Kristina Endres

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.

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)

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.

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.

Regulation of alpha-secretase ADAM10 expression and activity

The amyloid precursor protein (APP) has a pivotal role in pathogenesis of Alzheimer’s disease (AD) via its beta- and gamma-secretase-derived cleavage products—the A-beta peptides. An alternative processing pathway provided by the alpha-secretase prevents formation of those toxic peptides and gives rise to the neurotrophic and neuroprotective cleavage product APPs-alpha. The molecular identity of the alpha-secretase has been confirmed recently, and there is consistency about ADAM10 being the most relevant and physiological enzyme of this class. It is not clear to what extent a deficiency in the catalytic activity of ADAM10 contributes to AD pathology and whether a decline occurs in aging humans. Nevertheless, ADAM10 has been suggested as a valuable target for prevention and/or for treatment of Alzheimer’s disease. This review focuses on our knowledge about regulation of ADAM10 on different levels of cell physiology, such as transcription and translation, as well as protein–protein interactions and how this especially in the case of transcriptional regulation by retinoic acids might lead to the development of new therapeutic approaches.

Upregulation of the α‐secretase ADAM10 – risk or reason for hope?

A decade ago, a disintegrin and metalloproteinase 10 (ADAM10) was identified as an α‐secretase and as a key proteinase in the processing of the amyloid precursor protein. Accordingly, the important role that it plays in Alzheimer’s disease was manifested. Animal models with an overexpression of ADAM10 revealed a beneficial profile of the metalloproteinase with respect to learning and memory, plaque load and synaptogenesis. Therefore, ADAM10 presents a worthwhile target with respect to the treatment of a neurodegenerative disease such as Morbus Alzheimer. Initially, ADAM10 was suggested to be an enzyme, shaping the extracellular matrix by cleavage of collagen type IV, or to be a tumour necrosis factor α convertase. In a relatively short time, a wide variety of additional substrates (with amyloid precursor protein probably being the most prominent) has been identified and the search is still ongoing. Hence, any side effects concerning the therapeutic enhancement of ADAM10 α‐secretase activity have to be considered. The present review summarizes our knowledge about the structure and function of ADAM10 and highlights the opportunities for enhancing the expression and/or activity of the α‐secretase as a therapeutic target.

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

In Alzheimers 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‐β‐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.—Reinhardt, S., Schuck, F., Grösgen, S., Riemenschneider, M., Hartmann, T., Postina, R., Grimm, M., Endres, K. Unfolded protein response signaling by transcription factor XBP‐1 regulates ADAM10 and is affected in Alzheimers disease. FASEB J. 28, 978–997 (2014). www.fasebj.org

Computational identification and experimental validation of microRNAs binding to the Alzheimer-related gene ADAM10

BackgroundMicroRNAs (miRNAs) are post-transcriptional regulators involved in numerous biological processes including the pathogenesis of Alzheimer’s disease (AD). A key gene of AD, ADAM10, controls the proteolytic processing of APP and the formation of the amyloid plaques and is known to be regulated by miRNA in hepatic cancer cell lines. To predict miRNAs regulating ADAM10 expression concerning AD, we developed a computational approach.MethodsMiRNA binding sites in the human ADAM10 3 untranslated region were predicted using the RNA22, RNAhybrid and miRanda programs and ranked by specific selection criteria with respect to AD such as differential regulation in AD patients and tissue-specific expression. Furthermore, target genes of miR-103, miR-107 and miR-1306 were derived from six publicly available miRNA target site prediction databases. Only target genes predicted in at least four out of six databases in the case of miR-103 and miR-107 were compared to genes listed in the AlzGene database including genes possibly involved in AD. In addition, the target genes were used for Gene Ontology analysis and literature mining. Finally, we used a luciferase assay to verify the potential effect of these three miRNAs on ADAM10 3UTR in SH-SY5Y cells.ResultsEleven miRNAs were selected, which have evolutionary conserved binding sites. Three of them (miR-103, miR-107, miR-1306) were further analysed as they are linked to AD and most strictly conserved between different species. Predicted target genes of miR-103 (p-value = 0.0065) and miR-107 (p-value = 0.0009) showed significant overlap with the AlzGene database except for miR-1306. Interactions between miR-103 and miR-107 to genes were revealed playing a role in processes leading to AD. ADAM10 expression in the reporter assay was reduced by miR-1306 (28%), miR-103 (45%) and miR-107 (52%).ConclusionsOur approach shows the requirement of incorporating specific, disease-associated selection criteria into the prediction process to reduce the amount of false positive predictions. In summary, our method identified three miRNAs strongly suggested to be involved in AD, which possibly regulate ADAM10 expression and hence offer possibilities for the development of therapeutic treatments of AD.

Increased CSF APPs-α levels in patients with Alzheimer disease treated with acitretin

Objective: We investigated induction of α-secretase A disintegrin and metalloprotease 10 (ADAM10) by the synthetic retinoid acitretin (Neotigason; Actavis, München-Riem, Germany) in patients with mild to moderate Alzheimer disease (AD) via measurement of CSF content of α-secretase–derived amyloid precursor protein (APPs-α). Methods: Twenty-one patients clinically diagnosed with mild to moderate AD received acitretin (30 mg per day) or placebo in a 4-week double-blind study. Primary endpoint was the difference of CSF APPs-α ratios calculated from the APPs-α levels after treatment and at baseline. We monitored safety and tolerability of the treatment. In addition, we assessed biomarkers such as β-amyloid 42 (Aβ42) under treatment conditions. Results: The acitretin group showed a significant increase in CSF APPs-α levels compared with the placebo group (difference 0.38, 95% confidence interval 0.03–0.72, p = 0.035) within this rather short treatment period. The synthetic retinoid acitretin was overall safe and well tolerated. Conclusions: Our pilot study highlights that acitretin is able to enhance the nonamyloidogenic APP processing in human patients. Clinical consequences of this regulation should be investigated in larger and longer trials in patients with AD to evaluate acitretins potential to serve as a novel therapeutic drug. Classification of evidence: This study provides Class III evidence that in patients with AD, oral acitretin increases CSF APPs-α levels.

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.