Brf1 post-transcriptionally regulates pluripotency and differentiation responses downstream of Erk MAP Kinase

FGF/Erk MAP Kinase Signaling is a central regulator of mouse embryonic stem cell (mESC) self-renewal, pluripotency and differentiation. However, the mechanistic connection between this signaling pathway activity and the gene circuits stabilizing mESCs in vitro remain unclear. Here we show that FGF signaling post-transcriptionally regulates the mESC transcription factor network by controlling the expression of Brf1 (zfp36l1), an AU-rich element mRNA binding protein. Changes in Brf1 level disrupts the expression of core pluripotency-associated genes and attenuates mESC self-renewal without inducing differentiation. These regulatory effects are mediated by rapid and direct destabilization of Brf1 targets, such as Nanog mRNA. Interestingly, enhancing Brf1 expression does not compromise mESC pluripotency, but does preferentially regulate differentiation to mesendoderm by accelerating the expression of primitive streak markers. Together, these studies demonstrate that FGF signals utilize targeted mRNA degradation by Brf1 to enable rapid post-transcriptional control of gene expression.

Delivery of Targeted Nanoparticles Across the Blood-Brain Barrier Using a Detachable Targeting Ligand

Chronic diseases of the central nervous system are poorly treated due to the inability of most therapeutics to cross the blood-brain barrier. The blood-brain barrier is an anatomical and physiological barrier that severely restricts solute influx, including most drugs, from the blood to the brain. One promising method to overcome this obstacle is to use endogenous solute influx systems at the blood-brain barrier to transport drugs. Therapeutics designed to enter the brain through transcytosis by binding the transferrin receptor, however, are restricted within endothelial cells. The focus of this work was to develop a method to increase uptake of transferrin-containing nanoparticles into the brain by overcoming these restrictive processes.

To accomplish this goal, nanoparticles were prepared with surface transferrin molecules bound through various liable chemical bonds. These nanoparticles were designed to shed the targeting molecule during transcytosis to allow increased accumulation of nanoparticles within the brain.

Transferrin was added to the surface of nanoparticles through either redox or pH sensitive chemistry. First, nanoparticles with transferrin bound through disulfide bonds were prepared. These nanoparticles showed decreased avidity for the transferrin receptor after exposure to reducing agents and increased ability to enter the brain in vivo compared to those lacking the disulfide link.

Next, transferrin was attached through a chemical bond that cleaves at mildly acidic pH. Nanoparticles containing a cleavable link between transferrin and gold nanoparticle cores were found to both cross an in vitro model of the blood-brain barrier and accumulate within the brain in significantly higher numbers than similar nanoparticles lacking the cleavable bond. Also, this increased accumulation was not seen when using this same strategy with an antibody to transferrin receptor, indicating that behavior of nanoparticles at the blood-brain barrier varies depending on what type of targeting ligand is used.

Finally, polymeric nanoparticles loaded with dopamine and utilizing a superior acid-cleavable targeting chemistry were investigated as a potential treatment for Parkinson’s disease. These nanoparticles were capable of increasing dopamine quantities in the brains of healthy mice, highlighting the therapeutic potential of this design. Overall, this work describes a novel method to increase targeted nanoparticle accumulation in the brain.

Never-resting microglia: physiological roles in the healthy brain and pathological implications

Microglia are largely known as the major orchestrators of the brain inflammatory response. As such, they have been traditionally studied in various contexts of disease, where their activation has been assumed to induce a wide range of detrimental effects. In the last few years, a series of discoveries have challenged the current view of microglia, showing their active and positive contribution to normal brain function. This Research Topic will review the novel physiological roles of microglia in the developing, mature and aging brain, under non-pathological conditions. In particular, this Research Topic will discuss the cellular and molecular mechanisms by which microglia contribute to the formation, pruning and plasticity of synapses; the maintenance of the blood brain barrier; the regulation of adult neurogenesis and hippocampal learning; and neuronal survival, among other important roles. Because these novel findings defy our understanding of microglial function in health as much as in disease, this Research Topic will also summarize the current view of microglial nomenclature, phenotypes, origin and differentiation, sex differences, and contribution to various brain pathologies. Additionally, novel imaging approaches and molecular tools to study microglia in their non-activated state will be discussed. In conclusion, this Research Topic seeks to emphasize how the current research in neuroscience is challenged by never-resting microglia.

Cytokine pathway disruption in a mouse model of schizophrenia induced by Munc18-1a overexpression in the brain

Background: An accumulating body of evidence points to the significance of neuroinflammation and immunogenetics in schizophrenia, and an imbalance of cytokines in the central nervous system (CNS) has been suggested to be associated with the disorder. Munc18-overexpressing mice (Munc18-OE) have provided a model for the study of the alterations that may underlie the symptoms of subjects with schizophrenia. The aim of the present study was to elucidate the involvement of neuroinflammation and cytokine imbalance in this model. Methods: Cytokines were evaluated in the cortex and the striatum of Munc18-OE and wild-type (WT) mice by enzyme-linked immunosorbent assay (ELISA). Protein levels of specific microglia and macrophage, astrocytic and neuroinflammation markers were quantified by western blot in the cortex and the striatum of Munc18-OE and WT mice. Results: Each cytokine evaluated (Interferon-gamma (IFN-gamma), Tumor Necrosis Factor-alpha (TNF-alpha), Interleukin-2 (IL-2) and CCL2 chemokine) was present at higher levels in the striatum of Munc18-OE mice than WT. Cortical TNF-alpha and IL-2 levels were significantly lower in Munc18-OE mice than WT mice. The microglia and macrophage marker CD11b was lower in the cortexes of Munc18-OE mice than WT, but no differences were observed in the striatum. Glial Fibrillary Acidic Protein (GFAP) and Nuclear Factor-kappaB (NF-kappa B)p65 levels were not different between the groups. Interleukin-1beta (IL-1 beta) and IL-6 levels were beneath detection limits. Conclusions: The disrupted levels of cytokines detected in the brain of Munc18-OE mice was found to be similar to clinical reports and endorses study of this type for analysis of this aspect of the disorder. The lower CD11b expression in the cortex but not in the striatum of the Munc18-OE mice may reflect differences in physiological activity. The cytokine expression pattern observed in Munc18-OE mice is similar to a previously published model of schizophrenia caused by maternal immune activation. Together, these data suggest a possible role for an immune imbalance in this disorder.

Altered expression of Alzheimer's disease-related genes in the cerebellum of autistic patients: a model for disrupted brain connectome and therapy

Autism and Alzheimer's disease (AD) are, respectively, neurodevelopmental and degenerative diseases with an increasing epidemiological burden. The AD-associated amyloid-beta precursor protein-alpha has been shown to be elevated in severe autism, leading to the 'anabolic hypothesis' of its etiology. Here we performed a focused microarray analysis of genes belonging to NOTCH and WNT signaling cascades, as well as genes related to AD and apoptosis pathways in cerebellar samples from autistic individuals, to provide further evidence for pathological relevance of these cascades for autism. By using the limma package from R and false discovery rate, we demonstrated that 31% (116 out of 374) of the genes belonging to these pathways displayed significant changes in expression (corrected P-values <0.05), with mitochondria- related genes being the most downregulated. We also found upregulation of GRIN1, the channel-forming subunit of NMDA glutamate receptors, and MAP3K1, known activator of the JNK and ERK pathways with anti-apoptotic effect. Expression of PSEN2 (presinilin 2) and APBB1 (or F65) were significantly lower when compared with control samples. Based on these results, we propose a model of NMDA glutamate receptor-mediated ERK activation of alpha-secretase activity and mitochondrial adaptation to apoptosis that may explain the early brain overgrowth and disruption of synaptic plasticity and connectome in autism. Finally, systems pharmacology analyses of the model that integrates all these genes together (NOWADA) highlighted magnesium (Mg2+) and rapamycin as most efficient drugs to target this network model in silico. Their potential therapeutic application, in the context of autism, is therefore discussed.

Alcohol-Related Brain Damage in Humans

Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann's area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin beta II, and alpha- and beta-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in a-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in beta-spectrin protein levels, and a significant increase in transmembranous alpha 3 (catalytic) subunit of the Na+, K+-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of a-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic alpha-and beta-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics

Differential Neuroprotective Effects of 5 '-Deoxy-5 '-Methylthioadenosine

Background: 5'-deoxy-5'-methylthioadenosine (MTA) is an endogenous compound produced through the metabolism of polyamines. The therapeutic potential of MTA has been assayed mainly in liver diseases and, more recently, in animal models of multiple sclerosis. The aim of this study was to determine the neuroprotective effect of this molecule in vitro and to assess whether MTA can cross the blood brain barrier (BBB) in order to also analyze its potential neuroprotective efficacy in vivo. Methods: Neuroprotection was assessed in vitro using models of excitotoxicity in primary neurons, mixed astrocyte-neuron and primary oligodendrocyte cultures. The capacity of MTA to cross the BBB was measured in an artificial membrane assay and using an in vitro cell model. Finally, in vivo tests were performed in models of hypoxic brain damage, Parkinson's disease and epilepsy. Results: MTA displays a wide array of neuroprotective activities against different insults in vitro. While the data from the two complementary approaches adopted indicate that MTA is likely to cross the BBB, the in vivo data showed that MTA may provide therapeutic benefits in specific circumstances. Whereas MTA reduced the neuronal cell death in pilocarpine-induced status epilepticus and the size of the lesion in global but not focal ischemic brain damage, it was ineffective in preserving dopaminergic neurons of the substantia nigra in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-mice model. However, in this model of Parkinson's disease the combined administration of MTA and an A(2A) adenosine receptor antagonist did produce significant neuroprotection in this brain region. Conclusion: MTA may potentially offer therapeutic neuroprotection.

Inibição do receptor de glutamato do tipo NMDA em um modelo de hipóxia-isquemia prenatal: avaliação morfofuncional do cerebelo

Lesões sistêmicas peri e pré-natais alteram o desenvolvimento do SNC, levando a problemas cognitivos e motores em crianças que podem perdurar por toda a vida. Um tipo particular de lesão é a hipóxia-isquemia (HI), caracterizada pela interrupção momentânea ou permanente do fluxo sanguíneo. Um dos mecanismos propostos para as lesões decorrentes da HI é a excitotoxicidade glutamatérgica. O uso de inibidores da neurotransmissão glutamatérgica tem sido estudados em diversos modelos de HI. Neste trabalho, avaliamos os efeitos morfofuncionais da administração de um antagonista não-competitivo do receptor de glutamato NMDA sobre o desenvolvimento do cerebelo. Ratas no 18 dia de gestação foram anestesiadas, os cornos uterinos expostos e as 4 artérias uterinas obstruídas por 45 minutos (Grupo H). Animais controle tiveram os úteros expostos, sem a obstrução (Grupo S). Após a cirurgia a gestação prosseguiu. Somente animais nascidos a termo foram utilizados. Um dia após o nascimento, metade de cada ninhada foi designada para receber MK801, 0,3mg/kg/dia, (grupos SM e HM) e a outra metade recebeu solução salina (grupos SS e HS), por 5 dias. Após anestesia e perfusão-fixação com paraformaldeído 4% aos 9, 23, 30 e 60 dias pós-natais, cortes parassagitais do cerebelo foram obtidos em criótomo e submetidos à imunohistoquímica para calbindina, GFAP, GLAST, PDGFRα e MBP. A partir de 45 dias de vida, os animais foram testados em vários de testes comportamentais: labirinto em cruz elevado (LCE), campo vazado (CV), ROTAROD, teste de caminhada sobre barras (ladder test) e teste do comprimento da passada (stride length). Aos 9 dias, a espessura da árvore dendrítica era menor nos animais SM, HS/HM, demonstrando efeitos deletérios tanto do MK801 quanto da HI. Menor número de células PDGFRα+ foi observado nos animais HS/HM, sem efeitos da administração de MK801. Aos 23 dias, maior número de células PDGFRα+ foi observado nos animais HM comparado aos outros 3 grupos, indicando efeito neuroprotetor do MK801. Nessa idade, menor número de fibras mielinizadas (MBP+) foi observada nos animais HS, e a administração de MK801 parece reverter estes efeitos. Aos 9 dias a distribuição de GLAST estava alterada nos animais HS, com os efeitos da HI parcialmente revertidos pelo MK801. Não foram observados efeitos da HI ou do MK801 sobre comportamentos relacionados a ansiedade pelo LCE, assim como na latência de queda no ROTAROD. HI piora a performance motora no ladder test. No teste do CV, não observamos efeitos da HI sobre a busca por novidade assim como sobre a atividade locomotora espontânea. No entanto, MK801 diminui comportamentos de autolimpeza e a atividade locomotora espontânea. Menor variação das passadas foi observada em decorrência da administração de MK801 no stride length, com nenhum efeito da HI. Nossos resultados demonstram que a inibição do receptor NMDA tem um efeito neuroprotetor sobre os progenitores de oligodendrócitos e mielinização, provavelmente pela manutenção da capacidade proliferativa por um período maior. A atividade do receptor NMDA exerce importante papel na diferenciação das células de Purkinje, assim como na distribuição do transportador GLAST, corroborando a importância deste receptor na gênese das lesões causadas pela HI.

Neuroprotective effects of resveratrol and docosahexaenoic acid antioxidants in perinatal hypoxic-ischemic brain injury in rats

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Efeitos da hipóxia-isquemia perinatal sobre o comportamento motor, distribuição da Tirosina Hidroxilase na substância negra e da NADPH diaforase no hipocampo durante o desenvolvimento em ratos

A hipóxia isquemia (HI) pré-natal é uma das principais causas de mortalidade e doenças neurológicas crônicas em neonatos, que podem apresentar déficits remanentes como: retardamento, paralisia cerebral, dificuldade de aprendizado ou epilepsia. Estes prejuízos, provavelmente, estão relacionados com o atraso no desenvolvimento neural, astrogliose e com a perda de neurônios e oligodendrócitos. Déficits funcionais e cognitivos estão associados à degeneração de vias dopaminérgicas e de estruturas hipocampais. A enzima tirosina hidroxilase (TH) é a enzima limitante na síntese de dopamina e seus níveis são alterados em eventos de HI. O óxido nítrico (NO) é um gás difusível que atua modulando diferentes sistemas, participando de eventos como plasticidade sináptica e neuromodulação no sistema nervoso central e é produzido em grandes quantidades em eventos de injúria e inflamação, como é o caso da HI. O presente estudo teve por objetivos avaliar, utilizando o modelo criado por Robinson e colaboradores em 2005, os efeitos da HI sobre o comportamento motor e avaliar o desenvolvimento de estruturas encefálicas relacionadas a este comportamento como a substância negra (SN) e o complexo hipocampal. A HI foi induzida a partir do clampeamento das artérias uterinas da rata grávida, por 45 minutos no décimo oitavo dia de gestação (grupo HI). Em um grupo de fêmeas a cirurgia foi realizada, mas não houve clampeamento das artérias (grupo SHAM). A avaliação do comportamento motor foi realizada com os testes ROTAROD e de campo aberto em animais de 45 dias. Os encéfalos foram processados histologicamente nas idades de P9, P16, P23 e P90, sendo então realizada imunohistoquímica para TH e histoquímica para NADPH diaforase (NADPH-d), para avaliação do NO. Nossos resultados demonstraram redução da imunorreatividade para a TH em corpos celulares na SN aos 16 dias no grupo HI e aumento na imunorreatividade das fibras na parte reticulada aos 23 dias, com a presença de corpos celulares imunorreativos nesta região no grupo HI. Demonstramos também aumento do número de células marcadas para NADPH-d no giro dentado nos animais HI, nas idades analisadas, assim como aumento na intensidade de reação no corno de Ammon (CA1 e CA3) aos 9 dias no grupo HI, e posterior redução nesta marcação aos 23 e 90dias neste mesmo grupo. Nos testes comportamentais, observamos diminuição da atividade motora no grupo HI com uma melhora do desempenho ao longo dos testes no ROTAROD, sem entretanto atingir o mesmo nível do grupo SHAM. Os animais HI não apresentaram maior nível de ansiedade em relação ao grupo SHAM, descartando a hipótese das alterações observadas nos testes de motricidade estarem relacionadas a fatores ansiogênicos. O modelo de clampeamento das artérias uterinas da fêmea se mostrou uma ferramenta importante no estudo das alterações decorrentes do evento de HI pré-natal, por produzir diversos resultados que são similares aos ocorridos em neonatos que passam por este evento.

Pio del Rio Hortega and the discovery of the oligodendrocytes

Rio del Rio Hortega (1882-1945) discovered microglia and oligodendrocytes (OLGs), and after Ramon y Cajal, was the most prominent figure of the Spanish school of neurology. He began his scientific career with Nicolas Achucarro from whom he learned the use of metallic impregnation techniques suitable to study non-neuronal cells. Later on, he joined Cajal's laboratory. and Subsequently, he created his own group, where he continued to develop other innovative modifications of silver staining methods that revolutionized the study of glial cells a century ago. He was also interested in neuropathology and became a leading authority on Central Nervous System (CNS) tumors. In parallel to this clinical activity, del Rio Hortega rendered the first systematic description of a major polymorphism present in a subtype of macroglial cells that he named as oligodendroglia and later OLGs. He established their ectodermal origin and suggested that they built the myelin sheath of CNS axons, just as Schwann cells did in the periphery. Notably, he also suggested the trophic role of OLGs for neuronal functionality, an idea that has been substantiated in the last few years. Del Rio Hortega became internationally recognized and established an important neurohistological school with outstanding pupils from Spain and abroad, which nearly disappeared after his exile due to the Spanish civil war. Yet, the difficulty of metal impregnation methods and their variability in results, delayed for some decades the confirmation of his great insights into oligodendrocyte biology until the development of electron microscopy and immunohistochemistry. This review aims at summarizing the pioneer and essential contributions of del Rio Hortega to the current knowledge of oligodendrocyte structure and function, and to provide a hint of the scientific personality of this extraordinary and insufficiently recognized man.

A novel brain partition highlights the modular skeleton shared by structure and function

Elucidating the intricate relationship between brain structure and function, both in healthy and pathological conditions, is a key challenge for modern neuroscience. Recent progress in neuroimaging has helped advance our understanding of this important issue, with diffusion images providing information about structural connectivity (SC) and functional magnetic resonance imaging shedding light on resting state functional connectivity (rsFC). Here, we adopt a systems approach, relying on modular hierarchical clustering, to study together SC and rsFC datasets gathered independently from healthy human subjects. Our novel approach allows us to find a common skeleton shared by structure and function from which a new, optimal, brain partition can be extracted. We describe the emerging common structure-function modules (SFMs) in detail and compare them with commonly employed anatomical or functional parcellations. Our results underline the strong correspondence between brain structure and resting-state dynamics as well as the emerging coherent organization of the human brain.

Natural Variation In Recovery From A Short Period Of Anoxia Due To A cGMP-Dependent Protein Kinase in Drosophila melanogaster

[EN] Protein Kinase G (PKG) or cGMP-dependent protein kinases (PKG) have been shown to play an important role in resistance to abiotic stressors such as high temperatures or oxygen deprivation in Drosophila melanogaster. In Drosophila, the foraging gene encodes a PKG; natural variants for this gene exist, which differ in the level of expression of PKG: rovers (forR allele) which express high PKG levels, and sitters (forS allele) which express lower PKG levels. This project explores the differences in recovery from short periods of anoxia between natural variants (focusing on forS2, flies with a sitter gene in a rover background), as well as mutants with insertions in the foraging gene and RNAi recombinants that show a reduced PKG expression. The parameters measured were time to recovery and level of activity after anoxia. The results showed lower activity after anoxia in sitters than in rovers, reflecting a worse recovery from the anoxic coma in flies with lower PKG levels.

What Can the Brain Teach Us about Winemaking? An fMRI Study of Alcohol Level Preferences

Over the last few decades, wine makers have been producing wines with a higher alcohol content, assuming that they are more appreciated by consumers. To test this hypothesis, we used functional magnetic imaging to compare reactions of human subjects to different types of wine, focusing on brain regions critical for flavor processing and food reward. Participants were presented with carefully matched pairs of high- and low- alcohol content red wines, without informing them of any of the wine attributes. Contrary to expectation, significantly greater activation was found for low- alcohol than for high- alcohol content wines in brain regions that are sensitive to taste intensity, including the insula as well as the cerebellum. Wines were closely matched for all physical attributes except for alcohol content, thus we interpret the preferential response to the low- alcohol content wines as arising from top-down modulation due to the low alcohol content wines inducing greater attentional exploration of aromas and flavours. The findings raise intriguing possibilities for objectively testing hypotheses regarding methods of producing a highly complex product such as wine.