Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells

Background Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo. Methodology/Principal Findings Using an in vivo biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination. Conclusions/Significance These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.

Distribuição da caspase-3 clivada no cerebelo de ratos durante o desenvolvimento em um modelo de hipóxia-isquemia sistêmica pré-natal

A Hipóxia-isquemia (HI) perinatal é um problema de saúde pública, e ocorrem aproximadamente 1,5 casos de encefalopatias por HI por 1000 nascidos vivos. Dos que sobrevivem 25-60% sofrem de deficiências permanentes do desenvolvimento neurológico, incluindo paralisia cerebral, convulsões, retardo mental, e dificuldade de aprender. Neurônios e oligodendrócitos, especialmente os progenitores, são os mais afetados pela HI. Existem vários modelos de HI, no entanto, poucos levam em consideração as intercorrências maternas, a importância da atividade placentária, e as trocas entre mãe-filho, que são clinicamente observadas em humanos. Robinson estabeleceu um modelo de HI sistêmica pré-natal transitório, onde o fluxo das artérias uterinas da rata grávida era obstruído por 45 minutos no décimo oitavo dia (E18) de gestação. Neste modelo foram observadas alterações que são similares às observadas em cérebros humanos que passaram por hipóxia perinatal, dentre as quais foram relatados aumento no nível de apoptose. Caspase-3 é descrita como uma enzima que atua na apoptose, e é amplamente utilizada como marcador para células apoptóticas. Vários autores vêm mostrando, entretanto, que a enzima caspase-3 pode estar ativada para fins não apoptóticos. No modelo de HI sistêmica pré-natal, foram observados astrogliose na substância branca, morte de oligodendrócitos, lesão em axônios tanto na substância branca como no córtex cerebral, e danos motores. Pouco se sabe da influencia do insulto HI no desenvolvimento do cerebelo, considerando que o cerebelo junto com o córtex motor, contribui para o controle motor. O objetivo desse trabalho foi avaliar a distribuição da caspase-3 clivada durante o desenvolvimento do cerebelo em um modelo de HI pré-natal. Os resultados deste trabalho demonstram que as células caspase-3 clivadas apresentaram duas morfologias distintas em ambos os grupos. Uma onde a caspase-3 foi observada apenas no núcleo, oscilando entre células com imunorreatividade fraca a intensa, e de células com a presença da caspase-3 no corpo celular, nos prolongamentos condensados e presença de fragmentos ao redor do soma, morfologia típica de célula em apoptose. A HI pré-natal, assim como nos hemisférios cerebrais, levou ao aumento de células caspase-3 clivadas com morfologia de progenitores de oligodendrócitos no cerebelo do grupo HI em P2, mas não em P9 e P23. Também foi demonstrado que a HI pré-natal não levou a uma ativação da apoptose em oligodendrócitos, neurônios e microglia (identificados por seus respectivos marcadores, CNPase, NeuN e ED1) apresentando marcação no núcleos de células GFAP+, na substância branca, camada granular e nas células da glia de Bergmann, em P9 e P23 no cerebelo. Podemos concluir que a HI pré-natal aumentou o número de células imunorreativas para a caspase-3 em um período crítico do desenvolvimento da oligodendroglia no cerebelo, e que a diminuição de progenitores de oligodendrócitos no cerebelo decorrente do insulto pré-natal visto em trabalhos anteriores, pode estar relacionada a morte celular por apoptose, embora não se possa descartar a hipótese da participação dessas células que apresentam caspase-3 clivada em outros eventos não apoptóticos desencadeados pela hipóxia-isquemia.

Avaliação morfofuncional do complexo hipocampal em ratos submetidos a um modelo de hipóxia-isquemia pré-natal

A diminuição do aporte de oxigênio e nutrientes na vida perinatal resulta em danos, como astrogliose, morte de neurônios e de células proliferativas. Déficits cognitivos podem estar relacionados a danos no hipocampo. Neste trabalho avaliamos a citoarquitetura do giro dentado (DG) durante o desenvolvimento e a memória de ratos submetidos à HI. Para tal, utilizamos a técnica de imunohistoquímica para marcador de proliferação celular (KI67), neurônio jovem (DCX), de astrócitos (GFAP) e de óxido nítrico sintase neuronal (NOSn). Para avaliar a memória de curta e de longa duração foi utilizado o teste de reconhecimento de objetos (RO). Ratas Wistar grávidas em E18 foram anestesiadas (tribromoetanol) e as quatro artérias uterinas foram ocluídas com grampos de aneurisma (Grupo HI). Após 45 minutos, os grampos foram removidos e foi feita a sutura por planos anatômicos. Os animais do grupo controle (SHAM) sofreram o mesmo procedimento, excetuando a oclusão das artérias. Os animais nasceram a termo. Animais com idades de 7 a 90 dias pós-natal (P7 a P90), foram anestesiados e perfundido-fixados com paraformaldeído a 4%, e os encéfalos submetidos ao processamento histológico. Cortes coronais do hipocampo (20m) foram submetidos à imunohistoquímica para KI67, DCX, GFAP e NOSn. Animais P90 foram submetidos ao RO. Os procedimentos foram aprovados pelo comitê de ética (CEA/019/2010). Observamos menor imunomarcação para KI67 no giro dentado de animais HI em P7. Para a marcação de DCX nesta idade não foi observada diferença entre os grupos. Animais HI em P15, P20 e P45 tiveram menor imunomarcação para DCX e Ki67 na camada granular. Animais P90 de ambos os grupos não apresentaram marcação para KI67 e DCX. Vimos aumento da imunomarcação para GFAP nos animais HI em todas as idades. A imunomarcação para NOSn nos animais HI foi menor em todas as idades. O maior número de células NOSn positivas foi visto em animais P7 em ambos os grupos na camada polimórfica. Em P15, animais HI apresentam células NOSn+ em todo o DG. Em P30 animais HI apresentam células NOSn+ nas camadas polimórfica e sub-granular. Animais adultos (P90) de ambos os grupos apresentam células NOSn positivas apenas nas camadas granular e sub-granular. Embora animais HI P90 não apresentaram déficits de memória, estes apresentaram menor tempo de exploração do objeto. Comportamento correspondente a déficits de atenção em humanos. Nossos resultados sugerem que HI perinatal diminui a população de células proliferativas, de neurônios jovens, de neurônios NOSn+, além de causar astrogliose e possivelmente déficits de atenção. O modelo demonstrou ser útil para a compreensão dos mecanismos celulares das lesões hipóxico-isquêmicas e pode ser usado para testar estratégias terapêuticas.

Adaptive evolution of MRGX2, a human sensory neuron specific gene involved in nociception

MRGX2, a G-protein-coupled receptor, is specifically expressed in the sensory neurons of the human peripheral nervous system and involved in nociception. Here, we studied DNA polymorphism patterns and evolution of the MRGX2 gene in world-wide human populations and the representative nonhuman primate species. Our results demonstrated that MRGX2 had undergone adaptive changes in the path of human evolution, which were likely caused by Darwinian positive selection. The patterns of DNA sequence polymorphisms in human populations showed an excess of derived substitutions, which against the expectation of neutral evolution, implying that the adaptive evolution of MRGX2 in humans was a relatively recent event. The reconstructed secondary structure of the human MRGX2 revealed that three of the four human-specific amino acid substitutions were located in the extra-cellular domains. Such critical substitutions may alter the interactions between MRGX2 protein and its ligand, thus, potentially led to adaptive changes of the pain-perception-related nervous system during human evolution. (c) 2005 Elsevier B.V. All rights reserved.

Nonparametric mixtures of factor analyzers

The mixtures of factor analyzers (MFA) model allows data to be modeled as a mixture of Gaussians with a reduced parametrization. We present the formulation of a nonparametric form of the MFA model, the Dirichlet process MFA (DPMFA). The proposed model can be used for density estimation or clustering of high dimensiona data. We utilize the DPMFA for clustering the action potentials of different neurons from extracellular recordings, a problem known as spike sorting. DPMFA model is compared to Dirichlet process mixtures of Gaussians model (DPGMM) which has a higher computational complexity. We show that DPMFA has similar modeling performance in lower dimensions when compared to DPGMM, and is able to work in higher dimensions. ©2009 IEEE.

Controlled adhesion and growth of long term glial and neuronal cultures on Parylene-c

This paper explores the long term development of networks of glia and neurons on patterns of Parylene-C on a SiO 2 substrate. We harvested glia and neurons from the Sprague-Dawley (P1-P7) rat hippocampus and utilized an established cell patterning technique in order to investigate cellular migration, over the course of 3 weeks. This work demonstrates that uncontrolled glial mitosis gradually disrupts cellular patterns that are established early during culture. This effect is not attributed to a loss of protein from the Parylene-C surface, as nitrogen levels on the substrate remain stable over 3 weeks. The inclusion of the anti-mitotic cytarabine (Ara-C) in the culture medium moderates glial division and thus, adequately preserves initial glial and neuronal conformity to underlying patterns. Neuronal apoptosis, often associated with the use of Ara-C, is mitigated by the addition of brain derived neurotrophic factor (BDNF). We believe that with the right combination of glial inhibitors and neuronal promoters, the Parylene-C based cell patterning method can generate structured, active neural networks that can be sustained and investigated over extended periods of time. To our knowledge this is the first report on the concurrent application of Ara-C and BDNF on patterned cell cultures. © 2011 Delivopoulos, Murray.

Composition and evolution of the V2r vorneronasal receptor gene repertoire in mice and rats

Pheromones are chemicals produced and detected by conspecifics to elicit social/sexual physiological and behavioral responses, and they are perceived primarily by the vomeronasal organ (VNO) in terrestrial vertebrates. Two large superfamilies of G protein-coupled receptors, V1rs and V2rs, have been identified as pheromone receptors in vomeronasal sensory neurons. Based on a computational analysis of the mouse and rat genome sequences, we report the first global draft of the V2r gene repertoire, composed of similar to 200 genes and pseudogenes. Rodent V2rs are subject to rapid gene births/deaths and accelerated amino acid substitutions, likely reflecting the species-specific nature of pheromones. Vertebrate V2rs appear to have originated twice prior to the emergence of the VNO in ancestral tetrapods, explaining seemingly inconsistent observations among different V2rs. The identification of the entire V2r repertoire opens the door to genomic-level studies of the structure, function, and evolution of this diverse group of sensory receptors. (c) 2005 Elsevier Inc. All rights reserved.

In vitro construction of a recombinant human embryonic brain-derived neurotrophin-4 gene and pEGFP-N1 vector

BACKGROUND: Neurotrophin-4 (NT-4) can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its ac

Effects of parylene-C photooxidation on serum-assisted glial and neuronal patterning.

The increasing use of patterned neural networks in multielectrode arrays and similar devices drives the constant development and evaluation of new biomaterials. Recently, we presented a promising technique to guide neurons and glia reliably and effectively. Parylene-C, a common hydrophobic polymer, was photolithographically patterned on silicon oxide (SiO(2)) and subsequently activated via immersion in serum. In this article, we explore the effects of ultraviolet (UV)-induced oxidation on parylene's ability to pattern neurons and glia. We exposed parylene-C stripe patterns to increasing levels of UV radiation and found a dose-dependent reduction in the total mass of patterned cells, as well as a gradual loss of glial and neuronal conformity to the patterns. In contrast, nonirradiated patterns had superior patterning results and increased presence of cells. The reduced cell adhesion and patterning after the formation of aldehyde and carboxyl groups on UV-radiated parylene-C supports our hypothesis that cell adhesion and growth on parylene is facilitated by hydrophobic adsorption of serum proteins. We conclude that unlike other cell patterning schemes, our technique does not rely on photooxidation of the polymer. Nonetheless, the precise control of oxygenated groups on parylene could pave the way for the differential binding of proteins and other molecules on the surface, aiding in the adhesion of alternative cell types. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Transplantable neural progenitor populations derived from Rhesus monkey embryonic stem cells

Cell-based therapies using embryonic stem cells (ESCs) in the treatment of neural disease will require the generation of homogenous donor neural progenitor (NP) populations. Here we describe an efficient culture system containing hepatocyte growth factor (HGF) and G5 supplement for the production of highly enriched (88.3% +/- 8.1%)populations of NPs from rhesus monkey ESCs. Additional purification resulted in NP preparations that were 98% nestin positive. Moreover, NPs, as monolayers or neurospheres, could be maintained for prolonged periods of time in media containing HGF+G5 or G5 alone. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes, and oligodendrocytes. The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, approximately 25% of transplanted NPs survived and 65% - 80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. Subcloning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro and in vivo in rat brains. These observations set the stage for obtaining highly enriched NPs and evaluating the efficacy of NP-based transplantation therapy in the nonhuman primate and will provide a platform for probing the molecular mechanisms that control neural induction.

异丙酚对大鼠海马CAl区神经元兴奋性突触传递的影响

目的研究异丙酚对大鼠海马CA1区神经元兴奋性突触后电流(EPSC)和自发性兴奋性突触后电流(sEPSC)的影响。方法 Wistar大鼠断头后分离海马脑组织,制成400μm厚度的海马脑片,脑片随机分为5组(n=10)。脂肪乳剂Ⅰ组、异丙酚Ⅰ组、SR95531+异丙酚组:记录EPSC 10 min (基础值)后分别加入10％脂肪乳剂90μl、1％异丙酚90μl(相当于100μmol／L)、10μmol／L SR95531+100 μmol／L异丙酚,继续记录EPSC 40 min,分析EPSC幅值的变化。脂肪乳剂Ⅱ组、异丙酚Ⅱ组:细胞破膜后稳定10-15 min,分别加入10％脂肪乳剂90μl和1％异丙酚90μl,记录sEPSC 40 min,分析sEPSC频率、幅值和半衰期的变化。膜钳制电压均为-70 mV。结果与基础值比较,给药后脂肪乳剂Ⅰ组和 SR95531+异丙酚组EPSC幅值差异无统计学意义,异丙酚Ⅰ组EPSC幅值降低;给药后异丙酚Ⅰ组 EPSC幅值比脂肪乳剂Ⅰ组降低(P<0．05)。与脂肪乳剂Ⅱ组比较,异丙酚Ⅱ组sEPSC的频率、幅值降低、半衰期缩短(P<0．05)。结论异丙酚主要通过增强大鼠海马CA...

丙泊酚对大鼠海马CA1 区兴奋性突触传递的影响

　目的　观察500μmol/ L 丙泊酚对大鼠海马CA1 区电刺激诱发的兴奋性突触后电流 ( EPSC) 的影响,分析丙泊酚的可能作用机制。方法　断头法分离Wistar 大鼠(13～19 d) 海马半脑, 用切片机切出400μm 厚度的海马脑片,全细胞膜片钳技术记录CA1 区锥体神经元EPSC。实验分 两组:脂肪乳剂组( n = 6) 和丙泊酚组( n = 10) 。先以50μmol/ L 印防己毒素预孵脑片30 min 后,记录 基础EPSC 10 min ,然后加入450μl 脂肪乳剂或丙泊酚(相当于500 μmol/ L ) , 继续记录EPSC 40 min ;继而以配对刺激代替单刺激,观察EPSC2/ EPSC1 比率的变化;改变膜钳制电压( - 80～ + 60 mV) ,观察电流2电压( I2V) 曲线的变化。结果　脂肪乳剂对EPSC 无影响,500μmol/ L 丙泊酚降低 大鼠海马CA1 区EPSC 值,25～30 min 左右达最大抑制效果,EPSC 幅值下降至基础值的6715 % ,明 显低于脂肪乳剂组( P < 0105) ;而且500μmol/ L 丙泊酚明显降低EPSC2/ EPSC1 比率,也使I2V 曲线 左移,降低反转电位至- 35 mV 左右。结论　500μmol/ L 丙泊酚对大鼠海马CA1 区兴奋性突触传 递产生抑制作用,这可能与其增强突触前膜、突触后膜GABAA 受体活性有关。

异丙酚对大鼠海马区突触传递可塑性的影响

目的研究异丙酚对海马区突触传递和可塑性的影响。方法断头分离大 鼠海马半脑, 制备加阿厚度海马脑片。张脑片分为六组。脂肪乳剂组和异丙酚组的脑片以印防 己毒素预孵而, 然后加人川脂肪乳剂或异丙酚相当于拌, 观察对兴奋性突触后电流 的影响。月旨肪乳剂长时程增强】」下组、脂肪乳剂长时程抑制组、异丙酚功下组、异丙酚 组的脑片以川脂肪乳剂或异丙酚相当于脚预孵而, 给予高频刺激或低频 刺激, 记录或的发生情况。结果脂肪乳剂对无影响脚异 丙酚使细胞下降至基础值的尸, 使细胞玲上升至基础值的 。脂肪乳剂组给予邓后玲值为基础值的, 脂肪乳剂汀〕组给 予⋯乃后值为基础值的异丙酚组给予后, 可以产生但不能维 持, 后值为基础值的, 异丙酚几组给予后值为基础值的 , 明显低于脂肪乳剂组尸。结论异丙酚对大鼠海马区突触传递 具有双重影响, 出现抑制和兴奋两种效果异丙酚损害大鼠海马区锥体神经元的维持而易 化。 【关键

丙泊酚对大鼠海马CA1 区长时程抑制的影响

　目的　观察丙泊酚对大鼠海马CA1 区锥体神经元产生的长时程抑制(L TD) 的影响,并 分析其可能机制。方法　断头法分离wistar 大鼠(13～19 d) 海马半脑,用切片机切出400μm 厚度的 海马脑片。实验分三组:脂肪乳剂组( I 组) ,丙泊酚组(P 组) ,SR95531 + 丙泊酚组( GP 组) 。I 组和P 组以90μL 脂肪乳剂或丙泊酚(相当于100μmol/ L) 预孵脑片60 min ,然后给予低频刺激(L FS) ,记录 L TD 的表达情况; GP 组先在循环液中加入10μmol/ L SR95531 预孵脑片30 min ,再加入100μmol/ L 丙泊酚继续孵育60 min ,继而给予L FS ,记录L TD 的表达情况。结果　I 组给予L FS 后,产生L TD , L FS 后10～40 min 的兴奋性突触后电流( EPSC) 值为基础值的57185 %;P 组给予L FS 后10～40 min 的EPSC 值为基础值的40182 % ,明显低于I 组( P < 0105) ; GP 组给予L FS 后10～40 min 的EPSC 值为基础值的56151 % ,与I 组比较差异无显著意义( P > 0105) ,与P 组比较差异有显著意义( P < 0105) 。结论　100μmol/ L 丙泊酚使大鼠海马CA1 区锥体神经元L TD 表达增强,这种作用与其增强 GABAA 受体功能有关;当阻断GABAA 受体后,这种易化作用消失。

Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca2+ permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASICla-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca2+ elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.