Jenny Apelt

β-Amyloid-induced glial expression of both pro- and anti-inflammatory cytokines in cerebral cortex of aged transgenic Tg2576 mice with Alzheimer plaque pathology

To elucidate the mechanisms involved in beta-amyloid-mediated inflammation in Alzheimers disease, transgenic Tg2576 mice containing as transgene the Swedish double mutation of human amyloid precursor protein 695, were examined for the expression pattern of various cytokines using double immunocytochemistry and laser scanning microscopy. Tg2576 mice studied at postnatal ages of 13, 16 and 19 months demonstrated an age-related accumulation of both senile and diffuse beta-amyloid plaques in neocortex and hippocampus. Reactive interleukin (IL)-1beta-immunoreactive astrocytes were found in close proximity to both fibrillary and diffuse beta-amyloid deposits detectable at very early stages of plaque development, while activated microglia appeared in and around fibrillary beta-amyloid plaques only. Subpopulations of reactive astrocytes also demonstrated immunolabeling for transforming growth factor (TGF)-beta1, TGF-beta3, and IL-10, already detectable in 13-month-old transgenic mouse brain, while a few IL-6-immunoreactive astrocytes were observed only at later stages of plaque development. The early beta-amyloid-mediated upregulation of IL-1beta, TGF-beta, and IL-10 in surrounding reactive astrocytes indicates the induction of both pro- and anti-inflammatory mechanisms. The transgenic approach used in this study may thus provide a useful tool to further disclose the in vivo mechanisms by which pro- and anti-inflammatory cytokines interact and/or contribute to the progression of Alzheimers disease.

Experimental traumatic brain injury in rats stimulates the expression, production and activity of Alzheimer’s disease β-secretase (BACE-1)

Summary.Traumatic brain injury (TBI) is a risk factor for the development of Alzheimer’s disease (AD). After a traumatic brain injury depositions of amyloid beta (Aβ) in the brain parenchyma were found. In this study we investigated the expression pattern of β-secretase (BACE-1) in ipsi- or contralateral hippocampus and cortex following controlled cortical TBI in rats. BACE-1 mRNA levels, estimated by real time RT-PCR, were elevated 24 h post injury, and persisting up to 72 h, in the ipsi- and contralateral hippocampus and cerebral cortex as compared to the sham-treated animals (p<0.01). The TBI-induced changes in BACE-1 mRNA are due to enhanced hippocampal and cortical expression of BACE-1 mRNA in neurons and reactive astrocytes as revealed by in situ hybridization. The alterations in hippocampal BACE-1 mRNA levels are accompanied by corresponding increases in BACE-1 protein levels in ipsi- and contralateral hippocampus and ipsilateral cortex as demonstrated by Western blot analysis. In contrast, in the contralateral cortex only a weak increase of traumatically induced BACE-1 protein production was found. The activity of BACE-1 as measured by the formation of the cleavage product of amyloid beta precursor protein, transiently increased up to 48 h after injury, but returned to basal level 7 days post injury. This study demonstrates that the β-secretase is stimulated following TBI and may suggest a mechanism for the temporal increase of Aβ levels observed in patients with brain trauma.

Insulin-sensitive GLUT4 glucose transporters are colocalized with GLUT3-expressing cells and demonstrate a chemically distinct neuron-specific localization in rat brain.

The insulin‐sensitive glucose transporter (GLUT) 4, expressed primarily in peripheral tissue, has recently been detected also in the brain, demonstrating a region‐specific distribution. To identify the chemical nature of neurons expressing GLUT4 and to disclose whether GLUT4‐containing neurons also express the GLUT3 isoform, combined in situ hybridization for GLUT3 mRNA and double‐labeling immunocytochemistry for GLUT4 and different cellular markers was performed in brain sections through rat basal forebrain, cerebral cortex, hippocampus, and cerebellum. In all brain regions examined, GLUT4 immunoreactivity was exclusively found in neurons, and GLUT4‐immunoreactive cells were colocalized with neurons expressing GLUT3 mRNA. In rat basal forebrain, cholinergic and parvalbumin‐containing γ‐aminobutyric acid‐ergic cells demonstrated GLUT4 immunoreactivity, whereas calretinin‐, calbindin‐D‐, and neuronal nitric oxide synthase‐containing neurons did not express GLUT4 protein. Because brain GLUT4 transporters have been suggested to play a role in rapidly providing additional glucose to neurons under conditions of high‐energy demand, the selective presence of GLUT4 in basal forebrain cholinergic cells may explain the specific vulnerability of these cells to a lack of glucose supply. J. Neurosci. Res. 57:693–705, 1999.

Aging-related increase in oxidative stress correlates with developmental pattern of beta-secretase activity and beta-amyloid plaque formation in transgenic Tg2576 mice with Alzheimer-like pathology.

The molecular mechanisms of β‐amyloidogenesis in sporadic Alzheimers disease are still poorly understood. To reveal whether aging‐associated increases in brain oxidative stress and inflammation may trigger onset or progression of β‐amyloid deposition, a transgenic mouse (Tg2576) that express the Swedish double mutation of human amyloid precursor protein (APP) was used as animal model to study the developmental pattern of markers of oxidative stress and APP processing. In Tg2576 mouse brain, cortical levels of soluble β‐amyloid (1–40) and (1–42) steadily increased with age, but significant deposition of fibrillary β‐amyloid in cortical areas did not occur before postnatal age of 10 months. The slope of increase in cerebral cortical β‐secretase (BACE1) activities in Tg2576 mice between ages of 9 and 13 months was significantly higher as compared to that of the α‐secretase, while the expression level of BACE1 protein and mRNA did not change with age. The activities of superoxide dismutase and glutathione peroxidase in cortical tissue from Tg2576 mice steadily increased from postnatal age 9–12 months. The levels of cortical nitric oxide, and reactive nitrogen species demonstrated peak values around 9 months of age, while the level of interleukin‐1β steadily increased from postnatal month 13 onwards. The developmental temporal coincidence of increased levels of reactive nitrogen species and antioxidative enzymes with the onset of β‐amyloid plaque deposition provides further evidence that developmentally and aging‐induced alterations in brain oxidative status exhibit a major factor in triggering enhanced production and deposition of β‐amyloid, and potentially predispose to Alzheimers disease.

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.

Aging-related down-regulation of neprilysin, a putative β-amyloid-degrading enzyme, in transgenic Tg2576 Alzheimer-like mouse brain is accompanied by an astroglial upregulation in the vicinity of β-amyloid plaques

Pathological accumulation of cortical beta-amyloid is an early and consistent feature of Alzheimers disease. Brain level of beta-amyloid is determined both by its production and by its catabolism. Neprilysin, a zinc metalloproteinase has been suggested as potential candidate of beta-amyloid-degrading enzyme in vivo. To address the question whether pathological accumulation of beta-amyloid peptides in transgenic Tg2576 mice with Alzheimer-like pathology may affect beta-amyloid catabolism, the expression of neprilysin was studied during postnatal maturation and aging. Neprilysin protein but mRNA levels decreased in mouse cerebral cortex with age (2-22 months), independently of transgene status. Immunocytochemistry revealed few neprilysin-positive dystrophic neurites around beta-amyloid plaques and an upregulation of neprilysin in plaque-surrounding reactive astrocytes which may suggest a role of plaque-mediated astrogliosis in beta-amyloid degradation.

Neuronal and glial β-secretase (BACE) protein expression in transgenic Tg2576 mice with amyloid plaque pathology

We measured tissue distribution and expression pattern of the beta‐site amyloid precursor protein (APP)‐cleaving enzyme (BACE) in the brains of transgenic Tg2576 mice that show amyloid pathology. BACE protein was expressed at high levels in brain; at lower levels in heart and liver; and at very low levels in pancreas, kidney, and thymus and was almost absent in spleen and lung when assayed by Western blot analysis. We observed strictly neuronal expression of BACE protein in the brains of nontransgenic control mice, with the most robust immunocytochemical labeling present in the cerebral cortex, hippocampal formation, thalamus, and cholinergic basal forebrain nuclei. BACE protein levels did not differ significantly between control and transgenic mice or as a result of aging. However, in the aged, 17‐month‐old Tg2576 mice there was robust amyloid plaque formation, and BACE protein was also present in reactive astrocytes present near amyloid plaques, as shown by double immunofluorescent labeling and confocal laser scanning microscopy. The lack of astrocytic BACE immunoreactivity in young transgenic Tg2576 mice suggests that it is not the APP overexpression but rather the amyloid plaque formation that stimulates astrocytic BACE expression in Tg2576 mice. Our data also suggest that the neuronal overexpression of APP does not induce the overexpression of its metabolizing enzyme in neurons. Alternatively, the age‐dependent accumulation of amyloid plaques in the Tg2576 mice does not require increased neuronal expression of BACE. Our data support the hypothesis that neurons are the primary source of β‐amyloid peptides in brain and that astrocytic β‐amyloid generation may contribute to amyloid plaque formation at later stages or under conditions when astrocytes are activated. J. Neurosci. Res. 64:437–446, 2001.

Impairment of cholinergic neurotransmission in adult and aged transgenic Tg2576 mouse brain expressing the Swedish mutation of human β-amyloid precursor protein

To address the question of whether beta-amyloid peptides also affect cholinergic neurotransmission in vivo, brain tissue from transgenic Tg2576 mice with Alzheimer plaque pathology at ages ranging from 7 to 24 months were examined by immuno- and histochemical staining for choline acetyltransferase (ChAT) and acetycholinesterase (AChE), by assaying cholinergic enzyme activities and high-affinity choline uptake as well muscarinic and nicotinic cholinergic receptor binding levels by quantitative autoradiography. Cortical and hippocampal activities of AChE and ChAT were not different between transgenic mice and non-transgenic littermates regardless of the postnatal ages examined. However, high-affinity choline uptake was reduced in the hippocampus of 21-month-old transgenic mice. In brains of 8-month-old transgenic mice which do not yet demonstrate cortical beta-amyloids, reduced binding levels of cortical and hippocampal M1-muscarinic cholinergic receptors were observed, which were still reduced in 17-month-old transgenic mouse brains with high plaque load as compared to non-transgenic littermates. M2-muscarinic cholinergic receptor binding was hardly affected in brains from 8-month-old transgenic mice, but in 17-month-old transgenic mice reduced cortical and hippocampal binding levels were observed as compared to non-transgenic controls. Decreased cortical nicotinic cholinergic receptor binding was detected in 17-month-old transgenic mice. The development of changes in cholinergic synaptic markers in transgenic Tg2576 mouse brain before the onset of progressive plaque deposition provides in vivo evidence of a modulatory role of soluble beta-amyloid on cholinergic neurotransmission and may be referred to the deficits in learning and memory also observed in these mice before significant plaque load.

Alterations in cholinergic and non-cholinergic neurotransmitter receptor densities in transgenic Tg2576 mouse brain with β-amyloid plaque pathology

Cholinergic deficits in Alzheimers disease are accompanied by a number of alterations in other transmitter systems including glutamate, noradrenaline and serotonin, suggesting the involvement also of other neurotransmitter systems in the pathogenesis of the disease. To address the question whether β‐amyloid may contribute to these deficits, brain tissue from transgenic Tg2576 mice with Alzheimer plaque pathology at ages of 5 (still no significant plaque load) and 17 months (moderate to high cortical β‐amyloid plaque load) were examined for a number of cholinergic and non‐cholinergic markers. Transgenic mice with no significant plaque load demonstrated reduced hemicholinium‐3 (HCh‐3) binding to choline uptake sites in anterior brain regions as compared to non‐transgenic littermates, while in aged transgenic mice with high number of plaque deposits decreased HCh‐3 binding levels were accompanied by increased vesicular acetylcholine transporter binding in selected cortical brain regions. In aged transgenic mice GABAA, NMDA, AMPA, kainate, and β‐adrenergic as well 5‐HT1A‐ and 5‐HT2A‐receptor binding levels were hardly affected, whereas α1‐ and α2‐adrenoceptor binding was increased in selected cerebral cortical regions as compared to non‐transgenic littermates. The development of changes in both cholinergic and non‐cholinergic markers in transgenic Tg2576 mouse brain already before the onset of progressive plaque deposition provides in vivo evidence of a modulatory role of soluble β‐amyloid on cortical neurotransmission and may be referred to the deficits in learning and memory observed in these mice also before significant plaque load.

Degeneration of β-amyloid-associated cholinergic structures in transgenic APPsw mice.

Abstract Cholinergic dysfunction is a consistent feature of Alzheimer’s disease, and the interrelationship between β-amyloid deposits, inflammation and early cholinergic cell loss is still not fully understood. To characterize the mechanisms by which β-amyloid and pro-inflammatory cytokines may exert specific degenerating actions on cholinergic cells ultrastructural investigations by electron microscopy were performed in brain sections from transgenic Tg2576 mice that express the Swedish double mutation of the human amyloid precursor protein and progressively develop β-amyloid plaques during aging. Both light and electron microscopical investigations of the cerebral cortex of 19-month-old transgenic mice revealed a number of pathological tissue responses in close proximity of β-amyloid plaques, such as activated microglia, astroglial proliferation, increased number of fibrous astrocytes, brain edema, degeneration of nerve cells, dendrites and axon terminals. Ultrastructural detection of choline acetyl transferase (ChAT)-immunostaining in cerebral cortical sections of transgenic mice clearly demonstrated degeneration of ChAT-immunoreactive fibres in the environment of β-amyloid plaques and activated glial cells suggesting a role of β-amyloid and/or inflammation in specific degeneration of cholinergic synaptic structures.