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.

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.

In the hippocampal formation of Alzheimer’s disease (AD) patients, both focal and diffuse deposits of Aβ peptides appear in a subregion- and layer-specific manner. Recently, pyroglutamate (pGlu or pE)-modified Aβ peptides were identified as a highly pathogenic and seeding Aβ peptide species. Since the pE modification is catalyzed by glutaminyl cyclase (QC) this enzyme emerged as a novel pharmacological target for AD therapy. Here, we reveal the role of QC in the formation of different types of hippocampal pE-Aβ aggregates. First, we demonstrate that both, focal and diffuse pE-Aβ deposits are present in defined layers of the AD hippocampus. While the focal type of pE-Aβ aggregates was found to be associated with the somata of QC-expressing interneurons, the diffuse type was not. To address this discrepancy, the hippocampus of amyloid precursor protein transgenic mice was analysed. Similar to observations made in AD, focal (i.e. core-containing) pE-Aβ deposits originating from QC-positive neurons and diffuse pE-Aβ deposits not associated with QC were detected in Tg2576 mouse hippocampus. The hippocampal layers harbouring diffuse pE-Aβ deposits receive multiple afferents from QC-rich neuronal populations of the entorhinal cortex and locus coeruleus. This might point towards a mechanism in which pE-Aβ and/or QC are being released from projection neurons at hippocampal synapses. Indeed, there are a number of reports demonstrating the reduction of diffuse, but not of focal, Aβ deposits in hippocampus after deafferentation experiments. Moreover, we demonstrate in neurons by live cell imaging and by enzymatic activity assays that QC is secreted in a constitutive and regulated manner. Thus, it is concluded that hippocampal pE-Aβ plaques may develop through at least two different mechanisms: intracellularly at sites of somatic QC activity as well as extracellularly through seeding at terminal fields of QC expressing projection neurons.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-011-0806-2) contains supplementary material, which is available to authorized users.

Glutaminyl cyclase (QC) converts N-terminal glutaminyl residues into pyroglutamate (pE), thereby stabilizing these peptides/proteins. Recently, we demonstrated that QC also plays a pathogenic role in Alzheimer's disease by generating the disease-associated pE-Abeta from N-terminally truncated Abeta peptides in vivo. This newly identified function makes QC an interesting pharmacological target for Alzheimer's disease therapy. However, the expression of QC in brain and peripheral organs, its cell type-specific and subcellular localization as well as developmental profiles in brain are not known. The present study was performed to address these issues in mice. In brain, QC mRNA expression was highest in hypothalamus, followed by hippocampus and cortex. In liver, QC mRNA concentration was almost as high as in brain while lower QC mRNA levels were detected in lung and heart and very low expression levels were found in kidney and spleen. In the developmental course, stable QC mRNA levels were detected in hypothalamus from postnatal day 5 to 370. On the contrary, in cortex and hippocampus QC mRNA levels were highest after birth and declined during ontogenesis by 20-25%. These results were corroborated by immunocytochemical analysis in mouse brain demonstrating a robust QC expression in a subpopulation of lateral and paraventricular hypothalamic neurons and the labeling of a significant number of small neurons in the hippocampal molecular layer, in the hilus of the dentate gyrus and in all layers of the neocortex. Hippocampal QC-immunoreactive neurons include subsets of parvalbumin-, calbindin-, calretinin-, cholecystokinin- and somatostatin-positive GABAergic interneurons. The density of QC labeled hippocampal neurons declined during postnatal development matching the decrease in QC mRNA expression levels. Subcellular double immunofluorescent analysis localized QC within the endoplasmatic reticulum, Golgi apparatus and secretory granules, consistent with a function of QC in protein maturation and/or modification. Our results are in compliance with a role of QC in hypothalamic hormone maturation and suggest additional, yet unidentified QC functions in brain regions relevant for learning and memory which are affected in Alzheimer's disease.

Glutaminyl cyclase (QC) was discovered recently as the enzyme catalyzing the pyroglutamate (pGlu or pE) modification of N-terminally truncated Alzheimer’s disease (AD) Aβ peptides in vivo. This modification confers resistance to proteolysis, rapid aggregation and neurotoxicity and can be prevented by QC inhibitors in vitro and in vivo, as shown in transgenic animal models. However, in mouse brain QC is only expressed by a relatively low proportion of neurons in most neocortical and hippocampal subregions. Here, we demonstrate that QC is highly abundant in subcortical brain nuclei severely affected in AD. In particular, QC is expressed by virtually all urocortin-1-positive, but not by cholinergic neurons of the Edinger–Westphal nucleus, by noradrenergic locus coeruleus and by cholinergic nucleus basalis magnocellularis neurons in mouse brain. In human brain, QC is expressed by both, urocortin-1 and cholinergic Edinger–Westphal neurons and by locus coeruleus and nucleus basalis Meynert neurons. In brains from AD patients, these neuronal populations displayed intraneuronal pE-Aβ immunoreactivity and morphological signs of degeneration as well as extracellular pE-Aβ deposits. Adjacent AD brain structures lacking QC expression and brains from control subjects were devoid of such aggregates. This is the first demonstration of QC expression and pE-Aβ formation in subcortical brain regions affected in AD. Our results may explain the high vulnerability of defined subcortical neuronal populations and their central target areas in AD as a consequence of QC expression and pE-Aβ formation.