Rolf Postina

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.

Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins

The L1 adhesion molecule plays an important role in axon guidance and cell migration in the nervous system. L1 is also expressed by many human carcinomas. In addition to cell surface expression, the L1 ectodomain can be released by a metalloproteinase, but the biological function of this process is unknown. Here we demonstrate that membrane-proximal cleavage of L1 can be detected in tumors and in the developing mouse brain. The shedding of L1 involved a disintegrin and metalloproteinase (ADAM)10, as transfection with dominant-negative ADAM10 completely abolishes L1 release. L1-transfected CHO cells (L1-CHO) showed enhanced haptotactic migration on fibronectin and laminin, which was blocked by antibodies to αvβ5 and L1. Migration of L1-CHO cells, but not the basal migration of CHO cells, was blocked by a metalloproteinase inhibitor, indicating a role for L1 shedding in the migration process. CHO and metalloproteinase-inhibited L1-CHO cells were stimulated to migrate by soluble L1-Fc protein. The induction of migration was blocked by αvβ5-specific antibodies and required Arg-Gly-Asp sites in L1. A 150-kD L1 fragment released by plasmin could also stimulate CHO cell migration. We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via αvβ5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.

Regulation of the α-secretase ADAM10 by its prodomain and proprotein convertases

Ectodomain shedding of the Alzheimers amyloid precursor protein is mediated by α‐ and β‐secretases, which, for their part, are also proteolytically processed. The disintegrin metalloproteinase ADAM10 is synthesized as a zymogen with a proprotein convertase (PC) recognition sequence between the prodomain and the catalytic domain. In this study, we investigated the role of the prodomain in the regulation of the α‐secretase activity of ADAM 10. Overexpression of the proprotein convertases PC7 and furin in human embryonic kidney 293 cells revealed an increased ADAM10 maturation resulting in enhanced α‐secretase‐mediated processing of amyloid precursor protein. Mutation of the PC recognition sequence in ADAM10 as well as the use of a PC inhibitor and of the furin‐deficient LoVo cell line confirmed the role of PCs, in particular, of PC7, in ADAM10 maturation and activation. Furthermore, we demonstrated that the prodomain of ADAM10 has a dual function. When coexpressed in trans as separate polypeptide, it inhibited the α‐secretase activity of wild‐type ADAM10. However, the prodomain acted as a chaperone and functionally rescued the α‐secretase activity of a former inactive ADAM10 mutant lacking the prodomain. The results of our study suggest new approaches to enhance the nonamyloidogenic α‐secretase pathway.

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.

Genomic structure and functional characterization of the human ADAM10 promoter

The ADAM10 gene encodes a membrane‐bound disintegrin‐metalloproteinase, which, after overexpression in an Alzheimer disease (AD) mouse model, prevents amyloid pathology and improves long‐term potentiation and memory. Because enhancing ADAM10 expression appears to be a reasonable approach for treatment of AD, we functionally analyzed the ADAM10 gene. Both human and mouse ADAM10 genes comprise ∼160 kbp, are composed of 16 exons, and are evolutionarily highly conserved within 500 bp upstream of either translation initiation site. By using luciferase reporter assays, we demonstrate that nucleotides −2179 to −1 upstream of the human ADAM10 translation initiation site represent a functional TATA‐less promoter. Within this region we identified and examined several single nucleotide polymorphisms, but did not detect significant differences in their appearance between AD and nondemented control subjects. By deletion analysis, site‐directed mutagenesis, transcription factor overexpression and electrophoretic mobility shift assays, we identified nucleotides −508 to −300 as the core promoter and found Sp1, USF, and retinoic acid‐responsive elements to modulate its activity. Finally, we identified vitamin A acid (RA) as an inducer of human ADAM10 promoter activity. This finding suggests that pharmacologic targeting of RA receptors may increase the expression of the α‐secretase ADAM10 with beneficial effects on AD pathology.

The Disintegrin ADAM9 Indirectly Contributes to the Physiological Processing of Cellular Prion by Modulating ADAM10 Activity

The cellular prion protein (PrPc) is physiologically cleaved in the middle of its 106–126 amino acid neurotoxic region at the 110/111↓112 peptidyl bond, yielding an N-terminal fragment referred to as N1. We recently demonstrated that two disintegrins, namely ADAM10 and ADAM17 (TACE, tumor necrosis factor alpha converting enzyme) participated in both constitutive and protein kinase C-regulated generation of N1, respectively. These proteolytic events were strikingly reminiscent of those involved in the so-called “α-secretase pathway” that leads to the production of secreted sAPPα from βAPP. We show here, by transient and stable transfection analyses, that ADAM9 also participates in the constitutive secretion of N1 in HEK293 cells, TSM1 neurons, and mouse fibroblasts. Decreasing endogenous ADAM9 expression by an antisense approach drastically reduces both N1 and sAPPα recoveries. However, we establish that ADAM9 was unable to increase N1 and sAPPα productions after transient transfection in fibroblasts depleted of ADAM10. Accordingly, ADAM9 is unable to cleave a fluorimetric substrate of membrane-bound α-secretase activity in ADAM10-/- fibroblasts. However, we establish that co-expression of ADAM9 and ADAM10 in ADAM10-deficient fibroblasts leads to enhanced membrane-bound and released fluorimetric substrate hydrolyzing activity when compared with that observed after ADAM10 cDNA transfection alone in ADAM10-/- cells. Interestingly, we demonstrate that shedded ADAM10 displays the ability to cleave endogenous PrPc in fibroblasts. Altogether, these data provide evidence that ADAM9 is an important regulator of the physiological processing of PrPc and βAPP but that this enzyme acts indirectly, likely by contributing to the shedding of ADAM10. ADAM9 could therefore represent, besides ADAM10, another potential therapeutic target to enhance the breakdown of the 106–126 and Aβ toxic domains of the prion and βAPP proteins.

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.

Shedding of the amyloid precursor protein-like protein APLP2 by disintegrin-metalloproteinases.

Cleavage of the amyloid precursor protein (APP) within the amyloid‐beta (Aβ) sequence by the α‐secretase prevents the formation of toxic Aβu2003peptides. It has been shown that the disintegrin‐metalloproteinases ADAM10 and TACE (ADAM17) act as α‐secretases and stimulate the generation of a soluble neuroprotective fragment of APP, APPsα. Here we demonstrate that the related APP‐like proteinu20032 (APLP2), which has been shown to be essential for development and survival of mice, is also a substrate for both proteinases. Overexpression of either ADAM10 or TACE in HEK293 cells increased the release of neurotrophic soluble APLP2 severalfold. The strongest inhibition of APLP2 shedding in neuroblastoma cells was observed with an ADAM10‐preferring inhibitor. Transgenic mice with neuron‐specific overexpression of ADAM10 showed significantly increased levels of soluble APLP2 and its C‐terminal fragments. To elucidate a possible regulatory mechanism of APLP2 shedding in the neuronal context, we examined retinoic acid‐induced differentiation of neuroblastoma cells. Retinoic acid treatment of two neuroblastoma cell lines upregulated the expression of both APLP2 and ADAM10, thus leading to an increased release of soluble APLP2.

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