH-1aL. We examined the effect of small interfering RNA-mediated knock down of APH-1a or APH-1b on APH-1 mRNA expression and presenilin complex protein expression. We found that knock down of APH-1a, but not APH-1b, resulted in impaired maturation of nicastrin and reduced expression of presenilin 1, presenilin 2, and PEN-2 proteins. These findings indicate that APH-1a plays an essential role in the formation of presenilin–γ-secretase complexes. [Copyright &y& Elsevier]

Abstract: Studies conducted in cell culture indicate that the γ-secretase involved in amyloid β-formation and Notch signaling is a multisubunit aspartic protease. Little is known, however, of the structure, function, or localization of γ-secretase in the adult brain, or possible effects of familial Alzheimer''s disease (FAD)-causing mutations on the brain protease. We report here that mouse brain contains a complex composed of γ-secretase subunits presenilin-1 N-terminal fragment, presenilin-1 C-terminal fragment, Nicastrin, Aph-1a and Pen-2. A homozygous FAD-linked Presenilin-1 knock-in mutation does not alter relative subunit levels. Immunocytochemical localization of γ-secretase subunits revealed overlapping but distinct regional and subcellular distributions. All subunits are expressed throughout the neuraxis predominantly in neurons, and are present in axons. Their distributions and levels of expression are unaffected by mutant presenilin-1. In a presenilin-1/amyloid precursor protein double knock-in mouse, subunits are associated with plaques, but are expressed at similar levels in amyloid-rich and -poor regions. γ-Secretase subunits are distributed much more extensively than circumscribed amyloid deposits, suggesting the importance of other factors for localized amyloid deposition. These results indicate a widespread neuronal function for γ-secretase in the adult brain, and suggest the pathogenic mechanism of FAD-linked mutations does not involve alterations in the composition, expression or brain distribution of the protease. The subcellular localization of γ-secretase subunits is consistent with a nerve terminal source for amyloid aggregates. [Copyright &y& Elsevier]

Abstract: The hypothesis that polymorphisms in the gene for nicastrin (NCSTN) are associated with differences in cognitive level and ageing was tested in 462 relatively healthy surviving participants of the Scottish Mental Survey 1932. None had a history of dementia. They were tested on the Moray House Test of verbal reasoning at age 11 in 1932 and at age 79 between 1999 and 2001. At age 79 they also took tests of non-verbal reasoning, short- and long-term verbal declarative memory, Verbal Fluency, and a short screening test for dementia. Subjects who possessed at least one copy of the NCSTN B haplotype (Hap B) had higher scores on the Moray House Test (a test principally of verbal reasoning) at age 11 (p=0.036) and age 79 (p=0.027). The effect of Hap B on cognition at age 79 was non-significant after adjusting for the effect at age 11. Therefore, the effect of Hap B in this sample is on the life-long stable trait of cognitive ability, and not on age-related cognitive change. The possibility that this result might be a selection effect was not supported by the samples being in Hardy–Weinberg equilibrium with respect to the distribution of NCSTN genotypes. [Copyright &y& Elsevier]

APH-1 (anterior pharynx defective) along with nicastrin and PEN-2 (presenilin enhancer) are essential components of the presenilin (PS)-dependent γ-secretase complex. There exist three murine Aph-1 alleles termed Aph-1a, Aph-1b, and Aph-1c that encode four distinct APH-1 isoforms: APH-1aL and APH-1aS derived from differential splicing of Aph-1a, APH-1b, and APH-1c. To determine the contributions of mammalian APH-1 homologs in formation of functional γ-secretase complexes, we generated Aph-1a-/- mice and derived immortalized fibroblasts from these embryos. Compared with littermate controls, the development of Aph-1a-/- embryos was dramatically retarded by embryonic day 9.5 and exhibited patterning defects that resemble, but are not identical to, those of Notch1, nicastrin, or PS null embryos. Moreover, in immortalized Aph-1a-/- fibroblasts, the levels of nicastrin, PS fragments, and PEN-2 were dramatically decreased. Consequently, deletion of Aph-1a resulted in significant reduction in levels of high-molecular-weight γ-secretase complex and secretion of β-amyloid (Aβ). Importantly, complementation analysis revealed that all mammalian APH-1 isoforms were capable of restoring the levels of nicastrin, PS, and PEN-2, as well as Aβ secretion in Aph-1a-/- cells. Together, our findings establish that APH-1a is the major mammalian APH-1 homolog present in PS-dependent γ-secretase complexes during embryogenesis and support the view that mammalian APH-1 isoforms define a set of distinct functional γ-secretase complexes. [ABSTRACT FROM AUTHOR]

?-Secretase is an intramembrane cleaving protease involved in Alzheimer's disease.?-Secretase occurs as a high molecular weight complex composed of presenilin (PS1/2), nicastrin (NCT), anterior pharynx-defective phenotype 1 and PS enhancer 2. Little is known about the cellular mechanisms of?-secretase assembly. Here we demonstrate that the cytoplasmic tail of PS1 fulfills several functions required for complex formation, retention of unincorporated PS1 and?-secretase activity. The very C-terminus interacts with the transmembrane domain of NCT and may penetrate into the membrane. Deletion of the last amino acid is sufficient to completely block?-secretase assembly and release of PS1 from the endoplasmic reticulum (ER). This suggests that unincorporated PS1 is actively retained within the ER. We identified a hydrophobic stretch of amino acids within the cytoplasmic tail of PS1 distinct from the NCT-binding site, which is required to retain unincorporated PS1 within the ER. Deletion of the retention signal results in the release of PS1 from the ER and the assembly of a nonfunctional?-secretase complex, suggesting that at least a part of the retention motif may also be required for the function of PS1. [ABSTRACT FROM AUTHOR]

This study investigated whether nicastrin can induce apoptotic cell death in SK-N-MC cells. MTT assays revealed the transfected cells expressing mutant nicastrin, compared with those expressing wild nicastrin or the control vector, showing significantly increased cell death. The mutant nicastrin transfectants were also observed to induce cytosolic cytochrome c release from the mitochondria, and Bax protein expression in response, to increased cell death. These observations suggested that nicastrin, as well as the APP and PS proteins, were also involved in the upregulated Bax mediated neuroblastoma cell death and the release of cytochrome c in the neuroblastoma. [ABSTRACT FROM AUTHOR]

The presenilin (PS) complex, including PS, nicastrin, APH-1 and PEN-2, is essential for γ-secretase activity, which is required for amyloid β-protein (Aβ) generation. However, the precise individual roles of the three cofactors in the PS complex in Aβ generation remain to be clarified. Here, to distinguish the roles of PS cofactors in γ-secretase activity from those in PS endoproteolysis, we investigated their roles in the γ-secretase activity reconstituted by the coexpression of PS N- and C-terminal fragments (NTF and CTF) in PS-null cells. We demonstrate that the coexpression of PS1 NTF and CTF forms the heterodimer and restores Aβ generation in PS-null cells. The generation of Aβ was saturable at a certain expression level of PS1 NTF/CTF, while the overexpression of PEN-2 alone resulted in a further increase in Aβ generation. Although PEN-2 did not enhance PS1 NTF/CTF heterodimer formation, PEN-2 expression reduced the IC50 of a specific γ-secretase inhibitor, a transition state analogue, for Aβ generation, suggesting that PEN-2 expression enhances the affinity or the accessibility of the substrate to the catalytic site. Thus, our results strongly suggest that PEN-2 is not only an essential component of the γ-secretase complex but also an enhancer of γ-cleavage after PS heterodimer formation. [ABSTRACT FROM AUTHOR]

Nodals are signaling factors of the transforming growth factor-ß (TGFß) superfamily with a key role in vertebrate development. They control a variety of cell fate decisions required for the establishment of the embryonic body plan. We have identified two highly conserved transmembrane proteins, Nicalin and Nomo (Nodal modulator, previously known as pM5), as novel antagonists of Nodal signaling. Nicalin is distantly related to Nicastrin, a component of the Alzheimer's disease-associated ?-secretase, and forms a complex with Nomo. Ectopic expression of both proteins in zebrafish embryos causes cyclopia, a phenotype that can arise from a defect in mesendoderm patterning mediated by the Nodal signaling pathway. Accordingly, downregulation of Nomo resulted in an increase in anterior axial mesendoderm and the development of an enlarged hatching gland. Inhibition of Nodal signaling by ectopic expression of Lefty was rescued by reducing Nomo levels. Furthermore, Nodal- as well as Activin-induced signaling was inhibited by Nicalin and Nomo in a cell-based reporter assay. Our data demonstrate that the Nicalin/Nomo complex antagonizes Nodal signaling during mesendodermal patterning in zebrafish. [ABSTRACT FROM AUTHOR]

γ-Secretase is a high molecular mass aspartyl protease complex composed of presenilin (PS1 or PS2), nicastrin (Nct), anterior pharynx-defective-1 (APH-1) and presenilin enhancer-2 (PEN-2). The complex mediates the intramem- braneous proteolysis of β-secretase cleaved β-amyloid pre- cursor protein (APP) leading to the secretion of the Alzheimer's disease-associated amyloid β-peptide (Aβ). In order to dissect functionally important domains of Nct required for γ-secretase complex assembly, maturation, and activity we mutated evolutionary conserved amino acids. The mutant Nct variants were expressed in a cellular background with signi- ficantly reduced endogenous Nct. Mutant Nct was functionally investigated by its ability to restore PS, APH-1 and PEN-2 expression as well as by monitoring the accumulation of the APP C-terminal fragments, the immediate substrates of γ-secretase. We identified three independent mutations within the ectodomain of Nct, which rescued expression of APH-1 but not of PEN-2 or PS and thus failed to restore c-secretase activity. Interestingly, these immature Nct variants selectively bound to APH-1, suggesting a stable Nct/APH)1 interaction independent of PS and PEN-2. Consistent with this finding, expression of APH-1 remained largely unaffected in the PS double knock-out and immature Nct co-immunoprecipitated with APH-1 in the absence of PS and PEN-2. Taken together, our findings suggest that immature Nct can stably interact with APH-1 to form a potential scaffold for binding of PS and PEN- 2. Moreover, binding of the latter two complex partners crit- ically depends on the integrity of the Nct ectodomain. [ABSTRACT FROM AUTHOR]

One of the hallmarks of Alzheimer's disease is the cerebral deposition of plaques composed of a 37-43 amino acid amyloid-beta (Abeta) peptide. Abeta is produced by the sequential proteolytic cleavage of an integral-membrane protein, amyloid beta-protein precursor (AbetaPP), first by beta-secretase (BACE), and then by gamma-secretase, a complex containing presenilin and Nicastrin. Although these cleavages were originally documented to occur in the endosomal/ lysosomal system, other lines of evidence suggest that the responsible proteins and activity reside in the ER or Golgi. This lack of intracellular co-localization of enzyme and substrate has been referred to as the spatial paradox of Alzheimer's disease. Here we will review the biology of the lysosome and the literature supporting the endosomal/ lysosomal production of Abeta. We will also examine some of the data supporting Abeta production in the biosynthetic compartments and demonstrate its compatibility with an endosomal/ lysosomal model. Finally, we will discuss the possible role of the acidic environment of the lysosome in the amyloidogenic process, and review the evidence for intracellular amyloidogenesis preceding amyloid plaque formation. [ABSTRACT FROM AUTHOR]

γ-Secretase is a proteolytic complex whose substrates include Notch, β-amyloid precursor protein (APP), and several other type I transmembrane proteins. Presenilin (PS) and nicastrin are known components of this high-molecular-weight complex, and recent genetic screens in invertebrates have identified two additional gene products, Aph1 and Pen-2, as key factors in γ-secretase activity. Here, we examined the interaction of the components of the γ-secretase complex in Chinese hamster ovary cells stably expressing human forms of APP, PS1, Aph1, and Pen-2. Subcellular fractionation of membrane vesicles and subsequent coimmunoprecipitation of individual γ-secretase components revealed that interactions among all proteins occurred in the Golgi/trans-Golgi network (TGN) compartments. Furthermore, incubation of the Golgi/TGN-enriched vesicles resulted in de novo generation of amyloid β-protein and APP intracellular domain. Immunofluorescent staining of the individual γ-secretase components supported our biochemical evidence that the γ-secretase components assemble into the proteolytically active γ-secretase complex in the Golgi/TGN compartment. [Copyright &y& Elsevier]

Abstract Presenilin (PS) in association with nicastrin (NICA) forms a γ-secretase complex that plays a crucial role in facilitating intramembranous processing of Notch, a signaling receptor that is essential for neuronal fate specification and differentiation. Loss of function studies have implicated a role for PS1 in regulating neuronal differentiation in association with the down-regulation of Notch signaling during neurogenesis. By using a system for stable, as well as tetracycline-inducible expression of interfering RNAs (RNAi), we studied the functions of PS1 during neuronal differentiation in the murine pluripotent p19 embryonic carcinoma cell line. After retinoic acid (RA) treatment and in the absence of doxycycline, neuronal progenitor cells in the p19 clone were found to extend their processes towards the neighboring colony to form network-like connections, as revealed by neuron-specific microtubule-associated protein 2 staining and laser scanning confocal microscopy. However, doxycycline-induced expression of PS1 small interfering RNA (siRNA) in the p19 clone resulted in a severe defect in the formation of network-like connections. Expression of the NICA and Notch down-stream effector genes Hes1 and Hes5 was unaffected in p19 cells expressing doxycycline-induced PS1 siRNA. In contrast to PS1, constitutive inactivation of NICA by siRNA in p19 cells resulted in premature and partial differentiation without RA treatment. In these NICA siRNA-expressing p19 cells the expression of the Notch1 down-stream effector Hes1 gene was substantially reduced. After RA treatment the NICA siRNA clone failed to differentiate completely into networks of neurons. These results taken together provide direct evidence that PS1 and NICA may participate in neuronal differentiation during neurogenesis in vitro . [ABSTRACT FROM AUTHOR]

Presenilins (PS) constitute a fascinating family of proteins that play crucial roles in several major signalling processes involved in key cellular functions and are also closely associated with dysfunction in Alzheimer''s disease (AD). Presenilin-dependent intramembrane cleavage of transmembrane proteins such as amyloid β precursor protein (AβPP) and Notch resides in a high-molecular-weight γ-secretase protein complex, of which at least five core components have now been identified. Remarkably, it has now become evident that presenilin-dependent γ-secretase activity extends beyond its role in AβPP and Notch cleavages to have a generic role in the regulated intramembranous cleavage of certain membrane proteins. Actually, a new picture is emerging in which cells can relay signals from the extracellular space to their interior through presenilin-dependent proteolysis within the membrane-spanning regions of type 1 integral membrane proteins to generate potential transcriptionally active intracellular fragments. This review deals with the complex biology of presenilins and focuses more specifically on recent developments regarding the composition, assembly and regulation of the γ-secretase protein complex, its substrates and its implications for cellular signalling. [Copyright &y& Elsevier]

A major component of the amyloid plaque core in Alzheimer’s disease (AD) is the 40–42-residue amyloid β peptide (Aβ). Mutations linked to AD such as those in presenilins 1 (PS1) and 2 (PS2) invariably increase the longer Aβ42 species that forms neurotoxic oligomers. It is believed that PS1/2 constitute the catalytic subunit of the γ-secretase responsible for the final step in Aβ biogenesis. Recent genetic studies have identified a number of additional genes encoding APH1a, APH1b, PEN2, and Nicastrin proteins, which are part of the γ-secretase complex with PS1. Further, knockout studies using RNAi showed that these components are essential for γ-secretase activity. However, the nature of γ-secretase and how the aforementioned proteins regulate its activity are still incompletely understood. Here we present evidence that unlike PS1, overexpression of these proteins can increase the levels of Aβ, suggesting that these proteins are limiting for γ-secretase activity. In addition, our studies also suggest that the presenilin partners regulate the relative levels of Aβ40 and Aβ42. [Copyright &y& Elsevier]

Nicastrin has been recently identified as part of the γ-secretase complex that includes presenilin and other proteins. It is involved in the degradation of amyloid precursor protein to produce β-amyloid peptides which are believed to be central to the pathophysiology of Alzheimer’s disease. Previous reports have localized presenilin and nicastrin to the endoplasmic reticulum. However, during a proteomics-based characterization of lysosomal membrane proteins, a major spot observed on silver-stained IEF/SDS–PAGE gels was identified by mass spectrometric sequencing as nicastrin. Its Mr corresponded to the reported mature Mr for nicastrin, indicating that it is stable in the lysosomal environment. Furthermore, protease protection assays confirmed that nicastrin is contained in the outer lysosomal membrane, rather than in an internalized vesicle awaiting degradation, and that it is properly orientated with its amino-terminus facing the lysosomal lumen with its carboxyl-terminus facing the cytosol. We conclude that nicastrin is a resident lysosomal membrane protein. [Copyright &y& Elsevier]

Presenilin 1 (PS1) and presenilin 2 play a critical role in the γ-secretase processing of amyloid precursor protein (APP) and Notch1. Here, we investigate maturation and intracellular trafficking of APP and other membrane proteins in cells expressing an experimental PS1 deletion mutant (ΔM1,2). Stable expression of ΔM1,2 impairs γ-secretase processing of Notch1 and delays Aβ secretion. Kinetic studies show enhanced O-glycosylation and sialylation of holo-APP and marked accumulation of APP COOH-terminal fragments (CTFs). Surface biotinylation, live staining, and trafficking studies show increased surface accumulation of holo-APP and CTFs in ΔM1,2 cells resulting from enhanced surface delivery of newly synthesized APP. Expression of a loss-of-function PS1 mutant (D385A) or incubation of cells with γ-secretase inhibitors also increases surface levels of holo-APP and CTFs. In contrast to APP, glycosylation and surface accumulation of another type I membrane protein, nicastrin, are markedly reduced in ΔM1,2 cells. Finally, expression of ΔM1,2 results in the increased assembly and surface expression of nicotinic acetylcholine receptors, illustrating that PS1''s influence on protein trafficking extends beyond APP and other type I membrane protein substrates of γ-secretase. Collectively, our findings provide evidence that PS1 regulates the glycosylation and intracellular trafficking of APP and select membrane proteins. [Copyright &y& Elsevier]

Seladin-1 is a gene recently shown to be down-regulated in brain regions selectively degenerated in Alzheimer’s disease. The sequence of seladin-1 shares similarities with flavin-adenine-dinucleotide-dependent oxidoreductases and it has been found to protect cells from apoptotic cell death. In this work, we show that the transcription of seladin-1 is selectively down-regulated in the brain areas affected in Alzheimer’s disease. The down-regulation in seladin-1 transcription was associated with hyperphosphorylated tau seen as linkage to immunohistochemically detected paired helical filament tau, neuritic plaques and neurofibrillary tangles. In contrast, no association was found between seladin-1 transcription and β-amyloid deposition when analyzing human samples or tissue from transgenic animals. Furthermore, the relative transcription of seladin-1 was found to fluctuate during aging in the transgenic mouse model of Alzheimer’s disease. The fluctuation was enhanced by Alzheimer’s disease causing mutations in presenilin-1 and amyloid precursor protein genes. Finally, seladin-1 transcription was found to be up-regulated in mouse N2a cells induced to undergo apoptosis with okadaic acid.The results presented here indicate that seladin-1 transcription is selectively down-regulated in brain regions vulnerable to Alzheimer’s disease and this down-regulation is associated with the hyperphosphorylation of tau protein. [Copyright &y& Elsevier]

The transmembrane glycoprotein nicastrin is a component of presenilin (PS) protein complex that is involved in γ-cleavage of βAPP and site-3 cleavage of Notch. PS undergoes endoproteolysis, and the proteolytic fragments are incorporated into the high molecular weight protein complexes that are highly stabilized. Here we show that Endo H-resistant, N-glycosylated form of nicastrin (p150-NCT) is highly stabilized and selectively bound to PS fragments. Moreover, loss-of-function mutations of nicastrin inhibited formation of fully glycosylated p150-NCT as well as stabilization of nicastrin, suggesting that glycosylation and stabilization of nicastrin polypeptides are tightly correlated with its function. [Copyright &y& Elsevier]

β-Amyloid peptide (Aβ) is generated through the proteolytic cleavage of β-amyloid precursor protein (APP) by β- and γ-secretases. The β-secretase, BACE1, initiates Aβ formation followed by γ-cleavage within the APP transmembrane domain. Although BACE1 localizes in the transGolgi network (TGN), its physiological substrates and modulators are not known. In addition, the relationship to other secretase(s) also remains unidentified. Here, we demonstrate that BACE1 binds to nicastrin, a component of γ-secretase complexes, in vitro, and that nicastrin activates β-secretase activity in COS-7 cells. [Copyright &y& Elsevier]

Nicastrin acts as a key regulator for presenilin (PS)-mediated γ-secretase cleavage of β-amyloid precursor protein by forming a functional complex with PS1 and PS2. Both TNF-α and IL-1, aberrantly produced by activated microglia and astrocytes, play a role in amyloidogenesis and neurodegeneration in the brains of Alzheimer's disease (AD) patients, while BDNF synthesized chiefly by neurons has been found to be substantially reduced in AD brains. To investigate the constitutive and cytokine/neurotrophic factor-regulated expression of nicastrin in human neural cells, its mRNA levels were studied by RT-PCR and northern blot analysis in SK-N-SH neuroblastoma cells, IMR-32 neuroblastoma cells, U-373MG astrocytoma cells, and NTera2 teratocarcinoma-derived differentiated neurons (NTera2-N) following exposure to TNF-α, IL-1β, BDNF, dibutyryl cyclic AMP, or phorbol 12-myristate 13-acetate. Nicastrin mRNA expression was identified in all human neural and non-neural cell lines and tissues examined. The levels of nicastrin mRNA, however, were unaltered in SK-N-SH, IMR-32, U-373MG, and NTera2-N cells by exposure to the factors tested, and unchanged in NTera2 cells during retinoic acid-induced neuronal differentiation. These results indicate that nicastrin mRNA is expressed constitutively in human neural cell lines, where its expression is not regulated at the transcriptional level by a battery of cytokines and growth/differentiation factors which are supposed to be involved in amyloidogenesis, neurodegeneration or neuroprotection in AD brains. [ABSTRACT FROM AUTHOR]

Epigenetic regulation plays substantial roles in human pathophysiology, which provides opportunities for intervention in human disorders through the targeting of epigenetic pathways. Recently, emerging evidence from preclinical studies suggested the potential in developing therapeutics of Alzheimer's disease (AD) by targeting bromodomain containing protein 4 (BRD4), an epigenetic regulatory protein. However, further characterization of AD-related pathological events is urgently required. Here, we investigated the effects of pharmacological degradation or inhibition of BRD4 on AD cell models. Interestingly, we found that both degradation and inhibition of BRD4 by ARV-825 and JQ1, respectively, robustly increased the levels of amyloid-beta (Aβ), which has been associated with the neuropathology of AD. Subsequently, we characterized the mechanisms by which downregulation of BRD4 increases Aβ levels. We found that both degradation and inhibition of BRD4 increased the levels of BACE1, the enzyme responsible for cleavage of the amyloid-beta protein precursor (APP) to generate Aβ. Consistent with Aβ increase, we also found that downregulation of BRD4 increased AD-related phosphorylated Tau (pTau) protein in our 3D-AD human neural cell culture model. Therefore, our results suggest that downregulation of BRD4 would not be a viable strategy for AD intervention. Collectively, our study not only shows that BRD4 is a novel epigenetic component that regulates BACE1 and Aβ levels, but also provides novel and translational insights into the targeting of BRD4 for potential clinical applications., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The article discusses research on the first nicastrin (NCSTN) mutation observed in a patient with pyoderma gangrenosum, acne, and suppurative hidradenitis (PASH) syndrome. Topics explored include various gene mutations observed in individuals with PASH syndrome, results of the Sanger sequencing and genotyping procedures performed, and the clinical manifestations of PASH observed in the patient with NCSTN mutation.

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