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Glioblastoma is the most life‐threatening tumour of the central nervous system. Temozolomide (TMZ) is the first‐choice oral drug for the treatment of glioblastoma, although it shows low efficacy. Silver nanoparticles (AgNPs) have been shown to exhibit biocidal activity in a variety of microorganisms, including some pathogenic microorganisms. Herein, the antiproliferative effect of AgCl‐NPs on glioblastoma cell lines (GBM02 and GBM11) and on astrocytes was evaluated through automated quantitative image‐based analysis (HCA) of the cells. The cells were treated with 0.1‐5.0 μg/ml AgCl‐NPs or with 9.7‐48.5 μg/ml TMZ. Cells that received combined treatment were also analysed. At a maximum tested concentration of AgCl‐NPs, GBM02 and GBM11, the growth decreased by 93% and 40%, respectively, following 72 h of treatment. TMZ treatment decreased the proliferation of GBM02 and GBM11 cells by 58% and 34%, respectively. Combinations of AgCl‐NPs and TMZ showed intermediate antiproliferative effects; the lowest concentrations caused an inhibition similar to that obtained with TMZ, and the highest concentrations caused inhibition similar to that obtained with AgCl‐NPs alone. No significant changes in astrocyte proliferation were observed. The authors’ findings showed that HCA is a fast and reliable approach that can be used to evaluate the antiproliferative effect of the nanoparticles at the single‐cell level and that AgCl‐NPs are promising agents for glioblastoma treatment.

Cognitive dysfunction is often reported in post-COVID patients, but its underlying mechanisms remain unknown. While some evidence indicate that SARS-CoV-2 can reach and directly impact the brain, others suggest viral neuroinvasion as a rare event. Independently of brain viral infection, the ability of SARS-CoV-2 spike (S) protein to cross the BBB and reach memory-related brain regions has already been shown. Here, we demonstrate that brain infusion of S protein in mice induces late cognitive impairment and increases serum levels of neurofilament light chain (NFL), which recapitulates post-COVID features. Neuroinflammation, hippocampal microgliosis and synapse loss are induced by S protein. Increased engulfment of hippocampal presynaptic terminals late after S protein brain infusion were found to temporally correlate with cognitive deficit in mice. Blockage of TLR4 signaling prevented S-associated detrimental effects on synapse and memory loss. In a cohort of 86 patients recovered from mild COVID-19, genotype GG TLR4 -2604G>A (rs10759931) was associated with poor cognitive outcome. Collectively, these findings indicate that S protein directly impacts the brain and suggest that TLR4 is a potential target to prevent post-COVID cognitive dysfunction.One Sentence SummaryTLR4 mediates long-term cognitive impairment in mice and its genetic variant increases the risk of poor cognitive outcome in post-COVID patients.

The aggregation of α-synuclein (aSyn) is believed to be mechanistically linked to the degeneration of dopamine (DA)-producing neurons in Parkinson's disease (PD). In this respect, one crucial question that yet remains unsolved is whether aSyn aggregation is associated with either a gain- or loss-of-function of the protein in neuronal cells. Herein, we investigated the effect of monomeric versus fibrillar aSyn on mesencephalic dopaminergic neurons in primary cultures challenged with the neurotoxic catechols: salsolinol (SALSO; 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) and 3,4-dihydroxyphenylacetaldehyde (DOPAL). aSyn monomer protected cells against either SALSO- or DOPAL-induced toxicity via inhibition of caspase-3-mediated apoptosis. While fibrillar aSyn failed in attenuating SALSO neurotoxicity, it increased the viability of DOPAL-treated cells, which was apparently not associated with the inhibition of caspase-3 cleavage. The fact that DOPAL-derived aSyn adducts exhibit lower toxicity compared with DOPAL itself raises the question of whether the generation of these adducts could be part of or a collateral effect of aSyn-mediated protection in neurons exposed to DOPAL. Overall, our work provides important evidence on the impact of the fibrillation of aSyn on its protective role in neuronal cells exposed to the toxic catechols SALSO and DOPAL.

The misfolding and aggregation of the protein α-synuclein (aSyn) into potentially neurotoxic oligomers is believed to play a pivotal role in the neuropathogenesis of Parkinson's disease (PD). Herein, we explore how apomorphine (Apo), a nonselective dopamine D1 and D2 receptor agonist utilized in the therapy for PD, affects the aggregation and toxicity of aSyn in vitro. Our data indicated that Apo inhibits aSyn fibrillation leading to the formation of large oligomeric species (Apo-aSyn-O), which exhibit remarkable toxicity in mesencephalic dopaminergic neurons in primary cultures. Interestingly, purified Apo-aSyn-O, even at very low concentrations, seems to be capable of converting unmodified aSyn monomer into neurotoxic species. Collectively, our findings warn for a possible dangerous effect of Apo on aSyn misfolding/aggregation pathway.

The guanine-based purines (GBPs) have essential extracellular functions such as modulation of glutamatergic transmission and trophic effects on neurons and astrocytes. We previously showed that GBPs, such as guanosine-5'-monophosphate (GMP) or guanosine (GUO), promote the reorganization of extracellular matrix proteins in astrocytes, and increase the number of neurons in a neuron-astrocyte co-culture protocol. To delineate the molecular basis underlying these effects, we isolated cerebellar neurons in culture and treated them with a conditioned medium derived from astrocytes previously exposed to GUO or GMP (GBPs-ACM) or, directly, with GUO or GMP. Agreeing with the previous studies, there was an increase in the number of β-tubulin III-positive neurons in both conditions, compared with controls. Interestingly, the increase in the number of neurons in the neuronal cultures treated directly with GUO or GMP was more prominent, suggesting a direct interaction of GBPs on cerebellar neurons. To investigate this issue, we assessed the role of adenosine and glutamate receptors and related intracellular signaling pathways after GUO or GMP treatment. We found an involvement of A2A adenosine receptors, ionotropic glutamate N-methyl-D-aspartate (NMDA), and non-NMDA receptors in the increased number of cerebellar neurons. The signaling pathways extracellular-regulated kinase (ERK), calcium-calmodulin-dependent kinase-II (CaMKII), protein kinase C (PKC), phosphatidilinositol-3'-kinase (PI3-K), and protein kinase A (PKA) are also potentially involved with GMP and GUO effect. Such results suggest that GMP and GUO, and molecules released in GBPs-ACM promote the survival or maturation of primary cerebellar neurons or both via interaction with adenosine and glutamate receptors.

Parkinson's disease (PD) is characterized by selective death of dopaminergic neurons in the substantia nigra, degeneration of the nigrostriatal pathway, increases in glutamatergic synapses in the striatum and aggregation of α-synuclein. Evidence suggests that oligomeric species of α-synuclein (αSO) are the genuine neurotoxins of PD. Although several studies have supported the direct neurotoxic effects of αSO on neurons, their effects on astrocytes have not been directly addressed. Astrocytes are essential to several steps of synapse formation and function, including secretion of synaptogenic factors, control of synaptic elimination and stabilization, secretion of neural/glial modulators, and modulation of extracellular ions, and neurotransmitter levels in the synaptic cleft. Here, we show that αSO induced the astrocyte reactivity and enhanced the synaptogenic capacity of human and murine astrocytes by increasing the levels of the known synaptogenic molecule transforming growth factor beta 1 (TGF-β1). Moreover, intracerebroventricular injection of αSO in mice increased the number of astrocytes, the density of excitatory synapses, and the levels of TGF-β1 in the striatum of injected animals. Inhibition of TGF-β1 signaling impaired the effect of the astrocyte-conditioned medium on glutamatergic synapse formation in vitro and on striatal synapse formation in vivo, whereas addition of TGF-β1 protected mesencephalic neurons against synapse loss triggered by αSO. Together, our data suggest that αSO have important effects on astrocytic functions and describe TGF-β1 as a new endogenous astrocyte-derived molecule involved in the increase in striatal glutamatergic synaptic density present in early stages of PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14514.

A new series of 1,4-disubstituted-1,2,3-triazole derivatives were synthesized through the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (Click chemistry) and their inhibitory activities were evaluated against different human glioblastoma (GBM) cell lines, including highly drug-resistant human cell lines GBM02, GBM95. The most effective compounds were 9d, containing the methylenoxy moiety linked to triazole and the tosyl-hydrazone group, and the symmetrical bis-triazole 10a, also containing methylenoxy moiety linked to triazole. Single crystal X-ray diffraction analysis was employed for structural elucidation of compound 9d. In silico analyses of physicochemical, pharmacokinetic, and toxicological properties suggest that compounds 8a, 8b, 8c, 9d, and 10a are potential candidates for central nervous system-acting drugs.

Arboviruses pose a major threat throughout the world and represent a great burden in tropical countries of South America. Although generally associated with moderate febrile illness, in more severe cases they can lead to neurological outcomes, such as encephalitis, Guillain-Barré syndrome, and Congenital Syndromes. In this context astrocytes play a central role in production of inflammatory cytokines, regulation of extracellular matrix, and control of glutamate driven neurotoxicity in the central nervous system. Here, we presented a comprehensive genome-wide transcriptome analysis of human primary astrocytes infected with Chikungunya, Mayaro, Oropouche, or Zika viruses. Analyses of differentially expressed genes (DEGs), pathway enrichment, and interactomes have shown that Alphaviruses up-regulated genes related to elastic fiber formation and N-glycosylation of glycoproteins, with down-regulation of cell cycle and DNA stability and chromosome maintenance genes. In contrast, Oropouche virus up-regulated cell cycle and DNA maintenance and condensation pathways while down-regulated extracellular matrix, collagen metabolism, glutamate and ion transporters pathways. Zika virus infection only up-regulated eukaryotic translation machinery while down-regulated interferon pathways. Reactome and integration analysis revealed a common signature in down-regulation of innate immune response, antiviral response, and inflammatory cytokines associated to interferon pathway for all arboviruses tested. Validation of interferon stimulated genes by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) corroborated our transcriptome findings. Altogether, our results showed a co-evolution in the mechanisms involved in the escape of arboviruses to antiviral immune response mediated by the interferon (IFN) pathway.

Despite advances in treatment of lethal prostate cancer, the incidence of prostate cancer brain metastases is increasing. In this sense, we analyzed the molecular profile, as well as the functional consequences involved in the reciprocal interactions between prostate tumor cells and human astrocytes. We observed that the DU145 cells, but not the LNCaP cells or the RWPE-1 cells, exhibited more pronounced, malignant and invasive phenotypes along their interactions with astrocytes. Moreover, global gene expression analysis revealed several genes that were differently expressed in our co-culture models with the overexpression of GLIPR1 and SPARC potentially representing a molecular signature associated with the invasion of central nervous system by prostate malignant cells. Further, these results were corroborated by immunohistochemistry and in silico analysis. Thus, we conjecture that the data here presented may increase the knowledge about the molecular mechanisms associated with the invasion of CNS by prostate malignant cells.

Neural precursor cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in neural precursor cell morphology along their distinct differentiation fates. Both cells maintained in culture as neural precursors as well as those plated in neurobasal medium reveal a decrease in membrane tension over the first hours of culture followed by stabilization, with no change in bending modulus. During astrocyte differentiation, membrane tension initially decreases and then increases after 72 h, accompanied by consolidation of glial fibrillary acidic protein expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours, but their values subsequently decrease, correlating with a shift from oligodendrocyte marker O4 to myelin basic protein expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, thus contributing to a better understanding of neural differentiation from a mechanobiological perspective.

Neural stem cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in cell morphology along differentiation into neurons, astrocytes and oligodendrocytes. Both neurons and undifferentiated cells reveal a decrease in membrane tension over the first hours of differentiation followed by stabilization, with no change in bending modulus. Astrocytes membrane tension initially decreases and then increases after 72h, accompanied by consolidation of GFAP expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours but their values subsequently decrease, correlating with a shift from O4 to MBP expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, contributing to a better understanding of neural stem cell differentiation from a mechanobiological perspective.

The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is believed to play a central role in Parkinson’s disease neurodegeneration by stabilizing potentially toxic oligomers of the presynaptic protein α-Synuclein (aSyn). Besides the formation of covalent DOPAL-Lys adducts, DOPAL treatment promotes the oxidation of Met residues of aSyn, which is also a common oxidative post-translational modification found in the protein in vivo. Herein we set out to address the role of Met residues into the oligomerization and neurotoxic properties of DOPAL-modified aSyn. Our data indicate that DOPAL promotes the formation of two distinct types of aSyn oligomers: large and small (dimer and trimers) oligomers, which seem to be generated by independent mechanisms and cannot be interconverted by using denaturing agents. Interestingly, H(2)O(2)-treated aSyn monomer, which exhibits all-four Met residues oxidized to Met-sulfoxide, exhibited a reduced ability to form large oligomers upon treatment with DOPAL, with no effect on the population of small oligomers. In this context, triple Met-Val mutant M5V/M116V/M127V exhibited an increased population of large aSyn-DOPAL oligomers in comparison with the wild-type protein. Interestingly, the stabilization of large rather than small oligomers seems to be associated with an enhanced toxicity of DOPAL-aSyn adducts. Collectively, these findings indicate that Met residues may play an important role in modulating both the oligomerization and the neurotoxic properties of DOPAL-derived aSyn species.

Glioblastomas (GBM) are aggressive brain tumors with very poor prognosis. While silver nanoparticles represent a potential new strategy for anticancer therapy, the silver/silver chloride nanoparticles (Ag/AgCl-NPs) have microbicidal activity, but had not been tested against tumor cells. Here, we analyzed the effect of biogenically produced Ag/AgCl-NPs (from yeast cultures) on the proliferation of GBM02 glioblastoma cells (and of human astrocytes) by automated, image-based high-content analysis (HCA). We compared the effect of 0.1–5.0 µg mL(−1) Ag/AgCl-NPs with that of 9.7–48.5 µg mL(−1) temozolomide (TMZ, chemotherapy drug currently used to treat glioblastomas), alone or in combination. At higher concentrations, Ag/AgCl-NPs inhibited GBM02 proliferation more effectively than TMZ (up to 82 and 62% inhibition, respectively), while the opposite occurred at lower concentrations (up to 23 and 53% inhibition, for Ag/AgCl-NPs and TMZ, respectively). The combined treatment (Ag/AgCl-NPs + TMZ) inhibited GBM02 proliferation by 54–83%. Ag/AgCl-NPs had a reduced effect on astrocyte proliferation compared with TMZ, and Ag/AgCl-NPs + TMZ inhibited astrocyte proliferation by 5–42%. The growth rate and population doubling time analyses confirmed that treatment with Ag/AgCl-NPs was more effective against GBM02 cells than TMZ (~ 67-fold), and less aggressive to astrocytes, while Ag/AgCl-NP + TMZ treatment was no more effective against GBM02 cells than Ag/AgCl-NPs monotherapy. Taken together, our data indicate that 2.5 µg mL(−1) Ag/AgCl-NPs represents the safest dose tested here, which affects GBM02 proliferation, with limited effect on astrocytes. Our findings show that HCA is a useful approach to evaluate the antiproliferative effect of nanoparticles against tumor cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10616-018-0253-1) contains supplementary material, which is available to authorized users.

Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular heterogeneity and capability to communicate with microenvironment components, such as microglia. Microglia-glioblastoma interaction contributes to an increase in tumor invasiveness, and Wnt signaling pathway is one of the main cascades related to tumor progression through changes in cell migration and invasion. However, very little is known about the role of canonical Wnt signaling during microglia-glioblastoma crosstalk. Here, we show for the first time that Wnt3a is one of the factors that regulate interactions between microglia and glioblastoma cells. Wnt3a activates the Wnt/β-catenin signaling of both glioblastoma and microglial cells. Glioblastoma-conditioned medium not only induces nuclear translocation of microglial β-catenin but also increases microglia viability and proliferation as well as Wnt3a, cyclin-D1, and c-myc expression. Moreover, glioblastoma-derived Wnt3a increases microglial ARG-1 and STI1 expression, followed by an upregulation of IL-10 mRNA levels, and a decrease in IL1β gene expression. The presence of Wnt3a in microglia-glioblastoma co-cultures increases the formation of membrane nanotubes accompanied by changes in migration capability. In vivo, tumors formed from Wnt3a-stimulated glioblastoma cells presented greater microglial infiltration and more aggressive characteristics such as growth rate than untreated tumors. Thus, we propose that Wnt3a belongs to the arsenal of factors capable of stimulating the induction of M2-like phenotype on microglial cells, which contributes to the poor prognostic of glioblastoma, reinforcing that Wnt/β-catenin pathway can be a potential therapeutic target to attenuate glioblastoma progression.

Previous research advocates that exercise is a non-pharmacological therapy for Parkinson's disease (PD). However, few studies have investigated the effects of exercise on central nervous system structures other than the nigrostriatal pathway by using PD animal models. This study investigated the effects of exercise on tyrosine hydroxylase (TH)- and cerebral dopamine neurotrophic factor (CDNF)-containing spinal-cord neurons. Male Swiss mice were divided into 4 groups: sedentary control (SEDCONT), exercise control (EXERCONT), sedentary Parkinson (SEDPD), and exercise Parkinson (EXERPD). The PD groups were submitted to a surgical procedure for stereotaxic bilateral injection of 6-hydroxydopamine into the striatum. TH- and CDNF-containing spinal-cord neurons were evaluated in all groups, using immunohistochemistry and western-blotting. TH content in the ventral horn differed notably between the SEDPD and EXERPD groups. CDNF content was highest in the EXERPD group. SEDPD and EXERPD groups differed the most, as shown by immunohistochemistry and western-blotting. The EXERPD group showed the most intense labeling in immunohistochemistry compared to the SEDCONT and EXERCONT groups. Therefore, we showed here that exercise increased the content of both TH and CDNF in the spinal-cord neurons of a bilateral PD mouse model. We may assume that the spinal cord is affected in a PD model, and therefore this central nervous system region deserves more attention from researchers dealing with PD.

α-Synuclein protein (α-syn) is a central player in Parkinson's disease (PD) and in a spectrum of neurodegenerative diseases collectively known as synucleinopathies. These diseases are characterized by abnormal motor symptoms, such as tremor at rest, slowness of movement, rigidity of posture, and bradykinesia. Histopathological features of PD include preferential loss of dopaminergic neurons in the substantia nigra and formation of fibrillar intraneuronal inclusions called Lewy bodies and Lewy neurites, which are composed primarily of the α-syn protein. Currently, it is well accepted that α-syn oligomers (αSO) are the main toxic agent responsible for the etiology of PD. Glutamatergic excitotoxicity is associated with several neurological disorders, including PD. Excess glutamate in the synaptic cleft can be taken up by the astrocytic glutamate transporters GLAST and GLT-1. Although this event is the main defense against glutamatergic excitotoxicity, the molecular mechanisms that regulate this process have not yet been investigated in an early sporadic model of synucleinopathy. Here, using an early sporadic model of synucleinopathy, we demonstrated that the treatment of astrocytes with αSO increased glutamate uptake. This was associated with higher levels of GLAST and GLT-1 in astrocyte cultures and in a mouse model of synucleinopathy 24 h and 45 days after inoculation with αSO, respectively. Pharmacological inhibition of the TGF-β1 (transforming growth factor beta 1) pathway in vivo reverted GLAST/GLT-1 enhancement induced by αSO injection. Therefore, our study describes a new neuroprotective role of astrocytes in an early sporadic model of synucleinopathy and sheds light on the mechanisms of glutamate transporter regulation for neuroprotection against glutamatergic excitotoxicity in synucleinopathy.

Background Salsolinol (SALSO), a product from the reaction of dopamine (DA) with acetaldehyde, is found increased in dopaminergic neurons of Parkinson's disease (PD) patients. The administration of SALSO in rats causes myenteric neurodegeneration followed by the formation of deposits of the protein α-synuclein (aS), whose aggregation is intimately associated to PD. Methods NMR, isothermal titration calorimetry and MS were used to evaluate the interaction of SALSO with aS. The toxicity of SALSO and in vitro-produced aS-SALSO species was evaluated on mesencephalic primary neurons from mice. Results SALSO, under oxidative conditions, stabilizes the monomeric state besides a minor population of oligomers of aS, resulting in a strong inhibition of the fibrillation process. SALSO does not promote any chemical modification of the protein. Instead, the interaction of SALSO with aS seems to occur via hydrophobic effect, likely mediated by the NAC (non-amyloid component) domain of the protein. aS-SALSO species were found to be innocuous on primary neurons, while SALSO alone induces apoptosis via caspase-3 activation. Importantly, exogenous aS monomer was capable of protecting neurons against SALSO toxicity irrespective whether the protein was co-administered with SALSO or added until 2 h after SALSO, as evidenced by DAPI and cleaved-caspase 3 assays. Similar protective action of aS was found by pre-incubating neurons with aS before the administration of SALSO. Conclusions Interaction of SALSO with aS leads to the formation of fibril-incompetent and innocuous adducts. SALSO toxicity is attenuated by aS monomer. Significance aS could exhibit a protective role against the neurotoxic effects of SALSO in dopaminergic neuron.

Glioblastoma multiform (GBM) is the most common and devastating type of primary brain tumor, being considered the deadliest of human cancers. In this context, extensive efforts have been undertaken to develop new drugs that exhibit both antiproliferation and antimetastasis effects on GBM. 1,4-Naphthoquinone (1,4-NQ) scaffold has been found in compounds able to inhibit important biological targets associated with cancer, which includes DNA topoisomerase, Hsp90 and monoamine oxidase. Among potential antineoplastic 1,4-NQs is the plant-derived lapachol (2-hydroxy-3-prenyl-1,4-naphthoquinone) that was found to be active against the Walker-256 carcinoma and Yoshida sarcoma. In the present study, we examined the effect of polyamine (PA)-conjugated derivatives of lapachol, nor-lapachol and lawsone on the growth and invasion of the human GBM cells. The conjugation with PA (a spermidine analog) resulted in dose-dependent and time-dependent increase of cytotoxicity of the 1,4-NQs. In addition, in-vitro inhibition of GBM cell invasion by lapachol was increased upon PA conjugation. Previous biochemical experiments indicated that these PA-1,4-NQs are capable of inhibiting DNA human topoisomerase II-α (topo2α), a major enzyme involved in maintaining DNA topology. Herein, we applied molecular docking to investigate the binding of PA-1,4-NQs to the ATPase site of topo2α. The most active molecules preferentially bind at the ATP-binding site of topo2α, which is energetically favored by the conjugation with PA. Taken together, these findings suggested that the PA-1,4-NQ conjugates might represent potential molecules in the development of new drugs in chemotherapy for malignant brain tumors.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor and still lacks effective therapeutic strategies. It has already been shown that old drugs like sulfasalazine (SAS) and valproic acid (VPA) present antitumoral activities in glioma cell lines. SAS has also been associated with a decrease of intracellular glutathione (GSH) levels through a potent inhibition of xc- glutamate/cystine exchanger leading to an antioxidant deprotection. In the same way, VPA was recently identified as a histone deacetylase (HDAT) inhibitor capable of activating tumor suppression genes. As both drugs are widely used in clinical practice and their profile of adverse effects is well known, the aim of our study was to investigate the effects of the combined treatment with SAS and VPA in GBM cell lines. We observed that both drugs were able to reduce cell viability in a dose-dependent manner and the combined treatment potentiated these effects. Combined treatment also increased cell death and inhibited proliferation of GBM cells, while having no effect on human and rat cultured astrocytes. Also, we observed high protein expression of the catalytic subunit of xc- in all the examined GBM cell lines, and treatment with SAS blocked its activity and decreased intracellular GSH levels. Noteworthy, SAS but not VPA was also able to reduce the [14C]-ascorbate uptake. Together, these data indicate that SAS and VPA exhibit a substantial effect on GBM cell’s death related to an intracellular oxidative response imbalance, making this combination of drugs a promising therapeutic strategy.

Protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. It has been shown that lysine residues play a key role in the formation of these aggregates. Thus, the ability to disrupt aggregate formation by covalently modifying lysine residues could lead to the discovery of therapeutically relevant antiamyloidogenesis compounds. Herein, we demonstrate that an ortho-iminoquinone (IQ) can be utilized to inhibit amyloid aggregation. Using alpha-synuclein and Aβ1–40 as model amyloidogenic proteins, we observed that IQ was able to react with lysine residues and reduce amyloid aggregation. We also observed that IQ reacted with free amines within the amyloid fibrils preventing their dissociation and seeding capacity.

Astrocytes play a critical role in the development and homeostasis of the central nervous system (CNS). Astrocyte dysfunction results in several neurological and degenerative diseases. However, a major challenge to our understanding of astrocyte physiology and pathology is the restriction of studies to animal models, human post-mortem brain tissues, or samples obtained from invasive surgical procedures. Here, we report a protocol to generate human functional astrocytes from cerebral organoids derived from human pluripotent stem cells. The cellular isolation of cerebral organoids yielded cells that were morphologically and functionally like astrocytes. Immunolabelling and proteomic assays revealed that human organoid-derived astrocytes express the main astrocytic molecular markers, including glutamate transporters, specific enzymes and cytoskeletal proteins. We found that organoid-derived astrocytes strongly supported neuronal survival and neurite outgrowth and responded to ATP through transient calcium wave elevations, which are hallmarks of astrocyte physiology. Additionally, these astrocytes presented similar functional pathways to those isolated from adult human cortex by surgical procedures. This is the first study to provide proteomic and functional analyses of astrocytes isolated from human cerebral organoids. The isolation of these astrocytes holds great potential for the investigation of developmental and evolutionary features of the human brain and provides a useful approach to drug screening and neurodegenerative disease modelling.

Parkinson's disease (PD) is characterized by motor dysfunction, which is preceded by a number of non-motor symptoms including olfactory deficits. Aggregation of α-synuclein (α-syn) gives rise to Lewy bodies in dopaminergic neurons and is thought to play a central role in PD pathology. However, whether amyloid fibrils or soluble oligomers of α-syn are the main neurotoxic species in PD remains controversial. Here, we performed a single intracerebroventricular (i.c.v.) infusion of α-syn oligomers (α-SYOs) in mice and evaluated motor and non-motor symptoms. Familiar bedding and vanillin essence discrimination tasks showed that α-SYOs impaired olfactory performance of mice, and decreased TH and dopamine levels in the olfactory bulb early after infusion. The olfactory deficit persisted until 45days post-infusion (dpi). α- SYO-infused mice behaved normally in the object recognition and forced swim tests, but showed increased anxiety-like behavior in the open field and elevated plus maze tests 20 dpi. Finally, administration of α-SYOs induced late motor impairment in the pole test and rotarod paradigms, along with reduced TH and dopamine content in the caudate putamen, 45 dpi. Reduced number of TH-positive cells was also seen in the substantia nigra of α-SYO-injected mice compared to control. In conclusion, i.c.v. infusion of α-SYOs recapitulated some of PD-associated non-motor symptoms, such as increased anxiety and olfactory dysfunction, but failed to recapitulate memory impairment and depressive-like behavior typical of the disease. Moreover, α-SYOs i.c.v. administration induced motor deficits and loss of TH and dopamine levels, key features of PD. Results point to α-syn oligomers as the proximal neurotoxins responsible for early non-motor and motor deficits in PD and suggest that the i.c.v. infusion model characterized here may comprise a useful tool for identification of PD novel therapeutic targets and drug screening.

The balance between excitatory and inhibitory synaptic inputs is critical for the control of brain function. Astrocytes play important role in the development and maintenance of neuronal circuitry. Whereas astrocytes-derived molecules involved in excitatory synapses are recognized, molecules and molecular mechanisms underlying astrocyte-induced inhibitory synapses remain unknown. Here, we identified transforming growth factor beta 1 (TGF-β1), derived from human and murine astrocytes, as regulator of inhibitory synapse in vitro and in vivo. Conditioned media derived from human and murine astrocytes induce inhibitory synapse formation in cerebral cortex neurons, an event inhibited by pharmacologic and genetic manipulation of the TGF-β pathway. TGF-β1-induction of inhibitory synapse depends on glutamatergic activity and activation of CaM kinase II, which thus induces localization and cluster formation of the synaptic adhesion protein, Neuroligin 2, in inhibitory postsynaptic terminals. Additionally, intraventricular injection of TGF-β1 enhanced inhibitory synapse number in the cerebral cortex. Our results identify TGF-β1/CaMKII pathway as a novel molecular mechanism underlying astrocyte control of inhibitory synapse formation. We propose here that the balance between excitatory and inhibitory inputs might be provided by astrocyte signals, at least partly achieved via TGF-β1 downstream pathways. Our work contributes to the understanding of the GABAergic synapse formation and may be of relevance to further the current knowledge on the mechanisms underlying the development of various neurological disorders, which commonly involve impairment of inhibitory synapse transmission.

Oxidative stress and the formation of cytotoxic aggregates of the presynaptic protein α-synuclein (AS) are two important events associated with the pathogenesis of Parkinson's disease (PD) and several other neurodegenerative diseases. In this context, extensive efforts have been made to elucidate the molecular basis of the cytotoxic synergy between oxidative stress and AS aggregation. In this study, we demonstrate that the exposure of AS to oxidative stress induced by UV radiation (ASUV) blocks the protein fibrillation, leading to the formation of highly toxic fibril-incompetent oligomers. In addition, ASUV exhibited stronger anti-fibrillogenic properties than H2O2-treated AS, inhibiting the fibrillation of unmodified AS at notably low concentrations. Mass spectrometry indicated that Met5 oxidation to Met-sulfoxide was the only modification promoted by UV exposure, which is reinforced by NMR data indicating that Met5 is the only residue whose amide resonance completely disappeared from the (1)H-(15)N HSQC spectrum after UV exposure. This result is supported by previous data that indicate that C-terminal Met residues (Met116 and Met127) and N-terminal Met1 are less susceptible to oxidation than Met5 because of the residual structure of the disordered AS monomer. Overall, our findings suggest that specific oxidation of Met5 might be sufficient to promote the formation of highly neurotoxic oligomers of AS.

Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So far, all clinically available alternatives for treating GBM are limited, evoking the development of novel treatment strategies that can more effectively manage these tumors. Extensive effort is being dedicated to characterize the molecular basis of GBM resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. Cytolysins are toxins that form pores in target cell membranes, modifying ion homeostasis and leading to cell death. These pore-forming toxins might be used, therefore, to enhance the efficiency of conventional chemotherapeutic drugs, facilitating their entrance into the cell. In this study, we show that a non-cytotoxic concentration of equinatoxin II (EqTx-II), a pore-forming toxin from the sea anemone Actinia equina, potentiates the cytotoxicity induced by temozolomide (TMZ), a first-line GBM treatment, and by etoposide (VP-16), a second- or third-line GBM treatment. We also suggest that this effect is selective to GBM cells and occurs via PI3K/Akt pathway inhibition. Finally, Magnetic resonance imaging (MRI) revealed that a non-cytotoxic concentration of EqTx-II potentiates the VP-16-induced inhibition of GBM growth in vivo. These combined therapies constitute a new and potentially valuable tool for GBM treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy.

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.

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.

Thyroid hormones (THs) are essential for brain development, where they regulate gliogenesis, myelination, cell proliferation and protein synthesis. Hypothyroidism severely affects neuronal growth and establishment of synaptic connections. Triiodothyronine (T3), the biologically active form of TH, has a central function in these activities. So, Myosin-Va (Myo-Va), a molecular motor protein involved in vesicle and RNA transport, is a good candidate as a target for T3 regulation. Here, we analyzed Myo-Va expression in euthyroid and hypothyroid adult rat brains and synaptosomes. We observed a reduction of Myo-Va expression in cultured neural cells from newborn hypothyroid rat brain, while immunocytochemical experiments showed a punctate distribution of this protein in the cytoplasm of cells. Particularly, Myo-Va co-localized with microtubules in neurites, especially in their varicosities. Myo-Va immunostaining was stronger in astrocytes and neurons of controls when compared with hypothyroid brains. In addition, supplementation of astrocyte cultures with T3 led to increased expression of Myo-Va in cells from both euthyroid and hypothyroid animals, suggesting that T3 modulates Myo-Va expression in neural cells both in vivo and in vitro. We have further analyzed Myo-Va expression in U373 cells, a human glioblastoma line, and found the same punctate cytoplasmic protein localization. As in normal neural cells, this expression was also increased by T3, suggesting that the modulatory mechanism exerted by T3 over Myo-Va remains active on astrocyte tumor cells. These data, coupled with the observation that Myo-Va is severely affected in hypothyroidism, support the hypothesis that T3 activity regulates neural motor protein expression, taking Myo-Va as a model. As a consequence, reduced T3 activity could supposedly affect axonal transport and synaptic function, and could therefore explain disturbances seen in the hypothyroid brain.

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.

International audience; Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9-107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10-100 and 111-157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1-9), followed by the N-terminal portion of α-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by α-synuclein oligomers. This advises its use against physiological damages caused by α-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.

Parkinson's disease (PD) is characterized by the presence of cytoplasmic inclusions composed of alpha-synuclein (alpha-syn) in dopaminergic neurons. This suggests a pivotal role of dopamine (DA) on PD development. Here, we show that DA modulates differently the stability of protofibrils (PF) and fibrils (F) composed of wild type or variants of alpha-syn (A30P and A53T) as probed by high hydrostatic pressure (HHP). While in the absence of DA, all alpha-syn PF exhibited identical stability, in its presence, the variant-composed PF acquired a greater stability (DAPFwt < DAPFA30P = DAPFA53T), implying that they would last longer, which could shed light onto why these mutations are so aggressive. When alpha-syn was incubated for long times (18 days) in the presence of DA, we observed the formation of F by electronic microscopy, suggesting that the PF trapped in the presence of DA in short times can evolve into F. The stability of F was also altered by DA. DAFwt was more labile than Fwt, indicating that the former would be more susceptible to breakage. PFA30P and DAPFA30P, when added to mesencephalic and cortical neurons in culture, decreased the number and length of neurites and increased the number of apoptotic cells. Surprisingly, these toxic effects of PFA30P and DAPFA30P were practically abolished with HHP treatment, which was able to break the PF into smaller aggregates, as seen by atomic force microscopy. These results suggest that strategies aimed at breaking and/or clearing these aggregates is promising in alleviating the symptoms of PD.

The harmonious development of the central nervous system depends on the interactions of the neuronal and glial cells. Extracellular matrix elements play important roles in these interactions, especially laminin produced by astrocytes, which has been shown to be a good substrate for neuron growth and axonal guidance. Glioblastomas are the most common subtypes of primary brain tumors and may be astrocytes in origin. As normal laminin-producing glial cells are the preferential substrate for neurons, and glial tumors have been shown to produce laminin, we questioned whether glioblastoma retained the same normal glial–neuron interactive properties with respect to neuronal growth and differentiation. Then, rat neurons were co-cultured onto rat normal astrocytes or onto three human glioblastoma cell lines obtained from neurosurgery. The co-culture confirmed that human glioblastoma cells as well as astrocytes maintained the ability to support neuritogenesis, but non-neural normal or tumoral cells failed to do so. However, glioblastoma cells did not distinguish embryonic from post-natal neurons in relation to neurite pattern in the co-cultures, as normal astrocytes did. Further, the laminin organization on both normal and tumoral glial cells was altered from a filamentous arrangement to a mixed punctuate/filamentous pattern when in co-culture with neurons. Together, these results suggest that glioblastoma cells could identify neuronal cells as partners, to support their growth and induce complex neurites, but they lost the normal glia property to distinguish neuronal age. In addition, our results show for the first time that neurons modulate the organization of astrocytes and glioblastoma laminin on the extracellular matrix.

O artigo aborda a polêmica surgida entre a década de 1990 e a atual em torno de dois grupos de memórias contrapostas sobre um dos acontecimentos históricos-chave da formação do Estado Nacional argentino no século XIX, a denominada "Conquista do Deserto" (1878-1885), e seu principal comandante, o Gen. Julio Argentino Roca (1843-1914). Tal polêmica tem como principal disparador uma série de monumentos em homenagem a Roca presentes nos espaços públicos de diversas cidades da Argentina, dentre as quais, San Carlos de Bariloche, na Patagônia, onde parte da população local alega que a estátua alude não a um passado glorioso, mas ao massacre genocida que teve por alvo principalmente o povo indígena Mapuche que habitava originalmente aquela região. Aqui será exposto como tal monumento tornou-se objeto de uma disputa simbólica que revela, em seu conteúdo e origens, a emergência no espaço urbano de demandas específicas por terra, por autonomia territorial, por condições dignas de vida, reconhecimento identitário e, por fim, pela consolidação de novos espaços de representação social e política para os indígenas argentinos

Background Glioblastoma (GBM) is the most common primary brain tumor and the most aggressive glial tumor. This tumor is highly heterogeneous, angiogenic, and insensitive to radio- and chemotherapy. Here we have investigated the progression of GBM produced by the injection of human GBM cells into the brain parenchyma of immunocompetent mice. Methods Xenotransplanted animals were submitted to magnetic resonance imaging (MRI) and histopathological analyses. Results Our data show that two weeks after injection, the produced tumor presents histopathological characteristics recommended by World Health Organization for the diagnosis of GBM in humans. The tumor was able to produce reactive gliosis in the adjacent parenchyma, angiogenesis, an intense recruitment of macrophage and microglial cells, and presence of necrosis regions. Besides, MRI showed that tumor mass had enhanced contrast, suggesting a blood–brain barrier disruption. Conclusions This study demonstrated that the xenografted tumor in mouse brain parenchyma develops in a very similar manner to those found in patients affected by GBM and can be used to better understand the biology of GBM as well as testing potential therapies. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-923) contains supplementary material, which is available to authorized users.

The tumor microenvironment has a dynamic and usually cancer-promoting function during all tumorigenic steps. Glioblastoma (GBM) is a fatal tumor of the central nervous system, in which a substantial number of non-tumoral infiltrated cells can be found. Astrocytes neighboring these tumor cells have a particular reactive phenotype and can enhance GBM malignancy by inducing aberrant cell proliferation and invasion. The tumor suppressor p53 has a potential non-cell autonomous function by modulating the expression of secreted proteins that influence neighbor cells. In this work, we investigated the role of p53 on the crosstalk between GBM cells and astrocytes. We show that extracellular matrix (ECM) from p53(+/-) astrocytes is richer in laminin and fibronectin, compared with ECM from p53(+/+) astrocytes. In addition, ECM from p53(+/-) astrocytes increases the survival and the expression of mesenchymal markers in GBM cells, which suggests haploinsufficient phenotype of the p53(+/-) microenvironment. Importantly, conditioned medium from GBM cells blocks the expression of p53 in p53(+/+) astrocytes, even when DNA was damaged. These results suggest that GBM cells create a dysfunctional microenvironment based on the impairment of p53 expression that in turns exacerbates tumor endurance.

Semiconductor quantum dots based on II–VI materials may be prepared to develop good biolabeling properties. In this study we present some well-succeeded results related to the preparation, functionalization and bioconjugation of CdY (Y = S, Se and Te) to biological systems (live cells and fixed tissues). These nanostructured materials were prepared using colloidal synthesis in aqueous media resulting nanoparticles with very good optical properties and an excellent resistance to photodegradation.

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.

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.

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 (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 24 h 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 24 h 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 24 h 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 24 h 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.

Background Tumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies. Methods We 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. Results A 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. Conclusions Our 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.

Until about 100 years ago, astroglial cells were regarded solely as passive components of the nervous system. During the past few years, however, increasing knowledge of these cells has completely changed this scenario: rather than mere inert “brain glue”, astroglial cells are now considered active partners of neurons, and they seem to be much more actively involved in brain function than was formerly thought. Recent evidence shows that astroglia, in addition to their previously known roles in neurotransmitter clearance, ion buffering, and neuronal trophic support, are also involved in other functions, such as synapse development and neurogenesis. In this review, we will focus on the role of astroglial cells, mainly radial glia and astrocytes, in several events of nervous system development such as cell fate commitment, neuronal and astrocyte maturation, and synapse formation. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership. Finally, we will briefly discuss the emerging view of astrocytes as essential actors in neurodegenerative diseases and neurological disorders, and how a better understanding of glial development might open new avenues to develop therapeutic approaches to these pathologies.

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-beta1 (transforming growth factor beta 1) secretion in vitro. To access the effect of glutamate on GFAP gene, we used transgenic mice bearing the beta-Galactosidase (beta-Gal) reporter gene under the regulation of the GFAP gene promoter. We report that 100 muM glutamate activates the GFAP gene promoter of astrocytes from cerebral cortex revealed by a significant increase in the number of beta-Gal positive astrocytes. Neutralizing antibodies against TGF-beta completely prevented glutamate and neuronal-induction of GFAP gene, thus indicating that this event is mediated by TGF-beta1. Further, induction of GFAP gene in response to glutamate was followed by nuclear translocation of the Smad transcription factor, a hallmark of TGF-beta1 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-beta1 direct action on GFAP gene although they did not inhibit Smad-2 phosphorylation, suggesting that TGF-beta1-induced GFAP gene activation might involve cooperation between the canonical and non-canonical TGF-beta pathways. Together, our results suggest that glial metabotropic glutamate 2/3 receptor activation by neurons induces TGF-beta1 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.

Guanine derivatives (GD) have been shown to exert relevant extracellular effects as intercellular messengers, neuromodulators in the central nervous system, and trophic effects on astrocytes and neurons. Astrocytes have been pointed out as the major source of trophic factors in the nervous system, however, several trophic effects of astrocytic-released soluble factors are mediated through modulation of extracellular matrix (ECM) proteins. In this study, we investigated the effects of guanosine-5′-monophosphate (GMP) and guanosine (GUO) on the expression and organization of ECM proteins in cerebellar astrocytes. Moreover, to evaluate the effects of astrocytes pre-treated with GMP or GUO on cerebellar neurons we used a neuron-astrocyte coculture model. GMP or GUO alters laminin and fibronectin organization from a punctate to a fibrillar pattern, however, the expression levels of the ECM proteins were not altered. Guanine derivatives-induced alteration of ECM proteins organization is mediated by activation of mitogen activated protein kinases (MAPK), CA2+-calmodulin-dependent protein kinase II (CaMK-II), protein kinase C (PKC), and protein kinase A (PKA) pathways. Furthermore, astrocytes treated with GMP or GUO promoted an increased number of cerebellar neurons in coculture, without altering the neuritogenesis pattern. No proliferation of neurons or astrocytes was observed due to GMP or GUO treatment. Our results show that guanine derivatives promote a reorganization of the ECM proteins produced by astrocytes, which might be responsible for a better interaction with neurons in cocultures. © 2007 Wiley-Liss, Inc.

Parkinson's disease (PD) is characterized by the presence of cytoplasmic inclusions composed of alpha-synuclein (alpha-syn) in dopaminergic neurons. This suggests a pivotal role of dopamine (DA) on PD development. Here, we show that DA modulates differently the stability of protofibrils (PF) and fibrils (F) composed of wild type or variants of alpha-syn (A30P and A53T) as probed by high hydrostatic pressure (HHP). While in the absence of DA, all alpha-syn PF exhibited identical stability, in its presence, the variant-composed PF acquired a greater stability (DAPFwtDAPFA30P = DAPFA53T), implying that they would last longer, which could shed light onto why these mutations are so aggressive. When alpha-syn was incubated for long times (18 days) in the presence of DA, we observed the formation of F by electronic microscopy, suggesting that the PF trapped in the presence of DA in short times can evolve into F. The stability of F was also altered by DA. DAFwt was more labile than Fwt, indicating that the former would be more susceptible to breakage. PFA30P and DAPFA30P, when added to mesencephalic and cortical neurons in culture, decreased the number and length of neurites and increased the number of apoptotic cells. Surprisingly, these toxic effects of PFA30P and DAPFA30P were practically abolished with HHP treatment, which was able to break the PF into smaller aggregates, as seen by atomic force microscopy. These results suggest that strategies aimed at breaking and/or clearing these aggregates is promising in alleviating the symptoms of PD.

In this work we present the preparation, characterization and conjugation of colloidal core shell CdS-Cd(OH) 2 quantum dots to health and cancer glial rats living cells in culture media. The particles were obtained via colloidal synthesis in aqueous medium, with final pH=7.3-7.4. Laser Scan Confocal Microscopy (LSCM) and Fluorescence Microscopy were used to evaluate fluorescence intensities and patterns of health and cancer (glioblastoma) glial cells labeled with the quantum dots in different time intervals. Health and cancer glial cells clearly differ in their fluorescence intensities and patterns. These different fluorescence intensities and patterns may be associated to differences concerning cellular membrane and metabolic features of health and cancer cells. The results obtained indicate the potential of the methodology for fast and precise cancer diagnostics.