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The molecular determinants underlying the failure of axons to regenerate in the CNS after injury were studied in an in vitro model of astrogliosis and neuronal coculture. Mechanically lesioned neuron-astrocyte mouse cortical cocultures were treated with antisense glial fibrillary acidic protein (GFAP)-mRNA in order to inhibit the formation of gliofilaments that occurs in response to injury. This inhibition relieves the blockage of neuron migration and neuritic outgrowth observed after lesion, and migrating neurons reappeared, supported by a laminin-labeled extracellular network (permissive conditions). We then questioned the relationship between this permissivity and laminin production. Follow-up studies on the concentration of laminin indicated that, after antisense treatment, the laminin level was increased in the cocultures and was under the control of astrocyte-neuron interactions. The addition of exogenous laminin favored neuronal migration and neurite outgrowth, whereas neutralizing laminin bioavailability with antibodies recognizing the astroglial laminin resulted in an inhibition of both neuronal access to the lesion site and neurite outgrowth, suggesting an active role for laminin in the permissive process. This permissive process could be associated with modulation of extracellular matrix (ECM) molecule degradation by proteinases. Among the latter, matrix metalloproteinases (MMPs) are involved in the breakdown of the ECM component. Our investigation showed a net decrease of the matrix metalloproteinase MMP-2 expression and activity and an increase of its endogenous inhibitor TIMP-2 expression. Both proteins associated with permissivity should be involved in the laminin stabilization and cell-matrix interactions. High levels of laminin and laminin bioavailability, consequent to a reduction in astrogliosis, may be important permissive elements for neuronal migration and neurite outgrowth postlesion.

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Neurodegenerative disorders have a common characteristic that is the involvement of different cell types, typically the reactivity of astrocytes and microglia, characterizing gliosis, which in turn contributes to the neuronal dysfunction and or death. Flavonoids are secondary metabolites of plant origin widely investigated at present and represent one of the most important and diversified among natural products phenolic groups. Several biological activities are attributed to this class of polyphenols, such as antitumor activity, antioxidant, antiviral, and anti-inflammatory, among others, which give significant pharmacological importance. Our group have observed that flavonoids derived from Brazilian plants Dimorphandra mollis Bent., Croton betulaster Müll. Arg., e Poincianella pyramidalis Tul., botanical synonymous Caesalpinia pyramidalis Tul. also elicit a broad spectrum of responses in astrocytes and neurons in culture as activation of astrocytes and microglia, astrocyte associated protection of neuronal progenitor cells, neuronal differentiation and neuritogenesis. It was observed the flavonoids also induced neuronal differentiation of mouse embryonic stem cells and human pluripotent stem cells. Moreover, with the objective of seeking preclinical pharmacological evidence of these molecules, in order to assess its future use in the treatment of neurodegenerative disorders, we have evaluated the effects of flavonoids in preclinical in vitro models of neuroinflammation associated with Parkinson's disease and glutamate toxicity associated with ischemia. In particular, our efforts have been directed to identify mechanisms involved in the changes in viability, morphology, and glial cell function induced by flavonoids in cultures of glial cells and neuronal cells alone or in interactions and clarify the relation with their neuroprotective and morphogetic effects.

Prosopis juliflora is used for feeding cattle and humans. Intoxication with the plant has been reported, and is characterized by neuromuscular alterations and gliosis. Total alkaloidal extract (TAE) was obtained using acid/basic-modified extraction and was fractionated. TAE and seven alkaloidal fractions, at concentrations ranging 0.03-30 microg/ml, were tested for 24h on astrocyte primary cultures derived from the cortex of newborn Wistar rats. The MTT test and the measure of LDH activity on the culture medium, revealed that TAE and fractions F29/30, F31/33, F32 and F34/35 were cytotoxic to astrocytes. The EC(50) values for the most toxic compounds, TAE, F31/33 and F32 were 2.87 2.82 and 3.01 microg/ml, respectively. Morphological changes and glial cells activation were investigated through Rosenfeld's staining, by immunocytochemistry for the protein OX-42, specific of activated microglia, by immunocytochemistry and western immunoblot for GFAP, the marker of reactive and mature astrocytes, and by the production of nitric oxide (NO). We observed that astrocytes exposed to 3 microg/ml TAE, F29/30 or F31/33 developed compact cell body with many processes overexpressing GFAP. Treatment with 30 microg/ml TAE and fractions, induced cytotoxicity characterized by a strong cell body contraction, very thin and long processes and condensed chromatin. We also observed that when compared with the control (+/-1.34%), the proportion of OX-42 positive cells was increased in cultures treated with 30 microg/ml TAE or F29/30, F31/33, F32 and F34/35, with values raging from 7.27% to 28.74%. Moreover, incubation with 3 microg/ml F32, 30 microg/ml TAE, F29/30, F31/33 or F34/35 induced accumulation of nitrite in culture medium indicating induction of NO production. Taken together these results show that TAE and fractionated alkaloids from P. juliflora act directly on glial cells, inducing activation and/or cytotoxicity, stimulating NO production, and may have an impact on neuronal damages observed on intoxicated animals.

Flavonoids have key functions in the regulation of multiple cellular processes; however, their effects have been poorly examined in pluripotent stem cells. Here, we tested the hypothesis that neurogenesis induced by all-trans retinoic acid (RA) is enhanced by agathisflavone (FAB, Caesalpinia pyramidalis Tull). Mouse embryonic stem (mES) cells and induced pluripotent stem (miPS) cells growing as embryoid bodies (EBs) for 4 days were treated with FAB (60 μM) and/or RA (2 μM) for additional 4 days. FAB did not interfere with the EB mitotic rate of mES cells, as evidenced by similar percentages of mitotic figures labeled by phospho-histone H3 in control (3.4% ± 0.4%) and FAB-treated groups (3.5% ± 1.1%). Nevertheless, the biflavonoid reduced cell death in both control and RA-treated EBs from mES cells by almost 2-fold compared with untreated EBs. FAB was unable, by itself, to induce neuronal differentiation in EBs after 4 days of treatment. On the other hand, FAB enhanced neuronal differentiation induced by RA in both EBs of mES and miPS. FAB increased the percentage of nestin-labeled cells by 2.7-fold (mES) and 2.4 (miPS) and β-tubulin III-positive cells by 2-fold (mES) and 2.7 (miPS) in comparison to RA-treated EBs only. FAB increased the expression of RA receptors α and β in mES EBs, suggesting that the availability of RA receptors is limiting RA-induced neurogenesis in pluripotent stem cells. This is the first report to describe that naturally occurring biflavonoids regulate apoptosis and neuronal differentiation in pluripotent stem cells.

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