Glutaminyl cyclase contributes to the formation of focal and diffuse pyroglutamate (pGlu)-Aβ deposits in hippocampus via distinct cellular mechanisms

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

Cell Type-Specific Human APP Transgene Expression by Hippocampal Interneurons in the Tg2576 Mouse Model of Alzheimer’s Disease

Amyloid precursor protein (APP) transgenic animal models of Alzheimer’s disease have become versatile tools for basic and translational research. However, there is great heterogeneity of histological, biochemical, and functional data between transgenic mouse lines, which might be due to different transgene expression patterns. Here, the expression of human APP (hAPP) by GABAergic hippocampal interneurons immunoreactive for the calcium binding proteins parvalbumin, calbindin, calretinin, and for the peptide hormone somatostatin was analyzed in Tg2576 mice by double immunofluorescent microscopy. Overall, there was no GABAergic interneuron subpopulation that did not express the transgene. On the other hand, in no case all neurons of such a subpopulation expressed hAPP. In dentate gyrus molecular layer and in stratum lacunosum moleculare less than 10% of hAPP-positive interneurons co-express any of these interneuron markers, whereas in stratum oriens hAPP-expressing neurons frequently co-express these interneuron markers to different proportions. We conclude that these neurons differentially contribute to deficits in young Tg2576 mice before the onset of Abeta plaque pathology. The detailed analysis of distinct brain region and neuron type-specific APP transgene expression patterns is indispensable to understand particular pathological features and mouse line-specific differences in neuronal and systemic functions

Endogenous mouse huntingtin is highly abundant in cranial nerve nuclei, co-aggregates to Abeta plaques and is induced in reactive astrocytes in a transgenic mouse model of Alzheimer’s disease

Pathogenic variants of the huntingtin (HTT) protein and their aggregation have been investigated in great detail in brains of Huntington’s disease patients and HTT-transgenic animals. However, little is known about the physiological brain region- and cell type-specific HTT expression pattern in wild type mice and a potential recruitment of endogenous HTT to other pathogenic protein aggregates such as amyloid plaques in cross seeding events. Employing a monoclonal anti-HTT antibody directed against the HTT mid-region and using brain tissue of three different mouse strains, we detected prominent immunoreactivity in a number of brain areas, particularly in cholinergic cranial nerve nuclei, while ubiquitous neuronal staining appeared faint. The region-specific distribution of endogenous HTT was found to be comparable in wild type rat and hamster brain. In human amyloid precursor protein transgenic Tg2576 mice with amyloid plaque pathology, similar neuronal HTT expression patterns and a distinct association of HTT with Abeta plaques were revealed by immunohistochemical double labelling. Additionally, the localization of HTT in reactive astrocytes was demonstrated for the first time in a transgenic Alzheimer’s disease animal model. Both, plaque association of HTT and occurrence in astrocytes appeared to be age-dependent. Astrocytic HTT gene and protein expression was confirmed in primary cultures by RT-qPCR and by immunocytochemistry. We provide the first detailed analysis of physiological HTT expression in rodent brain and, under pathological conditions, demonstrate HTT aggregation in proximity to Abeta plaques and Abeta-induced astrocytic expression of endogenous HTT in Tg2576 mice

Distinct glutaminyl cyclase expression in Edinger–Westphal nucleus, locus coeruleus and nucleus basalis Meynert contributes to pGlu-Aβ pathology 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

Identification of thyrotropin-releasing hormone as hippocampal glutaminyl cyclase substrate in neurons and reactive astrocytes

Recently, Aβ peptide variants with an N-terminal truncation and pyroglutamate modification were identified and shown to be highly neurotoxic and prone to aggregation. This modification of Aβ is catalyzed by glutaminyl cyclase (QC) and pharmacological inhibition of QC diminishes Aβ deposition and accompanying gliosis and ameliorates memory impairment in transgenic mouse models of Alzheimer's disease (AD). QC expression was initially described in the hypothalamus, where thyrotropin-releasing hormone (TRH) is one of its physiological substrates. In addition to its hormonal role, a novel neuroprotective function of TRH following excitotoxicity and Aβ-mediated neurotoxicity has been reported in the hippocampus. Functionally matching this finding, we recently demonstrated QC expression by hippocampal interneurons in mouse brain. Here, we detected neuronal co-expression of QC and TRH in the hippocampus of young adult wild type mice using double immunofluorescence labeling. This provides evidence for TRH being a physiological QC substrate in hippocampus. Additionally, in neocortex of aged but not of young mice transgenic for amyloid precursor protein an increase of QC mRNA levels was found compared to wild type littermates. This phenomenon was not observed in hippocampus, which is later affected by Aβ pathology. However, in hippocampus of transgenic - but not of wild type mice - a correlation between QC and TRH mRNA levels was revealed. This co-regulation of the enzyme QC and its substrate TRH was reflected by a co-induction of both proteins in reactive astrocytes in proximity of Aβ deposits. Also, in primary mouse astrocytes a co-induction of QC and TRH was demonstrated upon Aβ stimulation

Immunohistochemical Demonstration of the pGlu79 α-Synuclein Fragment in Alzheimer’s Disease and Its Tg2576 Mouse Model

The deposition of β-amyloid peptides and of α-synuclein proteins is a neuropathological hallmark in the brains of Alzheimer’s disease (AD) and Parkinson’s disease (PD) subjects, respectively. However, there is accumulative evidence that both proteins are not exclusive for their clinical entity but instead co-exist and interact with each other. Here, we investigated the presence of a newly identified, pyroglutamate79-modified α-synuclein variant (pGlu79-aSyn)—along with the enzyme matrix metalloproteinase-3 (MMP-3) and glutaminyl cyclase (QC) implicated in its formation—in AD and in the transgenic Tg2576 AD mouse model. In the human brain, pGlu79-aSyn was detected in cortical pyramidal neurons, with more distinct labeling in AD compared to control brain tissue. Using immunohistochemical double and triple labelings and confocal laser scanning microscopy, we demonstrate an association of pGlu79-aSyn, MMP-3 and QC with β-amyloid plaques. In addition, pGlu79-aSyn and QC were present in amyloid plaque-associated reactive astrocytes that were also immunoreactive for the chaperone heat shock protein 27 (HSP27). Our data are consistent for the transgenic mouse model and the human clinical condition. We conclude that pGlu79-aSyn can be generated extracellularly or within reactive astrocytes, accumulates in proximity to β-amyloid plaques and induces an astrocytic protein unfolding mechanism involving HSP27

Defined astrocytic expression of human amyloid precursorprotein in Tg2576 mouse brain

Transgenic Tg2576 mice expressing human amyloid precursor protein (hAPP) with the Swedish mutation are among the most frequently used animal models to study the amyloid pathology related to Alzheimer's disease (AD). The transgene expression in this model is considered to be neuron‐specific. Using a novel hAPP‐specific antibody in combination with cell type‐specific markers for double immunofluorescent labelings and laser scanning microscopy, we here report that—in addition to neurons throughout the brain—astrocytes in the corpus callosum and to a lesser extent in neocortex express hAPP. This astrocytic hAPP expression is already detectable in young Tg2576 mice before the onset of amyloid pathology and still present in aged Tg2576 mice with robust amyloid pathology in neocortex, hippocampus, and corpus callosum. Surprisingly, hAPP immunoreactivity in cortex is restricted to resting astrocytes distant from amyloid plaques but absent from reactive astrocytes in close proximity to amyloid plaques. In contrast, neither microglial cells nor oligodendrocytes of young or aged Tg2576 mice display hAPP labeling. The astrocytic expression of hAPP is substantiated by the analyses of hAPP mRNA and protein expression in primary cultures derived from Tg2576 offspring. We conclude that astrocytes, in particular in corpus callosum, may contribute to amyloid pathology in Tg2576 mice and thus mimic this aspect of AD pathology

Glutaminyl cyclase in human cortex: Correlation with (pGlu)-amyloid-β load and cognitive decline in Alzheimer's disease

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