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

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

Glycoprotein NMB: a novel Alzheimer’s disease associated marker expressed in a subset of activated microglia

Abstract Alzheimer’s disease (AD) is an irreversible, devastating neurodegenerative brain disorder characterized by the loss of neurons and subsequent cognitive decline. Despite considerable progress in the understanding of the pathophysiology of AD, the precise molecular mechanisms that cause the disease remain elusive. By now, there is ample evidence that activated microglia have a critical role in the initiation and progression of AD. The present study describes the identification of Glycoprotein nonmetastatic melanoma protein B (GPNMB) as a novel AD-related factor in both transgenic mice and sporadic AD patients by expression profiling, immunohistochemistry and ELISA measurements. We show that GPNMB levels increase in an age-dependent manner in transgenic AD models showing profound cerebral neuron loss and demonstrate that GPNMB co-localizes with a distinct population of IBA1-positive microglia cells that cluster around amyloid plaques. Our data further indicate that GPNMB is part of a microglia activation state that is only present under neurodegenerative conditions and that is characterized by the up-regulation of a subset of genes including TREM2, APOE and CST7. In agreement, we provide in vitro evidence that soluble Aβ has a direct effect on GPNMB expression in an immortalized microglia cell line. Importantly, we show for the first time that GPNMB is elevated in brain samples and cerebrospinal fluid (CSF) of sporadic AD patients when compared to non-demented controls. The current findings indicate that GPNMB represents a novel disease-associated marker that appears to play a role in the neuroinflammatory response of AD

Evaluation of cerebrospinal fluid glycoprotein NMB (GPNMB) as a potential biomarker for Alzheimer’s disease

Background!#!Alzheimer's disease (AD) is a neurodegenerative disorder associated with extracellular amyloid-β peptide deposition and progressive neuron loss. Strong evidence supports that neuroinflammatory changes such as the activation of astrocytes and microglia cells are important in the disease process. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a transmembrane glycoprotein that has recently been associated with an emerging role in neuroinflammation, which has been reported to be increased in post-mortem brain samples from AD and Parkinson's disease patients.!##!Methods!#!The present study describes the partial 'fit for purpose' validation of a commercially available immunoassay for the determination of GPNMB levels in the cerebrospinal fluid (CSF). We further assessed the applicability of GPNMB as a potential biomarker for AD in two different cohorts that were defined by biomarker-supported clinical diagnosis or by neuroimaging with amyloid positron emission tomography, respectively.!##!Results!#!The results indicated that CSF GPNMB levels could not distinguish between AD or controls with other neurological diseases but correlated with other parameters such as aging and CSF pTau levels.!##!Conclusions!#!The findings of this study do not support GPNMB in CSF as a valuable neurochemical diagnostic biomarker of AD but warrant further studies employing healthy control individuals

SAR-studies of γ-secretase modulators with PPARγ-agonistic and 5-lipoxygenase-inhibitory activity for Alzheimer's disease

We present the design, synthesis and biological evaluation of compounds containing a 2-(benzylidene)hexanoic acid scaffold as multi-target directed γ-secretase-modulators. Broad structural variations were undertaken to elucidate the structure-activity-relationships at the 5-position of the aromatic core. Compound 13 showed the most potent activity profile with IC50 values of 0.79μM (Aβ42), 0.3μM (5-lipoxygenase) and an EC50 value of 4.64μM for PPARγ-activation. This derivative is the first compound exhibiting low micromolar to nanomolar activities for these three targets. Combining γ-secretase-modulation, PPARγ-agonism and inhibition of 5-lipoxygenase in one compound could be a novel disease-modifying multi-target-strategy for Alzheimer's disease to concurrently address the causative amyloid pathology and secondary pathologies like chronic brain inflammation

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

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.status: publishe

The metalloprotease ADAMTS4 generates N-truncated A4-x species and marks oligodendrocytes as a source of amyloidogenic peptides in Alzheimer's disease

Brain accumulation and aggregation of amyloid- (A) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length A peptides (mainly A1-40 and A1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by - and -secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble A peptides are truncated at the N-terminus, with A4-x peptides being particularly abundant. A4-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 A peptide sequence, which facilitates A4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of A4-40 but unchanged levels of A1-x peptides. In the 5xFAD mouse model of amyloidosis, A4-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, A4-40 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 A species, but A4-40 peptides were absent in cultures derived from ADAMTS4(-/-) mice indicating that the enzyme was essential for A4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of A4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic A peptides

Blood Platelets in the Progression of Alzheimer’s Disease

<div><p>Alzheimer’s disease (AD) is characterized by neurotoxic amyloid-<i>ß</i> plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-<i>ß (</i>A<i>ß)</i> peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of <i>Aß</i> peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by A<i>ß</i> in platelets. Treatment of platelets with A<i>ß</i> led to platelet activation and enhanced generation of reactive oxygen species (ROS) and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble A<i>ß</i> into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow <i>ex vivo</i> and <i>in vivo</i> and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke.</p></div

Incorporation of synthetic A<i>ß</i> fibrils by platelets.

<p><b>(</b>A) Viable platelets do not absorb synthetic A<i>ß</i> fibrils. Different platelet concentrations were incubated with 0.5 µM pre-aggregated FAM-labeled A<i>ß</i> and fluorecsence intensity was measured (upper panel). 10 µM Cytochalasin D (Cyt D) was used as inhibitor of phagocytosis. THP-1 cells served as positive control. Presence of platelets was controlled by DiOC₆ labeling (100 nM, lower left panel) and by phase contrast microscopy (lower right panel). N = 6; *p<0.05, **p<0.01 and ***p<0.001; n.s. = not significant. (B) Synthetic A<i>ß</i> was found on the plasma membrane of vital platelets as detected by confocal microscopy. FAM-A<i>ß</i> (green), platelet specific marker GPIb (red). Scale bar 20 µm. (C) Apoptotic platelets are able to incorporate pre-aggregated FAM-labeled A<i>ß</i>. Platelets were pre-treated with 1 and 5 µM ABT-737 to induce apoptosis. Fluorescence intensity of FAM-Aß was measured as indicated. Bar graphs depict mean values ±SEM (n = 6), *p<0.05, **p<0.01.</p

Strongly enhanced platelet adhesion under flow conditions <i>ex vivo</i> and on the injured carotid artery upon A<i>ß</i> stimulation of platelets <i>in vivo</i>.

<p>(A) Representative phase contrast images show platelet adhesion on collagen under flow conditions at a shear rate of 1000 sec⁻¹ (upper panel) and 1700 sec⁻¹ (lower panel) are shown. Scale bar indicates 20 µm. Bar graphs depict mean values ±SEM of the number of adherent cells per visual field [212×229 µm]. The platelet agonist ADP served as positive control, (n = 5 per group), **p<0.01 and ***p<0.001. (B) Platelets adhere to immobilized A<i>ß</i> under static and flow conditions (shear rate 1700 sec⁻¹). Coverslips coated with 250 µg/ml collagen and 1 mg/ml fibrinogen served as positive control. Representative phase contrast images are shown. (C) Representative images show platelet thrombus formation on collagen and on collagen/A<i>ß</i>, respectively, at a shear rate of 1000 sec⁻¹. Scale bar indicates 20 µm. Bar graphs depict mean values ±SEM of surface coverage, n = 5 per group, *p<0.05 and ***p<0.001. (D) Platelet adhesion at the injured carotid artery 10 min after injury. DCF-labeled platelets of C57BL/6J mice were incubated with vehicle or 50 µg/ml A<i>ß</i> for 30 min and injected into a C57BL/6J recipient mouse. Scale bar 50 µm. (E) Bar graphs depict mean values ±SEM showing the number of firmly adherent platelets at the vessel wall per mm² after 5, 10 and 30 min after injury of seven independent experiments. *p<0.05, **p<0.01.</p