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.

Consequences of Amyloid-ß Deficiency for the Liver.

The hepatic content of amyloid beta (Aß) decreases drastically in human and rodent cirrhosis highlighting the importance of understanding the consequences of Aß deficiency in the liver. This is especially relevant in view of recent advances in anti-Aß therapies for Alzheimer's disease (AD). Here, it is shown that partial hepatic loss of Aß in transgenic AD mice immunized with Aß antibody 3D6 and its absence in amyloid precursor protein (APP) knockout mice (APP-KO), as well as in human liver spheroids with APP knockdown upregulates classical hallmarks of fibrosis, smooth muscle alpha-actin, and collagen type I. Aß absence in APP-KO and deficiency in immunized mice lead to strong activation of transforming growth factor-ß (TGFß), alpha secretases, NOTCH pathway, inflammation, decreased permeability of liver sinusoids, and epithelial-mesenchymal transition. Inversely, increased systemic and intrahepatic levels of Aß42 in transgenic AD mice and neprilysin inhibitor LBQ657-treated wild-type mice protect the liver against carbon tetrachloride (CCl4)-induced injury. Transcriptomic analysis of CCl4-treated transgenic AD mouse livers uncovers the regulatory effects of Aß42 on mitochondrial function, lipid metabolism, and its onco-suppressive effects accompanied by reduced synthesis of extracellular matrix proteins. Combined, these data reveal Aß as an indispensable regulator of cell-cell interactions in healthy liver and a powerful protector against liver fibrosis.

Evidence for Enhanced Efficacy of Passive Immunotherapy against Beta-Amyloid in CD33-Negative 5xFAD Mice.

Passive immunotherapy is a very promising approach for the treatment of Alzheimer's disease (AD). Among the different antibodies under development, those targeting post-translationally modified Aß peptides might combine efficient reduction in beta-amyloid accompanied by lower sequestration in peripheral compartments and thus anticipated and reduced treatment-related side effects. In that regard, we recently demonstrated that the antibody-mediated targeting of isoD7-modified Aß peptides leads to the attenuation of AD-like amyloid pathology in 5xFAD mice. In order to assess novel strategies to enhance the efficacy of passive vaccination approaches, we investigated the role of CD33 for Aß phagocytosis in transgenic mice treated with an isoD7-Aß antibody. We crossbred 5xFAD transgenic mice with CD33 knock out (CD33KO) mice and compared the amyloid pathology in the different genotypes of the crossbreds. The knockout of CD33 in 5xFAD mice leads to a significant reduction in Aß plaques and concomitant rescue of behavioral deficits. Passive immunotherapy of 5xFAD/CD33KO showed a significant increase in plaque-surrounding microglia compared to 5xFAD treated with the antibody. Additionally, we observed a stronger lowering of Aß plaque load after passive immunotherapy in 5xFAD/CD33KO mice. The data suggest an additive effect of passive immunotherapy and CD33KO in terms of lowering Aß pathology. Hence, a combination of CD33 antagonists and monoclonal antibodies might represent a strategy to enhance efficacy of passive immunotherapy in AD.

CD33-directed immunotherapy with third-generation chimeric antigen receptor T cells and gemtuzumab ozogamicin in intact and CD33-edited acute myeloid leukemia and hematopoietic stem and progenitor cells.

Immunotherapies, such as chimeric antigen receptor (CAR) modified T cells and antibody-drug conjugates (ADCs), have revolutionized the treatment of cancer, especially of lymphoid malignancies. The application of targeted immunotherapy to patients with acute myeloid leukemia (AML) has been limited in particular by the lack of a tumor-specific target antigen. Gemtuzumab ozogamicin (GO), an ADC targeting CD33, is the only approved immunotherapeutic agent in AML. In our study, we introduce a CD33-directed third-generation CAR T-cell product (3G.CAR33-T) for the treatment of patients with AML. 3G.CAR33-T cells could be expanded up to the end-of-culture, that is, 17 days after transduction, and displayed significant cytokine secretion and robust cytotoxic activity when incubated with CD33-positive cells including cell lines, drug-resistant cells, primary blasts as well as normal hematopoietic stem and progenitor cells (HSPCs). When compared to second-generation CAR33-T cells, 3G.CAR33-T cells exhibited higher viability, increased proliferation and stronger cytotoxicity. Also, GO exerted strong antileukemia activity against CD33-positive AML cells. Upon genomic deletion of CD33 in HSPCs, 3G.CAR33-T cells and GO preferentially killed wildtype leukemia cells, while sparing CD33-deficient HSPCs. Our data provide evidence for the applicability of CD33-targeted immunotherapies in AML and its potential implementation in CD33 genome-edited stem cell transplantation approaches.

Tetrahydroimidazo[4,5-c]pyridine-Based Inhibitors of Porphyromonas gingivalis Glutaminyl Cyclase.

Periodontitis is a severe yet underestimated oral disease. Since it is linked to several systemic diseases, such as diabetes, artheriosclerosis, and even Alzheimer's disease, growing interest in treating periodontitis has emerged recently. The major cause of periodontitis is a shift in the oral microbiome. A keystone pathogen that is associated with this shift is Porphyromonas gingivalis. Hence, targeting P. gingivalis came into focus of drug discovery for the development of novel antiinfective compounds. Among others, glutaminyl cyclases (QCs) of oral pathogens might be promising drug targets. Here, we report the discovery and structure-activity relationship of a novel class of P. gingivalis QC inhibitors according to a tetrahydroimidazo[4,5-c]pyridine scaffold. Some compounds exhibited activity in the lower nanomolar range and thus were further characterized with regard to their selectivity and toxicity.

Combination of the Glutaminyl Cyclase Inhibitor PQ912 (Varoglutamstat) and the Murine Monoclonal Antibody PBD-C06 (m6) Shows Additive Effects on Brain Aß Pathology in Transgenic Mice.

Compelling evidence suggests that pyroglutamate-modified Aß (pGlu3-Aß; AßN3pG) peptides play a pivotal role in the development and progression of Alzheimer's disease (AD). Approaches targeting pGlu3-Aß by glutaminyl cyclase (QC) inhibition (Varoglutamstat) or monoclonal antibodies (Donanemab) are currently in clinical development. Here, we aimed at an assessment of combination therapy of Varoglutamstat (PQ912) and a pGlu3-Aß-specific antibody (m6) in transgenic mice. Whereas the single treatments at subtherapeutic doses show moderate (16-41%) but statistically insignificant reduction of Aß42 and pGlu-Aß42 in mice brain, the combination of both treatments resulted in significant reductions of Aß by 45-65%. Evaluation of these data using the Bliss independence model revealed a combination index of ≈1, which is indicative for an additive effect of the compounds. The data are interpreted in terms of different pathways, in which the two drugs act. While PQ912 prevents the formation of pGlu3-Aß in different compartments, the antibody is able to clear existing pGlu3-Aß deposits. The results suggest that combination of the small molecule Varoglutamstat and a pE3Aß-directed monoclonal antibody may allow a reduction of the individual compound doses while maintaining the therapeutic effect.

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.

Acute targeting of pre-amyloid seeds in transgenic mice reduces Alzheimer-like pathology later in life.

Amyloid-ß (Aß) deposits are a relatively late consequence of Aß aggregation in Alzheimer's disease. When pathogenic Aß seeds begin to form, propagate and spread is not known, nor are they biochemically defined. We tested various antibodies for their ability to neutralize Aß seeds before Aß deposition becomes detectable in Aß precursor protein-transgenic mice. We also characterized the different antibody recognition profiles using immunoprecipitation of size-fractionated, native, mouse and human brain-derived Aß assemblies. At least one antibody, aducanumab, after acute administration at the pre-amyloid stage, led to a significant reduction of Aß deposition and downstream pathologies 6 months later. This demonstrates that therapeutically targetable pathogenic Aß seeds already exist during the lag phase of protein aggregation in the brain. Thus, the preclinical phase of Alzheimer's disease-currently defined as Aß deposition without clinical symptoms-may be a relatively late manifestation of a much earlier pathogenic seed formation and propagation that currently escapes detection in vivo.

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.

Development of the clinical candidate PBD-C06, a humanized pGlu3-Aß-specific antibody against Alzheimer's disease with reduced complement activation.

In clinical trials with early Alzheimer's patients, administration of anti-amyloid antibodies reduced amyloid deposits, suggesting that immunotherapies may be promising disease-modifying interventions against Alzheimer's disease (AD). Specific forms of amyloid beta (Aß) peptides, for example post-translationally modified Aß peptides with a pyroglutamate at the N-terminus (pGlu3, pE3), are attractive antibody targets, due to pGlu3-Aß's neo-epitope character and its propensity to form neurotoxic oligomeric aggregates. We have generated a novel anti-pGlu3-Aß antibody, PBD-C06, which is based on a murine precursor antibody that binds with high specificity to pGlu3-Aß monomers, oligomers and fibrils, including mixed aggregates of unmodified Aß and pGlu3-Aß peptides. PBD-C06 was generated by first grafting the murine antigen binding sequences onto suitable human variable light and heavy chains. Subsequently, the humanized antibody was de-immunized and site-specific mutations were introduced to restore original target binding, to eliminate complement activation and to improve protein stability. PBD-C06 binds with the same specificity and avidity as its murine precursor antibody and elimination of C1q binding did not compromise Fcγ-receptor binding or in vitro phagocytosis. Thus, PBD-C06 was specifically designed to target neurotoxic aggregates and to avoid complement-mediated inflammatory responses, in order to lower the risk for vasogenic edemas in the clinic.

Effector function of anti-pyroglutamate-3 Aß antibodies affects cognitive benefit, glial activation and amyloid clearance in Alzheimer's-like mice.

BACKGROUND: Pyroglutamate-3 Aß (pGlu-3 Aß) is an N-terminally truncated and post-translationally modified Aß species found in Alzheimer's disease (AD) brain. Its increased peptide aggregation propensity and toxicity make it an attractive emerging treatment strategy for AD. We address the question of how the effector function of an anti-pGlu-3 Aß antibody influences the efficacy of immunotherapy in mouse models with AD-like pathology. METHODS: We compared two different immunoglobulin (Ig) isotypes of the same murine anti-pGlu-3 Aß mAb (07/1 IgG1 and 07/2a IgG2a) and a general N-terminal Aß mAb (3A1 IgG1) for their ability to clear Aß and protect cognition in a therapeutic passive immunotherapy study in aged, plaque-rich APPSWE/PS1ΔE9 transgenic (Tg) mice. We also compared the ability of these antibodies and a CDC-mutant form of 07/2a (07/2a-k), engineered to avoid complement activation, to clear Aß in an ex vivo phagocytosis assay and following treatment in APPSLxhQC double Tg mice, and to activate microglia using longitudinal microPET imaging with TSPO-specific 18F-GE180 tracer following a single bolus antibody injection in young and old Tg mice. RESULTS: We demonstrated significant cognitive improvement, better plaque clearance, and more plaque-associated microglia in the absence of microhemorrhage in aged APPSWE/PS1ΔE9 Tg mice treated with 07/2a, but not 07/1 or 3A1, compared to PBS in our first in vivo study. All mAbs cleared plaques in an ex vivo assay, although 07/2a promoted the highest phagocytic activity. Compared with 07/2a, 07/2a-k showed slightly reduced affinity to Fcγ receptors CD32 and CD64, although the two antibodies had similar binding affinities to pGlu-3 Aß. Treatment of APPSLxhQC mice with 07/2a and 07/2a-k mAbs in our second in vivo study showed significant plaque-lowering with both mAbs. Longitudinal 18F-GE180 microPET imaging revealed different temporal patterns of microglial activation for 3A1, 07/1, and 07/2a mAbs and no difference between 07/2a-k and PBS-treated Tg mice. CONCLUSION: Our results suggest that attenuation of behavioral deficits and clearance of amyloid is associated with strong effector function of the anti-pGlu-3 Aß mAb in a therapeutic treatment paradigm. We present evidence that antibody engineering to reduce CDC-mediated complement binding facilitates phagocytosis of plaques without inducing neuroinflammation in vivo. Hence, the results provide implications for tailoring effector function of humanized antibodies for clinical development.

Passive Aß Immunotherapy: Current Achievements and Future Perspectives.

Passive immunotherapy has emerged as a very promising approach for the treatment of Alzheimer's disease and other neurodegenerative disorders, which are characterized by the misfolding and deposition of amyloid peptides. On the basis of the amyloid hypothesis, the majority of antibodies in clinical development are directed against amyloid ß (Aß), the primary amyloid component in extracellular plaques. This review focuses on the current status of Aß antibodies in clinical development, including their characteristics and challenges that came up in clinical trials with these new biological entities (NBEs). Emphasis is placed on the current view of common side effects observed with passive immunotherapy, so-called amyloid-related imaging abnormalities (ARIAs), and potential ways to overcome this issue. Among these new ideas, a special focus is placed on molecules that are directed against post-translationally modified variants of the Aß peptide, an emerging approach for development of new antibody molecules.

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.

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.

Neuropeptide Y (NPY) in cerebrospinal fluid from patients with Huntington's Disease: increased NPY levels and differential degradation of the NPY1-30 fragment.

Huntington's disease (HD) is an inherited and fatal polyglutamine neurodegenerative disorder caused by an expansion of the CAG triplet repeat coding region within the HD gene. Progressive dysfunction and loss of striatal GABAergic medium spiny neurons (MSNs) may account for some of the characteristic symptoms in HD patients. Interestingly, in HD, MSNs expressing neuropeptide Y (NPY) are spared and their numbers is even up-regulated in HD patients. Consistent with this, we report here on increased immuno-linked NPY (IL-NPY) levels in human cerebrospinal fluid (hCSF) from HD patients (Control n = 10; early HD n = 9; mid HD n = 11). As this antibody-based detection of NPY may provide false positive differences as a result of the antibody-based detections of only fragments of NPY, the initial finding was validated by investigating the proteolytic stability of NPY in hCSF using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective inhibitors. A comparison between resulting NPY-fragments and detailed epitope analysis verified significant differences in IL-NPY1-36/3-36 and NPY1-30 levels between HD patients and control subjects with no significant differences between early vs mid HD cases. Ex vivo degradomics analysis demonstrated that NPY is initially degraded to NPY1-30 by cathepsin D in both HD patients and control subjects. Yet, NPY1-30 is then further differentially hydrolyzed by thimet oligopeptidase (TOP) in HD patients and by neprilysin (NEP) in control subjects. Furthermore, altered hCSF TOP-inhibitor Dynorphin A1-13 (Dyn-A1-13 ) and TOP-substrate Dyn-A1-8 levels indicate an impaired Dyn-A-TOP network in HD patients. Thus, we conclude that elevated IL-NPY-levels in conjunction with TOP-/NEP-activity/protein as well as Dyn-A1-13 -peptide levels may serve as a potential biomarker in human CSF of HD. Huntington's disease (HD) patients' cerebrospinal fluid (CSF) exhibits higher neuropeptide Y (NPY) levels. Further degradomics studies show that CSF-NPY is initially degraded to NPY1-30 by Cathepsin D. The NPY1-30 fragment is then differentially degraded in HD vs control involving Neprilysin (NEP), Thimet Oligopeptidase (TOP), and TOP-Dynorphin-A network. Together, these findings may help in search for HD biomarkers.

Identifying neuropeptide Y (NPY) as the main stress-related substrate of dipeptidyl peptidase 4 (DPP4) in blood circulation.

BACKGROUND: Dipeptidyl peptidase 4 (DPP4; EC 3.4.14.5; CD26) is a membrane-bound or shedded serine protease that hydrolyzes dipeptides from the N-terminus of peptides with either proline or alanine at the penultimate position. Substrates of DPP4 include several stress-related neuropeptides implicated in anxiety, depression and schizophrenia. A decline of DPP4-like activity has been reported in sera from depressed patient, but not fully characterized regarding DPP4-like enzymes, therapeutic interventions and protein. METHODS: Sera from 16 melancholic- and 16 non-melancholic-depressed patients were evaluated for DPP4-like activities and the concentration of soluble DPP4 protein before and after treatment by anti-depressive therapies. Post-translational modification of DPP4-isoforms and degradation of NPY, Peptide YY (PYY), Galanin-like peptide (GALP), Orexin B (OrxB), OrxA, pituitary adenylate cyclase-activating polypeptide (PACAP) and substance P (SP) were studied in serum and in ex vivo human blood. N-terminal truncation of biotinylated NPY by endothelial membrane-bound DPP4 versus soluble DPP4 was determined in rat brain perfusates and spiked sera. RESULTS: Lower DPP4 activities in depressed patients were reversed by anti-depressive treatment. In sera, DPP4 contributed to more than 90% of the overall DPP4-like activity and correlated with its protein concentration. NPY displayed equal degradation in serum and blood, and was equally truncated by serum and endothelial DPP4. In addition, GALP and rat OrxB were identified as novel substrates of DPP4. CONCLUSION: NPY is the best DPP4-substrate in blood, being truncated by soluble and membrane DPP4, respectively. The decline of soluble DPP4 in acute depression could be reversed upon anti-depressive treatment. Peptidases from three functional compartments regulate the bioactivity of NPY in blood.

Enhanced Fibril Fragmentation of N-Terminally Truncated and Pyroglutamyl-Modified Aß Peptides.

N-terminal truncation and pyroglutamyl (pE) formation are naturally occurring chemical modifications of the Aß peptide in Alzheimer's disease. We show herein that these two modifications significantly reduce the fibril length and the transition midpoint of thermal unfolding of the fibrils, but they do not substantially perturb the fibrillary peptide conformation. This observation implies that the N terminus of the unmodified peptide protects Aß fibrils against mechanical stress and fragmentation and explains the high propensity of pE-modified peptides to form small and particularly toxic aggregates.

An anti-pyroglutamate-3 Aß vaccine reduces plaques and improves cognition in APPswe/PS1ΔE9 mice.

Pyroglutamate-3 amyloid-beta (pGlu-3 Aß) is an N-terminally truncated Aß isoform likely playing a decisive role in Alzheimer's disease pathogenesis. Here, we describe a prophylactic passive immunization study in APPswe/PS1ΔE9 mice using a novel pGlu-3 Aß immunoglobulin G1 (IgG1) monoclonal antibody, 07/1 (150 and 500 µg, intraperitoneal, weekly) and compare its efficacy with a general Aß IgG1 monoclonal antibody, 3A1 (200 µg, intraperitoneal, weekly) as a positive control. After 28 weeks of treatment, plaque burden was reduced and cognitive performance of 07/1-immunized Tg mice, especially at the higher dose, was normalized to wild-type levels in 2 hippocampal-dependent tests and partially spared compared with phosphate-buffered saline-treated Tg mice. Mice that received 3A1 had reduced plaque burden but showed no cognitive benefit. In contrast with 3A1, treatment with 07/1 did not increase the concentration of Aß in plasma, suggesting different modes of Aß plaque clearance. In conclusion, early selective targeting of pGlu-3 Aß by immunotherapy may be effective in lowering cerebral Aß plaque burden and preventing cognitive decline in the clinical setting. Targeting this pathologically modified form of Aß thereby is unlikely to interfere with potential physiologic function(s) of Aß that have been proposed.

Proteolytic degradation of neuropeptide Y (NPY) from head to toe: Identification of novel NPY-cleaving peptidases and potential drug interactions in CNS and Periphery.

The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application. The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application.