Joice Stipursky

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.

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.

Neuron-glia signaling: Implications for astrocyte differentiation and synapse formation

Glial cells are currently viewed as active partners of neurons in synapse formation. The close proximity of astrocytes to the synaptic cleft implicates that they strongly influence synapse function as well as suggests that these cells might be potential targets for neuronal-released molecules. In this review, we discuss the signaling pathways of astrocyte generation and the role of astrocyte-derived molecules in synapse formation in the central nervous system. Further, we discuss the role of the excitatory neurotransmitter, glutamate and transforming growth factor beta 1 (TGF-β1) pathway in astrocyte generation and differentiation. We provide evidence that astrocytes surrounding synapses are target of neuronal activity and shed light into the role of astroglial cells into neurological disorders associated with glutamate neurotoxicity.

Neuron-astroglial interactions in cell-fate commitment and maturation in the central nervous system.

Neuron–astroglia interactions play a key role in several events of brain development, such as neuronal generation, migration, survival, and differentiation; axonal growth; and synapse formation and function. While there is compelling evidence of the effects of astrocyte factors on neurons, their effects on astrocytes have not been fully determined. In this review, we will focus on the role of neurons in astrocyte generation and maturation. Further, we highlight the great heterogeneity and diversity of astroglial and neural progenitors such as radial glia cells, and discuss the importance of the variety of cellular interactions in controlling the structural and functional organization of the brain. Finally, we present recent data on a new role of astrocytes in neuronal maturation, as mediators of the action of biolipids in the cerebral cortex. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership, by briefly discussing the emerging view of how neuron-astrocyte dysfunctions might be associated with neurodegenerative diseases and neurological disorders.

Activation of MAPK/PI3K/SMAD pathways by TGF-β(1) controls differentiation of radial glia into astrocytes in vitro.

The major neural stem cell population in the developing cerebral cortex is the radial glia cells, which generate neurons and glial cells. The mechanisms that modulate the maintenance of the radial glia stem cell phenotype, or its differentiation, are not completely elucidated. We previously demonstrated that transforming growth factor-β1 (TGF-β1) promotes radial glia differentiation into astrocytes in vitro [Glia 2007;55:1023–1033]. Here we investigated the intracellular signaling pathways involved in the TGF-β1-induced radial glia fate commitment. We demonstrate that the mechanisms underlying the TGF-β1 effect on radial glia cell differentiation or progenitor potential maintenance diverge. Whereas radial glia differentiation into astrocytes is mediated by the activation of the MAPK signaling pathway, neurogenesis is modulated by different levels of PI3K and SMAD2/3 activity. Our work demonstrates that radial glia cells are a heterogeneous population and a potential target of TGF-β1, and suggests that its effect on radial glia fate commitment is mediated by the recruitment of a complex multipathway mechanism that controls astrocyte and neuronal generation in the developing cerebral cortex.

Effects of the flavonoid casticin from Brazilian Croton betulaster in cerebral cortical progenitors in vitro: Direct and indirect action through astrocytes

Neurodegenerative diseases are a major constraint on the social and economic development of many countries. Evidence has suggested that phytochemicals have an impact on brain pathology; however, both their mechanisms of action and their cell targets are incompletely known. Here, we investigated the effects of the flavonoid casticin, extracted from Croton betulaster, a common plant in the state of Bahia in Brazil, on rat cerebral cortex neurons in vitro. Treatment of neural progenitors with 10 μM casticin increased the neuronal population positive for the neuronal marker β‐tubulin III and the neuronal transcriptional factor Tbr2 by approximately 20%. This event was followed by a 50% decrease in neuronal death. Pools of astrocyte (GFAP and S100β), neural (nestin), and oligodendrocyte (Olig2 and NG2) progenitors were not affected by casticin. Neither neuronal commitment nor proliferation of progenitors was affected by casticin, suggesting a neuroprotective effect of this compound. Culture of neural progenitors on casticin‐treated astrocyte monolayers increased the neuronal population by 40%. This effect was reproduced by conditioned medium derived from casticin‐treated astrocytes, suggesting the involvement of a soluble factor. ELISA assays of the conditioned medium revealed a 20% increase in interleukin‐6 level in response to casticin. In contrast to the direct effect, neuronal death was unaffected, but a 52% decrease in the death of nestin‐positive progenitors was observed. Together our data suggest that casticin influences the neuronal population by two mechanisms: 1) directly, by decreasing neuronal death, and 2) indirectly, via astrocytes, by modulating the pool of neuronal progenitors.

Flavonoids and Astrocytes Crosstalking: Implications for Brain Development and Pathology

Flavonoids are naturally occurring polyphenolic compounds that are present in a variety of fruits, vegetables, cereals, tea, and wine, and are the most abundant antioxidants in the human diet. Evidence suggests that these phytochemicals might have an impact on brain pathology and aging; however, neither their mechanisms of action nor their cell targets are completely known. In the mature mammalian brain, astroglia constitute nearly half of the total cells, providing structural, metabolic, and trophic support for neurons. During the past few years, increasing knowledge of these cells has indicated that astrocytes are pivotal characters in neurodegenerative diseases and brain injury. Most of the physiological benefits of flavonoids are generally thought to be due to their antioxidant and free-radical scavenging effects; however, emerging evidence has supported the hypothesis that their mechanism of action might go beyond these properties. In this review, we focus on astrocytes as targets for flavonoids and their implications in brain development, neuroprotection, and glial tumor formation. Finally, we will briefly discuss the emerging view of astrocytes as essential characters in neurodegenerative diseases, and how a better understanding of the action of flavonoids might open new avenues to develop therapeutic approaches to these pathologies.

Thyroid hormone treated astrocytes induce maturation of cerebral cortical neurons through modulation of proteoglycan levels.

Proper brain neuronal circuitry formation and synapse development is dependent on specific cues, either genetic or epigenetic, provided by the surrounding neural environment. Within these signals, thyroid hormones (T3 and T4) play crucial role in several steps of brain morphogenesis including proliferation of progenitor cells, neuronal differentiation, maturation, migration, and synapse formation. The lack of thyroid hormones during childhood is associated with several impair neuronal connections, cognitive deficits, and mental disorders. Many of the thyroid hormones effects are mediated by astrocytes, although the mechanisms underlying these events are still unknown. In this work, we investigated the effect of 3, 5, 3′-triiodothyronine-treated (T3-treated) astrocytes on cerebral cortex neuronal differentiation. Culture of neural progenitors from embryonic cerebral cortex mice onto T3-treated astrocyte monolayers yielded an increment in neuronal population, followed by enhancement of neuronal maturation, arborization and neurite outgrowth. In addition, real time PCR assays revealed an increase in the levels of the heparan sulfate proteoglycans, Glypican 1 (GPC-1) and Syndecans 3 e 4 (SDC-3 e SDC-4), followed by a decrease in the levels of the chondroitin sulfate proteoglycan, Versican. Disruption of glycosaminoglycan chains by chondroitinase AC or heparanase III completely abolished the effects of T3-treated astrocytes on neuronal morphogenesis. Our work provides evidence that astrocytes are key mediators of T3 actions on cerebral cortex neuronal development and identified potential molecules and pathways involved in neurite extension; which might eventually contribute to a better understanding of axonal regeneration, synapse formation, and neuronal circuitry recover.

TGF-β1 promotes cerebral cortex radial glia-astrocyte differentiation in vivo

The major neural stem cell population in the developing cerebral cortex is composed of the radial glial cells, which generate glial cells and neurons. The mechanisms that modulate the maintenance of the radial glia (RG) stem cell phenotype, or its differentiation, are not yet completely understood. We previously demonstrated that the transforming growth factor-β1 (TGF-β1) promotes RG differentiation into astrocytes in vitro (Glia 2007; 55:1023-33) through activation of multiple canonical and non-canonical signaling pathways (Dev Neurosci 2012; 34:68-81). However, it remains unknown if TGF-β1 acts in RG-astrocyte differentiation in vivo. Here, we addressed the astrogliogenesis induced by TGF-β1 by using the intraventricular in utero injection in vivo approach. We show that injection of TGF-β1 in the lateral ventricles of E14,5 mice embryos resulted in RG fibers disorganization and premature gliogenesis, evidenced by appearance of GFAP positive cells in the cortical wall. These events were followed by decreased numbers of neurons in the cortical plate (CP). Together, we also described that TGF-β1 actions are region-dependent, once RG cells from dorsal region of the cerebral cortex demonstrated to be more responsive to this cytokine compared with RG from lateral cortex either in vitro as well as in vivo. Our work demonstrated that TGF-β1 is a critical cytokine that regulates RG fate decision and differentiation into astrocytes in vitro and in vivo. We also suggest that RG cells are heterogeneous population that acts as distinct targets of TGF-β1 during cerebral cortex development.

PEMF fails to enhance nerve regeneration after sciatic nerve crush lesion

Abstract The use of electromagnetic fields has been reported to enhance peripheral nerve regeneration. This study aimed to identify the effects of a prolonged protocol of low‐frequency pulsed electromagnetic field (PEMF) on peripheral nerve regeneration. Thirty‐four male Swiss mice (Mus musculus) were divided into PEMF (n = 17) and control (n = 17) groups. All animals underwent a unilateral sciatic‐crush lesion, and the PEMF group was exposed to a 72‐Hz, 2‐G electromagnetic field for 30 min, five days a week, for three weeks. Functional analysis was carried out weekly. After three weeks, the animals were euthanized, and histological, morphometric, oxidative stress, and TGF‐β1 analyses were performed. Functional analysis showed no differences between the groups. Histological appearance was similar between PEMF and control nerves. Morphometric assessment showed that the PEMF nerves trended toward decreased regeneration. The levels of free radicals were more pronounced in PEMF nerves, but were not associated with an increase in the content of the TGF‐β1/Smad signaling pathway. Prolonged PEMF regimen leads to delayed histological peripheral nerve regeneration and increased oxidative stress but no loss of function recovery.