Flávia Carvalho Alcantara Gomes

Glial fibrillary acidic protein (GFAP): modulation by growth factors and its implication in astrocyte differentiation

Intermediate filament (IF) proteins constitute an extremely large multigene family of developmentally and tissue-regulated cytoskeleton proteins abundant in most vertebrate cell types. Astrocyte precursors of the CNS usually express vimentin as the major IF. Astrocyte maturation is followed by a switch between vimentin and glial fibrillary acidic protein (GFAP) expression, with the latter being recognized as an astrocyte maturation marker. Levels of GFAP are regulated under developmental and pathological conditions. Upregulation of GFAP expression is one of the main characteristics of the astrocytic reaction commonly observed after CNS lesion. In this way, studies on GFAP regulation have been shown to be useful to understand not only brain physiology but also neurological disease. Modulators of GFAP expression include several hormones such as thyroid hormone, glucocorticoids and several growth factors such as FGF, CNTF and TGF beta, among others. Studies of the GFAP gene have already identified several putative growth factor binding domains in its promoter region. Data obtained from transgenic and knockout mice have provided new insights into IF protein functions. This review highlights the most recent studies on the regulation of IF function by growth factors and hormones.

Emerging roles for TGF-β1 in nervous system development

Transforming growth factor betas (TGF‐βs) are known as multifunctional growth factors, which participate in the regulation of key events of development, disease and tissue repair. In central nervous system (CNS), TGF‐β1 has been widely recognized as an injury‐related cytokine, specially associated with astrocyte scar formation in response to brain injury. TGF‐βs family is represented by three isoforms: TGF‐β1, ‐β2 and ‐β3, all produced by both glial and neuronal cells. They are involved in essential tissue functions, including cell‐cycle control, regulation of early development and differentiation, neuron survival and astrocyte differentiation. TGF‐β signaling is mediated mainly by two serine threonine kinase receptors, TGFRI and TGFRII, which activate Smad 2/3 and Smad 4 transcription factors. Phosphorylation and activation of these proteins is followed by formation of Smad 2/3–4 complex, which translocates to the nucleus regulating transcriptional responses to TGF‐β. Very few data are available concerning the intracellular pathway required for the effect of TGF‐β in brain cells. Recently, emerging data on TGF‐β1 and its signaling molecules have been suggesting that besides its role in brain injury, TGF‐β1 might be a crucial regulator of CNS development. In this review, we will focus on TGF‐βs members, specially TGF‐β1, in neuron and astrocyte development. We will discuss some advances concerning the emerging scenario of TGF‐β1 and its signaling pathways as putative modulators of astrocyte biology and their implications as a novel mediator of cellular interactions in the CNS.

Neuritogenesis induced by thyroid hormone-treated astrocytes is mediated by epidermal growth factor/mitogen-activated protein kinase-phosphatidylinositol 3-kinase pathways and involves modulation of extracellular matrix proteins

Thyroid hormone (T3) plays a crucial role in several steps of cerebellar ontogenesis. By using a neuron-astrocyte coculture model, we have investigated the effects of T3-treated astrocytes on cerebellar neuronal differentiation in vitro. Neurons plated onto T3-astrocytes presented a 40–60% increase on the total neurite length and an increment in the number of neurites. Treatment of astrocytes with epidermal growth factor (EGF) yielded similar results, suggesting that this growth factor might mediate T3-induced neuritogenesis. EGF and T3 treatment increased fibronectin and laminin expression by astrocytes, suggesting that astrocyte neurite permissiveness induced by these treatments is mostly due to modulation of extracellular matrix (ECM) components. Such increase in ECM protein expression as well as astrocyte permissiveness to neurite outgrowth was reversed by the specific EGF receptor tyrosine kinase inhibitor, tyrphostin. Moreover, studies using selective inhibitors of several transduction-signaling cascades indicated that modulation of ECM proteins by EGF is mainly through a synergistic activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. In this work, we provide evidence of a novel role of EGF as an intermediary factor of T3 action on cerebellar ontogenesis. By modulating the content of ECM proteins, EGF increases neurite outgrowth. Our data reveal an important role of astrocytes as mediators of T3-induced cerebellar development and partially elucidate the role of EGF and mitogen-activated protein kinase/phosphatidylinositol 3-kinase pathways on this process.

Astrocyte-induced Synaptogenesis Is Mediated by Transforming Growth Factor β Signaling through Modulation of d-Serine Levels in Cerebral Cortex Neurons

Background: Synapse formation and function is modulated by intrinsic and extrinsic non-autonomous factors. Results: Astrocytes induce synapse formation through TGF-β1 pathway. TGF-β1 synaptogenic property is dependent on d-serine signaling. Conclusion: TGF-β induces excitatory glutamatergic synapses in vertebrates. Significance: This is a novel molecular mechanism that might impact synaptic function and shed light on new potential therapeutic targets for synaptic deficit diseases. Assembly of synapses requires proper coordination between pre- and postsynaptic elements. Identification of cellular and molecular events in synapse formation and maintenance is a key step to understand human perception, learning, memory, and cognition. A key role for astrocytes in synapse formation and function has been proposed. Here, we show that transforming growth factor β (TGF-β) signaling is a novel synaptogenic pathway for cortical neurons induced by murine and human astrocytes. By combining gain and loss of function approaches, we show that TGF-β1 induces the formation of functional synapses in mice. Further, TGF-β1-induced synaptogenesis involves neuronal activity and secretion of the co-agonist of the NMDA receptor, d-serine. Manipulation of d-serine signaling, by either genetic or pharmacological inhibition, prevented the TGF-β1 synaptogenic effect. Our data show a novel molecular mechanism that might impact synaptic function and emphasize the evolutionary aspect of the synaptogenic property of astrocytes, thus shedding light on new potential therapeutic targets for synaptic deficit diseases.

Cerebellar astrocytes treated by thyroid hormone modulate neuronal proliferation.

Thyroid hormones are important for neurogenesis and gliogenesis during brain development. We have previously demonstrated that triiodothyronine (T3) treatment induced proliferation in primary culture astrocytes derived from the cerebellum of neonatal rats. Conditioned medium obtained from those T3‐treated astrocytes (T3CM) mimicked the effect of hormonal treatment on these cells. Because neuron–glia interaction plays an important role in brain development, we tested the ability of such T3‐glial CM to influence neuronal physiology. With that aim, neurons from 19‐day embryonic cerebella were cultivated for 24 h in the presence of CM obtained from T3‐treated cerebellar astrocytes. Interestingly, the cerebellar neuronal population increased by 60–80% in T3CM. Addition of 5 μM forskolin enhanced the responsiveness of cerebellar neurons to astrocytes T3CM, but it did not interfere with neuronal survival in control medium. Conversely, inhibition of adenylate cyclase by its specific inhibitor, SQ22536, reversed the T3CM effect on neurons. These data strongly suggest that cAMP signal transduction pathways might be implicated in such an event. Analysis of bromodeoxyuridil incorporation revealed that the increase in neuron number in T3CM was partially due to neuron proliferation, because the proliferation index was three times higher in T3CM than in control medium. Neutralizing antibody assays demonstrated that T3CM effects on neurons are due, at least in part, to the presence of tumor necrosis factor‐β and epidermal growth factor. Thus, we report here a novel molecular mechanism of action of thyroid hormone on cerebellar neuronal cells: Thyroid hormone induces astrocytes to secrete growth factors that can interfere with neuronal proliferation via a paracrine pathway. GLIA 25:247–255, 1999.

Neuro–glia interaction effects on GFAP gene: a novel role for transforming growth factor-β1

Central nervous system (CNS) development is highly guided by microenvironment cues specially provided by neuron–glia interactions. By using a transgenic mouse bearing part of the gene promoter of the astrocytic maturation marker GFAP (glial fibrillary acidic protein) linked to the β‐galactosidase (β‐Gal) reporter gene, we previously demonstrated that cerebral cortical neurons increase transgenic β‐Gal astrocyte number and activate GFAP gene promoter by secretion of soluble factors in vitro. Here, we identified TGF‐β1 as the major mediator of this event. Identification of TGF‐β1 in neuronal and astrocyte extracts revealed that both cell types might synthesize this factor, however, addition of neurons to astrocyte monolayers greatly increased TGF‐β1 synthesis and secretion by astrocytes. Further, by exploiting the advantages of cell culture system we investigated the influence of neuron and astrocyte developmental stage on such interaction. We demonstrated that younger neurons derived from 14 embryonic days wild‐type mice were more efficient in promoting astrocyte differentiation than those derived from 18 embryonic days mice. Similarly, astrocytes also exhibited timed‐schedule developed responsiveness to neuronal influence with embryonic astrocytes being more responsive to neurons than newborn and late postnatal astrocytes. RT‐PCR assays identified TGF‐β1 transcripts in young but not in old neurons, suggesting that inability to induce astrocyte differentiation is related to TGF‐β1 synthesis and secretion. Our work reveals an important role for neuron–glia interactions in astrocyte development and strongly implicates the involvement of TGF‐β1 in this event.

Cognitive Dysfunction Is Sustained after Rescue Therapy in Experimental Cerebral Malaria, and Is Reduced by Additive Antioxidant Therapy

Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders.

TGF‐β1/SMAD signaling induces astrocyte fate commitment in vitro: Implications for radial glia development

Radial glial (RG) cells are specialized type of cell, which functions as neuronal precursors and scaffolding guides to migrating neurons during cerebral cortex development. After neurogenesis and migration are completed, most of RG cells transform into astrocytes. Mechanism and molecules involved in this process are not completely elucidated. We previously demonstrated that neurons activate the promoter of the astrocyte maturation marker GFAP in astrocytes by secretion of transforming growth factor beta 1 (TGF‐β1) in vitro. Here, we studied the role of neurons and TGF‐β1 pathway in RG differentiation. To address this question, we employed cortical progenitor cultures enriched in GLAST/nestin double‐labeled cells, markers of RG cells. TGF‐β1 and conditioned medium derived from neuron‐astrocyte cocultures (CM) decreased the number of cells expressing the precursor marker nestin and increased that expressing GFAP in cortical progenitor cultures. These events were impaired by addition of neutralizing antibodies against TGF‐β1. Increase in the number of GFAP positive cells was associated with Smads 2/3 nuclear translocation, a hallmark of TGF‐β1 pathway activation. PCR‐assays revealed a decrease in the levels of mRNA for the RG marker, BLBP (brain lipid binding protein), due to TGF‐β1 and CM treatment. We further identified TGF‐β1 receptor in cortical progenitor cultures suggesting that these cells might be target for TGF‐β1 during development. Our work provides strong evidence that TGF‐β1 might be a novel factor involved in RG‐astrocyte transformation and highlights the role of neuron‐glia interaction in this process.

Cross-talk between neurons and glia: highlights on soluble factors.

The development of the nervous system is guided by a balanced action between intrinsic factors represented by the genetic program and epigenetic factors characterized by cell-cell interactions which neural cells might perform throughout nervous system morphogenesis. Highly relevant among them are neuron-glia interactions. Several soluble factors secreted by either glial or neuronal cells have been implicated in the mutual influence these cells exert on each other. In this review, we will focus our attention on recent advances in the understanding of the role of glial and neuronal trophic factors in nervous system development. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership.

Lysophosphatidic Acid Receptor-dependent Secondary Effects via Astrocytes Promote Neuronal Differentiation *□

Lysophosphatidic acid (LPA) is a simple phospholipid derived from cell membranes that has extracellular signaling properties mediated by at least five G protein-coupled receptors referred to as LPA1–LPA5. In the nervous system, receptor-mediated LPA signaling has been demonstrated to influence a range of cellular processes; however, an unaddressed aspect of LPA signaling is its potential to produce specific secondary effects, whereby LPA receptor-expressing cells exposed to, or “primed,” by LPA may then act on other cells via distinct, yet LPA-initiated, mechanisms. In the present study, we examined cerebral cortical astrocytes as possible indirect mediators of the effects of LPA on developing cortical neurons. Cultured astrocytes express at least four LPA receptor subtypes, known as LPA1–LPA4. Cerebral cortical astrocytes primed by LPA exposure were found to increase neuronal differentiation of cortical progenitor cells. Treatment of unprimed astrocyte-progenitor cocultures with conditioned medium derived from LPA-primed astrocytes yielded similar results, suggesting the involvement of an astrocyte-derived soluble factor induced by LPA. At least two LPA receptor subtypes are involved in LPA priming, since the priming effect was lost in astrocytes derived from LPA receptor double-null mice (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{LPA}_{1}^{(-{/}-)}{/}\mathrm{LPA}_{2}^{(-{/}-)}\) \end{document}). Moreover, the loss of LPA-dependent differentiation in receptor double-null astrocytes could be rescued by retrovirally transduced expression of a single deleted receptor. These data demonstrate that receptor-mediated LPA signaling in astrocytes can induce LPA-dependent, indirect effects on neuronal differentiation.