Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N-terminus of the polypeptide. Among those, the N-terminus of monocyte chemoattractant protein 1 (CCL2 and MCP-1) is modified to a pyroglutamate (pE-) residue protecting against degradation in vivo. Here, we show that the N-terminal pE-formation depends on glutaminyl cyclase activity. The pE-residue increases stability against N-terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro. Genetic ablation of the glutaminyl cyclase iso-enzymes QC (QPCT) or isoQC (QPCTL) revealed a major role of isoQC for pE1-CCL2 formation and monocyte infiltration. Consistently, administration of QC-inhibitors in inflammatory models, such as thioglycollate-induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC-inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2-driven disorders such as atherosclerosis/restenosis and fibrosis.

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 Aβ(1–42) and pGlu-Aβ(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 Aβ(1–42) and pGlu-Aβ(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 Aβ 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 Aβ and pGlu-Aβ, 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 Aβ species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development.

Secretory peptides and proteins are frequently modified by pyroglutamic acid (pE, pGlu) at their N-terminus. This modification is catalyzed by the glutaminyl cyclases QC and isoQC. Here, we decipher the roles of the isoenzymes by characterization of IsoQC-/- mice. These mice show a significant reduction of glutaminyl cyclase activity in brain and peripheral tissue, suggesting ubiquitous expression of the isoQC enzyme. An assay of substrate conversion in vivo reveals impaired generation of the pGlu-modified C-C chemokine ligand 2 (CCL2, MCP-1) in isoQC-/- mice. The pGlu-formation was also impaired in primary neurons, which express significant levels of QC. Interestingly, however, the formation of the neuropeptide hormone thyrotropin-releasing hormone (TRH), assessed by immunohistochemistry and hormonal analysis of hypothalamic-pituitary-thyroid axis, was not affected in isoQC-/-, which contrasts to QC-/-. Thus, the results reveal differential functions of isoQC and QC in the formation of the pGlu-peptides CCL2 and TRH. Substrates requiring extensive prohormone processing in secretory granules, such as TRH, are primarily converted by QC. In contrast, protein substrates such as CCL2 appear to be primarily converted by isoQC. The results provide a new example, how subtle differences in subcellular localization of enzymes and substrate precursor maturation might influence pGlu-product formation.

Background Amyloid β (Aβ)-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 Aβ peptides might play a decisive role in onset and progression of AD and first clinical trials targeting such Aβ variants have been initiated. Modified Aβ represents a small fraction of deposited material in plaques compared to pan-Aβ epitopes, opening up pathways for tailored approaches of immunotherapy. Here, we generated the first monoclonal antibodies that recognize l-isoaspartate-modified Aβ (isoD7-Aβ) 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. Aβ 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-Aβ monoclonal antibody characterization by surface plasmon resonance, immunohistochemical staining of human and transgenic mouse brain, and the development and application of isoD7-Aβ ELISA as well as different non-modified Aβ ELISA. For antibody treatment studies, 12 mg/kg anti-isoD7-Aβ 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-Aβ compared to other Aβ variants. By using this antibody, we demonstrated that formation of isoD7-Aβ may occur after formation of aggregates; hence, the presence of the isoD7-modification differentiates aged Aβ 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-Aβ in cell culture. The presence of isoD7-Aβ 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-Aβ and total Aβ 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-Aβ epitope, the application of anti-isoD7-Aβ antibodies showed comparable treatment efficacy in terms of reduction of brain amyloid and spatial learning but did not result in an increase of plasma Aβ concentration as observed with 3D6 treatment. Conclusions The present study demonstrates, for the first time, that the antibody-mediated targeting of isoD7-modified Aβ peptides leads to attenuation of AD-like amyloid pathology. In conjunction with previously published data on antibodies directed against pGlu-modified Aβ, the results highlight the crucial role of modified Aβ peptides in AD pathophysiology. Hence, the results also underscore the therapeutic potential of targeting modified amyloid species for defining tailored approaches in AD therapy.