Lack of the protein tyrosine phosphatase SHP-1 results in decreased numbers of glia within the motheaten (me/me) mouse brain.

Mice that are homozygous for the autosomal recessive motheaten allele (me/me) lack the protein tyrosine phosphatase SHP-1. Loss of SHP-1 leads to many hematopoietic abnormalities, as well as defects such as infertility and low body weight. However, little is known regarding the role SHP-1 plays in the development of the central nervous system (CNS). To define the role of SHP-1 in CNS development and differentiation, we examined the brains of me/me mice at various times after birth for neuronal and glial abnormalities. Although the brains of me/me mice are slightly smaller than age-matched wild-type littermates, both me/me and wild-type brains are similar in weight, possess an intact blood-brain barrier, and have largely normal neuronal architecture. Significantly, the current study reveals that me/me brain shows decreases in the number of glial fibriallary acidic protein (GFAP)+ astrocytes and F480+ microglia compared with wild-type mice. In addition, decreased immunostaining for the myelin-synthesizing enzyme CNPase was observed in me/me mice, confirming the loss of myelin in these animals, as reported (Massa et al. [2000] Glia 29:376-385). It is particularly significant that there is a decreased number of immunolabeled glia of all subtypes and that this deficit in glial number is not restricted to a particular class of glia. This suggests that SHP-1 is necessary for the normal differentiation and distribution of astrocytes, microglia, and oligendrocytes within the murine CNS.     

Neuron‐glia interactions: Molecular basis of alzheimer’s disease and applications of neuroproteomics

Neurodegenerative disorders present with progressive and irreversible degeneration of the neurons. Alzheimer's disease (AD) is one of the most common neurodegenerative disorders affecting 50 million people worldwide (2017), expected to be doubled every 20 years. Primarily affected by age, AD is the cause for old‐age dementia, progressive memory loss, dysfunctional thoughts, confusion, cognitive impairment and personality changes. Neuroglia formerly understood as “glue” of the brain neurons consists of macroglia (astrocytes and oligodendrocyte), microglia and progenitors NG2‐glia, and constitute a large fraction of the mammalian brain. The primary functions of glial cells are to provide neurons with metabolic and structural support in the healthy brain; however, they attain a “reactive” state from the “resting” state upon challenged with a pathological insult such as a neurodegenerative cascade. Failure or defects in their homoeostatic functions (i.e. concentration of ions, neurotransmitters) ultimately jeopardize neurons with excitotoxicity and oxidative stress. Moreover, the most common clinical outcome of AD is the cognitive impairment and memory loss, which are attributed mainly by the accumulation of Aβ. Failure of glial cells to remove the Aβ toxic proteins accelerates the AD progression. The rapidly emerging proteomic techniques such as mass spectrometry (MS), cross‐linking mass spectrometry, hydrogen deuterium trade mass spectrometry, protein foot printing and 2‐DGE combined with LC–MS/MS present wide array of possibilities for the identification of differentially expressed proteins in AD.     

Expression of differential immune factors in temporal cortex and cerebellum: The role of α-1-antichymotrypsin,apolipoprotein E,and reactive glia in the progression of Alzheimer's disease

A variety of factors and processes have been implicated in the development and progression of the pathology of Alzheimer's Disease (AD), including amyloid fragment deposition, reactive gliosis, α-1-antichymotrypsin (ACT), and apolipoprotein E (APOE). Carriers of the APOE 4 allele have been shown to have an enhanced risk of developing AD, and the ACT signal peptide A/A genotype may modify the APOEϵ4 risk. The protein products of these genes have been shown to enhance conversion of diffuse β amyloid (Aβ) fibrils, which are found in diffuse plaques, to the fibrillar form found in neuritic plaques. In affected regions of AD brain, ACT and APOE colocalize with Aβ deposits and reactive microglia and astrocytes. We examined the regional distribution of ACT, APOE, and reactive glia in temporal cortex, where neuritic plaques are abundant, and cerebellum (in areas where diffuse plaques but not neuritic plaques accumulate) to examine the relationship of these markers to the deposition of Aβ. In temporal cortex, ACT and APOE staining was localized to plaque-like profiles, reactive astrocytes, and blood vessels; human leukocyte antigen-DR (HLA-DR) and glial fibrillary acidic protein (GFAP) staining revealed focal clusters of reactive microglia and astrocytes. In cerebellum, ACT and APOE immunoreactivity was never localized to plaque-like profiles but was weakly localized to unreactive astrocytes; weak HLA-DR and GFAP immunoreactivity was present on quiescent microglia throughout the cerebellum. The lack of fibrillar amyloid deposits in cerebellum, despite the presence of well-characterized markers thought to mediate the production of Aβ, suggests that this brain region may be lacking certain factors necessary for fibril formation or that the cerebellum responds differently to stimuli that successfully mediate inflammation in affected cortex. J. Comp. Neurol. 396:511–520, 1998. © 1998 Wiley-Liss, Inc.     

NG2‐glia crosstalk with microglia in health and disease

Neurodegenerative diseases are increasingly becoming a global problem. However, the pathological mechanisms underlying neurodegenerative diseases are not fully understood. NG2‐glia abnormalities and microglia activation are involved in the development and/or progression of neurodegenerative disorders, such as multiple sclerosis, Alzheimer''s disease, Parkinson''s disease, and cerebrovascular diseases. In this review, we summarize the present understanding of the interaction between NG2‐glia and microglia in physiological and pathological states and discuss unsolved questions concerning their fate and potential fate. First, we introduce the NG2‐glia and microglia in health and disease. Second, we formulate the interaction between NG2‐glia and microglia. NG2‐glia proliferation, migration, differentiation, and apoptosis are influenced by factors released from the microglia. On the other hand, NG2‐glia also regulate microglia actions. We conclude that NG2‐glia and microglia are important immunomodulatory cells in the brain. Understanding the interaction between NG2‐glia and microglia will help provide a novel method to modulate myelination and treat neurodegenerative disorders.     

LPS-induced expression of a novel chemokine receptor (L-CCR) in mouse glial cells in vitro and in vivo

There is increasing evidence that chemokines, specialized regulators of the peripheral immune system, are also involved in the physiology and pathology of the CNS. It is known that glial cells (astrocytes and microglia) express various chemokine receptors like CCR1, -3, -5, and CXCR4. We have investigated the possible expression of the known CC chemokine receptors (CCR1-8 and D6) in murine glial cells. In addition, we examined possible glial expression of the orphan CC chemokine receptor L-CCR that has been identified previously in murine macrophages. We report here expression of L-CCR mRNA in murine astrocytes and microglia. Furthermore, L-CCR mRNA expression was strongly induced after application of bacterial lipopolysaccharide (LPS), both in vitro and in vivo. Functional studies and binding experiments using biotinylated monocyte chemoattractant protein (MCP)-1 (CCL2) indicate that CCL2 could be a candidate chemokine ligand for glial L-CCR. Based on the data presented, it is suggested that L-CCR is a functional glial chemokine receptor that is important in neuroimmunology.     

αB‐crystallin and HSP27 in glial cells in tauopathies

Tauopathies are neurodegenerative diseases characterized by hyper‐phosphorylated tau deposition in neurons and glial cells. Chaperones, such as small heat shock proteins αB‐crystallin and HSP27 highly expressed in normal glial cells, have been postulated as putative molecules preventing abnormal deposition and folding in glial cells in tauopathies. The objective of this work was to assess the expression of αB‐crystallin, phosphorylated αB‐crystallin at Ser59 and HSP27 in glial cells with and without tau deposits in progressive supranuclear palsy, corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Pick's disease (PiD), Alzheimer's disease, frontotemporal lobar degeneration associated with mutations in the tau gene (FTLD‐tau), globular glial tauopathy (GGT) and tauopathy in the elderly. Immunohistochemistry, and double‐labeling immunofluorescence and confocal microscopy have been used for this purpose. Increased expression of αB‐crystallin and phosphorylated αB‐crystallin at Ser59 occurs in a subpopulation of glial cells with and without hyper‐phosphorylated tau deposition in all the analyzed tauopathies, but their expression in neurons is restricted to ballooned neurons in CBD, AGD and PiD. HSP27 barely co‐localizes with tau and with phosphorylated αB‐crystallin at Ser59, thus making the formation of active dimers operating as chaperones unlikely. Results suggest a limited function of αB‐crystallin and HSP27 in preventing abnormal tau protein deposition in glial cells and neurons; in addition, the expression of αB‐crystallin phosphorylated at Ser59 may act as a protective factor in glial cells.     

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.     

A novel method to establish microglia-free astrocyte cultures: comparison of matrix metalloproteinase expression profiles in pure cultures of astrocytes and microglia

Increased matrix metalloproteinase (MMP) proteolytic activity contributes to the pathogenesis of many neuroinflammatory and neurodegenerative conditions in the CNS. To fully understand this process, it is important to define the MMP expression profile of specific cell types, including the CNS-resident cells astrocytes and microglia. While previous studies have characterized astrocyte MMP expression by using mixed glial cultures, these results are likely complicated by the presence of contaminating microglia within these cultures. In the current study, we sought to clarify this complexity, by taking a novel approach to prepare pure astrocyte cultures entirely devoid of microglia, by promoting neural stem cell (NSC) differentiation into astrocytes. The MMP expression profile of mixed glial cultures, neurosphere-derived astrocytes, and pure microglia was characterized by RNase protection assay. This revealed that MMP gene expression is largely cell-type specific. Astrocytes constitutively expressed MMP-11, MMP-14, and MMP-2 and showed induction of MMP-3 in response to IL-1beta but did not respond to lipopolysaccharide (LPS). In contrast, microglia constitutively expressed high levels of MMP-12 and showed strong induction of MMP-9 and MMP-14 in response to LPS. Gelatin zymography confirmed that LPS and TNF-alpha induced strong expression of MMP-9 in microglia but not astrocytes. In summary, these studies demonstrate that neurosphere-derived astrocytes represent an attractive alternative system in which to study astrocyte behavior in vitro. Using this system, we have shown that astrocytes and microglia express distinct sets of MMP genes and that microglia, not astrocytes, are the major source of MMP-9 in response to LPS or TNF-alpha.     

Enhanced IL‐1β production in response to the activation of hippocampal glial cells impairs neurogenesis in aged mice

A variety of mechanisms that contribute to the accumulation of age‐related damage and the resulting brain dysfunction have been identified. Recently, decreased neurogenesis in the hippocampus has been recognized as one of the mechanisms of age‐related brain dysfunction. However, the molecular mechanism of decreased neurogenesis with aging is still unclear. In the present study, we investigated whether aging decreases neurogenesis accompanied by the activation of microglia and astrocytes, which increases the expression of IL‐1β in the hippocampus, and whether in vitro treatment with IL‐1β in neural stem cells directly impairs neurogenesis. Ionized calcium‐binding adaptor molecule 1 (Iba1)‐positive microglia and glial fibrillary acidic protein (GFAP)‐positive astrocytes were increased in the dentate gyrus of the hippocampus of 28‐month‐old mice. Furthermore, the mRNA level of IL‐1β was significantly increased without related histone modifications. Moreover, a significant increase in lysine 9 on histone H3 (H3K9) trimethylation at the promoter of NeuroD (a neural progenitor cell marker) was observed in the hippocampus of aged mice. In vitro treatment with IL‐1β in neural stem cells prepared from whole brain of E14.5 mice significantly increased H3K9 trimethylation at the NeuroD promoter. These findings suggest that aging may decrease hippocampal neurogenesis via epigenetic modifications accompanied by the activation of microglia and astrocytes with the increased expression of IL‐1β in the hippocampus. Synapse 64:721–728, 2010. © 2010 Wiley‐Liss, Inc.     

Methamphetamine‐induced neuroinflammation and neuronal dysfunction in the mice hippocampus: preventive effect of indomethacin

Methamphetamine (METH) causes irreversible damage to brain cells leading to neurological and psychiatric abnormalities. However, the mechanisms underlying life‐threatening effects of acute METH intoxication remain unclear. Indeed, most of the hypotheses focused on intra‐neuronal events, such as dopamine oxidation, oxidative stress and excitotoxicity. Yet, recent reports suggested that glia may contribute to METH‐induced neuropathology. In the present study, we investigated the hippocampal dysfunction induced by an acute high dose of METH (30 mg/kg; intraperitoneal injection), focusing on the inflammatory process and changes in several neuronal structural proteins. For that, 3‐month‐old male wild‐type C57BL/6J mice were killed at different time‐points post‐METH. We observed that METH caused an inflammatory response characterized by astrocytic and microglia reactivity, and tumor necrosis factor (TNF) system alterations. Indeed, glial fibrillary acidic protein (GFAP) and CD11b immunoreactivity were upregulated, likewise TNF‐α and TNF receptor 1 protein levels. Furthermore, the effect of METH on hippocampal neurons was also investigated, and we observed a downregulation in beta III tubulin expression. To clarify the possible neuronal dysfunction induced by METH, several neuronal proteins were analysed. Syntaxin‐1, calbindin D28k and tau protein levels were downregulated, whereas synaptophysin was upregulated. We also evaluated whether an anti‐inflammatory drug could prevent or diminish METH‐induced neuroinflammation, and we concluded that indomethacin (10 mg/kg; i.p.) prevented METH‐induced glia activation and both TNF system and beta III tubulin alterations. In conclusion, we demonstrated that METH triggers an inflammatory process and leads to neuronal dysfunction in the hippocampus, which can be prevented by an anti‐inflammatory treatment.     

Hyperactive glial cells contribute to axonal pathologies in the spinal cord of Npc1 mutant mice

Niemann‐Pick disease type C1 (NPC1) is a neurodegenerative disease with various progressive pathological features, for example, neuronal loss, dysmyelination, abnormal axon swelling, and gliosis, in the brain. Pathological activation of p38‐mitogen‐activated protein kinase (MAPK) results in hyperphosphorylation of tau protein, which contributes to the development of neurodegenerative diseases. In this study, axonal varicosities or spheroids and presynaptic aggregates in the spinal cord of the Npc1 mutant mice were found from postnatal day (P) 35 onwards, as indicated by the increased hyperphosphorylated neurofilament and synaptophysin immunoreactivity as well as the findings from electron microscopy. However, activities of astrocytes and microglia in the Npc1 mutant spinal cord were progressively increased earlier from P10 onwards, accompanied by increased expression of interleukin‐1β and apolipoprotein E, as well as up‐regulated p38‐MAPK activity and enhanced phosphorylated tau protein, but not cyclin‐dependent kinase 5/p35 complex and glycogen synthase kinase‐3β. Taken together, our data suggest that the axonal pathologies in the Npc1 mutant spinal cord are strongly correlated with the increase of activated glial cells, which produce IL‐1β and ApoE, resulting in the activation of p38‐MAPK signaling pathway and enhanced phosphorylated tau protein. GLIA 2014;62:1024–1040     

Glial cytoplasmic inclusions and tissue injury in multiple system atrophy: A quantitative study in white matter (olivopontocerebellar system) and gray matter (nigrostriatal system)

Glial cytoplasmic inclusions (GCIs) and microglia were quantified in 12 cases of multiple system atrophy (MSA) with special reference to their association with histologically defined lesion severity. The targets of the analysis were white matter (cerebellum, pontine base) and gray matter (putamen, substantia nigra). First, the lesion severity was defined: for white matter, the degree of demyelination and tissue rarefaction were semi‐quantified on Klüver‐Barrera (KB) sections as grade I (mildly injured), II (moderately injured), and III (severely injured); for gray matter, neurons and astrocytes were counted on KB and glial fibrillary acidic protein‐immunostained sections, respectively. Next, the GCI burden was quantified on sections immunostained for α‐synuclein, phosphorylated α‐synuclein, and ubiquitin and the microglial burden was quantified on sections immunostained for HLA‐DR. In white matter, the GCI and microglial burdens were the greatest when the tissue injury was mild and/or moderate (grade I and/or grade II), and they became less prominent when the tissue injury became more severe (grade III). In gray matter, in contrast, the GCI and microglial burdens failed to show significant correlations with the lesion severity. Our result suggests that the amount of GCIs as well as that of microglia is reduced when the tissue injury becomes severe in vulnerable white matter areas, but not in vulnerable gray matter areas, of MSA. It also suggests that there seems to be a difference between gray matter and white matter in the way GCIs and microglia participate in the degenerative process of MSA.     

Four-kilobase sequence of the mouse CNP gene directs spatial and temporal expression of lacZ in transgenic mice

The gene encoding 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) is one of the earliest myelin genes to be expressed in the brain. It is expressed at basal levels in some non-neural tissues but at much higher levels in the nervous system, and its relevance and mechanism are unknown. Using transgenic mice, we examined the expression pattern conferred by a 4-kilobase (-kb) 5′-flanking sequence of the mouse CNP gene coupled to the bacterial lacZ reporter gene. Here we report that this 4-kb fragment contains sufficient information to direct expression of the transgene to the tissue and/or cell type, in which CNP is normally expressed. In the central nervous system (CNS), CNP-lacZ expression was regulated in a temporal manner, consistent with endogenous CNP expression. Transgene expression was detected in embryonic brain and spinal cord in immature oligodendrocytes, and it significantly increased with age. In adult mice, β-galactosidase activity (which appeared to be oligodendrocyte specific) was found essentially in white matter areas of the CNS. Moreover, the transgene was expressed in peripheral nervous system, testis, and thymus—tissues that normally express CNP. Taken together, our results provide strong evidence that cis-acting regulatory elements, necessary to direct spatial and temporal expression of the transgene in oligodendrocytes, are located within the 4-kb 5′-flanking sequence of the mouse CNP gene. This promoter could be a valuable tool to target specific expression of other transgenes to oligodendrocytes, and may provide important new insights into myelination or dysmyelination. J. Neurosci. Res. 53:393–404, 1998. © 1998 Wiley-Liss, Inc.     

The role and therapeutic potential of monocytic cells in Alzheimer's disease

Alzheimer's disease (AD) is a dementing neurodegenerative disorder without a cure. The abnormal parenchymal accumulation of β‐amyloid (Aβ) is associated with inflammatory reactions involving microglia and astrocytes. Increased levels of Aβ and Aβ deposition in the brain are thought to cause neuronal dysfunction and underlie dementia. Microglia, the brain resident cells of monocytic origin, have a potential ability to phagocytose Aβ but they also react to Aβ by increased production of proinflammatory toxic agents. Microglia originate from hemangioblastic mesoderm during early embryonic stages and from bone marrow (BM)‐derived monocytic cells that home the brain throughout the neonatal stage of development. Recent studies indicate that BM or blood‐derived monocytes are recruited to the diseased AD brain, associate with the Aβ depositions, and are more efficient phagocytes of Aβ compared with resident microglia. The clearance of Aβ deposition by these cells has been recently under intensive investigation and can occur through several different mechanisms. Importantly, peripheral monocytic cells of patients with AD appear to be deficient in clearing Aβ. This review will summarize the findings on the role of blood‐derived cells in AD and discuss their therapeutic potential for treating patients suffering from this devastating disease. © 2010 Wiley‐Liss, Inc.     

Glial expression of the 90-kDa heat shock protein (HSP90) and the 94-kDa glucose-regulated protein (GRP94) following an excitotoxic lesion in the mouse hippocampus

Heat shock proteins (HSPs) are immediately expressed in neuronal and glial cells under various stressful conditions and play a protective role through molecular chaperones. Although several studies have been focused on the expression of HSPs, little is known about HSP90s expression in glial cells under neuropathological conditions. In this study, we evaluated the expression pattern of the glial cell-related HSP90 and GRP94 proteins, following the induction of an excitotoxic lesion in the mouse brain. Adult mice received an intracerebroventricular injection of kainic acid; the brain tissue was then analyzed immunohistochemically for HSPs and double labeling using glial markers. HSPs expression was quantified by Western blot analysis. Excitotoxic damage was found to cause pyramidal cell degeneration in the CA3 region of the hippocampus. In the injured hippocampus, reactive microglia/macrophages expressed HSP90 from 12 h until 7 days postlesion (PL), showing maximal levels at day 1. In parallel, hippocampal reactive astrocytes showed the expression of GRP94 from 12 h until 7 days PL. In general, HSPs expression was transient, peaked at 1-3 days PL and reached basal levels by day 7. For the first time, our data demonstrate the injury-induced expression of HSP90 and GRP94 in glial cells, which may contribute to the mechanism of glial cell protection and adaptation in response to damage, thereby playing an important role in the evolution of the glial response and the excitotoxic lesion outcome. HSP90 may provide antioxidant protective mechanisms against microglia/macrophages, whereas GRP94 may stabilize the astroglial cytoskeleton and participate in astroglial antioxidant mechanisms.