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.

Fibrillar Aß triggers microglial proteome alterations and dysfunction in Alzheimer mouse models.

Microglial dysfunction is a key pathological feature of Alzheimer's disease (AD), but little is known about proteome-wide changes in microglia during the course of AD and their functional consequences. Here, we performed an in-depth and time-resolved proteomic characterization of microglia in two mouse models of amyloid ß (Aß) pathology, the overexpression APPPS1 and the knock-in APP-NL-G-F (APP-KI) model. We identified a large panel of Microglial Aß Response Proteins (MARPs) that reflect heterogeneity of microglial alterations during early, middle and advanced stages of Aß deposition and occur earlier in the APPPS1 mice. Strikingly, the kinetic differences in proteomic profiles correlated with the presence of fibrillar Aß, rather than dystrophic neurites, suggesting that fibrillar Aß may trigger the AD-associated microglial phenotype and the observed functional decline. The identified microglial proteomic fingerprints of AD provide a valuable resource for functional studies of novel molecular targets and potential biomarkers for monitoring AD progression or therapeutic efficacy.

Alzheimer's disease is a progressive, irreversible brain disorder. Patients with Alzheimer's have problems with memory and other mental skills, which lead to more severe cognitive decline and, eventually, premature death. This is due to increasing numbers of nerve cells in the brain dying over time. A distinctive feature of Alzheimer's is the abnormally high accumulation of a protein called amyloid-ß, which forms distinctive clumps in the brain termed 'plaques'. The brain has a type of cells called the microglia that identify infections, toxic material and damaged cells, and prevent these from building up by clearing them away. In Alzheimer's disease, however, the microglia do not work properly, which is thought to contribute to the accumulation of amyloid-ß plaques. This means that people with mutations in the genes important for the microglia activity are also at higher risk of developing the disease. Although problems with the microglia play an important role in Alzheimer's, researchers still do not fully understand why microglia stop working in the first place. It is also not known exactly when and how the microglia change as Alzheimer's disease progresses. To unravel this mystery, Sebastian Monasor, Müller et al. carried out a detailed study of the molecular 'fingerprints' of microglia at each key stage of Alzheimer's disease. The experiments used microglia cells from two different strains of genetically altered mice, both of which develop the hallmarks of Alzheimer's disease, including amyloid-ß plaques, at similar rates. Analysis of the proteins in microglia cells from both strains revealed distinctive, large-scale changes corresponding to successive stages of the disease ­ reflecting the gradual accumulation of plaques. Obvious defects in microglia function also appeared soon after plaques started to build up. Microscopy imaging of the brain tissue showed that although amyloid-ß plaques appeared at the same time, they looked different in each mouse strain. In one, plaques were more compact, while in the other, plaques appeared 'fluffier', like cotton wool. In mice with more compacted plaques, microglia recognized the plaques earlier and stopped working sooner, suggesting that plaque structure and microglia defects could be linked. These results shed new light on the role of microglia and their changing protein 'signals' during the different stages of Alzheimer's disease. In the future, this information could help identify people at risk for the disease, so that they can be treated as soon as possible, and to design new therapies to make microglia work again.

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.

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.