Maike Hartlage-Rübsamen

Astrocytic expression of the Alzheimer's disease β-secretase (BACE1) is stimulus-dependent

The beta‐site APP‐cleaving enzyme (BACE1) is a prerequisite for the generation of β‐amyloid peptides, which give rise to cerebrovascular and parenchymal β‐amyloid deposits in the brain of Alzheimers disease patients. BACE1 is neuronally expressed in the brains of humans and experimental animals such as mice and rats. In addition, we have recently shown that BACE1 protein is expressed by reactive astrocytes in close proximity to β‐amyloid plaques in the brains of aged transgenic Tg2576 mice that overexpress human amyloid precursor protein carrying the double mutation K670N‐M671L. To address the question whether astrocytic BACE1 expression is an event specifically triggered by β‐amyloid plaques or whether glial cell activation by other mechanisms also induces BACE1 expression, we used six different experimental strategies to activate brain glial cells acutely or chronically. Brain sections were processed for the expression of BACE1 and glial markers by double immunofluorescence labeling and evaluated by confocal laser scanning microscopy. There was no detectable expression of BACE1 protein by activated microglial cells of the ameboid or ramified phenotype in any of the lesion paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1‐immunoreactive astrocytes in proximity to β‐amyloid plaques in the brains of aged Tg2576 mice and Alzheimers disease patients. GLIA 41:169–179, 2003.

Region and lamina-specific distribution of extracellular matrix proteoglycans, hyaluronan and tenascin-R in the mouse hippocampal formation

The extracellular matrix is known to show region-specific characteristics in the adult brain. Our comparative cytochemical study is focused on the laminar organisation of major extracellular matrix constituents in the murine hippocampal formation, including the regions CA1, CA2 and CA3 of the hippocampus proper, the dentate gyrus, the subiculum and the presubiculum. Components related to chondroitin sulphate proteoglycans were detected by N-acetylgalactosamine-binding Wisteria floribunda agglutinin, colloidal iron staining, and antibodies to different proteoglycan domains, including the Cat-301 and Cat-315 epitopes of aggrecan, as well as neurocan, brevican and phosphacan. The distribution patterns of these components were correlated with the patterns revealed for hyaluronan and the brain-specific extracellular matrix glycoprotein, tenascin-R, known to be ligands of extracellular matrix proteoglycans. Lectin binding clearly labelled perineuronal nets of the extracellular matrix around interneurons, which were preferentially located within or near the principal cell layers in all regions. In the hippocampus proper, the CA2 subfield showed an intense labelling of the neuropil around pyramidal cell bodies and the neuropil zones in the strata oriens and radiatum. These patterns were also seen after immunoreaction for chondroitin proteoglycan domains, brevican and phosphacan, as well as after detection of hyaluronan and tenascin-R. Characteristic laminar and intralaminar patterns were additionally expressed in the neuropil in all regions. In the dentate gyrus, the staining intensity for brevican, phosphacan and tenascin-R was predominant in the middle molecular layer, and for Cat-315 in the inner molecular layer, whereas immunoreactivity for neurocan increased within the outer molecular layer towards the hippocampal fissure. Our findings indicate that proteoglycans, hyaluronan and tenascin-R show differential patterns of co-expression in the individual regions and laminae of the hippocampal formation. The inhomogeneous composition of these major components suggests that the extracellular matrix is specifically adapted to the functional domains of intrahippocampal connections and afferent fibre systems.

Interleukin-1β, inducible nitric oxide synthase, and nuclear factor-κB are induced in morphologically distinct microglia after rat hippocampal lipopolysaccharide/interferon-γ injection

In a number of pathological states of the brain, the activation of the inducible nitric oxide synthase (iNOS) plays a major role. Interleukin (IL)‐1β is believed to be an essential factor in the induction of iNOS. However, little is known about the cascade of events culminating in iNOS expression in vivo. To identify the morphological as well as temporal relationship of lipopolysaccharide (LPS)/interferon‐γ (IFN‐γ) ‐induced microglial iNOS‐ and IL‐1β expression, a mixture of LPS and IFN‐γ was injected into the rat hippocampus. IL‐1β immunoreactivity was detected as early as 3 hr following surgery in ramified microglia in the lesioned hippocampus and in distal cortical layers adjacent to the pia mater. By 12 hr post‐injection, IL‐1β immunoreactive, ramified microglia with swollen processes were widely distributed throughout hippocampal and neocortical areas, and staining was observed up to 48 hr after treatment. In contrast, iNOS immunostaining was seen in activated amoeboid microglia/macrophages in the ipsilateral hippocampus and around blood vessels but not earlier than 12 hr post‐surgery. The temporal pattern of iNOS and IL‐1β expression corresponded to newly induced transcriptional activity as revealed by RT‐PCR. Activation of NF‐κB was restricted to brain regions in which IL‐1β was expressed and was detected both in microglia and astrocytes. A number of LPS/IFN‐γ‐stimulated, IL‐1β‐expressing microglia exhibited co‐staining for activated NF‐κB. The finding that IL‐1β precedes iNOS expression is consistant with a role of IL‐1β in the intercellular signaling events leading to microglial iNOS‐induction. Co‐localization of IL‐1β and NF‐κB suggests an association between IL‐1β and NF‐κB induction. J. Neurosci. Res. 57:388–398, 1999.

Differential injury-dependent glial expression of interleukins-1α, β, and interleukin-6 in rat brain

Interleukins (IL)‐1α, β and IL‐6 may play essential roles in early inflammatory processes in response to degenerating cholinergic cells observed in the basal forebrain of Alzheimer patients. To address this question in vivo, two distinct lesion paradigms were used. A specific and selective basal forebrain cholinergic cell loss was achieved by a single intracerebroventricular application of the cholinergic immunotoxin, 192IgG‐saporin. Intrahippocampal injection of lipopolysaccharide and interferon‐γ was used to produce an exogenously‐induced acute inflammation in the brain. In order to disclose the lesion‐induced temporal cascade of the expression pattern of IL‐1α, IL‐1β, and IL‐6, and the cell types expressing IL‐1α, β/IL‐6 mRNA, Western analysis, RT‐PCR, and double labeling immunocytochemistry were applied. In the intact brain, IL‐6, IL‐1α and IL‐1β demonstrated a constitutive expression in neurons. Following cholinergic lesion neither IL‐1β nor IL‐6 expression could be detected in any of the activated glial cell types, whereas IL‐1α was found to be expressed in astroglial cells only. In contrast, hippocampal administration of lipopolysaccharides/interferon‐γ resulted in expression of IL‐1α in microglial but not astroglial cells. These in vivo studies clearly demonstrate that the cellular expression of IL‐1α, IL‐1β, and IL‐6 in the brain is differentially regulated depending on the kind of injury producing the inflammatory response in the brain. The data suggest that each glial cell seems to be equally capable of expressing a number of various cytokines, but it depends on the kind of stimulus which temporal and cellular cascade of cytokine expression pattern is initiated under a particular pathological condition in the brain. GLIA 27:75–87, 1999.

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.

Formation of perineuronal nets in organotypic mouse brain slice cultures is independent of neuronal glutamatergic activity

Perineuronal nets (PNs) are a specialized form of the extracellular matrix and cover specific sets of neurons in distinct brain areas. Animal experiments on sensory visual deprivation have demonstrated that the generation of PNs around neurons of the visual cortex is dependent on neuronal activity during the critical period of visual experience. The importance of the activity of specific neurotransmitter systems for PN formation has, however, not yet been demonstrated. Based on the predominantly glutamatergic innervation of the visual cortex we hypothesized that reduced glutamatergic activity impairs the development of PNs. To address this question, genetic mouse models with compromised glutamate release [Munc13‐1‐knockout (KO) and Munc13‐1/2 double‐KO (DKO)] and chronic pharmacological treatments interfering with specific steps of glutamatergic transmission were used. Under experimental conditions of glutamatergic hypofunction PN formation was studied in organotypic brain slice cultures with Wisteria floribunda lectin binding and with aggrecan immunohistochemistry. After cultivation for 21 days a regular PN formation was observed in brain slices (i) derived from Munc13‐1‐KO and Munc13‐1/2‐DKO mice, (ii) after blockade of metabotropic and ionotropic glutamate receptors with MCPG and kynurenate, and (iii) after suppression of glutamate release by blockade of presynaptic Ca++ channels with riluzole. Nonselective suppression of neuronal activity by blockade of voltage‐gated sodium channels with tetrodotoxin clearly inhibited PN formation. These results indicate that neuronal activity is required but that the glutamatergic system is not essential for PN development.

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

Brains of Alzheimers 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.

Tenascin-R promotes assembly of the extracellular matrix of perineuronal nets via clustering of aggrecan

Perineuronal nets (PNs) in the brains of tenascin-R-deficient (tn-r−/−) mice develop in temporal concordance with those of wild-type (tn-r+/+) mice. However, the histological appearance of PNs is abnormal in adult tn-r−/− mice. Here, we investigated whether similar defects are also seen in dissociated and organotypic cultures from hippocampus and forebrain of tn-r−/− mice and whether the structure of PNs could be normalized. In tn-r−/− cultures, accumulations of several extracellular matrix molecules were mostly associated with somata, whereas dendrites were sparsely covered, compared with tn-r+/+ mice. Experiments to normalize the structure of PNs in tn-r−/− organotypic slice cultures by depolarization of neurons, or by co-culturing tn-r+/+ and tn-r−/− brain slices failed to restore a normal PN phenotype. However, formation of dendritic PNs in cultures was improved by the application of tenascin-R protein and rescued by polyclonal antibodies to aggrecan and a bivalent, but not monovalent form of the lectin Wisteria floribunda agglutinin. These results show that tenascin-R and aggrecan are decisive contributors to formation and stabilization of PNs and that tenascin-R may implement these functions by clustering of aggrecan. Proposed approaches for restoration of normal PN structure are noteworthy in the context of PN abnormalities in neurological disorders, such as epilepsy, schizophrenia and addiction.

Fibrillary beta-amyloid deposits are closely associated with atrophic nitric oxide synthase (NOS)-expressing neurons but do not upregulate the inducible NOS in transgenic Tg2576 mouse brain with Alzheimer pathology

Transgenic mice (Tg2576) that express the Swedish double mutation of human amyloid precursor protein and develop Alzheimer-like beta-amyloid deposits in the aged brain, were used to study the effect of beta-amyloid deposition on expression of both neuronal (nNOS) and inducible nitric oxide synthase (iNOS) in cells surrounding beta-amyloid plaques. Nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and double immunofluorescent labeling revealed that most of the fibrillary, thioflavine-S-positive cortical beta-amyloid deposits in 13-, 17-, and 21-month-old transgenic animals were closely associated with dystrophic nNOS-positive neurons, while nNOS-bearing neurons located more distal to plaques appeared to be unaffected. There was no significant expression of iNOS in transgenic mouse brain. The data suggest enhanced vulnerability of nNOS-containing neocortical neurons to beta-amyloid toxicity. Alternatively, expression of nNOS may also be a response to plaque-mediated damage of neurons, consistent with a neuroprotective role of nitric oxide.

Developmental expression and subcellular localization of glutaminyl cyclase in mouse brain.

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 Alzheimers 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 Alzheimers 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 Alzheimers disease.