Luciana Romão

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.

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.

Glioblastoma: therapeutic challenges, what lies ahead.

Glioblastoma (GBM) is one of the most aggressive human cancers. Despite current advances in multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome for patients with high grade glioma remains fatal. The knowledge of how glioma cells develop and depend on the tumor environment might open opportunities for new therapies. There is now a growing awareness that the main limitations in understanding and successfully treating GBM might be bypassed by the identification of a distinct cell type that has defining properties of somatic stem cells, as well as cancer-initiating capacity - brain tumor stem cells, which could represent a therapeutic target. In addition, experimental studies have demonstrated that the combination of antiangiogenic therapy, based on the disruption of tumor blood vessels, with conventional chemotherapy generates encouraging results. Emerging reports have also shown that microglial cells can be used as therapeutic vectors to transport genes and/or substances to the tumor site, which opens up new perspectives for the development of GBM therapies targeting microglial cells. Finally, recent studies have shown that natural toxins can be conjugated to drugs that bind to overexpressed receptors in cancer cells, generating targeted-toxins to selectively kill cancer cells. These targeted-toxins are highly effective against radiation- and chemotherapy-resistant cancer cells, making them good candidates for clinical trials in GBM patients. In this review, we discuss recent studies that reveal new possibilities of GBM treatment taking into account cancer stem cells, angiogenesis, microglial cells and drug delivery in the development of new targeted-therapies.

CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors

BackgroundTumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies.MethodsWe screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas.ResultsA long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide.ConclusionsOur results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

Glial fibrillary acidic protein gene promoter is differently modulated by transforming growth factor-beta 1 in astrocytes from distinct brain regions.

The expression of glial fibrillary acidic protein (GFAP), the major intermediate filament protein of mature astrocytes, is regulated under developmental and pathological conditions. Recently, we have investigated GFAP gene modulation by using a transgenic mouse bearing part of the GFAP gene promoter linked to the β‐galactosidase reporter gene. We demonstrated that cerebral cortex neurons activate the GFAP gene promoter, inducing transforming growth factor‐beta 1 (TGF‐β1) secretion by astrocytes. Here, we report that cortical neurons or conditioned medium derived from them do not activate the GFAP gene promoter of transgenic astrocytes derived from midbrain and cerebellum suggesting a neuroanatomical regional specificity of this phenomenon. Surprisingly, they do induce synthesis of TGF‐β1 by these cells. Western blot and immunocytochemistry assays revealed wild distribution of TGF receptor in all subpopulations of astrocytes and expression of TGF‐β1 in neurons derived from all regions, thus indicating that the unresponsiveness of the cerebellar and midbrain GFAP gene to TGF‐β1 is not due to a defect in TGF‐β1 signalling. Together, our data highlight the great complexity of neuron–glia interactions and might suggest a distinct mechanism underlying modulation of the GFAP gene in the heterogeneous population of astrocytes throughout the central nervous system.

The anti-Parkinsonian drug selegiline delays the nucleation phase of α-synuclein aggregation leading to the formation of nontoxic species.

Parkinsons disease (PD) is a movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of intraneuronal inclusions called Lewy bodies, which are composed mainly of α-synuclein (α-syn). Selegiline (Sel) is a noncompetitive monoamino oxidase B inhibitor that has neuroprotective effects and has been administered to PD patients as monotherapy or in combination with l-dopa. Besides its known effect of increasing the level of dopamine (DA) by monoamino oxidase B inhibition, Sel induces other effects that contribute to its action against PD. We evaluated the effects of Sel on the in vitro aggregation of A30P and wild-type α-syn. Sel delays fibril formation by extending the lag phase of aggregation. In the presence of Sel, electron microscopy reveals amorphous heterogeneous aggregates, including large annular species, which are innocuous to a primary culture enriched in dopaminergic neurons, while their age-matched counterparts are toxic. The inhibitory effect displayed by Sel is abolished when seeds (small fibril pieces) are added to the aggregation reaction, reinforcing the hypothesis that Sel interferes with early nuclei formation and, to a lesser extent, with fibril elongation. NMR experiments indicate that Sel does not interact with monomeric α-syn. Interestingly, when added in combination with DA (which favors the formation of toxic protofibrils), Sel overrides the inhibitory effect of DA and favors fibrillation. Additionally, Sel blocks the formation of smaller toxic aggregates by perturbing DA-dependent fibril disaggregation. These effects might be beneficial for PD patients, since the sequestration of protofibrils into fibrils or the inhibition of fibril dissociation could alleviate the toxic effects of protofibrils on dopaminergic neurons. In nondopaminergic neurons, Sel might slow the fibrillation, giving rise to the formation of large nontoxic aggregates.

Glutamate activates GFAP gene promoter from cultured astrocytes through TGF‐β1 pathways

Glial cells are currently viewed as active partners of neurons in synapse formation. The close proximity of astrocytes to the synaptic cleft suggests that these cells might be potential targets for neuronal‐released molecules although this issue has been less addressed. Here, we evaluated the role of the excitatory neurotransmitter, glutamate, in astrocyte differentiation. We recently demonstrated that cortical neurons activate the gene promoter of the astrocyte maturation marker, GFAP (glial fibrillary acidic protein) of cerebral cortex astrocytes by inducing TGF‐β1 (transforming growth factor beta 1) secretion in vitro. To access the effect of glutamate on GFAP gene, we used transgenic mice bearing the β‐Galactosidase (β‐Gal) reporter gene under the regulation of the GFAP gene promoter. We report that 100 μM glutamate activates the GFAP gene promoter of astrocytes from cerebral cortex revealed by a significant increase in the number of β‐Gal positive astrocytes. Neutralizing antibodies against TGF‐β completely prevented glutamate and neuronal‐induction of GFAP gene, thus indicating that this event is mediated by TGF‐β1. Further, induction of GFAP gene in response to glutamate was followed by nuclear translocation of the Smad transcription factor, a hallmark of TGF‐β1 pathway activation. The antagonist of the metabotropic glutamate receptor, MCPG, inhibited neuronal effect suggesting that neuronal activation of GFAP gene promoter involves glutamate metabotropic receptors. MAPK (PD98059) and PI3K (LY294002) inhibitors fully prevented activation of GFAP gene promoter by all treatments. Surprisingly, these inhibitors also abrogated TGF‐β1 direct action on GFAP gene although they did not inhibit Smad‐2 phosphorylation, suggesting that TGF‐β1‐induced GFAP gene activation might involve cooperation between the canonical and non‐canonical TGF‐β pathways. Together, our results suggest that glial metabotropic glutamate 2/3 receptor activation by neurons induces TGF‐β1 secretion, leading to GFAP gene activation and astrocyte differentiation and involves Smad and MAPK/PI3K pathways. Our work provides evidence that astrocytes surrounding synapses are target of neuronal activity and might shed light into the role of glial cells into neurological disorders associated with glutamate neurotoxicity.

Membrane Elastic Properties and Cell Function

Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function.

Microglial stress inducible protein 1 promotes proliferation and migration in human glioblastoma cells.

Microglial activation is a key event in the progression and infiltration of tumors. We have previously demonstrated that the co-chaperone stress inducible protein 1 (STI1), a cellular prion protein (PrP(C)) ligand, promotes glioblastoma (GBM) proliferation. In the present study, we examined the influence of microglial STI1 in the growth and invasion of the human glioblastoma cell line GBM95. We demonstrated that soluble factors secreted by microglia into the culture medium (microglia conditioned medium; MG CM) caused a two-fold increase in the proliferation of GBM95 cells. This effect was reversed when STI1 was removed from the MG CM. In this context, we have shown that microglial cells synthesize and secrete STI1. Interestingly, no difference was observed in proliferation rates when GBM cells were maintained in MG CM or MG CM containing an anti-PrP(C) neutralizing antibody. Moreover, rec STI1 and rec STI1(Δ230-245), which lack the PrP(C) binding site, both promoted similar levels of GBM95 proliferation. In the migration assays, MG CM favored the migration of GBM95 cells, but migration failed when STI1 was removed from the MG CM. We detected metalloproteinase 9 (MMP-9) activity in the MG CM, and when cultured microglia were treated with an anti-STI1 antibody, MMP-9 activity decreased. Our results suggest that STI1 is secreted by microglia and favors tumor growth and invasion through the participation of MMP-9 in a PrP(C)-independent manner.

Homocysteine induces cytoskeletal remodeling and production of reactive oxygen species in cultured cortical astrocytes

Homocysteine (Hcy) is an excitatory amino acid which markedly enhances the vulnerability of neuronal cells to excitotoxicity and oxidative injury. Patients with severe hyperhomocysteinemia exhibit a wide range of clinical manifestations including neurological abnormalities such as mental retardation, cerebral atrophy, and seizures. In this study we treated cortical astrocytes and neurons in culture with 10 and 100 μM Hcy and after 24h exposure cytoskeletal remodeling was analyzed by immunocytochemistry. We observed dramatically altered actin cytoskeleton in astrocytes exposed to 100 μM Hcy, with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm. Moreover, we observed disruption of the glial fibrillary acidic protein (GFAP) meshwork, supporting misregulation of actin cytoskeleton. Induction of reactive oxygen species (ROS) in astrocytes showed fluctuating levels along 24h exposure to both Hcy concentrations. Actin remodeling induced by 100 μM Hcy was prevented by the antioxidants folate (5 μM) or trolox (80 μM). Unlike astrocyte cytoskeleton, results evidence little susceptibility of neuron cytoskeleton until 24h of treatment, since immunocytochemical analysis showed that 10 and 100 μM Hcy-treated neurons presented unaltered neurite arborization. Moreover, alterations in astrocyte and neuron viability were not observed along the 24h of exposure to Hcy. Neuron/astrocyte co-cultures evidence an anchorage dependence for neuronal survival over long exposure to Hcy. Taken together, these findings indicate, that the cytoskeleton of cortical astrocytes, but not of neurons in culture, is a target to Hcy and such effects are mediated by redox signaling. Astrocytes were able to respond to Hcy (100 μM) reorganizing their cytoskeleton, surviving, and protecting neurons from Hcy damage. Moreover our results suggest a protective role for astrocytes remodeling the cytoskelon, and probably generating signals that would assure neuronal survival in response to the damage induced by Hcy.