Active Aβ vaccination fails to enhance amyloid clearance in a mouse model of Alzheimer's disease with Aβ42-driven pathology

S100B is a Ca(2+)-binding protein expressed in the nervous system. Increased levels of S100B in the extracellular space have been detected in several neurological disorders. We investigated the response of human astrocytes to micromolar chronic concentrations of this protein measuring the expression of some costimulatory molecules, such as CD137, CD137-L, CD40, CD40-L, the chemokine RANTES and two growth factors FGF-2 and TGF-β2. Our findings suggest that high levels of S100B in the brain parenchyma may modulate the activation status of astrocytes decreasing their neuroprotective role and modifying their interaction with microglia and other inflammatory cells. This effect may contribute to evolution of some neurological disorders.     

FGF-18 is a neuron-derived glial cell growth factor expressed in the rat brain during early postnatal development

We examined the expression of fibroblast growth factor-18 (FGF-18) in the rat brain during postnatal development by in situ hybridization. FGF-18 was transiently expressed at the early postnatal stages in various regions of the rat brain including the cerebral cortex and hippocampus. FGF-18 in the brain was preferentially expressed in neurons but not in glial cells. To elucidate the role of FGF-18 in the brain, we examined the ligand-specificity of FGF-18 by the BIAcore system. FGF-18 was found to bind to FGF receptors (FGFRs)-3c and -2c but not to FGFR-1c, suggesting that FGF-18 acts on glial cells but not on neurons. Therefore, we examined the mitogenic activity of FGF-18 for cultured rat astrocytes and microglia. FGF-18 was found to have mitogenic activity for both astrocytes and microglia. We also examined the neurotrophic activity of FGF-18 for cultured rat cortical neurons. FGF-18 was found to have no neurotrophic activity. The present findings indicated that FGF-18 is a unique FGF that plays a role as a neuron-derived glial cell growth factor in early postnatal development when gliogenesis occurs.     

Functional expression of the seven-transmembrane HIV-1 co-receptor APJ in neural cells

APJ is a recently described seven-transmembrane (7TM) receptor that is abundantly expressed in the central nervous system (CNS). This suggests an important role for APJ in neural development and/or function, but neither its cellular distribution nor its function have been defined. APJ can also serve as a co-receptor with CD4 for fusion and infection by some strains of human immunodeficiency virus (HIV-1) in vitro, suggesting a role in HIV neuropathogenesis if it were expressed on CD4-positive CNS cells. To address this, we examined APJ expression in cultured neurons, astrocytes, oligodendrocytes, microglia and monocyte-derived macrophages utilizing both immunocytochemical staining with a polyclonal anti-APJ antibody and RT - PCR. We also analyzed the ability of a recently identified APJ peptide ligand, apelin, to induce calcium elevations in cultured neural cells. APJ was expressed at a high level in neurons and oligodendrocytes, and at lower levels in astrocytes. In contrast, APJ was not expressed in either primary microglia or monocyte-derived macrophages. Several forms of the APJ peptide ligand induced calcium elevations in neurons. Thus, APJ is selectively expressed in certain CNS cell types and mediates intracellular signals in neurons, suggesting that APJ may normally play a role in signaling in the CNS. However, the absence of APJ expression in microglia and macrophages, the prinicpal CD4-positive cell types in the brain, indicates that APJ is unlikely to mediate HIV-1 infection in the CNS.     

Temporal expression of osteopontin and CD44 in rat brains with experimental cryolesions

Expression of osteopontin and CD44 in the brain was studied after cryolesioning to understand how osteopontin and its receptor, CD44, are involved in processes in the brains of rats with cryolesions. Western blot analysis showed that osteopontin increased significantly at days 4 and 7 post-injury and declined slightly thereafter in cryolesioned brains in comparison with levels in sham-operated controls. An immunohistochemical study localized osteopontin in activated microglia/macrophages in the core lesions, where the majority of macrophages proliferate. Osteopontin was also detected temporarily in some neurons and a few astrocytes in the lesion periphery on days 4 and 7 post-injury, but the immunoreactivity in macrophages, neurons, and astrocytes disappeared by day 14 post-injury. There was some CD44, a receptor for osteopontin, in the brain cells of sham-operated rats. After injury, intense CD44 immunostaining was seen in the majority of macrophages and in reactive astrocytes, but not in neurons, in the ipsilateral lesions after day 4 post-injury, and this immunoreactivity remained on day 14 post-injury. These findings suggest that activated microglia/macrophages and some neurons are major sources of osteopontin during the early stage of brain damage induced by a cryolesion and that osteopontin interacts with CD44 expressed on astrocytes and activated microglia/macrophages in the damaged cerebral cortex, possibly mediating cell migration after cryolesioning in the rat brain.     

Reconstitution of human immunodeficiency virus-induced neurodegeneration using isolated populations of human neurons, astrocytes, and microglia and neuroprotection mediated by insulin-like growth factors

Primary human neuron cultures are an important in vitro model system for studies on mechanisms involved in human immunodeficiency virus (HIV)-associated dementia (HAD) and other neurological disorders. Here, more than 80 cell surface antigens were screened to identify a marker that could readily distinguish between neurons and astrocytes and found that neurons lack CD44 surface expression, whereas astrocytes and other cell types in brain are CD44⁺. Neurons and astrocytes were isolated from human fetal brain based on differential expression of CD44. Using purified neurons cocultured with astrocytes and/or microglia, it was demonstrated that HIV infection of microglia induces cellular activation and production of soluble factors that activate uninfected microglia and astrocytes and induce neuronal cell death. Activated astrocytes promoted HIV replication in microglia, thereby amplifying HIV-induced neurotoxicity. A screen for 120 cytokine/proteins detected upregulation of insulin-like growth factor (IGF)-binding protein (IGFBP)-2, interleukin (IL)-6, and CCL8/MCP-2 (monocyte chemoattractant protein 2) in supernatants of HIV-infected brain cell cultures. IGF-1 and -2 increased neuronal survival in HIV-infected brain cell cultures, whereas IGFBP-2 inhibited prosurvival effects of these growth factors. These findings identify CD44 as a marker that can be used to sort neurons from other cell types in brain, suggest the importance of microglia-astrocyte interactions in neurodegenerative mechanisms associated with HIV infection, and indicate a role for insulin-like growth factors in neuroprotection from HIV-induced neurodegeneration. The ability to reconstitute brain cultures using isolated populations of neurons, astrocytes, and microglia will be valuable for studies on pathogenic mechanisms in HAD and other neurological disorders, and will also facilitate neuroactive drug discovery.     

Expression of indoleamine 2,3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons

There is good evidence that the kynurenine pathway (KP) and one of its end products, quinolinic acid (QUIN) play a role in the pathogenesis of several major neurological diseases. While QUIN has been shown to be produced in neurotoxic concentrations by macrophages and microglia, the capacity of astrocytes and neurons to produce QUIN is controversial. Using interferon gamma (IFN-gamma)-stimulated primary cultures of human mixed brain cells, we assayed expression of the KP regulatory enzyme indoleamine 2,3-dioxygenase (IDO) and QUIN production by immunocytochemistry. Using IFN-gamma-stimulated purified cultures of neurons, astrocytes, microglia and macrophages, we studied IDO expression by RT-PCR and production of QUIN using mass spectrometry. We found that astrocytes, neurons, and microglia expressed IDO but only microglia were able to produce detectable amounts of QUIN. However, astrocytes and neurons had the ability to catabolize QUIN. This study also provides the first evidence of IDO expression and lack of production of QUIN in culture of primary human neurons.     

Differential expression of fibroblast growth factor-2 and receptor by glial cells in experimental autoimmune encephalomyelitis (EAE)

To assess the expression pattern of basic fibroblast growth factor (FGF-2) and one of its receptors (FGFR-1/flg) during autoimmune inflammation of the CNS, FGF-2, and FGFR1/flg peptide and mRNA levels were examined by immunocytochemistry, by in situ hybridisation and by Northern blot analysis in T cell-mediated EAE of the Lewis rat. In naive control animals as well as in animals injected with nonencephalitogenic, PPD-reactive T lymphocytes, FGF-2 immunoreactivity was low and confined to blood vessels and to a few spinal cord neurons. In rats injected with encephalitogenic, MBP-reactive T lymphocytes, however, FGF-2-immunoreactive cells were detected from day 4 after T cell transfer onward, i.e., from the onset of clinical symptoms. The number of FGF-2 immunoreactive cells was highest between days 6 and 10 after T cell transfer. Increased FGF-2 peptide expression was paralleled by increased FGF-2 mRNA expression on macrophages/microglia in the spinal cord. By 21 days after T cell transfer, i.e. after complete recovery, FGF-2 peptide and mRNA expression had fully subsided. Based on morphological criteria and on double labeling with the macrophage/microglia-binding lectin GSI-B₄ two cell types expressed FGF-2: 1) round macrophages within the core, and 2) activated microglia at the edges of white and grey matter perivascular lesions. Paralleling the temporal and spatial expression pattern of FGF-2, FGFR-1/flg immunoreactivity was induced on activated macrophages/microglia but also on reactive astrocytes bordering perivascular inflammatory lesions. In situ hybridisation analysis furthermore showed that macrophages/microglia expressed the FGFR-1/flg mRNA, and that receptor mRNA expression paralleled ligand mRNA expression. Macrophage/microglia-derived FGF-2 could serve two main functions in EAE: 1) regulate microglial activation in an autocrine fashion, and 2) help to target astrocyte-derived insulin-like growth factor-I (IGF-I) to potentially injured oligodendrocytes in demyelination. © 1996 Wiley-Liss, Inc.     

Expression and localization of absent in melanoma 2 in the injured spinal cord

In traumatic brain injury, absent in melanoma 2(AIM2) has been demonstrated to be involved in pyroptotic neuronal cell death. Although the pathophysiological mechanism of spinal cord injury is similar to that of brain injury, the expression and cellular localization of AIM2 after spinal cord injury is still not very clear. In the present study, we used a rat model of T9 spinal cord contusive injury, produced using the weight drop method. The rats were randomly divided into 1-hour, 6-hour, 1-day, 3-day and 6-day(post-injury time points) groups. Sham-operated rats only received laminectomy at T9 without contusive injury. Western blot assay revealed that the expression levels of AIM2 were not significantly different among the 1-hour, 6-hour and 1-day groups. The expression levels of AIM2 were markedly higher in the 1-hour, 6-hour and 1-day groups compared with the sham, 3-day and 7-day groups. Double immunofluorescence staining demonstrated that AIM2 was expressed by NeuN+(neurons), GFAP+(astrocytes), CNPase+(oligodendrocytes) and CD11 b+(microglia) cells in the sham-operated spinal cord. In rats with spinal cord injury, AIM2 was also found in CD45+(leukocytes) and CD68+(activated microglia/macrophages) cells in the spinal cord at all time points. These findings indicate that AIM2 is mainly expressed in neurons, astrocytes, microglia and oligodendrocytes in the normal spinal cord, and that after spinal cord injury, its expression increases because of the infiltration of leukocytes and the activation of astrocytes and microglia/macrophages.     

Control of glial immune function by neurons

The immune status of the central nervous system (CNS) is strictly regulated. In the healthy brain, immune responses are kept to a minimum. In contrast, in a variety of inflammatory and neurodegenerative diseases, including multiple sclerosis, infections, trauma, stroke, neoplasia, and Alzheimer's disease, glial cells such as microglia gain antigen-presenting capacity through the expression of major histocompatibility complex (MHC) molecules. Further, proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF), interleukin-1beta (IL-1beta), and interferon-gamma (IFN-gamma), as well as chemokines, are synthesized by resident brain cells and T lymphocytes invade the affected brain tissue. The proinflammatory cytokines stimulate microglial MHC expression in the lesioned CNS areas only. However, the induction of brain immunity is strongly counterregulated in intact CNS areas. For instance, recent work demonstrated that microglia are kept in a quiescent state in the intact CNS by local interactions between the microglia receptor CD200 and its ligand, which is expressed on neurons. Work done in our laboratory showed that neurons suppressed MHC expression in surrounding glial cells, in particular microglia and astrocytes. This control of MHC expression by neurons was dependent on their electrical activity. In brain tissue with intact neurons, the MHC class II inducibility of microglia and astrocytes by the proinflammatory cytokine IFN-gamma was reduced. Paralysis of neuronal electric activity by neurotoxins restored the induction of MHC molecules on microglia and astrocytes. Loss of neurons or their physiological activity would render the impaired CNS areas recognizable by invading T lymphocytes. Thus, immunity in the CNS is inhibited by the local microenvironment, in particular by physiologically active neurons, to prevent unwanted immune mediated damage of neurons.     

Quantitation of ATP-binding cassette subfamily-A transporter gene expression in primary human brain cells

Five ATP-binding cassette (ABC) subfamily-A transporters (ABCA1, ABCA2, ABCA3, ABCA7 and ABCA8) are expressed in the brain. These transporters may regulate brain lipid transport; however, their relative expression level in isolated human brain cells is unknown. We developed real-time polymerase chain reaction assays to quantify the expression of these genes in human neurons, astrocytes, oligodendrocytes, microglia and cell lines. Neurons expressed predominantly ABCA1 and ABCA3; astrocytes ABCA1, ABCA2 and ABCA3; microglia ABCA1 and oligodendrocytes ABCA2 and ABCA3. Although ABCA7 and ABCA8 expression was relatively low in all cells, the highest expression occurred in microglia and neurons, respectively. ABCA gene expression in the NTERA-2 and MO3.13 cell lines closely resembled the ABCA expression pattern of primary neurons and oligodendrocytes, respectively.     

Expression of osteopontin and its ligand, CD44, in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

The expression of osteopontin (OPN) and one of its ligands, CD44, was studied in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that osteopontin significantly increased at the early and peak stage of EAE and slightly declined thereafter. Osteopontin was constitutively expressed in some astrocytes adjacent to pia mater and neurons in normal rats, and was shown to be increased in the same cells and also in some inflammatory cells including macrophages at the early and peak stage of EAE. CD44, a ligand for osteopontin, was constitutively expressed in astrocytes in normal and control spinal cords and was also expressed in inflammatory cells, as well as increased expression in astrocytes in EAE. These findings suggest that inflammatory cells as well as reactive astrocytes are major sources of osteopontin in rat EAE, and osteopontin may interact with its ligand CD44 on astrocytes and inflammatory cells in EAE, possibly mediating autoimmune central nervous system (CNS) diseases in rats.     

Rat oligodendrocytes and astrocytes preferentially express fibroblast growth factor receptor-2 and -3 mRNAs

Fibroblast growth factors (FGFs) exert various effects on glial cells as well as on neurons in the brain. The mRNAs for four FGF receptors (FGFR-1-FGFR-4) are expressed in the brain. Although FGFR-1 and -4 mRNAs are preferentially expressed in neurons, FGFR-2 and -3 mRNAs are preferentially expressed in glial cells. However, the glial cells that express these receptors remained to be identified. In this study, we found that oligodendrocytes and astrocytes in the brain preferentially expressed FGFR-2 and FGFR-3 mRNAs, respectively. The isoforms of immunoglobulin-like domain III (IIIb and IIIc) of the receptors have crucial roles in ligand binding. We also determined the isoforms of FGFR-2 and FGFR-3 expressed in glial cells to be of type IIIc. The expression of FGFR-2 IIIc and FGFR-3 IIIc with different ligand specificities might play important roles in the various effects of FGFs on oligodendrocytes and astrocytes. © 1996 Wiley-Liss, Inc.     

Closed head injury induces up-regulation of ErbB-4 receptor at the site of injury

ErbB-4 receptor tyrosine kinase and its ligand neu differentiation factor (NDF/neuregulin) are widely expressed in the brain. The closed head injury model was used to investigate the possible role of ErbB-4 receptor in neurodegeneration. It is demonstrated that levels of ErbB-4 are dramatically increased at the site of injury. Activated microglia/macrophages constitute the major population of cells with the highest receptor levels at the site of injury. In addition ErbB-4 expression after injury is elevated also in neurons but not in astrocytes. Confocal microscopy analysis suggests that the high level of ErbB-4 protein in activated microglia/macrophages is probably due to phagocytosis of neuronal cells. These findings show for the first time that ErbB-4 receptors play a role in brain responses to head trauma. Overexpression of ErbB-4 receptors may be important for directing activated microglia/macrophages to the lesion site.     

The majority of infiltrating CD8 T lymphocytes in multiple sclerosis lesions is insensitive to enhanced PD-L1 levels on CNS cells

Central nervous system (CNS) cells locally modulate immune responses using numerous molecules that are not fully elucidated. Engagement of programmed death-1 (PD-1), expressed on activated T cells, by its ligands (PD-L1 or PD-L2) suppresses T-cell responses. Enhanced CNS PD-1 and PD-L1 expression has been documented in inflammatory murine models; however, human CNS data are still incomplete. We determined that human primary cultures of astrocytes, microglia, oligodendrocytes, or neurons expressed low or undetectable PD-L1 under basal conditions, but inflammatory cytokines significantly induced such expression, especially on astrocytes and microglia. Blocking PD-L1 expression in astrocytes using specific siRNA led to significantly increased CD8 T-cell responses (proliferation, cytokines, lytic enzyme). Thus, our results establish that inflamed human glial cells can express sufficient and functional PD-L1 to inhibit CD8 T cell responses. Extensive immunohistochemical analysis of postmortem brain tissues demonstrated a significantly greater PD-L1 expression in multiple sclerosis (MS) lesions compared with control tissues, which colocalized with astrocyte or microglia/macrophage cell markers. However, more than half of infiltrating CD8 T lymphocytes in MS lesions did not express PD-1, the cognate receptor. Thus, our results demonstrate that inflamed human CNS cells such as in MS lesions express significantly elevated PD-L1, providing a means to reduce CD8 T cell responses, but most of these infiltrating immune cells are devoid of PD-1 and thus insensitive to PD-L1/L2. Strategies aimed at inducing PD-1 on deleterious activated human CD8 T cells that are devoid of this receptor could provide therapeutic benefits since PD-L1 is already increased in the target organ.     

Expression of stem cell factor and c-kit receptor in neural cells after brain injury

Previously it has been shown that c-kit receptor (c-kitR) and its ligand, stem cell factor (SCF), are expressed in the central nervous system. We have reported that SCF in cultures regulates mouse microglial function. Here we demonstrate that SCF/c-kitR signaling also takes place in situ. We used a penetrating stab wound injury as a model and analyzed the SCF and c-kitR expression in neural cells by immunohistochemistry and in situ hybridization. We found that microglia activated by injury up-regulated c-kitR expression, whereas some astrocytes in the vicinity of the wound expressed SCF mRNA in addition to neurons. This observation suggests that SCF/c-kitR signaling between neurons, astrocytes and microglia also occurs in situ. Received: 21 July 1998 / Revised, accepted: 20 October 1998     

Cellular localization of messenger RNA encoding amyloid-beta-protein in normal tissue and in Alzheimer disease

Amyloid precursor protein (APP) gene encodes the short peptide fragment amyloid-beta-protein present in senile plaque cores, cerebrovascular amyloid, and intracellular neurofibrillary tangles in Alzheimer disease (AD). Using in situ hybridization with biotin-labeled RNA probes, we found distinctive patterns of APP gene expression in different regions of postmortem human brain. Strong hybridization signal for APP messenger RNA (mRNA) was detected in specific classes of neurons, fascicular oligodendroglia, satellite glia, and presumptive microglia. Weaker signal was seen in other neuronal classes, fascicular astrocytes, and vascular endothelial cells, but no signal was seen in protoplasmic astrocytes. Human thymus also shows a restricted pattern of hybridization with high signal in reticular epithelial cells, and much lower signal in lymphocytes. In AD patients, neuronal hybridization for APP mRNA was specifically increased in hippocampus, but not cerebellar and visual cortex when compared to hybridization for neuron-specific enolase mRNA. Most neurons with neurofibrillary tangles had strong APP mRNA signal. These results suggest that APP gene expression is highly regulated in normal tissue, that many different cell classes in brain express the APP gene, and that neuronal expression may increase specifically in brain regions where widespread injury occurs in AD. Amyloid deposits in brains of AD patients might be explained by local production of precursor protein in endothelial cells, neurons or glia.     

Neural stem cells in the subventricular zone are a source of astrocytes and oligodendrocytes,but not microglia

The developmental origin of microglia remains a controversial subject. While it is generally accepted that primitive fetal macrophages that migrate from the yolk sac to the brain become microglia, it also has been argued that there is a second source of microglia that are of neuroectodermal lineage. To determine whether progenitors in the dorsolateral subventricular zone (SVZDL) are capable of producing microglia as well as macroglia, we infected perinatal rat SVZDL cells with a mixture of two replication-deficient retroviruses, placed these progenitors in vitro and then varied the media formulations to promote microglial differentiation. Mixed macroglial clones were obtained, but no heterogeneous clones containing microglia were observed, regardless of the media components. Among the macroglial clones, we observed every possible combination of type 1 astrocyte and O-2A lineage cells. Some clones were homogeneous and contained cells belonging to a single macroglial lineage. Other clonal clusters were heterogeneous and were comprised of type 1 astrocytes and oligodendrocytes, type 1 and type 2 astrocytes, or type 2 astrocytes and oligodendrocytes. Of 130 clones examined, where we used triple immunofluorescence with antibodies that recognize microglia, 2 clonal clusters contained OX-42+ microglia that were retrovirally labeled, but all of the cells in those clones expressed the microglial marker and none expressed either GFAP or O4. In addition, we isolated neural stem cells from the perinatal SVZDL and assessed their capacity to generate macroglia and microglia. Confirming and extending our previous analyses, neural stem cells generated homogeneous and heterogeneous macroglial clones, but they did not generate microglia. We conclude that brain macroglia and microglia do not share a common precursor, even though the neural stem cells in the SVZDL cells can produce neurons, astrocytes and oligodendrocytes. Therefore, the microglia that reside in the SVZDL are immigrants from nonneural precursors.     

Expression of alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) in rat microglial cells

Low density lipoprotein receptor-related protein (LRP) participates in the uptake and degradation of several ligands implicated in neuronal pathophysiology including apolipoprotein E (apoE), activated alpha(2) -macroglobulin (alpha(2)M*) and beta-amyloid precursor protein (APP). The receptor is expressed in a variety of tissues. In the brain LRP is present in pyramidal-type neurons in cortical and hippocampal regions and in astrocytes that are activated as a result of injury or neoplasmic transformation. As LRP is expressed in the monocyte/macrophage cell system, we were interested in examining whether LRP is expressed in microglia. We isolated glial cells from the brain of neonatal rats and LRP was immunodetected both in microglial cells and in astrocytes expressing glial fibrillar acidic protein (GFAP). Microglial cells were able to bind and internalize LRP-specific ligand, alpha(2)M*. The internalization was inhibitable by RAP, with a Kd of 1.7 nM. The expression of LRP was up-regulated by dexamethasone, and down-regulated by lipopolysaccharide (LPS), gamma interferon (IFN-gamma) or a combination of both. LRP was less sensitive to dexamethasone in activated astrocytes than in microglia. We provided the first analysis of LRP expression and regulation in microglia. Our results open the possibility that microglial cells could be related to the participation of LRP and its ligands in different pathophysiological states in brain.