Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Abeta peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimer's disease. We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo. Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimer's disease and correlated with the appearance of pE-modified Abeta. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Abeta(3(pE)-42) burden in two different transgenic mouse models of Alzheimer's disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Abeta(x-40/42), diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Abeta(3(pE)-42) acts as a seed for Abeta aggregation by self-aggregation and co-aggregation with Abeta(1-40/42). Therefore, Abeta(3(pE)-40/42) peptides seem to represent Abeta forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Abeta by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimer's disease and provides implications for other amyloidoses, such as familial Danish dementia.

Extracellular plaques of β-amyloid (Aβ) and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease (AD). Plaques comprise Aβ fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of AD. Despite the significance of plaques to AD, oligomers are considered to be the principal toxic forms of Aβ 1,2 . Interestingly, many adverse responses to Aβ, such as cytotoxicity 3 , microtubule loss 4 , impaired memory and learning 5 , and neuritic degeneration 6 , are greatly amplified by tau expression. N-terminally truncated, pyroglutamylated (pE) forms of Aβ 7,8 are strongly associated with AD, are more toxic than Aβ 1-42 and Aβ , and have been proposed as initiators of AD Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms * Correspondence: gsb4g@virginia.edu. **Correspondence: Hans-Ulrich.Demuth@probiodrug.de. J.M.N and S.S. contributed equally to the paper.Full Methods and relevant references will be available in the online Supplementary Information accompanying this paper at http:// www.nature.com/nature.Author Contributions: J.M.N. performed most of the biochemical and cell biological experiments; S.S. was the principal force behind the experiments involving hAPP SL /hQC and TBA2.1/tau KO mice, and was aided by B.H.-P., H.C.; A.S. and T.W. fractionated and analyzed human brain extracts; E.S., K.Y. and B.W. performed the peri-hippocampal injection experiments; A.H. and C.G.G. produced and characterized the M64 and M87 antibodies; R.R. and K.R. performed the electrophysiology experiments; A.A., W.J. and S.G. performed and analyzed the immunohistochemical experiments on TBA2.1 and Tau-KO/TBA2.1 mice; G.S.B. and H.-U.D. initiated and directed the project; G.S.B. was the principal writer of the paper; all of the authors participated in the design and analysis of experiments, and in editing of the paper. Fig. 2) to the oligomers. HHS Public AccessAt 5 μM peptide, 5% pE-Aβ aggregated faster than Aβ 3(pE)-42 or Aβ 1-42 alone based on thioflavin T fluorescence shifts 15 ( Supplementary Fig. 3). The OD 450 /OD 490 ratio for Aβ 3(pE)-42 rose and peaked more rapidly than for Aβ 1-42 , but peaked at an ~25% lower level. The fastest rise in the OD 450 /OD 490 ratio was for 5% pE-Aβ, which peaked similarly to Aβ 3(pE)-42 . Aβ 3(pE)-42 , Aβ 1-42 and 5% pE-Aβ thus oligomerized by different pathways.To test whether distinct biological activities were coupled to these oligomerization differences, we compared cytotoxicity of the peptides towards cultured neurons or glia using calcein-AM and fluorescence microscopy 16 . Twelve hours of Aβ 1-42 exposure had little effect on cell viability for wild type (WT) or tau knockout (KO) neurons, or WT glial cells (Fig. 1a). Contrastingly, most WT neurons died and detached from the substrate after exposur...

Posttranslational amyloid-␤ (A␤) modification is considered to play an important role in Alzheimer's disease (AD)etiology. An N-terminally modified A␤ species, pyroglutamate-amyloid-␤ (pE3-A␤), has been described as a major constituent of A␤ deposits specific to human AD but absent in normal aging. Formed via cyclization of truncated A␤ species by glutaminyl cyclase (QC; QPCT) and/or its isoenzyme (isoQC; QPCTL), pE3-A␤ aggregates rapidly and is known to seed additional A␤ aggregation. To directly investigate pE3-A␤ toxicity in vivo, we generated and characterized transgenic TBA2.1 and TBA2.2 mice, which express truncated mutant human A␤. Along with a rapidly developing behavioral phenotype, these mice showed progressively accumulating A␤ and pE3-A␤ deposits in brain regions of neuronal loss, impaired long-term potentiation, microglial activation, and astrocytosis. Illustrating a threshold for pE3-A␤ neurotoxicity, this phenotype was not found in heterozygous animals but in homozygous TBA2.1 or double-heterozygous TBA2.1/2.2 animals only. A significant amount of pE3-A␤ formation was shown to be QC-dependent, because crossbreeding of TBA2.1 with QC knock-out, but not isoQC knock-out, mice significantly reduced pE3-A␤ levels. Hence, lowering the rate of QC-dependent posttranslational pE3-A␤ formation can, in turn, lower the amount of neurotoxic A␤ species in AD.

It is well established that only a fraction of Ab peptides in the brain of Alzheimer's disease (AD) patients start with N-terminal aspartate (Ab 1D ) which is generated by proteolytic processing of amyloid precursor protein (APP) by BACE. N-terminally truncated and pyroglutamate modified Ab starting at position 3 and ending with amino acid 42 [Ab 3(pE)-42 ] have been previously shown to represent a major species in the brain of AD patients. When compared with Ab 1-42 , this peptide has stronger aggregation propensity and increased toxicity in vitro. Although it is unknown which peptidases remove the first two N-terminal amino acids, the cyclization of Ab at N-terminal glutamate can be catalyzed in vitro. Here, we show that Ab 3(pE)-42 induces neurodegeneration and concomitant neurological deficits in a novel mouse model (TBA2 transgenic mice). Although TBA2 transgenic mice exhibit a strong neuronal expression of Ab 3-42 predominantly in hippocampus and cerebellum, few plaques were found in the cortex, cerebellum, brain stem and thalamus. The levels of converted Ab 3(pE)-42 in TBA2 mice were comparable to the APP/PS1KI mouse model with robust neuron loss and associated behavioral deficits. Eight weeks after birth TBA2 mice developed massive neurological impairments together with abundant loss of Purkinje cells. Although the TBA2 model lacks important AD-typical neuropathological features like tangles and hippocampal degeneration, it clearly demonstrates that intraneuronal Ab 3(pE)-42 is neurotoxic in vivo.

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.

Brains of Alzheimer's disease (AD) patients are characterized in part by the formation of high molecular weight aggregates of amyloid-β (Aβ) peptides, which interfere with neuronal function and provoke neuronal cell death. The pyroglutamate (pGlu) modification of Aβ was demonstrated to be catalyzed by the enzyme glutaminyl cyclase (QC) and to enhance pathogenicity and neurotoxicity. Here, we addressed the role of QC in AD pathogenesis in human cortex. Two sets of human postmortem brain tissue from a total of 13 non-demented controls and 11 AD cases were analyzed by immunohistochemistry and unbiased stereology, quantitative RT-PCR, and enzymatic activity assays for the expression level of QC in temporal and entorhinal cortex. Additionally, cortical Aβ and pGlu-Aβ concentrations were quantified by ELISA. Data on QC expression and Aβ peptide concentrations were correlated with each other and with the Mini-Mental State Examination (MMSE) of individual cases. In control cases, QC expression was higher in the more vulnerable entorhinal cortex than in temporal cortex. In AD brains, QC mRNA expression and the immunoreactivity of QC were increased in both cortical regions and frequently associated with pGlu-Aβ deposits. The analyses of individual cases revealed significant correlations between QC mRNA levels and the concentration of insoluble pGlu-Aβ aggregates, but not of unmodified Aβ peptides. Elevated pGlu-Aβ load showed a better correlation with the decline in MMSE than elevated concentration of unmodified Aβ. Our observations provide evidence for an involvement of QC in AD pathogenesis and cognitive decline by QC-catalyzed pGlu-Aβ formation.

Amyloid-β (Aβ) peptides, starting with pyroglutamate at the third residue (pyroGlu-3 Aβ), are a major species deposited in the brain of Alzheimer disease (AD) patients. Recent studies suggest that this isoform shows higher toxicity and amyloidogenecity when compared to full-length Aβ peptides. Here, we report the first comprehensive and comparative IHC evaluation of pyroGlu-3 Aβ deposition in humans and animal models. PyroGlu-3 Aβ immunoreactivity (IR) is abundant in plaques and cerebral amyloid angiopathy of AD and Down syndrome patients, colocalizing with general Aβ IR. PyroGlu-3 Aβ is further present in two nontransgenic mammalian models of cerebral amyloidosis, Caribbean vervets, and beagle canines. In addition, pyroGlu-3 Aβ deposition was analyzed in 12 different AD-like transgenic mouse models. In contrast to humans, all transgenic models showed general Aβ deposition preceding pyroGlu-3 Aβ deposition. The findings varied greatly among the mouse models concerning age of onset and cortical brain region. In summary, pyroGlu-3 Aβ is a major species of β-amyloid deposited early in diffuse and focal plaques and cerebral amyloid angiopathy in humans and nonhuman primates, whereas it is deposited later in a subset of focal and vascular amyloid in AD-like transgenic mouse models. Given the proposed decisive role of pyroGlu-3 Aβ peptides for the development of human AD pathology, this study provides insights into the usage of animal models in AD studies.

Compelling evidence suggests that N-terminally truncated and pyroglutamyl-modified amyloid-beta (Abeta) peptides play a major role in the development of Alzheimer's disease. Posttranslational formation of pyroglutamic acid (pGlu) at position 3 or 11 of Abeta implies cyclization of an N-terminal glutamate residue rendering the modified peptide degradation resistant, more hydrophobic, and prone to aggregation. Previous studies using artificial peptide substrates suggested the potential involvement of the enzyme glutaminyl cyclase in generation of pGlu-Abeta. Here we show that glutaminyl cyclase (QC) catalyzes the formation of Abeta 3(pE)-40/42 after amyloidogenic processing of APP in two different cell lines, applying specific ELISAs and Western blotting based on urea-PAGE. Inhibition of QC by the imidazole derivative PBD150 led to a blockage of Abeta 3(pE)-42 formation. Apparently, the QC-catalyzed formation of N-terminal pGlu is favored in the acidic environment of secretory compartments, which is also supported by double-immunofluorescence labeling of QC and APP revealing partial colocalization. Finally, initial investigations focusing on the molecular pathway leading to the generation of truncated Abeta peptides imply an important role of the amino acid sequence near the beta-secretase cleavage site. Introduction of a single-point mutation, resulting in an amino acid substitution, APP(E599Q), i.e., at position 3 of Abeta, resulted in significant formation of Abeta 3(pE)-40/42. Introduction of the APP KM595/596NL "Swedish" mutation causing overproduction of Abeta, however, surprisingly diminished the concentration of Abeta 3(pE)-40/42. The study provides new cell-based assays for the profiling of small molecule inhibitors of QC and points to conspicuous differences in processing of APP depending on sequence at the beta-secretase cleavage site.