RESUMO
Complement component C1q is a protein complex of the innate immune system with well-characterized binding partners that constitutes part of the classical complement pathway. In addition, C1q was recently described in the central nervous system as having a role in synapse elimination both in the healthy brain and in neurodegenerative diseases. However, the molecular mechanism of C1q-associated synapse phagocytosis is still unclear. Here, we designed monomer and multimer protein constructs, which comprised the globular interaction recognition parts of mouse C1q (globular part of C1q [gC1q]) as single-chain molecules (sc-gC1q proteins) lacking the collagen-like effector region. These molecules, which can competitively inhibit the function of C1q, were expressed in an Escherichia coli expression system, and their structure and capabilities to bind known complement pathway activators were validated by mass spectrometry, analytical size-exclusion chromatography, analytical ultracentrifugation, CD spectroscopy, and ELISA. We further characterized the interactions between these molecules and immunoglobulins and neuronal pentraxins using surface plasmon resonance spectroscopy. We demonstrated that sc-gC1qs potently inhibited the function of C1q. Furthermore, these sc-gC1qs competed with C1q in binding to the embryonal neuronal cell membrane. We conclude that the application of sc-gC1qs can reveal neuronal localization and functions of C1q in assays in vivo and might serve as a basis for engineering inhibitors for therapeutic purposes.
Assuntos
Complemento C1q , Via Clássica do Complemento , Animais , Ensaio de Imunoadsorção Enzimática , CamundongosRESUMO
In synapses that show signs of local apoptosis and mitochondrial stress and undergo neuro-immunological synapse pruning, an increase in the levels of the presynaptic protein, neuronal-specific septin-3 can be observed. Septin-3 is a member of the septin GTPase family with the ability to form multimers and contribute to the cytoskeleton. However, the function of septin-3 remains elusive. Here, we provide evidence that septin-3 is capable of binding the most-studied autophagy protein Atg8 homolog microtubule-associated protein 1 light chain 3B (LC3B), besides another homolog, GABA receptor-associated protein-like 2 (GABARAPL2). Moreover, we demonstrate that colocalization of septin-3 and LC3B increases upon chemical autophagy induction in primary neuronal cells. Septin-3 is accumulated in primary neurons upon autophagy enhancement or blockade, similar to autophagy proteins. Using electron microscopy, we also show that septin-3 localizes to LC3B positive membranes and can be found at mitochondria. However, colocalization results of septin-3 and the early mitophagy marker PTEN-induced kinase 1 (PINK1) do not support that binding of septin-3 to mitochondria is mitophagy related. We conclude that septin-3 correlates with synaptic/neuronal autophagy, binds Atg8 and localizes to autophagic membranes that can be enhanced with chemical autophagy induction. Based on our results, elevated septin-3 levels might indicate enhanced or impeded autophagy in neurons.
Assuntos
Autofagossomos , Septinas , Autofagossomos/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Mitofagia , Neurônios/metabolismo , Septinas/metabolismoRESUMO
Synaptic functional disturbances with concomitant synapse loss represent central pathological hallmarks of Alzheimer's disease. Excessive accumulation of cytotoxic amyloid oligomers is widely recognized as a key event that underlies neurodegeneration. Certain complement components are crucial instruments of widespread synapse loss because they can tag synapses with functional impairments leading to their engulfment by microglia. However, an exact understanding of the affected synaptic functions that predispose to complement-mediated synapse elimination is lacking. Therefore, we conducted systematic proteomic examinations on synaptosomes prepared from an amyloidogenic mouse model of Alzheimer's disease (APP/PS1). Synaptic fractions were separated according to the presence of the C1q-tag using fluorescence-activated synaptosome sorting and subjected to proteomic comparisons. The results raised the decline of mitochondrial functions in the C1q-tagged synapses of APP/PS1 mice based on enrichment analyses, which was verified using flow cytometry. Additionally, proteomics results revealed extensive alterations in the level of septin protein family members, which are known to dynamically form highly organized pre- and postsynaptic supramolecular structures, thereby affecting synaptic transmission. High-resolution microscopy investigations demonstrated that synapses with considerable amounts of septin-3 and septin-5 show increased accumulation of C1q in APP/PS1 mice compared to the wild-type ones. Moreover, a strong positive correlation was apparent between synaptic septin-3 levels and C1q deposition as revealed via flow cytometry and confocal microscopy examinations. In sum, our results imply that deterioration of synaptic mitochondrial functions and alterations in the organization of synaptic septins are associated with complement-dependent synapse loss in Alzheimer's disease.
Assuntos
Doença de Alzheimer/genética , Amiloide/metabolismo , Proteoma/genética , Sinapses/genética , Doença de Alzheimer/patologia , Amiloide/toxicidade , Proteínas Amiloidogênicas/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Microglia/metabolismo , Microglia/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Oligopeptídeos/genética , Placa Amiloide/genética , Placa Amiloide/patologia , Septinas/genética , Sinapses/metabolismo , Sinapses/patologia , Sinaptossomos/metabolismo , Sinaptossomos/patologiaRESUMO
C1q, a member of the immune complement cascade, is implicated in the selective pruning of synapses by microglial phagocytosis. C1q-mediated synapse elimination has been shown to occur during brain development, while increased activation and complement-dependent synapse loss is observed in neurodegenerative diseases. However, the molecular mechanisms underlying C1q-controlled synaptic pruning are mostly unknown. This study addresses distortions in the synaptic proteome leading to C1q-tagged synapses. Our data demonstrated the preferential localization of C1q to the presynapse. Proteomic investigation and pathway analysis of C1q-tagged synaptosomes revealed the presence of apoptotic-like processes in C1q-tagged synapses, which was confirmed experimentally with apoptosis markers. Moreover, the induction of synaptic apoptotic-like mechanisms in a model of sensory deprivation-induced synaptic depression led to elevated C1q levels. Our results unveiled that C1q label-based synaptic pruning is triggered by and directly linked to apoptotic-like processes in the synaptic compartment.
Assuntos
Apoptose/fisiologia , Complemento C1q/metabolismo , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Idoso , Ativação do Complemento/fisiologia , Humanos , Masculino , Microglia/metabolismo , Microglia/fisiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Fagocitose/fisiologia , Proteoma/metabolismo , Proteômica/métodos , Sinapses/metabolismoRESUMO
An increasing number of studies have revealed associations between pre- and perinatal immune activation and the development of schizophrenia and autism spectrum disorders (ASDs). Accordingly, neuroimmune crosstalk has a considerably large impact on brain development during early ontogenesis. While a plethora of heterogeneous abnormalities have already been described in established maternal immune activation (MIA) rodent and primate animal models, which highly correlate to those found in human diseases, the underlying molecular background remains obscure. In the current study, we describe the long-term effects of MIA on the neocortical pre- and postsynaptic proteome of adolescent rat offspring in detail. Molecular differences were revealed in sub-synaptic fractions, which were first thoroughly characterized using independent methods. The widespread proteomic examination of cortical samples from offspring exposed to maternal lipopolysaccharide administration at embryonic day 13.5 was conducted via combinations of different gel-based proteomic techniques and tandem mass spectrometry. Our experimentally validated proteomic data revealed more pre- than postsynaptic protein level changes in the offspring. The results propose the relevance of altered synaptic vesicle recycling, cytoskeletal structure and energy metabolism in the presynaptic region in addition to alterations in vesicle trafficking, the cytoskeleton and signal transduction in the postsynaptic compartment in MIA offspring. Differing levels of the prominent signaling regulator molecule calcium/calmodulin-dependent protein kinase II in the postsynapse was validated and identified specifically in the prefrontal cortex. Finally, several potential common molecular regulators of these altered proteins, which are already known to be implicated in schizophrenia and ASD, were identified and assessed. In summary, unexpectedly widespread changes in the synaptic molecular machinery in MIA rats were demonstrated which might underlie the pathological cortical functions that are characteristic of schizophrenia and ASD.
Assuntos
Córtex Pré-Frontal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/imunologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Proteoma/metabolismo , Sinapses/metabolismo , Sinaptossomos/metabolismo , Animais , Transtorno do Espectro Autista/etiologia , Modelos Animais de Doenças , Feminino , Lipopolissacarídeos/farmacologia , Masculino , Gravidez , Proteômica/métodos , Ratos , Ratos Wistar , Esquizofrenia/etiologia , Sinapses/patologia , Sinaptossomos/patologiaRESUMO
S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. It binds to the nonmuscle myosin IIA (NMIIA) tail near the assembly competence domain (ACD) promoting filament disassembly, which could be associated with increasing metastatic potential of tumor cells. Here, we investigate the mechanism of S100A4-NMIIA interaction based on binding studies and the crystal structure of S100A4 in complex with a 45-residue-long myosin heavy chain fragment. Interestingly, we also find that S100A4 binds as strongly to a homologous heavy chain fragment of nonmuscle myosin IIC as to NMIIA. The structure of the S100A4-NMIIA complex reveals a unique mode of interaction in the S100 family: A single, predominantly α-helical myosin chain is wrapped around the Ca(2+)-bound S100A4 dimer occupying both hydrophobic binding pockets. Thermal denaturation experiments of coiled-coil forming NMIIA fragments indicate that the coiled-coil partially unwinds upon S100A4 binding. Based on these results, we propose a model for NMIIA filament disassembly: Part of the random coil tailpiece and the C-terminal residues of the coiled-coil are wrapped around an S100A4 dimer disrupting the ACD and resulting in filament dissociation. The description of the complex will facilitate the design of specific drugs that interfere with the S100A4-NMIIA interaction.
Assuntos
Miosina não Muscular Tipo IIA/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas S100/química , Sítios de Ligação , Calorimetria , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Miosina não Muscular Tipo IIA/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/genética , Proteínas S100/metabolismoRESUMO
Peripheral injection of bacterial lipopolysaccharide (LPS) facilitates 8-10Hz spike-wave discharges (SWD) characterizing absence epilepsy in WAG/Rij rats. It is unknown however, whether peripherally administered LPS is able to alter the generator areas of epileptic activity at the molecular level. We injected 1mg/kg dose of LPS intraperitoneally into WAG/Rij rats, recorded the body temperature and EEG, and examined the protein expression changes of the proteome 12h after injection in the fronto-parietal cortex and thalamus. We used fluorescent two-dimensional differential gel electrophoresis to investigate the expression profile. We found 16 differentially expressed proteins in the fronto-parietal cortex and 35 proteins in the thalamus. It is known that SWD genesis correlates with the transitional state of sleep-wake cycle thus we performed meta-analysis of the altered proteins in relation to inflammation, epilepsy as well as sleep. The analysis revealed that all categories are highly represented by the altered proteins and these protein-sets have considerable overlap. Protein network modeling suggested that the alterations in the proteome were largely induced by the immune response, which invokes the NFkB signaling pathway. The proteomics and computational analysis verified the known functional interplay between inflammation, epilepsy and sleep and highlighted proteins that are involved in their common synaptic mechanisms. Our physiological findings support the phenomenon that high dose of peripheral LPS injection increases SWD-number, modifies its duration as well as the sleep-wake stages and decreases body temperature.
Assuntos
Encéfalo/metabolismo , Epilepsia Tipo Ausência/metabolismo , Inflamação/metabolismo , Proteoma , Animais , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia Tipo Ausência/fisiopatologia , Lipopolissacarídeos/toxicidade , Proteômica , Ratos , Ratos Endogâmicos , Ratos Wistar , Transdução de SinaisRESUMO
The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 Å resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme.
Assuntos
Lectina de Ligação a Manose da Via do Complemento , Serina Proteases Associadas a Proteína de Ligação a Manose/química , Inibidores de Proteases/química , Cristalografia por Raios X , Humanos , Serina Proteases Associadas a Proteína de Ligação a Manose/antagonistas & inibidores , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes. We carried out stepwise bilateral common carotid occlusions on rats and investigated long-term mitochondrial, mitochondria-associated membrane (MAM), and cerebrospinal fluid (CSF) proteome changes. Samples were studied by gel-based and mass spectrometry-based proteomic analyses. We found 19, 35, and 12 significantly altered proteins in the mitochondria, MAM, and CSF, respectively. Most of the changed proteins were involved in protein turnover and import in all three sample types. We confirmed decreased levels of proteins involved in protein folding and amino acid catabolism, such as P4hb and Hibadh in the mitochondria by western blot. We detected reduced levels of several components of protein synthesis and degradation in the CSF as well as in the subcellular fractions, implying that hypoperfusion-induced altered protein turnover of brain tissue can be detected in the CSF by proteomic analysis.
Assuntos
Isquemia Encefálica , Proteômica , Ratos , Animais , Proteostase , Mitocôndrias/metabolismo , Encéfalo/metabolismo , Isquemia Encefálica/metabolismoRESUMO
Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2' (Leu or Asp), and P4' (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2' (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4' (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (K(D) 20 pm) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a K(D) of 110 pm and a selectivity ranging from 225- to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys(51), Arg(56), and Arg(80)) on the surface of human CTRC that interact with the P4' acidic residue of the inhibitor. The acidic preference of CTRC at P4' is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation.
Assuntos
Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Biblioteca de Peptídeos , Peptídeos/metabolismo , Peptídeos/farmacologia , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Quimotripsina/química , Quimotripsina/genética , Análise Mutacional de DNA , Evolução Molecular Direcionada , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Inibidores de Proteases/química , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoRESUMO
The complement system, an essential part of the innate immune system, can be activated through three distinct routes: the classical, the alternative, and the lectin pathways. The contribution of individual activation pathways to different biological processes can be assessed by using pathway-selective inhibitors. In this paper, we report lectin pathway-specific short peptide inhibitors developed by phage display against mannose-binding lectin-associated serine proteases (MASPs), MASP-1 and MASP-2. On the basis of the selected peptide sequences, two 14-mer peptides, designated as sunflower MASP inhibitor (SFMI)-1 and SFMI-2, were produced and characterized. SFMI-1 inhibits both MASP-1 and MASP-2 with a K(I) of 65 and 1030 nM, respectively, whereas SFMI-2 inhibits only MASP-2 with a K(I) of 180 nM. Both peptides block the lectin pathway activation completely while leaving the classical and the alternative routes intact and fully functional, demonstrating that of all complement proteases only MASP-1 and/or MASP-2 are inhibited by these peptides. In a C4 deposition inhibitor assay using preactivated MASP-2, SFMI-2 is 10-fold more effective than SFMI-1 in accordance with the fact that SFMI-2 is a more potent inhibitor of MASP-2. Surprisingly, however, out of the two peptides, SFMI-1 is much more effective in preventing C3 and C4 deposition when normal human serum containing zymogen MASPs is used. This suggests that MASP-1 has a crucial role in the initiation steps of lectin pathway activation most probably by activating MASP-2. Because the lectin pathway has been implicated in several life-threatening pathological states, these inhibitors should be considered as lead compounds toward developing lectin pathway blocking therapeutics.
Assuntos
Lectina de Ligação a Manose da Via do Complemento/fisiologia , Ativação Enzimática/fisiologia , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Ativação Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Reação em Cadeia da PolimeraseRESUMO
Alzheimer disease is characterized by accumulation of ß-amyloid (Aß) and cognitive dysfunctions linked to early loss of cholinergic neurons. As estrogen-based hormone replacement therapy has beneficial effects on cognition of demented patients, and it may prevent memory impairments, we investigated the effect of estrogen-pretreatment on Aß-induced cholinergic neurodegeneration in the nucleus basalis magnocellularis (NBM). We tested which Aß species induces the more pronounced cholinotoxic effect in vivo. We injected different Aß assemblies in the NBM of mice, and measured cholinergic cell and cortical fiber loss. Spherical Aß oligomers had the most toxic effect. Pretreatment of ovariectomized mice with estrogen before Aß injection decreased cholinergic neuron loss and partly prevented fiber degeneration. By using proteomics, we searched for proteins involved in estrogen-mediated protection and in Aß toxicity 24 h following injection. The change in expression of, e.g., DJ-1, NADH ubiquinone oxidoreductase, ATP synthase, phosphatidylethanolamine-binding protein 1, protein phosphatase 2A and dimethylarginine dimethylaminohydrolase 1 support our hypothesis that Aß induces mitochondrial dysfunction, decreases MAPK signaling, and increases NOS activation in NBM. On the other hand, altered expression of, e.g., MAP kinase kinase 1 and 2, protein phosphatase 1 and 2A by Aß might increase MAPK suppression and NOS signaling in the cortical target area. Estrogen pretreatment reversed most of the changes in the proteome in both areas. Our experiments suggest that regulation of the MAPK pathway, mitochondrial pH and NO production may all contribute to Aß toxicity, and their regulation can be prevented partly by estrogen pretreatment.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Núcleo Basal de Meynert/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Fibras Colinérgicas/efeitos dos fármacos , Estradiol/farmacologia , Fármacos Neuroprotetores/farmacologia , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/química , Animais , Núcleo Basal de Meynert/patologia , Fibras Colinérgicas/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Tamanho da Partícula , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacosRESUMO
Nucleosides are neuromodulators that have a wide range of biological roles in the brain. In order to better understand the function of nucleosides in the human central nervous system (CNS), we constructed a nucleoside map showing the concentration of various nucleosides and their metabolites using post mortem samples from 61 human brain areas and 4 spinal cord areas. We evaluated in vivo tissue levels of four nucleosides (uridine, inosine, guanosine, and adenosine) and three of their metabolites (uracil, hypoxanthine, and xanthine). The concentrations of nucleosides were unevenly distributed across different brain regions, where the highest levels were found in the cerebral cortex and basal ganglia, whereas the lowest concentrations were located in the locus coeruleus, the zona incerta, the substantia nigra, and the inferior colliculus. The regional differences in nucleoside levels in the CNS may reflect the distinct physiological functions adopted by these compounds in different brain areas.
Assuntos
Sistema Nervoso Central/metabolismo , Nucleosídeos/metabolismo , Adenosina/metabolismo , Adulto , Encéfalo/anatomia & histologia , Química Encefálica , Mapeamento Encefálico , Cromatografia Líquida de Alta Pressão , Interpretação Estatística de Dados , Feminino , Guanosina/metabolismo , Humanos , Hipoxantina/metabolismo , Inosina/metabolismo , Masculino , Pessoa de Meia-Idade , Medula Espinal/anatomia & histologia , Medula Espinal/metabolismo , Uracila/metabolismo , Uridina/metabolismo , Xantinas/metabolismoRESUMO
Elements of the immune system particularly that of innate immunity, play important roles beyond their traditional tasks in host defense, including manifold roles in the nervous system. Complement-mediated synaptic pruning is essential in the developing and healthy functioning brain and becomes aberrant in neurodegenerative disorders. C1q, component of the classical complement pathway, plays a central role in tagging synapses for elimination; however, the underlying molecular mechanisms and interaction partners are mostly unknown. Neuronal pentraxins (NPs) are involved in synapse formation and plasticity, moreover, NP1 contributes to cell death and neurodegeneration under adverse conditions. Here, we investigated the potential interaction between C1q and NPs, and its role in microglial phagocytosis of synapses in adult mice. We verified in vitro that NPs interact with C1q, as well as activate the complement system. Flow cytometry, immunostaining and co-immunoprecipitation showed that synapse-bound C1q colocalizes and interacts with NPs. High-resolution confocal microscopy revealed that microglia-surrounded C1q-tagged synapses are NP1 positive. We have also observed the synaptic occurrence of C4 suggesting that activation of the classical pathway cannot be ruled out in synaptic plasticity in healthy adult animals. In summary, our results indicate that NPs play a regulatory role in the synaptic function of C1q. Whether this role can be intensified upon pathological conditions, such as in Alzheimer's disease, is to be disclosed.
Assuntos
Proteína C-Reativa/imunologia , Complemento C1q/imunologia , Microglia/imunologia , Proteínas do Tecido Nervoso/imunologia , Fagocitose , Sinapses/imunologia , Doença de Alzheimer/imunologia , Animais , Complemento C4/imunologia , Masculino , CamundongosRESUMO
INTRODUCTION: Placental Protein 1 (PP1), PP8, and PP22 were isolated from the placenta. Herein, we aimed to identify PP1, PP8, and PP22 proteins and their placental and trophoblastic expression patterns to reveal potential involvement in pregnancy complications. METHODS: We analyzed PP1, PP8, and PP22 proteins with LC-MS. We compared the placental behaviors of PP1, PP8, and PP22 to the predominantly placenta-expressed PP5/TFPI-2. Placenta-specificity scores were generated from microarray data. Trophoblasts were isolated from healthy placentas and differentiated; total RNA was isolated and subjected to microarray analysis. We assigned the placentas to the following groups: preterm controls, early-onset preeclampsia, early-onset preeclampsia with HELLP syndrome, term controls, and late-onset preeclampsia. After histopathologic examination, placentas were used for tissue microarray construction, immunostaining with anti-PP1, anti-PP5, anti-PP8, or anti-PP22 antibodies, and immunoscoring. RESULTS: PP1, PP8, and PP22 were identified as 'nicotinate-nucleotide pyrophosphorylase', 'serpin B6', and 'protein disulfide-isomerase', respectively. Genes encoding PP1, PP8, and PP22 are not predominantly placenta-expressed, in contrast with PP5. PP1, PP8, and PP22 mRNA expression levels did not increase during trophoblast differentiation, in contrast with PP5. PP1, PP8, and PP22 immunostaining were detected primarily in trophoblasts, while PP5 expression was restricted to the syncytiotrophoblast. The PP1 immunoscore was higher in late-onset preeclampsia, while the PP5 immunoscore was higher in early-onset preeclampsia. DISCUSSION: PP1, PP8, and PP22 are expressed primarily in trophoblasts but do not have trophoblast-specific regulation or functions. The distinct dysregulation of PP1 and PP5 expression in either late-onset or early-onset preeclampsia reflects different pathophysiological pathways in these preeclampsia subsets.
Assuntos
Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Proteínas da Gravidez/metabolismo , Adulto , Cromatografia Líquida , Feminino , Humanos , Espectrometria de Massas , Gravidez , ProteômicaRESUMO
BACKGROUND: The impairment of the pontine reticular formation (PRF) has recently been revealed to be histopathologically connected with focal-cortical seizure induced generalized convulsive status epilepticus. To elucidate whether the impairment of the PRF is a general phenomenon during status epilepticus, the focal-cortical 4-aminopyridine (4-AP) application was compared with other epilepsy models. The presence of "dark" neurons in the PRF was investigated by the sensitive silver method of Gallyas in rats sacrificed at 3 h after focal 4-AP crystal or systemic 4-AP, pilocarpine, or kainic acid application. The behavioral signs of the developing epileptic seizures were scored in all rats. The EEG activity was recorded in eight rats. RESULTS: Regardless of the initiating drug or method of administration, "dark" neurons were consistently found in the PRF of animals entered the later phases of status epilepticus. EEG recordings demonstrated the presence of slow oscillations (1.5-2.5 Hz) simultaneously with the appearance of giant "dark" neurons in the PRF. CONCLUSION: We argue that the observed slow oscillation corresponds to the late periodic epileptiform discharge phase of status epilepticus, and that the PRF may be involved in the progression of status epilepticus.
Assuntos
Rede Nervosa/fisiopatologia , Neurônios/fisiologia , Formação Reticular/fisiopatologia , Estado Epiléptico/fisiopatologia , 4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Forma Celular , Eletrodos Implantados , Eletroencefalografia , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Masculino , Agonistas Muscarínicos/farmacologia , Rede Nervosa/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Pilocarpina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Formação Reticular/efeitos dos fármacos , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Processamento de Sinais Assistido por Computador , Coloração pela Prata , Estado Epiléptico/induzido quimicamenteRESUMO
In relaxed wakefulness, the EEG exhibits robust rhythms in the alpha band (8-13 Hz), which decelerate to theta (approximately 2-7 Hz) frequencies during early sleep. In animal models, these rhythms occur coherently with synchronized activity in the thalamus. However, the mechanisms of this thalamic activity are unknown. Here we show that, in slices of the lateral geniculate nucleus maintained in vitro, activation of the metabotropic glutamate receptor (mGluR) mGluR1a induces synchronized oscillations at alpha and theta frequencies that share similarities with thalamic alpha and theta rhythms recorded in vivo. These in vitro oscillations are driven by an unusual form of burst firing that is present in a subset of thalamocortical neurons and are synchronized by gap junctions. We propose that mGluR1a-induced oscillations are a potential mechanism whereby the thalamus promotes EEG alpha and theta rhythms in the intact brain.
Assuntos
Potenciais de Ação/fisiologia , Ritmo alfa/métodos , Sincronização Cortical/métodos , Corpos Geniculados/fisiologia , Ritmo Teta/métodos , Animais , GatosRESUMO
It is now widely accepted that certain types of cognitive functions are intimately related to synchronized neuronal oscillations at both low (alpha/theta) (4-7/8-13 Hz) and high (beta/gamma) (18-35/30-70 Hz) frequencies. The thalamus is a key participant in many of these oscillations, yet the cellular mechanisms by which this participation occurs are poorly understood. Here we describe how, under appropriate conditions, thalamocortical (TC) neurons from different nuclei can exhibit a wide array of largely unrecognised intrinsic oscillatory activities at a range of cognitively-relevant frequencies. For example, both metabotropic glutamate receptor (mGluR) and muscarinic Ach receptor (mAchR) activation can cause rhythmic bursting at alpha/theta frequencies. Interestingly, key differences exist between mGluR- and mAchR-induced bursting, with the former involving extensive dendritic Ca2+ electrogenesis and being mimicked by a non-specific block of K+ channels with Ba2+, whereas the latter appears to be more reliant on proximal Na+ channels and a prominent spike afterdepolarization (ADP). This likely relates to the differential somatodendritic distribution of mGluRs and mAChRs and may have important functional consequences. We also show here that in similarity to some neocortical neurons, inhibiting large-conductance Ca2+-activated K+ channels in TC neurons can lead to fast rhythmic bursting (FRB) at approximately 40 Hz. This activity also appears to rely on a Na+ channel-dependent spike ADP and may occur in vivo during natural wakefulness. Taken together, these results show that TC neurons are considerably more flexible than generally thought and strongly endorse a role for the thalamus in promoting a range of cognitively-relevant brain rhythms.
Assuntos
Potenciais de Ação/fisiologia , Relógios Biológicos/fisiologia , Córtex Cerebral/fisiologia , Neurônios/fisiologia , Tálamo/fisiologia , Animais , Humanos , Canais Iônicos/fisiologia , Vias Neurais/fisiologia , Periodicidade , Receptores de Neurotransmissores/fisiologiaRESUMO
In spite of several studies showing specific physiological functions of changes in the extracellular level of the major excitatory and inhibitory transmitters, Glu and GABA within the brain ([Glu](EXT), [GABA](EXT)) the exact origin (neuronal vs. astroglial, synaptic vs. extrasynaptic) of Glu and GABA present in dialysate samples is still a matter of debate. For better understanding the significance of in vivo microdialysis data, here we discuss methodological details and problems in addition to regulation of [Glu](EXT) and [GABA](EXT). Changes in [Glu](EXT) and [GABA](EXT) under pathological conditions such as ischemia and epilepsy are also reviewed. Based on recent in vivo microdialysis data we argue that ambient [Glu](EXT) and [GABA](EXT)may have a functional role. It is suggested that specific changes in concentrations of Glu and GABA in dialysate samples together with their alterations independent of neuronal activity indicate the involvement of Glu and GABA in the information processing of the brain as essential signaling molecules of nonsynaptic transmission as well. Since various drugs are able to interfere with extrasynaptic signals in vivo, studying the extracellular cell-to-cell communication of brain cells represents a new aspect to improve drugs modulating Gluergic as well as GABAergic neurotransmission.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácido Glutâmico/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Espaço Extracelular/metabolismo , Humanos , Microdiálise , Espectrometria de FluorescênciaRESUMO
Long-range gamma band EEG oscillations mediate information transmission between distant brain regions. Gamma band-based coupling may not be restricted to cortex-to-cortex communication but may include extracortical parts of the visual system. The retinogram and visual event-related evoked potentials exhibit time-locked, forward propagating oscillations that are candidates of gamma oscillatory coupling between the retina and the visual cortex. In this study, we tested if this gamma coupling is present as indicated by the coherence of gamma-range (70-200 Hz) oscillatory potentials (OPs) recorded simultaneously from the retina and the primary visual cortex in freely moving, adult rats. We found significant retino-cortical OP coherence in a wide range of stimulus duration (0.01-1000 msec), stimulus intensity (800-5000 mcd/mm2), interstimulus interval (10-400 msec), and stimulus frequency (0.25-25 Hz). However, at low stimulus frequencies, the OPs were time-locked, flickering light at 25 Hz entrained continuous OP coherence (steady-state response, SSR). Our results suggest that the retina and the visual cortex exhibit oscillatory coupling at high-gamma frequency with precise time locking and synchronization of information transfer from the retina to the visual cortex, similar to cortico-cortical gamma coupling. The temporal fusion of retino-cortical gamma coherence at stimulus rates of theater movies may explain the mechanism of the visual illusion of continuity. How visual perception depends on early transformations of ascending sensory information is incompletely understood. By simultaneous measurement of flash-evoked potentials in the retina and the visual cortex in awake, freely moving rats, we demonstrate for the first time that time-locked gamma oscillatory potentials exhibit stable retino-cortical synchrony across a wide range of stimulus parameters and that the temporal continuity of coherence changes with stimulus frequency according to the expected change in the visual illusion of continuity.