RESUMO
Age-related macular degeneration (AMD) is a leading cause of visual loss. It has a strong genetic basis, and common haplotypes on chromosome (Chr) 1 (CFH Y402H variant) and on Chr10 (near HTRA1/ARMS2) contribute the most risk. Little is known about the early molecular and cellular processes in AMD, and we hypothesized that analyzing submacular tissue from older donors with genetic risk but without clinical features of AMD would provide biological insights. Therefore, we used mass spectrometrybased quantitative proteomics to compare the proteins in human submacular stromal tissue punches from donors who were homozygous for high-risk alleles at either Chr1 or Chr10 with those from donors who had protective haplotypes at these loci, all without clinical features of AMD. Additional comparisons were made with tissue from donors who were homozygous for high-risk Chr1 alleles and had early AMD. The Chr1 and Chr10 risk groups shared common changes compared with the low-risk group, particularly increased levels of mast cellspecific proteases, including tryptase, chymase, and carboxypeptidase A3. Histological analyses of submacular tissue from donors with genetic risk of AMD but without clinical features of AMD and from donors with Chr1 risk and AMD demonstrated increased mast cells, particularly the tryptase-positive/chymase-negative cells variety, along with increased levels of denatured collagen compared with tissue from lowgenetic risk donors. We conclude that increased mast cell infiltration of the inner choroid, degranulation, and subsequent extracellular matrix remodeling are early events in AMD pathogenesis and represent a unifying mechanistic link between Chr1- and Chr10-mediated AMD.
Assuntos
Cromossomos Humanos Par 10 , Cromossomos Humanos Par 1 , Degeneração Macular , Mastócitos , Peptídeo Hidrolases , Alelos , Corioide/enzimologia , Corioide/patologia , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 10/genética , Humanos , Degeneração Macular/genética , Degeneração Macular/patologia , Mastócitos/patologia , Peptídeo Hidrolases/genética , Proteômica , Risco , Triptases/metabolismoRESUMO
Amyotrophic lateral sclerosis is a fatal neurodegenerative disease causing upper and lower motor neuron loss and currently no effective disease-modifying treatment is available. A pathological feature of this disease is neuroinflammation, a mechanism which involves both CNS-resident and peripheral immune system cells. Regulatory T-cells are immune-suppressive agents known to be dramatically and progressively decreased in patients with amyotrophic lateral sclerosis. Low-dose interleukin-2 promotes regulatory T-cell expansion and was proposed as an immune-modulatory strategy for this disease. A randomized placebo-controlled pilot phase-II clinical trial called Immuno-Modulation in Amyotrophic Lateral Sclerosis was carried out to test safety and activity of low-dose interleukin-2 in 36 amyotrophic lateral sclerosis patients (NCT02059759). Participants were randomized to 1MIU, 2MIU-low-dose interleukin-2 or placebo and underwent one injection daily for 5 days every 28 days for three cycles. In this report, we describe the results of microarray gene expression profiling of trial participants' leukocyte population. We identified a dose-dependent increase in regulatory T-cell markers at the end of the treatment period. Longitudinal analysis revealed an alteration and inhibition of inflammatory pathways occurring promptly at the end of the first treatment cycle. These responses are less pronounced following the end of the third treatment cycle, although an activation of immune-regulatory pathways, involving regulatory T-cells and T helper 2 cells, was evident only after the last cycle. This indicates a cumulative effect of repeated low-dose interleukin-2 administration on regulatory T-cells. Our analysis suggested the existence of inter-individual variation amongst trial participants and we therefore classified patients into low, moderate and high-regulatory T-cell-responders. NanoString profiling revealed substantial baseline differences between participant immunological transcript expression profiles with the least responsive patients showing a more inflammatory-prone phenotype at the beginning of the trial. Finally, we identified two genes in which pre-treatment expression levels correlated with the magnitude of drug responsiveness. Therefore, we proposed a two-biomarker based regression model able to predict patient regulatory T-cell-response to low-dose interleukin-2. These findings and the application of this methodology could be particularly relevant for future precision medicine approaches to treat amyotrophic lateral sclerosis.
RESUMO
Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch's membrane, are essential for RPE cell health and function, but the signals induced by Bruch's membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch's membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and ß1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.
Assuntos
Integrina alfa5beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Comunicação Celular , Morte Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Imobilizadas/metabolismo , Ligação Proteica , Telomerase/metabolismoRESUMO
Age-related macular degeneration (AMD) is a leading cause of blindness. Genetic variants at the chromosome 1q31.3 encompassing the complement factor H (CFH, FH) and CFH related genes (CFHR1-5) are major determinants of AMD susceptibility, but their molecular consequences remain unclear. Here we demonstrate that FHR-4 plays a prominent role in AMD pathogenesis. We show that systemic FHR-4 levels are elevated in AMD (P-value = 7.1 × 10-6), whereas no difference is seen for FH. Furthermore, FHR-4 accumulates in the choriocapillaris, Bruch's membrane and drusen, and can compete with FH/FHL-1 for C3b binding, preventing FI-mediated C3b cleavage. Critically, the protective allele of the strongest AMD-associated CFH locus variant rs10922109 has the highest association with reduced FHR-4 levels (P-value = 2.2 × 10-56), independently of the AMD-protective CFHR1-3 deletion, and even in those individuals that carry the high-risk allele of rs1061170 (Y402H). Our findings identify FHR-4 as a key molecular player contributing to complement dysregulation in AMD.
Assuntos
Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Degeneração Macular/sangue , Polimorfismo de Nucleotídeo Único , Idoso , Apolipoproteínas/sangue , Capilares/metabolismo , Estudos de Casos e Controles , Ativação do Complemento , Fator H do Complemento/metabolismo , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Fígado/fisiologia , Degeneração Macular/genética , Degeneração Macular/patologia , Proteínas Musculares/metabolismo , Retina/metabolismo , Retina/patologiaRESUMO
It is important to understand how the disease process affects the metabolic pathways in amyotrophic lateral sclerosis and whether these pathways can be manipulated to ameliorate disease progression. To analyse the basis of the metabolic defect in amyotrophic lateral sclerosis we used a phenotypic metabolic profiling approach. Using fibroblasts and reprogrammed induced astrocytes from C9orf72 and sporadic amyotrophic lateral sclerosis cases we measured the production rate of reduced nicotinamide adenine dinucleotides (NADH) from 91 potential energy substrates simultaneously. Our screening approach identified that C9orf72 and sporadic amyotrophic lateral sclerosis induced astrocytes have distinct metabolic profiles compared to controls and displayed a loss of metabolic flexibility that was not observed in fibroblast models. This loss of metabolic flexibility, involving defects in adenosine, fructose and glycogen metabolism, as well as disruptions in the membrane transport of mitochondrial specific energy substrates, contributed to increased starvation induced toxicity in C9orf72 induced astrocytes. A reduction in glycogen metabolism was attributed to loss of glycogen phosphorylase and phosphoglucomutase at the protein level in both C9orf72 induced astrocytes and induced neurons. In addition, we found alterations in the levels of fructose metabolism enzymes and a reduction in the methylglyoxal removal enzyme GLO1 in both C9orf72 and sporadic models of disease. Our data show that metabolic flexibility is important in the CNS in times of bioenergetic stress.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Astrócitos/metabolismo , Proteína C9orf72/metabolismo , Mitocôndrias/metabolismo , Neurônios Motores/metabolismo , Adulto , Idoso , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Progressão da Doença , Metabolismo Energético , Feminino , Glicogênio Fosforilase/metabolismo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Oxidative stress is an early hallmark in neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. However, the critical biochemical effector mechanisms of oxidative neurotoxicity have remained surprisingly elusive. In screening various peroxides and potential substrates of oxidation for their effect on neuronal survival, we observed that intramembrane compounds were significantly more active than aqueous or amphiphilic compounds. To better understand this result, we synthesized a series of competitive and site-specific membrane protein oxidation inhibitors termed aminoacyllipids, whose structures were designed on the basis of amino acids frequently found at the protein-lipid interface of synaptic membrane proteins. Investigating the aminoacyllipids in primary neuronal culture, we found that the targeted protection of transmembrane tyrosine and tryptophan residues was sufficient to prevent neurotoxicity evoked by hydroperoxides, kainic acid, glutathione-depleting drugs, and certain amyloidogenic peptides, but ineffective against non-oxidative inducers of apoptosis such as sphingosine or Akt kinase inhibitors. Thus, the oxidative component of different neurotoxins appears to converge on neuronal membrane proteins, irrespective of the primary mechanism of cellular oxidant generation. Our results indicate the existence of a one-electron redox cycle based on membrane protein aromatic surface amino acids, whose disturbance or overload leads to excessive membrane protein oxidation and neuronal death. Membrane proteins have rarely been investigated as potential victims of oxidative stress in the context of neurodegeneration. This study provides evidence that excessive one-electron oxidation of membrane proteins from within the lipid bilayer, depicted in the graphic, is a functionally decisive step toward neuronal cell death in response to different toxins.
Assuntos
Proteínas de Membrana/metabolismo , Degeneração Neural/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteínas de Membrana/química , Oxirredução , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-DawleyRESUMO
GABAergic interneurons are crucial to controlling the excitability and responsiveness of cortical circuitry. Their developmental origin may differ between rodents and human. We have demonstrated the expression of 12 GABAergic interneuron-associated genes in samples from human neocortex by quantitative rtPCR from 8 to 12 postconceptional weeks (PCW) and shown a significant anterior to posterior expression gradient, confirmed by in situ hybridization or immunohistochemistry for GAD1 and 2, DLX1, 2, and 5, ASCL1, OLIG2, and CALB2. Following cortical plate (CP) formation from 8 to 9 PCW, a proportion of cells were strongly stained for all these markers in the CP and presubplate. ASCL1 and DLX2 maintained high expression in the proliferative zones and showed extensive immunofluorescent double-labeling with the cell division marker Ki-67. CALB2-positive cells increased steadily in the SVZ/VZ from 10 PCW but were not double-labeled with Ki-67. Expression of GABAergic genes was generally higher in the dorsal pallium than in the ganglionic eminences, with lower expression in the intervening ventral pallium. It is widely accepted that the cortical proliferative zones may generate CALB2-positive interneurons from mid-gestation; we now show that the anterior neocortical proliferative layers especially may be a rich source of interneurons in the early neocortex.
Assuntos
Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Neocórtex/embriologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Calbindina 2/genética , Divisão Celular , Neurônios GABAérgicos/citologia , Expressão Gênica , Glutamato Descarboxilase/genética , Proteínas de Homeodomínio/genética , Humanos , Interneurônios/citologia , Proteínas do Tecido Nervoso/genética , Fator de Transcrição 2 de Oligodendrócitos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes death within a mean of 2-3 years from symptom onset. There is no diagnostic test and the delay from symptom onset to diagnosis averages 12 months. The identification of prognostic and diagnostic biomarkers in ALS would facilitate earlier diagnosis and faster monitoring of treatments. Gene expression profiling (GEP) can help to identify these markers as well as therapeutic targets in neurological diseases. One source of genetic material for GEP in ALS is peripheral blood, which is routinely accessed from patients. However, a high proportion of globin mRNA in blood can mask important genetic information. A number of methods allow safe collection, storage and transport of blood as well as RNA stabilisation, including the PAXGENE and TEMPUS systems for the collection of whole blood and LEUKOLOCK which enriches for the leukocyte population. Here we compared these three systems and assess their suitability for GEP in ALS. We collected blood from 8 sporadic ALS patients and 7 controls. PAXGENE and TEMPUS RNA extracted samples additionally underwent globin depletion using GlobinClear. RNA was amplified and hybridised onto Affymetrix U133 Plus 2.0 arrays. Lists of genes differentially regulated in ALS patients and controls were created for each method using the R package PUMA, and RT-PCR validation was carried out on selected genes. TEMPUS/GlobinClear, and LEUKOLOCK produced high quality RNA with sufficient yield, and consistent array expression profiles. PAXGENE/GlobinClear yield and quality were lower. Globin depletion for PAXGENE and TEMPUS uncovered the presence of over 60% more transcripts than when samples were not depleted. TEMPUS/GlobinClear and LEUKOLOCK gene lists respectively contained 3619 and 3047 genes differentially expressed between patients and controls. Real-time PCR validation revealed similar reliability between these two methods and gene ontology analyses revealed similar pathways differentially regulated in disease compared to controls.
Assuntos
Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/metabolismo , Perfilação da Expressão Gênica/métodos , RNA/isolamento & purificação , Primers do DNA/genética , Ontologia Genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico/métodos , RNA/sangue , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Discovery of intronic hexanucleotide repeat expansions of the C9ORF72 gene in a significant proportion of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)(1,2) was an important step for research into these disorders. The C9ORF72 genetic variant is more common than other described mutations and, unlike patients with mutations in SOD1, C9ORF72-ALS clinically and pathologically resembles the more numerous sporadic form.(3) However, progress has been limited by lack of understanding of the function of the C9ORF72 locus in health and disease. It is unknown whether the expansion causes disease by a gain of toxicity, or whether it disrupts expression of the wild-type protein encoded by the C9ORF72 gene, or some combination of both mechanisms.(1,2,4.)
Assuntos
Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Proteínas/genética , Proteína C9orf72 , Análise Mutacional de DNA , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismoRESUMO
Umbilical cord blood contains a population of non-hematopoietic multipotent stem cells that are capable of neuronal differentiation in-vitro. These cells have shown great potential as a therapeutic tool for central nervous system diseases and disorders. However whether these cells are able to produce neurons with similar developmental and functional characteristics to indigenous neurons within the brain remains poorly investigated. In this study, we used purified umbilical cord blood non-hematopoietic stem cells to produced GABAergic neurons with similar developmental and functional characteristics to cortical GABAergic neurons. We analyzed the expression of transcription factors MASH1, DLX1 and DLX2 throughout the 24 days of a sequential neuronal induction protocol and found that their expression patterns resembled those reported in the developing human cortex. The derived neurons also expressed components of GABAergic neurotransmission including GABA regulatory enzymes, GABA receptor subunits and GABA transporters. Thus we have demonstrated that umbilical cord blood stem cells are capable of producing cortical-like GABAergic neurons in vitro. This highlights the potential of umbilical cord blood stem cells as a therapeutic tool for neural injuries and disorders.
Assuntos
Diferenciação Celular , Córtex Cerebral/citologia , Células Precursoras Eritroides/fisiologia , Sangue Fetal/citologia , Neurônios GABAérgicos/fisiologia , Potenciais de Ação/genética , Células Cultivadas , Citometria de Fluxo , Glutamato Descarboxilase/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Neurogenesis of excitatory neurons in the developing human cerebral neocortex is a complex and dynamic set of processes and the exact mechanisms controlling the specification of human neocortical neuron subtypes are poorly understood due to lack of relevant cell models available. It has been shown that the transcription factors Pax6, Tbr2 and Tbr1, which are sequentially expressed in the rodent neocortex, regulate and define corticogenesis of glutamatergic neocortical neurons. In humans the homologues of these genes are generally expressed in a similar pattern, but with some differences. In this study, we used purified human umbilical cord blood stem cells, expressing pluripotency marker genes (OCT4, SOX2 and NANOG), to model human neocortical neurogenesis in vitro. We analyzed the expression patterns of PAX6, TBR2 and TBR1, at both protein and mRNA levels, throughout the 24 days of a sequential neuronal induction protocol. Their expression patterns correlated with those found in the developing human neocortex where they define different developmental stages of neocortical neurons. The derived cord blood neuron-like cells expressed a number of neuronal markers. They also expressed components of glutamatergic neurotransmission including glutamate receptor subunits and transporters, and generated calcium influxes upon stimulation with glutamate. Thus we have demonstrated that it is possible to model neocortical neurogenesis using cord blood stem cells in vitro. This may allow detailed analysis of the molecular mechanisms regulating neocortical neuronal specification, thus aiding the development of potential therapeutic tools for diseases and injuries of the cerebral cortex.
Assuntos
Sangue Fetal/citologia , Modelos Biológicos , Neocórtex/citologia , Neurogênese , Células-Tronco/fisiologia , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Sinalização do Cálcio , Células Cultivadas , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feto/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ácido Glutâmico/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteína Homeobox Nanog , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
Developing neocortical progenitors express transcription factors in gradients that induce programs of region-specific gene expression. Our previous work identified anteriorly upregulated expression gradients of a number of corticofugal neuron-associated gene probe sets along the anterior-posterior axis of the human neocortex (8-12 postconceptional weeks [PCW]). Here, we demonstrate by real-time polymerase chain reaction, in situ hybridization and immunohistochemistry that 3 such genes, ROBO1, SRGAP1, and CTIP2 are highly expressed anteriorly between 8-12 PCW, in comparison with other genes (FEZF2, SOX5) expressed by Layer V, VI, and subplate neurons. All 3 were prominently expressed by early postmitotic neurons in the subventricular zone, intermediate zone, and cortical plate (CP) from 8 to 10 PCW. Between 12 and 15 PCW expression patterns for ER81 and SATB2 (Layer V), TBR1 (Layer V/VI) and NURR1 (Layer VI) revealed Layer V forming. By 15 PCW, ROBO1 and SRGAP1 expression was confined to Layer V, whereas CTIP2 was expressed throughout the CP anteriorly. We observed ROBO1 and SRGAP1 immunoreactivity in medullary corticospinal axons from 11 PCW onward. Thus, we propose that the coexpression of these 3 markers in the anterior neocortex may mark the early location of the human motor cortex, including its corticospinal projection neurons, allowing further study of their early differentiation.
Assuntos
Desenvolvimento Fetal/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neocórtex , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores Imunológicos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Fatores Etários , Feto , Proteínas Ativadoras de GTPase/genética , Humanos , Neocórtex/citologia , Neocórtex/embriologia , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/genética , Tratos Piramidais/embriologia , Tratos Piramidais/metabolismo , RNA Mensageiro/metabolismo , Receptores Imunológicos/genética , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Proteínas RoundaboutRESUMO
The subplate is a largely transient zone containing precocious neurons involved in several key steps of cortical development. The majority of subplate neurons form a compact layer in mouse, but are dispersed throughout a much larger zone in the human. In rodent, subplate neurons are among the earliest born neocortical cells, whereas in primate, neurons are added to the subplate throughout cortical neurogenesis. Magnetic resonance imaging and histochemical studies show that the human subplate grows in size until the end of the second trimester. Previous microarray experiments in mice have shown several genes that are specifically expressed in the subplate layer of the rodent dorsal cortex. Here we examined the human subplate for some of these markers. In the human dorsal cortex, connective tissue growth factor-positive neurons can be seen in the ventricular zone at 15-22 postconceptional weeks (PCW) (most at 17 PCW) and are present in the subplate at 22 PCW. The nuclear receptor-related 1 protein is mostly expressed in the subplate in the dorsal cortex, but also in lower layer 6 in the lateral and perirhinal cortex, and can be detected from 12 PCW. Our results suggest that connective tissue growth factor- and nuclear receptor-related 1-positive cells are two distinct cell populations of the human subplate. Furthermore, our microarray analysis in rodent suggested that subplate neurons produce plasma proteins. Here we demonstrate that the human subplate also expresses α2zinc-binding globulin and Alpha-2-Heremans-Schmid glycoprotein/human fetuin. In addition, the established subplate neuron marker neuropeptide Y is expressed superficially, whereas potassium/chloride co-transporter (KCC2)-positive neurons are localized in the deep subplate at 16 PCW. These observations imply that the human subplate shares gene expression patterns with rodent, but is more compartmentalized into superficial and deep sublayers. This increased complexity of the human subplate may contribute to differential vulnerability in response to hypoxia/ischaemia across the depth of the cortex. Combining knowledge of cell-type specific subplate gene expression with modern imaging methods will enable a better understanding of neuropathologies involving the subplate.
Assuntos
Córtex Cerebral/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Neurônios/metabolismo , Adulto , Animais , Biomarcadores/análise , Proteínas Sanguíneas/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Camundongos , Análise em Microsséries , Neuropeptídeo Y/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Simportadores/análise , alfa-2-Glicoproteína-HS , alfa-Fetoproteínas/metabolismo , Cotransportadores de K e Cl-RESUMO
The division of the neocortex into functional areas (the cortical map) differs little between individuals, although brain lesions in development can lead to substantial re-organization of regional identity. We are studying how the cortical map is established in the human brain as a first step towards understanding this plasticity. Previous work on rodent development has identified certain transcription factors (e.g. Pax6, Emx2) expressed in gradients across the neocortex that appear to control regional expression of cell adhesion molecules and organization of area-specific thalamocortical afferent projections. Although mechanisms may be shared, the human neocortex is composed of different and more complex local area identities. Using Affymetrix gene chips of human foetal brain tissue from 8 to 12.5 post-conceptional weeks [PCW, equivalent to Carnegie stage (CS) 23, to Foetal stage (F) 4], human material obtained from the MRC-Wellcome Trust Human Developmental Biology Resource (http://www.hdbr.org), we have identified a number of genes that exhibit gradients along the anterior-posterior axis of the neocortex. Gene probe sets that were found to be upregulated posteriorally compared to anteriorally, included EMX2, COUPTFI and FGF receptor 3, and those upregulated anteriorally included cell adhesion molecules such as cadherins and protocadherins, as well as potential motor cortex markers and frontal markers (e.g. CNTNAP2, PCDH17, ROBO1, and CTIP2). Confirmation of graded expression for a subset of these genes was carried out using real-time PCR. Furthermore, we have established a dissociation cell culture model utilizing tissue dissected from anteriorally or posteriorally derived developing human neocortex that exhibits similar gradients of expression of these genes for at least 72 h in culture.
Assuntos
Moléculas de Adesão Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Expressão Gênica , Neocórtex/embriologia , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Animais , Mapeamento Encefálico/métodos , Fator I de Transcrição COUP/genética , Caderinas/genética , Moléculas de Adesão Celular/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas de Membrana/genética , Análise em Microsséries , Proteínas do Tecido Nervoso/metabolismo , Ratos , Receptores Imunológicos/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Roedores/embriologia , Roedores/genética , Roedores/metabolismo , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Regulação para Cima/genética , Proteínas RoundaboutRESUMO
The transcription factors Emx2 and Pax6 are expressed in the proliferating zones of the developing rodent neocortex, and gradients of expression interact in specifying caudal and rostral identities. Pax6 is also involved in corticoneurogenesis, being expressed by radial glial progenitors that give rise to cells that also sequentially express Tbr2, NeuroD and Tbr1, genes temporally downstream of Pax6. In this study, using in situ hybridization, we analysed the expression of EMX2, PAX6, TBR2, NEUROD and TBR1 mRNA in the developing human cortex between 8 and 12 postconceptional weeks (PCW). EMX2 mRNA was expressed in the ventricular (VZ) and subventricular zones (SVZ), but also in the cortical plate, unlike in the rodent. However, gradients of expression were similar to that of the rodent at all ages studied. PAX6 mRNA expression was limited to the VZ and SVZ. At 8 PCW, PAX6 was highly expressed rostrally but less so caudally, as has been seen in the rodent, however this gradient disappeared early in corticogenesis, by 9 PCW. There was less restricted compartment-specific expression of TBR2, NEUROD and TBR1 mRNA than in the rodent, where the gradients of expression were similar to that of PAX6 prior to 9 PCW. The gradient disappeared for TBR2 by 10 PCW, and for NEUROD and TBR1 by 12 PCW. These data support recent reports that EMX2 but not PAX6 is more directly involved in arealization, highlighting that analysis of human development allows better spatio-temporal resolution than studies in rodents.
Assuntos
Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Neurogênese/genética , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Movimento Celular/genética , Córtex Cerebral/citologia , Regulação para Baixo/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neurônios/citologia , Neurônios/metabolismo , Fator de Transcrição PAX6 , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico/genética , RNA Mensageiro/metabolismo , Ratos , Roedores/embriologia , Roedores/genética , Roedores/metabolismo , Especificidade da Espécie , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
We have employed immunohistochemistry for multiple markers to investigate the structure and possible function of the different compartments of human cerebral wall from the formation of cortical plate at 8 postconceptional weeks (PCW) to the arrival of thalamocortical afferents at 17 PCW. New observations include the subplate emerging as a discrete differentiated layer by 10 PCW, characterized by synaptophysin and vesicular gamma-aminobutyric acid transporter expression also seen in the marginal zone, suggesting that these compartments may maintain a spontaneously active synaptic network even before the arrival of thalamocortical afferents. The subplate expanded from 13 to 17 PCW, becoming the largest compartment and differentiated further, with NPY neurons located in the outer subplate and KCC2 neurons in the inner subplate. Glutamate decarboxylase and calretinin-positive inhibitory neurons migrated tangentially and radially from 11.5 PCW, appearing in larger numbers toward the rostral pole. The proliferative zones, marked by Ki67 expression, developed a complicated structure by 12.5 PCW reflected in transcription factor expression patterns, including TBR2 confined to the inner subventricular and outer ventricular zones and TBR1 weakly expressed in the subventricular zone (SVZ). PAX6 was extensively expressed in the proliferative zones such that the human outer SVZ contained a large reservoir of PAX6-positive potential progenitor cells.
Assuntos
Envelhecimento/patologia , Envelhecimento/fisiologia , Ventrículos Cerebrais , Desenvolvimento Embrionário/fisiologia , Neocórtex , Proteínas do Tecido Nervoso/metabolismo , Biomarcadores/metabolismo , Ventrículos Cerebrais/citologia , Ventrículos Cerebrais/embriologia , Ventrículos Cerebrais/metabolismo , Feminino , Humanos , Masculino , Neocórtex/citologia , Neocórtex/embriologia , Neocórtex/metabolismoRESUMO
Steroids that activate glucocorticoid receptors (GRs) and mineralocorticoid receptors have important regulatory effects on neural development, plasticity, and the body's stress response. Here, we investigated the role of corticosteroids in regulating the expression of the glial glutamate transporters glial glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in rat primary astrocytes. The synthetic glucocorticoid dexamethasone provoked a marked increase of GLT-1 transcription and protein levels in cortical astrocytes, whereas GLAST expression remained unaffected. Up-regulation of GLT-1 expression was accompanied by an enhanced glutamate uptake, which could be blocked by the specific GLT-1 inhibitor dihydrokainate. The promoting effect of dexamethasone on GLT-1 gene expression and function was abolished by the GR antagonist mifepristone. A predominant role of the GR was further supported by the observation that corticosterone could elevate GLT-1 expression in a dose-dependent manner, whereas aldosterone, the physiological ligand of the mineralocorticoid receptor, exerted only weak effects even when applied at high concentrations. Moreover, we monitored brain region-specific differences, since all corticosteroids used in this study failed to alter the expression of GLT-1 in midbrain and cerebellar glia, although expression levels of both corticosteroid receptor subtypes were similar in all brain regions analyzed. Dexamethasone, however, modestly enhanced GLT-1 expression in cerebellar glia in combination with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine, suggesting that suppression of GLT-1 expression in cerebellar cultures may at least in part be epigenetically mediated by a DNA methylation-dependent process. Taken together, our data highlight a potential role for glucocorticoids in regulating GLT-1 gene expression during central nervous system development or pathophysiological processes including stress.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/biossíntese , Encéfalo/metabolismo , Glucocorticoides/química , Corticosteroides/metabolismo , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Western Blotting , Sistema Nervoso Central/patologia , Cerebelo/patologia , Corticosterona/metabolismo , Corticosterona/farmacologia , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Primers do DNA/química , Decitabina , Detergentes/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Corantes Fluorescentes/farmacologia , Imuno-Histoquímica , Ácido Caínico/análogos & derivados , Ácido Caínico/farmacologia , Ligantes , Luciferases/metabolismo , Microdomínios da Membrana/metabolismo , Mifepristona/farmacologia , Neuroglia/patologia , Ratos , Ratos Sprague-Dawley , Receptores de Mineralocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
Corticotropin-releasing hormone (CRH) modulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis, and has a key role in mediating neuroendocrine effects that occur in response to stressful stimuli. Disruption of the CRH system however has been shown to be closely associated with the progression of Alzheimer's disease (AD), and these observations prompted an investigation into the potential neuroprotective effects of the hormone. In addition to its regulatory affects on the molecular processes that underlie AD i.e., amyloid precursor protein (APP) processing and potentially tau phosphorylation, evidence is provided that the neuroprotective effects of CRH are mediated by a number of diverse mechanisms. These stem from activation of its high affinity receptor, the CRH type 1 receptor, and involve the induction of protective intracellular pathways including PKA-CREB that eventually lead to expression of neurotrophic factors. Conversely, inhibition of harmful events, such as caspase activation during apoptosis may also occur. Taken together, an impressive amount of evidence has accumulated recently, highlighting this new and potentially important function of CRH.
Assuntos
Envelhecimento/fisiologia , Doença de Alzheimer/fisiopatologia , Hormônio Liberador da Corticotropina/fisiologia , Sistema Hipotálamo-Hipofisário/fisiologia , Sistema Hipófise-Suprarrenal/fisiologia , Doença de Alzheimer/metabolismo , Animais , Humanos , Estresse Oxidativo/fisiologiaRESUMO
CRH receptor (CRHR) 1 and the cannabinoid receptor 1 (CB1) are both G protein-coupled receptors. Activation of CRHR1 leads to increases in cAMP production and phosphorylation of the transcription factor cAMP response element-binding protein (CREB). In contrast, CB1 is negatively coupled to the cAMP signaling cascade. In this study, we analyzed a putative interaction between these two systems focusing on the regulation of the expression of brain-derived neurotrophic factor (BDNF), a CREB-regulated gene. In situ hybridization revealed coexpression of CRHR1 and CB1 receptors in the granular layer of the cerebellum. Therefore, we analyzed the effects of CRH and the CB1 agonist WIN-55,212-2 on BDNF expression in primary cerebellar neurons from rats and mice. We observed that application of CRH for 48 h led to an increase in BDNF mRNA and protein levels. This effect was inhibited by WIN-55,212-2. At the level of intracellular signaling, short-term application of WIN-55,212-2 inhibited CRH-induced cAMP accumulation and CREB phosphorylation. Pharmacological analysis demonstrated that the CRHR1 antagonist R121919, the protein kinase A inhibitor H89, and the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester inhibited CRH-mediated BDNF expression. Moreover, depolarization-induced BDNF synthesis was also inhibited by long-term application of WIN-55,212-2 in wild-type mice but not in CB1-deficient mice. Thus, these data highlight an interaction between the CRH and the cannabinoid system in the regulation of BDNF expression by influencing cAMP and Ca2+ signaling pathways.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Moduladores de Receptores de Canabinoides/metabolismo , Hormônio Liberador da Corticotropina/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ácido Egtázico/análogos & derivados , Endocanabinoides , Transdução de Sinais , Animais , Benzoxazinas , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Cerebelo/patologia , Hormônio Liberador da Corticotropina/metabolismo , AMP Cíclico/metabolismo , Ácido Egtázico/farmacologia , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Hibridização In Situ , Isoquinolinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Morfolinas/farmacologia , Naftalenos/farmacologia , Neurônios/metabolismo , Ligação Proteica , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
Caveolae represent membrane microdomains acting as integrators of cellular signaling and functional processes. Caveolins are involved in the biogenesis of caveolae and regulate the activity of caveolae-associated proteins. Although caveolin proteins are found in the CNS, the regulation of caveolins in neural cells is poorly described. In the present study, we investigated different modes and mechanisms of caveolin gene regulation in primary rat astrocytes. We demonstrated that activation of cAMP-dependent signaling pathways led to a marked reduction in protein levels of caveolin-1/-2 in cortical astrocytes. Application of transforming growth factor-alpha (TGF-alpha) also resulted in a decrease of caveolin-1/-2 expression. Decreased caveolin protein levels were mirrored by diminished caveolin gene transcription. The repressive effect of TGF-alpha on caveolin-1 expression was MAP kinase-independent and partly mediated through the PI3-kinase pathway. Further downstream, inhibition of histone deacetylases abrogated TGF-alpha effects, suggesting that chromatin remodeling processes could contribute to caveolin-1 repression. Intriguingly, alterations of caveolin gene expression in response to cAMP or TGF-alpha coincided with reciprocal and brain-region specific changes in glial glutamate transporter GLT-1 expression. The reciprocal regulation of caveolin-1 and GLT-1 expression might be gated through a common PI3-kinase dependent pathway triggered by TGF-alpha. Finally, we showed that GLT-1 is located in non-caveolar lipid rafts of cortical astrocytes. In conclusion, this study highlights the occurrence of the reciprocal regulation of caveolin and GLT-1 expression during processes such as astrocyte differentiation via common signaling pathways. We also provide strong evidence that GLT-1 itself is concentrated in lipid rafts, inferring an important role for glial glutamate transporter function.