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Keywords:

  • Alzheimer’s disease;
  • ADAM proteases;
  • ectodomain shedding;
  • metalloproteases;
  • regulated intramembrane proteolysis;
  • sirtuins

Abstract

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

J. Neurochem. (2011) 116, 10–21.

Abstract

Ectodomain shedding of the amyloid precursor protein (APP) by the metalloprotease activity α-secretase is a key regulatory event preventing the generation of the Alzheimer’s disease (AD) amyloid β peptide. Proteases similar to α-secretase are essential for diverse physiological processes, such as embryonic development, cell adhesion and neuronal guidance. Previously, several proteases were suggested as candidate α-secretases for APP, in particular members of the ADAM family (a disintegrin and metalloprotease). Two recent studies analyzed primary neurons, which are the cell type affected in AD, and finally demonstrated that the constitutively cleaving α-secretase activity is selectively mediated by ADAM10. An increase in α-secretase cleavage is considered a therapeutic approach for AD. However, the molecular mechanisms regulating α-secretase cleavage remain only partly understood. Signaling pathways activating protein kinase C and MAP kinase play a central role in stimulating α-secretase cleavage of APP. Additionally, several recent publications demonstrate that ADAM10 expression and α-secretase cleavage of APP are tightly controlled at the level of transcription, e.g. by retinoic acid receptors and sirtuins, and at the level of translation and protein trafficking. This review focuses on the recent progress made in unraveling the molecular identity, regulation and therapeutic potential of α-secretase in Alzheimer’s disease.

Abbreviations used:
AD

Alzheimer’s disease

ADAM

a disintegrin and metalloprotease

APP

amyloid precursor protein

BACE

β-site APP cleaving enzyme

MMP

matrix metalloprotease

MT

membrane type

RAR

retinoic acid receptors

SAP97

synapse-associated protein-97

SIRT1

sirtuin 1

SweAPP

Swedish mutant form of APP

TSPAN12

tetraspanin 12

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder and affects more than 30 million patients worldwide. A neuropathological hallmark of the disease are the amyloid plaques, which are extracellular protein deposits found in the brain of AD patients. The major constituent of the plaques is the ∼4 kDa amyloid β peptide (Aβ), which are considered the culprit of the disease (Hardy and Selkoe 2002). Aβ is a 38–42 amino acid long peptide, which is hydrophobic and prone to aggregation. Small Aβ oligomers are neurotoxic and trigger a pathological cascade of events, including inflammation, inhibition of hippocampal long-term potentiation, synaptic dysfunction, neurofibrillary tangles, neuronal loss, the clinical onset of the disease and ultimately death (reviewed in Hardy and Selkoe 2002; Haass and Selkoe 2007). As the generation of Aβ is at the beginning of the cascade, the mechanisms of Aβ generation and Aβ prevention are the target of intensive research both in academia and in pharmaceutical companies.

Aβ is a proteolytic fragment of the ubiquitously expressed type I membrane protein amyloid precursor protein (APP). The biological function of APP is not yet clear, but may involve a role in cell adhesion or as a signaling receptor (reviewed in Jacobsen and Iverfeldt 2009). Formation of Aβ requires APP to be cleaved by the two proteases β- and γ-secretase (Fig. 1). β- and γ-secretase were identified ten years ago and are both membrane proteins. β-Secretase is the aspartyl protease β-site APP cleaving enzyme (BACE)1 (reviewed in Rossner et al. 2006; Cole and Vassar 2008) and cleaves APP at the N-terminus of the Aβ domain. This leads to the secretion (or shedding) of the large APP ectodomain (APPsβ) and the generation of a C-terminal APP fragment of 99 amino acids (C99). C99 is further cleaved by γ-secretase, which is a hetero-tetrameric protease complex consisting of the four subunits presenilin, nicastrin, Aph-1 (anterior pharynx defective) and Pen-2 (presenilin enhancer) (reviewed in Steiner et al. 2008). As a result of this cleavage, Aβ is secreted and the APP intracellular domain is released into the cytosol. A potential transcriptional role of APP intracellular domain is controversially discussed (Cao and Sudhof 2001, 2004; Hebert et al. 2006).

image

Figure 1.  Proteolytic processing of APP by α-, β- and γ-secretase. The type I membrane protein APP is proteolytically cleaved in two competing pathways. Sequential APP cleavage by β- and γ-secretase is referred to as the amyloidogenic pathway and generates Aβ. β-Secretase cleavage occurs within the ectodomain of APP close to the transmembrane domain, resulting in the shedding of the soluble APP ectodomain (APPsβ) and the formation of the membrane-bound C-terminal fragment C99 (C-terminal 99 amino acids of APP). γ-Secretase cleavage of C99 leads to Aβ secretion and the formation of the APP intracellular domain (AICD). In the alternative, non-amyloidogenic pathway, APP is first cleaved by the metalloprotease α-secretase. This cleavage yields the soluble APP ectodomain APPsα and a C-terminal fragment (C83), which is further processed by γ-secretase, leading to the secreted p3-peptide and AICD.

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β- and γ-secretase are obvious drug targets for AD, but the development of clinically suitable drugs targeting both proteases has been challenging. Nevertheless, the first inhibitors and modulators are being tested in clinical trials. A third protease, α-secretase, cleaves APP within the Aβ domain and is assumed to preclude Aβ generation (Fig. 1). In cell lines, APP is mainly cleaved by α-secretase and only to a lower extent by β-secretase, whereas the opposite is found in primary neurons, where β-secretase expression is particularly high (Simons et al. 1996). α-secretase generates a secreted form of APP (APPsα), which has been reported to have neurotrophic and neuroprotective properties (Furukawa et al. 1996; Meziane et al. 1998; Stein et al. 2004). In contrast, the slightly shorter form generated by β-secretase (APPsβ) seems to have a proapoptotic function (Nikolaev et al. 2009). The second product of α-secretase cleavage is the C-terminal APP fragment (C83). C83 is further cleaved by γ-secretase, giving rise to the p3 peptide, which lacks the amino-terminal 16 amino acids of Aβ. p3 is assumed to be benign and is not found in the compact amyloid plaques (reviewed in Dulin et al. 2008). α- and β-secretase appear to compete for the initial cleavage of APP, but have opposite effects on Aβ generation. Thus, an increase in α-secretase cleavage is considered as a therapeutic approach for AD (Fahrenholz 2007). However, the molecular mechanisms regulating α-secretase cleavage remain only partly understood. Additionally, different α-secretase candidates were suggested, but until recently the molecular identity of α-secretase was not fully resolved. Several recent publications shed new light on the regulation of α-secretase cleavage and finally clarified the identity of α-secretase, in particular in neurons. The focus of this review is the recent progress in our understanding of the molecular identity, regulation and therapeutic potential of α-secretase.

Molecular identity of the constitutive α-secretase

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

Exactly 20 years ago, Esch et al. (1990) described for the first time that APP is cleaved within the Aβ sequence by a then unknown protease, which was named secretase and which is now known as α-secretase. The initial protease cleavage occurs between amino acids lysine16 und leucine17 of the Aβ sequence (Fig. 1) and appears to be followed by an as yet unidentified carboxypeptidase cleavage selectively removing lysine16 (Esch et al. 1990).

APP α-secretase cleavage occurs constitutively (constitutive α-secretase) and can additionally be stimulated above its constitutive level by a heterogeneous group of molecules (reviewed in Bandyopadhyay et al. 2007). This stimulation was first shown for the activation of muscarinic acetylcholine receptors (Nitsch et al. 1992) and is generally referred to as the regulated α-secretase cleavage.

α-Secretase was shown to have characteristics of a metalloprotease (Roberts et al. 1994) and different metalloproteases were suggested as potential constitutive α-secretases, because they cleave APP-derived synthetic peptides in vitro and because their over-expression increased APP cleavage. The most frequently studied candidate α-secretases are members of the ADAM (a disintegrin and metalloprotease) family, in particular ADAM9, ADAM10 and ADAM17 (Koike et al. 1999; Lammich et al. 1999; Slack et al. 2001). ADAM10 shows coordinated expression with APP in human brain, which is less seen for ADAM17 (Marcinkiewicz and Seidah 2000). Additionally, ADAM19 was suggested as α-secretase. However, ADAM19 does not cleave an APP derived peptide in vitro, suggesting that it may indirectly modulate α-secretase cleavage (Tanabe et al. 2007). ADAM proteases are type I membrane proteins of the metzincin family and require a zinc ion for proteolytic activity (reviewed in Edwards et al. 2008). In contrast to the protease over-expression, the knock-down or knock-out of the candidate α-secretases gave less clear results about whether all or only one of them mediates APP α-secretase cleavage at endogenous expression conditions. RNAi-mediated knock-down of ADAM9, ADAM10 or ADAM17 reduced constitutive APP shedding by 20 to 60% (Asai et al. 2003; Allinson et al. 2004; Camden et al. 2005; Freese et al. 2009; Taylor et al. 2009). Moreover, cells derived from ADAM9, 10- or 17-deficient mice showed a largely unaltered level of APP shedding (Buxbaum et al. 1998; Hartmann et al. 2002; Weskamp et al. 2002). Only in a subset of ADAM10-deficient fibroblasts, APP α-secretase cleavage was altered to a variable degree (Hartmann et al. 2002). The cause of this variation is not yet known. Importantly, APP processing was analyzed in primary neurons derived from ADAM9 deficient-mice, but not from ADAM10 or ADAM17 knock-out mice. ADAM9-deficient neurons showed no change in the levels of Aβ or p3, but APPsα levels were not analyzed (Weskamp et al. 2002).

The finding that APP shedding was never fully suppressed has led to the conclusion that ADAM9, 10 and 17 may together constitute α-secretase activity and that in the absence of one of them the other proteases can still mediate APP α-secretase cleavage. This assumption is in clear contrast to other ADAM protease substrates, many of which are predominantly cleaved by a single ADAM protease, such as ephrins, N-cadherin and members of the epidermal growth factor family (Hattori et al. 2000; Janes et al. 2005; Reiss et al. 2005; Le Gall et al. 2009).

Given the inconsistencies regarding the identity of α-secretase, two very recent studies used novel reagents, primary neurons and conditional knock-out mice to resolve the identity of α-secretase (Jorissen et al. 2010; Kuhn et al. 2010). Both studies come to the same conclusion and report that ADAM10, but not ADAM9 or ADAM17, is the constitutive α-secretase in primary neurons, and thus in those cells which are mostly affected in AD. One of the studies systematically knocked-down expression of ADAM9, 10 or 17 by RNA interference and evaluated the effect on α-secretase cleavage of the endogenous APP in two different cell lines as well as in primary murine neurons (Kuhn et al. 2010). Using a new APPsα-specific antibody, the protease ADAM10, but not ADAM9 or 17, was found to be essential for α-secretase cleavage. Moreover, ADAM9 and ADAM17 were not able to compensate for ADAM10 in the constitutive α-secretase cleavage. The requirement for ADAM10 was further validated by mass spectrometric determination of APP cleavage products. The other study used conditional ADAM10 knock-out mice (Jorissen et al. 2010). In primary neurons prepared from these mice at E14.5, APPsα production was nearly completely abolished, again demonstrating that ADAM10 is required for constitutive APPsα cleavage in neurons.

At present, it remains unclear why earlier work reported that embryonic fibroblasts from ADAM10 knock-out mice (not the conditional knock-out) showed either no or a variable degree of reduction in α-secretase activity (Hartmann et al. 2002). Perhaps certain cells, such as mouse embryonic fibroblasts have the ability to partially compensate for the loss of ADAM10, while this was clearly not seen in primary neurons and several cell lines investigated (Jorissen et al. 2010; Kuhn et al. 2010).

One aspect for future studies is to identify whether knock-down or knock-out of ADAM10 induces an APPsα loss-of-function phenotype. The function of APPsα is not yet fully established. APPsα is sufficient to rescue the phenotype of APP-deficient mice (Ring et al. 2007) and has neuroprotective and neurotrophic effects (Furukawa et al. 1996; Meziane et al. 1998; Stein et al. 2004). Possibly, ADAM10-deficient neurons are more vulnerable to stress conditions or show an altered neurite length due to the absence of APPsα. However, a potential loss of APPsα function may be counterbalanced by other neuronal ADAM10 substrates, which may also affect the cellular stress response or neurite outgrowth. In fact, a previous study modulated neuronal ADAM10 activity in an in vivo model of acute excitotoxic stress (Clement et al. 2008). Under some conditions, ADAM10 activity provided a neurotrophic effect, whereas under other conditions it enhanced neurotoxicity or had no effect. Future studies are needed to resolve a potential loss of APPsα function in ADAM10-deficient mice.

Cell biology of ADAM10

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

ADAM10 is a type I membrane protein of about 750 amino acids. It has a large ectodomain, a transmembrane and a proline-rich cytoplasmic domain. The ectodomain comprises several distinct functional domains. It starts at the N-terminus with the signal peptide and the prodomain, which is followed by the zinc-binding metalloprotease domain with the conserved zinc-binding amino acid motif HEXGHXXGXXHD and a structural motif called the methionine turn in the active site helix. Further domains are a disintegrin domain and a cysteine-rich domain, whereas an epidermal growth factor-like domain, which is found in most ADAM proteases, is missing in ADAM10 (Janes et al. 2005). A more detailed description of the ADAM protease domains, their structure and specific functions can be found in recent reviews (Edwards et al. 2008; Reiss and Saftig 2009). During transport through the secretory pathway, ADAM10 is complex N-glycosylated (Escrevente et al. 2008). The prodomain of ADAM10 is removed by the proprotein convertases furin and PC7 (Anders et al. 2001; Lopez-Perez et al. 1999, 2001), resulting in the active protease, which mediates proteolysis in the late compartments of the secretory pathway and at the plasma membrane. Interestingly, ADAM10 also undergoes ectodomain shedding by ADAM9 and 15, which is followed by γ-secretase intramembrane proteolysis and translocation of the ADAM10 ICD into the nucleus, where it is found in nuclear speckles, thought to be involved in gene regulation (Parkin and Harris 2009; Tousseyn et al. 2009). Thus, ADAM10 may function as a signaling protein in addition to its role as protease.

Additional functions of ADAM10 beyond α-secretase cleavage of APP

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

α-Secretase cleavage of APP is one example for a more general cellular process, called ectodomain shedding or simply shedding. This proteolytic process converts an increasing number of single-span membrane proteins to their soluble counterparts and is an important way of regulating the biological activity of membrane proteins (Pruessmeyer and Ludwig 2009; Reiss and Saftig 2009). Similar to APP, other membrane proteins are shed within their ectodomain close to the transmembrane domain. Although for most protein substrates the corresponding shedding enzyme (sheddase) has not yet been identified, metalloproteases of the ADAM and matrix metalloprotease (MMP) family as well as the aspartyl proteases BACE1 and BACE2 are considered as predominant sheddases. Among these enzymes, ADAM10 has been particularly well studied and sheds over 30 different membrane proteins besides APP, such as Notch, betacellulin, klotho and N-cadherin (Pruessmeyer and Ludwig 2009; Reiss and Saftig 2009). More substrates are likely to be identified in the future. Importantly, ADAM protease-mediated shedding occurs for substrates on the same cell surface, but can also happen in trans, when ADAM10 resides on one cell surface and cleaves a substrate residing on the neighboring cell surface as shown for ephrin-Eph receptor signaling (Janes et al. 2005).

ADAM10 knock-out mice die at embryonic day E9.5 and show a typical loss-of-function phenotype of Notch signaling (Hartmann et al. 2002). This demonstrates that Notch, which is a cell surface receptor involved in cell differentiation, is a major ADAM10 substrate during development. Additional changes, which may result from the lack of cleavage of other ADAM10 substrates, have not yet been investigated in detail. Ligand-binding induces Notch shedding, which is followed by γ-secretase-mediated intramembrane proteolysis. This releases the Notch intracellular domain into the nucleus, where it acts as a transcriptional activator of Notch target genes, such as hairy and enhancer of split. Despite the clear Notch loss-of-function phenotype of ADAM10 knock-out mice, both ADAM10 and ADAM17 were suggested as the Notch sheddases. However, similar to the constitutive APP α-secretase cleavage, recent work established ADAM10 as the relevant protease for the physiological ligand-induced Notch1 shedding and signaling. Under certain ligand-independent conditions, including leukemia-linked Notch1 mutations, Notch1 cleavage can also be mediated by ADAM17 (Cagavi Bozkulak and Weinmaster 2009; van Tetering et al. 2009). Thus, similar to the constitutive and regulated α-secretase cleavage of APP, Notch can be cleaved in a ligand-dependent and -independent manner requiring different proteases.

Given that ADAM10 knock-out mice die early during embryonic development, efforts are being made to generate conditional ADAM10 knock-out mice. These should elucidate functions of ADAM10 later during development or in adulthood or in specific tissues. In fact, Notch-related signaling defects were also reported in two recent studies. An ADAM10 knock-out specifically in B cells demonstrated an essential function of ADAM10 in B-cell development, by initiating Notch2 signaling (Gibb et al. 2010). Another study suppressed ADAM10 expression in neural progenitor cells after day E9, resulting in perinatal lethality (Jorissen et al. 2010). The mice showed a disrupted neocortex because of premature neuronal differentiation resulting from a loss of Notch signaling. This demonstrates that ADAM10 is essential for the establishment of the brain cortex. It will be interesting to see whether Notch remains a major ADAM10 substrate even later in adulthood. Alternatively, a more complex phenotype may be observed, given the large number of ADAM10 substrates with different biological functions.

Taken together, ADAM10 is a major sheddase for membrane proteins and is involved in different physiological and pathophysiological processes, such as embryonic development, cell adhesion, signal transduction, the immune system, cancer and AD (Edwards et al. 2008; Reiss and Saftig 2009).

Identity of regulated α-secretase

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

A heterogeneous group of molecules can stimulate APP α-secretase shedding above its constitutive, ADAM10-mediated level (Bandyopadhyay et al. 2007). This increased cleavage is referred to as regulated α-secretase cleavage. For most stimuli, it has not yet been determined, which metalloprotease they activate. In fact, different metalloproteases, including ADAM9, ADAM10 and ADAM17 may contribute to the regulated α-secretase cleavage. For example, the neuropeptide pituitary adenylate cyclase-activating polypeptide stimulates the α-secretase cleavage of APP through ADAM10 (Kojro et al. 2006), suggesting that ADAM10 is not only the constitutive α-secretase, but also contributes to the regulated α-secretase activity. In contrast, the phorbol ester phorbol myristate acetate (PMA) requires ADAM17 to increase α-secretase cleavage (Buxbaum et al. 1998), and this is not dependent on ADAM10 (Kuhn et al. 2010). Because PMA does not naturally occur in the body, future studies need to address whether ADAM17 cleavage of APP also occurs under physiologically or pathophysiologically relevant conditions other than upon treatment with the synthetic phorbol ester PMA. In fact, one class of compounds activating ADAM17 may be M1 muscarinic agonists, such as AF267B, which increased α-secretase cleavage of APP as well as ADAM17 expression in an AD mouse model (Caccamo et al. 2006). This suggests that ADAM17 acts as the regulated α-secretase upon muscarinic stimulation, although the final proof is missing that the increased ADAM17 expression was responsible for the increased α-secretase cleavage.

Additional metalloproteases of the ADAM or MMP family may also contribute to regulated α-secretase cleavage. Based on over-expression studies, candidates for the regulated α-secretase are MMP9, membrane type-1 matrix metalloprotease (MT1-MMP), MT3-MMP, MT5-MMP and ADAM8 (Amour et al. 2002; Higashi and Miyazaki 2003; Ahmad et al. 2006; Naus et al. 2006; Talamagas et al. 2007; Vaisar et al. 2009). Several of these proteases do not cleave APP at the site of constitutive cleavage (Lys16-Leu17), but at multiple sites in the ectodomain (MT-MMPs). One site is the His14-Gln15 peptide bond within the Aβ domain, such that an increased expression or activity of these proteases can reduce Aβ generation and enhance α-secretase cleavage, leading to a slightly truncated APPsα. Knock-down or knock-out studies are required to understand the physiological relevance of these proteases as α-secretases. Interestingly, MMP9 is able to degrade Aβin vitro by cleaving at the α-secretase site (Lys16-Leu17) (Backstrom et al. 1996) and MMP9-deficient mice show elevated Aβ levels (Yin et al. 2006). Thus, even if MMP9 does not produce APPsα from full-length APP under physiological conditions, it might still be beneficial for AD by degrading Aβ.

Taken together, the identity of the regulated α-secretase remains to be fully clarified and may be mediated by different proteases. At least ADAM10 and ADAM17 can act as regulated α-secretases after specific stimuli, such as pituitary adenylate cyclase-activating polypeptide and PMA. Future studies analyzing the activation of APP α-secretase cleavage should always test which metalloprotease finally mediates the increased cleavage. This will allow us to obtain a more comprehensive picture of the physiologically and therapeutically relevant regulated α-secretase(s).

Competition between α- and β-secretase cleavage

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

In most studies where α-secretase cleavage was stimulated above its constitutive level (regulated α-secretase), the increased APPsα generation was accompanied by a reduction of both β-secretase cleavage and Aβ generation. This was shown by genetic means (e.g. over-expression of ADAM10) (Postina et al. 2004) and pharmacological approaches (e.g. muscarinic activation) (Bandyopadhyay et al. 2007) and has led to the concept that α- and β-secretase compete for APP as a substrate. As such, an activation of α-secretase may be a therapeutic approach for AD. The molecular mechanisms underlying the competition between α- and β-secretase are not yet fully understood, but may involve changes in the cellular compartments, where the cleavage typically takes place. α-Secretase cleavage occurs at the plasma membrane (Sisodia 1992), but also in the trans-Golgi network, at least upon stimulation with PMA (Skovronsky et al. 2000). In contrast, β-secretase cleavage of wild-type APP occurs mainly in the endosome and to a lower extent in the trans-Golgi network (Koo and Squazzo 1994; Vassar et al. 1999). Competition between α- and β-secretase cleavage is seen for example upon over-expression of the β-secretase BACE1. This strongly increased β-secretase cleavage and reduced α-secretase cleavage (Vassar et al. 1999; Kuhn et al. 2010), probably because over-expressed BACE1 artificially cleaves APP in early compartments of the secretory pathway, before APP reaches the plasma membrane and has access to α-secretase. A second condition with competition between α- and β-secretase cleavage, is the Swedish mutant form of APP (SweAPP), which is linked to an inherited form of AD. Compared with wild-type APP the SweAPP is more efficiently cleaved by β-secretase and is processed to more Aβ and less APPsα, presumably because SweAPP is already cleaved by β-secretase in the trans-Golgi network, before encountering α-secretase (Haass et al. 1995). Another example is the phorbol ester PMA, which increased APPsα and reduced APPsβ and Aβ in APP-transfected CHO cells (Skovronsky et al. 2000). The authors argued that PMA shifts APP α-secretase cleavage away from the plasma membrane towards the Golgi/trans-Golgi network, such that APP is cleaved earlier in the secretory pathway and less APP is available for β-secretase cleavage. It remains to be seen whether such a relocalization of α-secretase cleavage is also required for other activators of α-secretase cleavage.

In contrast to the regulated α-secretase cleavage, it is not yet clear whether a competition between α- and β-secretase cleavage is also seen under constitutive cleavage conditions by both proteases. The two recent studies analyzing the knock-down or knock-out of ADAM10 on APP α-secretase cleavage also investigated the consequence of the ADAM10 deficiency on β-secretase cleavage and Aβ generation, but come to different conclusions. The knock-down of ADAM10 in primary neurons mildly increased β-secretase cleavage and Aβ generation (Kuhn et al. 2010), which is in agreement with a previous study showing that transgenic expression of a dominant-negative ADAM10 mutant increased Aβ levels in mice (Postina et al. 2004). These findings indicate that there is a competition between α- and β-secretase for APP as a substrate. In contrast, the study using the conditional ADAM10 knock-out mice reported that the ADAM10 deficiency did not only reduce APPsα levels, but also APPsβ and Aβ (Jorissen et al. 2010). The reason for the discrepancy between these studies is not yet clear. Because the conditional ADAM10 knock-out induced premature neuronal differentiation and defects in neuronal migration, it will be important to analyze neurons where the ADAM10 knock-out starts at a later time point, when differentiation of neurons is complete. Ideally, these would be adult neurons. Interestingly, no competition between constitutive α- and β-secretase cleavage was observed in cell lines (HEK293, CHO and neuroblastoma SH-SY5Y) (Kim et al. 2008; Kuhn et al. 2010), raising the possibility that the competition between both proteases under constitutive cleavage conditions depends on the cell type. This may potentially be due to the fact that expression of the β-secretase BACE1 is high in neurons, but much lower in cell lines.

Even though it is not yet clear whether a knock-out or knock-down of ADAM10 increases Aβ levels, there is evidence that a reduction of α-secretase cleavage may contribute to AD pathogenesis, potentially by increasing Aβ or even by reducing the neuroprotective APPsα or by both mechanisms. Loss-of-function mutations in the prodomain of ADAM10 have been discovered in several families with late-onset forms of AD (Kim et al. 2009). These mutations reduce APPsα and increase Aβ. Additionally, some, but not all studies analyzing APPsα levels in CSF reported a reduction of APPsα in AD versus controls (Sennvik et al. 2000; Olsson et al. 2003; Fellgiebel et al. 2009). A similar result was found in a study, which additionaly reported reduced ADAM10 levels in platelets and reduced APPsα secretion from platelets of AD patients (Colciaghi et al. 2002). A more thorough investigation of these findings in larger patient cohorts is required, also for the purpose of testing APPsα as a potential biomarker for AD.

Although a competition between α- and β-secretase is not yet clear for the constitutive α-secretase cleavage, it is well established that the regulated component of α-secretase can compete with β-secretase and thereby reduces Aβ generation, in agreement with the idea that a pharmacological activation of α-secretase may be a therapeutic approach to AD (Fahrenholz 2007).

Regulation of α-secretase cleavage

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

Like many other cellular processes, the α-secretase cleavage of APP is tightly regulated. Despite good progress in the recent past, we are still lacking the complete picture of the cellular control mechanisms for α-secretase cleavage and as a consequence for Aβ generation. α-Secretase cleavage is controlled at the transcriptional, translational and post-translational level (Fig. 2). Here, only selected recent examples will be discussed in detail.

image

Figure 2.  Cellular control of APP α-secretase cleavage. α-Secretase cleavage can be controlled by different mechanisms, including transcription, translation, signaling, trafficking and others. Red bars indicate an inhibition of α-secretase cleavage. Green arrows indicate an activation through ADAM10, whereas black arrows symbolize an activation, where it is not yet clear, through which protease the increased α-secretase cleavage is mediated. Different ligands, such as growth factors, neurotransmitters, cytokines, muscarinic agonists, pituitary adenylate cyclase-activating polypeptide (PACAP) and the drug etazolate bind their respective receptors (e.g. G-protein coupled receptors or receptor tyrosine kinases). This stimulates different signaling pathways, most notably the MAPK, PI-3K and protein kinase C (PKC) pathways, or leads to calcium release. These pathways in turn increase α-secretase cleavage through yet to be identified mechanisms (question mark). The effect of PMA requires ADAM17, whereas PACAP peptides require ADAM10 to increase α-secretase cleavage. Transcription of ADAM10 is positively controlled by retinoic acid receptors (RAR), which are activated by their ligand retinoic acid (RA) or by the drug acitretin or through deacetylation by sirtuin 1 (SIRT1). Translation of ADAM10 is blocked by elements in the 5′ untranslated region (5′UTR) of its mRNA. Trafficking of α-secretase is promoted by nardilysin, tetraspanin 12 (TSPAN12) and SAP97. In these cases, an increase in α-secretase cleavage of APP is observed. APP endocytosis reduces α-secretase cleavage and enhances β-secretase cleavage in endosomes. Cholesterol reduces α-secretase cleavage. Tissue inhibitor of metalloproteases 3 (TIMP3) is a natural inhibitor of ADAM10 and ADAM17 and blocks α-secretase cleavage. Details and references are given in the text.

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At the transcriptional level, there appears to be a tight regulation of ADAM10 expression. The ADAM10 promoter contains several transcription factor binding-sites, including a retinoic acid-responsive element, where retinoic acid receptors (RAR) and retinoic X receptors bind (Prinzen et al. 2005; Tippmann et al. 2009). Accordingly, retinoic acid and other vitamin A derivatives increase ADAM10 expression, resulting in enhanced APP α-secretase cleavage and a reduction of Aβ generation. One of the vitamin A derivatives is acitretin, which is in clinical use for treatment of psoriasis and may thus be a promising drug candidate to be tested in a clinical trial for AD (Tippmann et al. 2009). A very recent study confirmed the retinoic acid inducible ADAM10 expression and extended it by identifying sirtuin 1 (SIRT1) as an upstream element in this signaling pathway (Donmez et al. 2010). Sirtuins are deacetylases which show antiaging properties in animal models and can protect from stress, in particular SIRT1 (Donmez and Guarente 2010). Using SIRT1-transgenic and SIRT1-deficient mice, this protein was found to deacetylate and thereby activate the RARβ transcription factor, which in turn increased ADAM10 expression, activated α-secretase cleavage and lowered Aβ levels (Donmez et al. 2010). As a consequence, amyloid plaque formation as well as several other markers of AD was reduced. Additionally, SIRT1 over-expression was found to increase signaling of the Notch receptor, which is another ADAM10 substrate, as described above. Although, in a therapeutic context, this may sound like an undesired side effect, the authors suggest that the up-regulation of Notch signaling may in fact be beneficial for repairing neuronal damage in AD. This possibility needs to be further investigated in future experiments. The mouse model used in this study expressed the Swedish mutant APP protein (and a mutant presenilin), which undergoes enhanced β-cleavage and reduced α-cleavage compared with wild-type APP. Thus, it will be interesting to see whether the SIRT1 effect on reducing Aβ generation will be as strong when mice are used expressing wild-type APP instead of Swedish APP.

At the translational level, ADAM10 expression is suppressed through mechanisms involving the 5′ untranslated region of the ADAM10 mRNA (Lammich et al. 2010). It remains to be tested whether this constitutes a permanent block of translation. Alternatively, the translation may be modulated by signaling pathways, as was recently demonstrated for a similar translational suppression of the β-secretase BACE1 (Lammich et al. 2004; O’Connor et al. 2008).

At the post-translational level, different mechanisms control α-secretase cleavage (Fig. 2). They can be broadly grouped into three different categories. First, an activation of certain receptors increases α-secretase cleavage. This includes stimulation by growth factors, cytokines and neurotransmitters. Analysis of the underlying signaling pathways revealed that protein kinase C and MAPK constitute two central signaling hubs for the regulation of α-secretase cleavage, but other kinases, such as PI-3 kinase are also involved. These pathways can mediate a rapid increase of α-secretase cleavage, which can be seen as soon as 15 min after activation. Despite a good knowledge of the upstream components of several of these pathways, it still remains unclear through which downstream molecules they finally mediate the increased α-secretase cleavage (Fig. 2). The different signaling pathways have been reviewed elsewhere and are not further discussed here (Bandyopadhyay et al. 2007). Second, mechanisms that control the intracellular trafficking of APP or ADAM10 tightly control the amount of α-secretase cleavage. This group will be discussed in more detail in the next chapter. Third, a heterogeneous group of molecules regulates α-secretase cleavage, including natural inhibitors, such as tissue inhibitor of metalloproteases 3 (Hoe et al. 2007), as well as changes in the membrane cholesterol concentration. α-Secretase cleavage takes place outside of the cholesterol-rich membrane microdomains (lipid rafts), such that an inhibition of cholesterol biosynthesis, for example by statins or the squalene synthase inhibitor zaragozic acid, stimulates α-secretase cleavage (Kojro et al. 2001, 2010).

Protein trafficking controls α-secretase cleavage

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

α-Secretase cleavage is also controlled by intracellular protein trafficking (Fig. 2). This is not surprising, given that ADAM10 and its substrate APP are membrane proteins and must meet at the same membrane for proteolysis to occur. For example, endocytic APP trafficking is a prime modulator of α-secretase cleavage (reviewed in Lichtenthaler 2006). This is explained by the cellular localization of the secretases. α-Secretase cleavage occurs at the plasma membrane, whereas β-secretase cleavage mostly occurs in endosomes. A reduction of APP endocytosis increases APP levels at the cell surface, resulting in enhanced APP cleavage by α-secretase and reduced Aβ levels. Conversely, enlarged endosomes, which are consistent with an increased APP endocytosis and β-secretase cleavage, are associated with early neuropathological changes observed in AD brains (Grbovic et al. 2003; Nixon 2005). Interestingly, a reduction of plasma membrane cholesterol, which increases α-secretase cleavage as described above, also reduces APP endocytosis (Kojro et al. 2001).

Trafficking of ADAM10 is also controlled, but less is known about the underlying mechanisms. A recent study identified tetraspanin 12 (TSPAN12) as a novel interaction partner of ADAM10. TSPAN12 is an integral membrane protein and was found to facilitate α-secretase cleavage of APP (Xu et al. 2009). TSPAN12 predominantly interacted with the mature form of ADAM10 and additionally promoted ADAM10 maturation, suggesting that it increases ADAM10 transport through the secretory pathway to the plasma membrane, where APP α-secretase cleavage takes place. Other tetraspanins were reported to affect the proteolytic activity of γ-secretase and its interaction with substrates, potentially by localizing γ-secretase to tetraspanin-containing membrane microdomains (Wakabayashi et al. 2009). It will be interesting to see whether distinct tetraspanins interact with different ADAM proteases and thereby allow their selective activation.

Another protein implicated in ADAM10 trafficking and APP α-secretase cleavage is nardilysin (N-arginine dibasic convertase) (Hiraoka et al. 2007). Although nardilysin is a metalloprotease, mutational studies demonstrated that the α-secretase enhancing activity did not require the proteolytic activity of nardilysin, suggesting that it acts as a scaffolding or transport protein. Further studies are needed to fully elucidate the exact molecular mechanism of action. As nardilysin binds different ADAM proteases and even the β-secretase BACE1, it does not only affect APP processing but also the shedding of additional membrane proteins (Nishi et al. 2006; Hiraoka et al. 2008; Ohno et al. 2009).

Another example links signaling pathways and changes in ADAM10 trafficking. Short-term activation of the NMDA receptor in primary neurons activates APP α-secretase cleavage. The underlying mechanism involves synapse-associated protein-97 (SAP97). With its SH3 domain, SAP97 binds to the proline-rich sequences in the cytosolic domain of ADAM10, thereby driving the protease to the post-synaptic membrane (Marcello et al. 2007). This results in increased APP α-secretase cleavage. Recent work showed that ADAM10 contains an ER retention signal, suggesting that as yet unidentified proteins are required for ER exit and transport of ADAM10 to the plasma membrane (Marcello et al. 2010). SAP97 was ruled out as a potential binding partner in this case.

Taken together, the recent studies show an increasing number of trafficking and adaptor proteins which control α-secretase cleavage by altering the trafficking of ADAM10. The example of SAP97 mentioned above demonstrates that the trafficking of ADAM10 and, as a consequence, its activity can be regulated by signaling pathways. It is tempting to speculate that also other previously known signaling pathways may activate α-secretase cleavage by promoting ADAM10 trafficking or its access to APP.

Activation of α-secretase cleavage as a therapeutic strategy

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

An activation of α-secretase cleavage may have two distinct beneficial effects for AD. First, it lowers the levels of the neurotoxic and pathogenic Aβ peptide. Second, it increases the amount of the neuroprotective APPsα. Thus, an activation of APP α-secretase cleavage is considered a therapeutic approach for AD (Fahrenholz 2007). Compelling evidence for the approach comes from the over-expression of the α-secretase ADAM10 in an AD mouse model, which reduced Aβ levels. As a consequence, amyloid plaque load and inflammation in the brain were strongly diminished and were accompanied by an improvement in long-term potentiation and cognitive functions (Postina et al. 2004). Importantly, ADAM10 expression was increased less than twofold compared with wild-type mice, revealing that a moderate increase in α-secretase level or activity is sufficient to block the main pathological features in this mouse model. Although ADAM10 over-expression induced mild expression changes in over 300 genes (Prinzen et al. 2009), the signaling of another ADAM10 substrate, the Notch receptor, was not affected, at least in adult mice (Postina et al. 2004). This indicates that a moderate activation of ADAM10 may be therapeutically acceptable (Endres and Fahrenholz 2010). However, a slightly different result was obtained upon transgenic expression of SIRT1 (described above), where ADAM10 levels were also increased by about twofold (Donmez et al. 2010). Under these conditions, Notch signaling was increased. Given that ADAM10 cleaves over 30 different substrates, it will be essential to carefully evaluate the consequences of chronic ADAM10 activation.

The over-expression of other candidate α-secretases was not yet explored in mice, but may have similar beneficial effects with regard to amyloid pathology as ADAM10 over-expression. Thus, pathways increasing their expression or activity should also be tested for their therapeutic potential.

The activation of APP α-secretase cleavage as a therapeutic strategy for AD has been further validated by the development of pharmacological α-secretase activators. In particular, a new generation of agonists of the M1 muscarinic acetylcholine receptor was tested in pre-clinical studies and in AD patients. The M1 receptor is required for α-secretase cleavage, as reported in a recent study using M1 receptor-deficient mice (Davis et al. 2010). The new M1 agonists are more specific and better tolerated than previous muscarinic agonists. One compound (AF267B, also known as NGX267) reduced Aβ and tau protein pathology in the hippocampus and cortex of an AD mouse model (Caccamo et al. 2006). The compound also improved cognitive deficits and increased the expression of ADAM17. AF267B was tested in a clinical phase II trial for AD, but results have not yet been reported. A related compound, AF102B, reduced Aβ levels in the CSF of AD patients (Nitsch et al. 2000), again pointing to the activation of α-secretase as a potential therapeutic strategy to lower Aβ levels.

Another small molecule currently being tested in a phase II clinical study is etazolate (EHT 0202), which selectively modulates the GABAA receptor. In guinea pig brains and in rat cortical neurons etazolate stimulated APPsα release at submicromolar concentrations. Additionally, it protected cortical neurons against Aβ-induced toxicity (Marcade et al. 2008). Interestingly, the compound did not affect Aβ levels and therefore cannot be classified as an Aβ-lowering drug. Its positive effect may predominantly result from the increased production of the neuroprotective APPsα. However, increased secretion of other ADAM10/α-secretase substrates may also contribute, which remains to be tested. Moreover, the exact mechanism of α-secretase activation by etazolate needs to be fully elucidated.

Although a therapeutic activation of α-secretase has not yet been intensively tested, the above studies demonstrate the potential of this approach. It is certain that more signaling pathways, including the SIRT1/RAR pathways of transcriptional ADAM10 activation, will be thoroughly tested for therapeutic suitability. Additionally, more compounds are likely to be tested in future clinical trials and may present a good addition to other therapeutic approaches against AD, such as immunotherapy and inhibitors/modulators of β- and γ-secretase.

Concluding remarks and outlook

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

The enzymatic α-secretase activity was first described 20 years ago. Over the past few years, several candidates were suggested, in particular ADAM9, 10 and 17. Very recent work finally established ADAM10 as the constitutive α-secretase in neurons. Together with other studies on ADAM10 from the past few years, this provides a firm basis for more intensive research on ADAM10 both from a biological and a medical point of view. For example, what other functions does ADAM10 have, in particular in the brain? What are the cellular pathways that control ADAM10 activity and APP α-secretase cleavage? Can they be pharmacologically influenced in the desired direction? Can such compounds be efficiently delivered into the brain? Are they safe, given that ADAM10 influences distinct biological processes, including differentiation, proliferation and cell adhesion? It would be desirable to find ways to selectively activate α-secretase cleavage of APP. Potentially, this may be possible with an as yet unidentified APP-ligand, similar to what is known for Notch, where ligand binding activates Notch receptor shedding by ADAM10. Alternatively, cellular pathways may exist which alter trafficking of APP but not of other substrates, and thus control the access of APP to ADAM10 and thereby α-secretase cleavage. Another topic is to determine the identity of physiologically relevant, regulated α-secretase(s), specifically in the brain. Is it possible to stimulate such proteases in vivo to increase α-secretase cleavage and reduce Aβ generation? An additional aspect to clarify is the contribution of APPsα to AD pathogenesis and therapy. Is it beneficial to increase APPsα without a concomitant decrease of Aβ, as suggested by the results for the drug etazolate? Despite the many open questions about α-secretase, the recent progress on the identity, cellular regulation and therapeutic potential shows that this is an exciting time for α-secretase research. As we learn more about this enzymatic activity, we do not only get further insights into AD pathology and potential therapeutic applications, but also into other cellular processes, where α-secretase like-enzymes are involved.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References

I thank Richard Page, Peer-Hendrik Kuhn and Bastian Dislich for helpful comments on the manuscript, Anna Münch for help with figure preparation and the following funding agencies for support: Deutsche Forschungsgemeinschaft (SFB596, TP B12) and BMBF (KNDD). I apologize to the colleagues that not all previous work on α-secretase could be included in this review on recent developments in the field.

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  1. Top of page
  2. Abstract
  3. Molecular identity of the constitutive α-secretase
  4. Cell biology of ADAM10
  5. Additional functions of ADAM10 beyond α-secretase cleavage of APP
  6. Identity of regulated α-secretase
  7. Competition between α- and β-secretase cleavage
  8. Regulation of α-secretase cleavage
  9. Protein trafficking controls α-secretase cleavage
  10. Activation of α-secretase cleavage as a therapeutic strategy
  11. Concluding remarks and outlook
  12. Acknowledgements
  13. References
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