Nuclear signalling by membrane protein intracellular domains: The AICD enigma

Cellular Signalling 24 (2012) 402–409

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Review

Nuclear signalling by membrane protein intracellular domains: The AICD enigma Caroline Beckett a, Natalia N. Nalivaeva a, b, Nikolai D. Belyaev a, Anthony J. Turner a,⁎ a b

Institute of Molecular & Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, St. Petersburg, Russia

a r t i c l e

i n f o

Article history: Received 3 October 2011 Accepted 10 October 2011 Available online 17 October 2011 Keywords: AICD Alzheimer's disease Amyloid Intramembrane proteolysis Neprilysin Secretase

a b s t r a c t Alzheimer's disease (AD) is a neurodegenerative illness and the leading cause of dementia in the elderly. The accumulation of amyloid-β peptide (Aβ) is a well-known pathological hallmark associated with the disease. However, Aβ is only one of several metabolites produced by β- and γ-secretase actions on the transmembrane protein, the amyloid precursor protein (APP). A proteolytic fragment termed the APP intracellular domain (AICD) is also produced. By analogy with the Notch signalling pathway, AICD has been proposed as a transcriptional regulator although its mechanism of action and the complement of genes regulated remain controversial. This review will focus on the contributions that studies of APP processing have brought to the understanding of a novel nuclear signalling pathway that may contribute to the pathology of AD and may provide new therapeutic opportunities. © 2011 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Intracellular domains of transmembrane proteins as signalling molecules 3. The APP family of proteins . . . . . . . . . . . . . . . . . . . . . . 4. APP processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Comparing AICD and NICD production . . . . . . . . . . . . . . . . . 6. Neprilysin is an AICD-regulated gene . . . . . . . . . . . . . . . . . 7. The missing link in AICD transcriptional regulation . . . . . . . . . . . 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Alzheimer's disease (AD) is the leading cause of dementia and is associated with a progressive and serious deterioration in mental functions including memory, language, orientation, judgement, behaviour and personality. It is estimated that there are currently some 36 million people worldwide living with AD and other forms Abbreviations: Aβ, amyloid β-peptide; AD, Alzheimer's disease; ADAM, a disintegrin and metalloprotease; AICD, APP intracellular domain; APP, amyloid precursor protein; GSAP, γ-secretase activating protein; HDAC, histone deacetylase; IDE, insulindegrading enzyme; KPI, Kunitz proteinase inhibitor; NEP, neprilysin; NICD, Notch intracellular domain; NMDA, N-methyl-D-aspartate; PSEN, presenilin; PTB, phosphotyrosine binding. ⁎ Corresponding author. Tel.: + 44 113 343 3131. E-mail address: [email protected] (A.J. Turner). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.10.007

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of dementia and, with an ever-ageing population, this number is expected to increase dramatically over the next decades [1]. Now twenty years has elapsed since the discovery of the first mutations in the human amyloid precursor protein (APP) gene leading to the development of early onset Alzheimer's disease [2]. This discovery laid the foundations for the amyloid cascade hypothesis of AD in which aberrant proteolytic metabolism of APP by “secretase” enzymes produces the amyloid β-peptide, which is proposed to trigger the cytotoxic chain of events that leads to neuronal cell death [3,4]. The hypothesis provided much optimism for the development of novel therapies for AD, and has driven much pharmaceutical and academic research in the intervening period, which has ranged from the development of secretase inhibitors to amyloid immunisation [5–7]. To date, no clinical trials based on these concepts have been successful [8] and the only drugs that are available remain

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acetylcholinesterase inhibitors (e.g. donepezil, rivastigmine, galantamine) and memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, which are all palliative rather than cures for the disease. This suggests a missing link in the chain from disease initiation to cognitive decline and death, and the need for a fuller understanding of the normal physiological functions of APP. Despite these setbacks, AD research has provided completely novel insight into a number of cellular signalling mechanisms, particularly those involving proteolysis, for example the phenomena of protein shedding and intra-membrane proteolysis [9–11]. APP is the precursor for the generation of multiple amyloid βpeptide (Aβ) species which, when aggregated, particularly in their soluble, oligomeric form, are neurotoxic [12]. Although Aβ production is a normal physiological event [13,14] its overproduction and the increased ratio of Aβ42 to Aβ40 form the basis underlying the amyloid cascade hypothesis of AD [4,15]. It was through the purification and sequencing of this 4 kDa peptide [15,16] that APP was originally cloned and identified [17]. Consequently, AD has traditionally been regarded as a protein misfolding disease rather than one of aberrant signalling. It is well established that mutations in one of three genes: APP, presenilin-1 (PSEN1) or presenilin-2 (PSEN2), as well as APP gene duplication [18] are causative of early-onset, autosomal dominant AD [19]. PSEN1 and PSEN2 proteins constitute the catalytic core of the γ-secretase complex that mediates intra-membrane proteolysis. Given the strong genetic links between mutations in APP or PSENs and the onset of AD, it is perhaps no surprise that research has focussed excessively on the amyloid cascade hypothesis but this has been at the expense of studies into the normal physiology of APP [20]. The dilemma remains — how far does the rare, familial form of the disease mirror the common late-onset disease in its pathology and biochemistry? Here we reassess the role of APP illuminating the controversies and issues surrounding the multiple APP metabolites, particularly its intracellular domain (AICD). The downstream pathways from these processing events include nuclear signalling and regulation of gene expression which may, in turn, provide important clues in the search for novel AD therapeutics.

2. Intracellular domains of transmembrane proteins as signalling molecules The intramembrane proteolysis of transmembrane proteins provides a relatively direct signalling pathway through which the intracellular domain, once cleaved from its protein precursor at the cell-surface by the presenilin/γ-secretase complex, is capable of translocating to the nucleus and selectively regulating gene expression [21]. This proteolytic concept was first developed for Notch signalling where the liberation of its intracellular domain (NICD), which binds directly to downstream transcription factors, can control many cellular processes determining intercellular signalling and cell fate [22]. This is a two-step process in which cleavage of the extracellular domain of Notch by a disintegrin and metalloproteinase (ADAM) is followed by the intramembranous cleavage by γ-secretase. In all, some 100 substrates of the γsecretase complex are now known and are thoroughly tabulated in [23]. In addition to the Notch and APP protein families, other examples include the Notch ligands Delta and Jagged, cell adhesion proteins such as cadherins, and a number of cytokines and their receptors. This multitude of substrates for γ-secretase enormously complicates the targeting of the enzyme as a treatment for AD, although substrateselective (“Notch-sparing”) γ-secretase inhibitors are a valid concept for further development [24,25]. The molecular enzymology of the γsecretase complex has been thoroughly reviewed recently [10] and will not be discussed in further detail here. Additionally, structural information on the complex is emerging from cryoelectron microscopy and single-particle image reconstruction studies [26,27].

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3. The APP family of proteins The APP family of proteins are a small group of ubiquitously expressed, type 1 integral membrane polypeptides which include APP, APLP1, APLP2 (Homo sapiens), Appl (Drosophila melanogaster) and apl-1 (Caenorhabditis elegans). The APP protein is approximately 110–135 kDa and contains a large extracellular domain and a short cytosolic domain [28]. Notably, it is the only family member whose sequence encodes the Aβ peptide. APP contains 19 exons and three main isoforms of the protein exist (APP695, APP751 APP770), attributable to alternative splicing of exons 7 and 8, which encode respectively a 57-amino acid serine protease inhibitor homologous to the Kunitz proteinase inhibitor (KPI) domain and a 19-amino acid thymusderived lymphoid OX-2 antigen homologue (Fig. 1) [29]. The isoforms differ in their cellular and tissue distributions with the smallest isoform, APP695, being the major neuronal species. APP770 and APP751, which both contain the KPI domain, are found abundantly in most peripheral tissues and astroglial cells [30]. Despite their distinct localisation and structural differences, the normal functions and any functional differences between the isoforms remain relatively unexplored. Studies suggest that the KPI domain may play a role in regulating blood coagulation [31] and, in AD, the balance between the KPI- and non-KPI containing isoforms has been suggested to play an important role in influencing Aβ deposition as there appears to be an increase in the proportion of KPI-containing isoforms to non-KPI containing forms in the AD brain [32]. Despite views suggesting this may be a pathological response advancing the severity of disease [33], recent evidence implies this may reflect a neuroprotective response as we have shown that amyloidogenic processing appears to occur preferentially from the APP695 isoform [34]. 4. APP processing APP can be proteolytically cleaved in two separate processing pathways termed amyloidogenic and non-amyloidogenic, generating a diverse metabolome of extracellular and intracellular peptide fragments. Once newly synthesised APP is inserted into the plasma membrane, it can either associate with various low-density lipoprotein receptors (LDLRs) such as SORL1 and become internalised and recycled via the endosomal pathway or it can enter the nonamyloidogenic pathway, which is the major, ubiquitous pathway for APP processing in all cells. It occurs at the plasma membrane [35] and begins with the cleavage of APP by α-secretases, namely ADAM9, 10 or 17 [36]. In primary neurons ADAM10 appears to be the physiologically relevant constitutive α-secretase [37]. As this cleavage takes place within the Aβ domain (Lys 16–Leu 17 bond of Aβ), it prevents the production and deposition of Aβ peptide. A soluble, N-terminal ectodomain fragment sAPPα is generated which has neuritogenic and neuroprotective functions, and also increases longterm potentiation and spatial memory [38,39]. The membrane bound C-terminal fragment (C89) is further cleaved by γ-secretase which is a complex containing the integral membrane proteins nicastrin (NCT), anterior pharynx defective 1 (Aph-1), presenilin enhancer 2 (PEN-2) in addition to the catalytic core containing the heterodimeric presenilin 1 and/or 2 (PSEN1/2) [40]. Its actions upon C89 produce a small, nontoxic p3 peptide and AICD (49–59 amino acids). By stimulating the αsecretase pathway, Aβ formation can be rapidly reduced and this concept has been explored as a possible therapeutic strategy [41]. AICD produced by consecutive α/γ cleavage is released directly into the cytoplasm where it is rapidly degraded, rendering it non-functional [42,43]. The alternative pathway of APP metabolism, the amyloidogenic pathway, requires the endocytosis of APP. Once internalised into late endosomes the β-secretase or BACE1, cleaves APP within lipid raft domains [34,44,45] to produce a large, soluble N-terminal ectodomain sAPPβ and a membrane bound C-terminal fragment (C99). Recent reports suggest sAPPβ may be further cleaved to

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Fig. 1. Schematic diagram of APP and its major derivatives APP695, APP751 and APP770. APP has a single transmembrane region, a large extracellular, N-terminal domain and a small intracellular C-terminal domain. There are three major derivatives APP695, APP751 and APP770. The largest isoform APP770 contains a 57 amino acid KPI domain and a 19 amino acid MRC OX-2 antigen whereas APP751 only contains the KPI domain and APP695 lacks both. A 17 residue signal peptide is present at the N-terminus. Two sites of N-glycosylation (\CHO) occur at residues 542 and 571.

generate a ligand capable of binding to the death receptor DR6, although the protease involved remains unidentified [46]. Not all effects of sAPPβ, however, are detrimental: this metabolite can stimulate neurite outgrowth like sAPPα, consistent with both secreted fragments sharing the necessary two domains for this activity which is mediated through the early growth response-1 (Egr-1) signalling pathway [47]. The C99 fragment produced by BACE1 cleavage is further cleaved intramembranously by γ-secretase, again within lipid rafts [48], generating the Aβ peptide and AICD. In contrast to the highly selective nature of Notch γ-secretase cleavage, APP processing is much less specific and multiple cleavage sites have been identified within the transmembrane domain. The production of AICD59 and AICD57 (named according to amino acid length) has been identified as well as AICD50 which corresponds to a downstream ε-cleavage site of γ-secretase analogous to the S3 cleavage of Notch [49,50]. While most research in this area has focused on γ-cleavage, relatively little attention has been paid to ε-cleavage. Despite both cleavage sites being mediated by presenilin-dependent γ-secretase, studies have shown that the precision of cleavage is dependent on subcellular localisation and conditions [51]. This suggests that the cleavage products may well function differently and be generated independently. While the insulin degrading enzyme can rapidly degrade the AICD fragment in the cytosol, recent evidence has shown that the endosomal/lysosomal protease, Cathepsin B, can also degrade APP CTFs such that specific inhibitors result in CTF and AICD accumulation [52]. Additional truncation of AICD has also been reported to occur by the action of caspase-3 producing a 31-amino acid peptide [53]. This has been shown to be increased in AD brain and is involved in the cell death pathway [54]. AICD levels have been reported both to be increased [55] or decreased [56] in Alzheimer brain. The competing APP processing pathways are illustrated in Fig. 2.

5. Comparing AICD and NICD production Notch and APP share some similar characteristics as γ-secretase substrates: they are both type 1 transmembrane proteins with large extracellular domains and both undergo sequential proteolysis, first of the ectodomain and then an intramembrane cleavage event. Despite initial reports of APP metabolism bearing ‘remarkable’ analogy to Notch signalling [57], subtle and important differences between AICD and NICD signalling mechanisms have emerged as the biochemical details have unfolded. Briefly, ectodomain cleavage mediated by ADAM metalloproteinases is a ligand-dependent event in the case of the Notch receptor requiring the binding of DSL ligands (Delta, Serrate/Jagged). This cleavage triggers the subsequent γ-secretase intramembrane cleavage and releases the intracellular domain, NICD. The γ-secretase activity is dependent on endocytosis of the ligand-activated Notch and monoubiquitination appears to be a dependent factor [58]. Several nuclear localisation signals then allow nuclear import via the classical importin pathway [59]. The ectodomain cleavage may occur within or outside of lipid raft domains [60]. Within the nucleus, NICD displaces repressor proteins that are associated with the CSL family of DNA-binding proteins (where CSL stands for CBF1, Su(H), Lag-1) [61] and forms a ternary complex together with CSL and Mastermind (Mam) that recruits further transcription factors activating gene expression. While the transcriptional role of NICD is firmly accepted, the mechanism of action of AICD, in contrast, is distinct in a number of features and a specific role in transcriptional regulation has been vigorously debated (see e.g. [62,63]). Part of the difficulty in establishing a physiological role for AICD has been its extreme lability, having a very rapid turnover, and pulse chase experiments have shown that its half life may be as short as 5 min [64]. This highly labile nature has been attributed to its rapid degradation by insulin degrading

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Fig. 2. The alternative processing pathways of APP. APP is cleaved either by α- or β-secretases to produce large, soluble N-terminal fragments sAPPα or sAPPβ. The remaining short, membrane bound C-terminal fragments are further cleaved by γ-secretases to release a non-toxic p3 peptide and AICD in the non-amyloidogenic pathway and Aβ and AICD in the amyloidogenic pathway. Both pathways have been shown to occur in distinct subcellular compartments, the amyloidogenic pathway requiring the endocytosis of APP, whereas APP is cleaved at the plasma membrane in the non-amyloidogenic pathway. AICD released in this pathway is rapidly degraded by IDE into smaller fragments and cathepsin B may also contribute. Alternatively, AICD is truncated into a 31 amino acid fragment (C31) by caspase-3. APP695 generated AICD can be translocated to the nucleus and in a complex with MED12, Fe65 and Tip60 can regulate gene expression.

enzyme (IDE), a thiol-dependent metalloprotease that degrades a number of peptide hormones and has also been shown to act as an efficient Aβ-degrading enzyme and to clear extracellular Aβ [42]. The proteasome may also degrade AICD [43] although this has been contested [65]. How, then, does APP/AICD signalling differ from Notch/NICD? The first description of the AICD fragment (then termed AID) and its presence in brain tissue from normal subjects and AD patients was provided by d'Adamio and colleagues. They also showed that this fragment could act as a positive regulator of apoptosis [66]. The 6.5 kDa AICD domain is a highly conserved region in all forms of APP from fly to human which suggests that it is functionally significant. In contrast to Notch there appear to be no ligands necessary to trigger the APP cleavage process. However, as transcriptionally functional AICD is generated from β- and not α-secretase cleavage [34], it is important that APP is endocytosed where the acidic interior favours the catalytic action of the aspartic proteinase, BACE1 [45,67]. AICD contains welldocumented phosphorylation sites at threonine residues 654 and 668 and serine residue 655 as well as a recognised 682YENPTY687 functional motif which has been reported to serve two important roles. The first

involves the motif being involved in clathrin-mediated endocytosis which regulates internalisation of APP and regulates its own intracellular sorting and the second is the involvement of AICD in transcriptional regulation. These processes are highly dependent on the interaction of binding partners which associate with the consensus motif via phosphotyrosine binding (PTB) domains and consequently connect APP to various intracellular molecular pathways. Over 20 binding partners have been reported to be associated with a range of processes including transcriptional regulation, apoptosis, development, synaptic plasticity and cytoskeletal dynamics. Characterised APP binding proteins include members of the Fe65 family (Fe65, Fe65L1) [68,69], X11 family (X11a, X11L, X11L2) [69], c-jun-N-Terminal kinase interacting protein (JIP) family (JIP1b, JIP2), SRC family (Shc A, Shc C), MINT (MINT1, MINT2, MINT3) as well as mammalian disabled-1 (mDab1), Numb and Numb-like proteins, kinesin light chain (KLC), clathrin and cAbl (reviewed in [70] and [71]). The phosphorylation status of AICD has been shown to affect the affinity of binding partners and therefore can influence the biological roles that AICD plays within the cell [72]. Similarly abnormal phosphorylation events have been associated with AD pathology [73]. Of particular relevance is the

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interaction between AICD and Fe65. The Fe65 family (Fe65, Fe65L1 and Fe65L2) is a group of multidomain scaffolding/adaptor proteins [74] with Fe65 being enriched in the brain whereas Fe65L1 and Fe65L2 are more peripherally expressed. The multifunctional protein complex is made up of three protein–protein interaction domains: an Nterminal WW domain and two C-terminal PTB domains. Through its PTB2 domain and in a tyrosine phosphorylation-independent manner, Fe65 is able to interact with the YENPTY motif on APP [68]. AICD detection in cells is elusive because of its lability but binding of AICD to Fe65 appears to stabilise the fragment analogous to NICD stabilisation by the CSL protein [75]. Fe65, present in both nuclear and non-nuclear fractions is able to interact with nuclear proteins and through its PTB1 domain has been shown to interact with the histone acetyltransferase tat-interactive protein (Tip60) [21,76]. Together with Fe65 and Tip60, AICD was originally proposed to form a transcriptionally active, multimeric complex (AFT complex) influencing gene expression [21]. Since this discovery in 2001, AICD has been increasingly investigated for its signalling capacity from plasma membrane to nucleus. The delivery of AICD to the nucleus occurs in a dynamin-mediated retrograde transport process where it forms its nuclear AFT complex with Fe65 and Tip60 observable as nuclear transcription factories, where Notch intracellular domains can also be detected [77]. In contrast, the X11 proteins, which bind competitively with Fe65, trap AICD in the cytosol in a non-functional state [78]. Among the genes reported to be upregulated by AICD were glycogen synthase kinase-3β (GSK-3β) [79], the metastasis suppressor gene, KAI1 (CD82) [80], the Aβ-degrading enzyme neprilysin (NEP) [81,82], the tumour suppressor protein, p53 (and hence indirectly the prion protein) [83,84] and aquaporin-1 [85]. APP itself and the β-secretase BACE-1 have also been reported to be positively regulated [78] whereas expression of at least one gene, the epidermal growth factor receptor gene, is down-regulated by AICD signalling [86]. The precise mechanisms regulating these cellular processes are still not validated in many cases and there are conflicting data on gene regulation by AICD with a lack of consistency between cell-based models (and cell types used), variants of APP constructs expressed in cell or transgenic models, and the results of transcriptomic profiling. For example, Hébért et al. [62] in their model systems failed to detect robust transactivation of several of the above target genes by AICD although Fe65 could transactivate a number of genes independent of AICD [87]. This has led to a number of differing views surrounding the signal transduction models followed by this APP metabolite. The initial hypothesis of AICD signalling, as described above, proposed that, after γ-secretase cleavage of APP, AICD binds to Fe65, translocates to the nucleus and associates with Tip60 forming a transactivating complex [21]. Subsequent studies [88], however, suggest an alternative model whereby AICD may function indirectly by modifying Fe65 which independently translocates to the nucleus and regulates transcription [88]. Although Fe65 may have a dominant role in nuclear signalling being able alone to trigger robust reporter activities, the knockdown of APP or the disruption of Fe65APP binding significantly reduces signalling [89]. Thus, Fe65 and AICD may function co-operatively in regulating only a relatively small subset of genes and APP may mediate nuclear signalling through AICD and also independently of this metabolite. For example, APP may regulate transcription of the transthyretin and klotho genes through one of its ectodomain fragments, sAPPβ, through an as yet undefined intercellular signalling mechanism [90]. An additional compounding factor is a novel γ-secretase activating protein (GSAP) which has recently been identified [91]. GSAP was shown to interact with both γ-secretase and its substrate, the APP Cterminal fragment, and thereby modulate Aβ and AICD production in vitro. GSAP appeared to be able to regulate γ- and ε-cleavage of the C-terminal fragment of APP differentially to form Aβ and AICD [89]. GSAP had no influence on Notch cleavage and therefore may represent an allosteric regulator modulating the substrate

specificity of the γ-secretase complex [91]. GSAP-immunoreactive deposits are observed both in the normal and the AD brain, however specific morphological features of GSAP may distinguish between healthy and pathological states [92]. Transgenic mice overexpressing the AICD fragment have also produced insight into the role of this peptide. These mice demonstrate that AICD is biologically relevant, causes significant alterations in cell signalling mediated through upregulation of GSK-3β, and may play a role in axonal elongation or pathfinding [93]. AICD-overexpressing mice also display a number of AD-like pathological features, for example hyperphosphorylation and aggregation of tau, neurodegeneration and working memory deficits but how far this model is representative of the normal physiological situation in regard to APP metabolism remains unclear [55]. At the molecular level, the Aβ-degrading enzyme neprilysin (NEP) is now one of the most established AICD-regulated genes. Since the original association of AICD with NEP expression [81], significant progress has been made in understanding this critical intracellular signalling mechanism at the nuclear level. 6. Neprilysin is an AICD-regulated gene NEP (neprilysin, neutral endopeptidase, enkephalinase or CD10) is a type II membrane protein, a member of the zinc metalloendopeptidase family (reviewed in [94]). The ubiquitously expressed protein has a broad specificity and a wide range of physiological substrates including enkephalins and substance P. It plays an important role in turning off peptide signalling events at the cell surface. Importantly, in the brain, NEP can inactivate Aβ, cleaving the peptide at multiple sites. This finding has resulted in NEP being intensively studied in relation to AD research and as NEP activity decreases significantly with age, the loss of this enzyme has been suggested to play a role in Aβ accumulation, plaque formation and the onset of dementia [95,96]. In transgenic animal models of AD, over-expression of NEP can facilitate the clearance of amyloid plaques and reverse cognitive and behavioural deficits [97]. NEP loss is also associated with the pathogenesis of prostate cancer [98] and hence understanding the mechanisms of transcriptional regulation of NEP is highly relevant to diseases of ageing and could contribute to new therapeutic approaches in prostate cancer as well as AD. The molecular details of AICD gene regulation have now been most clearly delineated in studies of regulation of NEP expression as first reported by Pardossi-Piquard et al. [81]. Following an initial observation that PS-deficient fibroblasts had markedly decreased levels of NEP selectively among Aβ-degrading proteases, it was shown that re-expression of PS restored NEP expression and activity. Furthermore, treatment of neuronal cells with γ-secretase inhibitors also reduced NEP levels. These studies clearly seemed to indicate a role for AICD in NEP regulation. However, in a subsequent study using broadly similar methodologies, Chen and Selkoe [63] failed to replicate these findings adding yet further to the controversies surrounding the signalling role of AICD. In order to resolve these discrepancies we focused on establishing whether AICD could directly affect transcription and, by using chromatin immunoprecipitation (ChIP) assays could detect a direct interaction of AICD with the NEP promoters in neuronal but not non-neuronal cells, an effect abolished by inhibition of APP metabolism by β- or γ-secretase inhibitors but not by an αsecretase inhibitor. Our data imply that the transcriptional activation was mediated selectively in neuronal cells operating through the amyloidogenic (β/γ-secretase pathway) but not via α/γ-secretase action [34,82]. Similar conclusions regarding the preferential use of the amyloidogenic pathway have been arrived at by Goodger et al. [67] although using somewhat different strategies. Repression of NEP expression correlated with replacement of AICD with the histone deacetylase HDAC1 on the NEP promoter consistent with regulation through chromatin remodelling [34,82]. Treatment with HDAC inhibitors restored AICD promoter binding as well as NEP expression, with the commonly used anticonvulsant sodium

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valproate producing the largest effect suggesting it may have potential therapeutic application in AD. In animal models in vivo, valproate treatment has also been shown to cause the replacement of HDACs with AICD on the NEP promoter in cortex and hippocampus and to upregulate NEP [99]. Indeed, in a number of neurodegenerative conditions an imbalance of histone acetylation has been suggested [100] and chromatin remodelling involving increased histone acetylation has been associated with the recovery of learning and memory [101]. In summary, AICD generated from amyloidogenic metabolism of APP, binds to the NEP promoter displacing HDACs and hence activates transcription providing a clear signalling pathway from the plasma membrane to the nucleus. A further complexity in the AICD signalling mechanism is provided by the existence of the multiple isoforms of APP which do not follow identical metabolic pathways. That distinct roles may exist for the three major isoforms of APP has largely been ignored over the years despite evidence that their relative expression levels may change during development and disease. We have recently demonstrated that it is predominantly the neuronal 695 isoform of APP that follows the amyloidogenic pathway leading to formation of Aβ and/or AICD and hence to regulation of NEP, and other, gene expression [34]. Furthermore, this pathway was cell-type specific operating in neuronal (e.g. SH-SY5Y, Neuro2a and primary neurons) but not non-neuronal cells (e.g. HUVEC, HEK cells). While the mechanism underlying this selectivity remains unclear, it is most likely that this reflects the differential cellular trafficking of the isoforms due to interactions with specific domains within the larger isoforms. In addition there may well be neuronalspecific cofactors that are required to facilitate the nuclear signalling pathway followed by AICD. 7. The missing link in AICD transcriptional regulation While regulation of NEP expression has provided unequivocal evidence of a nuclear signalling pathway operating via AICD, in which replacement of HDACs with AICD activates transcription, the molecular basis of the transcriptional complex involving AICD has remained nebulous. Xu et al. [102] have revealed an additional component essential for linking AICD to transcription. By using a yeast two-hybrid screen in a human foetal brain cDNA library, they identified APP family members as proteins that interacted with the C-terminus of MED12, a component of the multi-subunit Mediator complex. Furthermore, MED12 formed a complex with Fe65 and Tip60 but only in the presence of AICD, supporting the original model for AICD action proposed by Cao and Sudhof [21] (Fig. 2). Hence Mediator appears to be a direct physical target and functional transducer of AICD action. More generally, Mediator is able to link gene-specific transcription factors with RNA polymerase II hence altering global gene expression programmes that control development and differentiation. The MED12 subunit has previously been shown to play a role in neuronal development and cognition and hence polymorphisms in MED12 might contribute to the development of AD. The study by Xu et al. [102] also validated a number of other genes previously implicated in AICD-dependent nuclear signalling, for example aquaporin-1 [85], as well as NEP. Although the Mediator complex normally mediates transcriptional activation it can, in some cases, control transcriptional repression consistent with the reported down-regulation of EGFR by AICD [86]. An overall model for the AICD signalling pathway, based on currently available data, is shown in Fig. 2. Numerous studies have highlighted genes potentially regulated by AICD but most of these have focused on a single gene or group of genes. Table 1 summarises a range of such investigations through to recent whole gene transcriptomic studies in which a DNA microarray analysis in AICD overexpressing P19 cell lines led to more than a 10fold up-regulation of 277 genes and a down regulation of 341 genes [103]. In another transcriptome profiling study using APP knockout phenotypes, genes associated with plasticity as well as heat shock

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Table 1 Key genes reported to be regulated by AICD in various model systems. Regulated gene

Experimental evidence

PS-deficient cells and γ-secretase inhibitors reduce the transcription, expression, and activity of NEP; however activity can be restored by transient expression of PS1 or PS2 and by expression of AICD. NEP gene promoters are transactivated by AICDs and ChIP analysis reveals AICD binding to the NEP promoter enhancing histone acetylation. APP Clonal cell lines with inducible expression BACE1 constructs were generated differing only Tip60 in the expression of AICD and its putative downstream effector genes. AICD upregulated the expression of the APP genes, the gene encoding the β-site APP cleaving enzyme (BACE) and Tip60. In this model GSK-3β and KAI1 AICD-target genes were also confirmed. Glycogen synthase AICD transgenic mice show activation of kinase-3β (GSK-3β) GSK-3β and increased phosphorylation of a downstream substrate, CRMP2. Confirmed by above and confirmed by genome-wide analysis. Tetraspanin, AICD/Fe65/Tip60 complex was identified KAI1/CD82 on the KAI1 promoter and able to displace the N-CoR/TAB2/HDAC3 complex in the absence of interleukin-β signal causing gene activation. Confirmed by above and by genome-wide analysis. p53 PS deficiency, catalytically inactive PS mutants, γ-secretase inhibitors, and APP or APLP2 depletion all reduce the expression and activity of p53 and lower the transactivation of its promoter and mRNA expression. Aquaporin (AQP) AQP1 mRNA and protein were downregulated in fibroblasts lacking APP or PS2. AQP1 expression was restored by stable expression of full-length APP or PS2 but not by APP with C-terminal deletion. Levels of PS and EGFR in the skin tumours Epidermal growth of PS1(+/−)/PS2(−/−) mice and the factor receptor brains of PS1/2 conditional double KO (EGFR) mice are inversely correlated. Deficiency in PS/γ-secretase activity or APP expression results in a significant increase of EGFR in fibroblasts. AICD binds directly to the EGFR promoter and negatively regulates transcription. LRP1 Deletion of APP and APLP2, or components of the γ-secretase, enhance the expression and function of LRP1, which was reversed by expression of AICD. AICD/Fe65/Tip60 interacts with the LRP1 promoter and suppresses its transcription. SPT subunit (SPTLC2) Truncated AICD decreases SPTLC2 expression resulting in decreased SPT activity. Patched homologue In AICD overexpressing Neuro2A cells the 1 (PTCH1) expression of mRNA and protein levels Transient receptor were significantly increased for PTCH1 potential cation and decreased for TRPC5 indicating the channel (TRPC5) possibility that both proteins may be controlled in an AICD-dependent manner. Other genes Microarray studies have been used to implicated analyse transcriptome changes. Studies have used AICD-overexpressing cell lines, inducible AICD expression constructs and APP knockout models. The subsets of genes regulated differ considerably depending on particular cell types and conditions used. See main text for discussion Neprilysin (NEP)

Ref [81,82]

[78]

[78,93,112]

[78,80,112]

[83]

[85]

[86]

[113]

[114]

[115]

[103,104,112]

408

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proteins were down-regulated; however many previously reported AICD-regulated genes such as GSK-3β and KAI1 remained unchanged [104]. What is striking is the divergence among such studies which, we believe, reflects the transitory nature of AICD regulation, a feature originally highlighted by Kimberly et al. [105] who observed that endogenous AICD in primary neurons was detectable for only a short time period during differentiation in culture. More recently Bauer et al. [106] have shown that cell ageing and passage number also affect AICD levels and detection of AICD in cell extracts has certainly proven elusive. Although this is generally attributed to rapid AICD metabolism, it also reflects the diversity of cell types and constructs used in exploring AICD action, including transgenic models, as well as the difficulties in interpreting over-expression or knockdown studies. There are strategies that allow upregulation and stabilisation of AICD levels including the use of the tyrosine kinase inhibitor gleevec [107] although this has been disputed [108], and alkalisation of cells [109,110], stressing the importance of the pH, and hence the cellular compartment, in which AICD is generated. It also needs to be borne in mind that higher AICD levels do not necessarily reflect upregulation of gene expression since not all cellular pools of AICD are transcriptionally competent (e.g. cytosolic vs. endosomal). Even where nuclear AICD is detectable, for example in prostate cancer cell lines, this does not necessarily lead to transcriptional regulation where factors other than histone acetylation levels predominate, e.g. DNA methylation [111]. All these caveats, of course, make it difficult to compare directly different studies and emphasise the need for caution in interpreting data relating to this transient signalling pathway. 8. Conclusions This review has highlighted a novel signalling pathway from plasma membrane to nucleus involving the intracellular domain of the Alzheimer's APP, in part resembling Notch signalling yet differing in a number of crucial aspects. There are still numerous components in the pathway to identify, especially those that confer cell specificity and the timing of AICD responses. However, methodologies and limitations for detection of AICD are becoming much clearer, which should allow rapid progress in defining the AICD transcriptome in different cell types and its role from development to disease. Ultimately, understanding in fuller detail the mechanisms and complement of genes regulated by APP-dependent nuclear signalling should provide new avenues for AD research and potential therapeutics. Conflict of interests Authors declare no conflict of interests. Acknowledgements We thank the Medical Research Council and Alzheimer Research U.K. for support. References [1] H.W. Querfurth, F.M. LaFerla, The New England Journal of Medicine 362 (4) (2010) 329–344. [2] A. Goate, M.-C. Chartier-Harlin, M. Mullan, J. Brown, F. Crawford, L. Fidani, L. Giuffra, A. Haynes, N. Irving, L. James, R. Mant, P. Newton, K. Rooke, P. Roques, C. Talbot, M. Pericak-Vance, A. Roses, R. Williamson, M. Rossor, M. Owen, J. Hardy, Nature 349 (6311) (1991) 704–706. [3] D.J. Selkoe, Neuron 6 (4) (1991) 487–498. [4] J.A. Hardy, G.A. Higgins, Science 256 (5054) (1992) 184–185. [5] H.-N. Woo, S.-H. Baik, J.-S. Park, A.R. Gwon, S. Yang, Y.-K. Yun, D.-G. Jo, Biochemical and Biophysical Research Communications 404 (1) (2011) 10–15. [6] C.A. Lemere, E. Masliah, Nature Reviews. Neurology 6 (2) (2010) 108–119. [7] M. Citron, Nature Reviews. Drug Discovery 9 (5) (2010) 387–398. [8] E. Karran, M. Mercken, B.D. Strooper, Nature Reviews. Drug Discovery 10 (9) (2011) 698–712.

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