Mariana S. Silveira

Altered oxygen metabolism associated to neurogenesis of induced pluripotent stem cells derived from a schizophrenic patient.

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts, perceptions, and emotions, usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however, most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells, derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient, presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS), when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia, contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening.

Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

Background Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. Methodology/Principal Findings We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Conclusion Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

Control of programmed cell death by neurotransmitters and neuropeptides in the developing mammalian retina

It has long been known that a barrage of signals from neighboring and connecting cells, as well as components of the extracellular matrix, control cell survival. Given the extensive repertoire of retinal neurotransmitters, neuromodulators and neurotrophic factors, and the exhuberant interconnectivity of retinal interneurons, it is likely that various classes of released neuroactive substances may be involved in the control of sensitivity to retinal cell death. The aim of this article is to review evidence that neurotransmitters and neuropeptides control the sensitivity to programmed cell death in the developing retina. Whereas the best understood mechanism of execution of cell death is that of caspase-mediated apoptosis, current evidence shows that not only there are many parallel pathways to apoptotic cell death, but non-apoptotic programs of execution of cell death are also available, and may be triggered either in isolation or combined with apoptosis. The experimental data show that many upstream signaling pathways can modulate cell death, including those dependent on the second messengers cAMP-PKA, calcium and nitric oxide. Evidence for anterograde neurotrophic control is provided by a variety of models of the central nervous system, and the data reviewed here indicate that an early function of certain neurotransmitters, such as glutamate and dopamine, as well as neuropeptides such as pituitary adenylyl cyclase-activating polypeptide and vasoactive intestinal peptide is the trophic support of cell populations in the developing retina. This may have implications both regarding the mechanisms of retinal organogenesis, as well as pathological conditions leading to retinal dystrophies and to dysfunctional cellular behavior.

Neuritogenesis and neuronal differentiation promoted by 2,4-dinitrophenol, a novel anti-amyloidogenic compound

Neurite outgrowth is a critical event in neuronal development, formation, and remodeling of synapses, response to injury, and regeneration. We examined the effects of 2,4‐dinitrophenol (DNP), a recently described blocker of the aggregation and neurotoxicity of the β‐amyloid peptide, on neurite elongation of central neurons. Morphometric analysis of rat embryo hippocampal and cortical neuronal cultures showed that neurite outgrowth was stimulated by DNP. This effect was accompanied by increases in the neuronal levels of the microtubule‐associated protein tau and of cyclic adenosine 3′,5′ monophosphate (cAMP). DNP also promoted cAMP accumulation, increased tau level, neurite outgrowth, and neuronal differentiation in the mouse neuroblastoma cell line N2A. We show that DNP‐induced differentiation requires activation of the extracellular signal‐regulated kinase (ERK). The finding that DNP promotes neuritogenesis and neuronal differentiation suggests that, in addition to its anti‐amyloidogenic actions, it may be a useful lead compound in the development of novel therapeutic approaches targeting neurite dystrophy and synaptic dysfunction in neurodegenerative pathologies such as Alzheimers disease. Wasilewska‐Sampaio, A. P., Silveira, M. S., Holub, O., Goecking, R., Gomes, F. C. A., Neto, V. M., Linden, R., Ferreira, S. T., De Felice, F. G. Neuritogenesis and neuronal differentiation promoted by 2,4‐dinitrophenol, a novel anti‐amyloidogenic compound. FASEB J. 19, 1627–1636 (2005)

Macrophage migration inhibitory factor is critical to interleukin-5-driven eosinophilopoiesis and tissue eosinophilia triggered by Schistosoma mansoni infection

Macrophage migration inhibitory factor (MIF) participates in the pathogenesis of inflammatory diseases, including asthma, in which it enhances airway hypersensitivity and tissue eosinophilia. Herein, we investigated the role of MIF in eosinophilopoiesis and tissue eosinophilia using Schistosoma mansoni infection. MIF‐deficient (Mif−/−) mice had similar numbers of adult worms, eggs, and granulomas compared to wildtype mice, but the size of granulomas was strikingly reduced due to smaller numbers of eosinophils. MIF did not affect the acquired response to infection, as Mif−/− mice produced normal amounts of Th2 cytokines and IgE. Nevertheless, recombinant MIF (rMIF) behaved as a chemoattractant for eosinophils, what could partially explain the reduced eosinophilia in infected Mif−/− mice. Moreover, the percentage of eosinophils was reduced in bone marrows of Mif−/− mice chronically infected with S. mansoni compared to wild type. Mif−/− had impaired eosinophilopoiesis in response to interleukin (IL)‐5 and addition of rMIF to bone marrow cultures from IL‐5 transgenic mice enhanced the generation of eosinophils. In the absence of MIF, eosinophil precursors were unable to survive the IL‐5‐supplemented cell culture, and were ingested by macrophages. Treatment with pancaspase inhibitor z‐VAD or rMIF promoted the survival of eosinophil progenitors. Together, these results indicate that MIF participates in IL‐5‐driven maturation of eosinophils and in tissue eosinophilia associated with S. mansoni infection.—Magalhaes, E. S., Paiva, C. N., Souza, H. S. P., Pyrrho, A. S., Mourao‐Sa, D., Figueiredo, R. T., Vieira‐de‐Abreu, A., Dutra, H. S., Silveira, M. S., Gaspar‐Elsas, M. I. C., Xavier‐Elsas, P., Bozza, P. T., Bozza, M. T. Macrophage migration inhibitory factor is critical to interleukin‐5‐driven eosinophilopoiesis and tissue eosinophilia triggered by Schistosoma mansoni infection. FASEB J. 23, 1262–1271 (2009)

Pituitary adenylyl cyclase-activating polypeptide controls the proliferation of retinal progenitor cells through downregulation of cyclin D1.

During retinal development, cell proliferation and exit from the cell cycle must be precisely regulated to ensure the generation of the appropriate numbers and proportions of the various retinal cell types. Previously, we showed that pituitary adenylyl cyclase‐activating polypeptide (PACAP) exerts a neuroprotective effect in the developing retina of rats, through the cAMP–cAMP‐dependent protein kinase (protein kinase A) (PKA) pathway. Here, we show that PACAP also regulates the proliferation of retinal progenitor cells. PACAP, PACAP‐specific receptor (PAC1), and the receptors activated by both PACAP and vasoactive intestinal peptide (VIP), VPAC1 and VPAC2, are expressed during embryonic and postnatal development of the rat retina. Treatment of retinal explants with PACAP38 reduced the incorporation of [3H]thymidine as well as the number of 5‐bromo‐2′‐deoxyuridine‐positive and cyclin D1‐positive cells. Pharmacological experiments indicated that PACAP triggers this antiproliferative effect through the activation of both PAC1 and VPACs, and the cAMP–PKA pathway. In addition, PACAP receptor activation decreased both cyclin D1 mRNA and protein content. Altogether, the data support the hypothesis that PACAP is a cell‐extrinsic regulator with multiple roles during retinal development, including the regulation of proliferation in a subpopulation of retinal progenitor cells.

NMDA receptor activation modulates programmed cell death during early post-natal retinal development: a BDNF-dependent mechanism

Glutamate is a classical excitotoxin of the central nervous system (CNS), but extensive work demonstrates neuroprotective roles of this neurotransmitter in developing CNS. Mechanisms of glutamate‐mediated neuroprotection are still under scrutiny. In this study, we investigated mediators of glutamate‐induced neuroprotection, and tested whether this neurotransmitter controls programmed cell death in the developing retina. The protective effect of N‐methyl‐d‐aspartate (NMDA) upon differentiating cells of retinal explants was completely blocked by a neutralizing antibody to brain‐derived neurotrophic factor (BDNF), but not by an antibody to neurotrophin‐4 (NT‐4). Consistently, chronic activation of NMDA receptor increased the expression of BDNF and trkB mRNA, as well as BDNF protein content, but did not change the content of NT‐4 mRNA in retinal tissue. Furthermore, we showed that in vivo inactivation of NMDA receptor by intraperitoneal injections of MK‐801 increased natural cell death of specific cell populations of the post‐natal retina. Our results show that chronic activation of NMDA receptors in vitro induces a BDNF‐dependent neuroprotective state in differentiating retinal cells, and that NMDA receptor activation controls programmed cell death of developing retinal neurons in vivo.

Neuroprotection by cAMP

Programmed cell death occurs in the nervous system both in normal development as well as in pathologic conditions, and is a key issue related to both brain repair and neurodegenerative diseases. Modulation of cell death in the nervous system may involve neurotrophic factors and other peptides, neurotransmitters and neuromodulators, that activate various signal transduction pathways, which in turn interact with the cell death execution machinery. Here we discuss the role of the second messenger cyclic adenosine 3′5′-monophosphate (cAMP) in cell death, and summarize current evidence that cAMP is a nodal point of neuroprotective signaling pathways.

Pleiotropic Functions of Pituitary Adenylyl Cyclase-Activating Polypeptide on Retinal Ontogenesis: Involvement of KLF4 in the Control of Progenitor Cell Proliferation

We showed previously that the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP) negatively regulates proliferation of postnatal rat retinal progenitor cells through the downregulation of cyclin D1 in a cAMP/protein kinase A dependent manner. In the present study, we describe by microarray analysis several putative PACAP targets regulated by different transcription factor families. One of these families is the Sp/Klf family of transcriptional factors capable of regulating cyclin D1, and among members, we demonstrate by immunocytochemistry that KLF4 is expressed throughout rat retinal development by retinal progenitor cells and in most differentiated cell types. Using retinal explants preparations, PACAP treatment can transiently increase Klf4 mRNA levels; from electrophoretic mobility shift assays, PACAP is also able to increase the nuclear KLF4 content. From these results, we suggest that KLF4 may be involved in the anti-proliferative effects of PACAP as one mechanism regulating progenitor cell transition from proliferation to differentiation throughout retinal development.

Pituitary adenylyl cyclase-activating polypeptide receptor re-sensitization induces plastic changes in the dopaminergic phenotype in the mature avian retina

Pituitary Adenylyl Cyclase‐Activating Polypeptide (PACAP) is a neuroactive peptide present in the avian retina where it activates adenylyl cyclase (AC) since early in development via PACAP receptors. The synthesis of cAMP in response to PACAP is observed since embryonic day 8/9 (E8/9). After E12, signaling via PACAP receptors desensitizes, reaching very low levels in the mature tissue. We show here that chronic administration of PACAP in vitro desensitizes PACAP‐induced cAMP accumulation, while the administration of the PACAP antagonist (PACAP 6‐38) re‐sensitizes PACAP receptor/cyclase system in vitro and in vivo. Moreover, a twofold increase in the number of tyrosine hydroxylase positive (TH+) cells is observed after in vivo injection of PACAP6‐38. NURR1, a transcription factor associated with the differentiation of dopaminergic cells in the CNS, is present in the chick retina in all developmental stages studied. The presence of NURR1 positive cells in the mature tissue far exceeds the number of TH+ cells, suggesting that these NURR1‐positive cells might have the potential to express the dopaminergic phenotype. Our data show that if PACAP signaling is increased in mature retinas, plastic changes in dopaminergic phenotype can be achieved.