Crosstalk between angiotensin and the nonamyloidogenic pathway of Alzheimer's amyloid precursor protein.

The association between hypertension and an increased risk for Alzheimer's disease (AD) and dementia is well established. Many data suggest that modulation of the renin-angiotensin system may be meaningful for the prevention and therapy of neurodegenerative disorders, in particular AD. Proteolytic cleavage of the amyloid precursor protein (APP) by α-secretase precludes formation of neurotoxic Aß peptides and is expected to counteract the development of AD. An established approach for the up-regulation of α-secretase cleavage is the activation of G protein-coupled receptors (GPCRs). Therefore, our study aimed to analyze whether stimulation of angiotensin AT1 or AT2 receptors stably expressed in HEK cells influence the nonamyloidogenic pathway of APP processing. Treatment of both receptors with angiotensin II clearly showed that only activation of the AT1 receptor increased several fold the α-secretase-mediated shedding of APP. This effect was completely abolished by treatment with the AT1 receptor-specific antagonist telmisartan. Using the BIM-46187 inhibitor, we demonstrate that the Gαq protein-mediated pathway is involved in this stimulation process. Stimulation of AT1 receptors with the ß-arrestin-biased agonist SII was ineffective regarding α-secretase-mediated APP shedding. This result discloses that only the G protein-dependent pathway is involved in the Ang II-induced APP shedding. Blocking of Gßγ subunits by the inhibitor gallein completely prevented constitutive and Ang II-induced APP shedding. Our findings provide evidence that induction of APP shedding via Ang II/AT1 receptor stimulation is effected by G protein activation with Gßγ subunits playing important roles.

Dissecting the role of ADAM10 as a mediator of Staphylococcus aureus α-toxin action.

Staphylococcus aureus is a leading cause of bacterial infections in humans, including life-threatening diseases such as pneumonia and sepsis. Its small membrane-pore-forming α-toxin is considered an important virulence factor. By destroying cell-cell contacts through cleavage of cadherins, the metalloproteinase ADAM10 (a disintegrin and metalloproteinase 10) critically contributes to α-toxin-dependent pathology of experimental S. aureus infections in mice. Moreover, ADAM10 was proposed to be a receptor for α-toxin. However, it is unclear whether the catalytic activity or specific domains of ADAM10 are involved in mediating binding and/or subsequent cytotoxicity of α-toxin. Also, it is not known how α-toxin triggers ADAM10's enzymatic activity, and whether ADAM10 is invariably required for all α-toxin action on cells. In the present study, we show that efficient cleavage of the ADAM10 substrate epithelial cadherin (E-cadherin) requires supra-cytotoxic concentrations of α-toxin, leading to significant increases in intracellular [Ca(2+)]; the fall in cellular ATP levels, typically following membrane perforation, became observable at far lower concentrations. Surprisingly, ADAM10 was dispensable for α-toxin-dependent xenophagic targeting of S. aureus, whereas a role for α-toxin attack on the plasma membrane was confirmed. The catalytic site of ADAM10, furin cleavage site, cysteine switch and intracellular domain of ADAM10 were not required for α-toxin binding and subsequent cytotoxicity. In contrast, an essential role for the disintegrin domain and the prodomain emerged. Thus, co-expression of the prodomain with prodomain-deficient ADAM10 reconstituted binding of α-toxin and susceptibility of ADAM10-deficient cells. The results of the present study may help to inform structural analyses of α-toxin-ADAM10 interactions and to design novel strategies to counteract S. aureus α-toxin action.

Amphiphilic Copolymers Shuttle Drugs Across the Blood-Brain Barrier.

Medical treatment of diseases of the central nervous system requires transport of drugs across the blood-brain barrier (BBB). Here, it is extended previously in vitro experiments with a model compound to show that the non-water-soluble and brain-impermeable drug domperidone (DOM) itself can be enriched in the brain by use of an amphiphilic copolymer as a carrier. This carrier consists of poly(N-(2-hydroxypropyl)-methacrylamide), statistically copolymerized with 10 mol% hydrophobic lauryl methacrylate, into whose micellar aggregates DOM is noncovalently absorbed. As tested in a BBB model efficient transport of DOM across, the BBB is achievable over a wide range of formulations, containing 0.8 to 35.5 wt% domperidone per copolymer. In neither case, the polymer itself is translocated across the BBB model. In vivo experiments in mice show that already 10 min after intraperitoneal injection of the polymer/domperidone (PolyDOM) formulation, domperidone can be detected in blood and in the brain. Highest serum and brain levels of domperidone are detected 40 min after injection. At that time point serum domperidone is increased 48-fold. Most importantly, domperidone is exclusively detectable in high amounts in the brain of PolyDOM injected mice and not in mice injected with bare domperidone.

Melatonin stimulates the nonamyloidogenic processing of ßAPP through the positive transcriptional regulation of ADAM10 and ADAM17.

Melatonin controls many physiological functions including regulation of the circadian rhythm and clearance of free radicals and neuroprotection. Importantly, melatonin levels strongly decrease as we age and patients with Alzheimer's disease (AD) display lower melatonin than age-matched controls. Several studies have reported that melatonin can reduce aggregation and toxicity of amyloid-ß peptides that are produced from the ß-amyloid precursor protein (ßAPP). However, whether melatonin can directly regulate the ßAPP-cleaving proteases ('secretases') has not been investigated so far. In this study, we establish that melatonin stimulates the α-secretase cleavage of ßAPP in cultured neuronal and non-neuronal cells. This effect is fully reversed by ADAM10- and ADAM17-specific inhibitors and requires both plasma membrane-located melatonin receptor activation, and ERK1/2 phosphorylation. Moreover, we demonstrate that melatonin upregulates both ADAM10 and ADAM17 catalytic activities and endogenous protein levels. Importantly, genetic depletion of one or the other protease in mouse embryonic fibroblasts prevents melatonin stimulating constitutive and PKC-regulated sAPPα secretion and ADAM10/ADAM17 catalytic activities. Furthermore, we show that melatonin induces ADAM10 and ADAM17 promoter transactivation, and we identify the targeted promoter regions. Finally, we correlate melatonin-dependent sAPPα production with a protection against staurosporine-induced apoptosis. Altogether, our results provide the first demonstration that melatonin upregulates the nonamyloidogenic ADAM10 and ADAM17 proteases through melatonin receptor activation, ERK phosphorylation and the transactivation of some specific regions of their promoters and further underline the preventive rather than curative nature of melatonin regarding AD treatment.

Unfolded protein response signaling by transcription factor XBP-1 regulates ADAM10 and is affected in Alzheimer's disease.

In Alzheimer's disease (AD), disturbed homeostasis of the proteases competing for amyloid precursor protein processing has been reported: a disintegrin and metalloproteinase 10 (ADAM10), the physiological α-secretase, is decreased in favor of the amyloid-ß-generating enzyme BACE-1. To identify transcription factors that modulate the expression of either protease, we performed a screening approach: 48 transcription factors significantly interfered with ADAM10/BACE-1-promoter activity. One selective inducer of ADAM10 gene expression is the X-box binding protein-1 (XBP-1). This protein regulates the unfolded protein-response pathway. We demonstrate that particularly the spliced XBP-1 variant dose dependently regulates ADAM10 expression, which can be synergistically enhanced by 100 nM insulin. Analysis of 2 different transgenic mouse models (APP/PS1 and 5xFAD) revealed that at early time points in pathology XBP-1 metabolism is induced. This is accompanied by a 2-fold augmented ADAM10 amount as compared with nontransgenic littermates (P=0.011). Along with aging of the mice, the system is counterregulated, and XBP-1 together with ADAM10 expression level decreased to ∼50% as compared with control animals. Analyses of expression levels in human AD brains showed that ADAM10 mRNA correlated with active XBP-1 (r=0.3120), but expression did not reach levels of healthy age-matched controls, suggesting deregulation of XBP-1 signaling. Our results demonstrate that XBP-1 is a driver of ADAM10 gene expression and that disturbance of this pathway might contribute to development or progression of AD.

Statins stimulate the production of a soluble form of the receptor for advanced glycation end products.

The beneficial effects of statin therapy in the reduction of cardiovascular pathogenesis, atherosclerosis, and diabetic complications are well known. The receptor for advanced glycation end products (RAGE) plays an important role in the progression of these diseases. In contrast, soluble forms of RAGE act as decoys for RAGE ligands and may prevent the development of RAGE-mediated disorders. Soluble forms of RAGE are either produced by alternative splicing [endogenous secretory RAGE (esRAGE)] or by proteolytic shedding mediated by metalloproteinases [shed RAGE (sRAGE)]. Therefore we analyzed whether statins influence the production of soluble RAGE. Lovastatin treatment of either mouse alveolar epithelial cells endogenously expressing RAGE or HEK cells overexpressing RAGE caused induction of RAGE shedding, but did not influence secretion of esRAGE from HEK cells overexpressing esRAGE. Lovastatin-induced secretion of sRAGE was also evident after restoration of the isoprenylation pathway, demonstrating a correlation of sterol biosynthesis and activation of RAGE shedding. Lovastatin-stimulated induction of RAGE shedding was completely abolished by a metalloproteinase ADAM10 inhibitor. We also demonstrate that statins stimulate RAGE shedding at low physiologically relevant concentrations. Our results show that statins, due to their cholesterol-lowering effects, increase the soluble RAGE level by inducing RAGE shedding, and by doing this, might prevent the development of RAGE-mediated pathogenesis.

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin ß and ADAM10.

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and ß we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin ß through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin ß, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

Synthesis of maleimide-functionalyzed HPMA-copolymers and in vitro characterization of the aRAGE- and human immunoglobulin (huIgG)-polymer conjugates.

Herein the synthesis of antibody-polymer conjugates, with a quite narrow dispersity based on the polymer HPMA, are reported. These conjugates are synthesized by coupling antibodies to maleimide-functionalized poly(N-(2-hydroxypropyl)-methacrylamide) (poly-HPMA) copolymers derived through reversible addition-fragmentation chain transfer (RAFT) polymerization of pentafluorophenyl methacrylate via the intermediate step of an activated ester polymer. We develop a protocol that allows the attachment of two different model antibodies, monoclonal anti-RAGE (receptor for advanced glycation end-products) antibody, and polyclonal human immunoglobulin (huIgG). Modification of the antibody and conjugation is monitored by SDS-PAGE electrophoresis. Preserved affinity is demonstrated by Western Blott and cell-uptake analysis, for example, to cells of the immune system.

Amphiphilic HPMA-LMA copolymers increase the transport of Rhodamine 123 across a BBB model without harming its barrier integrity.

The successful non-invasive treatment of diseases associated with the central nervous system (CNS) is generally limited by poor brain permeability of various developed drugs. The blood-brain barrier (BBB) prevents the passage of therapeutics to their site of action. Polymeric drug delivery systems are promising solutions to effectively transport drugs into the brain. We recently showed that amphiphilic random copolymers based on the hydrophilic p(N-(2-hydroxypropyl)-methacrylamide), pHPMA, possessing randomly distributed hydrophobic p(laurylmethacrylate), pLMA, are able to mediate delivery of domperidone into the brain of mice in vivo. To gain further insight into structure-property relations, a library of carefully designed polymers based on p(HPMA) and p(LMA) was synthesized and tested applying an in vitro BBB model which consisted of human brain microvascular endothelial cells (HBMEC). Our model drug Rhodamine 123 (Rh123) exhibits, like domperidone, a low brain permeability since both substances are recognized by efflux transporters at the BBB. Transport studies investigating the impact of the polymer architecture in relation to the content of hydrophobic LMA revealed that random p(HPMA)-co-p(LMA) having 10mol% LMA is the most auspicious system. The copolymer significantly increased the permeability of Rh123 across the HBMEC monolayer whereas transcytosis of the polymer was very low. Further investigations on the mechanism of transport showed that integrity and barrier function of the BBB model were not harmed by the polymer. According to our results, p(HPMA)-co-p(LMA) copolymers are a promising delivery system for neurological therapeutics and their application might open alternative treatment strategies.

Induction of RAGE shedding by activation of G protein-coupled receptors.

The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimers disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cß, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cß, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca(2+) signaling, PKCα/PKCßI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNA-mediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount of soluble RAGE in the mouse lung. Our findings suggest that pharmacological stimulation of RAGE shedding might open alternative treatment strategies for Alzheimers disease and diabetes-induced inflammation.

Activation of α-secretase cleavage.

Alpha-secretase-mediated cleavage of the amyloid precursor protein (APP) releases the neuroprotective APP fragment sαAPP and prevents amyloid ß peptide (Aß) generation. Moreover, α-secretase-like cleavage of the Aß transporter 'receptor for advanced glycation end products' counteracts the import of blood Aß into the brain. Assuming that Aß is responsible for the development of Alzheimer's disease (AD), activation of α-secretase should be preventive. α-Secretase-mediated APP cleavage can be activated via several G protein-coupled receptors and receptor tyrosine kinases. Protein kinase C, mitogen-activated protein kinases, phosphatidylinositol 3-kinase, cAMP and calcium are activators of receptor-induced α-secretase cleavage. Selective targeting of receptor subtypes expressed in brain regions affected by AD appears reasonable. Therefore, the PACAP receptor PAC1 and possibly the serotonin 5-HT(6) receptor subtype are promising targets. Activation of APP α-secretase cleavage also occurs upon blockade of cholesterol synthesis by statins or zaragozic acid A. Under physiological statin concentrations, the brain cholesterol content is not influenced. Statins likely inhibit Aß production in the blood by α-secretase activation which is possibly sufficient to inhibit AD development. A disintegrin and metalloproteinase 10 (ADAM10) acts as α-secretase on APP. By targeting the nuclear retinoic acid receptor ß, the expression of ADAM10 and non-amyloidogenic APP processing can be enhanced. Excessive activation of ADAM10 should be avoided because ADAM10 and also ADAM17 are not APP-specific. Both ADAM proteins cleave various substrates, and therefore have been associated with tumorigenesis and tumor progression.

Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) slows down Alzheimer's disease-like pathology in amyloid precursor protein-transgenic mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has neuroprotective and neurotrophic properties and is a potent α-secretase activator. As PACAP peptides and their specific receptor PAC1 are localized in central nervous system areas affected by Alzheimer's disease (AD), this study aims to examine the role of the natural peptide PACAP as a valuable approach in AD therapy. We investigated the effect of PACAP in the brain of an AD transgenic mouse model. The long-term intranasal daily PACAP application stimulated the nonamyloidogenic processing of amyloid precursor protein (APP) and increased expression of the brain-derived neurotrophic factor and of the antiapoptotic Bcl-2 protein. In addition, it caused a strong reduction of the amyloid ß-peptide (Aß) transporter receptor for advanced glycation end products (RAGE) mRNA level. PACAP, by activation of the somatostatin-neprilysin cascade, also enhanced expression of the Aß-degrading enzyme neprilysin in the mouse brain. Furthermore, daily PAC1-receptor activation via PACAP resulted in an increased mRNA level of both the PAC1 receptor and its ligand PACAP. Our behavioral studies showed that long-term PACAP treatment of APP[V717I]-transgenic mice improved cognitive function in animals. Thus, nasal application of PACAP was effective, and our results indicate that PACAP could be of therapeutic value in treating AD.

Statins and the squalene synthase inhibitor zaragozic acid stimulate the non-amyloidogenic pathway of amyloid-beta protein precursor processing by suppression of cholesterol synthesis.

Cholesterol-lowering drugs such as statins influence the proteolytic processing of the amyloid-beta protein precursor (AbetaPP) and are reported to stimulate the activity of alpha-secretase, the major preventive secretase of Alzheimer's disease. Statins can increase the alpha-secretase activity by their cholesterol-lowering properties as well as by impairment of isoprenoids synthesis. In the present study, we elucidate the contribution of these pathways in alpha-secretase activation. We demonstrate that zaragozic acid, a potent inhibitor of squalene synthase which blocks cholesterol synthesis but allows synthesis of isoprenoids, also stimulates alpha-secretase activity. Treatment of human neuroblastoma cells with 50 microM zaragozic acid resulted in a approximately 3 fold increase of alpha-secretase activity and reduced cellular cholesterol by approximately 30%. These effects were comparable to results obtained from cells treated with a low lovastatin concentration (2 microM). Zaragozic acid-stimulated secretion of alpha-secretase-cleaved soluble AbetaPP was dose dependent and saturable. Lovastatin- or zaragozic acid-stimulated increase of alpha-secretase activity was completely abolished by a selective ADAM10 inhibitor. By targeting the alpha-secretase ADAM10 to lipid raft domains via a glycosylphosphatidylinositol anchor, we demonstrate that ADAM10 is unable to cleave AbetaPP in a cholesterol-rich environment. Our results indicate that inhibition of cholesterol biosynthesis by a low lovastatin concentration is sufficient for alpha-secretase activation.

Ectodomain shedding of the receptor for advanced glycation end products: a novel therapeutic target for Alzheimer's disease.

Receptor for advanced glycation end products (RAGE) mediates diverse physiological and pathological effects and is involved in the pathogenesis of Alzheimer's disease (AD). RAGE is a receptor for amyloid beta peptides (Ab), mediates Abeta neurotoxicity and also promotes Abeta influx into the brain and contributes to Abeta aggregation. Soluble RAGE (sRAGE), a secreted RAGE isoform, acts as a decoy receptor to antagonize RAGE-mediated damages. Accumulating evidence has suggested that sRAGE represents a promising pharmaceutic against RAGE-mediated disorders. Recent studies revealed proteolysis of RAGE as a previously unappreciated means of sRAGE production. In this review we summarize these findings on the proteolytic cleavage of RAGE and discuss the underlying regulatory mechanisms of RAGE shedding. Furthermore, we propose a model in which proteolysis of RAGE could restrain AD development by reducing Abeta transport intothe brain and Abeta production via BACE. Thus, the modulation of RAGE proteolysis provides a novel intervention strategy for AD.

Regulated proteolysis of RAGE and AbetaPP as possible link between type 2 diabetes mellitus and Alzheimer's disease.

Epidemiological studies have linked type 2 diabetes mellitus (T2DM) with an increased risk of developing Alzheimer's disease (AD). In T2DM, the elevated blood glucose level promotes formation of advanced glycation end products (AGEs). The receptor for AGEs (RAGE) is a type I membrane-protein and is also able to import amyloid-beta (Abeta) from the blood across the blood-brain-barrier into the brain. Oligomeric Abeta peptides disturb synaptic function in the brain and are believed to contribute to the development of AD. Abeta peptides are released from the amyloid-beta protein precursor (AbetaPP) after sequential proteolysis by beta- and gamma-secretases but alpha-secretase-mediated cleavage of AbetaPP prevents Abeta generation. Insulin influences Abeta production by modulating alpha-secretase activity and Abeta degradation. Recent publications demonstrate that RAGE is subjected to protein ectodomain shedding. Proteolysis of RAGE occurs constitutively and is inducible by activation of protein kinase C. Alpha-secretase-like enzymes release the ligand binding domain of RAGE from the cell surface and after that gamma-secretase processes the membrane-remaining part of RAGE. Proteolysis of RAGE may represent a regulatory mechanism in RAGE signal transduction and in addition may prevent Abeta peptide transport across the blood-brain-barrier. Current data suggest that the sequential proteolysis of RAGE is homologous to AbetaPP processing.

Differential gene expression in ADAM10 and mutant ADAM10 transgenic mice.

BACKGROUND: In a transgenic mouse model of Alzheimer disease (AD), cleavage of the amyloid precursor protein (APP) by the alpha-secretase ADAM10 prevented amyloid plaque formation, and alleviated cognitive deficits. Furthermore, ADAM10 overexpression increased the cortical synaptogenesis. These results suggest that upregulation of ADAM10 in the brain has beneficial effects on AD pathology. RESULTS: To assess the influence of ADAM10 on the gene expression profile in the brain, we performed a microarray analysis using RNA isolated from brains of five months old mice overexpressing either the alpha-secretase ADAM10, or a dominant-negative mutant (dn) of this enzyme. As compared to non-transgenic wild-type mice, in ADAM10 transgenic mice 355 genes, and in dnADAM10 mice 143 genes were found to be differentially expressed. A higher number of genes was differentially regulated in double-transgenic mouse strains additionally expressing the human APP[V717I] mutant.Overexpression of proteolytically active ADAM10 affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. Although ADAM10 has been implicated in Notch and beta-catenin signaling, no significant changes in the respective target genes were observed in adult ADAM10 transgenic mice.Real-time RT-PCR confirmed a downregulation of genes coding for the inflammation-associated proteins S100a8 and S100a9 induced by moderate ADAM10 overexpression. Overexpression of the dominant-negative form dnADAM10 led to a significant increase in the expression of the fatty acid-binding protein Fabp7, which also has been found in higher amounts in brains of Down syndrome patients. CONCLUSION: In general, there was only a moderate alteration of gene expression in ADAM10 overexpressing mice. Genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. Even a decrease of inflammation markers was observed. These results are further supportive for the strategy to treat AD by increasing the alpha-secretase activity.

Receptor for advanced glycation end products is subjected to protein ectodomain shedding by metalloproteinases.

The receptor for advanced glycation end products (RAGE) is a 55-kDa type I membrane glycoprotein of the immunoglobulin superfamily. Ligand-induced up-regulation of RAGE is involved in various pathophysiological processes, including late diabetic complications and Alzheimer disease. Application of recombinant soluble RAGE has been shown to block RAGE-mediated pathophysiological conditions. After expression of full-length RAGE in HEK cells we identified a 48-kDa soluble RAGE form (sRAGE) in the culture medium. This variant of RAGE is smaller than a 51-kDa soluble version derived from alternative splicing. The release of sRAGE can be induced by the phorbol ester PMA and the calcium ionophore calcimycin via calcium-dependent protein kinase C subtypes. Hydroxamic acid-based metalloproteinase inhibitors block the release of sRAGE, and by RNA interference experiments we identified ADAM10 and MMP9 to be involved in RAGE shedding. In protein biotinylation experiments we show that membrane-anchored full-length RAGE is the precursor of sRAGE and that sRAGE is efficiently released from the cell surface. We identified cleavage of RAGE to occur close to the cell membrane. Ectodomain shedding of RAGE simultaneously generates sRAGE and a membrane-anchored C-terminal RAGE fragment (RAGE-CTF). The amount of RAGE-CTF increases when RAGE-expressing cells are treated with a gamma-secretase inhibitor, suggesting that RAGE-CTF is normally further processed by gamma-secretase. Identification of these novel mechanisms involved in regulating the availability of cell surface-located RAGE and its soluble ectodomain may influence further research in RAGE-mediated processes in cell biology and pathophysiology.

A closer look at alpha-secretase.

Accumulation of amyloid beta-peptides (Abeta) in the brain is believed to contribute to the development of Alzheimer disease (AD). Abeta, a 40-42 amino acid-comprising proteolytical fragment of the amyloid precursor protein (APP), is released from APP by sequential cleavages via beta- and gamma-secretases. However, the predominant route of APP processing consists of successive cleavages by alpha- and gamma-secretases. Alpha-secretase attacks APP inside the Abeta sequence, and therefore prevents formation of neurotoxic Abeta. After cleavage by alpha-secretase, the soluble N-terminal domain of APP, which possesses neurotrophic and neuroprotective properties, is released. In AD patients, a decrease in alpha-secretase processing of APP has been found and therefore, strategies to improve alpha-secretase activity are obvious. Several years after descriptive reports on alpha-secretase, the responsible enzymes have been identified to belong to the family of A Disintegrin And Metalloproteinase (ADAM). Three of these membrane-anchored zinc-dependent metalloproteinases, ADAM10 as well as ADAM17 and presumably also ADAM9 display alpha-secretase activity. Since the individual knock-out of these proteinases in neither case completely prevented alpha-secretase processing of APP, it seems likely that different ADAMs are compensating mutually, and under different conditions may contribute to alpha-secretase cleavage of APP. In addition to ADAMs, perhaps other membrane-associated metalloproteinases contribute to the shedding of APP. Stimulation of alpha-secretase activities can be achieved via several signaling cascades including phospholipase C, phosphatidylinositol 3-kinase and serine/threonine-specific kinases such as protein kinases C, and mitogen activated protein kinases. Direct activation of protein kinase C and stimulation of distinct G protein-coupled receptors are known to increase alpha-secretase processing of APP. Agonists for M1 muscarinic acetylcholine receptors and serotonin 5-HT4 receptors are currently in clinical trials to test their efficiency in the treatment of AD.

Alpha-secretase activation--an approach to Alzheimer's disease therapy.

The nonamyloidogenic pathway of processing the amyloid precursor protein (APP) involves the cleavage within the amyloid-beta peptide sequence, and thus precludes amyloid-beta formation. The identification of a member of the disintegrin and metalloproteinase family, ADAM10, as an alpha-secretase that prevents plaque formation and hippocampal deficits in vivo gave us the possibility to examine the alpha-secretase as a potential target for the therapy of Alzheimer's disease. Within the priority program Cellular Mechanisms of Alzheimer's Disease, we investigated several approaches to stimulate the alpha-secretase pathway. Two protein convertases were found to be responsible for the removal of the prodomain, and for the formation of the mature enzyme with alpha-secretase activity. The cloning and characterization of the human ADAM10 promoter provided the basis to examine ADAM10 gene expression. We found a common upregulation of ADAM10, APP, and APP-like protein 2 during differentiation of neuronal cells by retinoic acid, and increased alpha-secretase cleavage of the two substrates. Other approaches for enhancing alpha-secretase activity are the reduction of cellular cholesterol and the stimulation of G protein-coupled neuropeptide receptors. Our results suggest medications and dietary regiments which enhance the nonamyloidogenic pathway of APP processing to be a valuable approach to Alzheimer's disease therapy.

The neuropeptide PACAP promotes the alpha-secretase pathway for processing the Alzheimer amyloid precursor protein.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has neurotrophic as well as anti-apoptotic properties and is involved in learning and memory processes. Its specific G protein-coupled receptor PAC1 is expressed in several central nervous system (CNS) regions, including the hippocampal formation. Here we examined the effect of PAC1 receptor activation on alpha-secretase cleavage of the amyloid precursor protein (APP) and the production of secreted APP (APPsalpha). Stimulation of endogenously expressed PAC1 receptors with PACAP in human neuroblastoma cells increased APPsalpha secretion, which was completely inhibited by the PAC1 receptor specific antagonist PACAP-(6-38). In HEK cells stably overexpressing functional PAC1 receptors, PACAP-27 and PACAP-38 strongly stimulated alpha-secretase cleavage of APP. The PACAP-induced APPsalpha production was dose dependent and saturable. This increase of alpha-secretase activity was completely abolished by hydroxamate-based metalloproteinase inhibitors, including a preferential ADAM 10 inhibitor. By using several specific protein kinase inhibitors, we show that the MAP-kinase pathway [including extracellular-regulated kinase (ERK) 1 and ERK2] and phosphatidylinositol 3-kinase mediate the PACAP-induced alpha-secretase activation. Our findings provide evidence for a role of the neuropeptide PACAP in stimulation of the nonamyloidogenic pathway, which might be related to its neuroprotective properties.