Amburana cearensis seed extract stimulates astrocyte glutamate homeostatic mechanisms in hippocampal brain slices and protects oligodendrocytes against ischemia.

BACKGROUND: Stroke is a leading cause of death and disability worldwide. A major factor in brain damage following ischemia is excitotoxicity caused by elevated levels of the neurotransmitter glutamate. In the brain, glutamate homeostasis is a primary function of astrocytes. Amburana cearensis has long been used in folk medicine and seed extract obtained with dichloromethane (EDAC) have previously been shown to exhibit cytoprotective activity in vitro. The aim of the present study was to analyse the activity of EDAC in hippocampal brain slices. METHODS: We prepared a dichloromethane extract (EDAC) from A. cearensis seeds and characterized the chemical constituents by 1H and 13C-NMR. Hippocampal slices from P6-8 or P90 Wistar rats were used for cell viability assay or glutamate uptake test. Hippocampal slices from P10-12 transgenic mice SOX10-EGFP and GFAP-EGFP and immunofluorescence for GS, GLAST and GLT1 were used to study oligodendrocytes and astrocytes. RESULTS: Astrocytes play a critical role in glutamate homeostasis and we provide immunohistochemical evidence that in excitotoxicity EDAC increased expression of glutamate transporters and glutamine synthetase, which is essential for detoxifying glutamate. Next, we directly examined astrocytes using transgenic mice in which glial fibrillary acidic protein (GFAP) drives expression of enhanced green fluorescence protein (EGFP) and show that glutamate excitotoxicity caused a decrease in GFAP-EGFP and that EDAC protected against this loss. This was examined further in the oxygen-glucose deprivation (OGD) model of ischemia, where EDAC caused an increase in astrocytic process branching, resulting in an increase in GFAP-EGFP. Using SOX10-EGFP reporter mice, we show that the acute response of oligodendrocytes to OGD in hippocampal slices is a marked loss of their processes and EDAC protected oligodendrocytes against this damage. CONCLUSION: This study provides evidence that EDAC is cytoprotective against ischemia and glutamate excitotoxicity by modulating astrocyte responses and stimulating their glutamate homeostatic mechanisms.

Protective Effects of Flavonoid Rutin Against Aminochrome Neurotoxicity.

Causes of dopaminergic neuronal loss in Parkinson's disease (PD) are subject of investigation and the common use of models of acute neurodegeneration induced by neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine, and rotenone contributed to advances in the study of PD. However, the use of study models more similar to the pathophysiology of PD is required for advances in early diagnosis and translational pharmacology. Aminochrome (AMI), a compound derived from dopamine oxidation and a precursor of neuromelanin, is able to induce all the mechanisms associated with neurodegeneration. Previously, we showed AMI is cytotoxic in primary culture of mesencephalic cells (PCMC) and induces in vitro and in vivo neuroinflammation. On the other hand, the effect of rutin in central nervous system cells has revealed anti-inflammatory, antioxidative, and neuroprotective potential. However, there have been no data studies on the effect of rutin against aminochrome neurotoxicity. Here, we show that rutin prevents lysosomal dysfunction and aminochrome-induced cell death in SHSY-5Y cells, protects PCMC against aminochrome cytotoxicity, and prevents in vivo loss of dopaminergic neurons in substantia nigra pars compacta (SNPc), as well as microgliosis and astrogliosis. Additionally, we show that rutin decreases levels of interleukin-1ß (IL-1ß) mRNA and increases levels of glia-derived neurotrophic factor (GDNF) and nerve-derived neurotrophic factor (NGF) mRNA. We evidence for the first time the protective effect of rutin on PD aminochrome-induced models and suggest the potential role of the anti-inflammatory activity and upregulation of NGF and GDNF in the mechanism of rutin action against aminochrome neurotoxicity.

Aminochrome Induces Neuroinflammation and Dopaminergic Neuronal Loss: A New Preclinical Model to Find Anti-inflammatory and Neuroprotective Drugs for Parkinson's Disease.

Studies have suggested aminochrome as an endogenous neurotoxin responsible for the dopaminergic neuron degeneration in Parkinson's disease (PD). However, neuroinflammation, an important alteration in PD pathogenesis, has been strictly induced in vitro by aminochrome. The aim of this study was to characterize the neuroinflammation induced in vivo by aminochrome. Wistar rats (male, 250-270 g) received a unilateral single dose by stereotaxic injection of saline into three sites in the striatum in the negative control group, or 32 nmol 6-hydroxydopamine (6-OHDA) in the positive control, or 6 nmol aminochrome. After 14 days, histological and molecular analyses were performed. We observed by immunofluorescence that aminochrome, as well as 6-OHDA, induced an increase in the number of Iba-1+ cells and in the number of activated (Iba-1+/ CD68+) microglia. An increase in the number of S100b+ cells and in the GFAP expression were also evidenced in the striatum and the SNpc of animals from aminochrome and positive control group. Dopaminergic neuronal loss was marked by reduction of TH+ cells and confirmed with reduction in the number of Nissl-stained neurons in the SNpc of rats from aminochrome and positive control groups. In addition, we observed by qPCR that aminocrhome induced an increase in the levels of IL-1ß, TNF-α, NLRP3, CCL5 and CCR2 mRNA in the SNpc. This work provides the first evidence of microgliosis, astrogliosis and neuroinflammation induced by aminochrome in an in vivo model. Since aminochrome is an endogenous molecule derived from dopamine oxidation present in the targeted neurons in PD, these results reinforce the potential of aminochrome as a useful preclinical model to find anti-inflammatory and neuroprotective drugs for PD. Aminochrome induced dopaminergic neuronal loss, microglial activation, astroglial activation and neuroinflammation marked by an increase in NLRP3, IL1ß, TNF-α, CCL2, CCL5 and CCR2.

JM-20, a Benzodiazepine-Dihydropyridine Hybrid Molecule, Inhibits the Formation of Alpha-Synuclein-Aggregated Species.

Studies showed that JM-20, a benzodiazepine-dihydropyridine hybrid molecule, protects against rotenone and 6-hydroxydopamine neurotoxicity. However, its protective effects against cytotoxicity induced by endogenous neurotoxins involved in Parkinson's disease (PD) pathogenesis have never been investigated. In this study, we evaluated the ability of JM-20 to inhibit alpha-synuclein (aSyn) aggregation. We also evaluated the interactions of JM-20 with aSyn by molecular docking and molecular dynamics and assessed the protective effect of JM-20 against aminochrome cytotoxicity. We demonstrated that JM-20 induced the formation of heterogeneous amyloid fibrils, which were innocuous to primary cultures of mesencephalic cells. Moreover, JM-20 reduced the average size of aSyn positive inclusions in H4 cells transfected with SynT wild-type and synphilin-1-V5, but not in HEK cells transfected with synphilin-1-GFP. In silico studies showed the interaction between JM-20 and the aSyn-binding site. Additionally, we showed that JM-20 protects SH-SY5Y cells against aminochrome cytotoxicity. These results reinforce the potential of JM-20 as a neuroprotective compound for PD and suggest aSyn as a molecular target for JM-20.

Astrocyte Reaction to Catechol-Induced Cytotoxicity Relies on the Contact with Microglia Before Isolation.

Astrocytes preserve the brain microenvironment homeostasis in order to protect other brain cells, mainly neurons, against damages. Glial cells have specific functions that are important in the context of neuronal survival in different models of central nervous system (CNS) diseases. Microglia are among these cells, secreting several molecules that can modulate astrocyte functions. Although 1,2-dihydroxybenzene (catechol) is a neurotoxic monoaromatic compound of exogenous origin, several endogenous molecules also present the catechol group. This study compared two methods to obtain astrocyte-enriched cultures from newborn Wistar rats of both sexes. In the first technique (P1), microglial cells began to be removed early 48 h after primary mixed glial cultures were plated. In the second one (P2), microglial cells were late removed 7 to 10 days after plating. Both cultures were exposed to catechol for 72 h. Catechol was more cytotoxic to P1 cultures than to P2, decreasing cellularity and changing the cell morphology. Microglial-conditioned medium (MCM) protected P1 cultures and inhibited the catechol autoxidation. P2 cultures, as well as P1 in the presence of 20% MCM, presented long, dense, and fibrillary processes positive for glial fibrillary acidic protein, which retracted the cytoplasm when exposed to catechol. The Ngf and Il1beta transcription increased in P1, meanwhile astrocytes expressed more Il10 in P2. Catechol decreased Bdnf and Il10 in P2 cultures, and it decreased the expression of Il1beta in both conditions. A prolonged contact with microglia before isolation of astrocyte-enriched cultures modifies astrocyte functions and morphology, protecting these cells against catechol-induced cytotoxicity.

Agathisflavone modulates astrocytic responses and increases the population of neurons in an in vitro model of traumatic brain injury.

Traumatic brain injury (TBI) is a critical health problem worldwide, with a high incidence rate and potentially severe long-term consequences. Depending on the level of mechanical stress, astrocytes react with complex morphological and functional changes known as reactive astrogliosis. In cases of severe tissue injury, astrocytes proliferate in the area immediately adjacent to the lesion to form the glial scar, which is a major barrier to neuronal regeneration in the central nervous system. The flavonoid agathisflavone has been shown to have neuroprotective, neurogenic, and immunomodulatory effects and could have beneficial effects in situations of TBI. In this study, we investigated the effects of agathisflavone on modulating the responses of astrocytes and neurons to injury, using the in vitro scratch wound model of TBI in primary cultures of rat cerebral cortex. In control conditions, the scratch wound induced an astroglial injury response, characterized by upregulation of glial fibrillary acidic protein (GFAP) and hypertrophy, together with the reduction in proportion of neurons within the lesion site. Treatment with agathisflavone (1 µM) decreased astroglial GFAP expression and hypertrophy and induced an increase in the number of neurons and neurite outgrowth into the lesion site. Agathisflavone also induced increased expression of the neurotrophic factors NGF and GDNF, which are associated with the neuroprotective profile of glial cells. These results demonstrate that in an in vitro model of TBI, the flavonoid agathisflavone modulates the astrocytic injury response and glial scar formation, stimulating neural recomposition.

The flavonoid rutin and its aglycone quercetin modulate the microglia inflammatory profile improving antiglioma activity.

Microglia cells are the immune effector in the Central Nervous System (CNS). However, studies have showed that they contribute more to glioma progression than to its elimination. Rutin and its aglycone quercetin are flavonoids present in many fruits as well as plants and have been demonstrated to bear anti-inflammatory, antioxidant and antitumor properties also to human glioblastoma cell lines. Previous studies also demonstrated that rutin, isolated from the Brazilian plant Dimorphandra mollis Bent., presents immunomodulatory effect on astrocytes and microglia. In this study, we investigate the antitumor and immunomodulatory properties of rutin and its aglycone quercetin on the viability of glioma cells alone and under direct and indirect interaction with microglia. Flavonoid treatment of rat C6 glioma cells induced inhibition of proliferation and migration, and also induced microglia chemotaxis that was associated to the up regulation of tumor necrosis factor (TNF) and the down regulation of Interleukin 10 (IL-10) at protein and mRNA expression levels, regulation of mRNA expression for chemokines CCL2, CCL5 and CX3CL1, and Heparin Binding Growth Factor (HDGF), Insulin-like growth factor (IGF) and Glial cell-derived neurotrophic factor (GDNF) growth factors. Treatment of human U251 and TG1 glioblastoma cells with both flavonoids also modulated negatively the expression of mRNA for IL-6 and IL-10 and positively the expression of mRNA for TNF characterizing changes to the immune regulatory profile. Treatment of microglia and C6 cells either in co-cultures or during indirect interaction, via conditioned media from glioma cells treated with flavonoids or via conditioned media from microglia treated with flavonoids reduced proliferation and migration of glioma cells. It also directed microglia towards an inflammatory profile with increased expression of mRNA for IL-1ß, IL-6, IL-18 and decreased expression of mRNA for nitric oxide synthase 2 (NOS2) and prostaglandin-endoperoxide synthase 2 (PTGS2), arginase and transforming growth factor beta (TGF-ß), as well as Insulin-like growth factor (IGF). Treatment of U251 cells with flavonoids also reduced tumorigenesis when the cells were xenotransplanted in rat brains, and directed microglia and also astrocytes in the microenvironment of tumor cell implantation as well as in the brain parenchyma to a not favorable molecular inflammatory profile to the glioma growth, as observed in cultures. Together these results demonstrate that the flavonoid rutin and its aglycone quercetin present antiglioma effects related to the property of modulating the microglial inflammatory profile and may be considered for molecular and preclinical studies as adjuvant molecules for treatment of gliomas.

Neuroprotection During Neospora caninum Infection Is Related To the Release of Neurotrophic Factors BDNF and NGF.

Neospora caninum is a parasite that infects many animal species and has tropism for various tissues, particularly the nervous system, where it generally remains in cysts. Under N. caninum infection, glial cells activate immune responses by a Th2 profile, suggesting an immunologically privileged environment that controls parasite proliferation, with neuronal preservation. In this study, we investigated the role of soluble neurotrophic factors released by glial cells on neuronal integrity during N. caninum infection in vitro. Primary cultures of rat glial cells enriched in astrocytes were infected with N. caninum tachyzoites (1:1) for 24 hr. Neuron-glia co-cultures were cultured for 24 hr with conditioned medium from glial cells infected with N. caninum (CMNc) and from uninfected cultures (control). Cell viability was determined through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test; astrocyte morphology and reactivity were determined through immunocytochemistry for glial fibrillar acid protein (GFAP) and the integrity of neurons through immunocytochemistry for ß-tubulin III. Expression of inflammatory cytokines and neurotrophic factors was determined through RT-qPCR. The MTT test demonstrated that 1:1 was the best parasite/host cell ratio, considering that it was enough to increase metabolism of glial cells when compared with control cultures and was not cytotoxic after 48 hr infection. N. caninum-infected glial cultures responded with astrogliosis characterized by an increase in GFAP expression and increase in IL-10 (2-fold), BDNF (1.6-fold), and NGF (1.7-fold) gene expression. In the neuron/glia co-cultures, it was observed that treatment with CMNc induced neuritis outgrowth without toxicity. Together, these results show that modulatory mechanisms by neurotrophic factors derived from glial cells, primarily astrocytes during the N. caninum infection, can favor neuroprotection.

Role of IFN-γ and LPS on neuron/glial co-cultures infected by Neospora caninum.

Neospora caninum causes cattle abortion and neurological symptoms in dogs. Although infection is usually asymptomatic, classical neurological symptoms of neosporosis may be associated with encephalitis. This parasite can grow in brain endothelial cells without markedly damages, but it can modulate the cellular environment to promote its survival in the brain. In previous studies, we described that IFN-γ decreased the parasite proliferation and down regulated nitric oxide (NO) production in astrocyte/microglia cultures. However, it remains unclear how glial cells respond to N. caninum in the presence of neurons. Therefore, we evaluated the effect of 300 IU/mL IFN-γ or 1.0 mg/mL of LPS on infected rat neuron/glial co-cultures. After 72 h of infection, LPS did not affect the mitochondrial dehydrogenase activity. However, IFN-γ decreased this parameter by 15.5 and 12.0% in uninfected and infected cells, respectively. The number of tachyzoites decreased 54.1 and 44.3% in cells stimulated with IFN-γ and LPS, respectively. Infection or LPS treatment did not change NO production. On the other hand, IFN-γ induced increased nitrite release in 55.7%, but the infection reverted this induction. IL-10 levels increased only in infected cultures (treated or not), meanwhile PGE2 release was improved in IFN-γ/infected or LPS/infected cells. Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001), this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%). The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-γ stimulus. This observation contributes to understand the immune mediated mechanisms of neosporosis in central nervous system (CNS).