β-Amyloid precursor protein metabolism: focus on the functions and degradation of its intracellular domain

Pharmacological Research 62 (2010) 308–317

Contents lists available at ScienceDirect

Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

Review

␤-Amyloid precursor protein metabolism: focus on the functions and degradation of its intracellular domain Erica Buoso a , Cristina Lanni a , Gennaro Schettini b , Stefano Govoni a , Marco Racchi a,∗ a b

Department of Experimental and Applied Pharmacology, Center of Excellence in Applied Biology, University of Pavia, Viale Taramelli 14, 27100 Pavia, Italy Department of Oncology, Biology and Genetics, University of Genova, Italy

a r t i c l e

i n f o

Article history: Received 12 April 2010 Received in revised form 10 May 2010 Accepted 10 May 2010 Keywords: APP AICD Fe65 Proteasome Insulin degrading enzyme

a b s t r a c t Alzheimer’s disease (AD) is a neurodegenerative disorder that represents the most common type of dementia in the elderly. One of the hallmarks of this disease is a progressive accumulation of amyloid fibrils in senile plaques (SPs), which are composed principally of amyloid-␤ peptides (A␤). The 4-kDa ␤-amyloid peptides are produced from the ␤-amyloid precursor protein (APP) through sequential processing by ␤- and ␥-secretase enzymes in the amyloidogenic pathway. By an alternative nonamyloidogenic pathway, mediated by ␣- and ␥-secretases enzymes, APP is processed within the A␤ domain. Both processing pathways may result in the generation of a fragment called APP intracellular C-terminal domain (AICD) which is hypothesized to contribute to the pathophysiology of AD. Experimental evidence highlights that biological functions of AICD are mediated by interactions between its YENPTY motif and specific binding factors. We critically reviewed literature concerning physiological function of this proteolitic fragment, mainly focusing on their degradation by the two best characterized systems, proteasome and IDE (insulin degrading enzyme). Our work is aimed to analyse the functional role of AICD, integrating also the AICD degradation processes, to better define a potential role of AICD in signal transduction. © 2010 Elsevier Ltd. All rights reserved.

Contents 1.

2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. APP intracellular C-terminal domain (AICD) binding factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1. X11 family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.2. Jip family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3. mDab1 protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.4. Shc family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.5. Fe65 family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Possible role of aicd in Fe65 and Tip60 complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AICD degradation processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Proteasome and its possible implication in AICD degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Insulin degrading enzyme (IDE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder that represents the most common cause of dementia in the elderly.

∗ Corresponding author. Tel.: +39 0382 987738; fax: +39 0382 987405. E-mail address: [email protected] (M. Racchi). 1043-6618/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2010.05.002

308 310 310 310 310 310 310 311 312 312 313 313 315 315

The brain tissue of AD patients shows the presence of neuropathologic markers such as neurofibrillary tangles and neuritic or senile plaques. The former are mainly composed by a cytoskeletal protein, named tau, which becomes hyperphosphorylated and self-aggregates into neurofibrillary tangles. The latter are characterized by the accumulation of proteins in the form of ␤-pleated sheet fibrils, which are composed mainly of a 39–43 amino acids peptide known as ␤-amyloid (A␤) [1]. The 4-kDa ␤-amyloid pro-

E. Buoso et al. / Pharmacological Research 62 (2010) 308–317

309

Fig. 1. Schematic representation of APP proteolitic catabolism. The APP proteolitic catabolism includes two different pathway: an amyloidogenic pathway and a nonamyloidogenic pathway. The figure illustrate these two pathways with the different APP fragments which are generated after secretase activities. In the figure are also indicated the APP C-terminal binding domain (see text for details).

tein is derived from the ␤-amyloid precursor protein (APP) [2] by a physiological intracellular processing [3]. The amyloid hypothesis of AD is focused on the potential toxic role of an excessive formation of A␤ derived either from an increase production of A␤ or from its reduced catabolism [4]. APP, encoded by a gene located on chromosome 21 [2], is an important cell constituent that may play a role in the recognition of extracellular signals, cell adhesion and apoptosis. In neurons, APP is required for synaptogenesis, synapse remodeling and neurite outgrowth [5]. APP expression is increased during neuronal maturation and differentiation [6] and during traumatic brain injury [7]. APP is a type I transmembrane protein with a large extracellular domain, a membrane anchoring domain and a short intracellular C-terminal tail. During synthesis APP undergoes several post-translational modifications, including N-glycosylation, O-glycosylation and Tyr sulfation to give rise to the mature form of APP which is processed by at least two distinct proteolytic pathways (Fig. 1). One pathway involves cleavage by the enzyme ␣-secretase, which cuts APP within the A␤ sequence, thereby preventing the formation of A␤ [8]. This step produces a secreted form of APP (sAPP␣) and a C-terminal fragment (C83 or ␣-CTF), which remains associated to the membrane. Several transmembrane proteases belonging to the metalloproteases, such as the tumor necrosis factor ␣-converting enzyme (TACE/ADAM17) and ADAM-10 have been proposed as an ␣secretase [9,10]. A second pathway involves cleavage of APP by an enzyme referred to as ␤-secretase. ␤-Secretase cleaves APP at the Nterminal side of Asp1 of the A␤ sequence. This is the major site of cleavage for the ␤-secretase, although some cleavage adjacent to Glu11 can also occur. Cleavage at Asp1 results in the production of a N-terminally truncated form of APP (sAPP␤) that is released from the membrane [11] and a C-terminal membrane-associated fragment (C99 or ␤-CTF). The ␤-secretase was identified by several groups as the transmembrane aspartic protease BACE [12–16]. BACE1 has been shown to be the ␤-secretase; it can cleave APP at both ␤-secretase cleavage sites within the A␤ sequence [14] and has high levels of expression in neurons [13,14].

Following cleavage by ␣ or ␤ secretase, the ␥-secretase complex cleaves, inside the membrane, the remaining C-terminal fragments of APP, C83 and C99, via a mechanism referred to as regulated intramembrane proteolysis (RIP). ␥-Secretase complex consists of nicastrin (NCSTN or APH2), anterior pharynx defective 1 (APH1), presenilin enhancer 2 (PEN2) and presenilin 1 (PSEN1) and/or presenilin 2 (PSEN2) [17]. C83 and C99 cleavage generates p3 and A␤ respectively together with APP intracellular C-terminal domain (AICD). Due to the heterogenous cleavage of ␥-secretase, the AICD length varies generally from less than 57–59 amino acids. AICD generated by other activities like those of ␧ and ␨-cleavages (53 and 50 amino acids respectively) have also been reported [18] (Fig. 1). The existence of AICD remained elusive until its presence was first documented in guinea pig brain [19]. The generation of AICD, like that of A␤, takes place in membrane compartments upstream of endoplasmic reticulum (ER) and is dependent upon presenilins [20]. The potential importance of AICD has been emphasized by the recognition of similarities between APP and another type I transmembrane protein called Notch [21]. Notch undergoes two cleavages, the first of which is performed by the metalloprotease ADAM-17. It is subsequently cut by the presenilin-dependent ␥-secretase, releasing the Notch intracellular domain (NICD), a fragment analogous to AICD. After being released from the membrane, the NICD fragment associates with the cellular factor CSL (for CBF1, Suppressor of Hairless and Lag-1) and translocates into the nucleus, where it modulates gene transcription. This analogy of APP processing to Notch receptor signalling suggested a possible function for AICD in nuclear signalling [22,23]. Different proteins have been reported to interact with AICD including some that are necessary for AICD-dependent function in signal transduction, apoptosis or in modulation of cytoskeletal dynamics. AICD is difficult to study because of its small size and its rapid degradation. In this regard, proteasome and insulin degrading enzyme (IDE) are reported to be putatively involved in AICD degradation. In this work our aim is to integrate AICD degradation processes with different possible signal transduction models.

310

E. Buoso et al. / Pharmacological Research 62 (2010) 308–317

1.1. APP intracellular C-terminal domain (AICD) binding factors The APP intracellular domain contains at least three functionally important motifs able to interact with different intracellular adaptor proteins. Two different groups of these proteins have been identified: the first is pertussis toxin-sensitive heterotrimeric GTPase G0 [24,25], while the second comprises cytoplasmic adaptors without enzyme activity. The first AICD domain is the 653-YTSI-656 motif (amino acid numbering according to human APP695 isoform). This motif is the basolateral sorting signal which is bound by the microtubule binding protein, PAT1 (protein interacting with APP tail 1). The PAT1–AICD interaction mediates APP intracellular transport through the secretory pathway. Central to this functional APP domain are Thr654 and Ser655 , two consensus residues for phosphorylation known to be phosphorylated in vitro by PKC and calcium and calmodulin dependent protein kinase II (CaMKII) in several cell lines and in vivo in rat brain [26]. The second motif, 667-VTPEER-672, contains the phosphorylation site Thr668 . This residue can be phosphorylated by neuronal cyclin-dependent kinase 5 (Cdk5) in neurons [27], by Cdc2 kinase in dividing cells [28,26], by glycogen synthase kinase 3␤ (GSK3␤) and by stress-activated protein kinase 1␤ in vitro [29,30]. Phosphorylation at Thr668 in mature APP occurs only in the brain suggesting that phosphorylation at this residue might be involved in a neuron-specific aspect of APP metabolism and function [31,27]. The 14-3-3␥ protein, a well studied protein kinase C inhibitor, which is highly expressed in brain, skeletal muscle and heart, has been reported to bind to this AICD motif [32]. The third intracellular domain contains the amino acid sequence 682-YENPTY-687, encompassing an NPXY element, which is a typical internalization signal via clathrin-coated pits for membrane-associated receptor proteins [33–35]. This motif is recognized by proteins containing phosphotyrosine interaction domain (PID or PTB), such as the X11 family (X11␣ or Mint1, X11␤ or Mint2 and X11␥ or Mint3), the Jip (c-Jun N-terminal kinase interacting protein) family (Jip1b and Jip2), the Fe65 family (Fe65, Fe65L1 and Fe65L2), the Shc family (Shc A and Shc C) as well as mDab1 (mammalian disabled-1), Numb and Numb-like proteins, KLC (kinesin light chain) and Abl non-receptor tyrosine kinase. 1.1.1. X11 family X11 is a neuronal cytosolic adaptor protein which is a part of the tripartite complex X11␣/CASK/Veli involved in basolateral protein sorting. Because of this involvement, X11 is thought to be implicated in APP metabolism; in fact X11␣ has been suggested to slow APP metabolism and to prolong its half-life, resulting in the reduction of soluble A␤1-40 and A␤1-42. X11 also possesses a transinhibition function, since X11␣ and ␤ are able to strongly inhibit transcriptional transactivation mediated by AICD [36]. 1.1.2. Jip family The JNK-interacting proteins, JIP1b and JIP2, associate with the cytoplasmic domain of the amyloid precursor protein. This interaction involves the JIP1b or JIP2 carboxyl-terminal PTB domain and the YENPTY motif in the APP cytoplasmic domain. Jips are a scaffolding protein family of the JNK pathway kinases, which have been implicated in various signaling pathways including neuronal apoptosis. The most JIPs established function consists of axonal transport through binding to the tetratricopeptide repeat (TPR) of the kinesin-1 light-chain (Klcl1). AICD dimerization triggers neuronal cell death mediated by the complex ASK (apoptosis signal-regulating kinase)-1/JNK. ASK-

1 forms a complex with AICD through Jip1b which associates with JNK signaling pathway. Another JIPs function is their different ability to phosphorylate APP at Thr668 residue through JNK activation. This phosphorylation has been involved in APP putative functions and metabolism; therefore this JIPs action may bear physiological importance. The expression of JIP1b stabilizes immature APP and suppresses the production of a secreted large extracellular amino-terminal domain of APP, the generation of a cleaved intracellular carboxylterminal fragment of APP and the secretion of ␤-amyloid 40 and 42. Deletion of the PTB domain or alteration of its amino acid residues prevents JIP1b from interacting with APP and affecting its metabolism, whereas deletion of the JNK-binding domain of JIP1b has no effect. JIP2, a weaker APP-binding protein, does not affect the processing of APP, although it is known that JIP1b and JIP2 equally regulate the JNK signaling cascade. These results suggest that JIP1b can directly modulate APP metabolism by interacting with the APP cytoplasmic domain, independent of its regulation of the JNK signaling cascade [37]. 1.1.3. mDab1 protein The mDab1 is an adaptor protein which participates in nervous system development. It has been reported in a mouse model, that it binds the YENPTY domain of the AICD through its PTB domain. mDab1 becomes phosphorylated during embriogenesis and it is responsible for the correct neuron positioning within laminar structures throughout the brain, functioning as the downstream transducer of the reelin pathway (reelin-VLDLR/apoER2-mDab1). Phosphorylated mDab1 increases cellular levels of mature APP; mDab1 also inhibits APP endocytosis enhancing its cell surface expression probably by competing with other endocytic regulators. When X11␣ and mDab1 are co-expressed in equal amounts, the former is able to overcome the influence of the latter on cellular APP [38]. 1.1.4. Shc family The APP cytoplasmic tail undergoes phosphorylation at threonine and tyrosine residues mediated by Cdk5, glycogen synthase and c-Jun N-terminal kinase-3. Tyrosine phosphorylation of APP acts mainly as the docking site for the proteins ShcA and Grb2. Shc adaptors family and Grb2 usually connect growth factor receptors to specific signaling pathways and are involved in oncogenic proliferation, neuronal development, cell differentiation and apoptosis. In light of these observations it is likely that post-translational modifications, such as selective phosphorylation or dephosphorylation of APP-CTF-AICD, may act as a coupling event for different cellular pathways [39]. 1.1.5. Fe65 family The Fe65s are a family of adaptor/scaffolding proteins mediating multimolecular complexes assembly through a variety of protein interaction domains. There are three members: Fe65, Fe65 like-1 (Fe65L1) and Fe65 like-2 (Fe65L2); Fe65 expression is enriched in the brain whereas both Fe65L1 and Fe65L2 are more ubiquitously expressed [40–43]. Full-length Fe65 (p97Fe65) can undergo proteolytic cleavage close to the WW domain to generate a 65-kDa C-terminal fragment (p65Fe65) with substantially greater affinity for APP than full-length Fe65 [44]. The three Fe65s have a common structure and all contain three protein interaction domains: an N-terminal WW domain and two contiguous C-terminal phosphotyrosine binding (PTB) domains (PTB1 and PTB2). The single WW domain typically binds to proline-rich sequences [45]. Several binding partners have been identified: Mena and Evl, members of the Ena/VASP family of actin cytoskeleton regulatory proteins, the c-Abl tyrosine kinase and the ionotropic P2X2 receptor subunit [46–49]. Together, APP and Fe65 have been implicated

E. Buoso et al. / Pharmacological Research 62 (2010) 308–317

in the regulation of actin-based cell motility and have been found to colocalize in neuronal growth cones and interact in synaptic terminals, suggesting potential roles for APP/Fe65/Mena in synaptic plasticity [50,51]. The first Fe65 PTB domain (PTB1) binds to two transcription factors, CP2/LSF/LBP1 and the histone acetyltransferase Tip60 [52–54]; it also binds an NPXY motif in the intracellular domain of the low-density lipoprotein receptor-related protein (LRP) and couples LRP to APP in the trimeric complex LRP/Fe65/APP [55–57]. Through the second PTB domain (PTB2), all three members of this family bind to the YENPTY reinternalization motif within AICD [40–42,58–61] although in a tyrosine phosphorylationindependent manner. The interaction between Fe65 and AICD is regulated by Thr668 within the Fe65-binding region of AICD (numbering for APP695 ). Phosphorylating or mutating Thr668 impedes binding of Fe65, presumably through altering the conformation of AICD [62–64]. These final considerations suggest that AICD, through its association with Fe65 adaptor protein, may be an element of a multimeric complex made of proteins with transcription-related activities. 1.2. Possible role of aicd in Fe65 and Tip60 complex The possible AICD role in signal transduction is to date one of the most controversially discussed mechanism; thus different models have been hypothesized to explain the potential AICD role within Fe65/Tip60 complex. Tip60 is a histone acetyltransferase identified as a Fe65 binding partner which APP ears to act as a co-activator for the AICD-Fe65 complex; Tip60 is also part of a large complex with DNA-binding, ATPase and DNA helicase activity [65]. A ternary complex consisting of AICD, Fe65 and Tip60 was observed in spherical nuclear spots of HEK293 cells, suggesting its co-localization with sites of active transcription [66]. When Tip60 is fused to the DNA-binding domain of yeast transcription factor Gal4, it cannot induce expression of a Gal4dependent reporter gene, suggesting that Tip60, although a nuclear protein that is part of a DNA-binding protein complex, is transcriptionally inactive. Co-expression of either APP or Fe65 with Gal4-Tip60 also fails to activate reporter gene expression. However, co-expression of both APP and Fe65 with Gal4-Tip60 leads to dramatically enhance expression of the reporter gene, indicating that APP and Fe65 may play a role in activating gene transcription [53]. It has been found that Tip60/Fe65/AICD ternary complex is able to displace the N-CoR repressive complex from the KAI gene promoter region [67], a putative target of APP-mediated transcription. This complex was also shown to stimulate the activity of the neprilysin gene promoter and to increase the protein level in HEK293 cells [68]. AICD has also been involved in transcription regulation of other genes, such as p53 [69,70], EGFR, LRP and APP itself. Cao and Südhof suggested that Fe65 binding to the AICD has to take place close to the membrane in order to stimulate Fe65 activation; the major evidence for this proposal consists in the observation that overexpression of the soluble AICD alone, in any form, does not cause major Fe65-dependent transactivation of Gal4-Tip60; while as soon as the AICD is placed into the context of a membrane protein that is a ␥-secretase substrate, it becomes a potent transactivator [71]. Subsequently the APP ␥-cleavage is required to release activated Fe65 from the membrane, which then translocates into the nucleus where its WW domain interacts with other transcription factors to activate gene expression [51,72,73]. After ␥-cleavage, the AICD fragment could accompany Fe65 into the nucleus or, as some authors argue, it could be immediately degradated.

311

Other authors have proposed an alternative model in which AICD nuclear translocation takes place without Fe65 association. They argue that the phosphorylation of the APP intracellular domain at threonine 668 (Thr668 ) is essential for its nuclear translocation and its following binding to Fe65 [64]. However, other authors have demonstrated that phosphorylation or amino acid substitution of APP and AICD at Thr668 suppresses their association with Fe65 [62], because this phosphorylation affects the aminoterminal helix capping-box structure composed of VTPEER and the helical state of the following amino acid sequence that includes the YENPTY Fe65-binding motif [74]. Nakaya and Suzuki [63] have analysed, in a mutant mouse, the Thr668 residue role in regulating Fe65 and AICD nuclear translocation and Fe65-dependent gene transactivation mediated by AICD. In this mutant mouse, AICDa, in which AICD Thr668 was replaced with Ala, was detected in the nuclear fraction, although it has been reported that both the Thr substitution for Ala and the phosphorylation of Thr suppressed the interaction with Fe65 [62]. They have also shown that AICD is transported into the nucleus independently of Fe65 and its own phosphorylation. These observations suggest that AICD phosphorylation and Fe65 association are not essential for the nuclear translocation of AICD in vivo [63]. One remarkable difference is the process of AICD generation. Endogenous AICD is generated from APP by cleavage in the juxtamembrane region, whereas exogenous AICD is synthesized in the cytoplasm as a cytoplasmic protein. This difference may cause the alternative fate in AICD metabolism, although the reason why only exogenous AICD may be stabilized by Fe65 expression is still under investigation [75,21,76]; therefore endogenous AICD metabolism behaves differently from that of exogenous AICD in cells. This observation is also consistent with data reported by Waldron et al. [77]. They demonstrated, by modifying environmental factors, such as intracellular pH, an accumulation of endogenous AICD, which in turn has been hypothesized to remain anchored in membrane or free in cytosol, rather than to tranlocate into nucleus and activate target gene transcription [77]. Finally experiments using embryonic stem cells deficient in both presenilin-1 and presenilin-2 [presenilin-1/2 knockout (PS-KO) cells] have shown that APP can activate Tip60-mediated transcription in the absence of presenilin-mediated ␥-secretase cleavage, contrary to current dogma. Additionally, in other cell lines, in the presence of ␥-secretase inhibitors at concentrations that prevent production of A␤ and generation of the AICD fragment, APP retained the ability to activate Tip60. These results suggest an alternative model of APP signaling in which holo-APP recruits to the membrane not only Fe65 but also Tip60 to induce Tip60 phosphorylation and complex formation with Fe65, leading to signal transduction activation. This phosphorylation event appears to be carried out by a Cdk, given that signaling could be blocked by mutating the known Cdk sites in Tip60 or by the Cdk inhibitor roscovitine. A key aspect of this model is that the signaling role of APP may lie in its ability to recruit proteins to the microdomain of the membrane, where particular kinases, such as Cdk5, are active [78]. However, ␥-secretase cleavage appears to facilitate signaling in embryonic stem cells, presumably through the release of the Fe65-Tip60 complex from the membrane. One potential ␥secretase-independent mechanism for the dissociation of Fe65 and Tip60 from APP and the membrane could be phosphorylation of APP at threonine 668, which has been shown to decrease the interaction between Fe65 and APP. Interestingly, Cdk5 has been shown to phosphorylate APP at this site [27], raising the possibility that Cdk5 could play the dual role of activating Tip60 and releasing the complex from the membrane.

312

E. Buoso et al. / Pharmacological Research 62 (2010) 308–317

Considering these conflicting observations, Müller et al. [79] summarized different models: • Model I: AICD recruits Fe65 and changes its conformation. Subsequently, Fe65 translocates into the nucleus and binds to Tip60; • Model II: Fe65 and AICD translocate into the nucleus independently building up the ternary complex AICD/FE65/Tip60 in the nucleus; • Model III: suggests a ␥-secretase-independent mechanism in which APP recruits Tip60 through Fe65 resulting in the phosphorylation, stabilization and activation of Tip60 by Cdk. As a result, the Tip60-Fe65 complex translocates into the nucleus; • Model IV: AICD/Fe65 complex generates at the membrane and subsequently translocates into the nucleus. Following translocation, Tip60 enters the complex to generate the transcription factor. 2. AICD degradation processes In this section AICD degradation will be reviewed focusing on the two best characterized degradation processes, proteasome and IDE. 2.1. Proteasome and its possible implication in AICD degradation Proteasome is a large protein complex existing inside all eukaryotes and archaea, as well as in some bacteria. In eukaryotes, it is located both in the nucleus and in the cytoplasm [80]. The main cellular function of the proteasome is to degrade by proteolysis misfolded, damaged and unneeded proteins. The proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, the regulation of gene expression and responses to oxidative stress because it regulates protein concentration in cells. Proteins are tagged for degradation by a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules. The result is a polyubiquitin chain that is bound by the proteasome, allowing it to degrade the tagged protein [81]. The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin-proteasome system. Skovronsky et al. [82] examined the effects of MG132, a potent cell permeable and selective proteasome inhibitor, on the production of secreted and intracellular A␤ species in NT2N neuronal cells. They found that MG132 inhibited secretion of A␤1-40 nearly 2-fold more than secretion of A␤1-42 from human NT2N neuronal cells. While decreasing secretion of A␤1-40, MG132 led to the accumulation of APP C-terminal fragments. C83, C88 and C99 levels were all increased to similar extents, suggesting that all three of the major APP C-terminal fragments are substrates for the same or similar ␥secretase(s). These results are consistent with several studies that have examined the effects of peptide-aldehyde protease inhibitors on A␤ secretion [83,84]. Furthermore, the bulk of these fragments were generated in post-ER/IC (intermediate compartment). The accumulation of C99 in the context of selective inhibition of A␤140 secretion suggests that cleavage to generate secreted A␤1-40 accounts for at least a fraction of the turnover of this fragment. These results are sustain previous studies that have reported selective inhibition of A␤1-40 (relative to A␤1-42) by MG132 and related peptide aldehydes in non-neuronal cells [83]. However, in contrast to these studies, they did not observe an increase in A␤1-42 secretion at any concentration of MG132 tested. This may reflect the use of ␥-secretases with different properties by neuronal versus non-neuronal cells. Given that the overproduction of A␤1-42 is

implicated in the pathogenesis of AD, they investigated the proteasome role using a more specific proteasome inhibitor, lactacystin, which induced a similar increase in recovery of ER/IC generated A␤. These results suggest that the proteasome is potentially involved in the normal ␥-secretase metabolism (or a ␥-secretase cofactor) itself. They conclude suggesting that proteasome inhibition leads to an increased ␥-secretase activity but they also propose a possible proteasome role in C99 and other APP C-terminal fragments degradation. A degradative turnover of an APP transmembrane probe has been observed in the ER of HEK293 cells [85]. Thus, the involvement of the proteasome in APP processing may have important consequences for the regulation of APP intracellular fragment production and the pathogenesis of AD. To investigate the nature of the proteasome-dependent degradation of APP-CTF␤, a recombinant APP cytoplasmic domain protein (APP cyt) was incubated in the presence or absence of recombinant 20S proteasome for up to 24 h. After 24 h, little breakdown of APP cyt had occurred in the absence of proteasome. However, in the presence of the proteasome, the amount of APP cyt was significantly decreased. This decrease in APP cyt was reduced in the presence of MG132, indicating that the effect of the proteasome had been produced via a proteolytic cleavage mechanism. MG132, a nonselective protease inhibitor, which has also been shown to inhibit ␥-secretase, caused a large accumulation of APP-CTF␤, by inhibiting both ␥-secretase and the proteasome. Other proteasome inhibitors such as ALLN and lactacystin did not act directly on ␥secretase and thus caused an increase in both APP-CTF␤ and A␤ levels, providing more substrate available for ␥-secretase cleavage [86]. These results have given the first evidence that the proteasome can cleave the APP cytoplamic domain and directly influence A␤ production. APP-CTFs degradation may occur through the early secretory compartment because proteasome inhibition increases APP processing through the ␥-secretase pathway, a result that would not have occurred if proteasome-dependent degradation took place subsequently to ␥-secretase cleavage in the secretory pathway. The fact that ␥-secretase cleavage is thought to occur first in the ER or Golgi complex, suggests that the proteasomedependent processing may occur when APP-CTF␤ is in a similar compartment. To identify proteasome cleavage sites in APP, two peptides homologous to the APP C-terminal tail were incubated with recombinant 20S proteasome and their cleavage was monitoreted by reversed phase high-performance liquid chromatography and mass spectrometry. Proteasome cleaved the APP C-terminal peptides at several sites, including a region around the YENPTY sequence which interacs with several APP-binding proteins [87]. The loss of this sequence may affect the APP-CTFs ability to bind to proteins necessary for trafficking to the ␥-secretase complex. However, proteasomal degradation of the YENPTY sequence within the APP cytoplasmic domain prevents binding and thereby hinders translocation to the ␥-secretase decreasing its activity and consequently inhibiting A␤ production [88]. So far, the protein responsible for this mechanism has not been identified. Clearly, this protein could be an important target for drug development in AD. Inhibition of its binding to APP-CTF␤ would be expected to decrease A␤ production and thereby reduce the amyloid load in the brain. Published evidence suggests that the proteasome system may be impaired in AD [89,90]; thus it is possible that a smaller portion of the C-terminal fragment of APP is degraded via this pathway in the AD brain. This decrease in the proteasome-dependent processing would be expected to cause an increase in APP-CTF␤ available for ␥-secretase processing, thereby increasing A␤ production. Thus, a defective proteasome function could directly contribute to increase A␤ production in cases of sporadic AD [86].

E. Buoso et al. / Pharmacological Research 62 (2010) 308–317

2.2. Insulin degrading enzyme (IDE) Insulin degrading enzyme (IDE) is a neutral thiol metalloprotease, which requires both a free thiol and bivalent cations for its activity as a protease. It is a single polypeptide with a molecular weight of 110 kDa, and dimers or trimers of it have been purified under nondenaturating conditions [91]. Zn2+ is the metal bound to IDE. The IDE active site consists of the sequence His-Glu-aaaa-His (HEXXH) in which the two histidines coordinate the binding of the zinc atom and the glutamate plays an essential role in catalysis [92]. IDE was co-isolated with the multicatalytic proteinase, suggesting that IDE might be involved in a protein complex [93]. IDE is ubiquitously expressed, with its highest expression in the liver, testes, muscle and brain [94]. Its expression is regulated during cell differentiation and growth with IDE mRNA level increased in the brain and testes when development proceeds [95]. Furthermore, IDE expression is affected by aging, with IDE activity significantly decreased in both the muscles and the liver of old animals as compared to young animals [96]. The subcellular localization shows that IDE is abundant in cytosol and peroxisomes [92]. In addition, IDE is also found in rough endoplasmic reticulum (RER), plasma membrane as well as in the extracellular compartment [97]. Although the mechanism of IDE to locate outside the cells is unclear, it has been identified intact in human CSF, further indicating that IDE does exist in extracellular fluid under physiological condition. Biochemical studies have shown the presence of IDE in the soluble fractions from human brains, which contain both extracellular and cytosolic compartments [98,99]. The subcellular location of IDE seems to be regulated during development and differentiation. In undifferentiated neuronal PC12 cells, IDE has been found on the cell surface as well as released into the extracellular space, whereas IDE is no longer secreted when the cells differentiate in response to growth factor [100]. Several short peptides with molecular weights of 3–10 kDa have been shown to serve as the substrates of IDE, including insulin [101], insulin-like growth factors I and II [102], amylin [103] and A␤ [96,97,104,105]. The peptide substrates share similar secondary structure and amyloidogenic character. Among all secreted proteases from the cells, only IDE has been demonstrated to degrade A␤. Therefore, IDE likely plays a role in catabolic regulation, especially in preventing formation of amyloid deposits by cleaving the component peptides. It has been found that under physiological conditions IDE is secreted at high levels from the microglial cells and degrades A␤ extracellularly [96]. Purified IDE from rat liver and brain was shown to degrade A␤ effectively. Primary cultured neurons were also shown to clear A␤ via extracellular IDE as well as IDE on the cell surface [100]. From brain homogenates IDE degrades different forms of A␤: A␤1-40, A␤1-42 and an A␤ mutant in one type of AD (Dutch Variant 1-40 Q). In AD brains A␤ degrading activity by IDE was shown to be lower than in the controls; moreover, the amount of hippocampal IDE protein was also found to be reduced in AD brains as compared to the controls. Finally, when the IDE gene was deleted in mouse model, A␤ levels in the brain were elevated, suggesting IDE activity is critical in determining the amount of brain A␤ in vivo. More significantly, enhanced IDE activity in the IDE and APP double transgenic mice decreased their brain A␤ levels, and prevented the formation of AD pathology [106]. IDE has multiple substrates in vivo with different Km. They can compete with each other to be degraded by IDE. One working hypothesis is that the imbalance of the substrates could affect the degradation process by IDE, thus influencing the pathogenesis of AD. IDE has also been implicated in AICD removal. Edbauer et al. have demonstrated that AICD can efficiently be generated in vitro in a ␥-secretase dependent manner from crude membrane fractions. The in vitro generated AICD is rapidly degraded and the addition of

313

a protease inhibitor mix containing EDTA is necessary to stabilize AICD. Interestingly, the protease inhibitor mix without EDTA did not block AICD degradation, suggesting the involvement of a divalent metal ion-dependent proteolytic activity in AICD generation [107]. They next investigated whether the metalloprotease activity involved in AICD degradation was cytosolic or membrane-bound, arriving to the conclusion that a cytoplasmic metalloprotease present in peripheral as well as in neuronal cells was involved in AICD degradation. Based on the inhibition profile and the cytoplasmic localization of the AICD-degrading activity, they searched candidate AICD-degrading enzymes. This search revealed that three cytoplasmic candidate proteases from two metalloprotease families (M3A and M16A families) could be responsible for the degradation of AICD: thimet oligopeptidase (TOP, M3A family), neurolysin (M3A family) and IDE (insulysin, M16A family). Their data suggested that IDE was a major AICD-degrading enzyme. In fact, biochemical properties of IDE make it an ideal candidate for AICD degradation: it is highly expressed in the same cellular compartment as AICD, it is a common enzyme in brain tissue, where abundant APP expression is observed, and it preferentially degrades small cytoplasmic peptides of about 20–50 amino acids in lenght. Furthermore, through the application of Edman degradation, Venugopal et al. [108] demonstrated that IDE can cleave AICD at multiple sites, even if in a non-specific manner, to generate small peptides ranging from 5 to 14 amino acids. Farris Mansourian et al. [109] analysed whether IDE mediates AICD catabolism in vivo. Endogenous brain AICD levels were quantified in six IDE −/− and six +/+ mice by immunoprecipitation with antibody raised against the APP C terminus. Two specifically immunoreactive AICD proteins were routinely detected in freshly prepared brain homogenates. Both these proteins were absent in the brains of mice lacking APP expression and increased in the brains of transgenic mice over-expressing human APP. There was a clear and consistent increase in the lower AICD band in the IDE-deficient compared with wild-type brains, but no increase in the upper band. Dephosphorylation of the homogenates with calf intestinal phospatase eliminated the upper band in both mouse genotypes and increased the density of the lower band, without changing its migration, thus indicating that the former is a phosphorylated form and the latter a nonphosphorylated form of AICD. Thus, IDE appears in vivo to selectively regulate the levels of unphosphorylated AICD, but not its minor phosphorylated form. Although IDE has been identified as a major AICD-degrading enzyme in non-neuronal and neuronal cell lysates, it cannot exclude the possibility that other mechanisms may degrade AICD in cells where IDE activity or its expression is low. This observation is also reinforced by experiments on knockout mice lacking IDE, which show in the brain the presence of AICD in a minor extent of their precursor, thus indicating the existence of an alternative turnover [108]. 3. Conclusion The AICD signal transduction, within Fe65 and Tip60 complex, is a very controversially discussed mechanism. In 2001 Cao and Südhof [53] attributed to the AICD an important biological role related to its ability to promote the expression regulation of some genes including p53. On the basis of different data presented in the literature, Müller and colleagues have summarized in four models the different assumptions about the transactivation mechanism that involves these intracellular domains. The analysis of these models, in relation to the degradation process, highlights the contrast among mechanisms responsible of AICD localization, function and degradation.

314

E. Buoso et al. / Pharmacological Research 62 (2010) 308–317

Fig. 2. Hypothesis about AICD function and degradation. The figure shows two possible AICD mechanisms. (a) After a specific stimulus Fe65 binds AICD preventing its proteasome degradation. AICD/Fe65 complex, alone or together with Tip60 translocate to the nucleus where, in association with Tip60, regulate gene transcription AICD transcriptionally active form). (b) A different stimulus induces AICD phosphorylation which impedes Fe65 bind and promote its cytosol release where it can become an IDE substrate (see text for details).

On the basis of these considerations, the analysis of model I shows that the degradation of these fragments is primarily due to the proteasome action. Cao and Südhof [71] in fact have suggested that the AICD generation occurs to the membrane as a consequence of Fe65 binding to the YENPTY domain. Furthermore, Nunan and coworkers argue that the binding of a protein to this domain is responsible for preventing proteasome-mediated degradation. This consideration on the role of proteasome can be valid even for the model presented by Hass and Yankner [78], in which this APP domain acts as an assembly site for the Fe65/Tip60 complex. Also in this case, the Fe65 binding would be able to prevent degradation mediated by proteasome of the membrane-bound AICD, as suggested by Nunan et al. [86]. In these models, the AICD acts as an anchor site which is essential for Fe65 activation; in addiction, after ␥-secretase cleavage, either AICD or Fe65 or Fe65/Tip60 complexes are released in the cytosol. The AICD becames an IDE substrate, whereas Fe65 alone or in complex with Tip60 translocates to the nucleus. In light of these considerations both models suggest that either proteasome or IDE are involved in AICD degradation, even if they operate at two different levels. Degradation mediated by proteasome impedes the complex formation and consequently blocks gene regulation promoted by this latter. IDE plays a role in AICD removing, once it has exploited their function to activate Fe65 or the entire complex Fe65/Tip60. With regard to IV model, the AICD plays a double role: the Fe65 activation and the association with Tip60 at nuclear level. As in the other models, the Fe65 binding to the AICD domain impedes their degradation promoted by proteasome. However, in this case, IDE is not implicated in AICD removal, because these fragments are not released in the cytosol, but, when complexed with Fe65, they translocate to the nucleus. The last model, presented by Nakaya and Suzuki [63], argues that the AICD are released at the cytosol level and then translocate independently to the nucleus. In this model the AICD do not act as an anchoring site but acquire the faculty to regulate gene expression in association with Fe65/Tip60 complex. Considering this as putative mechanism of action of AICD, IDE is the only process involved in AICD degradation.

Despite all these considerations, we cannot exclude the possibility that an alternative mechanism in which IDE and proteasome are involved in a different way in AICD degradation may exist. We might speculate that IDE acts when the AICD do not play their role in gene expression regulation. This hypothesis suggests that, within cells, a signaling mechanism, that allows the regulation of APP proteolysis in order to generate a transcriptionally active fragment from a non-active one, may exist (Fig. 2). Following a specific stimulus, Fe65 may bind AICD that is anchored to membrane. Following ␥-secretase cleavage, the Fe65/AICD complex is activated and can move to the nucleus in order to regulate gene transcription. Whereas in the second case, we might hypothesize that IDE intervene when Fe65 protein do not bind AICD domain. In this regard, some literature data suggest that when AICD are phosphorilated at Thr668 (APP695 numbering), the bound between Fe65 and AICD is prevented. Moreover, a recent published work indicates that this phosphorylation facilitates BACE1 association to APP, thus favoring its processing [110], with consequent A␤ and AICD release. AICD released into the cytosol may become IDE substrate. Although other recent studies have demonstrated that serine/threonine phosphorylation, specifically at amino acid residue Thr668 , regulates APP processing, other works have investigated the possibility that APP tyrosine phosphorylation at different sites can regulate APP processing [48,111]. It has also been found that APP phosphorylation at Tyr687 , the major tyrosine kinase phosphorylation site, regulates APP processing by ␣- and ␥-secretases, influencing AICD expression level [112]. Altogether these results suggest that proteasome degradation may be hindered not only by protein binding, as Nunan proposed, but also by the phosphorylation state of APP domain, which seems to promote APP processing with consequent AICD release into the cytosol where they become an IDE substrate. The role of AICD in the pathogenesis of AD is still under investigation. Recently, a putative contribution of these proteolytic fragments has been demonstrated in transgenic mice over-expressing AICD [113], where their increased levels initiate a series of events that begins with activation of GSK-3␤ and culminates in neurodegeneration. The reason of their increased levels in AD brain is still unknown, even if a possibility is suggested by the

E. Buoso et al. / Pharmacological Research 62 (2010) 308–317

fact that proteasome degradation pathways have been found to be deficient in AD [114]. Acknowledgements This work was supported by the contribution of grants from CARIPLO to M.R. We are grateful to Marie Moutel for careful editing and proofreading of the manuscript. References [1] Racchi M, Mazzucchelli M, Porrello E, Lanni C, Govoni S. Acetylcholinesterase inhibitors: novel activities of old molecules. Pharmacol Res 2004;50(4):441–51. [2] Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik KH, et al. The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature 1987;325(6106):733–6. [3] Haass C, Schlossmacher MG, Hung AY, Vigo-Pelfrey C, Mellon A, Ostaszewski BL, et al. Amyloid ␤-peptide is produced by cultured cells during normal metabolism. Nature 1992;359:322–5. [4] Hardy J, Selkoe DJ. The Amyloid Hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002:297–353. [5] Zheng H, Koo EH. The amyloid precursor protein: beyond amyloid. Mol Neurodegener 2006;3(1):5. [6] Hung AY, Koo EH, Haass C, Selkoe DJ. Increased expression of beta-amyloid precursor protein during neuronal differentiation is not accompanied by secretory cleavage. Proc Natl Acad Sci USA 1992;89(20):9439–43. [7] Ciallella JR, Ikonomovic MD, Paljug WR, Wilbur YI, Dixon CE, Kochanek PM, et al. Changes in expression of amyloid precursor protein and interleukin1beta after experimental traumatic brain injury in rats. J Neurotrauma 2002;19(12):1555–67. [8] Esch FS, Keim PS, Beattie EC, Blacher RW, Culwell AR, Oltersdorf T, et al. Cleavage of amyloid beta peptide during constitutive processing of its precursor. Science 1990;248(4959):1122–4. [9] Buxbaum JD, Thinakaran G, Koliatsos V, O’Callahan J, Slunt HH, Price DL, et al. Alzheimer amyloid protein precursor in the rat hippocampus: transport and processing through the perforant path. J Neurosci 1998;18(23):9629–37. [10] Asai M, Hattori C, Szabó B, Sasagawa N, Maruyama K, Tanuma S, et al. Putative function of ADAM9, ADAM10, and ADAM17 as APP alpha-secretase. Biochem Biophys Res Commun 2003;301(1):231–5. [11] Seubert P, Oltersdorf T, Lee MG, Barbour R, Blomquist C, Davis DL, et al. Secretion of beta-amyloid precursor protein cleaved at the amino terminus of the beta-amyloid peptide. Nature 1993;361(6409):260–3. [12] Hussain I, Powell D, Howlett DR, Tew DG, Meek TD, Chapman C, et al. Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol Cell Neurosci 1999;14(6):419–27. [13] Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, et al. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 1999;402(6761):537–40. [14] Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, et al. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 1999;286(5440):735–41. [15] Yan R, Bienkowski MJ, Shuck ME, Miao H, Tory MC, Pauley AM, et al. Membrane-anchored aspartyl protease with Alzheimer’s disease betasecretase activity. Nature 1999;402(6761):533–7. [16] Lin X, Koelsch G, Wu S, Downs D, Dashti A, Tang J. Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci USA 2000;97(4):1456–60. [17] Edbauer D, Winkler E, Regula JT, Pesold B, Steiner H, Haass C. Reconstitution of gamma-secretase activity. Nat Cell Biol 2003;5(5):486–8. [18] Weidemann A, Eggert S, Reinhard FB, Vogel M, Paliga K, Baier G, et al. A novel epsilon-cleavage within the transmembrane domain of the Alzheimer amyloid precursor protein demonstrates homology with Notch processing. Biochemistry 2002;41(8):2825–35. [19] Pinnix I, Musunuru U, Tun H, Sridharan A, Golde T, Eckman C, et al. A novel gamma-secretase assay based on detection of the putative Cterminal fragment-gamma of amyloid beta protein precursor. J Biol Chem 2001;276(1):481–7. [20] Bergman A, Religa D, Karlström H, Laudon H, Winblad B, Lannfelt L, et al. APP intracellular domain formation and unaltered signaling in the presence of familial Alzheimer’s disease mutations. Exp Cell Res 2003;287(1):1–9. [21] Kimberly WT, Zheng JB, Guénette SY, Selkoe DJ. The intracellular domain of the beta-amyloid precursor protein is stabilized by Fe65 and translocates to the nucleus in a notch-like manner. J Biol Chem 2001;276(43):40288–92. [22] De Strooper B, Annaert W, Cupers P, Saftig P, Craessaerts K, Mumm JS, et al. A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 1999;398(6727):518–22. [23] Schroeter EH, Kisslinger JA, Kopan R. Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 1998;393(6683):382–6. [24] Nishimoto I, Okamoto T, Matsuura Y, Takahashi S, Okamoto T, Murayama Y, et al. Alzheimer amyloid protein precursor complexes with brain GTP-binding protein G(o). Nature 1993;362(6415):75–9.

315

[25] Brouillet E, Trembleau A, Galanaud D, Volovitch M, Bouillot C, Valenza C, et al. The amyloid precursor protein interacts with Go heterotrimeric protein within a cell compartment specialized in signal transduction. J Neurosci 1999;19(5):1717–27. [26] Oishi M, Nairn AC, Czernik AJ, Lim GS, Isohara T, Gandy SE, et al. The cytoplasmic domain of Alzheimer’s amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells. Mol Med 1997;3(2):111–23. [27] Iijima K, Ando K, Takeda S, Satoh Y, Seki T, Itohara S, et al. Neuron-specific phosphorylation of Alzheimer’s beta-amyloid precursor protein by cyclindependent kinase 5. J Neurochem 2000;75(3):1085–91. [28] Suzuki T, Oishi M, Marshak DR, Czernik AJ, Nairn AC, Greengard P. Cell cycle-dependent regulation of the phosphorylation and metabolism of the Alzheimer amyloid precursor protein. EMBO J 1994;13(5):1114–22. [29] Aplin AE, Gibb GM, Jacobsen JS, Gallo JM, Anderton BH. In vitro phosphorylation of the cytoplasmic domain of the amyloid precursor protein by glycogen synthase kinase-3beta. J Neurochem 1996;67(2):699–707. [30] Standen CL, Brownlees J, Grierson AJ, Kesavapany S, Lau KF, McLoughlin DM, et al. Phosphorylation of thr(668) in the cytoplasmic domain of the Alzheimer’s disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3). J Neurochem 2001;76(1):316–20. [31] Ando K, Oishi M, Takeda S, Iijima K, Isohara T, Nairn AC, et al. Role of phosphorylation of Alzheimer’s amyloid precursor protein during neuronal differentiation. J Neurosci 1999;19(11):4421–7. [32] Horie Y, Wolf R, Chervenak RP, Jennings SR, Granger DN. T-lymphocytes contribute to hepatic leukostasis and hypoxic stress induced by gut ischemiareperfusion. Microcirculation 1999;6(4):267–80. [33] Chen WJ, Goldstein JL, Brown MS. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J Biol Chem 1990;265(6):3116–23. [34] Koo EH, Squazzo SL. Evidence that production and release of amyloid betaprotein involves the endocytic pathway. J Biol Chem 1994;269(26):17386–9. [35] Lai A, Sisodia SS, Trowbridge IS. Characterization of sorting signals in the beta-amyloid precursor protein cytoplasmic domain. J Biol Chem 1995;270(8):3565–73. [36] Biederer T, Cao X, Südhof TC, Liu X. Regulation of APP -dependent transcription complexes by Mint/X11s: differential functions of Mint isoforms. J Neurosci 2002;22(17):7340–51. [37] Taru H, Kirino Y, Suzuki. Differential roles of JIP scaffold proteins in the modulation of amyloid precursor protein metabolism. J Biol Chem T 2002;277(30):27567–74. [38] Parisiadou L, Efthimiopoulos S. Expression of mDab1 promotes the stability and processing of amyloid precursor protein and this effect is counteracted by X11alpha. Neurobiol Aging 2007;28(3):377–88. [39] Russo C, Venezia V, Repetto E, Nizzari M, Violani E, Carlo P, et al. The amyloid precursor protein and its network of interacting proteins: physiological and pathological implications. Brain Res Brain Res Rev 2005;48(2):257– 64. [40] Bressler SL, Gray MD, Sopher BL, Hu Q, Hearn MG, Pham DG, et al. cDNA cloning and chromosome mapping of the human Fe65 gene: interaction of the conserved cytoplasmic domains of the human beta-amyloid precursor protein and its homologues with the mouse Fe65 protein. Hum Mol Genet 1996;5(10):1589–98. [41] Guénette S, Chang Y, Hiesberger T, Richardson JA, Eckman CB, Eckman EA, et al. Essential roles for the FE65 amyloid precursor protein-interacting proteins in brain development. EMBO J 2006;25(2):420–31. [42] Duilio A, Faraonio R, Minopoli G, Zambrano N, Russo T. Fe65L2: a new member of the Fe65 protein family interacting with the intracellular domain of the Alzheimer’s beta-amyloid precursor protein. Biochem J 1998;330(Pt 1):513–9. [43] Tanahashi H, Tabira T. Characterization of an amyloid precursor proteinbinding protein Fe65L2 and its novel isoforms lacking phosphotyrosineinteraction domains. Biochem J 2002;367(Pt3):687–95. [44] Hu Q, Wang L, Yang Z, Cool BH, Zitnik G, Martin GM. Endoproteolytic cleavage of FE65 converts the adaptor protein to a potent suppressor of the sAPP alpha pathway in primates. J Biol Chem 2005;280(13):12548–58. [45] Sudol M, Sliwa K, Russo T. Functions of WW domains in the nucleus. FEBS Lett 2001;490(3):190–5. [46] Ermekova KS, Zambrano N, Linn H, Minopoli G, Gertler F, Russo T, et al. The WW domain of neural protein FE65 interacts with proline-rich motifs in Mena, the mammalian homolog of Drosophila enabled. J Biol Chem 1997;272(52):32869–77. [47] Lambrechts A, Kwiatkowski AV, Lanier LM, Bear JE, Vandekerckhove J, Ampe C, et al. cAMP-dependent protein kinase phosphorylation of EVL, a Mena/VASP relative, regulates its interaction with actin and SH3 domains. J Biol Chem 2000;275(46):36143–51. [48] Zambrano N, Bruni P, Minopoli G, Mosca R, Molino D, Russo C, et al. The beta-amyloid precursor protein APP is tyrosine-phosphorylated in cells expressing a constitutively active form of the Abl protoncogene. J Biol Chem 2001;276(23):19787–92. [49] Masin M, Kerschensteiner D, Dümke K, Rubio ME, Soto F. Fe65 interacts with P2X2 subunits at excitatory synapses and modulates receptor function. J Biol Chem 2006;281(7):4100–8. [50] Sabo SL, Ikin AF, Buxbaum JD, Greengard PJ. The Alzheimer amyloid precursor protein (APP) and FE65, an ABPP-binding protein, regulate cell movement. Cell Biol 2001;153(7):1403–14.

316

E. Buoso et al. / Pharmacological Research 62 (2010) 308–317

[51] Sabo SL, Ikin AF, Buxbaum JD, Greengard P. The amyloid precursor protein and its regulatory protein, Fe65, in growth cones and synapses in vitro and in vivo. J Neurosci 2003;23(13):5407–15. [52] Zambrano N, Minopoli G, de Candia P, Russo T. The Fe65 adaptor protein interacts through its PID1 domain with the transcription factor CP2/LSF/LBP1. J Biol Chem 1998;273(32):20128–33. [53] Cao X, Südhof TC. A transcriptionally active complex of APP with Fe65 and histone acetyltransferase Tip60. Science 2001;293(5527):115–20. [54] Kim HS, Kim EM, Lee JP, Park CH, Kim S, Seo JH, et al. C-terminal fragments of amyloid precursor protein exert neurotoxicity by inducing glycogen synthase kinase-3beta expression. FASEB J 2003;17(13):1951–3. [55] Trommsdorff M, Borg JP, Margolis B, Herz J. Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein. J Biol Chem 1998;273(50):33556–60. [56] Kinoshita A, Whelan CM, Smith CJ, Mikhailenko I, Rebeck GW, Strickland DK, et al. Demonstration by fluorescence resonance energy transfer of two sites of interaction between the low-density lipoprotein receptor-related protein and the amyloid precursor protein: role of the intracellular adapter protein Fe65. J Neurosci 2001;21(21):8354–61. [57] Pietrzik CU, Yoon IS, Jaeger S, Busse T, Weggen S, Koo EH. FE65 constitutes the functional link between the low-density lipoprotein receptor-related protein and the amyloid precursor protein. J Neurosci 2004;24(17):4259–65. [58] Fiore F, Zambrano N, Minopoli G, Donini V, Duilio A, Russo T. The regions of the Fe65 protein homologous to the phosphotyrosine interaction/phosphotyrosine binding domain of Shc bind the intracellular domain of the Alzheimer’s amyloid precursor protein. J Biol Chem 1995;270(52):30853–6. [59] McLoughlin DM, Miller CC. The intracellular cytoplasmic domain of the Alzheimer’s disease amyloid precursor protein interacts with phosphotyrosine-binding domain proteins in the yeast two-hybrid system. FEBS Lett 1996;397(2–3):197–200. [60] Borg JP, Ooi J, Levy E, Margolis B. The phosphotyrosine interaction domains of X11 and Fe65 bind to distinct sites on the YENPTY motif of amyloid precursor protein. Mol Cell Biol 1996;16(11):6229–41. [61] Tanahashi H, Tabira T. Molecular cloning of human Fe65L2 and its interaction with the Alzheimer’s beta-amyloid precursor protein. Neurosci Lett 1999;261(3):143–6. [62] Ando K, Iijima KI, Elliott JI, Kirino Y, Suzuki T. Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production of beta-amyloid. J Biol Chem 2001;276(43):40353–61. [63] Nakaya T, Suzuki T. Role of APP phosphorylation in Fe65-dependent gene transactivation mediated by AICD. Genes Cells 2006;11(6):633–45. [64] Chang KA, Kim HS, Ha TY, Ha JW, Shin KY, Jeong YH, et al. Phosphorylation of amyloid precursor protein (APP) at Thr668 regulates the nuclear translocation of the APP intracellular domain and induces neurodegeneration. Mol Cell Biol 2006;26(11):4327–38. [65] Ikura T, Ogryzko VV, Grigoriev M, Groisman R, Wang J, Horikoshi M, et al. Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis. Cell 2000;102(4):463–73. [66] Von Rotz RC, Kohli BM, Bosset J, Meier M, Suzuki T, Nitsch RM, et al. The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor. J Cell Sci 2004;117(Pt 19):4435–48. [67] Baek SH, Ohgi KA, Rose DW, Koo EH, Glass CK, Rosenfeld MG. Exchange of NCoR corepressor and Tip60 coactivator complexes links gene expression by NF-kA␤PP aB and beta-amyloid precursor protein. Cell 2002;110(1):55–67. [68] Pardossi-Piquard R, Petit A, Kawarai T, Sunyach C, Alves da Costa C, Vincent B, et al. Presenilin-dependent transcriptional control of the Abeta-degrading enzyme neprilysin by intracellular domains of beta APP and APLP. Neuron 2005;46(4):541–54. [69] Ozaki T, Li Y, Kikuchi H, Tomita T, Iwatsubo T, Nakagawara A. The intracellular domain of the amyloid precursor protein (AICD) enhances the p53-mediated apoptosis. Biochem Biophys Res Commun 2006;351(1):57–63. [70] Alves da Costa C, Sunyach C, Pardossi-Piquard R, Sévalle J, Vincent B, Boyer N, et al. Presenilin-dependent gamma-secretase-mediated control of p53associated cell death in Alzheimer’s disease. J Neurosci 2006;26(23):6377–85. [71] Cao X, Südhof TC. Dissection of amyloid-beta precursor protein-dependent transcriptional transactivation. J Biol Chem 2004;279(23):24601–11. [72] Sabo SL, Lanier LM, Ikin AF, Khorkova O, Sahasrabudhe S, Greengard P, et al. Regulation of beta-amyloid secretion by Fe65, an amyloid protein precursorbinding protein. J Biol Chem 1999;274(12):7952–7. [73] Minopoli G, de Candia P, Bonetti A, Faraonio R, Zambrano N, Russo T. The beta-amyloid precursor protein functions as a cytosolic anchoring site that prevents Fe65 nuclear translocation. J Biol Chem 2001;276(9):6545–50. [74] Ramelot TA, Nicholson LK. Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR. J Mol Biol 2001;307(3):871–84. [75] Cupers P, Orlans I, Craessaerts K, Annaert W, De Strooper B. The amyloid precursor protein (APP)-cytoplasmic fragment generated by gamma-secretase is rapidly degraded but distributes partially in a nuclear fraction of neurones in culture. J Neurochem 2001;78(5):1168–78. [76] Kinoshita A, Whelan CM, Smith CJ, Berezovska O, Hyman BT. Direct visualization of the gamma secretase-generated carboxyl-terminal domain of the amyloid precursor protein: association with Fe65 and translocation to the nucleus. J Neurochem 2002;82(4):839–47. [77] Waldron E, Isbert S, Kern A, Jaeger S, Martin AM, Hébert SS, et al. Increased AICD generation does not result in increased nuclear translocation or activation of target gene transcription. Exp Cell Res 2008;314(13):2419–33.

[78] Hass MR, Yankner BA. A {gamma}-secretase-independent mechanism of signal transduction by the amyloid precursor protein. J Biol Chem 2005;280(44):36895–904. [79] Müller T, Meyer HE, Egensperger R, Marcus K. The amyloid precursor protein intracellular domain (AICD) as modulator of gene expression, apoptosis, and cytoskeletal dynamics-relevance for Alzheimer’s disease. Prog Neurobiol 2008;85(4):393–406. [80] Peters JM. Proteasomes: protein degradation machines of the cell. Trends Biochem Sci 1994;19(9):377–82. [81] Lodish HF, Rodriguez RK, Klionsky DJ. Points of view: lectures: can’t learn with them, can’t learn without them. Cell Biol Educ 2004;3(4):202–11. [82] Skovronsky DM, Pijak DS, Doms RW, Lee VM. A distinct ER/IC gammasecretase competes with the proteasome for cleavage of APP. Biochemistry 2000;39(4):810–7. [83] Klafki H, Abramowski D, Swoboda R, Paganetti PA, Staufenbiel M. The carboxyl termini of beta-amyloid peptides 1-40 and 1-42 are generated by distinct gamma-secretase activities. J Biol Chem 1996;271(45):28655–9. [84] Yamazaki N, Suzuki H, Kibayashi C. Nucleophilic alkylation on anti-bredt iminium ions. Facile entry to the synthesis of 1-alkylated 2-azabicyclo [3.3.1] nonanes (Morphans) and 5-azatricyclo[6.3.1.0(1,5)] dodecane. J Org Chem 1997;62(24):8280–1. [85] Bunnell WL, Pham HV, Glabe CG. Gamma-secretase cleavage is distinct from endoplasmic reticulum degradation of the transmembrane domain of the amyloid precursor protein. J Biol Chem 1998;273(48):31947–55. [86] Nunan J, Shearman MS, Checler F, Cappai R, Evin G, Beyreuther K, et al. The C-terminal fragment of the Alzheimer’s disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from gammasecretase. Eur J Biochem 2001;268(20):5329–36. [87] Nunan J, Williamson NA, Hill AF, Sernee MF, Masters CL, Small DH. Proteasome-mediated degradation of the C-terminus of the Alzheimer’s disease beta-amyloid protein precursor: effect of C-terminal truncation on production of beta-amyloid protein. J Neurosci Res 2003;74(3):378–85. [88] Kerr ML, Small DH. Cytoplasmic domain of the beta-amyloid protein precursor of Alzheimer’s disease: function, regulation of proteolysis, and implications for drug development. J Neurosci Res 2005;80(2):151–9. [89] Keller JN, Hanni KB, Markesbery WR. Impaired proteasome function in Alzheimer’s disease. J Neurochem 2000;75(1):436–9. [90] Lam YA, Pickart CM, Alban A, Landon M, Jamieson C, Ramage R, et al. Inhibition of the ubiquitin-proteasome system in Alzheimer’s disease. Proc Natl Acad Sci USA 2000;97(18):9902–6. [91] Ogawa W, Shii K, Yonezawa K, Baba S, Yokono K. Affinity purification of insulin-degrading enzyme and its endogenous inhibitor from rat liver. J Biol Chem 1992;267(2):1310–6. [92] Authier F, Cameron PH, Taupin V. Association of insulin-degrading enzyme with a 70 kDa cytosolic protein in hepatoma cells. Biochem J 1996;319(Pt 1):149–58. [93] Bennett RG, Hamel FG, Duckworth WC. Identification and isolation of a cytosolic proteolytic complex containing insulin degrading enzyme and the multicatalytic proteinase. Biochem Biophys Res Commun 1994;202(2):1047–53. [94] Kuo WL, Montag AG, Rosner MR. Insulin-degrading enzyme is differentially expressed and developmentally regulated in various rat tissue. Endocrinology 1993;132:604–11. [95] Baumeister H, Müller D, Rehbein M, Richter D. The rat insulin-degrading enzyme. Molecular cloning and characterization of tissue-specific transcripts. FEBS Lett 1993;317(3):250–4. [96] Runyan K, Duckworth WC, Kitabchi AE, Huff G. The effect of age on insulindegrading activity in rat tissue. Diabetes 1979;28(4):324–5. [97] Qiu WQ, Walsh DM, Ye Z, Vekrellis K, Zhang J, Podlisny MB, et al. Insulindegrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J Biol Chem 1998;273(49):32730–8. [98] McDermott JR, Gibson AM. Degradation of Alzheimer’s beta-amyloid protein by human and rat brain peptidases: involvement of insulin-degrading enzyme. Neurochem Res 1997;22(1):49–56. ˜ EM. Degradation of soluble amyloid [99] Pérez A, Morelli L, Cresto JC, Castano beta-peptides 1-40, 1-42, and the Dutch variant 1-40Q by insulin degrading enzyme from Alzheimer disease and control brains. Neurochem Res 2000;25(2):247–55. [100] Vekrellis K, Ye Z, Qiu WQ, Walsh D, Hartley D, Chesneau V, et al. Neurons regulate extracellular levels of amyloid beta-protein via proteolysis by insulin-degrading enzyme. J Neurosci 2000;20(5):1657–65. [101] Kirschner RJ, Goldberg AL. A high molecular weight metalloendoprotease from the cytosol of mammalian cells. J Biol Chem 1983;258(2):967–76. [102] Luchsinger JA, Tang MX, Stern Y, Shea S, Mayeux R. Mellitus and risk of Alzheimer’s disease and dementia with stroke in a multiethnic cohort. Am J Epidemiol 2001;154(7):635–41. [103] Bennett RG, Duckworth WC, Hamel FG. Degradation of amylin by insulindegrading enzyme. J Biol Chem 2000;275(47):36621–5. [104] Kurochkin IV, Goto S. Alzheimer’s beta-amyloid peptide specifically interacts with and is degraded by insulin degrading enzyme. FEBS Lett 1994;345(1):33–7. [105] Mukherjee A, Song E, Kihiko-Ehmann M, Goodman Jr JP, Pyrek JS, Estus S, et al. Insulysin hydrolyzes amyloid beta peptides to products that are neither neurotoxic nor deposit on amyloid plaques. J Neurosci 2000;20(23):8745–9. [106] Leissring MA, Farris W, Chang AY, Walsh DM, Wu X, Sun X, et al. Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque forma-

E. Buoso et al. / Pharmacological Research 62 (2010) 308–317

[107]

[108] [109]

[110]

tion, secondary pathology, and premature death. Neuron 2003;40(6):1087– 93. Edbauer D, Willem M, Lammich S, Steiner H, Haass C. Insulin-degrading enzyme rapidly removes the ␤-amyloid precursor protein intracellular domain (AICD). J Biol Chem 2002;277:13389–21339. Venugopal C, Pappolla MA, Sambamurti K. Insulysin cleaves the APP Cytoplasmic Fragment at multiple sites. Neurochem Res 2007;32:2225–34. Farris Mansourian S, Chang Y, Lindsley L, Eckman EA, Frosch MP, Eckman CB, et al. Insulin-degrading enzyme regulates the levels of insulin, amyloid betaprotein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci USA 2003;100(7):4162–416. Lee MS, Kao SC, Lemere CA, Xia W, Tseng HC, Zhou Y, et al. APP processing is regulated by cytoplasmic phosphorylation. J Cell Biol 2003;163(1):83–95.

317

[111] Russo C, Dolcini V, Salis S, Venezia V, Violani E, Carlo P, et al. Signal transduction through tyrosine-phosphorylated carboxy-terminal fragments of APP via an enhanced interaction with Shc/Grb2 adaptor proteins in reactive astrocytes of Alzheimer’s disease brain. Ann N Y Acad Sci 2002;973:323– 33. [112] Takahashi K, Niidome T, Akaike A, Kihara T, Sugimoto H. Phosphorylation of amyloid precursor protein (APP) at Tyr687 regulates APP processing by alphaand gamma-secretase. Biochem Biophys Res Commun 2008;377(2):544–9. [113] Ghosal K, Vogt DL, Liang M, Shen Y, Lamb BT, Pimplikar SW. Alzheimer’s disease-like pathological features in transgenic mice expressing the APP intracellular domain. PNAS 2009;106(43):18367–72. [114] Oddo S. The ubiquitin–proteasome system in Alzheimer’s disease. J Cell Mol Med 2008;12:363–73.