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Zinc deficiency affects the development of the central nervous system (CNS) through mechanisms only partially understood. We previously showed that zinc deficiency causes CNS oxidative stress, damaging microtubules and impairing protein nuclear shuttling. STAT1 and STAT3 transcription factors, which require nuclear import for their functions, play major roles in CNS development. Thus, we investigated whether zinc deficiency disrupts STAT1 and STAT3 signaling pathways in the developing fetal CNS, characterizing the involvement of oxidative stress and the cytoskeleton in the adverse effects. Maternal (gestation day 0-19) marginal zinc deficiency (MZD) reduced STAT1 and STAT3 tyrosine phosphorylation and their nuclear translocation in the embryonic day 19 (E19) rat brain. Similar effects were observed in zinc depleted IMR-32 neuroblastoma cells, with an associated decrease in STAT1- and STAT3-dependent gene transactivation. Zinc deficiency caused oxidative stress (increased 4-hydroxynonenal-protein adducts) in E19 brain and IMR-32 cells, which was prevented in cells by supplementation with 0.5mM α-lipoic acid (LA). In zinc depleted IMR-32 cells, the low tyrosine phosphorylation of STAT1, but not that of STAT3, recovered upon incubation with LA. STAT1 and STAT3 nuclear transports were also restored by LA. Accordingly, chemical disruption of the cytoskeleton partially reduced STAT1 and STAT3 nuclear levels. In summary, the redox-dependent tyrosine phosphorylation, and oxidant-mediated disruption of the cytoskeleton are involved in the deleterious effects of zinc deficit on STAT1 and STAT3 activation and nuclear translocation. Therefore, disruption of the STAT1 and STAT3 signaling pathways may in part explain the deleterious effects of maternal MZD on fetal brain development.

Background: Obesity and consumption of high-fat diets (HFD) are associated with intestinal permeabilization and increased paracellular transport of endotoxins, which can promote neuroinflammation. Inflammation can affect the hypothalamic pituitary adrenal (HPA) axis, which controls responses to stress and downregulates the brain-derived neurotrophic factor (BDNF), which can promote anxiety and depression, conditions frequently found in obesity. We previously showed that consumption of anthocyanins (AC) mitigate HFD-induced insulin resistance, intestinal permeability, and inflammation., Objectives: This study investigated if a dietary supplementation with a cyanidin- and delphinidin-rich extract (CDRE) could counteract HFD/obesity-induced hippocampal inflammation in mice., Methods: C57BL/6J male mice were fed for 14 wk on one of the following diets: 1) a control diet containing 10% total calories from fat (C), 2) a control diet supplemented with 40 mg AC/kg body weight (BW) (CAC), 3) a HFD containing 60% total calories from fat (lard) (HF), or 4) the HFD supplemented with 2, 20, or 40 mg AC/kg BW (HFA2, HFA20, and HFA40, respectively). In plasma and in the hippocampus, parameters of neuroinflammation and the underlying cause (endotoxemia) and consequences (alterations to the HPA and BDNF downregulation) were measured., Results: Consumption of the HFD caused endotoxemia. Accordingly, hippocampal Tlr4 mRNA levels were 110% higher in the HF group, which were both prevented by CDRE supplementation. Consumption of the HFD also caused: 1) microgliosis and increased expression of genes involved in neuroinflammation, that is, Iba-1, Nox4, Tnfα, and Il-1β, 2) alterations of HPA axis regulation, that is, with low expression of mineralocorticoid (MR) and glucocorticoid (GR) receptors; and 3) decreased Bdnf expression. Supplementation of HFD-fed mice with CDRE mitigated neuroinflammation, microgliosis, and MR and BDNF decreases., Conclusions: CDRE supplementation mitigates the negative effects associated with HFD consumption and obesity in mouse hippocampus, in part by decreasing inflammation, improving glucocorticoid metabolism, and upregulating BDNF., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Multiple sclerosis (MS) is an immune-mediated central nervous system (CNS) disease characterized by demyelination resulting from oligodendrocyte loss and inflammation. Cuprizone (CPZ) administration experimentally replicates MS pattern-III lesions, generating an inflammatory response through microgliosis and astrogliosis. Potentially remyelinating agents include oligodeoxynucleotides (ODN) with a specific immunomodulatory sequence consisting of the active motif PyNTTTTGT. In this work, the remyelinating effects of ODN IMT504 were evaluated through immunohistochemistry and qPCR analyses in a rat CPZ-induced demyelination model. Subcutaneous IMT504 administration exacerbated the pro-inflammatory response to demyelination and accelerated the transition to an anti-inflammatory state. IMT504 reduced microgliosis in general and the number of phagocytic microglia in particular and expanded the population of oligodendroglial progenitor cells (OPCs), later reflected in an increase in mature oligodendrocytes. The intracranial injection of IMT504 and intravenous inoculation of IMT504-treated B lymphocytes rendered comparable results. Altogether, these findings unveil potentially beneficial properties of IMT504 in the regulation of neuroinflammation and oligodendrogenesis, which may aid the development of therapies for demyelinating diseases such as MS., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Oligodendrocytes are responsible for myelin production in the central nervous system (CNS). Hypomyelination may slow saltatory nerve signal conduction and affect motor performance and behavior in adults. Gestational marginal zinc deficiency in rats significantly decreases proliferation of neural stem cells (NSCs) in the offspring brain., Objectives: Given that NSCs are precursors of oligodendrocytes, this study investigated if marginal zinc deficiency during early development in rats affects oligodendrogenesis in the offspring's CNS., Methods: Rat dams were fed an adequate (25 μg zinc/g diet) (C) or a marginal zinc diet (MZD) (10 μg zinc/g diet), from gestation day zero until postnatal day (P) 20, and subsequently all offspring was fed the control diet until P60. Oligodendrogenesis was evaluated in the offspring at P2, P5, P10, P20, and P60, by measuring parameters of oligodendrocyte progenitor cells (OPCs) proliferation, differentiation, maturation, and of myelination., Results: The expression of 1) proteins that regulate OPC proliferation (Shh, Sox10, Olig2); 2) OPC markers (NG2, PDGFRα); 3) myelin proteins (MBP, MAG, MOG, PLP) were lower in the brain cortex from MZD than C offspring at various stages in development. The amount of myelin after zinc replenishment continued to be low in the MZD young adult at P60. Accordingly, parameters of motor performance and behavior [grip strength, rotarod, elevated T-maze (ETM), and open-field tests] were impaired in the MZD offspring at P60., Conclusions: Results support the concept that maternal and early postnatal exposure to MZD affects oligodendrogenesis causing long-lasting effects on myelination and on motor performance in the young adult offspring., (Copyright © 2023. Published by Elsevier Inc.)

We previously observed that developmental marginal zinc deficiency affects neurogenesis. Maternal phthalate exposure could disrupt fetal zinc homeostasis by triggering an acute phase response, causing maternal liver zinc retention that limits zinc availability to the fetus. Thus, we currently investigated whether exposure to di-2-ethylhexyl phthalate (DEHP) during gestation in rats alters fetal brain neurogenesis by impairing zinc homeostasis. Dams consumed an adequate (25 μg zinc/g diet) (C) or a marginal zinc deficient (MZD) (10 μg zinc/g diet) diet, without or with DEHP (300 mg/kg BW) (C + DEHP, MZD + DEHP) from embryonic day (E) 0 to E19. To evaluate neurogenesis we measured parameters of neural progenitor cells (NPC) proliferation and differentiation. Maternal exposure to DEHP and/or zinc deficiency lowered fetal brain cortical tissue (CT) zinc concentrations. Transcription factors involved in NPC proliferation (PAX6, SOX2, EMX1), differentiation (TBR2, TBR1) and mature neurons (NeuN) were lower in MZD, MZD + DEHP and C + DEHP than in C E19 brain CT, being the lowest in the MZD + DEHP group. VGLUT1 levels, a marker of glutamatergic neurons, showed a similar pattern. Levels of a marker of GABAergic neurons, GAD65, did not vary among groups. Phosphorylated ERK1/2 levels were reduced by both MZD and DEHP, and particularly in the MZD + DEHP group. MEHP-treated human neuroblastoma IMR-32 cells and E19 brains from DEHP-treated dams showed that the zinc-regulated phosphatase PP2A can be in part responsible for DEHP-mediated ERK1/2 downregulation and impaired neurogenesis. Overall, gestational exposure to DEHP caused secondary zinc deficiency and impaired neurogenesis. These harmful effects could have long-term consequences on the adult offspring brain structure and function., (Copyright © 2022 Elsevier Inc. All rights reserved.)

We previously showed that zinc (Zn) deficiency affects the STAT3 signaling pathway in part through redox-regulated mechanisms. Given that STAT3 is central to the process of astrogliogenesis, this study investigated the consequences of maternal marginal Zn deficiency on the developmental timing and key mechanisms of STAT3 activation, and its consequences on astrogliogenesis in the offspring. This work characterized the temporal profile of cortical STAT3 activation from the mid embryonic stage up to young adulthood in the offspring from dams fed a marginal Zn deficient diet (MZD) throughout gestation and until postnatal day (P) 2. All rats were fed a Zn sufficient diet (control) from P2 until P56. Maternal zinc deficiency disrupted cortical STAT3 activation at E19 and P2. This was accompanied by altered activation of JAK2 kinase due to changes in PTP1B phosphatase activity. The underlying mechanisms mediating the adverse impact of a decreased Zn availability on STAT3 activation in the offspring brain include: (i) impaired PTP1B degradation via the ubiquitin/proteasome pathway; (ii) tubulin oxidation, associated decreased interactions with STAT3 and consequent impaired nuclear translocation; and (iii) decreased nuclear STAT3 acetylation. Zn deficiency-associated decreased STAT3 activation adversely impacted astrogliogenesis, leading to a lower astrocyte number in the early postnatal and adult brain cortex. Thus, a decreased availability of Zn during early development can have a major and irreversible adverse effect on astrogliogenesis, in part via multistep alterations in the STAT3 pathway., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Dietary proanthocyanidins (PAC) consumption is associated with a decreased risk for colorectal cancer (CRC). Dysregulation of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway is frequent in human cancers, including CRC. We previously showed that hexameric PAC (Hex) exert anti-proliferative and pro-apoptotic actions in human CRC cells. This work investigated if Hex could exert anti-CRC effects through its capacity to regulate the EGFR pathway. In proliferating Caco-2 cells, Hex acted attenuating EGF-induced EGFR dimerization and NADPH oxidase-dependent phosphorylation at Tyr 1068, decreasing EGFR location at lipid rafts, and inhibiting the downstream activation of pro-proliferative and anti-apoptotic pathways, i.e. Raf/MEK/ERK1/2 and PI3K/Akt. Hex also promoted EGFR internalization both in the absence and presence of EGF. While Hex decreased EGFR phosphorylation at Tyr 1068, it increased EGFR Tyr 1045 phosphorylation. The latter provides a docking site for the ubiquitin ligase c-Cbl and promotes EGFR degradation by lysosomes. Importantly, Hex acted synergistically with the EGFR-targeted chemotherapeutic drug Erlotinib, both in their capacity to decrease EGFR phosphorylation and inhibit cell growth. Thus, dietary PAC could exert anti-CRC actions by modulating, through both redox- and non-redox-regulated mechanisms, the EGFR pro-oncogenic signaling pathway. Additionally, Hex could also potentiate the actions of EGFR-targeted drugs., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The gastrointestinal (GI) tract can play a critical role in the development of pathologies associated with overeating, overweight and obesity. We previously observed that supplementation with anthocyanins (AC) (particularly glycosides of cyanidin and delphinidin) mitigated high fat diet (HFD)-induced development of obesity, dyslipidemia, insulin resistance and steatosis in C57BL/6J mice. This paper investigated whether these beneficial effects could be related to AC capacity to sustain intestinal monolayer integrity, prevent endotoxemia, and HFD-associated dysbiosis. The involvement of redox-related mechanisms were further investigated in Caco-2 cell monolayers. Consumption of a HFD for 14 weeks caused intestinal permeabilization and endotoxemia, which were associated with a decreased ileum expression of tight junction (TJ) proteins (occludin, ZO-1 and claudin-1), increased expression of NADPH oxidase (NOX1 and NOX4) and NOS2 and oxidative stress, and activation of redox sensitive signals (NF-κB and ERK1/2) that regulate TJ dynamics. AC supplementation mitigated all these events and increased GLP-2 levels, the intestinal hormone that upregulates TJ protein expression. AC also prevented, in vitro, tumor necrosis factor alpha-induced Caco-2 monolayer permeabilization, NOX1/4 upregulation, oxidative stress, and NF-κB and ERK activation. HFD-induced obesity in mice caused dysbiosis and affected the levels and secretion of MUC2, a mucin that participates in intestinal cell barrier protection and immune response. AC supplementation restored microbiota composition and MUC2 levels and distribution in HFD-fed mice. Thus, AC, particularly delphinidin and cyanidin, can preserve GI physiology in HFD-induced obesity in part through redox-regulated mechanisms. This can in part explain AC capacity to mitigate pathologies, i.e. insulin resistance and steatosis, associated with HFD-associated obesity., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Multiple sclerosis (MS) is an immune-mediated central nervous system disease mostly affecting young people. Multiple sclerosis and other neurodegenerative and white matter disorders involve oligodendrocyte (OL) damage and demyelination. Therefore, elucidating the signaling pathways involved in the remyelination process through the maturation of OL progenitor cells (OPCs) may contribute to the development of new therapeutic approaches. In this context, this paper further characterizes toxic cuprizone (CPZ)-induced demyelination and spontaneous remyelination in rats and investigates the role of ligand-dependent Notch signaling activation along demyelination/remyelination both in vivo and in vitro. Toxic treatment generated an inflammatory response characterized by both microgliosis and astrogliosis. Interestingly, early demyelination revealed an increase in the proportion of Jagged1+/GFAP+ cells, which correlated with an increase in Jagged1 transcript and concomitant Jagged1-driven Notch signaling activation, particularly in NG2+ OPCs, in both the corpus callosum (CC) and subventricular zone (SVZ). The onset of remyelination then exhibited an increase in the proportion of F3/contactin+/NG2+ cells, which correlated with an increase in F3/contactin transcript during ongoing remyelination in the CC. Moreover, neurosphere cultures revealed that neural progenitor cells present in the brain SVZ of CPZ-treated rats recapitulate in vitro the mechanisms underlying the response to toxic injury observed in vivo, compensating for mature OL loss. Altogether, the present results offer strong evidence of cell-type and ligand-specific Notch signaling activation and its time- and area-dependent participation in toxic demyelination and spontaneous remyelination., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

During pregnancy, a decreased availability of zinc to the fetus can disrupt the development of the central nervous system leading to defects ranging from severe malformations to subtle neurological and cognitive effects. We previously found that marginal zinc deficiency down-regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and affects neural progenitor cell (NPC) proliferation. This study investigated if marginal zinc deficiency during gestation in rats could disrupt fetal neurogenesis and affect the number and specification of neurons in the adult offspring brain cortex. Rats were fed a marginal zinc deficient or adequate diet throughout gestation and until postnatal day (P) 2, and subsequently the zinc adequate diet until P56. Neurogenesis was evaluated in the offspring at embryonic day (E)14, E19, P2, and P56 measuring parameters of NPC proliferation and differentiation by Western blot and/or immunofluorescence. At E14 and E19, major signals (i.e., ERK1/2, Sox2, and Pax6) that stimulate NPC proliferation and self-renewal were markedly downregulated in the marginal zinc deficient fetal brain. These alterations were associated to a lower number of Ki67 positive cells in the ventricular (VZs) and subventricular zones (SVZs). Following the progression of NPCs into intermediate progenitor cells (IPCs) and into neurons, Pax6, Tbr2 and Tbr1 were affected in the corresponding areas of the brain at E19 and P2. The above signaling alterations led to a lower density of neurons and a selective decrease of glutamatergic neurons in the young adult brain cortex exposed to maternal marginal zinc deficiency from E14 to P2. Current results supports the concept that marginal zinc deficiency during fetal development can disrupt neurogenesis and alter cortical structure potentially leading to irreversible neurobehavioral impairments later in life.

Increased permeability of the intestinal barrier is proposed as an underlying factor for obesity-associated pathologies. Consumption of high fat diets (HFD) is associated with increased intestinal permeabilization and increased paracellular transport of endotoxins which can promote steatosis and insulin resistance. This study investigated whether dietary (-)-epicatechin (EC) supplementation can protect the intestinal barrier against HFD-induced permeabilization and endotoxemia, and mitigate liver damage and insulin resistance. Mechanisms leading to loss of integrity and function of the tight junction (TJ) were characterized. Consumption of a HFD for 15 weeks caused obesity, steatosis, and insulin resistance in male C57BL/6J mice. This was associated with increased intestinal permeability, decreased expression of ileal TJ proteins, and endotoxemia. Supplementation with EC (2-20mg/kg body weight) mitigated all these adverse effects. EC acted modulating cell signals and the gut hormone GLP-2, which are central to the regulation of intestinal permeability. Thus, EC prevented HFD-induced ileum NOX1/NOX4 upregulation, protein oxidation, and the activation of the redox-sensitive NF-κB and ERK1/2 pathways. Supporting NADPH oxidase as a target of EC actions, in Caco-2 cells EC and apocynin inhibited tumor necrosis alpha (TNFα)-induced NOX1/NOX4 overexpression, protein oxidation and monolayer permeabilization. Together, our findings demonstrate protective effects of EC against HFD-induced increased intestinal permeability and endotoxemia. This can in part underlie EC capacity to prevent steatosis and insulin resistance occurring as a consequence of HFD consumption., (Copyright © 2017. Published by Elsevier B.V.)

Adult neural progenitor cells (NPCs) are capable of differentiating into neurons, astrocytes, and oligodendrocytes throughout life. Notch and transforming growth factor β1 (TGF-β) signaling pathways play critical roles in controlling these cell fate decisions. TGF-β has been previously shown to exert pro-neurogenic effects on hippocampal and subventricular zone (SVZ) NPCs in vitro and to interact with Notch in different cellular types. Therefore, the aim of our work was to study the effect of TGF-β on adult rat brain SVZ NPC glial commitment and its interaction with Notch signaling. Initial cell characterization revealed a large proportion of Olig2+, Nestin+, and glial fibrillary acidic protein (GFAP+) cells, a low percentage of platelet-derived growth factor receptor α (PDGFRα+) or NG2+ cells, and <1% Tuj1+ cells. Immunocytochemical analyses showed a significant increase in the percentage of PDGFRα+, NG2+, and GFAP+ cells upon four-day TGF-β treatment, which demonstrates the pro-gliogenic effect of this growth factor on adult brain SVZ NPCs. Real-time polymerase chain reaction analyses showed that TGF-β induced the expression of Notch ligand Jagged1 and downstream gene Hes1. Notch signaling inhibition in cultures treated with TGF-β produced a decrease in the proportion of PDGFRα+ cells, while TGF-β receptor II (TβRII) inhibition also rendered a decrease in the proportion of PDGFRα+ cells, concomitantly with a decrease in Jagged1 levels. These findings demonstrate the participation of Notch signaling in TGF-β effects and illustrate the impact of TGF-β on glial cell fate decisions of adult brain SVZ NPCs, as well as on oligodendroglial progenitor cell proliferation and maturation., (© 2017 Wiley Periodicals, Inc.)

Zinc deficiency affects the development of the central nervous system (CNS) through mechanisms only partially understood. We previously showed that zinc deficiency causes CNS oxidative stress, damaging microtubules and impairing protein nuclear shuttling. STAT1 and STAT3 transcription factors, which require nuclear import for their functions, play major roles in CNS development. Thus, we investigated whether zinc deficiency disrupts STAT1 and STAT3 signaling pathways in the developing fetal CNS, characterizing the involvement of oxidative stress and the cytoskeleton in the adverse effects. Maternal (gestation day 0-19) marginal zinc deficiency (MZD) reduced STAT1 and STAT3 tyrosine phosphorylation and their nuclear translocation in the embryonic day 19 (E19) rat brain. Similar effects were observed in zinc depleted IMR-32 neuroblastoma cells, with an associated decrease in STAT1- and STAT3-dependent gene transactivation. Zinc deficiency caused oxidative stress (increased 4-hydroxynonenal-protein adducts) in E19 brain and IMR-32 cells, which was prevented in cells by supplementation with 0.5mM α-lipoic acid (LA). In zinc depleted IMR-32 cells, the low tyrosine phosphorylation of STAT1, but not that of STAT3, recovered upon incubation with LA. STAT1 and STAT3 nuclear transports were also restored by LA. Accordingly, chemical disruption of the cytoskeleton partially reduced STAT1 and STAT3 nuclear levels. In summary, the redox-dependent tyrosine phosphorylation, and oxidant-mediated disruption of the cytoskeleton are involved in the deleterious effects of zinc deficit on STAT1 and STAT3 activation and nuclear translocation. Therefore, disruption of the STAT1 and STAT3 signaling pathways may in part explain the deleterious effects of maternal MZD on fetal brain development., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

This paper investigated if marginal zinc nutrition during gestation could affect fetal exposure to glucocorticoids as a consequence of a deregulation of placental 11βHSD2 expression. Placenta 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) plays a central role as a barrier protecting the fetus from the deleterious effects of excess maternal glucocorticoids. Rats were fed control (25 μg zinc per g diet) or marginal (10 μg zinc per g diet, MZD) zinc diets from day 0 through day 19 (GD19) of gestation. At GD19, corticosterone concentration in plasma, placenta, and amniotic fluid was similar in both groups. However, protein and mRNA levels of placenta 11βHSD2 were significantly higher (25% and 58%, respectively) in MZD dams than in controls. The main signaling cascades modulating 11βHSD2 expression were assessed. In MZD placentas the activation of ERK1/2 and of the downstream transcription factor Egr-1 was low, while p38 phosphorylation and SP-1-DNA binding were low compared to the controls. These results point to a central role of ERK1/Egr-1 in the regulation of 11βHSD2 expression under the conditions of limited zinc availability. In summary, results show that an increase in placenta 11βHSD2 expression occurs as a consequence of gestational marginal zinc nutrition. This seems to be due to a low tissue zinc-associated deregulation of ERK1/2 rather than to exposure to high maternal glucocorticoid exposure. The deleterious effects on brain development caused by diet-induced marginal zinc deficiency in rats do not seem to be due to fetal exposure to excess glucocorticoids.

This study investigated if a marginal zinc deficiency during gestation in rats could affect fetal neural progenitor cell (NPC) proliferation through a down-regulation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. Rats were fed a marginally zinc-deficient or adequate diet from the beginning of gestation until embryonic day (E)19. The proportion of proliferating cells in the E19 fetal ventricular zone was decreased by marginal zinc deficiency. Immunostaining for phosphorylated ERK1/2 in the cerebral cortex was decreased in the marginal zinc fetuses, and this effect was strongest in the ventricular zone. Furthermore, phosphorylation of the upstream mitogen-activated ERK kinases (MEK1/2) was not affected, suggesting that marginal zinc deficiency could have increased ERK-directed phosphatase activity. Similar findings were observed in cultured rat embryonic cortical neurons and in IMR-32 neuroblastoma cells, in which zinc-deficiency decreased ERK1/2 phosphorylation without affecting MEK1/2 phosphorylation. Indeed, zinc deficiency increased the activity of the ERK-directed phosphatase protein phosphatase 2A (PP2A) in the fetal cortex and IMR-32 cells. Inhibition of PP2A with okadaic acid prevented the decrease in ERK phosphorylation and proliferation of zinc-deficient IMR-32 cells. Together these results demonstrated that decreased zinc availability reduces ERK1/2 signaling and decreased NPC proliferation as a consequence of PP2A activation. Disruption of fetal neurogenesis could underlie irreversible neurobehavioral impairments observed after even marginal zinc nutrition during a critical period of early brain development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This work investigated the blood pressure (BP)-lowering effect of the flavanol (-)-epicatechin in a model of metabolic syndrome. Rats were fed a regular chow diet without (Control) or with 10% (w/v) fructose in the drinking water (high fructose, HF) for 8 weeks. A subgroup of the HF-fed rats was supplemented with (-)-epicatechin 20 mg/kg body weight (HF-EC). Dietary (-)-epicatechin reverted the increase in BP caused by the fructose treatment. In aorta, superoxide anion production and the expression of the NADPH oxidase (NOX) subunits p47(phox) and p22(phox) were enhanced in the HF-fed rats. The increase was prevented by (-)-epicatechin. Similar profile was observed for NOX4 expression. The activity of aorta nitric oxide synthase (NOS) was increased in the HF group and was even higher in the HF-EC rats. These effects were paralleled by increased endothelial NOS phosphorylation at the activation site Ser1177. Among the more relevant mitogen-activated protein kinase pathways in vascular tissue, c-Jun-N-terminal kinase was shown to be activated in the aorta of the HF-fed rats, and (-)-epicatechin supplementation mitigated this activation. Thus, the results suggest that dietary (-)-epicatechin supplementation prevented hypertension in HF-fed rats, decreasing superoxide anion production and elevating NOS activity, favoring an increase in NO bioavailability., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Growth-associated protein 43 (GAP-43) is a neuronal phosphoprotein associated with initial axonal outgrowth and synaptic remodeling and recent work also suggests its involvement in cell cycle control. The complex expression of GAP-43 features transcriptional and posttranscriptional components. However, in some conditions, GAP-43 gene expression is controlled primarily by the interaction of stabilizing or destabilizing RNA-binding proteins (RBPs) with adenine and uridine (AU)-rich instability elements (AREs) in its 3'UTR. Like GAP-43, many proteins involved in cell proliferation are encoded by ARE-containing mRNAs, some of which codify cell-cycle-regulating proteins including cyclin D1. Considering that GAP-43 and cyclin D1 mRNA stabilization may depend on similar RBPs, this study evaluated the participation of GAP-43 in cell cycle control and its underlying mechanisms, particularly the possible role of its 3'UTR, using GAP-43-transfected NIH-3T3 fibroblasts. Our results show an arrest in cell cycle progression in the G0/G1 phase. This arrest may be mediated by the competition of GAP-43 3'UTR with cyclin D1 3'UTR for the binding of Hu proteins such as HuR, which may lead to a decrease in cyclin D1 expression. These results might lead to therapeutic applications involving the use of sequences in the B region of GAP-43 3'UTR to slow down cell cycle progression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Demyelination is a pathological process characterized by the loss of myelin around axons. In the central nervous system, oligodendroglial damage and demyelination are common pathological features characterizing white matter and neurodegenerative disorders. Remyelination is a regenerative process by which myelin sheaths are restored to demyelinated axons, resolving functional deficits. This process is often deficient in demyelinating diseases such as multiple sclerosis (MS), and the reasons for the failure of repair mechanisms remain unclear. The characterization of these mechanisms and the factors involved in the proliferation, recruitment, and differentiation of oligodendroglial progenitor cells is key in designing strategies to improve remyelination in demyelinating disorders. First, a very dynamic combination of different molecules such as growth factors, cytokines, chemokines, and different signaling pathways is tightly regulated during the remyelination process. Second, factors unrelated to this pathology, i.e., age and genetic background, may impact disease progression either positively or negatively, and in particular, age-related remyelination failure has been proven to involve oligodendroglial cells aging and their intrinsic capacities among other factors. Third, nutrients may either help or hinder disease progression. Experimental evidence supports the anti-inflammatory role of omega-6 and omega-3 polyunsaturated fatty acids through the competitive inhibition of arachidonic acid, whose metabolites participate in inflammation, and the reduction in T cell proliferation. In turn, vitamin D intake and synthesis have been associated with lower MS incidence levels, while vitamin D-gene interactions might be involved in the pathogenesis of MS. Finally, dietary polyphenols have been reported to mitigate demyelination by modulating the immune response.

Oligodendroglial damage and demyelination are common pathological features characterizing white matter and neurodegenerative disorders. Identifying the signaling pathways involved in myelin repair through oligodendroglial progenitor maturation is essential for the development of new therapies. This article investigated the role of the Notch signaling pathway in CNS demyelination and apotransferrin-induced remyelination in a focal lysolecithin-induced demyelination model in rats. Notch was found activated in Nestin-expressing neural progenitor cells and in NG2-expressing oligodendroglial precursor cells in the subventricular zone and corpus callosum of lysolecithin-demyelinated rats. Notch activation seemed to be driven by Jagged1, which led to a high expression of downstream gene Hes5 in the subventricular zone of demyelinated rats. Apotransferrin injection induced remyelination, while the injection of the γ-secretase inhibitor reversed this effect. In addition, 24 h after apotransferrin injection, evidence showed Notch activation concomitantly with an increase in F3/contactin levels and the up-regulation of the myelin-associated glycoprotein gene in the subventricular zone and corpus callosum of demyelinated rats. Collected evidence supports the participation of both canonical and non-canonical Notch signaling pathways in demyelination/remyelination. Notch activation was found to trigger Hes5 expression as a consequence of focal demyelination, which might promote oligodendroglial precursor cell proliferation. During apotransferrin-induced remyelination, Notch activation seemed to be mediated by the expression of F3/contactin, which might induce apotransferrin-mediated oligodendroglial maturation. Evidence of the participation of Notch signaling in the demyelination/remyelination process will help further understand demyelinating disorders such as Multiple Sclerosis and the use of aTf should be taken into consideration as a possible therapeutic intervention., (© 2013 International Society for Neurochemistry.)

Epidemiological and intervention studies have shown that the intake of certain chocolates or cocoa products decreases blood pressure (BP) in humans. (-)-Epicatechin is the most abundant flavanol present in cocoa seeds and its derived foods. This work investigates the effects of dietary (-)-epicatechin on BP in rats that received N(ω)-nitro-l-arginine methyl ester (L-NAME) for 4 days. (-)-Epicatechin administration prevented the 42mm Hg increase in BP associated with the inhibition of NO production in a dose-dependent manner (0.2-4.0g/kg diet). This BP effect was associated with a reduction in L-NAME-mediated increase in the indexes of oxidative stress (plasma TBARS and GSSG/GSH(2) ratio) and with a restoration of the NO concentration. At the vascular level, none of the treatments modified NOS expression, but (-)-epicatechin administration avoided the L-NAME-mediated decrease in eNOS activity and increase in both superoxide anion production and NOX subunit p47(phox) expression. In summary, (-)-epicatechin was able to prevent the increase in BP and in oxidative stress and restored NO bioavailability. The fact that (-)-epicatechin is present in several plants usually consumed by humans gives the possibility of developing diets rich in those plants or pharmacological strategies using that flavonoid to diminish BP in hypertensive subjects., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Zinc is essential for normal brain development. Gestational severe zinc deficiency can lead to overt fetal brain malformations. Although not teratogenic, suboptimal zinc nutrition during gestation can have long-term effects on the offspring's nervous system. This article will review current knowledge on the role of zinc in modulating neurogenesis and neuronal apoptosis as well as the proposed underlying mechanisms. A decrease in neuronal zinc causes cell cycle arrest, which in part involves a deregulation of select signals (ERK1/2, p53, and NF-kappaB). Zinc deficiency also induces apoptotic neuronal death through the intrinsic (mitochondrial) pathway, which can be triggered by the activation of the zinc-regulated enzyme caspase-3, and as a consequence of abnormal regulation of prosurvival signals (ERK1/2 and NF-kappaB). Alterations in the finely tuned processes of neurogenesis, neuronal migration, differentiation, and apoptosis, which involve the developmental shaping of the nervous system, could have a long-term impact on brain health. Zinc deficiency during gestation, even at the marginal levels observed in human populations, could increase the risk for behavioral/neurological disorders in infancy, adolescence, and adulthood.

Although a requirement of zinc (Zn) for normal brain development is well documented, the extent to which Zn can modulate neuronal proliferation and apoptosis is not clear. Thus, we investigated the role of Zn in the regulation of these two critical events. A low Zn availability leads to decreased cell viability in human neuroblastoma IMR-32 cells and primary cultures of rat cortical neurons. This occurs in part as a consequence of decreased cell proliferation and increased apoptotic cell death. In IMR-32 cells, Zn deficiency led to the inhibition of cell proliferation through the arrest of the cell cycle at the G0/G1 phase. Zn deficiency induced apoptosis in both proliferating and quiescent neuronal cells via the intrinsic apoptotic pathway. Reductions in cellular Zn triggered a translocation of the pro-apoptotic protein Bad to the mitochondria, cytochrome c release, and caspase-3 activation. Apoptosis is the resultant of the inhibition of the prosurvival extracellular-signal-regulated kinase, the inhibition of nuclear factor-kappa B, and associated decreased expression of antiapoptotic proteins, and to a direct activation of caspase-3. A deficit of Zn during critical developmental periods can have persistent effects on brain function secondary to a deregulation of neuronal proliferation and apoptosis.

The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with (32)P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.

Although treatment of Hodgkin's lymphoma (HL) with a multi-drug approach has been very successful, its toxicity becomes evident after several years as secondary malignancies and cardiovascular disease. Therefore, the current goal in HL treatment is to find new therapies that specifically target the deregulated signaling cascades, such as NF-kappaB and STAT3, which cause Hodgkin and Reed-Sternberg (H-RS) cell proliferation and resistance of apoptosis. Based on the above information, we investigated the capacity of curcumin to inhibit NF-kappaB and STAT3 in H-RS cells, characterizing the functional consequences. Curcumin is incorporated into H-RS cells and acts inhibiting both NF-kappaB and STAT3 activation, leading to a decreased expression of proteins involved in cell proliferation and apoptosis, e.g. Bcl-2, Bcl-xL, cFLIP, XIAP, c-IAP1, survivin, c-myc and cyclin D1. Interestingly, curcumin caused cell cycle arrest in G2-M and a significant reduction (80-97%) in H-RS cell viability. Furthermore, curcumin triggered cell death by apoptosis, as evidenced by the activation of caspase-3 and caspase-9, changes in nuclear morphology and phosphatidylserine translocation. The above findings provide a mechanistic rationale for the potential use of curcumin as a therapeutic agent for patients with HL., ((c) 2008 Wiley-Liss, Inc.)

Due to long-term toxicity of current Hodgkin's lymphoma (HL) treatment, the present challenge is to find new therapies that specifically target deregulated signaling cascades, including NF-kappaB, which are involved in Hodgkin (H) and Reed-Sternberg (RS) cell proliferation and resistance to apoptosis. We previously presented evidence that dimeric procyanidin B2 (B2) can interact with NF-kappaB proteins inhibiting the binding of NF-kappaB to DNA. Herein, we investigated if B2, acting at a late event in NF-kappaB signaling cascade, could be effective in inhibiting NF-kappaB in H-RS cells with different mechanisms of constitutive NF-kappaB activation. B2 caused a concentration-dependent inhibition of NF-kappaB-DNA binding to a similar extent (41-48% inhibition at 25 microM B2) in all the tested H-RS cell lines (L-428, KM-H2, L-540, L-1236 and HDML-2). This was associated with the inhibition of NF-kappaB-driven gene expression, including cytokines (IL-6, TNFalpha and RANTES) and anti-apoptotic proteins (Bcl-xL, Bcl-2, XIAP and cFLIP). The finding of similar amounts of RelA and p50 proteins in the nucleus, but decreased NF-kappaB-DNA binding, even in those H-RS cells characterized by mutations in the inhibitory IkappaB proteins, supports that B2 acts by preventing the binding of NF-kappaB to DNA. B2 did not inhibit AP-1 and STAT3 constitutive activation in H-RS cells, indicating that the moderate effects of B2 on cell viability are due to the complex signaling aberrations in HL. Thus, several signaling pathways should be targeted when designing therapeutics for HL. In this regard, the capacity of B2 to inhibit NF-kappaB could be valuable in a multi-drug approach.

Twenty-one-day-old Wistar rats were fed a diet containing 0.6% cuprizone for 2 weeks. Studies carried out after withdrawal of cuprizone showed histological evidences of marked demyelination in the corpus callosum. Biochemical studies of isolated myelin showed a marked decrease in myelin proteins, phospholipids, and galactocerebrosides as well as a marked decrease in myelin yield. Treatment of these animals with a single intracranial injection of 350 ng of apotransferrin at the time of withdrawal of cuprizone induced a marked increase in myelin deposition resulting in a significantly improved remyelination, evaluated by histological, immunocytochemical, and biochemical parameters, in comparison to what was observed in spontaneous recovery. Immunocytochemical studies of cryotome sections to analyze developmental parameters of the oligodendroglial cell population at the time of termination of cuprizone and at different times thereafter showed that in the untreated animals, there was a marked increase in the number of NG2-BrdU-positive precursor cells together with a marked decrease in MBP expression at the peak of cuprizone-induced demyelination. As expected, the amount of precursor cells decreased markedly during spontaneous remyelination and was accompanied by an increase in MBP reactivity. In the apotransferrin-treated animals, these phenomena occurred much faster, and remyelination was much more efficient than in the untreated controls. The results of this study suggest that apotransferrin is a very active promyelinating agent which could be important for the treatment of certain demyelinating conditions.

Growth-associated protein-43 (GAP-43) is a phosphoprotein whose expression in neurons is related to the initial establishment and remodeling of neural connections. GAP-43 gene expression is known to be regulated at both the transcriptional and the postranscriptional levels. However, very little is known about the cellular mechanism involved in the degradation of this protein. Ubiquitin (Ub) is well known for its role in targeting cytoplasmic proteins for degradation by the 26S proteasome. The ubiquitin-proteasome system (UPS) consists of a conserved cascade of three enzymatic components that attach Ub covalently to various substrates and control the degradation of protein involved in several important cellular processes. In this study, we investigated the degradation of GAP-43 in transfected NIH 3T3 cells and neuronal cultures. We found that the proteasome inhibitors, lactacystin and MG132 increased the cellular GAP-43 level, leading to the accumulation of polyubiquitinated forms of this protein in transfected cells and that the Ub-proteasome pathway is also involved in the turnover of this protein in neurons. We conclude based on our findings that GAP-43 is a substrate of the UPS., (Copyright (c) 2005 Wiley-Liss, Inc.)

We have previously reported that restraint stress applied to the gestant mother results in long-lasting effects in the offspring that show an increase in the number of dopamine D2-type receptors in limbic areas on the adult rat brain cortex. Evidence that stress during pregnancy results in activation of the hypothalamic-pituitary-adrenal (HPA) axis has been extensively demonstrated. Therefore, high levels of corticosterone secreted in response to stress by the gestant mother might be one of the predisposing factors for the changes observed in dopamine receptors in the adult rat brain. In this study we addressed the question whether corticosterone would directly up-regulate D2-type receptors in vitro. We have investigated the effect of different concentrations of corticosterone on D4 dopamine receptor in immortalized cell lines from cerebral cortex of normal mouse fetuses, detected by immunocytochemistry employing polyclonal antibodies generated against synthetic peptides homologous to an extracellular domain of D4 receptor. The results show that corticosterone in vitro decreases the number of dopamine D4 receptors, suggesting that the increase of D2-type receptors in adult rats following prenatal stress is not related to a direct action of corticosterone on receptor expression.

2,4-Dichlorophenoxyacetic acid (2,4-D) and derivatives are herbicides widely used in Argentina and other parts of the world. Exposure to 2,4-D, its ester and salt formulations, have been associated with a range of adverse health effects in humans and different animal species, from embryotoxicity and teratogenicity to neurotoxicity. In this work, we demonstrate that after 24 hs of treatment with 1 and 2 mM 2,4-D there is an induction of apoptosis in cerebellar granule cells (CGC) in culture. However, with 2 mM 2,4-D one population of CGC developed features of apoptosis while another appeared to die by necrosis. This process is associated with an increase in caspase-3 activity after 12 hs of treatment with the herbicide, which is preceded by cytochrome c release from the mitochondria. Treatment of CGC with 2,4-D appears to induce apoptosis by a direct effect on mitochondria producing cytochrome c release and consequently activation of caspase-3, being mitochondrial damage sufficient for triggering the events that may cause apoptosis.

Mutations in the presenilin 1 (PS1) gene have been associated to familial Alzheimer disease although the exact pathogenic mechanism is unclear. We report that stable overexpression of wild type PS1 led to a decrease in cyclin-dependent kinase 4 (CDK 4) activity and retinoblastoma tumor suppressor protein (pRb) phosphorylation that correlated with decreased levels of beta-catenin and cyclin D1. PS1 mutant D385A also precipitated a similar effect suggesting that gamma-secretase cleavage is not essential for PS1-mediated CDK 4 inhibition. We postulate that PS1 overexpression may balance the hyperphosphorylation of pRb associated with death of post mitotic neurons after injury.

We analyzed the influence of presenilins on the genetic cascades that control neuronal differentiation in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduced the number of N-tubulin+ primary neurons and modulated Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increased shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduced shh expression. Both shh and presenilin promoted cell proliferation and apoptosis, but the effects of shh were widely distributed, while those resulting from presenilin injection coincided with the range of shh signaling. We suggest that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling.

We have recently shown that sustained neonatal hyperthyroidism in the rat activates apoptosis of oligodendroglial cells (OLGc) and that inhibition of the proteasome-ubiquitin (Ub) pathway by lactacystin produces increased apoptosis in cerebellar granule cells (CGC). In the present study we have analyzed the relationship between the activation of the Ub-dependent pathway, the expression of the Ub genes and programmed cell death in neurons of the rat cerebellum and cerebral cortex and in OLGc. This study was carried out in normal animals, in rats submitted to sustained neonatal hyperthyroidism and in cell cultures treated with an excess of thyroid hormones. In neurons of the cerebral cortex, thyroid hormone produces an increase of Ub-protein conjugates, an enhancement in the expression of the Ub genes and an increase in apoptosis, while the opposite results are obtained in CGC. These results indicate that in neurons, the changes in the cell death program produced by thyroid hormone run in parallel with those occurring in the Ub-dependent pathway. In OLGc, thyroid hormone increases apoptosis but does not produce changes in the Ub pathway. Preliminary studies indicate that in coincidence with what occurs in optic nerves, the sciatic nerves both in controls and in hyperthyroid animals are unable to form Ub-protein conjugates. These results indicate that in cells of the CNS such as neurons, in which the Ub-dependent pathway is actively expressed, it appears to be closely correlated with apoptosis.

The multicatalytic protease complex or proteasome is a fundamental nonlysosomal tool that the cell uses to process or degrade proteins at a fast rate through the ubiquitin and ATP-dependent proteolytic pathway. Examples of these important proteins include the tumor suppressor protein p53, various cyclins, the cyclin-dependent kinase inhibitor p27, NFkappaB, IkappaB, c-fos, and c-jun. The activation of proteolytic enzymes, including certain cystein-proteases of the ced-3/ICE (interleukin-1beta-converting enzyme) family, is a characteristic feature of the apoptotic program. However, the role of the multicatalytic protease complex in apoptosis is not well known. In order to obtain further information regarding the participation of the ubiquitin-mediated pathway in the decision of the cell to execute the cell death program, we have used a specific inhibitor of the multicatalytic protease complex, lactacystin, in cultured cerebellar granule cells. Cells were obtained from the cerebellum of 6- to 8-day-old Wistar rats and cultured in Neurobasal medium supplemented with B-27. Addition of lactacystin to the cultures induced apoptosis of the granule cells in a time-dependent fashion. The morphological changes produced by the proteasome inhibitor included nuclear condensation and DNA fragmentation measured by the diphenylamine test, as well as a positive labeling by the TUNEL (terminal deoxynucleotidyltransferase mediated-dUTP nick end labeling) assay, all of them typical features of apoptosis. Concomitant with apoptosis, there were changes in the expression of the ubiquitin mRNA, a progressive depletion in the free ubiquitin pool, and an increase in the high molecular weight ubiquitin-protein conjugates. Caspase-3, a member of the ced-3/ICE family of cystein-proteases, showed a marked increase in activity in the lactacystin-treated cells. In flow cytometry studies, the amount of cells in the S phase of the cell cycle was smaller in the lactacystin-treated cells than in controls, suggesting that apoptosis could be due, in part, to an alteration of the cell cycle., (Copyright 2000 Wiley-Liss, Inc.)

Ubiquitin (Ub) modification of different proteins plays an important role in many cellular processes. However, the best studied function of Ub is the labeling of proteins committed to rapid degradation, by an ATP-dependent pathway. We previously found that this pathway is operative in the central nervous system (CNS) of adult rats (Adamo et al. [1994] J. Neurosci. Res. 38:358-364). In the present study, we examined the changes in the capacity to form high-molecular-weight Ub protein conjugates (UbPC) and the changes in the production of 2-thiobarbituric acid-reactive substances (TBARS), in the content of protein-associated carbonyl groups (PAC), and in the activity of glutamine synthetase produced by in vitro peroxidation of the cell cytosolic proteins and of the mitochondrial fraction isolated from rat brain. Under these experimental conditions, there was an increase in PAC and TBARS in the cytosol, indicating that damage to certain cellular components had occurred. Simultaneously there was a marked increase in UbPC in comparison with the nonoxidized controls. These conjugates showed an active turnover and accumulated when Ub-mediated proteolysis was inhibited. In vitro peroxidation of the mitochondrial fractions resulted in an increase in the production of PAC and in an enhancement in the formation of UbPC. These results demonstrate that the oxidized proteins can be recognized by the ubiquitylating system and that in the CNS the Ub-dependent proteolytic pathway is one of the possible mechanisms involved in the removal of cytosolic and mitochondrial fraction damaged proteins., (Copyright 1999 Wiley-Liss, Inc.)

We have carried out a study of the effects of sustained neonatal hyperthyroidism on myelin and on the oligodendroglial cells, in an effort to obtain further insight into the molecular mechanisms underlying the action of thyroid hormones on the central nervous system (CNS). Expression of the mRNAs of myelin basic protein (MBP) myelin proteolipid protein (PLP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), transferrin, and c-Jun was investigated in 10- and 17-day-old normal and hyperthyroid rats, using Northern blot analysis. At 10 days of age, the levels of all the explored mRNAs were markedly higher in the experimental animals. The mRNA of transferrin showed a ninefold increase over control values, suggesting the possibility that this putative trophic factor might act as one of the mediators in the action of thyroid hormones. At 17 days of age on the other hand, the levels of all the mRNAs decreased markedly, reaching values below control, except for c-Jun, which remained higher than in normals. At 70 days of age, hyperthyroid rats showed clear evidence of myelin deficit, in agreement with previous results of our laboratories (Pasquini et al.: J Neurochem 57: Suppl S124, 1991). Immunocytochemistry of 70-day-old rat brain tissue sections showed a substantial reduction in the amount of MBP-reacting structures and a marked decrease in the number of oligodendroglial cells. Although the above-mentioned results could be the consequence, as proposed by Barres et al. (Development 120:1097-1108, 1994) and Baas et al. (Glia 19:324-332, 1997) of a premature arrest in oligodendroglial cell proliferation followed by early differentiation, the persistent high levels of expression of c-Jun, together with the dramatic decrease in the number of oligodendrocytes, suggested the possibility that prolonged hyperthyroidism could activate apoptotic mechanisms in the myelin forming cells. Using propidium iodide-labeled isolated oligodendroglial cells, we found, by flow cytometry, a significant increase in the number of apoptotic/hypo-diploid propidium iodide-positive cells. These results indicate that one of the actions of sustained levels of thyroid hormones in the neonate rat is to increase oligodendroglial cell death by apoptosis.

Using in situ hybridization techniques with an RNA probe coding for approximately 3.5 repeats of ubiquitin, corresponding to the polyubiquitin genes, we were able to demonstrate that under normal conditions the expression of the ubiquitin genes predominates specially in regions CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus and in Purkinje cells of the cerebellum, being less prominent in neuronal cell bodies of the cerebral cortex. When the animals were submitted to an acute oxidative stress by injection of Fe/Dextran, the hybridization signal was apparently increased in the above mentioned regions of the hippocampus and in the cerebral cortex. On the other hand, the animals chronically injected with Fe/Dextran showed a highly intense gene expression in the cerebral cortex and in the cerebellum, particularly in the granular cell layer of this structure. The hybridization signal of the transcripts was absent in the Purkinje cells. The results suggest that the expression of the ubiquitin genes by CNS neurons depends on the anatomical location of the cells and that it increases as a consequence of the oxidative stress conditions to which they are submitted.

The capacity to form ubiquitin (Ub)-protein conjugates was investigated in the cytosol of different structures of the rat central nervous system (CNS) in order to confirm the presence of this extralysosomal, adenosine triphosphate (ATP)-dependent, protein degradation system as well as its structural localization. Using 125I-Ub, we found that in the presence of ATP, the cytosol obtained from whole brains was able to form high molecular weight Ub-protein conjugates. These conjugates could be detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. The formation of these conjugates was much higher in the cerebral cortex than in the brain stem, which is mainly constituted by white matter, being intermediate in the cytosol isolated from whole brain total homogenates. These results suggested to us that under normal conditions the capacity to form Ub-protein conjugates was mainly located in structures containing neuronal cell bodies. Strong support for this contention was obtained when the cytosol isolated from rat optic nerves or from oligodendroglial cells isolated from whole brain was found to be totally unable to form Ub-protein conjugates. The inability of certain CNS structures to form conjugates with Ub could be attributed, among other reasons, to the lack of enzymes catalyzing the various steps of the Ub degradation system, to the absence of short half-life (target) proteins in those structures, or to the lack of activity of the enzymes catalyzing the reaction due to regulatory control mechanisms operating under normal conditions.

Thyroid hormones have a significant influence on the development and maturation of the central nervous system. Among their actions, T3 and T4 have effects on the differentiation of various cell types in the rat brain and cerebellum as well as on the process of myelination. Recently, several investigators have shown effects of thyroid hormones on myelin protein gene expression. Thyroid hormones seem to have a regulatory role with regard to life span. Hyperthyroid animals appear to have a shorter life and, at advanced age, show a myelin deficit. This may be due to the damage produced by the oxidative stress generated by an excess of thyroid hormones.

The effect of neonatal hyperthyroidism produced by injection of tri-iodothyronine (T3) on myelination and on the microperoxisomal population of the brain was studied in young rats. Data on the lipid composition of myelin show that myelinogenesis starts earlier in treated animals. In coincidence with the early appearance of myelin, there is an increase in the population of brain microperoxisomes, indicated by the increase in the activity of two enzymes that have been shown to be located in these organelles: catalase and acyl CoA-dihydroxyacetone phosphate acyl transferase. Double-label experiments using (1,2,3-3H) and (2-3H) glycerol to study the synthesis of glycerophospholipids through the dihydroxyacetone phosphate (DHAP) pathway give further support to the above-mentioned findings and suggest that there is an active participation of microperoxisomes in the synthesis of myelin lipids during the period of myelin formation.

Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr.

Newborn Wistar rats were made hyperthyroid by injection of tri-iodothyronine and assayed for survival, brain oxygen uptake, brain chemiluminescence and activity of antioxidant enzymes. Brain chemiluminescence was measured (1) by removing the parietal bones or (2) through the translucid parietal bones. Control animals showed a brain chemiluminescence of 130 +/- 12 c.p.s./cm2 and 99 +/- 10 c.p.s./cm2 for procedures (1) and (2) respectively. Hyperthyroid rats showed increases in the spontaneous brain photoemission of 46 and 70% compared with controls, measured by procedures 1 and 2 respectively. The hyperthyroid state did not modify the oxygen-dependent chemiluminescence of brain homogenates. The hyperthyroid animals showed a 30% increase in the oxygen uptake of brain slices and a dramatic shortening of life-span to about 16 weeks. Superoxide dismutase (the Cu-Zn enzyme), catalase and Se-dependent glutathione peroxidase activities of brain homogenates were increased by 18, 36 and 30% respectively in the hyperthyroid animals. Isolated brain mitochondria produced 0.18-0.20 nmol of H2O2/min per mg of protein in state 4 in the presence of succinate as substrate. No difference was observed between control and hyperthyroid animals. It is concluded that hyperthyroidism leads to hypermetabolism and oxidative stress in the brain. The increased levels of oxygen and peroxyl radicals may contribute to premature ageing in these animals.

Using catalase activity as a marker enzyme of microperoxisomes we determined the presence of these organelles in rat cerebral cortex (grey matter) and in brain stem (white matter) throughout development. While in grey matter the values of specific activity of catalase remained constant during all the period studied, in white matter the values increased up to 17 days of age, remained constant up to 31 days and decreased thereafter. Studies carried out in isolated oligodendroglial cells confirmed the results obtained in white matter. The results give support to the hypothesis of a possible relationship between the increase in the population of microperoxisomes and the appearance of myelin.