Mutagenesis mapping of the presenilin 1 calcium leak conductance pore

Missense mutations in presenilin 1 (PS1) and presenilin 2 (PS2) proteins are a major cause of familial Alzheimer disease. Presenilins are proteins with nine transmembrane (TM) domains that function as catalytic subunits of the γ-secretase complex responsible for the cleavage of the amyloid precursor protein and other type I transmembrane proteins. The water-filled cavity within presenilin is necessary to mediate the intramembrane proteolysis reaction. Consistent with this idea, cysteine-scanning mutagenesis and NMR studies revealed a number of water-accessible residues within TM7 and TM9 of mouse PS1. In addition to γ-secretase function, presenilins also demonstrate a low conductance endoplasmic reticulum Ca(2+) leak function, and many familial Alzheimer disease presenilin mutations impair this function. To map the potential Ca(2+) conductance pore in PS1, we systematically evaluated endoplasmic reticulum Ca(2+) leak activity supported by a series of cysteine point mutants in TM6, TM7, and TM9 of mouse PS1. The results indicate that TM7 and TM9, but not TM6, could play an important role in forming the conductance pore of PS1. These results are consistent with previous cysteine-scanning mutagenesis and NMR analyses of PS1 and provide further support for our hypothesis that the hydrophilic catalytic cavity of presenilins may also constitute a Ca(2+) conductance pore.     

Protease digestion indicates that endogenous presenilin 1 is present in at least two physical forms

The membrane-bound protein complex γ-secretase is an intramembranous protease whose substrates are a number of type I transmembrane proteins including the β-amyloid precursor protein (APP). A presenilin molecule is thought to be the catalytic unit of γ-secretase and either of two presenilin homologues, PS1 or PS2, can play this role. Mutations in the presenilins, apparently leading to aberrant processing of APP, have been genetically linked to early-onset familial Alzheimer’s disease. To look for possible molecular heterogeneity in presenilin/γ-secretase we examined the ability of proteinase K (PK) to digest endogenously expressed presenilins in intact endoplasmic reticulum vesicles. We demonstrate the existence of two physically different forms of γ-secretase-associated PS1, one that is relatively PK-sensitive and one that is significantly more PK-resistant. A similarly PK-resistant form of PS2 was not observed. We speculate that the structural heterogeneity we observe may underlie, at least in part, previous observations indicating the physical and functional heterogeneity of γ-secretase. In particular, our results suggest that there are significant differences between γ-secretase complexes incorporating PS1 and PS2. This difference may underlie the more dominant role of PS1 in the generation of β-amyloid peptides and in familial Alzheimer’s disease.     

Presenilins: how much more than γ-secretase?!

AD (Alzheimer's disease) is a neurodegenerative disease characterized by a gradual loss of neurons and the accumulation of neurotoxic Aβ (amyloid β-peptide) and hyperphosphorylated tau. The discovery of mutations in three genes, PSEN1 (presenilin 1), PSEN2 (presenilin 2) and APP (amyloid precursor protein), in patients with FAD (familial AD) has made an important contribution towards an understanding of the disease aetiology; however, a complete molecular mechanism is still lacking. Both presenilins belong to the γ-secretase complex, and serve as the catalytic entity needed for the final cleavage of APP into Aβ. PSEN only functions within the γ-secretase complex through intra- and inter-molecular interactions with three other membrane components, including nicastrin, Aph-1 (anterior pharynx defective-1) and Pen-2 (PSEN enhancer-2). However, although the list of γ-secretase substrates is still expanding, other non-catalytic activities of presenilins are also increasing the complexity behind its molecular contribution towards AD. These γ-secretase-independent roles are so far mainly attributed to PSEN1, including the transport of membrane proteins, cell adhesion, ER (endoplasmic reticulum) Ca(2+) regulation and cell signalling. In the present minireview, we discuss the current understanding of the γ-secretase-independent roles of PSENs and their possible implications in respect of AD.     

The presenilins

The presenilins are evolutionarily conserved transmembrane proteins that regulate cleavage of certain other proteins in their transmembrane domains. The clinical significance of this regulation is shown by the contribution of presenilin mutations to 20-50% of early-onset cases of inherited Alzheimer's disease. Although the precise molecular mechanism underlying presenilin function or dysfunction remains elusive, presenilins are thought to be part of a complex of proteins that has 'γ-secretase cleavage' activity, which is clearly central in the pathogenesis of Alzheimer's disease. Mutations in presenilins increase the production of the longer isoforms of amyloid β peptide, which are neurotoxic and prone to self-aggregation. Biochemical studies indicate that the presenilins do not act alone but operate within large heteromeric protein complexes, whose components and enzymatic core are the subject of much study and controversy; one essential component is nicastrin. The presenilin primary sequence is remarkably well conserved in eukaryotes, suggesting some functional conservation; indeed, defects caused by mutations in the nemotode presenilin homolog can be rescued by human presenilin.     

Toward the structure of presenilin/γ-secretase and presenilin homologs

Presenilin is the catalytic component of the γ-secretase complex, a membrane-embedded aspartyl protease that plays a central role in biology and in the pathogenesis of Alzheimer’s disease. Upon assembly with its three protein cofactors (nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis into two subunits, each of which contributes one of the catalytic aspartates to the active site. A family of presenilin homologs, including signal peptide peptidase, possess proteolytic activity without the need for other protein factors, and these simpler intramembane aspartyl proteases have given insight into the action of presenilin within the γ-secretase complex. Cellular and molecular studies support a nine-transmembrane topology for presenilins and their homologs, and small-molecule inhibitors and cysteine scanning with crosslinking have suggested certain presenilin residues and regions that contribute to substrate recognition and handling. Identification of partial complexes has also offered clues to protein–protein interactions within the γ-secretase complex. Biophysical methods have allowed 3D views of the γ-secretase complex and presenilins. Most recently, the crystal structure of a microbial presenilin homolog has confirmed a nine-transmembrane topology and intramembranous location and proximity of the two conserved and essential aspartates. The crystal structure also provides a platform for the formulation of specific hypotheses regarding substrate interaction and catalysis as well as the pathogenic mechanism of Alzheimer-causing presenilin mutations. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Role of presenilin 1 in structural plasticity of cortical dendritic spines in vivo

Mutations in presenilins are the major cause of familial Alzheimer's disease (FAD), leading to impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Presenilins are the catalytic subunits of γ-secretase, which itself is critically involved in the processing of amyloid precursor protein to release neurotoxic amyloid β (Aβ). Besides Aβ generation, there is growing evidence that presenilins play an essential role in the formation and maintenance of synapses. To further elucidate the effect of presenilin1 (PS1) on synapses, we performed longitudinal in vivo two-photon imaging of dendritic spines in the somatosensory cortex of transgenic mice over-expressing either human wild-type PS1 or the FAD-mutated variant A246E (FAD-PS1). Interestingly, the consequences of transgene expression were different in two subtypes of cortical dendrites. On apical layer 5 dendrites, we found an enhanced spine density in both mice over-expressing human wild-type presenilin1 and FAD-PS1, whereas on basal layer 3 dendrites only over-expression of FAD-PS1 increased the spine density. Time-lapse imaging revealed no differences in kinetically distinct classes of dendritic spines nor was the shape of spines affected. Although γ-secretase-dependent processing of synapse-relevant proteins seemed to be unaltered, higher expression levels of ryanodine receptors suggest a modified Ca(2+) homeostasis in PS1 over-expressing mice. However, the conditional depletion of PS1 in single cortical neurons had no observable impact on dendritic spines. In consequence, our results favor the view that PS1 influences dendritic spine plasticity in a gain-of-function but γ-secretase-independent manner.     

SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production

In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca(2+) is maintained at low levels by pumping Ca(2+) into stores in the endoplasmic reticulum (ER) via the sarco ER Ca(2+)-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca(2+), whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca(2+) clearance and exaggerating inositol 1,4,5-trisphosphate-mediated Ca(2+) liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid beta production. Our results point to a physiological role for the presenilins in Ca(2+) signaling via regulation of the SERCA pump.     

Presenilins: A novel link between intracellular calcium signaling and lysosomal function?

Mutations in presenilins (PS), transmembrane proteins encoding the catalytic subunit of γ-secretase, result in familial Alzheimer's disease (FAD). Several studies have identified lysosomal defects in cells lacking PS or expressing FAD-associated PS mutations, which have been previously attributed to a function for PS in lysosomal acidification. Now, in this issue, Coen et al. (2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201201076) provide a series of results that challenge this idea and propose instead that presenilins play a role in calcium-mediated lysosomal fusion.     

Upregulated function of mitochondria-associated ER membranes in Alzheimer disease

Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by γ-secretase, via an unknown mechanism. We recently showed that presenilin-1 and -2, the catalytic components of γ-secretase, and γ-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER–mitochondrial communication—as measured by cholesteryl ester and phospholipid synthesis, respectively—are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and γ-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER–mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD.     

Presenilins function in ER calcium leak and Alzheimer's disease pathogenesis

Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide and is at present, incurable. The accumulation of toxic amyloid-beta (Aβ) peptide aggregates in AD brain is thought to trigger the extensive synaptic loss and neurodegeneration linked to cognitive decline, an idea that underlies the ‘amyloid hypothesis’ of AD etiology in both the familal (FAD) and sporadic forms of the disease. Genetic mutations causing FAD also result in the dysregulation of neuronal calcium (Ca²⁺) handling and may contribute to AD pathogenesis, an idea termed the ‘calcium hypothesis’ of AD. Mutations in presenilin proteins account for the majority of FAD cases. Presenilins function as catalytic subunits of γ-secretase involved in the generation of Aβ peptide. Recently, we discovered that presenilns function as low-conductance, passive ER Ca²⁺ leak channels, independent of γ-secretase activity. We further discovered that many FAD mutations in presenilins results in the loss of ER Ca²⁺ leak function activity and Ca²⁺ overload in the ER. These results provided potential explanation for abnormal Ca²⁺ signaling observed in FAD cells with mutations in presenilns. The implications of these findings for understanding AD pathogenesis are discussed in this article.     

The amyloid precursor protein: beyond amyloid

Mutations in PSEN1 and PSEN2 genes account for the majority of cases of early-onset familial Alzheimer disease. Since the first prediction of a genetic link between PSEN1 and PSEN2 with Alzheimer's disease, many research groups from both academia and pharmaceutical industry have sought to unravel how pathogenic mutations in PSEN cause presenile dementia. PSEN genes encode polytopic membrane proteins termed presenilins (PS1 and PS2), which function as the catalytic subunit of γ-secretase, an intramembrane protease that has a wide spectrum of type I membrane protein substrates. Sequential cleavage of amyloid precursor protein by BACE and γ-secretase releases highly fibrillogenic β-amyloid peptides, which accumulate in the brains of aged individuals and patients with Alzheimer's disease. Familial Alzheimer's disease-associated presenilin variants are thought to exert their pathogenic function by selectively elevating the levels of highly amyloidogenic Aβ42 peptides. In addition to Alzheimer's disease, several recent studies have linked PSEN1 to familiar frontotemporal dementia. Here, we review the biology of PS1, its role in γ-secretase activity, and discuss recent developments in the cell biology of PS1 with respect to Alzheimer's disease pathogenesis.     

Identifying genes that interact with Drosophila presenilin and amyloid precursor protein

The γ‐secretase complex is involved in cleaving transmembrane proteins such as Notch and one of the genes targeted in Alzheimer's disease known as amyloid precursor protein (APP). Presenilins function within the catalytic core of γ‐secretase, and mutated forms of presenilins were identified as causative factors in familial Alzheimer's disease. Recent studies show that in addition to Notch and APP, numerous signal transduction pathways are modulated by presenilins, including intracellular calcium signaling. Thus, presenilins appear to have diverse roles. To further understand presenilin function, we searched for Presenilin‐interacting genes in Drosophila by performing a genetic modifier screen for enhancers and suppressors of Presenilin‐dependent Notch‐related phenotypes. We identified 177 modifiers, including known members of the Notch pathway and genes involved in intracellular calcium homeostasis. We further demonstrate that 53 of these modifiers genetically interacted with APP. Characterization of these genes may provide valuable insights into Presenilin function in development and disease. genesis 47:246–260, 2009. © 2009 Wiley‐Liss, Inc.     

Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."     

Processive proteolysis by γ-secretase and the mechanism of Alzheimer's disease

Abstract γ-Secretase is a membrane-embedded protease complex with presenilin as the catalytic component. Cleavage within the transmembrane domain of the amyloid β-protein precursor (APP) by γ-secretase produces the C-terminus of the amyloid β-peptide (Aβ), a proteolytic product prone to aggregation and strongly linked to Alzheimer's disease (AD). Presenilin mutations are associated with early-onset AD, but their pathogenic mechanisms are unclear. One hypothesis is that these mutations cause AD through a toxic gain of function, changing γ-secretase activity to increase the proportion of 42-residue Aβ over the more soluble 40-residue form. A competing hypothesis is that the mutations cause AD through a loss of function, by reducing γ-secretase activity. However, γ-secretase apparently has two types of activities, an endoproteolytic function that first cuts APP to generate a 48/49-residue form of Aβ, and a carboxypeptidase activity that processively trims these longer Aβ intermediates approximately every three residues to form shorter, secreted forms. Recent studies suggest a resolution of the gain-of-function vs. loss-of-function debate: presenilin mutations may increase the proportion of longer, more aggregation-prone Aβ by specifically decreasing the trimming activity of γ-secretase. That is, the reduction of this particular proteolytic function of presenilin, not its endoproteolytic activity, may lead to the neurotoxic gain of function.     

Presenilins in synaptic function and disease

The presenilin genes harbor approximately 90% of mutations linked to early-onset familial Alzheimer's disease (FAD), but how these mutations cause the disease is still being debated. Genetic analysis in Drosophila and mice demonstrate that presenilin plays essential roles in synaptic function, learning and memory, as well as neuronal survival in the adult brain, and the FAD-linked mutations alter the normal function of presenilin in these processes. Presenilin has also been reported to regulate the calcium homeostasis of intracellular stores, and presynaptic presenilin controls neurotransmitter release and long-term potentiation through modulation of calcium release from intracellular stores. In this review, we highlight recent advances in deciphering the role of presenilin in synaptic function, calcium regulation and disease, and pose key questions for future studies.     

早老蛋白(Presenilins)功能研究进展

编码早老蛋白（Ｐｒｅｓｅｎｉｌｉｎｓ,ＰＳｓ）的基因ｐｓ－１、ｐｓ－２被认为是构成早发家族性老年痴呆（ｅａｒｌｙ－ｏｎｓｅｔ ｆａｍｉｌｉａｌ Ａｌｚｈｅｉｍｅｒ’ｓ ｄｉｓｅａｓｅ,ＦＡＤ）的主要致病基因。早老蛋白具有８个跨膜区的结构,在神经系统广泛表达。其功能尚未完全明确,最近几年的研究主要集中在早老蛋白与淀粉样沉淀前体蛋白（ａｍｙｌｏｉｄ ｐｒｅｃｕｒｓｏｒ ｐｒｏｔｅｉｎ,ＡＰＰ）的切割、     

Involvement of presenilin holoprotein upregulation in calcium dyshomeostasis of Alzheimer's disease

Mutations in presenilins (PS1 and PS2) account for the vast majority of early onset familial Alzheimer's disease cases. Beside the well investigated role of presenilins as the catalytic unit in γ‐secretase complex, their involvement in regulation of intracellular calcium homeostasis has recently come into more focus of Alzheimer's disease research. Here we report that the overexpression of PS1 full‐length holoprotein forms, in particular familial Alzheimer's disease‐causing forms of PS1, result in significantly attenuated calcium release from thapsigargin‐ and bradykinin‐sensitive stores. Interestingly, treatment of HEK293 cells with γ‐secretase inhibitors also leads to decreased amount of calcium release from endoplasmic reticulum (ER) accompanying elevated PS1 holoprotein levels. Similarly, the knockdown of PEN‐2 which is associated with deficient PS1 endoproteolysis and accumulation of its holoprotein form also leads to decreased ER calcium release. Notably, we detected enhanced PS1 holoprotein levels also in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. Taken together, the conditions in which the amount of full length PS1 holoprotein is increased result in reduction of calcium release from ER. Based on these results, we propose that the disturbed ER calcium homeostasis mediated by the elevation of PS1 holoprotein levels may be a contributing factor to the pathogenesis of Alzheimer's disease.     

Apoptotic activities of wild-type and Alzheimer's disease-related mutant presenilins in Drosophila melanogaster.

Mutant human presenilins cause early-onset familial Alzheimer's disease and render cells susceptible to apoptosis in cultured cell models. We show that loss of presenilin function in Drosophila melanogaster increases levels of apoptosis in developing tissues. Moreover, overexpression of presenilin causes apoptotic and neurogenic phenotypes resembling those of Presenilin loss-of-function mutants, suggesting that presenilin exerts a dominant negative effect when expressed at high levels. In Drosophila S2 cells, Psn overexpression leads to reduced Notch receptor synthesis affecting levels of the intact approximately 300-kD precursor and its approximately 120-kD processed COOH-terminal derivatives. Presenilin-induced apoptosis is cell autonomous and can be blocked by constitutive Notch activation, suggesting that the increased cell death is due to a developmental mechanism that eliminates improperly specified cell types. We describe a genetic model in which the apoptotic activities of wild-type and mutant presenilins can be assessed, and we find that Alzheimer's disease-linked mutant presenilins are less effective at inducing apoptosis than wild-type presenilin.     

Identification of a novel family of putative methyltransferases that interact with human and Drosophila presenilins.

Mutations in the presenilin genes have been shown to cause the majority of cases of early-onset familial Alzheimer's disease (AD). In addition to their role in AD, presenilins are also known to function during development by interacting with the Notch pathway. To determine if presenilins have additional functions during development and AD we have used a yeast two-hybrid approach to search for proteins that can bind to presenilins. Here, we show the identification and characterization of a novel putative methyltransferase (Metl) that interacts with the loop region of Drosophila presenilin as well as human presenilin-1 and presenilin-2, suggesting that this interaction is evolutionarily conserved and functionally important. Metl appears to be a member of a conserved family of methyltransferases that share homology with, but are distinct from, the UbiE family of methyltransferases involved in ubiquinone and menaquinone biosynthesis. In Drosophila, the metl gene gives rise to two major isoforms by alternative splicing that are broadly expressed throughout development and found in the central nervous system in an overlapping pattern with Drosophila presenilin. Finally, we show that two independent dominant adult phenotypes produced by overexpression of presenilin can be enhanced by overexpression of metl in the same tissue. Taken together, these results suggest that presenilin and Metl functionally and genetically interact during development.