Elzbieta Kojro

Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the α-secretase ADAM 10

Biochemical, epidemiological, and genetic findings demonstrate a link between cholesterol levels, processing of the amyloid precursor protein (APP), and Alzheimers disease. In the present report, we identify the α-secretase ADAM 10 (a disintegrin and metalloprotease) as a major target of the cholesterol effects on APP metabolism. Treatment of various peripheral and neural cell lines with either the cholesterol-extracting agent methyl-β-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin resulted in a drastic increase of secreted α-secretase cleaved soluble APP. This strong stimulatory effect was in the range obtained with phorbol esters and was further increased in cells overexpressing ADAM 10. In cells overexpressing APP, the increase of α-secretase activity resulted in a decreased secretion of Aβ peptides. Several mechanisms were elucidated as being the basis of enhanced α-secretase activity: increased membrane fluidity and impaired internalization of APP were responsible for the effect observed with methyl-β-cyclodextrin; treatment with lovastatin resulted in higher expression of the α-secretase ADAM 10. Our results demonstrate that cholesterol reduction promotes the nonamyloidogenic α-secretase pathway and the formation of neuroprotective α-secretase cleaved soluble APP by several mechanisms and suggest approaches to prevention of or therapy for Alzheimers disease.

A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model

Alzheimer disease (AD) is characterized by excessive deposition of amyloid beta-peptides (A beta peptides) in the brain. In the nonamyloidogenic pathway, the amyloid precursor protein (APP) is cleaved by the alpha-secretase within the A beta peptide sequence. Proteinases of the ADAM family (adisintegrin and metalloproteinase) are the main candidates as physiologically relevant alpha-secretases, but early lethality of knockout animals prevented a detailed analysis in neuronal cells. To overcome this restriction, we have generated transgenic mice that overexpress either ADAM10 or a catalytically inactive ADAM10 mutant. In this report we show that a moderate neuronal overexpression of ADAM10 in mice transgenic for human APP([V717I]) increased the secretion of the neurotrophic soluble alpha-secretase-released N-terminal APP domain (APPs alpha), reduced the formation of A beta peptides, and prevented their deposition in plaques. Functionally, impaired long-term potentiation and cognitive deficits were alleviated. Expression of mutant catalytically inactive ADAM10 led to an enhancement of the number and size of amyloid plaques in the brains of double-transgenic mice. The results provide the first in vivo evidence for a proteinase of the ADAM family as an alpha-secretase of APP, reveal activation of ADAM10 as a promising therapeutic target, and support the hypothesis that a decrease in alpha-secretase activity contributes to the development of AD.

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 γ-secretase inhibitor, suggesting that RAGE-CTF is normally further processed by γ-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.

The Non-Amyloidogenic Pathway: Structure and Function of α-Secretases

The amyloid cascade hypothesis is the most accepted explanation for the pathogenesis of Alzheimers disease (AD). APP is the precursor of the amyloid beta peptide (Abeta), the principal proteinaceous component of amyloid plaques in brains of Alzheimers disease patients. Proteolytic cleavage of APP by the alpha-secretase within the Abeta sequence precludes formation of amyloidogenic peptides and leads to a release of soluble APPsalpha which has neuroprotective properties. In several studies, a decreased amount of APPsalpha in the cerebrospinal fluid of AD patients has been observed. Three members of the ADAM family (a disintegrin and metalloproteinase) ADAM-10, ADAM-17 (TACE) and ADAM-9 have been proposed as alpha-secretases. We review the evidence for each of these enzymes acting as a physiologically relevant alpha-secretase. In particular, we focus on ADAM-10, which recently was shown in a transgenic mouse model for AD, to act as an alpha-secretase in vivo. We also discuss the pharmacological up-regulation of alpha-secretases as a possible therapeutic treatment for AD.

The neuropeptide PACAP promotes the α-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 ?‐secretase cleavage of the amyloid precursor protein (APP) and the production of secreted APP (APPs?). Stimulation of endogenously expressed PAC1 receptors with PACAP in human neuroblastoma cells increased APPs? secretion, which was completely inhibited by the PAC1 receptor specific antagonist PACAP‐(6–38). In HEK cells, stably overexpressing the functional PAC1 receptors PACAP‐27 and PACAP‐38 strongly stimulated ?‐secretase cleavage of APP. The PACAP‐induced APPs? production was dose dependent and saturable. This increase of ?‐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 ?‐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

Separate agonist and peptide antagonist binding sites of the oxytocin receptor defined by their transfer into the V2 vasopressin receptor.

The neurohypophyseal nonapeptide oxytocin (OT) is the main hormone responsible for the initiation of labor; uterus contraction can be enhanced by application of oxytocin or suppressed by oxytocin antagonists. By transfer of domains from the G protein-coupled OT receptor into the related V2 vasopressin receptor, chimeric “gain in function” V2/OT receptors were produced that were able to bind either OT receptor agonists or a competitive peptide antagonist with high affinity. The binding site for the OT antagonist d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr9]vasotocin was found to be formed by transmembrane helices 1, 2, and 7 with a major contribution to binding affinity by the upper part of helix 7. These transmembrane receptor regions could be excluded from participating in OT binding. For agonist binding and selectivity the first three extracellular receptor domains were most important. The interaction of the N-terminal domain and of the first extracellular loop of the OT receptor with the linear C-terminal tripeptidic part of oxytocin was demonstrated. Furthermore, the second extracellular loop of the OT receptor could be identified to interact with the cyclic hormone part. These three domains contribute to OT binding by synergistic interaction with oxytocin but not with the competitive antagonist. Our results provide evidence for the existence of separate domains and different conformations of a peptide hormone receptor involved in binding and selectivity for agonists and peptide antagonists.

Cloning and nucleotide sequence of the structural genes encoding the formate dehydrogenase of Wolinella succinogenes

The formate dehydrogenase of Wolinella succinogenes is a membraneous molybdo-enzyme which is involved in phosphorylative electron transport. The gene (fdhA) encoding the largest subunit was isolated from a gene bank by immunological screening. The fdhA gene was located in an apparent transcriptional unit (fdh A, B, C. D) together with three more structural genes. The N-terminal sequences of three polypeptides present in the isolated enzyme were found to map within the fdhA, B and C structural genes. A polypeptide corresponding to fdhD was not detected in the enzyme preparation. This suggested that the functional formate dehydrogenase was made up of three or four different subunits.The genes fdhA and C encode larger preproteins which differ from the corresponding mature proteins by N-terminal signal peptides. The N-terminal half of the mature FdhA is homologous to the larger subunits of the formate dehydrogenases of E. coli (formate-hydrogenlyase linked) and Methanobacterium formicicum as well as to three bacterial reductases containing molybdenum. It harbours a conserved cysteine cluster and two more domains which may be involved in binding the molybdenum cofactor. FdhB may represent an iron-sulphur protein, twelve cysteine residues of which are arranged in two clusters which are typical of ligands of the iron-sulfur centers in ferredoxins. FdhC is a hydrophobic protein with four predicted transmembrane segments, which appears to be identical with the cytochrome b present in the isolated formate dehydrogenase. It may form the membrane anchor of the enzyme and react with the bacterial menaquinone.

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 Alzheimers 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 β‐peptide (Aβ) transporter receptor for advanced glycation end products (RAGE) mRNA level. PACAP, by activation of the somatostatin‐neprilysin cascade, also enhanced expression of the Aβ‐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.—Rat, D., Schmitt, U., Tippmann, F., Dewachter, I., Theunis, C., Wieczerzak, E, Postina, R., van Leuven, F., Fahrenholz, F., Kojro, E. Neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP) slows down Alzheimers disease‐like pathology in amyloid precursor protein‐transgenic mice. FASEB J. 25, 3208‐3218 (2011). www.fasebj.org

α‐Secretase Activity of the Disintegrin Metalloprotease ADAM 10: Influences of Domain Structure

Abstract: Disintegrin metalloproteases from different organisms form the ADAM (adisintegrin and metalloprotease) family. All members display a common domain organization and possess four potential functions: proteolysis, cell adhesion, cell fusion, and cell signaling. Members of the ADAM family are responsible for the proteolytic cleavage of transmembrane proteins and release of their extracellular domain. The proteolytic process is referred to as ectodomain shedding, which is activated by phorbol esters and inhibited by hydroxamic acid‐based inhibitors. We have shown that the disintegrin metalloprotease ADAM 10 has both constitutive and regulated α‐secretase activity. Expression of a dominant negative mutant of ADAM 10 in HEK cells decreases the secretion of APPsα. In order to investigate the influence of distinct protein domains of ADAM 10 on α‐secretase activity, several deletion mutants of ADAM 10 were constructed. Our findings demonstrate that the deletion of the disintegrin domain results in a mutant ADAM 10 with remaining α‐secretase activity, whereas the deletion of the prodomain destroys the proteolytic activity of ADAM 10.

Influence of ADAM10 on prion protein processing and scrapie infectiosity in vivo

Both the cellular prion protein (PrP(c)) and the amyloid precursor protein (APP) are physiologically subjected to complex proteolytic processing events. While for APP the proteinases involved--alpha-, beta- and gamma-secretase--have been identified in vitro and in vivo, the cleavage of PrP(c) by now has been linked only to the shedding activity of the metalloproteinase ADAM10 and/or ADAM17 in cell culture. Here we show that neuronal overexpression of the alpha-secretase ADAM10 in mice reduces all PrP(c) species detected in the brain instead of leading to enhanced amounts of specific cleavage products of PrP(c). Additionally, the incubation time of mice after scrapie infection is significantly increased in mice moderately overexpressing ADAM10. This indicates that overexpression of ADAM10 rather influences the amount of the cellular prion protein than its processing in vivo.