Astrocytic Neuroimmunological Roles Interacting with Microglial Cells in Neurodegenerative Diseases.

Both astrocytic and microglial functions have been extensively investigated in healthy subjects and neurodegenerative diseases. For astrocytes, not only various sub-types were identified but phagocytic activity was also clarified recently and is making dramatic progress. In this review paper, we mostly focus on the functional role of astrocytes in the extracellular matrix and on interactions between reactive astrocytes and reactive microglia in normal states and in neurodegenerative diseases, because the authors feel it is necessary to elucidate the mechanisms among activated glial cells in the pathology of neurological diseases in order to pave the way for drug discovery. Finally, we will review cyclic phosphatidic acid (cPA), a naturally occurring phospholipid mediator that induces a variety of biological activities in the brain both in vivo and in vitro. We propose that cPA may serve as a novel therapeutic molecule for the treatment of brain injury and neuroinflammation.

Integrin Signaling in the Central Nervous System in Animals and Human Brain Diseases.

The integrin family is involved in various biological functions, including cell proliferation, differentiation and migration, and also in the pathogenesis of disease. Integrins are multifunctional receptors that exist as heterodimers composed of α and ß subunits and bind to various ligands, including extracellular matrix (ECM) proteins; they are found in many animals, not only vertebrates (e.g., mouse, rat, and teleost fish), but also invertebrates (e.g., planarian flatworm, fruit fly, nematodes, and cephalopods), which are used for research on genetics and social behaviors or as models for human diseases. In the present paper, we describe the results of a phylogenetic tree analysis of the integrin family among these species. We summarize integrin signaling in teleost fish, which serves as an excellent model for the study of regenerative systems and possesses the ability for replacing missing tissues, especially in the central nervous system, which has not been demonstrated in mammals. In addition, functions of astrocytes and reactive astrocytes, which contain neuroprotective subpopulations that act in concert with the ECM proteins tenascin C and osteopontin via integrin are also reviewed. Drug development research using integrin as a therapeutic target could result in breakthroughs for the treatment of neurodegenerative diseases and brain injury in mammals.

Metabolic Plasticity of Astrocytes and Aging of the Brain.

As part of the blood-brain-barrier, astrocytes are ideally positioned between cerebral vasculature and neuronal synapses to mediate nutrient uptake from the systemic circulation. In addition, astrocytes have a robust enzymatic capacity of glycolysis, glycogenesis and lipid metabolism, managing nutrient support in the brain parenchyma for neuronal consumption. Here, we review the plasticity of astrocyte energy metabolism under physiologic and pathologic conditions, highlighting age-dependent brain dysfunctions. In astrocytes, glycolysis and glycogenesis are regulated by noradrenaline and insulin, respectively, while mitochondrial ATP production and fatty acid oxidation are influenced by the thyroid hormone. These regulations are essential for maintaining normal brain activities, and impairments of these processes may lead to neurodegeneration and cognitive decline. Metabolic plasticity is also associated with (re)activation of astrocytes, a process associated with pathologic events. It is likely that the recently described neurodegenerative and neuroprotective subpopulations of reactive astrocytes metabolize distinct energy substrates, and that this preference is supposed to explain some of their impacts on pathologic processes. Importantly, physiologic and pathologic properties of astrocytic metabolic plasticity bear translational potential in defining new potential diagnostic biomarkers and novel therapeutic targets to mitigate neurodegeneration and age-related brain dysfunctions.

Neuroimmunological Implications of AQP4 in Astrocytes.

The brain has high-order functions and is composed of several kinds of cells, such as neurons and glial cells. It is becoming clear that many kinds of neurodegenerative diseases are more-or-less influenced by astrocytes, which are a type of glial cell. Aquaporin-4 (AQP4), a membrane-bound protein that regulates water permeability is a member of the aquaporin family of water channel proteins that is expressed in the endfeet of astrocytes in the central nervous system (CNS). Recently, AQP4 has been shown to function, not only as a water channel protein, but also as an adhesion molecule that is involved in cell migration and neuroexcitation, synaptic plasticity, and learning/memory through mechanisms involved in long-term potentiation or long-term depression. The most extensively examined role of AQP4 is its ability to act as a neuroimmunological inducer. Previously, we showed that AQP4 plays an important role in neuroimmunological functions in injured mouse brain in concert with the proinflammatory inducer osteopontin (OPN). The aim of this review is to summarize the functional implication of AQP4, focusing especially on its neuroimmunological roles. This review is a good opportunity to compile recent knowledge and could contribute to the therapeutic treatment of autoimmune diseases through strategies targeting AQP4. Finally, the author would like to hypothesize on AQP4's role in interaction between reactive astrocytes and reactive microglial cells, which might occur in neurodegenerative diseases. Furthermore, a therapeutic strategy for AQP4-related neurodegenerative diseases is proposed.

Aquaporin 4-dependent expression of glial fibrillary acidic protein and tenascin-C in activated astrocytes in stab wound mouse brain and in primary culture.

We previously reported that aquaporin 4 (AQP4) has a neuroimmunological function via astrocytes and microglial cells involving osteopontin. AQP4 is a water channel localized in the endofoot of astrocytes in the brain, and its expression is upregulated after a stab wound to the mouse brain or the injection of methylmercury in common marmosets. In this study, the correlation between the expression of AQP4 and the expression of glial fibrillary acidic protein (GFAP) or tenascin-C (TN-C) in reactive astrocytes was examined in primary cultures and brain tissues of AQP4-deficient mice (AQP4/KO). In the absence of a stab wound to the brain or of any stimulation of the cells, the expressions of both GFAP and TN-C were lower in astrocytes from AQP4/KO mice than in those from wild-type (WT) mice. High levels of GFAP and TN-C expression were observed in activated astrocytes after a stab wound to the brain in WT mice; however, the expressions of GFAP and TN-C were insignificant in AQP4/KO mice. Furthermore, lipopolysaccharide (LPS) stimulation activated primary culture of astrocytes and upregulated GFAP and TN-C expression in cells from WT mice, whereas the expressions of GFAP and TN-C were slightly upregulated in cells from AQP4/KO mice. Moreover, the stimulation of primary culture of astrocytes with LPS also upregulated inflammatory cytokines in cells from WT mice, whereas modest increases were observed in cells from AQP4/KO mice. These results suggest that AQP4 expression accelerates GFAP and TN-C expression in activated astrocytes induced by a stab wound in the mouse brain and LPS-stimulated primary culture of astrocytes.

Immunological function of aquaporin-4 in stab-wounded mouse brain in concert with a pro-inflammatory cytokine inducer, osteopontin.

During injury to the central nervous system (CNS), astrocytes and microglia proliferate and migrate around the lesion sites. Recently, it has been reported that one of the water channels, aquaporin-4 (AQP4) is seemed to have a role in astroglial migration and glial scar formation caused by brain injury, although its molecular mechanism is largely unknown. In the present study, we examined the expression profiles in wild-type (WT) and AQP4-deficient (AQP4/KO) mice after a stab wound to the cerebral cortex. Three days after the stab wound, AQP4 expression was observed in activated microglia around the lesion site as well as in astrocytes. A microarray analysis revealed that 444 genes around the lesion site were upregulated 3 days after the wounding in WT mice. Surprisingly, most of these up-regulations were significantly attenuated in AQP4/KO mice. Real-time RT-PCR and immunofluorescence showed that osteopontin (OPN) expression around the lesion site was much lower in AQP4/KO mice than in WT mice. Moreover, the up-regulation of pro-inflammatory cytokines was significantly attenuated in AQP4/KO mice. Taken together, these results suggest that AQP4 plays an important role in immunological function in concert with OPN under pathological conditions in the CNS.

An astrocyte-specific enhancer of the aquaporin-4 gene functions through a consensus sequence of POU transcription factors in concert with multiple upstream elements.

Aquaporin-4, a predominant water channel in the brain, is specifically expressed in astrocyte endfeet and plays a central role in water homeostasis, neuronal activity, and cell migration in the brain. It has two dominant isoforms called M1 and M23, whose mRNA is driven by distinct promoters located upstream of exons 0 and 1 of the aquaporin-4 gene, respectively. To identify cis-acting elements responsible for the astrocyte-specific transcription of M1 mRNA, the promoter activity of the 5'-flanking region upstream of exon 0 in primary cultured mouse astrocytes was examined by luciferase assay, and sequences, where nuclear factors bind, were identified by electrophoretic mobility shift assay. An astrocyte-specific activity enhancing transcription from the M1 promoter was observed within ∼2 kb from the transcriptional start sites of M1 mRNA. At least five elements clustered within the 286-bp region were found to function as a novel astrocyte-specific enhancer. Among the five elements, a consensus sequence of Pit-1/Oct/Unc-86 (POU) transcription factors was indispensable to the astrocyte-specific enhancer since disruption of the POU motif completely abolished the enhancer activity in astrocytes. However, the POU motif alone had little activity, indicating the requirement for cooperation with other upstream elements to exert full enhancer activity.

The neuroprotective function of 2-carba-cyclic phosphatidic acid: Implications for tenascin-C via astrocytes in traumatic brain injury.

We examined the mechanism how 2-carba-cyclic phosphatidic acid (2ccPA), a lipid mediator, regulates neuronal apoptosis in traumatic brain injury (TBI). First, we found 2ccPA suppressed neuronal apoptosis after the injury, and increased the immunoreactivity of tenascin-C (TN-C), an extracellular matrix protein by 2ccPA in the vicinity of the wound region. 2ccPA increased the mRNA expression levels of Tnc in primary cultured astrocytes, and the conditioned medium of 2ccPA-treated astrocytes suppressed the apoptosis of cortical neurons. The neuroprotective effect of TN-C was abolished by knockdown of TN-C. These results indicate that 2ccPA contributes to neuroprotection via TN-C from astrocytes in TBI.

Cloning and expression of cDNA for interleukin 4 from the MSKR inbred strain of Mus musculus molossinus.

The 5'-upsteam region of the genomic gene and cDNA encoding interleukin 4 (IL-4) has been isolated and sequenced from the inbred mouse strain MSKR derived from a Japanese wild mouse using polymerase chain reaction (PCR) methodology. The cDNA sequence of IL-4 of MSKR was found to contain several nucleotide alterations, which result in amino acid substitutions, in comparison with that of inbred mouse BALB/c. In MSKR, IL-4 was expressed at high levels in thymus and spleen as revealed by reverse transcriptase PCR method and Northern hybridization. The tissue-specific expression profile was quite similar to that of laboratory mice.

Astrocytic endfeet re-cover blood vessels after removal by laser ablation.

The astrocyte, one of the glial cells, plays many functional roles. These include provision of nutrients from blood vessels to neurons, supply of neurotransmitters and support of blood-brain barrier (BBB) integrity. Astrocytes are known to support the integrity of BBB through maintenance of the tight junction between endothelial cells of blood vessels. However, evidence of its direct contribution to BBB is lacking owing to technical limitations. In this study, astrocytic endfeet covering blood vessels were removed by the laser ablation method with two photon laser scanning microscopy in in vivo mouse brain, and the re-covering of blood vessels with the astrocytic endfeet was observed in about half of the cases. Blood vessels kept their integrity without astrocytic endfoot covers: leakage of plasma marker dyes, Evans Blue or dextran-conjugated fluorescein, was not observed from stripped blood vessels, while ablation of vascular walls induced extravasation of Evans Blue. These results suggest that the astrocytic endfeet covering blood vessels do not contribute to the immediate BBB barrier.

Selective ablation of an astroglial subset by toxic gene expression driven by tenascin promoter.

OBJECTIVE: In vitro culture, one of the astroglia-derived extracellular matrix proteins, tenascin, expressed highly in fibrous astrocytes, whereas it expressed only low levels in protoplasmic astrocytes. We devised a method of selectively altering the population of astroglial subsets in primary culture of astrocytes derived from embryonic mouse brains using toxic gene expression driven by the tenascin promoter. METHODS: We have identified that the segment of 512-bases of 5'-flanking plus 243-bases leader sequences of the mouse tenascin gene contains maximum promoter activity in primary culture of astrocytes by deletion analysis of 5'-upstream region. This promoter element was used to specifically express the herpes simplex virus thymidine kinase (HSV-TK) gene in tenascin-positive astrocytes. RESULTS: This strategy allowed us to selectively decrease tenascin-positive astrocytes at the optimal concentration of ganciclovir, which is cytotoxic in HSV-TK-expressing cells. DISCUSSION: This approach should be useful for examining the role of the tenascin-negative astroglial subset in the development and regeneration of the central nervous system.

Alteration of inflammatory cytokine production in the injured central nervous system of tenascin-deficient mice.

Although tenascin-C (TN) is highly up-regulated during the proliferation of reactive astrocytes, little is known about the function of TN at injury sites in the central nervous system (CNS). Here, the function of TN-expressing astrocytes in the injured brain was investigated by analyzing TN-deficient mice with stab-wound injuries of the cerebral cortex. Glial fibrillary acid protein expression after injury was down-regulated earlier in TN-deficient mice than in wild-type (WT) mice. To evaluate immune responses in the injured CNS in the absence of TN, inflammatory cytokine production was examined after unilateral stab injuries of the cerebral cortex in TN-deficient and WT mice. The expression of interleukin (IL)-1beta, tumor necrosis factor-a and IL-6 was higher in TN-deficient mice, whereas levels of IL-4 and granulocyte colony-stimulating factor were lower in TN-deficient mice than WT mice. Our findings suggest that TN helps to regulate production of inflammatory cytokines in the injured brain.

Osteopontin is indispensable for activation of astrocytes in injured mouse brain and primary culture.

OBJECTIVES: Osteopontin (OPN) is an inflammatory cytokine inducer involved in cell proliferation and migration in inflammatory diseases or tumors. To investigate the function of OPN in astrocyte activation during brain injury, we compared OPN-deficient (OPN/KO) with wild-type (WT) mouse brains after stab wound injury and primary culture of astrocytes. METHODS: Primary cultures of astrocytes were prepared from either WT or OPN/KO postnatal mouse brains. Activation efficiency of astrocytes in primary culture was accessed using Western blotting by examining the protein levels of glial fibrillary acidic protein (GFAP) and tenascin-C (TN-C), which are markers for reactive astrocytes, following lipopolysaccharide (LPS) stimulation. Furthermore, the stab wound injury on the cerebral cortex as a brain traumatic injury model was used, and activation of astrocytes and microglial cells was investigated using immunofluorescent analysis on fixed brain sections. RESULTS: Primary cultures of astrocytes prepared from WT or OPN/KO postnatal mouse brains showed that only 25% of normal shaped astrocytes in a flask were produced in OPN/KO mice. The expression levels of both GFAP and TN-C were downregulated in the primary culture of astrocytes from OPN/KO mice compared with that from WT mice. By the immunofluorescent analysis on the injured brain sections, glial activation was attenuated in OPN/KO mice compared with WT mice. DISCUSSION: Our data suggest that OPN is essential for proper astrocytic generation in vitro culture prepared from mouse cerebral cortex. OPN is indispensable for astrocyte activation in the mouse brain injury model and in LPS stimulated primary culture. ABBREVIATIONS: AQP4: aquaporin 4; BBB: blood brain barrier; BrdU: bromo-deoxy uridine; CNS: central nervous system; GFAP: glial fibllirary acidic protein; IgG: immunoglobulin G; LPS: lipopolysaccharide; OPN: osteopontin; OPN/KO: osteopontin-deficient; TN-C: tenascin-C.

2-carba cyclic phosphatidic acid suppresses inflammation via regulation of microglial polarisation in the stab-wounded mouse cerebral cortex.

Traumatic brain injury (TBI) is caused by physical damage to the brain and it induces blood-brain barrier (BBB) breakdown and inflammation. To diminish the sequelae of TBI, it is important to decrease haemorrhage and alleviate inflammation. In this study, we aimed to determine the effects of 2-carba-cyclic phosphatidic acid (2ccPA) on the repair mechanisms after a stab wound injury as a murine TBI model. The administration of 2ccPA suppressed serum immunoglobulin extravasation after the injury. To elucidate the effects of 2ccPA on inflammation resulting from TBI, we analysed the mRNA expression of inflammatory cytokines. We found that 2ccPA prevents a TBI-induced increase in the mRNA expression of Il-1ß, Il-6, Tnf-α and Tgf-ß1. In addition, 2ccPA reduces the elevation of Iba1 levels. These data suggest that 2ccPA attenuates the inflammation after a stab wound injury via the modulation of pro-inflammatory cytokines release from microglial cells. Therefore, we focused on the function of 2ccPA in microglial polarisation towards M1 or M2 phenotypes. The administration of 2ccPA decreased the number of M1 and increased the number of M2 type microglial cells, indicating that 2ccPA modulates the microglial polarisation and shifts them towards M2 phenotype. These data suggest that 2ccPA treatment suppresses the extent of BBB breakdown and inflammation after TBI.

Tenascin-C is required for proliferation of astrocytes in primary culture.

Astrocytes in primary culture can be classified morphologically into two types: fibrous astrocytes and protoplasmic astrocytes. To examine the role of tenascin-C (TN) in an in vitro astrocyte culture, primary cultures of astrocytes prepared from the brains of wild-type and of TN-deficient embryonic mice were analyzed. In primary culture of astrocytes from TN-deficient mice fibrous astrocytes did not appear and astrocytes did not become tile-shaped when they came in contact with each other. The rate of 5-bromo-2'-deoxyuridine incorporation in a cell proliferation assay was much lower for astrocytes from TN-deficient mice than for astrocytes from wild-type mice. These results suggest that TN is an essential molecule for maintaining the proliferation and proper morphology of astrocytes in primary culture.

The dual effects of the astrocyte-specific enhancer of the AQP4 M1 promoter.

Aquaporin-4, a predominant water channel in the central nervous system, has two isoforms, M1 and M23, whose transcripts are driven by distinct promoters. Using a reporter assay, we found that a fragment located between exons 0 and 1 of the mouse aquaporin-4 gene, which had been thought to be the promoter for M23, lacked enhancers functioning in astrocytes. When the astrocyte-specific enhancer (ASE) of the M1 promoter is connected to the putative M23 core promoter, it also works in astrocytes. Importantly, the ASE inhibits downstream promoter activity in NIH3T3 cells, indicating that the ASE also functions as a silencer in cells lacking aquaporin-4.

Vitronectin Regulates the Fibrinolytic System during the Repair of Cerebral Cortex in Stab-Wounded Mice.

Vitronectin (VN), one of the serum proteins, is known to be involved in the regulation of blood coagulation, fibrinolysis, and cell migration. It has been proposed that the regulation of fibrinolysis by VN promotes the blood-brain barrier (BBB) recovery from brain injuries such as traumatic injury and subarachnoid hemorrhage. The effects of VN on fibrinolysis in the injured brain remain unclear, however. We examined the effects of VN on the fibrinolytic system in the stab-wounded cerebral cortex of VN-knockout (KO) mice. First, hemorrhage and recovery from BBB breakdown in the wounded regions were assessed by serum immunoglobulin G (IgG) extravasation. The level of IgG extravasation increased 3-7 days after the stab wound (D3-7) in the cortex of VN-KO mice, compared with that in wild type mice, indicating that VN deficiency inhibited the recovery from BBB breakdown. The VN deficiency decreased fibrin fiber deposition at D1-3, suggesting that VN deficiency tilts the balance between fibrinogenesis and fibrinolysis toward fibrinolysis. Next, the effects of VN deficiency on the fibrinolytic factors were analyzed in the stab-wounded cortex. The VN deficiency impaired the activity of plasminogen activator inhibitor-1, an inhibitor of the fibrinolytic system, at D3-5. Further, VN deficiency up-regulated the mRNA and protein expression levels of tissue-type plasminogen activator, and urokinase-type plasminogen activator. These results demonstrate that VN contributes to the regulation of the fibrinolytic system and recovery from BBB breakdown in the wounded brain.

Correlation between astrocyte activity and recovery from blood-brain barrier breakdown caused by brain injury.

Glial activation is associated with cell proliferation and upregulation of astrocyte marker expression following traumatic injury in the brain. However, the biological significance of these processes remains unclear. In the present study, astrocyte activation was investigated in a murine brain injury model. Brain injury induces blood-brain barrier (BBB) breakdown and immunoglobulin G (IgG) leak into the brain parenchyma. The recovery of BBB breakdown was evaluated by analyzing immunofluorescent staining with mouse IgG antibody. IgG leakage was greatest at 1 day after stab wound injury and decreased thereafter, and almost diminished after 7 days. Bromodeoxy uridine incorporation was used, and astrocyte proliferation rates were examined by coimmunostaining with anti-bromodeoxy uridine and anti-glial fibrillary acid protein antibodies. Consistent with IgG leakage assays, astrocyte activation was the highest at day 3 and decreased after 7 days. Moreover, in reverse transcriptase-quantitative-PCR experiments, genes associated with BBB integrity were downregulated immediately after BBB breakdown and recovered to basal expression levels within 7 days. These data indicated that astrocyte activation correlated with BBB recovery from breakdown following brain injury.

Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4.

Neuromyelitis optica is a demyelinating disease characterized by a disease-specific autoantibody designated as NMO-IgG that specifically recognizes aquaporin-4, and the binding of NMO-IgG to AQP4 causes the progress of the disease. Prevention of the binding of NMO-IgG, therefore, may alleviate the disease. Here we have developed monoclonal antibodies against AQP4 with a baculovirus display system in order to obtain high affinity monoclonal antibodies against the extracellular domains of AQP4. Our monoclonal antibodies can block the binding of NMO-IgG in spite of their heterogeneity. Taken together, we propose that our monoclonal antibodies can be applied in clinical therapy for NMO patients.

Increased expression of aquaporin-4 with methylmercury exposure in the brain of the common marmoset.

The relationship between methylmercury (MeHg) exposure and aquaporin (AQP) expression in the brain is currently unknown. To investigate this, we used a common marmoset model of acute MeHg exposure to examine AQP1, AQP4 and AQP11 gene expression. MeHg (1.5 mg Hg/kg/day p.o.) was given to three marmosets for 14 days, followed by 14 days without. All treated marmosets showed slight akinesia before sacrifice. In the frontal lobe, occipital lobe and cerebellum, total mercury concentrations following MeHg administration were 26.7, 31.4, and 22.6 µg/g, respectively. Slight apoptosis was observed in the occipital lobe. Immunohistochemistry showed increased expression of glial fibrillary acidic protein, its mRNA and Iba1 with MeHg, indicating that neuronal injury activated astrocytes and microglia. There was no significant difference between control and MeHg-administered groups in AQP1 protein or AQP11 mRNA in the frontal lobe, occipital lobe or cerebellum. The ratio of AQP4 mRNA expression in MeHg-administered marmosets to the mean AQR4 expression in the controls (n = 3) were 1.3, 1.5 and 1.2, 1.7, 1.9 and 1.5, and 1.5, 1.6 and 1.2 for the frontal lobe, occipital lobe and cerebellum, respectively. Western blotting showed significantly increased AQP4 protein in the occipital lobe and cerebellum with MeHg administration, but no obvious up-regulation in the frontal lobe. Immunofluorescence analysis with double staining revealed low AQP4 expression in the cell body of reactive astrocytes in the MeHg-administered group. These results indicate that AQP4 expression might be stimulated by MeHg exposure in astrocytes in the occipital lobe and cerebellum, suggesting a role for AQP4 in MeHg neurotoxicity via astrocyte dysfunction.