Contribution of amino acids in the active site of dipeptidyl peptidase 4 to the catalytic action of the enzyme.

Dipeptidyl peptidase 4 (DP4)/CD26 regulates the biological function of various peptide hormones by releasing dipeptides from their N-terminus. The enzyme is a prominent target for the treatment of type-2 diabetes and various DP4 inhibitors have been developed in recent years, but their efficacy and side effects are still an issue. Many available crystal structures of the enzyme give a static picture about enzyme-ligand interactions, but the influence of amino acids in the active centre on binding and single catalysis steps can only be judged by mutagenesis studies. In order to elucidate their contribution to inhibitor binding and substrate catalysis, especially in discriminating the P1 amino acid of substrates, the amino acids R125, N710, E205 and E206 were investigated by mutagenesis studies. Our studies demonstrated, that N710 is essential for the catalysis of dipeptide substrates. We found that R125 is not important for dipeptide binding but interacts in the P1`position of the peptide backbone. In contrast to dipeptide substrates both amino acids play an essential role in the binding and arrangement of long natural substrates, particularly if lacking proline in the P1 position. Thus, it can be assumed that the amino acids R125 and N710 are important in the DP4 catalysed substrate hydrolysis by interacting with the peptide backbone of substrates up- and downstream of the cleavage site. Furthermore, we confirmed the important role of the amino acids E205 and E206. However, NP Y, displaying proline in P1 position, is still processed without the participation of E205 or E206.

A novel human tau knock-in mouse model reveals interaction of Abeta and human tau under progressing cerebral amyloidosis in 5xFAD mice.

BACKGROUND: Hyperphosphorylation and intraneuronal aggregation of the microtubule-associated protein tau is a major pathological hallmark of Alzheimer's disease (AD) brain. Of special interest is the effect of cerebral amyloid beta deposition, the second main hallmark of AD, on human tau pathology. Therefore, studying the influence of cerebral amyloidosis on human tau in a novel human tau knock-in (htau-KI) mouse model could help to reveal new details on their interplay. METHODS: We studied the effects of a novel human htau-KI under fast-progressing amyloidosis in 5xFAD mice in terms of correlation of gene expression data with human brain regions, development of Alzheimer's-like pathology, synaptic transmission, and behavior. RESULTS: The main findings are an interaction of human beta-amyloid and human tau in crossbred 5xFADxhtau-KI observed at transcriptional level and corroborated by electrophysiology and histopathology. The comparison of gene expression data of the 5xFADxhtau-KI mouse model to 5xFAD, control mice and to human AD patients revealed conspicuous changes in pathways related to mitochondria biology, extracellular matrix, and immune function. These changes were accompanied by plaque-associated MC1-positive pathological tau that required the htau-KI background. LTP deficits were noted in 5xFAD and htau-KI mice in contrast to signs of rescue in 5xFADxhtau-KI mice. Increased frequencies of miniature EPSCs and miniature IPSCs indicated an upregulated presynaptic function in 5xFADxhtau-KI. CONCLUSION: In summary, the multiple interactions observed between knocked-in human tau and the 5xFAD-driven progressing amyloidosis have important implications for future model development in AD.

Structure and Dynamics of Meprin ß in Complex with a Hydroxamate-Based Inhibitor.

The astacin protease Meprin ß represents an emerging target for drug development due to its potential involvement in disorders such as acute and chronic kidney injury and fibrosis. Here, we elaborate on the structural basis of inhibition by a specific Meprin ß inhibitor. Our analysis of the crystal structure suggests different binding modes of the inhibitor to the active site. This flexibility is caused, at least in part, by movement of the C-terminal region of the protease domain (CTD). The CTD movement narrows the active site cleft upon inhibitor binding. Compared with other astacin proteases, among these the highly homologous isoenzyme Meprin α, differences in the subsites account for the unique selectivity of the inhibitor. Although the inhibitor shows substantial flexibility in orientation within the active site, the structural data as well as binding analyses, including molecular dynamics simulations, support a contribution of electrostatic interactions, presumably by arginine residues, to binding and specificity. Collectively, the results presented here and previously support an induced fit and substantial movement of the CTD upon ligand binding and, possibly, during catalysis. To the best of our knowledge, we here present the first structure of a Meprin ß holoenzyme containing a zinc ion and a specific inhibitor bound to the active site. The structural data will guide rational drug design and the discovery of highly potent Meprin inhibitors.

Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.

Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer Disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures  of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.

Targeting isoaspartate-modified Aß rescues behavioral deficits in transgenic mice with Alzheimer's disease-like pathology.

BACKGROUND: Amyloid ß (Aß)-directed immunotherapy has shown promising results in preclinical and early clinical Alzheimer's disease (AD) trials, but successful translation to late clinics has failed so far. Compelling evidence suggests that post-translationally modified Aß peptides might play a decisive role in onset and progression of AD and first clinical trials targeting such Aß variants have been initiated. Modified Aß represents a small fraction of deposited material in plaques compared to pan-Aß epitopes, opening up pathways for tailored approaches of immunotherapy. Here, we generated the first monoclonal antibodies that recognize L-isoaspartate-modified Aß (isoD7-Aß) and tested a lead antibody molecule in 5xFAD mice. METHODS: This work comprises a combination of chemical and biochemical techniques as well as behavioral analyses. Aß peptides, containing L-isoaspartate at position 7, were chemically synthesized and used for immunization of mice and antibody screening methods. Biochemical methods included anti-isoD7-Aß monoclonal antibody characterization by surface plasmon resonance, immunohistochemical staining of human and transgenic mouse brain, and the development and application of isoD7-Aß ELISA as well as different non-modified Aß ELISA. For antibody treatment studies, 12 mg/kg anti-isoD7-Aß antibody K11_IgG2a was applied intraperitoneally to 5xFAD mice for 38 weeks. Treatment controls implemented were IgG2a isotype as negative and 3D6_IgG2a, the parent molecule of bapineuzumab, as positive control antibodies. Behavioral studies included elevated plus maze, pole test, and Morris water maze. RESULTS: Our advanced antibody K11 showed a KD in the low nM range and > 400fold selectivity for isoD7-Aß compared to other Aß variants. By using this antibody, we demonstrated that formation of isoD7-Aß may occur after formation of aggregates; hence, the presence of the isoD7-modification differentiates aged Aß from newly formed peptides. Importantly, we also show that the Tottori mutation responsible for early-onset AD in a Japanese pedigree is characterized by massively accelerated formation of isoD7-Aß in cell culture. The presence of isoD7-Aß was verified by K11 in post mortem human cortex and 5xFAD mouse brain tissue. Passive immunization of 5xFAD mice resulted in a significant reduction of isoD7-Aß and total Aß in brain. Amelioration of cognitive impairment was demonstrated by Morris water maze, elevated plus maze, pole, and contextual fear conditioning tests. Interestingly, despite the lower abundance of the isoD7-Aß epitope, the application of anti-isoD7-Aß antibodies showed comparable treatment efficacy in terms of reduction of brain amyloid and spatial learning but did not result in an increase of plasma Aß concentration as observed with 3D6 treatment. CONCLUSIONS: The present study demonstrates, for the first time, that the antibody-mediated targeting of isoD7-modified Aß peptides leads to attenuation of AD-like amyloid pathology. In conjunction with previously published data on antibodies directed against pGlu-modified Aß, the results highlight the crucial role of modified Aß peptides in AD pathophysiology. Hence, the results also underscore the therapeutic potential of targeting modified amyloid species for defining tailored approaches in AD therapy.

Hydrazides Are Potent Transition-State Analogues for Glutaminyl Cyclase Implicated in the Pathogenesis of Alzheimer's Disease.

Amyloidogenic plaques are hallmarks of Alzheimer's disease (AD) and typically consist of high percentages of modified Aß peptides bearing N-terminally cyclized glutamate residues. The human zinc(II) enzyme glutaminyl cyclase (QC) was shown in vivo to catalyze the cyclization of N-terminal glutamates of Aß peptides in a pathophysiological side reaction establishing QC as a druggable target for therapeutic treatment of AD. Here, we report crystallographic snapshots of human QC catalysis acting on the neurohormone neurotensin that delineate the stereochemical course of catalysis and suggest that hydrazides could mimic the transition state of peptide cyclization and deamidation. This hypothesis is validated by a sparse-matrix inhibitor screening campaign that identifies hydrazides as the most potent metal-binding group compared to classic Zn binders. The structural basis of hydrazide inhibition is illuminated by X-ray structure analysis of human QC in complex with a hydrazide-bearing peptide inhibitor and reveals a pentacoordinated Zn complex. Our findings inform novel strategies in the design of potent and highly selective QC inhibitors by employing hydrazides as the metal-binding warhead.

Amyloid-Beta Peptides Trigger Aggregation of Alpha-Synuclein In Vitro.

Alzheimer's disease (AD) and Parkinson's disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of α-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of Aß(1-42) and pGlu-Aß(3-42) on the aggregation of α-synuclein in vitro. The aggregation of human recombinant α-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of α-synuclein in the presence of minor concentrations of Aß(1-42) and pGlu-Aß(3-42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of α-synuclein and Aß in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of α-synuclein and Aß and pGlu-Aß, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides α-synuclein and Aß species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.

QUINT: Workflow for Quantification and Spatial Analysis of Features in Histological Images From Rodent Brain.

Transgenic animal models are invaluable research tools for elucidating the pathways and mechanisms involved in the development of neurodegenerative diseases. Mechanistic clues can be revealed by applying labelling techniques such as immunohistochemistry or in situ hybridisation to brain tissue sections. Precision in both assigning anatomical location to the sections and quantifying labelled features is crucial for output validity, with a stereological approach or image-based feature extraction typically used. However, both approaches are restricted by the need to manually delineate anatomical regions. To circumvent this limitation, we present the QUINT workflow for quantification and spatial analysis of labelling in series of rodent brain section images based on available 3D reference atlases. The workflow is semi-automated, combining three open source software that can be operated without scripting knowledge, making it accessible to most researchers. As an example, a brain region-specific quantification of amyloid plaques across whole transgenic Tg2576 mouse brain series, immunohistochemically labelled for three amyloid-related antigens is demonstrated. First, the whole brain image series were registered to the Allen Mouse Brain Atlas to produce customised atlas maps adapted to match the cutting plan and proportions of the sections (QuickNII software). Second, the labelling was segmented from the original images by the Random Forest Algorithm for supervised classification (ilastik software). Finally, the segmented images and atlas maps were used to generate plaque quantifications for each region in the reference atlas (Nutil software). The method yielded comparable results to manual delineations and to the output of a stereological method. While the use case demonstrates the QUINT workflow for quantification of amyloid plaques only, the workflow is suited to all mouse or rat brain series with labelling that is visually distinct from the background, for example for the quantification of cells or labelled proteins.

Endogenous mouse huntingtin is highly abundant in cranial nerve nuclei, co-aggregates to Abeta plaques and is induced in reactive astrocytes in a transgenic mouse model of Alzheimer's disease.

Pathogenic variants of the huntingtin (HTT) protein and their aggregation have been investigated in great detail in brains of Huntington's disease patients and HTT-transgenic animals. However, little is known about the physiological brain region- and cell type-specific HTT expression pattern in wild type mice and a potential recruitment of endogenous HTT to other pathogenic protein aggregates such as amyloid plaques in cross seeding events. Employing a monoclonal anti-HTT antibody directed against the HTT mid-region and using brain tissue of three different mouse strains, we detected prominent immunoreactivity in a number of brain areas, particularly in cholinergic cranial nerve nuclei, while ubiquitous neuronal staining appeared faint. The region-specific distribution of endogenous HTT was found to be comparable in wild type rat and hamster brain. In human amyloid precursor protein transgenic Tg2576 mice with amyloid plaque pathology, similar neuronal HTT expression patterns and a distinct association of HTT with Abeta plaques were revealed by immunohistochemical double labelling. Additionally, the localization of HTT in reactive astrocytes was demonstrated for the first time in a transgenic Alzheimer's disease animal model. Both, plaque association of HTT and occurrence in astrocytes appeared to be age-dependent. Astrocytic HTT gene and protein expression was confirmed in primary cultures by RT-qPCR and by immunocytochemistry. We provide the first detailed analysis of physiological HTT expression in rodent brain and, under pathological conditions, demonstrate HTT aggregation in proximity to Abeta plaques and Abeta-induced astrocytic expression of endogenous HTT in Tg2576 mice.

Dipeptidyl-Peptidase Activity of Meprin ß Links N-truncation of Aß with Glutaminyl Cyclase-Catalyzed pGlu-Aß Formation.

The formation of amyloid-ß (Aß) peptides is causally involved in the development of Alzheimer's disease (AD). A significant proportion of deposited Aß is N-terminally truncated and modified at the N-terminus by a pGlu-residue (pGlu-Aß). These forms show enhanced neurotoxicity compared to full-length Aß. Although the truncation may occur by aminopeptidases after formation of Aß, recently discovered processing pathways of amyloid-ß protein precursor (AßPP) by proteases such as meprin ß may also be involved. Here, we assessed a role of meprin ß in forming Aß3-40/42, which is the precursor of pGlu-Aß3-40/42 generated by glutaminyl cyclase (QC). Similar to QC, meprin ß mRNA is significantly upregulated in postmortem brain from AD patients. A histochemical analysis supports the presence of meprin ß in neurons and astrocytes in the vicinity of pGlu-Aß containing deposits. Cleavage of AßPP-derived peptides by meprin ß in vitro results in peptides Aß1-x, Aß2-x, and Aß3-x. The formation of N-truncated Aß by meprin ß was also corroborated in cell culture. A subset of the generated peptides was converted into pGlu-Aß3-40 by an addition of glutaminyl cyclase, supporting the preceding formation of Aß3-40. Further analysis of the meprin ß cleavage revealed a yet unknown dipeptidyl-peptidase-like activity specific for the N-terminus of Aß1-x. Thus, our data suggest that meprin ß contributes to the formation of N-truncated Aß by endopeptidase and exopeptidase activity to generate the substrate for QC-catalyzed pGlu-Aß formation.

Continuous assays for meprin alpha and beta using prolyl tripeptidyl aminopeptidase (PtP) from Porphyromonas gingivalis.

Common assays for endoprotease activity of meprin α and ß are based on cleavage of internally quenched substrates. Although direct and convenient, for meprins these assays bear disadvantages such as, e.g., significant substrate inhibition or potential fluorescence quenching by compounds applied in inhibitor analysis. Here, we present a novel continuous assay by introducing an auxiliary enzyme, prolyl tripeptidyl aminopeptidase (PtP) and the chromogenic substrate KKGYVADAP-p-nitroanilide. We provide a quick strategy for expression and one-step-purification of the auxiliary enzyme. The enzyme kinetic data for meprin α and ß suggest hyperbolic v/S-characteristics, the kinetic parameters of substrate conversion by meprin ß were Km = 184 ±â€¯32 µM and kcat = 20 ±â€¯4 s-1. We also present conditions for the use of the fluorogenic substrate KKGYVADAP-AMC to assess meprin ß activity. The assays were applied for determination of inhibitory parameters of the natural inhibitor actinonin and two recently published hydroxamates. Hence, we present two novel methods, which can be applied to assess inhibitory mechanism and potency with the attractive current drug targets meprin α and ß. Furthermore, the assay might also provide implications for analysis of other endoproteases as well as their inhibitors.

Kallikrein-related peptidases are activators of the CC chemokine CCL14.

Chemokine CCL14 is inactive in its proform. Here, we show that inflammation- and cancer-associated kallikrein-related peptidases KLK5 and KLK8 remove the N-terminal eight amino acids from the proform thereby converting CCL14 to its active state. Activity of the chemokine is demonstrated by migration of myeloid cells expressing relevant receptors.

Immunohistochemical Evidence from APP-Transgenic Mice for Glutaminyl Cyclase as Drug Target to Diminish pE-Abeta Formation.

Oligomeric assemblies of neurotoxic amyloid beta (Abeta) peptides generated by proteolytical processing of the amyloid precursor protein (APP) play a key role in the pathogenesis of Alzheimer's disease (AD). In recent years, a substantial heterogeneity of Abeta peptides with distinct biophysical and cell biological properties has been demonstrated. Among these, a particularly neurotoxic and disease-specific Abeta variant is N-terminally truncated and modified to pyroglutamate (pE-Abeta). Cell biological and animal experimental studies imply the catalysis of this modification by the enzyme glutaminyl cyclase (QC). However, direct histopathological evidence in transgenic animals from comparative brain region and cell type-specific expression of transgenic hAPP and QC, on the one hand, and on the formation of pE-Abeta aggregates, on the other, is lacking. Here, using single light microscopic, as well as triple immunofluorescent, labeling, we report the deposition of pE-Abeta only in the brain regions of APP-transgenic Tg2576 mice with detectable human APP and endogenous QC expression, such as the hippocampus, piriform cortex, and amygdala. Brain regions showing human APP expression without the concomitant presence of QC (the anterodorsal thalamic nucleus and perifornical nucleus) do not display pE-Abeta plaque formation. However, we also identified brain regions with substantial expression of human APP and QC in the absence of pE-Abeta deposition (the Edinger-Westphal nucleus and locus coeruleus). In these brain regions, the enzymes required to generate N-truncated Abeta peptides as substrates for QC might be lacking. Our observations provide additional evidence for an involvement of QC in AD pathogenesis via QC-catalyzed pE-Abeta formation.

Comprehensive Characterization of the Pyroglutamate Amyloid-ß Induced Motor Neurodegenerative Phenotype of TBA2.1 Mice.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and is being intensively investigated using a broad variety of animal models. Many of these models express mutant versions of human amyloid-ß protein precursor (AßPP) that are associated with amyloid-ß protein (Aß)-induced early onset familial AD. Most of these models, however, do not develop bold neurodegenerative pathology and the respective phenotypes. Nevertheless, this may well be essential for their suitability to identify therapeutically active compounds that have the potential for a curative or at least disease-modifying therapy in humans. In this study, the new transgenic mouse model TBA2.1 was explored in detail to increase knowledge about the neurodegenerative process induced by the presence of pyroglutamate modified human Aß3-42 (pEAß3-42). Analysis of the sensorimotor phenotype, motor coordination, Aß pathology, neurodegeneration, and gliosis revealed formation and progression of severe pathology and phenotypes including massive neuronal loss in homozygous TBA2.1 mice within a few months. In contrast, the start of a slight phenotype was observed only after 21 months in heterozygous mice. These data highlight the role of pEAß3-42 in the disease development and progression of AD. Based on the findings of this study, homozygous TBA2.1 mice can be utilized to gain deeper understanding in the underlying mechanisms of pEAß3-42 and might be suitable as an animal model for treatment studies targeting toxic Aß species, complementary to the well described transgenic AßPP mouse models.

Concerted action of dipeptidyl peptidase IV and glutaminyl cyclase results in formation of pyroglutamate-modified amyloid peptides in vitro.

Compelling evidence suggests a crucial role of amyloid beta peptides (Aß(1-40/42)) in the etiology of Alzheimer's disease (AD). The N-terminal truncation of Aß(1-40/42) and their modification, e.g. by glutaminyl cyclase (QC), is expected to enhance the amyloid toxicity. In this work, the MALDI-TOF mass spectrometry application proved N-terminal cleavage of Aß(1-40/42) by purified dipeptidyl peptidase IV (DPPIV) in vitro observed earlier. The subsequent transformation of resulted Aß(3-40/42) to pE-Aß(3-40/42) in QC catalyzed glutamate cyclization was manifested. Hence, consecutive conversion of Aß(1-40/42) by DPPIV and QC can be assumed as a potential mechanism of formation of non-degrading pyroglutamated pE-Aß(3-40/42), which might accumulate and contribute to AD progression. The in vitro acceleration of Aß(1-40) aggregation in the simultaneous presence of DPPIV and QC was shown also.

Aß oligomer eliminating compounds interfere successfully with pEAß(3-42) induced motor neurodegenerative phenotype in transgenic mice.

Currently, there are no causative or disease modifying treatments available for Alzheimer's disease (AD). Previously, it has been shown that D3, a small, fully d-enantiomeric peptide is able to eliminate low molecular weight Aß oligomers in vitro, enhance cognition and reduce plaque load in AD transgenic mice. To further characterise the therapeutic potential of D3 towards N-terminally truncated and pyroglutamated Aß (pEAß(3-42)) we tested D3 and its head-to-tail tandem derivative D3D3 both in vitro and in vivo in the new mouse model TBA2.1. These mice produce human pEAß(3-42) leading to a strong, early onset motor neurodegenerative phenotype. In the present study, we were able to demonstrate 1) strong binding affinity of both D3 and D3D3 to pEAß(3-42) in comparison to Aß(1-42) and 2) increased affinity of the tandem derivative D3D3 in comparison to D3. Subsequently we tested the therapeutic potentials of both peptides in the TBA2.1 animal model. Truly therapeutic, non-preventive treatment with D3 and D3D3 clearly slowed the progression of the neurodegenerative TBA2.1 phenotype, indicating the strong therapeutic potential of both peptides against pEAß(3-42) induced neurodegeneration.

N-terminal pyroglutamate formation in CX3CL1 is essential for its full biologic activity.

CX3CL1 (fractalkine) is a unique member of the CX3C chemokine family and mediates both adhesion and cell migration in inflammatory processes. Frequently, the activity of chemokines depends on a modified N-terminus as described for the N-terminus of CCL2 modified to a pGlu- (pyroglutamate) residue by QC (glutaminyl cyclase) activity. Here, we assess the role of the pGlu-modified residue of the CX3CL1 chemokine domain in human endothelial and smooth muscle cells. For the first time, we demonstrated using MS that QC (QPCT, gene name of QC) or its isoenzyme isoQC (iso-glutaminyl cyclase) (QPCTL, gene name of isoQC) catalyse the formation of N-terminal-modified pGlu-CX3CL1. Expression of QPCT is co-regulated with its substrates CCL2 and CX3CL1 in HUVECs (human umbilical vein endothelial cells) and HCASMCs (human coronary artery smooth muscle cells) upon stimulation with TNF-α and IL-1ß whereas QPCTL expression is not affected. By contrast, inhibition of the NF-κB pathway using an IKK2 inhibitor decreased the expression of the co-regulated targets QPCT, CCL2, and CX3CL1 Furthermore, RNAi-mediated inhibition of QPCT expression resulted in a reduction in CCL2 and CX3CL1 mRNA. In HCASMCs, N-terminal-modified pGlu1-CX3CL1 induced a significant stronger effect on phosphorylation of ERK (extracellular signal regulated kinase) 1/2, Akt (protein kinase B), and p38 (p38 mitogen-activated protein kinase) kinases than the immature Gln1-CX3CL1 in a time- and concentration-dependent manner. Furthermore, pGlu1-CX3CL1 affected the expression of CCL2, CX3CL1, and the adhesion molecule ICAM1/CD54 (intercellular adhesion molecule-1) inducing in higher expression level compared with its Gln1-variant in both HCASMCs and HUVECs. These results strongly suggest that QC-catalysed N-terminal pGlu formation of CX3CL1 is important for the stability or the interaction with its receptor and opens new insights into the function of QC in inflammation.

Structural and functional analyses of pyroglutamate-amyloid-ß-specific antibodies as a basis for Alzheimer immunotherapy.

Alzheimer disease is associated with deposition of the amyloidogenic peptide Aß in the brain. Passive immunization using Aß-specific antibodies has been demonstrated to reduce amyloid deposition both in vitro and in vivo Because N-terminally truncated pyroglutamate (pE)-modified Aß species (AßpE3) exhibit enhanced aggregation potential and propensity to form toxic oligomers, they represent particularly attractive targets for antibody therapy. Here we present three separate monoclonal antibodies that specifically recognize AßpE3 with affinities of 1-10 nm and inhibit AßpE3 fibril formation in vitro. In vivo application of one of these resulted in improved memory in AßpE3 oligomer-treated mice. Crystal structures of Fab-AßpE3 complexes revealed two distinct binding modes for the peptide. Juxtaposition of pyroglutamate pE3 and the F4 side chain (the "pEF head") confers a pronounced bulky hydrophobic nature to the AßpE3 N terminus that might explain the enhanced aggregation properties of the modified peptide. The deep burial of the pEF head by two of the antibodies explains their high target specificity and low cross-reactivity, making them promising candidates for the development of clinical antibodies.

Posttranslational modification impact on the mechanism by which amyloid-ß induces synaptic dysfunction.

Oligomeric amyloid-ß (Aß) 1-42 disrupts synaptic function at an early stage of Alzheimer's disease (AD). Multiple posttranslational modifications of Aß have been identified, among which N-terminally truncated forms are the most abundant. It is not clear, however, whether modified species can induce synaptic dysfunction on their own and how altered biochemical properties can contribute to the synaptotoxic mechanisms. Here, we show that a prominent isoform, pyroglutamated Aß3(pE)-42, induces synaptic dysfunction to a similar extent like Aß1-42 but by clearly different mechanisms. In contrast to Aß1-42, Aß3(pE)-42 does not directly associate with synaptic membranes or the prion protein but is instead taken up by astrocytes and potently induces glial release of the proinflammatory cytokine TNFα. Moreover, Aß3(pE)-42-induced synaptic dysfunction is not related to NMDAR signalling and Aß3(pE)-42-induced impairment of synaptic plasticity cannot be rescued by D1-agonists. Collectively, the data point to a scenario where neuroinflammatory processes together with direct synaptotoxic effects are caused by posttranslational modification of soluble oligomeric Aß and contribute synergistically to the onset of synaptic dysfunction in AD.