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Study and Identification of ADAM10 as Neuronal α-secretase in relation to Alzheimer’s disease

Final Report Summary - ADAMNEURON (Study and Identification of ADAM10 as Neuronal a-secretase in relation to Alzheimer’s disease)

Project final report

Grant Agreement number: 224847

Project acronym: ADAMNEURON

Project title: Study and Identification of ADAM10 as Neuronal alpha-secretase in relation to Alzheimer's disease

Funding Scheme: FP7-PEOPLE-2007-4-3-IRG

Period covered: from [01.04.2008] to [31.03.2012]

Name of the scientific representative of the project's co-ordinator , Title and Organisation:

Bart De Strooper, M.D. Ph.D.

Research director VIB Center for the Biology of Disease

Arthur Bax and Anna Vanluffelen chair for Alzheimer's Disease research

co-Director of LIND, Leuven institute for neurodegenerative diseases

Tel: +32 - 16 37 31 01

Fax: +32 - 16 37 27 00

E-mail: Bart.DeStrooper@cme.vib-kuleuven.be

Project website7 address: -

4.1 Final publishable summary report
- Executive summary

Worldwide the incidence of Alzheimer's disease (AD) is increasing rapidly, while cures remain unavailable. The cleavage of the amyloid precursor protein (APP) at the beta- and gamma-secretase cleavage site and subsequent formation of Abeta peptides plays an important role in the disease process. APP is also cleaved by alpha-secretase, which cleaves APP in the middle of the Abeta peptide and hence precludes the formation of Abeta. Most therapeutic strategies for AD are currently focused on decreasing Abeta by lowering/modulating the beta- and gamma-secretase activity. Increasing alpha-secretase activity could be an alternative strategy, although one of the main problems is that the exact nature of alpha-secretase remains unknown. The overall goal of this proposal was to determine whether the metalloprotease and major alpha-secretase candidate ADAM10, is indeed responsible for beta-secretase activity in neurons, whether it functions in neuronal survival, and whether growth and neuroprotective factors can induce its expression or activity. ADAM10 is responsible for the shedding of numerous proteins including Cadherins, Ephrins and Notch receptors, which are important during development. Not surprisingly, Adam10-/- mice die at embryonic day 9.5 due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knockout (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPC) and NPC-derived neurons and glial cells. We found that alpha-secretase mediated processing of APP was largely reduced in neurons derived from embryonic cKO mice, demonstrating that ADAM10 represents indeed the most important APP alpha-secretase in brain. Unfortunately, the cKO mice die perinatally. In addition we generated and validated a neuroblastoma screening system that allowed us to screen for factors and downstream signaling molecules influencing the regulation of alpha-secretase. This will aid in elucidating the mechanism of alpha-secretase regulation. In conclusion, our study demonstrated that ADAM10 plays a central role in the constitutive neuronal surface shedding of APP and indicates that complex signaling mechanisms regulate inducible alpha-secretase.
- Project context and objectives

Worldwide, the population of people 65 years and older is expected to double in the next 50 years, and for the first time in history, the elderly will outnumber the children. With age being a major risk factor, neurodegenerative diseases that result in dementia, will become more and more common. Currently, over 12 million people worldwide are suffering from AD, the most common neurodegenerative disease. This number is expected to triple by 2050, and will further increase the socio-economic burden associated with the care of patients. Currently, there are no cures and few treatments for AD. To effectively design cures for this disease, it is key to understand the molecular processes involved in the pathogenesis of AD, as these will help design treatments that can stop, prevent or even revert the disease process.

AD is characterised by increased production of the amyloid-ß (Aß) peptide and its aggregation into oligomers and amyloid plaques, which are believed to cause neurotoxicity and degeneration. Aß is generated by amyloidogenic processing of the larger amyloid precursor protein (APP) via a- and beta-secretase, and produced at low levels in the healthy brain. The majority of APP is cleaved within the Aß domain by alpha-secretase, which precludes the formation of intact Aß peptide and releases the larger ectodomain of APP, called alpha-cleaved soluble APP (sAPPalpha), from the cell surface. In AD, sAPPalpha levels in cerebrospinal fluid are reduced, and temporal cortex brain homogenates show reduced beta-secretase activity.

The exact nature of neuronal beta-secretase remained unknown, and indirect evidence suggested both a constitutive and an inducible activity. The goal of this proposal was to determine whether ADAM10 is responsible for beta-secretase activity in neurons, and to determine the nature and regulation of inducible beta-secretase.

To obtain this goal it was broken down into the following specific aims:
- To determine whether neuronal loss of ADAM10 in vivo results in decreased sAPPalpha, and increased Aß production and deposition
- To determine whether ADAM10 would be responsible for inducible alpha-secretase activity
- To determine which downstream signalling molecules would be responsible in inducing alpha-secretase

Since Aß seems central to AD pathogenesis, large efforts are being directed at developing small molecule inhibitors for alpha- and beta-secretases to decrease Aß production. Developing such inhibitors with high specificity and no unwanted side effects seems to be a particular challenge. Since alpha-secretase cleaves within the Aß peptide domain, it may be an alternative target and its activation may even have the double advantage of: 1) preventing the formation of neurotoxic Aß; and 2) increasing the amount of neuroprotective sAPPa.

Therefore it is important to determine whether ADAM10 is responsible for ß-cleavage of APP in vivo and whether other proteins exist with similar beta-secretase activity. The conditional ADAM10 deficient mouse will allow us to determine whether ADAM10 exerts beta-secretase activity in neurons in vivo.

Identifying specific factors that induce or regulate alpha-secretase activity in the brain will be of importance for the development of new AD therapeutics and may lead to new possibilities for pharmacological intervention in AD.
- Main published S&T results/foregrounds

Generation of Adam10 conditional knock-out mice

To establish this objective, we needed to circumvent the early embryonic lethality of adam10-/- mice and generate conditional Adam10 knock-out (Adam10 cKO) mice by employing the Cre/loxP recombination system. A mouse cosmid clone containing the 5' region of the Adam10 gene with exon 2 was isolated from a 129/ola cosmid library (RZPD clone ID F11344Q2). For construction of the targeting vector, a 8.2-kb KpnI-PstI DNA restriction fragment of Adam10 covering exon 2, 7.3 kb 5' sequence, and 5.8 kb 3' downstream sequence was subcloned into the plasmid vector pUC-18. The hygromycin B resistance gene, driven by the phosphoglycerate kinase (PGK) promoter was flanked with two FRT sequences. One loxP sequence was inserted downstream of the hygromycin B resistance gene in the NotI-ApaI site in intron1. A second loxP sequence was inserted into the SphI-SpeI site in intron 2. The targeting vector was linearised with KpnI and introduced into the ES cell line E14 129 SvEv by electroporation (ITL ingenious targeting laboratory, inc, NY). Hygromycin B-resistant (100µg/ml) colonies were screened by Southern blot analysis. Genomic DNA of ADAM10 ES cells was digested with Pst1, BanI and hybridised either with a 5' external gDNA probe (KpnI-BglII 767 bp fragment), an internal hygromycin probe and a 3' external gDNA probe (5' TCT TGG CAG AGC TTG AGC A 3'- 5' ACA CAG GGG CTG GAA TAC TTC TA 3'). Mutated ES cell lines were microinjected into blastocysts of C57BL/6J mice. Chimeric males were obtained and mated with C57BL/6J females to transmit the modified Adam10 alleles to the germ line. Homozygous floxed Adam10Fl/Fl mice and the Adam10Fl/+ hemizygous mice were viable and fertile and genotypes of tail-clips were determined by PCR.

To restrict ADAM10 inactivation to the central nervous system (CNS), Adam10Fl/Fl were first crossed with Nestin-Cre transgenic mice, which express Cre in neural precursor cells under the control of the Nestin promoter (10). Expression of Cre transcripts in Nestin-cre transgenic mice begins at E9.0 in the spinal cord and the forebrain-midbrain junction, and then extends to the entire CNS by E10.5 (11). The expression of Cre in the neural progenitor cells localised in the ventricular zone was demonstrated previously (12). Mice heterozygous for the floxed Adam10 allele and hemizygous for the Nestin-Cre allele were then crossed with Adam10Fl/Fl mice to obtain Adam10 cKO mice. Western blotting established strongly decreased levels of ADAM10 protein in total brain extracts of Adam10 cKO mice, with a very low residual expression level likely caused by non-neuronal cells. Adam10Fl/Fl, Adam10Fl/+ or Nestin-Cre;Adam10Fl/+ were used as a control.

ADAM10 is the most important constitutive neuronal alpha-secretase

The generation of Adam10 cKO mice provided us with the unique opportunity to generate ADAM10 deficient neurons and address the function of ADAM10 in the physiological processing of APP. In order to analyze human APP cleavage, 3- to 5-day-old primary neuronal cultures from E14.5 Adam10 cKO embryos and control Adam10Fl/Fl littermates were infected with pSFV-1 virus bearing human wild type APP. The APP ectodomain (sAPPa, sAPPß), full length (APP-FL), membrane-bound CTFs (APP-CTFa, APP-CTFß) and total Aß were analysed from supernatants and cell extract of dissociated neurons (Fig. 2). Taken together, the diminished appearance of the membrane-bound alpha-secretase–cleaved APP CTF (CTFa), and the observed 90 % reduction in the levels of secreted sAPPa indicate that ADAM10 is the most important APP alpha-secretase in these neurons. Surprisingly we also observed a paradoxical reduction in CTFß, sAPPß and total Aß peptide production and in the amount of Aß1–40 and Aß1–42 in the Adam10 cKO. Comparison of the Aß spectra between control and Adam10 cKO showed no shift towards longer Aß peptide species (Aß1–42) or shorter Aß peptides (Aß1–37, Aß1–38, Aß1–40), but confirmed the general decrease in all the Aß species in the cK.

Some residual alpha-secretase activity could be observed in the cKO neurons. Although it is possible that the other alpha-secretase candidates, including ADAM9 and ADAM17 contribute to a certain extent to APP processing in neurons, their contribution appears to be quantitatively less significant. Surprisingly we found in the ADAM10 KO neurons a paradoxical significant reduction in the beta-secretase cleavage, which is in contradiction to the general dogma that BACE1 and alpha-secretase are in competition for ectodomain cleavage of APP (13). The molecular basis of this 'competition' between the two secretase pathways is not very well understood, and could involve as well direct competition between the two proteases for the substrate, as effects on subcellular trafficking of substrate or protease. In our experiments, in which ADAM10 is taken away in primary neurons, we see an opposite effect, i.e. a decrease in beta-secretase activity (although beta-secretase levels are not decreased, results not shown). We speculate that ADAM10 deficiency and the effects on APP processing result in a change in localization or subcellular trafficking of either BACE1 or APP but further research is clearly needed to explain this intriguing phenomenon.
- Potential socio-economic impact and wider societal implications of the project and exploitation of results

Identification of neuronal alpha-secretase and characterization of the regulation of alpha-secretase and its' downstream signaling cascade will provide important insight in the regulation of APP cleavage, one of the key molecules in AD pathology. This regulation could give rise to the development of alternative therapeutic strategies aimed at regulating alpha-secretase cleavage. This could shift the cleavage of APP away from the more pathogenic beta-secretase cleavage and generation of pathogenic amyloid-ß. In addition there are indications that soluble alpha-cleaved APP can have neuroprotective action.

From a more applied point of view, our SH-SY5Y cell-based assay could be used in medium- to high throughput screening assays to identify novel regulators of alpha-secretase and could thereby provide an alternative strategy to identify targets modulating AD pathogenesis. These efforts could then lead to novel disease-modifying therapeutics for AD.