Early association of reactive astrocytes with senile plaques in Alzheimer's disease

The fibrillar beta-amyloid protein (A beta) plaques of Alzheimer's disease (AD) are associated with reactive astrocytes and dystrophic neurites and have been suggested to contribute to neurodegenerative events in the disease. We recently reported parallel in vitro and in situ findings, suggesting that the adoption of a reactive phenotype and the colocalization of astrocytes with plaques in AD may be mediated in large part by aggregated A beta. Thus, A beta-mediated effects on astrocytes may directly affect disease progression by modifying the degenerative plaque environment. Alternatively, plaque-associated reactive astrocytosis may primarily represent a glial response to the neural injury associated with plaques and not significantly contribute to AD pathology. To investigate the validity of these two positions, we examined the differential colocalization of reactive astrocytes and dystrophic neurites with plaques. Hippocampal sections from AD brains--ranging in neuropathology from mild to severe--were triple-labeled with antibodies recognizing A beta protein, reactive astrocytes, and dystrophic neurites. We observed not only plaques containing both or neither cell type, but also plaques containing (1) reactive astrocytes but not dystrophic neurites and (2) dystrophic neurites but not reactive astrocytes. The relative proportion of plaques colocalized with reactive astrocytes in the absence of dystrophic neurites is relatively high in mild AD but significantly decreases over the course of the disease, suggesting that plaque-associated astrocytosis may be an early and perhaps contributory event in AD pathology rather than merely a response to neuronal injury. These data underscore the potentially significant contributions of reactive astrocytosis in modifying the plaque environment in particular and disease progression in general.     

Disruption of the blood-brain interface in neonatal rat neocortex induces a transient expression of metallothionein in reactive astrocytes

Exposure of the adult rat brain parenchyma to zinc induces an increase in the intracerebral expression of the metal-binding protein, metallothionein, which is normally confined to astrocytes, ependymal cells, choroid plexus epithelial cells, and brain endothelial cells. Metallothionein is expressed only in diminutive amounts in astrocytes of the neonatal rat brain, which could imply that neonatal rats are devoid of the capacity to detoxify free metals released from a brain wound. In order to examine the influence of a brain injury on the expression of metallothionein in the neonatal brain, PO rats were subjected to a localized freeze lesion of the neocortex of the right temporal cortex. This lesion results in a disrupted blood-brain interface, leading to extravasation of plasma proteins. From 16 h, reactive astrocytosis, defined as an increase in the number and size of cells expressing GFAP and vimentin, was observed surrounding the neocortical lesion site. Astrocytes and pial cells situated adjacent to the area of injury also became positively stained for metallothionein. At 3-6 days post-lesion, the highest level of reactive astrocytes expressing metallothionein was observed. Neo-Timm staining revealed that histochemically reactive zinc had disappeared from the lesion site. Extracellular albumin and metallothionein-positive astrocytes were absent approximately 2 weeks after the lesion, whereas reactive astrocytosis was still observed. These results show that a lesion of the neonatal rat brain induces a transient expression of metallothionein in reactive astrocytes, probably as a response to metals released from the site of the brain injury.     

Inappropriate apoptosis of salivary and lacrimal gland epithelium of immunodeficient NOD-scid mice

Reactive gliosis, which occurs in response to any damage or disturbance to the central nervous system, has been recognized for many years, but is still not completely understood. The hallmark is the increased expression of glial fibrillary acidic protein (GFAP), yet studies in GFAP knockout mice suggest that GFAP may not be required for an astrocyte to become hypertrophic. In this review, we describe a series of tissue culture models that have been established in order to address: 1) the biochemical phenotype of reactive astrocytes; 2) the factor and/or cell responsible for induction of gliosis; 3) the mechanisms by which one might block the induction. These models range from cultures of astrocytes, both neonatal and adult, to co-cultures of astrocytes with either neurons or microglia, to organ cultures. None is ideal: each addresses a different set of questions, but taken together, they are beginning to provide useful information which should allow a better understanding of the plasticity response of astrocytes to brain injury.     

Glial fibrillary acidic protein-apolipoprotein E (apoE) transgenic mice: astrocyte-specific expression and differing biological effects of astrocyte-secreted apoE3 and apoE4 lipoproteins

The epsilon4 allele of apolipoprotein E (apoE) is associated with increased risk for Alzheimer's disease (AD) and poor outcome after brain injury. In the CNS, apoE is expressed by glia, predominantly astrocytes. To define the potential biological functions of different human apoE isoforms produced within the brain, transgenic mice were generated in which human apoE3 and apoE4 expression is under control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. These animals were then bred back to apoE knock-out mice. Human apoE protein is found within astrocytes and the neuropil throughout development and into the adult period, as assessed by immunocytochemistry and immunoblot analysis in several GFAP-apoE3 and E4 lines. Cultured astrocytes from these mice secrete apoE3 and apoE4 in lipoproteins that are high-density lipoprotein-like in size. When primary hippocampal neurons are grown in the presence of astrocyte monolayers derived from these transgenic mice, there is significantly greater neurite outgrowth from neurons grown in the presence of apoE3-secreting astrocytes compared with apoE4-secreting or apoE knock-out astrocytes. These effects are not dependent on direct astrocyte-neuron contact and appear to require the low-density lipoprotein receptor-related protein. These data suggest that astrocyte-secreted, apoE3-containing lipoproteins have different biological effects than apoE4-containing lipoproteins. In addition to providing information regarding the role of astrocyte-secreted apoE lipoproteins in the normal brain, these animals will also be useful in models of both AD and CNS injury.     

Distinct expressions of three cytokines by IL-1-stimulated astrocytes in vitro and in AIDS brain

Interleukin-1 (IL-1) is elevated in brain tissue of individuals who died with acquired immunodeficiency syndrome (AIDS) and other diseases where this cytokine likely stimulates reactive astrocytosis. IL-1 stimulates, among others, production of interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha) in cultured astrocytes and astrocytoma cell lines. These and other cytokines may contribute to the neuropathogenesis after infection by human immunodeficiency virus type-1 (HIV-1). For example, concentration of TNF-alpha is increased in brain tissue of individuals who died with AIDS and correlates with the severity of AIDS Dementia Complex (ADC). TNF-alpha and IL-6 have been immunocytochemically detected in brain tissue but they have not been localized to astrocytes. We, therefore, examined the expression of IL-6, GM-CSF, and TNF-alpha in human primary astrocytes and astrocytoma cell lines U251 and 253 exposed to IL-1 in serum-free medium. In addition, we immunocytochemically assayed GM-CSF expression by astrocytes in brain tissue (n = 8). The three cytokines were differentially induced in cultured astrocytes by IL-1. The astrocytoma cell lines recapitulated cytokine-specific patterns of expression in astrocytes. The patterns were characterized by amounts produced, compartmentalization (intra- and/or extracellular), time courses, and optimal doses of IL-1 for induction. GM-SCF-like immunoreactivity was detected in some but not all, GFAP+ cells. GM-CSF+/GFAP+ cells were detected in only three of seven cases containing GM-CSF immunoreactivity. Thus, a discrepancy may exist between human astrocytic cytokine expression in vitro and in tissue. Novel methods therefore may need to be developed to recapitulate in vitro the heterogeneity of astrocytic cytokine expression in AIDS and other brain tissue.     

A role for astrocytes in glucose delivery to neurons?

The present paper examines the possible role of astrocytes in the delivery of glycogen-derived glucose for neuronal metabolism. Such a process would require astrocytic expression of glucose-6-phosphatase. The degree and significance of brain expression of glucose-6-phosphatase (EC 3.1.3.9) has been a subject of controversy. Published immunohistochemical data are consistent with expression of glucose-6-phosphatase by astrocytes, both in vivo and in vitro. In this paper additional confirmation of the expression of glucose-6-phosphatase mRNA in rat brain is presented. Although cultured astrocytes demonstrate glucose-6-phosphatase activity in vitro under assay conditions, there is very limited in vitro evidence that this activity confers a glucose-export capacity on astrocytes. Under most conditions in vitro, lactate export predominates, however this may relate to aspects of the in vitro phenotype. Data relating to astrocytic glucose and lactate export are considered in the context of hypotheses of trafficking by astrocytes of substrates for neuronal metabolism, hypotheses that imply and require compartmentation of these substances, in contrast with current formulations of glucose transport into and within brain that imply no glucose compartmentation. Microdialysis studies of the properties of the brain extracellular fluid (ECF) glucose pool in the freely moving rat were performed seeking evidence of glucose compartmentation. Results of these studies do imply compartmentalisation of brain glucose, and are consistent with a model envisaging the majority of glucose reaching the neuron via the astrocytic intracellular space and the ECF. In addition, such studies provide evidence that rises in ECF glucose concentration are not the direct result of local recruitment of cerebral blood flow, but suggest the influence of intermediate, astrocyte-based mechanisms. Astrocytic glucose-6-phosphatase may permit astrocytes to modulate the trans-astrocytic flux of glucose to adjacent neurons in response to signals reflecting increased neuronal demand.     

Anisotropic Heisenberg ferromagnetic model in two dimensions

Transformation of normal resting astrocytes to reactive astrocytes after injury is a well-known phenomenon. Using immunofluorescent labelling methods, astrocytes in the ischemically and retrogradely/anterogradely damaged adult forebrain nuclei were shown to express substance-P immunoreactivity. In contrast, astrocytes were not immunostained for substance-P in the normal brain or undamaged areas. Since substance-P has been shown to regulate inflammatory, wound-healing and immune responses in the peripheral tissues, it is likely that this aberrant expression of substance-P immunoreactivity in reactive astrocytes may relate to similar functions in the central nervous system as in the peripheral tissues after injury.     

The relation between rhinitis and allergic asthma. The action of antihistaminics on bronchial hyperreactivity

The intermediate filament nestin is highly expressed in multipotential stem cells of the developing central nervous system (CNS). During neuro- and gliogenesis, nestin is replaced by cell type-specific intermediate filaments, e.g. neurofilaments and glial fibrillary acidic protein (GFAP). In this study, we demonstrate that nestin expression is re-induced in reactive astrocytes in the lesioned adult brain. Following ischaemic and mechanical lesioning, a strong and sustained expression of nestin was noted in GFAP-positive cells surrounding the lesion site. Lesion experiments in transgenic mice carrying the lacZ gene under control of regulatory sequences from the nestin gene suggested that the upregulation of nestin in reactive astrocytes is mediated via the same sequences that control nestin expression during CNS development. These observations and recent data on the co-expression of glial and neuronal marker antigens in reactive astrocytes point to a close relationship between proliferating astrocytes and neuroepithelial precursor cells.     

CD95 (Fas/Apo-1) as a receptor governing astrocyte apoptotic or inflammatory responses: a key role in brain inflammation?

Astrocytes are a major cellular component of the brain that are capable of intense proliferation and metabolic activity during diverse inflammatory brain diseases (such as multiple sclerosis, Alzheimer's dementia, tumor, HIV encephalitis, or prion disease). In this biological process, called reactive gliosis, astrocyte apoptosis is frequently observed and could be an important mechanism of regulation. However, the factors responsible for apoptosis in human astrocytes are poorly defined. Here, we report that short term cultured astrocytes derived from different brain regions express significant levels of CD95 at their surface. Only late passage astrocytes are sensitive to CD95 ligation using either CD95 mAb or recombinant CD95 ligand. Blocking experiments using caspase inhibitors with different specificities (DEVD-CHO, z-VAD-fmk, and YVAD-cmk), an enzymatic activity assay, and immunoblotting show that CPP32/caspase-3 play a prominent role in CD95-induced astrocyte death. In contrast, early passage astrocytes are totally resistant to death, but a significant increase in astrocytic IL-8 secretion (p < 0.001, by Wilcoxon's test for paired samples) is observed after CD95 triggering. Production of IL-8 contributes to the resistance of astrocytes to CD95 ligation. Furthermore, in the presence of IFN-gamma, resistant astrocytes became sensitive to CD95-mediated death. These data suggest that microenvironmental factors can influence the consequences of CD95 ligation on astrocytes. Therefore, we propose that CD95 expressed by human astrocytes plays a pivotal role in the regulation of astrocyte life and death and may be a key factor in inflammatory processes in the brain, such as reactive gliosis.     

Macrophage colony-stimulating factor augments beta-amyloid-induced interleukin-1, interleukin-6, and nitric oxide production by microglial cells

In Alzheimer's disease (AD), a chronic cerebral inflammatory state is thought to lead to neuronal injury. Microglia, intrinsic cerebral immune effector cells, are likely to be key in the pathophysiology of this inflammatory state. We showed that macrophage colony-stimulating factor, a microglial activator found at increased levels in the central nervous system in AD, dramatically augments beta-amyloid peptide (betaAP)-induced microglial production of interleukin-1, interleukin-6, and nitric oxide. In contrast, granulocyte macrophage colony-stimulating factor, another hematopoietic cytokine found in the AD brain, did not augment betaAP-induced microglial secretory activity. These results indicate that increased macrophage colony-stimulating factor levels in AD could magnify betaAP-induced microglial inflammatory cytokine and nitric oxide production, which in turn could intensify the cerebral inflammatory state by activating astrocytes and additional microglia, as well as directly injuring neurons.     

Inflammatory processes and antiinflammatory drugs in Alzheimer's disease: a current appraisal

The study of risk factors and protective influences can yield clues to the pathogenesis of Alzheimer's disease (AD). Intervention on such factors can effect disease prevention or treatment while etiology remains unknown. Most known AD risk factors offer no prospect of prevention, but 14 of 15 relevant publications since 1987 suggest that the symptoms of AD are prevented or attenuated by antiinflammatory treatments. These findings are supported by numerous circumstantial findings suggesting a role for cytokines and acute phase reactants in the pathogenesis of AD. In particular, activated microglia and/or reactive astrocytes, found within or near all AD lesions, are thought to kill target cells by using either free radicals or the classical complement pathway. These mechanisms should be suppressed by glucocorticoids, but the available data suggest that nonsteroidal antiinflammatory drugs (NSAIDs) exert a stronger protective influence than steriods. NSAIDs (but not steroids) suppress the action of cyclooxygenases (COX), which catalyze synthesis of prostaglandins. The latter are intermediaries in the postsynaptic signal transduction cascade of cells with NMDA-type glutamate receptors. They may also potentiate glutamatergic transmission by inhibiting astrocytic reuptake of glutamate. Both mechanisms can potentiate excitotoxic cell death. Further work is needed to clarify whether steroids, NSAIDs, or both prevent or attenuate the symptoms of AD.     

Intravascular infusions of soluble beta-amyloid compromise the blood-brain barrier, activate CNS glial cells and induce peripheral hemorrhage

Vascular wall levels of soluble beta-amyloid1-40 (Abeta1-40) are elevated in Alzheimer's disease (AD). Moreover, plasma Abeta levels are increased in familial AD, as well as in some cases of sporadic AD. To determine the histopathologic and behavioral consequences of elevated vascular Abeta levels, Abeta1-40 (50 micrograms in distilled water) or vehicle was intravenously infused twice daily into 3-month old male Sprague-Dawley rats for 2 weeks. Intravenous Abeta infusions impaired blood-brain barrier integrity, as indicated by substantial perivascular and parenchyma IgG immunostaining within the brain. Also evident in Abeta-infused animals was an increase in GFAP immunostaining around cerebral blood vessels, and an enhancement of OX-42 microglial immunostaining in brain white matter. Gross pulmonary hemorrhage was noted in most Abeta-infused animals. All the observed changes occurred in the absence of Congo red birefringence. No significant cognitive deficits were present in Abeta-infused animals during water maze acquisition and retention testing, which was conducted during the second week of treatment. These results indicate that circulating Abeta can: (1) induce vessel dysfunction/damage in both the brain and the periphery without complex Abeta fibril formation/deposition, and (2) induce an activation of brain astrocytes and microglia. Taken together, our results suggest that if circulating Abeta is elevated in AD, it is likely to have a pathophysiologic role.     

A culture model of reactive astrocytes: increased nerve growth factor synthesis and reexpression of cytokine responsiveness

Reactive gliosis, which occurs in response to damage to the central nervous system, has been recognized for years but is not yet understood. We describe here a tissue culture model of reactive astrocytes used to characterize their properties. Cultures are prepared 1 week following 6-hydroxydopamine (6-OHDA) lesion of rat substantia nigra and compared with astrocytes cultured from normal adult rats or rats injected with saline only. Astrocytes from the 6-OHDA-lesioned side contained elevated levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA and were intensely immunoreactive for GFAP, vimentin, and two epitopes that in vivo are found only on reactive astrocytes. The basal content of nerve growth factor (NGF) mRNA and NGF in astrocytes from 6-OHDA-lesioned rats was significantly higher relative to control astrocytes. Two inflammatory cytokines, interleukin-1beta and interferon-gamma, increased synthesis of NGF up to 20-fold in the reactive cells, whereas there was no response in the normal adult astrocytes. Astrocytes from postnatal day 2 rats shared many of the properties of the reactive adult astrocytes. These cultures offer the possibility to characterize the cellular and molecular properties of reactive astrocytes and to determine the factors responsible for activation of astrocytes.     

Increased immunoreactivities for the basic fibroblast growth factor and its receptor in astrocytes at the site of cerebral lesions and oedematous change in SHR

1. To obtain information about changes of basic fibroblast growth factor (bFGF) in the brain in chronic hypertension, we immunohistochemically studied the distribution and level of bFGF and its receptor in the brain of stroke-prone spontaneously hypertensive rats (SHRSP). 2. In the control normotensive rats, immunoreactivity for bFGF was demonstrated in nerve cells, while there was almost no reactivity in astrocytes. 3. In SHRSP, there was a marked immunoreactivity in the densely accumulated reactive cells, particularly astrocytes, in and around cerebral cortical lesions. Slightly increased reaction for bFGF was found in the nerve cells around lesions. Astrocytes in the subcortical white matter on both ipsi- and contralateral sides of the cortical lesion also showed immunoreactivity for bFGF. The location of increased bFGF expression in SHRSP corresponded very well with the site of extravasated plasma fluid demonstrated by anti-fibrinogen antibody. Electron microscopically, bFGF was shown in astrocytes along the rough endoplasmic reticulum suggesting the growth factor to be produced in the cells and not to be taken up from the surroundings. Expression of FGF-receptor was also demonstrated in reactive astrocytes in the oedematous cortical portion around lesion and in the oedematous subcortical white matter. 4. These findings indicate the possibility that oedema and the simultaneously generated free radicals or some extravasated plasma components express bFGF in astrocytes and probably in nerve cells as well as FGF-receptor in astrocytes, and that the thus expressed bFGF and its receptor play some role in the sequence of developmental events of hypertensive cerebral lesions.     

Compartmental models for glycaemic prediction and decision-support in clinical diabetes care: promise and reality

Aggregated tau proteins constitute the basic matrix of neuronal inclusions specific to numerous neurodegenerative disorders. Monodimensional and two-dimensional Western blot analyses performed on cortical brain homogenates allowed discrimination between disease-specific tau protein profiles. These observations raised the issue of the physiopathological significance of such specificities. Alzheimer's disease (AD) pathological tau proteins (PTPs) (tau 74, 69, 64, 55) were compared with those of Pick's disease (PiD) (tau 64, 55) using a panel of antibodies against peptidic sequences of tau isoforms corresponding to exons 2, 3, and 10. AD and PiD could then be critically differentiated by the absence of translated tau isoforms with exon 10 in PiD PTPs, along with the absence of the phosphorylation site on Ser262. Immunohistochemical studies corroborate these findings. Indeed, Pick bodies were strongly immunostained by an anti-"exon 2" antibody but failed to reveal any anti-exon 10 reactive epitope. Tangles in AD contained exon 2, 3, and 10 epitopes. Altogether, our results demonstrated that Pick bodies develop within specific neuronal subsets that express specific patterns of 7 isoforms lacking exon 10 peptidic sequence. We conclude that neurodegenerative disorders imply attrition of selectively vulnerable neuronal subsets, a process revealed, and may be sustained by specific tau isoform patterns.     

Screening of potential transport systems for methyl mercury uptake in rat erythrocytes at 5 degrees by use of inhibitors and substrates

This report is devoted to the characterization of the apolipoprotein E (ApoE) in Microcebus murinus. Only one allele homologous to the human ApoE4 allele was evidenced. The distribution of the corresponding ApoE protein in the brain was found in association with the pathological proteins characteristic of Alzheimer's disease (AD). Immunocytochemistry revealed brain deposits of ApoE in: (1) the cortical amyloid plaques; (2) the neurones of the various cortical lobes, the hippocampus and the brainstem; (3) the glial cells, astrocytes of the cortical parenchym and oligodendrocytes of the corpus callosum; and (4) the vessel walls. Most ApoE, beta-amyloid protein, abnormally phosphorylated Tau proteins and gliofilament acid proteins were seen in the same cortical areas. These findings for ApoE report the view that Microcebus murinus, in captivity, presents a pathological profile very similar to that observed in AD.