Perceptual Information Integration: Hypothetical Role of Astrocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Astrocytes and human cognition: Modeling information integration and modulation of neuronal activity

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein

The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target cells. Strikingly, CDV transmission to remote cells could occur in less than 6 h, suggesting that a complete viral cycle with production of extracellular free particles was not essential in enabling CDV to spread in glial cells. Titration experiments and electron microscopy confirmed a very low CDV particle production despite higher titers of membrane-associated viruses. Interestingly, confocal laser microscopy and lentivirus transduction indicated expression and functionality of the viral fusion machinery, consisting of the viral fusion (F) and attachment (H) glycoproteins, at the cell surface. Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread. Furthermore, since we could not detect CD150/SLAM expression in brain cells, the presence of a yet non-identified glial receptor for CDV was suggested. Altogether, our findings indicate that persistence in CDV infection results from intracellular cell-to-cell transmission requiring the CDV-H protein. Viral transfer, happening selectively at the tip of astrocytic processes, may help the virus to cover long distances in the astroglial network, "outrunning" the host's immune response in demyelinating plaques, thus continuously eliciting new lesions.

Toxicity of Streptococcus pneumoniae in neurons, astrocytes, and microglia in vitro

The toxicity of pneumococci and endotoxin in primary cultures of rat neurons, astrocytes, and microglia and in a human astrocyte and two human glial cell lines was determined. Heat-inactivated, rough pneumococci (up to 10(8) cfu/mL) or their cell wall (up to 50 micrograms/mL) produced dose-dependent toxicity after 48 h in microglial cells and to a lesser extent in astrocytes but not in neurons. Toxicity was similar for equivalent doses of heat-inactivated organisms and pneumococcal cell wall, but time-course experiments showed significant differences between the two stimuli. Endotoxin at concentrations of up to 5 micrograms/mL did not induce significant toxicity in any of the cells. Thus, pneumococci can induce toxicity in two brain cell types, microglia and astrocytes, and the pneumococcal cell wall appears to mediate toxicity. Direct toxic effects of bacteria on brain cells may in part be responsible for brain injury during meningitis.

Agrin defines polarized distribution of orthogonal arrays of particles in astrocytes

Accumulating evidence indicates that agrin, a heparan sulphate proteoglycan of the extracellular matrix, plays a role in the organization and maintenance of the blood-brain barrier. This evidence is based on the differential effects of agrin isoforms on the expression and distribution of the water channel protein, aquaporin-4 (AQP4), on the swelling capacity of cultured astrocytes of neonatal mice and on freeze-fracture data revealing an agrin-dependent clustering of orthogonal arrays of particles (OAPs), the structural equivalent of AQP4. Here, we show that the OAP density in agrin-null mice is dramatically decreased in comparison with wild-types, by using quantitative freeze-fracture analysis of astrocytic membranes. In contrast, anti-AQP4 immunohistochemistry has revealed that the immunoreactivity of the superficial astrocytic endfeet of the agrin-null mouse is comparable with that in wild-type mice. Moreover, in vitro, wild-type and agrin-null astrocytes cultured from mouse embryos at embryonic day 19.5 differ neither in AQP4 immunoreactivity, nor in OAP density in freeze-fracture replicas. Analyses of brain tissue samples and cultured astrocytes by reverse transcription with the polymerase chain reaction have not demonstrated any difference in the level of AQP4 mRNA between wild-type astrocytes and astrocytes from agrin-null mice. Furthermore, we have been unable to detect any difference in the swelling capacity between wild-type and agrin-null astrocytes. These results clearly demonstrate, for the first time, that agrin plays a pivotal role for the clustering of OAPs in the endfoot membranes of astrocytes, whereas the mere presence of AQP4 is not sufficient for OAP clustering.

FoxM1B regulates NEDD4-1 expression, leading to cellular transformation and full malignant phenotype in immortalized human astrocytes.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

CSF from Parkinson disease patients differentially affects cultured microglia and astrocytes.

BACKGROUND: Excessive and abnormal accumulation of alpha-synuclein (α-synuclein) is a factor contributing to pathogenic cell death in Parkinson's disease. The purpose of this study, based on earlier observations of Parkinson's disease cerebrospinal fluid (PD-CSF) initiated cell death, was to determine the effects of CSF from PD patients on the functionally different microglia and astrocyte glial cell lines. Microglia cells from human glioblastoma and astrocytes from fetal brain tissue were cultured, grown to confluence, treated with fixed concentrations of PD-CSF, non-PD disease control CSF, or control no-CSF medium, then photographed and fluorescently probed for α-synuclein content by deconvolution fluorescence microscopy. Outcome measures included manually counted cell growth patterns from day 1-8; α-synuclein density and distribution by antibody tagged 3D model stacked deconvoluted fluorescent imaging. RESULTS: After PD-CSF treatment, microglia growth was reduced extensively, and a non-confluent pattern with morphological changes developed, that was not evident in disease control CSF and no-CSF treated cultures. Astrocyte growth rates were similarly reduced by exposure to PD-CSF, but morphological changes were not consistently noted. PD-CSF treated microglia showed a significant increase in α-synuclein content by day 4 compared to other treatments (p ≤ 0.02). In microglia only, α-synuclein aggregated and redistributed to peri-nuclear locations. CONCLUSIONS: Cultured microglia and astrocytes are differentially affected by PD-CSF exposure compared to non-PD-CSF controls. PD-CSF dramatically impacts microglia cell growth, morphology, and α-synuclein deposition compared to astrocytes, supporting the hypothesis of cell specific susceptibility to PD-CSF toxicity.

Astrocytes from the contused spinal cord inhibit oligodendrocyte differentiation of adult oligodendrocyte precursor cells by increasing the expression of bone morphogenetic proteins.

Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.

Brain-borne IL-1 adjusts glucoregulation and provides fuel support to astrocytes and neurons in an autocrine/paracrine manner.

It is still controversial which mediators regulate energy provision to activated neural cells, as insulin does in peripheral tissues. Interleukin-1β (IL-1β) may mediate this effect as it can affect glucoregulation, it is overexpressed in the 'healthy' brain during increased neuronal activity, and it supports high-energy demanding processes such as long-term potentiation, memory and learning. Furthermore, the absence of sustained neuroendocrine and behavioral counterregulation suggests that brain glucose-sensing neurons do not perceive IL-1β-induced hypoglycemia. Here, we show that IL-1β adjusts glucoregulation by inducing its own production in the brain, and that IL-1β-induced hypoglycemia is myeloid differentiation primary response 88 protein (MyD88)-dependent and only partially counteracted by Kir6.2-mediated sensing signaling. Furthermore, we found that, opposite to insulin, IL-1β stimulates brain metabolism. This effect is absent in MyD88-deficient mice, which have neurobehavioral alterations associated to disorders in glucose homeostasis, as during several psychiatric diseases. IL-1β effects on brain metabolism are most likely maintained by IL-1β auto-induction and may reflect a compensatory increase in fuel supply to neural cells. We explore this possibility by directly blocking IL-1 receptors in neural cells. The results showed that, in an activity-dependent and paracrine/autocrine manner, endogenous IL-1 produced by neurons and astrocytes facilitates glucose uptake by these cells. This effect is exacerbated following glutamatergic stimulation and can be passively transferred between cell types. We conclude that the capacity of IL-1β to provide fuel to neural cells underlies its physiological effects on glucoregulation, synaptic plasticity, learning and memory. However, deregulation of IL-1β production could contribute to the alterations in brain glucose metabolism that are detected in several neurologic and psychiatric diseases.Molecular Psychiatry advance online publication, 8 December 2015; doi:10.1038/mp.2015.174.

The role of reactive astrocytes in the resistance of melanoma brain metastasis to chemotherapy

Brain metastasis is resistant to chemotherapy while the leaky blood-brain-barrier in brain metastasis can not be the underlying reason. Metastatic tumor cells (“seed”) exploit the host microenvironment (“soil”) for survival advantages. Astrocytes which maintain the homeostasis of the brain microenvironment become reactive subsequent to brain damages and protect neurons from various injuries. We observed reactive astrocytes surrounding and infiltrating into brain metastasis in both clinical specimen and experimental animal model, thus raising a possibility that reactive astrocytes may protect tumor cells from cytotoxic chemotherapeutic drugs. ^ To test this hypothesis, we first generated an immortalized astrocyte cell line from H-2Kb-tsA58 mice. The immortal mouse astrocytes expressed specific markers including GFAP. Scanning electron microscopy demonstrated that astrocytes formed direct physical contact with tumor cells. Moreover, the expression of GFAP by astrocytes was up-regulated subsequent to co-culture with tumor cells, indicating that the co-culture of astrocytes and tumor cells may serve as a model to recapitulate the pathophysiological situation of brain metastasis. ^ In co-culture, astrocytes dramatically reduced apoptosis of tumor cells produced by various chemotherapeutic drugs. This protection effect was not because of culturing cells from different species since mouse fibroblasts did not protect tumor cells from chemotherapy. Furthermore, the protection by astrocytes was completely dependent on a physical contact. ^ Gap junctional communication (GJC) served as this physical contact. Tumor cells and astrocytes both expressed the major component of gap junctional channel—connexin 43 and formed functional GJC as evidenced by the “dye transfer” assay. The blockage of GJC between tumor cells and astrocytes by either specific chemical blocker carbenoxolone (CBX) or by genetically knocking down connexin 43 on astrocytes reversed the chemo-protection. ^ Calcium was the signal molecule transmitted through GJC that rescued tumor cells from chemotherapy. Accumulation of cytoplasmic calcium preceded the progress of apoptosis in tumor cells treated with chemotherapeutic drugs. Furthermore, chelation of accumulated cytoplasmic calcium inhibited the apoptosis of tumor cells treated with chemotherapeutic drugs. Most importantly, astrocytes could “shunt” the accumulated cytoplasmic calcium from tumor cells (treated with chemotherapeutic drug) through GJC. We also used gene expression micro-array to investigate global molecular consequence of tumor cells forming GJC with astrocytes. The data demonstrated that astrocytes (but not fibroblasts), through GJC, up-regulated the expressions of several well known survival genes in tumor cells. ^ In summary, this dissertation provides a novel mechanism underlying the resistance of brain metastasis to chemotherapy, which is due to protection by astrocytes through GJC. Interference with the GJC between astrocytes and tumor cells holds great promise in sensitizing brain metastasis to chemotherapy and improving the prognosis for patients with brain metastasis. ^

Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1β, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter–luciferase constructs identified a unique −555/−513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (−624/−556 bp) essential for PKC and cAMP stimulation. DNA–protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

Differential expression of dystrophin isoforms and utrophin during dibutyryl-cAMP-induced morphological differentiation of rat brain astrocytes

We have identified isoforms of dystrophin and utrophin, a dystrophin homologue, expressed in astrocytes and examined their expression patterns during dibutyryl-cAMP (dBcAMP)-induced morphological differentiation of astrocytes. Immunoblot and immunocytochemical analyses showed that full-length-type dystrophin (427 kDa), utrophin (395 kDa), and Dp71 (75 kDa), a small-type dystrophin isoform, were coexpressed in cultured nondifferentiated rat brain astrocytes and were found to be located in the cell membrane. During morphological differentiation of the astrocytes induced by 1 mM dBcAMP, the amount of Dp71 markedly increased, whereas that of dystrophin and utrophin decreased. Northern blot analyses revealed that dBcAMP regulates the mRNA levels of Dp71 and dystrophin but not that of utrophin. dBcAMP slightly increased the amount of the β-dystroglycan responsible for anchoring dystrophin isoforms and utrophin to the cell membrane. Immunocytochemical analyses showed that most utrophin was observed in the cytoplasmic area during astrocyte differentiation, whereas Dp71 was found along the cell membrane of the differentiated astrocytes. These findings suggest that most of the dystrophin/utrophin-dystroglycan complex on cell membrane in cultured astrocytes was replaced by the Dp71-dystroglycan complex during morphological differentiation. The cell biological roles of Dp71 are discussed.

A short segment within the cytoplasmic domain of the neural cell adhesion molecule (N-CAM) is essential for N-CAM-induced NF-κB activity in astrocytes

The neural cell adhesion molecule (N-CAM) is expressed on the surface of astrocytes, where its homophilic binding leads to the activation of the transcription factor NF-κB. Transfection of astrocytes with a construct encompassing the transmembrane region and the cytoplasmic domain of N-CAM (designated Tm-Cyto, amino acids 685–839 in the full-length molecule) inhibited this activation up to 40%, and inhibited N-CAM-induced translocation of NF-κB to the nucleus. N-CAM also activated NF-κB in astrocytes from N-CAM knockout mice, presumably through binding to a heterophile. This activation, however, was not blocked by Tm-Cyto expression, indicating that the inhibitory effect of the Tm-Cyto construct is specific for cell surface N-CAM. Deletions and point mutations of the cytoplasmic portion of the Tm-Cyto construct indicated that the region between amino acids 780 and 800 were essential for inhibitory activity. This region contains four threonines (788, 793, 794, and 797). Mutation to alanine of T788, T794, or T797, but not T793, abolished inhibitory activity, as did mutation of T788 or T797 to aspartic acid. A Tm-Cyto construct with T794 mutated to aspartic acid retained inhibitory activity but did not itself induce a constitutive NF-κB response. This result suggests that phosphorylation of T794 may be necessary but is not the triggering event. Overall, these findings define a short segment of the N-CAM cytoplasmic domain that is critical for N-CAM-induced activation of NF-κB and may be important in other N-CAM-mediated signaling.