Falk Fahrenholz

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.

Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles.

Here, to study lipid–protein interactions that contribute to the biogenesis of regulated secretory vesicles, we have developed new approaches by which to label proteins in vivo, using photoactivatable cholesterol and glycerophospholipids. We identify synaptophysin as a major specifically cholesterol-binding protein in PC12 cells and brain synaptic vesicles. Limited cholesterol depletion, which has little effect on total endocytic activity, blocks the biogenesis of synaptic-like microvesicles (SLMVs) from the plasma membrane. We propose that specific interactions between cholesterol and SLMV membrane proteins, such as synaptophysin, contribute to both the segregation of SLMV membrane constituents from plasma-membrane constituents, and the induction of synaptic-vesicle curvature.

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 receptor function by cholesterol.

Abstract. Cholesterol influences many of the biophysical properties of membranes and is nonrandomly distributed between cellular organelles, subdomains of membranes, and leaflets of the membrane bilayer. In combination with the high dynamics of cholesterol distribution, this offers many possibilities for regulation of membrane-embedded receptors. Depending on the receptor, cholesterol can have a strong influence on the affinity state, on the binding capacity, and on signal transduction. Most important, cholesterol may stabilize receptors in defined conformations related to their biological functions. This may occur by direct molecular interaction between cholesterol and receptors. In this review, we discuss the functional dependence of the nicotinic acetylcholine receptor as well as different G protein-coupled receptors on the presence of cholesterol.

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.

Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs

Collagen XVII, a type II transmembrane protein and epithelial adhesion molecule, can be proteolytically shed from the cell surface to generate a soluble collagen. Here we investigated the release of the ectodomain and identified the enzymes involved. After surface biotinylation of keratinocytes, the ectodomain was detectable in the medium within minutes and remained stable for >48 h. Shedding was enhanced by phorbol esters and inhibited by metalloprotease inhibitors, including hydroxamates and TIMP‐3, but not by inhibitors of other protease classes or by TIMP‐2. This profile implicated MMPs or ADAMs as candidate sheddases. MMP‐2, MMP‐9 and MT1‐MMP were excluded, but TACE, ADAM‐10 and ADAM‐9 were shown to be expressed in keratinocytes and to be actively involved. Transfection with cDNAs for the three ADAMs resulted in increased shedding and, vice versa, in TACE‐deficient cells shedding was significantly reduced, indicating that transmembrane collagen XVII represents a novel class of substrates for ADAMs. Functionally, release of the ectodomain of collagen XVII from the cell surface was associated with altered keratinocyte motility in vitro.

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.

Up-regulation of the α-secretase ADAM10 by retinoic acid receptors and acitretin

Late‐onset Alzheimers disease is often connected with nutritional misbalance, such as enhanced cholesterol intake, deficiency in polyunsaturated fatty acids, or hypovitaminosis. The α‐secretase ADAM10 has been found to be regulated by retinoic acid, the bioreactive metabolite of vitamin A. Here we show that retinoids induce gene expression of ADAM10 and α‐secretase activity by nonpermissive retinoid acid receptor/retinoid X receptor (RAR/RXR) heterodimers, whereby α‐and β‐isotypes of RAR play a major role. However, ligands of other RXR binding partners, such as the vitamin D receptor, do not stimulate β‐secretase activity. On the basis of these findings, we examined the effect of synthetic retinoids and found a strong enhancement of nonamyloidogenic processing of the amyloid precursor protein by the vitamin A analog acitretin: it stimulated ADAM10 promoter activity with an EC50 of 1.5 μ.M and led to an increase of mature ADAM10 protein that resulted in a two‐to three‐fold increase of the ratio between α‐and β‐secretase activity in neuroblastoma cells. The α‐secretase stimulation by acitretin was completely inhibited by the ADAM10‐specific inhibitor GI254023X. Intracerebral injection of acitretin in APP/PS1‐21 transgenic mice led to a reduction of Aβ40 and Aβ42. The results of this study may have clinical relevance because acitretin has been approved for the treatment of psoriasis since 1997 and found generally safe for long‐term use in humans.—Tippmann, F.,Hundt, J., Schneider, A., Endres, K., Fahrenholz, F. Up‐regulation of the α‐secretase ADAM10 by retinoic acid receptors and acitretin. FASEB J. 23, 1643–1654 (2009)