Bruno A. Silva

Tumor Necrosis Factor‐α Modulates Survival, Proliferation, and Neuronal Differentiation in Neonatal Subventricular Zone Cell Cultures

Tumor necrosis factor (TNF)‐α has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF‐α strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1–3 C57BL/6 mice. By using single‐cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF‐α resulted in increased differentiation of cells displaying a neuronal‐like profile of [Ca2+]i responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF‐α receptor, we found that the proneurogenic effect of 1 ng/ml TNF‐α is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein‐positive neurons was increased following exposure to mouse TNF‐α. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF‐α induced cell proliferation, whereas 10 and 100 ng/ml TNF‐α induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF‐α for 15 minutes or 6 hours caused an increase in the phospho‐stress‐activated protein kinase/c‐Jun N‐terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF‐α induces neurogenesis in neonatal SVZ cell cultures of mice. TNF‐α, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infection.

Neuropeptide Y Promotes Neurogenesis in Murine Subventricular Zone

Stem cells of the subventricular zone (SVZ) represent a reliable source of neurons for cell replacement. Neuropeptide Y (NPY) promotes neurogenesis in the hippocampal subgranular layer and the olfactory epithelium and may be useful for the stimulation of SVZ dynamic in brain repair purposes. We describe that NPY promotes SVZ neurogenesis. NPY (1 μM) treatments increased proliferation at 48 hours and neuronal differentiation at 7 days in SVZ cell cultures. NPY proneurogenic properties are mediated via the Y1 receptor. Accordingly, Y1 receptor is a major active NPY receptor in the mouse SVZ, as shown by functional autoradiography. Moreover, short exposure to NPY increased immunoreactivity for the phosphorylated form of extracellular signal‐regulated kinase 1/2 in the nucleus, compatible with a trigger for proliferation, whereas 6 hours of treatment amplified the phosphorylated form of c‐Jun‐NH2‐terminal kinase signal in growing axons, consistent with axonogenesis. NPY, as a promoter of SVZ neurogenesis, is a crucial factor for future development of cell‐based brain therapy.

Quercetin, kaempferol and biapigenin fromhypericum perforatum are neuroprotective against excitotoxic insults

In the present study we investigated the effects of phenolic compounds present inHypericum perforatum against neuronal excitotoxicity and mitochondrial dysfunction. Quercetin, kaemp-ferol and biapigenin significantly reduced neuronal death caused by 100 μM kainate plus 100 μMN-methyl-D-aspartate. The observed neuroprotection was correlated with prevention of delayed calcium deregulation and with the maintenance of mitochondrial transmembrane electric potential. The three compounds were able to reduce mitochondrial lipid peroxidation and loss of mitochondrial transmembrane electric potential caused by oxidative stress induced by ADP plus iron. Moreover, biapigenin was also able to significantly affect mitochondrial bioenergetics and decrease the capacity of mitochondria to accumulate calcium. Taken together, the results suggest that the neuroprotective action induced by quercetin and kaempferol are mainly mediated by antioxidant effects, whereas biapigenin mainly affects mitochondrial bioenergetics and calcium uptake.

Neuroprotective effect of H. perforatum extracts on beta-amyloid-induced neurotoxicity.

In the present study we assessed the neuroprotective role of aHypericum perforatum ethanolic extract and obtained fractions in amyloid-β peptide (Aβ)(25–35)-induced cell death in rat cultured hippocampal neurons. Lipid peroxidation was used as a marker of oxidative stress by following the formation of TBARS in rat cortical synaptosomes, after incubation with ascorbate/Fe2+, alone or in the presence of EC97 effective concentrations ofH. perforatum fractions. Induced lipid peroxidation was significantly inhibited by fractions containing flavonol glycosides, flavonol and biflavone aglycones, and by a fraction containing several phenols, mainly chlorogenic acid-type phenolics (21%,77%and 98%, respectively). Lipid peroxidation evaluated after incubation with 25 μM Aβ(25–35), was significantly inhibited byH. perforatum extract.Cell viability was assessed by use of the Syto-13/PI assay. The total ethanolic extract (TE) and fractions containing flavonol glycosides, flavonol and biflavone aglycones, reduced Aβ(25–35)-induced cell death (65%,58%and 59%,respectively). These results were further supported by morphological analysis of cells stained with cresyl violet. Peptide β-amyloid(25–35) induced a decrease in cell volume, chromatin condensation and nuclear fragmentation, alterations not evident in the presence of the TE and fractions containing hypericins (hypericin concentration = 11.02 μM), or fractions containing flavonoids (quercetin concentration = 21.13 μM). Dendritic lesion,an evidence of neurodegeneration, was observed by neuronal staining with cobalt following insult with Aβ(25–35), but prevented after exposure to the peptide plus the fractions referred above.The results of the present paper suggest thatH. perforatum extracts may be endowed with neuroprotective compounds able to prevent Aβ(25–35)-induced toxicity.

Neuroprotective effect ofH. perforatum extracts on β-amyloid-induced neurotoxicity

In the present study we assessed the neuroprotective role of aHypericum perforatum ethanolic extract and obtained fractions in amyloid-β peptide (Aβ)(25–35)-induced cell death in rat cultured hippocampal neurons. Lipid peroxidation was used as a marker of oxidative stress by following the formation of TBARS in rat cortical synaptosomes, after incubation with ascorbate/Fe2+, alone or in the presence of EC97 effective concentrations ofH. perforatum fractions. Induced lipid peroxidation was significantly inhibited by fractions containing flavonol glycosides, flavonol and biflavone aglycones, and by a fraction containing several phenols, mainly chlorogenic acid-type phenolics (21%,77%and 98%, respectively). Lipid peroxidation evaluated after incubation with 25 μM Aβ(25–35), was significantly inhibited byH. perforatum extract.Cell viability was assessed by use of the Syto-13/PI assay. The total ethanolic extract (TE) and fractions containing flavonol glycosides, flavonol and biflavone aglycones, reduced Aβ(25–35)-induced cell death (65%,58%and 59%,respectively). These results were further supported by morphological analysis of cells stained with cresyl violet. Peptide β-amyloid(25–35) induced a decrease in cell volume, chromatin condensation and nuclear fragmentation, alterations not evident in the presence of the TE and fractions containing hypericins (hypericin concentration = 11.02 μM), or fractions containing flavonoids (quercetin concentration = 21.13 μM). Dendritic lesion,an evidence of neurodegeneration, was observed by neuronal staining with cobalt following insult with Aβ(25–35), but prevented after exposure to the peptide plus the fractions referred above.The results of the present paper suggest thatH. perforatum extracts may be endowed with neuroprotective compounds able to prevent Aβ(25–35)-induced toxicity.

Response to histamine allows the functional identification of neuronal progenitors, neurons, astrocytes, and immature cells in subventricular zone cell cultures.

Subventricular zone (SVZ) cell cultures contain mixed populations of immature cells, neurons, astrocytes, and progenitors in different stages of development. In the present work, we examined whether cell types of the SVZ could be functionally discriminated on the basis of intracellular free calcium level ([Ca(2+)](i)) variations following KCl and histamine stimulation. For this purpose, [Ca(2+)](i) were measured in SVZ cell cultures from neonatal P1-3 C57Bl/6 donor mice, in single cells, after stimulation with 100 microM histamine or 50 mM KCl. MAP-2-positive neurons and doublecortin-positive neuroblasts were distinguished on the basis of their selective ratio of response to KCl and/or histamine stimulation. Moreover, we could distinguish immature cells on the basis of the selective response to histamine via the histamine 1 receptor activation. Exposure of SVZ cultures to the pro-neurogenic stem cell factor (SCF) induced an increase in the number of cells responding to KCl and a decrease in the number of cells responding to histamine, consistent with neuronal differentiation. The selective response to KCl/histamine in single cell calcium imaging analysis offers a rapid and efficient way for the functional discrimination of neuronal differentiation in SVZ cell cultures, opening new perspectives for the search of potential pro-neurogenic factors.

Effects of Starch/ Polycaprolactone-based Blends for Spinal Cord Injury Regeneration in Neurons/Glial Cells Viability and Proliferation

Spinal cord injury (SCI) leads to drastic alterations on the quality of life of afflicted individuals. With the advent of Tissue Engineering and Regenerative Medicine where approaches combining biomaterials, cells and growth factors are used, one can envisage novel strategies that can adequately tackle this problem. The objective of this study was to evaluate a blend of starch with poly(ε-caprolactone) (SPCL) aimed to be used for the development of scaffolds spinal cord injury (SCI) repair. SPCL linear parallel filaments were deposited on polystyrene coverslips and assays were carried out using primary cultures of hippocampal neurons and glial cells. Light and fluorescence microscopy observations revealed that both cell populations were not negatively affected by the SPCL-based biomaterial. MTS and total protein quantification indicated that both cell viability and proliferation rates were similar to controls. Both neurons and astrocytes occasionally contacted the surface of SPCL filaments through their dendrites and cytoplasmatic processes, respectively, while microglial cells were unable to do so. Using single cell [Ca2+ ]i imaging, hippocampal neurons were observed growing within the patterned channels and were functional as assessed by the response to a 30 mM KCl stimulus. The present data demonstrated that SPCL-based blends are potentially suitable for the development of scaffolds in SCI regenerative medicine.

Biapigenin Modulates the Activity of the Adenine Nucleotide Translocator in Isolated Rat Brain Mitochondria

In this study, we investigated the effects of biapigenin, a biflavone present in the extracts of Hypericum perforatum, in rat brain mitochondrial bioenergetics and calcium homeostasis. We found that biapigenin significantly decreased adenosine diphosphate (ADP)-induced membrane depolarization and increased repolarization (by 68 and 37%, respectively). These effects were blocked by atractyloside and bongkrekic acid, but not oligomycin. In the presence of biapigenin, an ADP-stimulated state 3 respiration was still noticeable, which did not happen in the presence of adenine nucleotide translocator (ANT) inhibitors. Taking in consideration the relevance of the ANT in the modulation of the mitochondrial permeability transition pore (mPTP), mitochondrial calcium homeostasis was evaluated alone or in the presence of biapigenin. We found that biapigenin reduces mitochondrial calcium retention by increasing calcium efflux, an effect that was blocked by ADP plus oligomycin, an efficient blocker of the mPTP in brain mitochondria. Taken together, the results in this article suggest that biapigenin modulates mPTP opening, possibly by modulating ANT function, contributing for enhanced mitochondrial calcium efflux, thereby reducing calcium burden and contributing for neuroprotection against excitotoxicity.

Mitochondria as Targets for Neuronal Protection Against Excitotoxicity: A Role for Phenolic Compounds?

Mitochondria are key players in the energetic metabolism, providing energy for almost every cellular process, playing also a central role in the maintenance of normal cellular function. The brain has a high energy demand; hence neurons are especially susceptible to disturbances in oxygen and nutrient availability. Such disturbances, as observed in pathologies such as ischemia/reperfusion or stroke, can represent an insult with irreversible consequences to cell viability. Mitochondrial dysfunction resulting from pathological events represents a serious threat to cellular viability. Since mitochondria are tightly related with a variety of cellular processes, the loss of mitochondrial function frequently represents a point of no return towards cell death. In this aspect, mitochondria can also play an important role in the decision of cellular fate - apoptosis versus necrosis. The search for compounds aiming at neuroprotection through the preservation of mitochondrial function might prove to be a suitable therapeutic approach for the treatment of neurodegenerative diseases. Examples of such molecules are phenolic compounds, which can be found in natural sources such as in plant extracts. Phenolics present in Hypericum perforatum are endowed with strong antioxidant and neuroprotective properties. In fact, the observed protection is suggested to be, at least in part, mediated through mitochondria-based effects, indicating a potential application for the use of such compounds or extracts in neuroprotection.