RESUMO
Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.
Assuntos
Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Doenças Neurodegenerativas , Humanos , Proteína ADAM10/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Proteínas Priônicas/metabolismo , Proteínas de Membrana/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , AnticorposRESUMO
In the CNS, insulin-like growth factor-1 (IGF-1) is mainly known for its trophic effect both during development and in adulthood. Here, we show than in adult rat supraoptic nucleus (SON), IGF-1 receptor immunoreactivity is present in neurons, whereas IGF-1 immunoreactivity is found principally in astrocytes and more moderately in neurons. In vivo application of IGF-1 within the SON acutely inhibits the activity of both vasopressin and oxytocin neurons, the two populations of SON neuroendocrine cells. Recordings of acutely isolated SON neurons showed that this inhibition occurs through two rapid and reversible mechanisms, both involving the neuronal IGF-1 receptor but different intracellular messengers. IGF-1 inhibits Gd3+-sensitive and osmosensitive mechanoreceptor cation current via phosphatidylinositol-3 (PI3) kinase activation. IGF-1 also potentiates taurine-activated glycine receptor (GlyR) Cl- currents by increasing the agonist sensitivity through a extremely rapid (within a second) PI3 kinase-independent mechanism. Both mechanoreceptor channels and GlyR, which form the excitatory and inhibitory components of SON neuron osmosensitivity, are active at rest, and their respective inhibition and potentiation will both be inhibitory, leading to strong decrease in neuronal activity. It will be of interest to determine whether IGF-1 is released by neurons, thus participating in an inhibitory autocontrol, or astrocytes, then joining the growing family of glia-to-neuron transmitters that modulate neuronal and synaptic activity. Through the opposite and complementary acute regulation of mechanoreceptors and GlyR, IGF-1 appears as a new important neuromodulator in the adult CNS, participating in the complex integration of neural messages that regulates the level of neuronal excitability.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Mecanorreceptores/fisiologia , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de Glicina/fisiologia , Núcleo Supraóptico/citologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Androstadienos/farmacologia , Animais , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Interações Medicamentosas , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Glicinérgicos/farmacologia , Imuno-Histoquímica/métodos , Técnicas In Vitro , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Neurônios/metabolismo , Ocitocina/metabolismo , Técnicas de Patch-Clamp/métodos , Ratos , Receptor IGF Tipo 1/metabolismo , Estricnina/farmacologia , Taurina/metabolismo , Taurina/farmacologia , Trítio/metabolismo , Vasopressinas/metabolismo , WortmaninaRESUMO
The trophoblast cell lineage is specified early at the blastocyst stage, leading to the emergence of the trophectoderm and the pluripotent cells of the inner cell mass. Using a double mRNA amplification technique and a comparison with transcriptome data on pluripotent stem cells, placenta, germinal and adult tissues, we report here some essential molecular features of the human mural trophectoderm. In addition to genes known for their role in placenta (CGA, PGF, ALPPL2 and ABCG2), human trophectoderm also strongly expressed Laminins, such as LAMA1, and the GAGE Cancer/Testis genes. The very high level of ABCG2 expression in trophectoderm, 7.9-fold higher than in placenta, suggests a major role of this gene in shielding the very early embryo from xenobiotics. Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression. A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta. The induction of this network could be recapitulated in an in vitro trophoblast differentiation model.
Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Transcriptoma , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Diferenciação Celular/genética , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Ectoderma/citologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Trofoblastos/metabolismo , Trofoblastos/fisiologiaRESUMO
In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts.
Assuntos
Ectoderma/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Transcriptoma , Trofoblastos/metabolismo , Blastocisto/citologia , Blastocisto/metabolismo , Linhagem da Célula/genética , Ectoderma/citologia , Feminino , Perfilação da Expressão Gênica , Humanos , Oócitos/citologia , Oócitos/metabolismo , Gravidez , Trofoblastos/citologiaRESUMO
Peptide nucleic acids (PNAs) constitute a remarkable new class of synthetic nucleic acids analogs, based on peptide-like backbone. This structure gives PNAs the capacity to hybridize with high affinity and specificity to complementary RNA and DNA sequences. Over the last few years, the use of PNAs has proven its efficacy in cytogenetics for the rapid in situ identification of human chromosomes. Multicolour PNA-FISH protocols have been described and their adaptation to human spermatozoa has allowed the development of a new and fast procedure, which can advantageously be used for the assessment of aneuploidy in male gametes.