Publications by authors named "Claus Pietrzik"

79 Publications

Brain endothelial LRP1 maintains blood-brain barrier integrity.

Fluids Barriers CNS 2021 Jun 19;18(1):27. Epub 2021 Jun 19.

Molecular Neurodegeneration, Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg University of Mainz, Duesbergweg 6, 55099, Mainz, Germany.

The entry of blood-borne molecules into the brain is restricted by the blood-brain barrier (BBB). Various physical, transport and immune properties tightly regulate molecule movement between the blood and the brain to maintain brain homeostasis. A recent study utilizing a pan-endothelial, constitutive Tie2-Cre showed that paracellular passage of blood proteins into the brain is governed by endocytic and cell signaling protein low-density lipoprotein receptor-related protein 1 (LRP1). Taking advantage of conditional Slco1c1-CreER specific to CNS endothelial cells and choroid plexus epithelial cells we now supplement previous results and show that brain endothelial Lrp1 ablation results in protease-mediated tight junction degradation, P-glycoprotein (P-gp) reduction and a loss of BBB integrity.
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http://dx.doi.org/10.1186/s12987-021-00260-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214794PMC
June 2021

Phosphorylation of meprin β controls its cell surface abundance and subsequently diminishes ectodomain shedding.

FASEB J 2021 07;35(7):e21677

Biochemical Institute, Unit for Degradomics of the Protease Web, University of Kiel, Kiel, Germany.

Meprin β is a zinc-dependent metalloprotease exhibiting a unique cleavage specificity with strong preference for acidic amino acids at the cleavage site. Proteomic studies revealed a diverse substrate pool of meprin β including the interleukin-6 receptor (IL-6R) and the amyloid precursor protein (APP). Dysregulation of meprin β is often associated with pathological conditions such as chronic inflammation, fibrosis, or Alzheimer's disease (AD). The extracellular regulation of meprin β including interactors, sheddases, and activators has been intensively investigated while intracellular regulation has been barely addressed in the literature. This study aimed to analyze C-terminal phosphorylation of meprin β with regard to cell surface expression and proteolytic activity. By immunoprecipitation of endogenous meprin β from the colon cancer cell line Colo320 and subsequent LC-MS analysis, we identified several phosphorylation sites in its C-terminal region. Here, T694 in the C-terminus of meprin β was the most preferred residue after phorbol 12-myristate 13-acetate (PMA) stimulation. We further demonstrated the role of protein kinase C (PKC) isoforms for meprin β phosphorylation and identified the involvement of PKC-α and PKC-β. As a result of phosphorylation, the meprin β activity at the cell surface is reduced and, consequently, the extent of substrate cleavage is diminished. Our data indicate that this decrease of the surface activity is caused by the internalization and degradation of meprin β.
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http://dx.doi.org/10.1096/fj.202100271RDOI Listing
July 2021

Is LRP2 Involved in Leptin Transport over the Blood-Brain Barrier and Development of Obesity?

Int J Mol Sci 2021 May 8;22(9). Epub 2021 May 8.

Institute for Experimental and Clinical Pharmacology and Toxicology, Center of Brain, Behavior and Metabolism, University of Lübeck, 23562 Lübeck, Germany.

The mechanisms underlying the transport of leptin into the brain are still largely unclear. While the leptin receptor has been implicated in the transport process, recent evidence has suggested an additional role of LRP2 (megalin). To evaluate the function of LRP2 for leptin transport across the blood-brain barrier (BBB), we developed a novel leptin-luciferase fusion protein (pLG), which stimulated leptin signaling and was transported in an in vitro BBB model based on porcine endothelial cells. The LRP inhibitor RAP did not affect leptin transport, arguing against a role of LRP2. In line with this, the selective deletion of LRP2 in brain endothelial cells and epithelial cells of the choroid plexus did not influence bodyweight, body composition, food intake, or energy expenditure of mice. These findings suggest that LRP2 at the BBB is not involved in the transport of leptin into the brain, nor in the development of obesity as has previously been described.
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http://dx.doi.org/10.3390/ijms22094998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125945PMC
May 2021

The Blood-Brain Barrier in Alzheimer's Disease.

Handb Exp Pharmacol 2021 Feb 13. Epub 2021 Feb 13.

Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.

The accumulation of neurotoxic amyloid-beta (Aβ) in the brain is one of the characteristic hallmarks of Alzheimer's disease (AD). Aβ-peptide brain homeostasis is governed by its production and various clearance mechanisms. The blood-brain barrier provides a large surface area for influx and efflux mechanisms into and out of the brain. Different transporters and receptors have been implicated to play crucial roles in Aβ clearance from brain. Besides Aβ transport, the blood-brain barrier tightly regulates the brain's microenvironment; however, vascular alterations have been shown in patients with AD. Here, we summarize how the blood-brain barrier changes during aging and in disease and focus on recent findings of how the ABC transporter P-glycoprotein (ABCB1/P-gp) and the receptor low-density lipoprotein receptor-related protein 1 (LRP1) play a role in Aβ clearance from brain.
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http://dx.doi.org/10.1007/164_2020_418DOI Listing
February 2021

Lipoprotein receptor loss in forebrain radial glia results in neurological deficits and severe seizures.

Glia 2020 12 24;68(12):2517-2549. Epub 2020 Jun 24.

Department of Cell Morphology and Molecular Neurobiology, Ruhr University Bochum, Bochum, Germany.

The Alzheimer disease-associated multifunctional low-density lipoprotein receptor-related protein-1 is expressed in the brain. Recent studies uncovered a role of this receptor for the appropriate functioning of neural stem cells, oligodendrocytes, and neurons. The constitutive knock-out (KO) of the receptor is embryonically lethal. To unravel the receptors' role in the developing brain we generated a mouse mutant by specifically targeting radial glia stem cells of the dorsal telencephalon. The low-density lipoprotein receptor-related protein-1 lineage-restricted KO female and male mice, in contrast to available models, developed a severe neurological phenotype with generalized seizures during early postnatal development. The mechanism leading to a buildup of hyperexcitability and emergence of seizures was traced to a failure in adequate astrocyte development and deteriorated postsynaptic density integrity. The detected impairments in the astrocytic lineage: precocious maturation, reactive gliosis, abolished tissue plasminogen activator uptake, and loss of functionality emphasize the importance of this glial cell type for synaptic signaling in the developing brain. Together, the obtained results highlight the relevance of astrocytic low-density lipoprotein receptor-related protein-1 for glutamatergic signaling in the context of neuron-glia interactions and stage this receptor as a contributing factor for epilepsy.
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http://dx.doi.org/10.1002/glia.23869DOI Listing
December 2020

Meprin β: A novel regulator of blood-brain barrier integrity.

J Cereb Blood Flow Metab 2021 01 16;41(1):31-44. Epub 2020 Feb 16.

Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.

The metalloprotease meprin β (Mep1b) is capable of cleaving cell-adhesion molecules in different tissues (e.g. skin, kidney and intestine) and is dysregulated in several diseases associated with barrier breakdown (Alzheimer´s disease, kidney disruption, inflammatory bowel disease). In this study, we demonstrate that Mep1b is a novel regulator of tight junction (TJ) composition and blood-brain barrier (BBB) integrity in brain endothelium. In Mep1b-transfected mouse brain endothelial cells (bEnd.3), we observed a reduction of the TJ protein claudin-5, decreased transendothelial electrical resistance (TEER) and an elevated permeability to paracellular diffusion marker [C]-inulin. Analysis of global Mep1b knock-out (Mep1b) mice showed increased TJ protein expression (claudin-5, occludin, ZO-1) in cerebral microvessels and increased TEER in cultivated primary mouse brain endothelial compared to wild-type (wt) mice. Furthermore, we investigated the IgG levels in cerebrospinal fluid (CSF) and the brain water content as additional permeability markers and detected lower IgG levels and reduced brain water content in Mep1b mice compared to wt mice. Showing opposing features in overexpression and knock-out, we conclude that Mep1b plays a role in regulating brain endothelial TJ-proteins and therefore affecting BBB tightness in vitro and in vivo.
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http://dx.doi.org/10.1177/0271678X20905206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7747169PMC
January 2021

Enzyme replacement therapy with recombinant pro-CTSD (cathepsin D) corrects defective proteolysis and autophagy in neuronal ceroid lipofuscinosis.

Autophagy 2020 05 16;16(5):811-825. Epub 2019 Jul 16.

Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany.

CTSD (cathepsin D) is one of the major lysosomal proteases indispensable for the maintenance of cellular proteostasis by turning over substrates of endocytosis, phagocytosis and autophagy. Consequently, CTSD deficiency leads to a strong impairment of the lysosomal-autophagy machinery. In mice and humans CTSD dysfunction underlies the congenital variant (CLN10) of neuronal ceroid lipofuscinosis (NCL). NCLs are distinct lysosomal storage disorders (LSDs) sharing various hallmarks, namely accumulation of protein aggregates and ceroid lipofuscin leading to neurodegeneration and blindness. The most established and clinically approved approach to treat LSDs is enzyme replacement therapy (ERT) aiming to replace the defective hydrolase with an exogenously applied recombinant protein. Here we reveal that recombinant human pro-CTSD produced in a mammalian expression system can be efficiently taken up by a variety of cell models, is correctly targeted to lysosomes and processed to the active mature form of the protease. In proof-of-principle experiments we provide evidence that recombinant human CTSD (rhCTSD) can improve the biochemical phenotype of CTSD-deficient hippocampal slice cultures and retinal cells . Furthermore, we demonstrate that dosing of rhCTSD in the murine CLN10 model leads to a correction of lysosomal hypertrophy, storage accumulation and impaired autophagic flux in the viscera and central nervous system (CNS). We establish that direct delivery of the recombinant protease to the CNS is required for improvement of neuropathology and lifespan extension. Together these data support the continuation of the pre-clinical studies for the application of rhCTSD in the treatment of NCL. AIF1/IBA1: allograft inflammatory factor 1; BBB: blood brain barrier; CNS: central nervous system; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; ERT: enzyme replacement therapy; GFAP: glial fibrillary acidic protein; INL: inner nuclear layer; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; LDL: low-density lipoprotein; LRP1: low density lipoprotein receptor-related protein 1; LSD: lysosomal storage disorder; MEFs: mouse embryonic fibroblasts; M6P: mannose 6-phosphate; mCTSD: mature CTSD; NCL: neuronal ceroid lipofuscinosis; ONL: outer nuclear layer; PB: phosphate buffer; proCTSD: pro-cathepsin D; LRPAP1: low density lipoprotein receptor-related protein associated protein 1; rhCTSD: human recombinant CTSD; SAPC: saposin C; SAPD: saposin D; ATP5G1: ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C1 (subunit 9); SQSTM1/p62: sequestosome 1; TPP1: tripeptidyl peptidase I.
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http://dx.doi.org/10.1080/15548627.2019.1637200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158922PMC
May 2020

Degradome of soluble ADAM10 and ADAM17 metalloproteases.

Cell Mol Life Sci 2020 Jan 17;77(2):331-350. Epub 2019 Jun 17.

Unit for Degradomics of the Protease Web, Biochemical Institute, University of Kiel, Kiel, Germany.

Disintegrin and metalloproteinases (ADAMs) 10 and 17 can release the extracellular part of a variety of membrane-bound proteins via ectodomain shedding important for many biological functions. So far, substrate identification focused exclusively on membrane-anchored ADAM10 and ADAM17. However, besides known shedding of ADAM10, we identified ADAM8 as a protease capable of releasing the ADAM17 ectodomain. Therefore, we investigated whether the soluble ectodomains of ADAM10/17 (sADAM10/17) exhibit an altered substrate spectrum compared to their membrane-bound counterparts. A mass spectrometry-based N-terminomics approach identified 134 protein cleavage events in total and 45 common substrates for sADAM10/17 within the secretome of murine cardiomyocytes. Analysis of these cleavage sites confirmed previously identified amino acid preferences. Further in vitro studies verified fibronectin, cystatin C, sN-cadherin, PCPE-1 as well as sAPP as direct substrates of sADAM10 and/or sADAM17. Overall, we present the first degradome study for sADAM10/17, thereby introducing a new mode of proteolytic activity within the protease web.
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http://dx.doi.org/10.1007/s00018-019-03184-4DOI Listing
January 2020

Regulation of the alternative β-secretase meprin β by ADAM-mediated shedding.

Cell Mol Life Sci 2019 Aug 14;76(16):3193-3206. Epub 2019 Jun 14.

Unit for Degradomics of the Protease Web, Biochemical Institute, University of Kiel, Kiel, Germany.

Alzheimer's Disease (AD) is the sixth-leading cause of death in industrialized countries. Neurotoxic amyloid-β (Aβ) plaques are one of the pathological hallmarks in AD patient brains. Aβ accumulates in the brain upon sequential, proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretases. However, so far disease-modifying drugs targeting β- and γ-secretase pathways seeking a decrease in the production of toxic Aβ peptides have failed in clinics. It has been demonstrated that the metalloproteinase meprin β acts as an alternative β-secretase, capable of generating truncated Aβ peptides that have been described to be increased in AD patients. This indicates an important β-site cleaving enzyme 1 (BACE-1)-independent contribution of the metalloprotease meprin β within the amyloidogenic pathway and may lead to novel drug targeting avenues. However, meprin β itself is embedded in a complex regulatory network. Remarkably, the anti-amyloidogenic α-secretase a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a direct competitor for APP at the cell surface, but also a sheddase of inactive pro-meprin β. Overall, we highlight the current cellular, molecular and structural understanding of meprin β as alternative β-secretase within the complex protease web, regulating APP processing in health and disease.
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http://dx.doi.org/10.1007/s00018-019-03179-1DOI Listing
August 2019

LRP1 Has a Predominant Role in Production over Clearance of Aβ in a Mouse Model of Alzheimer's Disease.

Mol Neurobiol 2019 Oct 19;56(10):7234-7245. Epub 2019 Apr 19.

Laboratory for Experimental Mouse Genetics, Department of Human Genetics, KU Leuven, Herestraat 49, Box 604, 3000, Leuven, Belgium.

The low-density lipoprotein receptor-related protein-1 (LRP1) has a dual role in the metabolism of the amyloid precursor protein (APP). In cellular models, LRP1 enhances amyloid-β (Aβ) generation via APP internalization and thus its amyloidogenic processing. However, conditional knock-out studies in mice define LRP1 as an important mediator for the clearance of extracellular Aβ from brain via cellular degradation or transcytosis across the blood-brain barrier (BBB). In order to analyze the net effect of LRP1 on production and clearance of Aβ in vivo, we crossed mice with impaired LRP1 function with a mouse model of Alzheimer's disease (AD). Analysis of Aβ metabolism showed that, despite reduced Aβ clearance due to LRP1 inactivation in vivo, less Aβ was found in cerebrospinal fluid (CSF) and brain interstitial fluid (ISF). Further analysis of APP metabolism revealed that impairment of LRP1 in vivo shifted APP processing from the Aβ-generating amyloidogenic cleavage by beta-secretase to the non-amyloidogenic processing by alpha-secretase as shown by a decrease in extracellular Aβ and an increase of soluble APP-α (sAPP-α). This shift in APP processing resulted in overall lower Aβ levels and a reduction in plaque burden. Here, we present for the first time clear in vivo evidence that global impairment of LRP1's endocytosis function favors non-amyloidogenic processing of APP due to its reduced internalization and subsequently, reduced amyloidogenic processing. By inactivation of LRP1, the inhibitory effect on Aβ generation overrules the simultaneous impaired Aβ clearance, resulting in less extracellular Aβ and reduced plaque deposition in a mouse model of AD.
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http://dx.doi.org/10.1007/s12035-019-1594-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728278PMC
October 2019

Tethering soluble meprin α in an enzyme complex to the cell surface affects IBD-associated genes.

FASEB J 2019 06 27;33(6):7490-7504. Epub 2019 Mar 27.

Unit for Degradomics of the Protease Web, Biochemical Institute, University of Kiel, Kiel, Germany.

Biologic activity of proteases is mainly characterized by the substrate specificity, tissue distribution, and cellular localization. The human metalloproteases meprin α and meprin β share 41% sequence identity and exhibit a similar cleavage specificity with a preference for negatively charged amino acids. However, shedding of meprin α by furin on the secretory pathway makes it a secreted enzyme in comparison with the membrane-bound meprin β. In this study, we identified human meprin α and meprin β as forming covalently linked membrane-tethered heterodimers in the early endoplasmic reticulum, thereby preventing furin-mediated secretion of meprin α. Within this newly formed enzyme complex, meprin α was able to be activated on the cell surface and detected by cleavage of a novel specific fluorogenic peptide substrate. However, the known meprin β substrates amyloid precursor protein and CD99 were not shed by membrane-tethered meprin α. On the other hand, being linked to meprin α, activation of or substrate cleavage by meprin β on the cell surface was not altered. Interestingly, proteolytic activity of both proteases was increased in the heteromeric complex, indicating an increased proteolytic potential at the plasma membrane. Because meprins are susceptibility genes for inflammatory bowel disease (IBD), and to investigate the physiologic impact of the enzyme complex, we performed transcriptome analyses of intestinal mucosa from meprin-knockout mice. Comparison of the transcriptional gene analysis data with gene analyses of IBD patients revealed that different gene subsets were dysregulated if meprin α was expressed alone or in the enzyme complex, demonstrating the physiologic and pathophysiological relevance of the meprin heterodimer formation.-Peters, F., Scharfenberg, F., Colmorgen, C., Armbrust, F., Wichert, R., Arnold, P., Potempa, B., Potempa, J., Pietrzik, C. U., Häsler, R., Rosenstiel, P., Becker-Pauly, C. Tethering soluble meprin α in an enzyme complex to the cell surface affects IBD-associated genes.
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http://dx.doi.org/10.1096/fj.201802391RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529335PMC
June 2019

The metalloprotease ADAMTS4 generates N-truncated Aβ4-x species and marks oligodendrocytes as a source of amyloidogenic peptides in Alzheimer's disease.

Acta Neuropathol 2019 02 13;137(2):239-257. Epub 2018 Nov 13.

Department of Neuropathology, Heinrich-Heine University, Moorenstrasse 5, 40225, Düsseldorf, Germany.

Brain accumulation and aggregation of amyloid-β (Aβ) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length Aβ peptides (mainly Aβ1-40 and Aβ1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble Aβ peptides are truncated at the N-terminus, with Aβ4-x peptides being particularly abundant. Aβ4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the Aβ peptide sequence, which facilitates Aβ4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of Aβ4-40 but unchanged levels of Aβ1-x peptides. In the 5xFAD mouse model of amyloidosis, Aβ4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4 knockout background, Aβ4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of Aβ species, but Aβ4-40 peptides were absent in cultures derived from ADAMTS4 mice indicating that the enzyme was essential for Aβ4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of Aβ4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic Aβ peptides.
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http://dx.doi.org/10.1007/s00401-018-1929-5DOI Listing
February 2019

The concerted amyloid-beta clearance of LRP1 and ABCB1/P-gp across the blood-brain barrier is linked by PICALM.

Brain Behav Immun 2018 10 21;73:21-33. Epub 2018 Jul 21.

Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany. Electronic address:

The accumulation of neurotoxic amyloid-beta (Aβ) in the brain is a characteristic hallmark of Alzheimer's disease (AD). The blood-brain barrier (BBB) provides a large surface area and has been shown to be an important mediator for removal of brain Aβ. Both, the ABC transporter P-glycoprotein (ABCB1/P-gp) and the receptor low-density lipoprotein receptor-related protein 1 (LRP1) have been implicated to play crucial roles in Aβ efflux from brain. Here, with immunoprecipitation experiments, co-immunostainings and dual inhibition of ABCB1/P-gp and LRP1, we show that both proteins are functionally linked, mediating a concerted transcytosis of Aβ through endothelial cells. Late-onset AD risk factor Phosphatidylinositol binding clathrin assembly protein (PICALM) is associated with both ABCB1/P-gp and LRP1 representing a functional link and guiding both proteins through the brain endothelium. Together, our results give more mechanistic insight on Aβ transport across the BBB and show that the functional interplay of different clearance proteins is needed for the rapid removal of Aβ from the brain.
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http://dx.doi.org/10.1016/j.bbi.2018.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7748946PMC
October 2018

Discovery and validation of 2-styryl substituted benzoxazin-4-ones as a novel scaffold for rhomboid protease inhibitors.

Bioorg Med Chem Lett 2018 05 9;28(8):1417-1422. Epub 2018 Feb 9.

Department of Neuropathology, Heinrich-Heine University Duesseldorf, Moorenstrasse 5, 40225 Duesseldorf, Germany. Electronic address:

Rhomboids are intramembrane serine proteases with diverse physiological functions in organisms ranging from archaea to humans. Crystal structure analysis has provided a detailed understanding of the catalytic mechanism, and rhomboids have been implicated in various disease contexts. Unfortunately, the design of specific rhomboid inhibitors has lagged behind, and previously described small molecule inhibitors displayed insufficient potency and/or selectivity. Using a computer-aided approach, we focused on the discovery of novel scaffolds with reduced liabilities and the possibility for broad structural variations. Docking studies with the E. coli rhomboid GlpG indicated that 2-styryl substituted benzoxazinones might comprise novel rhomboid inhibitors. Protease in vitro assays confirmed activity of 2-styryl substituted benzoxazinones against GlpG but not against the soluble serine protease α-chymotrypsin. Furthermore, mass spectrometry analysis demonstrated covalent modification of the catalytic residue Ser201, corroborating the predicted mechanism of inhibition and the formation of an acyl enzyme intermediate. In conclusion, 2-styryl substituted benzoxazinones are a novel rhomboid inhibitor scaffold with ample opportunity for optimization.
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http://dx.doi.org/10.1016/j.bmcl.2018.02.017DOI Listing
May 2018

The LepR-mediated leptin transport across brain barriers controls food reward.

Mol Metab 2018 02 7;8:13-22. Epub 2017 Dec 7.

Institute for Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany. Electronic address:

Objective: Leptin is a key hormone in the control of appetite and body weight. Predominantly produced by white adipose tissue, it acts on the brain to inhibit homeostatic feeding and food reward. Leptin has free access to circumventricular organs, such as the median eminence, but entry into other brain centers is restricted by the blood-brain and blood-CSF barriers. So far, it is unknown for which of its central effects leptin has to penetrate brain barriers. In addition, the mechanisms mediating the transport across barriers are unclear although high expression in brain barriers suggests an important role of the leptin receptor (LepR).

Methods: We selectively deleted LepR in brain endothelial and epithelial cells of mice (LepR). The expression of LepR in fenestrated vessels of the periphery and the median eminence as well as in tanycytes was not affected.

Results: Perfusion studies showed that leptin uptake by the brain depended on LepR in brain barriers. When being fed with a rewarding high-fat diet LepR mice gained more body weight than controls. The aggravated obesity of LepR mice was due to hyperphagia and a higher sensitivity to food reward.

Conclusions: The LepR-mediated transport of leptin across brain barriers in endothelial cells lining microvessels and in epithelial cells of the choroid plexus controls food reward but is apparently not involved in homeostatic control of feeding.
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http://dx.doi.org/10.1016/j.molmet.2017.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985039PMC
February 2018

Discovery and Biological Evaluation of Potent and Selective N-Methylene Saccharin-Derived Inhibitors for Rhomboid Intramembrane Proteases.

Biochemistry 2017 12 12;56(51):6713-6725. Epub 2017 Dec 12.

Department of Neuropathology, Heinrich-Heine University Duesseldorf , Moorenstrasse 5, 40225 Duesseldorf, Germany.

Rhomboids are intramembrane serine proteases and belong to the group of structurally and biochemically most comprehensively characterized membrane proteins. They are highly conserved and ubiquitously distributed in all kingdoms of life and function in a wide range of biological processes, including epidermal growth factor signaling, mitochondrial dynamics, and apoptosis. Importantly, rhomboids have been associated with multiple diseases, including Parkinson's disease, type 2 diabetes, and malaria. However, despite a thorough understanding of many structural and functional aspects of rhomboids, potent and selective inhibitors of these intramembrane proteases are still not available. In this study, we describe the computer-based rational design, chemical synthesis, and biological evaluation of novel N-methylene saccharin-based rhomboid protease inhibitors. Saccharin inhibitors displayed inhibitory potency in the submicromolar range, effectiveness against rhomboids both in vitro and in live Escherichia coli cells, and substantially improved selectivity against human serine hydrolases compared to those of previously known rhomboid inhibitors. Consequently, N-methylene saccharins are promising new templates for the development of rhomboid inhibitors, providing novel tools for probing rhomboid functions in physiology and disease.
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http://dx.doi.org/10.1021/acs.biochem.7b01066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040592PMC
December 2017

Endothelial LRP1 - A Potential Target for the Treatment of Alzheimer's Disease : Theme: Drug Discovery, Development and Delivery in Alzheimer's Disease Guest Editor: Davide Brambilla.

Pharm Res 2017 Dec 25;34(12):2637-2651. Epub 2017 Sep 25.

Molecular Neurodegeneration, Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg-University, Duesbergweg 6, 55099, Mainz, Germany.

The accumulation of the neurotoxin beta-amyloid (Aβ) is a major hallmark in Alzheimer's disease (AD). Aβ homeostasis in the brain is governed by its production and various clearance mechanisms. Both pathways are influenced by the ubiquitously expressed low-density lipoprotein receptor-related protein 1 (LRP1). In cerebral blood vessels, LRP1 is an important mediator for the rapid removal of Aβ from brain via transport across the blood-brain barrier (BBB). Here, we summarize recent findings on LRP1 function and discuss the targeting of LRP1 as a modulator for AD pathology and drug delivery into the brain.
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http://dx.doi.org/10.1007/s11095-017-2267-3DOI Listing
December 2017

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-β clearance in blood-brain barrier endothelial cells.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 Jan 20;1863(1):40-60. Epub 2017 Sep 20.

Institute of Pathophysiology and Immunology, Medical University of Graz, Graz, Austria. Electronic address:

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3×Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB.
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http://dx.doi.org/10.1016/j.bbalip.2017.09.008DOI Listing
January 2018

Expression of endogenous mouse APP modulates β-amyloid deposition in hAPP-transgenic mice.

Acta Neuropathol Commun 2017 06 20;5(1):49. Epub 2017 Jun 20.

Translational Neurodegeneration Research and Neuropathology Lab, University of Oslo (UiO) / Oslo Universiy Hospital (OUS), Postboks 4950 Nydalen, Oslo, 0424, Norway.

Amyloid-β (Aβ) deposition is one of the hallmarks of the amyloid hypothesis in Alzheimer's disease (AD). Mouse models using APP-transgene overexpression to generate amyloid plaques have shown to model only certain parts of the disease. The extent to which the data from mice can be transferred to man remains controversial. Several studies have shown convincing treatment results in reducing Aβ and enhancing cognition in mice but failed totally in human. One model-dependent factor has so far been almost completely neglected: the endogenous expression of mouse APP and its effects on the transgenic models and the readout for therapeutic approaches.Here, we report that hAPP-transgenic models of amyloidosis devoid of endogenous mouse APP expression (mAPP-knockout / mAPPko) show increased amounts and higher speed of Aβ deposition than controls with mAPP. The number of senile plaques and the level of aggregated hAβ were elevated in mAPPko mice, while the deposition in cortical blood vessels was delayed, indicating an alteration in the general aggregation propensity of hAβ together with endogenous mAβ. Furthermore, the cellular response to Aβ deposition was modulated: mAPPko mice developed a pronounced and age-dependent astrogliosis, while microglial association to amyloid plaques was diminished. The expression of human and murine aggregation-prone proteins with differing amino acid sequences within the same mouse model might not only alter the extent of deposition but also modulate the route of pathogenesis, and thus, decisively influence the study outcome, especially in translational research.
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http://dx.doi.org/10.1186/s40478-017-0448-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480119PMC
June 2017

LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State.

Front Mol Neurosci 2017 27;10:118. Epub 2017 Apr 27.

Division of Human Biology and Human Genetics, Technical University of KaiserslauternKaiserslautern, Germany.

The low-density lipoprotein receptor-related protein 1, LRP1, interacts with APP and affects its processing. This is assumed to be mostly caused by the impact of LRP1 on APP endocytosis. More recently, also an interaction of APP and LRP1 early in the secretory pathway was reported whereat retention of LRP1 in the ER leads to decreased APP cell surface levels and in turn, to reduced Aβ secretion. Here, we extended the biochemical and immunocytochemical analyses by showing via live cell imaging analyses in primary neurons that LRP1 and APP are transported only partly in common (one third) but to a higher degree in distinct fast axonal transport vesicles. Interestingly, co-expression of LRP1 and APP caused a change of APP transport velocities, indicating that LRP1 recruits APP to a specific type of fast axonal transport vesicles. In contrast lowered levels of LRP1 facilitated APP transport. We further show that monomeric and dimeric APP exhibit similar transport characteristics and that both are affected by LRP1 in a similar way, by slowing down APP anterograde transport and increasing its endocytosis rate. In line with this, a knockout of LRP1 in CHO cells and in primary neurons caused an increase of monomeric and dimeric APP surface localization and in turn accelerated shedding by meprin β and ADAM10. Notably, a choroid plexus specific LRP1 knockout caused a much higher secretion of sAPP dimers into the cerebrospinal fluid compared to sAPP monomers. Together, our data show that LRP1 functions as a sorting receptor for APP, regulating its cell surface localization and thereby its processing by ADAM10 and meprin β, with the latter exhibiting a preference for APP in its dimeric state.
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http://dx.doi.org/10.3389/fnmol.2017.00118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406469PMC
April 2017

Structure and Synaptic Function of Metal Binding to the Amyloid Precursor Protein and its Proteolytic Fragments.

Front Mol Neurosci 2017 31;10:21. Epub 2017 Jan 31.

Division of Human Biology and Human Genetics, Technical University of Kaiserslautern Kaiserslautern, Germany.

Alzheimer's disease (AD) is ultimately linked to the amyloid precursor protein (APP). However, current research reveals an important synaptic function of APP and APP-like proteins (APLP1 and 2). In this context various neurotrophic and neuroprotective functions have been reported for the APP proteolytic fragments sAPPα, sAPPβ and the monomeric amyloid-beta peptide (Aβ). APP is a metalloprotein and binds copper and zinc ions. Synaptic activity correlates with a release of these ions into the synaptic cleft and dysregulation of their homeostasis is linked to different neurodegenerative diseases. Metal binding to APP or its fragments affects its structure and its proteolytic cleavage and therefore its physiological function at the synapse. Here, we summarize the current data supporting this hypothesis and provide a model of how these different mechanisms might be intertwined with each other.
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http://dx.doi.org/10.3389/fnmol.2017.00021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5281630PMC
January 2017

The Metalloprotease Meprin β Is an Alternative β-Secretase of APP.

Front Mol Neurosci 2016 5;9:159. Epub 2017 Jan 5.

Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg-University Mainz Mainz, Germany.

The membrane bound metalloprotease meprin β is important for collagen fibril assembly in connective tissue formation and for the detachment of the intestinal mucus layer for proper barrier function. Recent proteomic studies revealed dozens of putative new substrates of meprin β, including the amyloid precursor protein (APP). It was shown that APP is cleaved by meprin β in distinct ways, either at the β-secretase site resulting in increased levels of Aβ peptides, or at the N-terminus releasing 11 kDa, and 20 kDa peptide fragments. The latter event was discussed to be rather neuroprotective, whereas the ectodomain shedding of APP by meprin β reminiscent to BACE-1 is in line with the amyloid hypothesis of Alzheimer's disease, promoting neurodegeneration. The N-terminal 11 kDa and 20 kDa peptide fragments represent physiological cleavage products, since they are found in human brains under different diseased or non-diseased states, whereas these fragments are completely missing in brains of meprin β knock-out animals. Meprin β is not only a sheddase of adhesion molecules, such as APP, but was additionally demonstrated to cleave within the prodomain of ADAM10. Activated ADAM10, the α-secretase of APP, is then able to shed meprin β from the cell surface thereby abolishing the β-secretase activity. All together meprin β seems to be a novel player in APP processing events, even influencing other enzymes involved in APP cleavage.
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http://dx.doi.org/10.3389/fnmol.2016.00159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215381PMC
January 2017

Altered Gut Microbiome Composition and Tryptic Activity of the 5xFAD Alzheimer's Mouse Model.

J Alzheimers Dis 2017 ;56(2):775-788

Clinic of Psychiatry and Psychotherapy, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.

The regulation of physiological gut functions such as peristalsis or secretion of digestive enzymes by the central nervous system via the Nervus vagus is well known. Recent investigations highlight that pathological conditions of neurological or psychiatric disorders might directly interfere with the autonomous neuronal network of the gut - the enteric nervous system, or even derive from there. By using a murine Alzheimer's disease model, we investigated a potential influence of disease-associated changes on gastrointestinal properties. 5xFAD mice at three different ages were compared to wild type littermates in regard to metabolic parameters and enzymes of the gut by fluorimetric enzyme assay and western blotting. Overexpression of human amyloid-β protein precursor (AβPP) within the gut was assessed by qPCR and IHC; fecal microbiome analysis was conducted by 16SrRNA quantitation of selected phyla and species. While general composition of fecal samples, locomotion, and food consumption of male 5xFAD animals were not changed, we observed a reduced body weight occurring at early pathological stages. Human AβPP was not only expressed within the brain of these mice but also in gut tissue. Analysis of fecal proteins revealed a reduced trypsin amount in the 5xFAD model mice as compared to the wild type. In addition, we observed changes in fecal microbiota composition along with age. We therefore suggest that the presence of the mutated transgenes (AβPP and PS1), which are per se the basis for the genetic form of Alzheimer's disease in humans, directly interferes with gut function as shown here for the disease model mice.
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http://dx.doi.org/10.3233/JAD-160926DOI Listing
February 2018

Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin β and promotes transendothelial cell migration.

FASEB J 2017 03 21;31(3):1226-1237. Epub 2016 Dec 21.

Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, Kiel, Germany;

The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin β and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin β with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin β would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin β induced cell migration of Lewis lung carcinoma cells in an TEM assay. Accordingly, deficiency of meprin β in mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin β could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin β inhibitor-treated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Köllmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin β and promotes transendothelial cell migration.
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http://dx.doi.org/10.1096/fj.201601113RDOI Listing
March 2017

Mice are not Men: ADAM30 Findings Emphasize a Broader Look Towards Murine Alzheimer's Disease Models.

EBioMedicine 2016 Jul 25;9:19-20. Epub 2016 Jun 25.

Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg University of Mainz, 55128 Mainz, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.ebiom.2016.06.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972564PMC
July 2016

Low-density lipoprotein receptor-related protein 1 is a novel modulator of radial glia stem cell proliferation, survival, and differentiation.

Glia 2016 08 3;64(8):1363-80. Epub 2016 Jun 3.

Department of Cell Morphology and Molecular Neurobiology, Faculty of Biology and Biotechnology, Ruhr-University, Bochum, D-44780, Germany.

The LDL family of receptors and its member low-density lipoprotein receptor-related protein 1 (LRP1) have classically been associated with a modulation of lipoprotein metabolism. Current studies, however, indicate diverse functions for this receptor in various aspects of cellular activities, including cell proliferation, migration, differentiation, and survival. LRP1 is essential for normal neuronal function in the adult CNS, whereas the role of LRP1 in development remained unclear. Previously, we have observed an upregulation of LewisX (LeX) glycosylated LRP1 in the stem cells of the developing cortex and demonstrated its importance for oligodendrocyte differentiation. In the current study, we show that LeX-glycosylated LRP1 is also expressed in the stem cell compartment of the developing spinal cord and has broader functions in the developing CNS. We have investigated the basic properties of LRP1 conditional knockout on the neural stem/progenitor cells (NSPCs) from the cortex and the spinal cord, created by means of Cre-loxp-mediated recombination in vitro. The functional status of LRP1-deficient cells has been studied using proliferation, differentiation, and apoptosis assays. LRP1 deficient NSPCs from both CNS regions demonstrated altered differentiation profiles. Their differentiation capacity toward oligodendrocyte progenitor cells (OPCs), mature oligodendrocytes and neurons was reduced. In contrast, astrocyte differentiation was promoted. Moreover, LRP1 deletion had a negative effect on NSPCs proliferation and survival. Our observations suggest that LRP1 facilitates NSPCs differentiation via interaction with apolipoprotein E (ApoE). Upon ApoE4 stimulation wild type NSPCs generated more oligodendrocytes, but LRP1 knockout cells showed no response. The effect of ApoE seems to be independent of cholesterol uptake, but is rather mediated by downstream MAPK and Akt activation. GLIA 2016 GLIA 2016;64:1363-1380.
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http://dx.doi.org/10.1002/glia.23009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033964PMC
August 2016

Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains.

Biol Chem 2016 09;397(9):857-69

Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified.
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http://dx.doi.org/10.1515/hsz-2016-0126DOI Listing
September 2016

Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains.

Biol Chem 2016 May 14. Epub 2016 May 14.

Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It was shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. Since several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified being present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified.
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http://dx.doi.org/10.1515/hsz-20-2016-0126DOI Listing
May 2016

Generation of aggregation prone N-terminally truncated amyloid β peptides by meprin β depends on the sequence specificity at the cleavage site.

Mol Neurodegener 2016 Feb 19;11:19. Epub 2016 Feb 19.

Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University of Mainz, Duesbergweg 6, 55128, Mainz, Germany.

Background: The metalloprotease meprin β cleaves the Alzheimer's Disease (AD) relevant amyloid precursor protein (APP) as a β-secretase reminiscent of BACE-1, however, predominantly generating N-terminally truncated Aβ2-x variants.

Results: Herein, we observed increased endogenous sAPPα levels in the brains of meprin β knock-out (ko) mice compared to wild-type controls. We further analyzed the cellular interaction of APP and meprin β and found that cleavage of APP by meprin β occurs prior to endocytosis. The N-terminally truncated Aβ2-40 variant shows increased aggregation propensity compared to Aβ1-40 and acts even as a seed for Aβ1-40 aggregation. Additionally, we observed that different APP mutants affect the catalytic properties of meprin β and that, interestingly, meprin β is unable to generate N-terminally truncated Aβ peptides from Swedish mutant APP (APPswe).

Conclusion: Concluding, we propose that meprin β may be involved in the generation of N-terminally truncated Aβ2-x peptides of APP, but acts independently from BACE-1.
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http://dx.doi.org/10.1186/s13024-016-0084-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759862PMC
February 2016

Endothelial LRP1 transports amyloid-β(1-42) across the blood-brain barrier.

J Clin Invest 2016 Jan 30;126(1):123-36. Epub 2015 Nov 30.

According to the neurovascular hypothesis, impairment of low-density lipoprotein receptor-related protein-1 (LRP1) in brain capillaries of the blood-brain barrier (BBB) contributes to neurotoxic amyloid-β (Aβ) brain accumulation and drives Alzheimer's disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in Aβ transport and the expression of LRP1 in brain endothelium, the role of LRP1 at the BBB is uncertain. As global Lrp1 deletion in mice is lethal, appropriate models to study the function of LRP1 are lacking. Moreover, the relevance of systemic Aβ clearance to AD pathology remains unclear, as no BBB-specific knockout models have been available. Here, we developed transgenic mouse strains that allow for tamoxifen-inducible deletion of Lrp1 specifically within brain endothelial cells (Slco1c1-CreER(T2) Lrp1(fl/fl) mice) and used these mice to accurately evaluate LRP1-mediated Aβ BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [125I] Aβ(1-42). Additionally, in the 5xFAD mouse model of AD, brain endothelial-specific Lrp1 deletion reduced plasma Aβ levels and elevated soluble brain Aβ, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic Aβ elimination via the BBB. Together, our results suggest that receptor-mediated Aβ BBB clearance may be a potential target for treatment and prevention of Aβ brain accumulation in AD.
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http://dx.doi.org/10.1172/JCI81108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701557PMC
January 2016
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