Luísa Cortes

Pollen proteases compromise the airway epithelial barrier through degradation of transmembrane adhesion proteins and lung bioactive peptides.

To cite this article: Vinhas R, Cortes L, Cardoso I, Mendes VM, Manadas B, Todo‐Bom A, Pires E, Veríssimo P. Pollen proteases compromise the airway epithelial barrier through degradation of transmembrane adhesion proteins and lung bioactive peptides. Allergy 2011; 66: 1088–1098.

Polymeric Nanoparticles to Control the Differentiation of Neural Stem Cells in the Subventricular Zone of the Brain

Herein, we report the use of retinoic acid-loaded polymeric nanoparticles as a potent tool to induce the neuronal differentiation of subventricular zone neural stem cells. The intracellular delivery of retinoic acid by the nanoparticles activated nuclear retinoic acid receptors, decreased stemness, and increased proneurogenic gene expression. Importantly, this work reports for the first time a nanoparticle formulation able to modulate in vivo the subventricular zone neurogenic niche. The work further compares the dynamics of initial stages of differentiation between SVZ cells treated with retinoic acid-loaded polymeric nanoparticles and solubilized retinoic acid. The nanoparticle formulation developed here may ultimately offer new perspectives to treat neurodegenerative diseases.

Controlling the Neuronal Differentiation of Stem Cells by the Intracellular Delivery of Retinoic Acid-Loaded Nanoparticles

The manipulation of endogenous stem cell populations from the subventricular zone (SVZ), a neurogenic niche, creates an opportunity to induce neurogenesis and influence brain regenerative capacities in the adult brain. Herein, we demonstrate the ability of polyelectrolyte nanoparticles to induce neurogenesis exclusively after being internalized by SVZ stem cells. The nanoparticles are not cytotoxic for concentrations equal or below 10 μg/mL. The internalization process is rapid, and nanoparticles escape endosomal fate in a few hours. Retinoic acid-loaded nanoparticles increase the number of neuronal nuclear protein (NeuN)-positive neurons and functional neurons responding to depolarization with KCl and expressing NMDA receptor subunit type 1 (NR1). These nanoparticles offer an opportunity for in vivo delivery of proneurogenic factors and neurodegenerative disease treatment.

Neuropeptide Y Modulation of Interleukin-1β (IL-1β)-induced Nitric Oxide Production in Microglia

Given the modulatory role of neuropeptide Y (NPY) in the immune system, we investigated the effect of NPY on the production of NO and IL-1β in microglia. Upon LPS stimulation, NPY treatment inhibited NO production as well as the expression of inducible nitric-oxide synthase (iNOS). Pharmacological studies with a selective Y1 receptor agonist and selective antagonists for Y1, Y2, and Y5 receptors demonstrated that inhibition of NO production and iNOS expression was mediated exclusively through Y1 receptor activation. Microglial cells stimulated with LPS and ATP responded with a massive release of IL-1β, as measured by ELISA. NPY inhibited this effect, suggesting that it can strongly impair the release of IL-1β. Furthermore, we observed that IL-1β stimulation induced NO production and that the use of a selective IL-1 receptor antagonist prevented NO production upon LPS stimulation. Moreover, NPY acting through Y1 receptor inhibited LPS-stimulated release of IL-1β, inhibiting NO synthesis. IL-1β activation of NF-κB was inhibited by NPY treatment, as observed by confocal microscopy and Western blotting analysis of nuclear translocation of NF-κB p65 subunit, leading to the decrease of NO synthesis. Our results showed that upon LPS challenge, microglial cells release IL-1β, promoting the production of NO through a NF-κB-dependent pathway. Also, NPY was able to strongly inhibit NO synthesis through Y1 receptor activation, which prevents IL-1β release and thus inhibits nuclear translocation of NF-κB. The role of NPY in key inflammatory events may contribute to unravel novel gateways to modulate inflammation associated with brain pathology.

Mitochondrial metabolism in Parkinson's disease impairs quality control autophagy by hampering microtubule-dependent traffic

Abnormal presence of autophagic vacuoles is evident in brains of patients with Parkinsons disease (PD), in contrast to the rare detection of autophagosomes in a normal brain. However, the actual cause and pathological significance of these observations remain unknown. Here, we demonstrate a role for mitochondrial metabolism in the regulation of the autophagy-lysosomal pathway in ex vivo and in vitro models of PD. We show that transferring mitochondria from PD patients into cells previously depleted of mitochondrial DNA is sufficient to reproduce the alterations in the autophagic system observed in PD patient brains. Although the initial steps of this pathway are not compromised, there is an increased accumulation of autophagosomes associated with a defective autophagic activity. We prove that this functional decline was originated from a deficient mobilization of autophagosomes from their site of formation toward lysosomes due to disruption in microtubule-dependent trafficking. This contributed directly to a decreased proteolytic flux of α-synuclein and other autophagic substrates. Our results lend strong support for a direct impact of mitochondria in autophagy as defective autophagic clearance ability secondary to impaired microtubule trafficking is driven by dysfunctional mitochondria. We uncover mitochondria and mitochondria-dependent intracellular traffic as main players in the regulation of autophagy in PD.

Combined effects of singlet oxygen and hydroxyl radical in photodynamic therapy with photostable bacteriochlorins: evidence from intracellular fluorescence and increased photodynamic efficacy in vitro.

Sulfonamides of halogenated bacteriochlorins bearing Cl or F substituents in the ortho positions of the phenyl rings have adequate properties for photodynamic therapy, including strong absorption in the near-infrared (λ(max) ≈ 750 nm, ε ≈ 10(5) M(-1) cm(-1)), controlled photodecomposition, large cellular uptake, intracellular localization in the endoplasmic reticulum, low cytotoxicity, and high phototoxicity against A549 and S91 cells. The roles of type I and type II photochemical processes are assessed by singlet oxygen luminescence and intracellular hydroxyl radical detection. Phototoxicity of halogenated sulfonamide bacteriochlorins does not correlate with singlet oxygen quantum yields and must be mediated both by electron transfer (superoxide ion, hydroxyl radicals) and by energy transfer (singlet oxygen). The photodynamic efficacy is enhanced when cellular death is induced by both singlet oxygen and hydroxyl radicals.

The Angiogenic Factor Angiopoietin-1 Is a Proneurogenic Peptide on Subventricular Zone Stem/Progenitor Cells

In the adult mammalian brain, the subventricular zone (SVZ) hosts stem cells constantly generating new neurons. Angiopoietin-1 (Ang-1) is an endothelial growth factor with a critical role in division, survival, and adhesion of endothelial cells via Tie-2 receptor activity. Expression of Tie-2 in nonendothelial cells, especially neurons and stem cells, suggests that Ang-1 may be involved in neurogenesis. In the present work, we investigated the putative role of Ang-1 on SVZ neurogenesis. Immature cells from SVZ-derived neurospheres express Ang-1 and Tie-2 mRNA, suggesting a role for the Ang-1/Tie-2 system in the neurogenic niche. Moreover, we also found that Tie-2 protein expression is retained on differentiation in neurons and glial cells. Ang-1 triggered proliferation via activation of the ERK1/2 (extracellular signal-regulated kinase 1/2) mitogen-activated protein kinase (MAPK) kinase pathway but did not induce cell death. Accordingly, coincubation with an anti-Tie-2 neutralizing antibody prevented the pro-proliferative effect of Ang-1. Furthermore, Ang-1 increased the number of NeuN (neuronal nuclear protein)-positive neurons in cultures treated for 7 d, as well as the number of functional neurons, as assessed by monitoring [Ca2+]i rises after application of specific stimuli for neurons and immature cells. The proneurogenic effect of Ang-1 is mediated by Tie-2 activation and subsequent mTOR (mammalian target of rapamycin kinase) mobilization. In agreement, neuronal differentiation significantly decreased after exposure to an anti-Tie-2 neutralizing antibody and to rapamycin. Moreover, Ang-1 elicited the activation of the SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) MAPK, involved in axonogenesis. Our work shows a proneurogenic effect of Ang-1, highlighting the relevance of blood vessel/stem cell cross talk in health and disease.

Neuropeptide Y inhibits interleukin-1β-induced phagocytosis by microglial cells.

BackgroundNeuropeptide Y (NPY) is emerging as a modulator of communication between the brain and the immune system. However, in spite of increasing evidence that supports a role for NPY in the modulation of microglial cell responses to inflammatory conditions, there is no consistent information regarding the action of NPY on microglial phagocytic activity, a vital component of the inflammatory response in brain injury. Taking this into consideration, we sought to assess a potential new role for NPY as a modulator of phagocytosis by microglial cells.MethodsThe N9 murine microglial cell line was used to evaluate the role of NPY in phagocytosis. For that purpose, an IgG-opsonized latex bead assay was performed in the presence of lipopolysaccharide (LPS) and an interleukin-1β (IL-1β) challenge, and upon NPY treatment. A pharmacological approach using NPY receptor agonists and antagonists followed to uncover which NPY receptor was involved. Moreover, western blotting and immunocytochemical studies were performed to evaluate expression of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27), in an inflammatory context, upon NPY treatment.ResultsHere, we show that NPY inhibits phagocytosis of opsonized latex beads and inhibits actin cytoskeleton reorganization triggered by LPS stimulation. Co-stimulation of microglia with LPS and adenosine triphosphate also resulted in increased phagocytosis, an effect inhibited by an interleukin-1 receptor antagonist, suggesting involvement of IL-1β signaling. Furthermore, direct application of LPS or IL-1β activated downstream signaling molecules, including p38 MAPK and HSP27, and these effects were inhibited by NPY. Moreover, we also observed that the inhibitory effect of NPY on phagocytosis was mediated via Y1 receptor activation.ConclusionsAltogether, we have identified a novel role for NPY in the regulation of microglial phagocytic properties, in an inflammatory context.

Neuropeptide Y inhibits interleukin-1 beta-induced microglia motility.

J. Neurochem. (2012) 120, 93–105.

Histamine Stimulates Neurogenesis in the Rodent Subventricular Zone

Neural stem/progenitor cells present in the subventricular zone (SVZ) are a potential source of repairing cells after injury. Therefore, the identification of novel players that modulate neural stem cells differentiation can have a huge impact in stem cell‐based therapies. Herein, we describe a unique role of histamine in inducing functional neuronal differentiation from cultured mouse SVZ stem/progenitor cells. This proneurogenic effect depends on histamine 1 receptor activation and involves epigenetic modifications and increased expression of Mash1, Dlx2, and Ngn1 genes. Biocompatible poly (lactic‐co‐glycolic acid) microparticles, engineered to release histamine in a controlled and prolonged manner, also triggered robust neuronal differentiation in vitro. Preconditioning with histamine‐loaded microparticles facilitated neuronal differentiation of SVZ‐GFP cells grafted in hippocampal slices and in in vivo rodent brain. We propose that neuronal commitment triggered by histamine per se or released from biomaterial‐derived vehicles may represent a new tool for brain repair strategies. STEM CELLS 2012; 30:773–784