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1.
J Neuroinflammation ; 16(1): 73, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953561

RESUMO

BACKGROUND: Autoantibodies against the paranodal protein contactin-1 have recently been described in patients with severe acute-onset autoimmune neuropathies and mainly belong to the IgG4 subclass that does not activate complement. IgG3 anti-contactin-1 autoantibodies are rare, but have been detected during the acute onset of disease in some cases. There is evidence that anti-contactin-1 prevents adhesive interaction, and chronic exposure to anti-contactin-1 IgG4 leads to structural changes at the nodes accompanied by neuropathic symptoms. However, the pathomechanism of acute onset of disease and the pathogenic role of IgG3 anti-contactin-1 is largely unknown. METHODS: In the present study, we aimed to model acute autoantibody exposure by intraneural injection of IgG of patients with anti-contacin-1 autoantibodies to Lewis rats. Patient IgG obtained during acute onset of disease (IgG3 predominant) and IgG from the chronic phase of disease (IgG4 predominant) were studied in comparison. RESULTS: Conduction blocks were measured in rats injected with the "acute" IgG more often than after injection of "chronic" IgG (83.3% versus 35%) and proved to be reversible within a week after injection. Impaired nerve conduction was accompanied by motor deficits in rats after injection of the "acute" IgG but only minor structural changes of the nodes. Paranodal complement deposition was detected after injection of the "acute IgG". We did not detect any inflammatory infiltrates, arguing against an inflammatory cascade as cause of damage to the nerve. We also did not observe dispersion of paranodal proteins or sodium channels to the juxtaparanodes as seen in patients after chronic exposure to anti-contactin-1. CONCLUSIONS: Our data suggest that anti-contactin-1 IgG3 induces an acute conduction block that is most probably mediated by autoantibody binding and subsequent complement deposition and may account for acute onset of disease in these patients. This supports the notion of anti-contactin-1-associated neuropathy as a paranodopathy with the nodes of Ranvier as the site of pathogenesis.


Assuntos
Contactina 1/imunologia , Síndrome de Guillain-Barré/complicações , Imunização Passiva/métodos , Imunoglobulina G/farmacologia , Transtornos Motores/fisiopatologia , Transtornos Motores/cirurgia , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Complemento C1q/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Síndrome de Guillain-Barré/etiologia , Humanos , Transtornos Motores/induzido quimicamente , Condução Nervosa/efeitos dos fármacos , Neurite Óptica/sangue , Neurite Óptica/imunologia , Nós Neurofibrosos/efeitos dos fármacos , Nós Neurofibrosos/metabolismo , Ratos , Ratos Endogâmicos Lew , Tempo de Reação/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Teste de Desempenho do Rota-Rod , Estatísticas não Paramétricas
2.
J Mol Neurosci ; 68(2): 261-274, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30949956

RESUMO

The level of miR-181a decreases rapidly in N2a cells following oxygen-glucose deprivation/reperfusion, but its role in this process is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-181a. We hypothesized that miR-181a reduces neuronal apoptosis and protects neurons by targeting reelin. Second mitochondria-derived activator of caspases (Smac) is a protein located in mitochondria that regulates apoptosis. The pro-apoptotic effect of Smac is achieved by reversing the effects of apoptosis-inhibiting proteins (IAPs), particularly X-linked inhibitor of apoptosis (XIAP). We also evaluated the effect of miR-181a on the Smac/IAP signaling pathway after oxygen-glucose deprivation and reperfusion in N2a cells. The miR-181a level, apoptosis rate, and the levels of reelin mRNA and protein, Smac, and XIAP were assessed in N2a cells subjected to oxygen-glucose deprivation for 4 h and reperfusion for 0, 4, 12, or 24 h with/without an miR-181a mimic, or mismatched control. Direct targeting of reelin by miR-181a was assessed in vitro by dual luciferase assay and immunoblotting. Pre-treatment with miR-181a mimicked the increase in the miR-181a level in N2a cells after oxygen-glucose deprivation/reperfusion, resulting in a significant decrease in the apoptosis rate. Changes in the miR-181a level in N2a cells were inversely correlated with reelin protein expression. Direct targeting of the reelin 3' untranslated region by miR-181a was verified by dual luciferase assay, which showed that miR-181a significantly inhibited luciferase activity. The Smac level was significantly lower in the miR-181a mimics than the normal control and mimics-cont groups (P < 0.01), whereas the level of XIAP was increased slightly. These findings suggest that miR-181a protects neurons from apoptosis by inhibiting reelin expression and regulating the Smac/IAP signaling pathway after oxygen-glucose deprivation/reperfusion injury.


Assuntos
Apoptose , MicroRNAs/genética , Neurônios/metabolismo , Oxigênio/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Glucose/deficiência , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , MicroRNAs/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
3.
Biol Pharm Bull ; 42(3): 354-356, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30828067

RESUMO

Reelin is a secreted protein that antagonizes the deposition and toxicity of amyloid ß peptide (Aß). Therefore, augmentation of Reelin activity may ameliorate Alzheimer's disease (AD). We have recently reported that a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS-3) cleaves and inactivates Reelin in the mouse brain. In the present study, we investigated the effect of reducing ADAMTS-3 on deposition of Aß by crossbreeding drug-inducible ADAMTS-3 conditional knock-out (cKO) mice with "next-generation" AD model mice. We found that reducing ADAMTS-3 inhibited deposition of Aß significantly in AppNL-F mice, which produce human wild-type Aß. On the other hand, reducing ADAMTS-3 had no effect in AppNL-G-F mice, which produce the Arctic mutant Aß (E22G) that forms protofibrils more efficiently than does wild-type Aß. Thus, the findings suggest that the administration of an inhibitor against ADAMTS-3 will prevent the progression of AD pathology caused by deposition of wild-type Aß.


Assuntos
Proteínas ADAMTS/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Proteínas ADAMTS/antagonistas & inibidores , Proteínas ADAMTS/genética , Doença de Alzheimer , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
4.
J Mol Neurosci ; 68(2): 171-180, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30888622

RESUMO

Synaptic cell adhesion molecules, including neurexins and neuroligins, mediate the formation and maintenance of connections between neuronal cells. Although neurexins and neuroligins are known to interact with each other in a calcium-dependent manner and several neuropeptides have been shown to act through G protein-coupled receptors to increase intracellular calcium levels, no studies have examined the role of the neuropeptide oxytocin in association with adhesion molecules. Given that oxytocin receptors are located on presynaptic and postsynaptic membranes and that oxytocin exerts direct effects on neuronal excitability, it could be hypothesized that oxytocin affects the expression of cell surface adhesion molecules. In the present study, we show that incubation in the presence of oxytocin (1 µM, 48 h) exerted cell-specific effects on the levels of neurexin 2α, neurexin 2ß, and neuroligin 3. Oxytocin significantly increased the mRNA expression levels of neurexin 2α, neurexin 2ß, and neuroligin 3 in SH-SY5Y, U-87MG, and primary cerebellar cells. The effect of inhibiting oxytocin receptors on the expression of neurexin 2ß was more dramatic in U-87MG cells than in SH-SY5Y cells. Oxytocin did not exert effects in primary corticohippocampal cells. Additionally, we measured the expression of selected GTPases to determine whether they could mediate the effects of oxytocin. Oxytocin induced a decrease in the mRNA level of Rac1 in U-87MG and primary cerebellar cells and exerted a stimulatory effect on the expression of RhoB at the gene and protein level in SH-SY5Y cells. These results suggest that the regulation of neurexins and neuroligins involves the activation of oxytocin receptors. These effects are likely mediated by the stimulation of RhoB GTPase, at least in certain types of cells.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Receptores de Ocitocina/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Cerebelo/citologia , Humanos , Hipotálamo/citologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Ocitocina/farmacologia , Ratos , Ratos Wistar , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoB de Ligação ao GTP/genética , Proteína rhoB de Ligação ao GTP/metabolismo
5.
eNeuro ; 6(1)2019 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30863790

RESUMO

CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.


Assuntos
Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Técnicas Genéticas , Neurônios/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Cultura Primária de Células , Distribuição Aleatória , Ratos Sprague-Dawley , Serina Endopeptidases/metabolismo , Transcrição Genética , Transcriptoma
6.
ACS Appl Mater Interfaces ; 11(8): 7764-7773, 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30707832

RESUMO

Over the recent years, the development of neural interface systems has stuck to using electrical cues to stimulate neurons and read out neural signals, although neurons relay signals via chemical release and recognition at synapses. In addition, conventional neural interfaces are vulnerable to cell migration and glial encapsulation due to the absence of connection anchoring the neuron into the device unlike synapses, which are firmly sustained by protein bonding. To close this discrepancy, we conducted an intensive investigation into the induced synapse interface by employing engineered synaptic proteins from a neural interface perspective. The strong potential of induced synaptic differentiation as an emerging neural interfacing technique is demonstrated by exploring its structural features, chemical release kinetics, robustness, and scalability to the brain tissue. We show that the exocytosis kinetics of induced synapses is similar to that of endogenous synapses. Moreover, induced synapses show remarkable stability, despite cell migration and growth. The synapse-inducing technique has broad applications to cultured hippocampal and cortex tissues and suggests a promising method to integrate neural circuits with digital elements.


Assuntos
Sinapses/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Embrião de Mamíferos/citologia , Exocitose , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Microscopia Eletrônica de Varredura , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Imagem com Lapso de Tempo
7.
Exp Mol Med ; 51(1): 7, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30700695

RESUMO

Osteoclasts (OCs) are bone-resorbing cells that originate from hematopoietic stem cells and develop through the fusion of mononuclear myeloid precursors. Dysregulation of OC development causes bone disorders such as osteopetrosis, osteoporosis, and rheumatoid arthritis. Although the molecular mechanisms underlying osteoclastogenesis have been well established, the means by which OCs maintain their survival during OC development remain unknown. We found that Ninjurin1 (Ninj1) expression is dynamically regulated during osteoclastogenesis and that Ninj1-/- mice exhibit increased trabecular bone volume owing to impaired OC development. Ninj1 deficiency did not alter OC differentiation, transmigration, fusion, or actin ring formation but increased Caspase-9-dependent intrinsic apoptosis in prefusion OCs (preOCs). Overexpression of Ninj1 enhanced the survival of mouse macrophage/preOC RAW264.7 cells in osteoclastogenic culture, suggesting that Ninj1 is important for the survival of preOCs. Finally, analysis of publicly available microarray data sets revealed a potent correlation between high NINJ1 expression and destructive bone disorders in humans. Our data indicate that Ninj1 plays an important role in bone homeostasis by enhancing the survival of preOCs.


Assuntos
Osso Esponjoso/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Fatores de Crescimento Neural/genética , Osteoclastos/metabolismo , Osteogênese , Animais , Apoptose , Osso Esponjoso/crescimento & desenvolvimento , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Humanos , Masculino , Camundongos , Fatores de Crescimento Neural/metabolismo , Osteoclastos/citologia , Células RAW 264.7
8.
Cancer Sci ; 110(5): 1644-1652, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30784169

RESUMO

Cancer tissues contain small populations of highly tumorigenic cells termed cancer stem cells (CSCs). Immortalized cell lines containing CSCs are valuable and powerful experimental tools for research into the characteristics of these stem cells. We previously reported that the hepatocellular carcinoma cell line Li-7 includes abundant CD13+ CD166- CSCs; however, the number of these cells decreases after long-term culture as a result of differentiation to non-CSC populations. To ensure consistent and reproducible results in experiments using Li-7 cells, it is important that the CSC population is maintained stably regardless of culture duration and passage. In the present study, we found that a commercially available culture medium for maintenance of embryonic stem cells and induced pluripotent stem cells, mTeSR1, effectively prevented spontaneous differentiation by CD13+ CD166- cells to CD13- CD166+ cells and therefore maintained the CSC population in Li-7 cell cultures. CD13+ CD166- CSCs maintained using this culture medium retained high tumorigenicity after transplantation into mice; they also showed the ability to differentiate in vitro into non-CSC populations in RPMI-1640 with 10% FBS medium. We analyzed gene expression profiles of CSC and non-CSC populations in Li-7 cultures using an RNA sequencing method. Genes such as FGFR, NOTCH1, and JAG1, that are associated with tumorigenicity and stemness, were upregulated in the CSC population. Our results suggest that CSCs can be maintained in immortalized cancer cell lines cultured over an extended period using a medium developed for culture of embryonic/induced pluripotent stem cells.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/metabolismo , Técnicas de Cultura de Células/métodos , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/transplante , Animais , Antígenos CD/metabolismo , Antígenos CD13/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultura/farmacologia , Proteínas Fetais/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteína Jagged-1/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Receptor Notch1/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Análise de Sequência de RNA , Regulação para Cima
9.
Biochem J ; 476(2): 293-306, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30602588

RESUMO

Retromer is an evolutionarily conserved endosomal trafficking complex that mediates the retrieval of cargo proteins from a degradative pathway for sorting back to the cell surface. To promote cargo recycling, the core retromer trimer of VPS (vacuolar protein sorting)26, VPS29 and VPS35 recognises cargo either directly, or through an adaptor protein, the most well characterised of which is the PDZ [postsynaptic density 95 (PSD95), disk large, zona occludens] domain-containing sorting nexin SNX27. Neuroligins (NLGs) are postsynaptic trans-synaptic scaffold proteins that function in the clustering of postsynaptic proteins to maintain synaptic stability. Here, we show that each of the NLGs (NLG1-3) bind to SNX27 in a direct PDZ ligand-dependent manner. Depletion of SNX27 from neurons leads to a decrease in levels of each NLG protein and, for NLG2, this occurs as a result of enhanced lysosomal degradation. Notably, while depletion of the core retromer component VPS35 leads to a decrease in NLG1 and NLG3 levels, NLG2 is unaffected, suggesting that, for this cargo, SNX27 acts independently of retromer. Consistent with loss of SNX27 leading to enhanced lysosomal degradation of NLG2, knockdown of SNX27 results in fewer NLG2 clusters in cultured neurons, and loss of SNX27 or VPS35 reduces the size and number of gephyrin clusters. Together, these data indicate that NLGs are SNX27-retromer cargoes and suggest that SNX27-retromer controls inhibitory synapse number, at least in part through trafficking of NLG2.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Lisossomos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Sinapses/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/genética , Proteína 4 Homóloga a Disks-Large/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Células HEK293 , Humanos , Lisossomos/genética , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Wistar , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
10.
Behav Brain Res ; 362: 7-20, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30605713

RESUMO

The cell adhesion molecule neuroligin2 (NLGN2) regulates GABAergic synapse development, but its role in neural circuit function in the adult hippocampus is unclear. We investigated GABAergic synapses and hippocampus-dependent behaviors following viral-vector-mediated overexpression of NLGN2. Transducing hippocampal neurons with AAV-NLGN2 increased neuronal expression of NLGN2 and membrane localization of GABAergic postsynaptic proteins gephyrin and GABAARγ2, and presynaptic vesicular GABA transporter protein (VGAT) suggesting trans-synaptic enhancement of GABAergic synapses. In contrast, glutamatergic postsynaptic density protein-95 (PSD-95) and presynaptic vesicular glutamate transporter (VGLUT) protein were unaltered. Moreover, AAV-NLGN2 significantly increased parvalbumin immunoreactive (PV+) synaptic boutons co-localized with postsynaptic gephyrin+ puncta. Furthermore, these changes were demonstrated to lead to cognitive impairments as shown in a battery of hippocampal-dependent mnemonic tasks and social behaviors.


Assuntos
Comportamento Animal/fisiologia , Moléculas de Adesão Celular Neuronais/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Animais , Masculino , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Terminações Pré-Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores , Ácido gama-Aminobutírico/metabolismo
11.
Transl Psychiatry ; 9(1): 29, 2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30664619

RESUMO

Deletions in the 15q11.2 region of the human genome are associated with neurobehavioral deficits, and motor development delay, as well as in some cases, symptoms of autism or schizophrenia. The cytoplasmic FMRP-interacting protein 1 (CYFIP1) is one of the four genes contained within this locus and has been associated with other genetic forms of autism spectrum disorders (ASD). In mice, Cyfip1 haploinsufficiency leads to alteration of dendritic spine morphology and defects in synaptic plasticity, two pathophysiological hallmarks of mouse models of ASD. At the behavioral level, however, Cyfip1 haploinsufficiency leads to minor phenotypes, not directly relevant for 15q11.2 deletion syndrome or ASD. A fundamental question is whether neuronal phenotypes caused by the mutation of Cyfip1 are relevant for the human condition. Here, we describe a synaptic cluster of ASD-associated proteins centered on CYFIP1 and the adhesion protein Neuroligin-3. Cyfip1 haploinsufficiency in mice led to decreased dendritic spine density and stability associated with social behavior and motor learning phenotypes. Behavioral training early in development resulted in alleviating the motor learning deficits caused by Cyfip1 haploinsufficiency. Altogether, these data provide new insight into the neuronal and behavioral phenotypes caused by Cyfip1 mutation and proof-of-concept for the development of a behavioral therapy to treat phenotypes associated with 15q11.2 syndromes and ASD.


Assuntos
Transtorno do Espectro Autista/genética , Haploinsuficiência/genética , Deficiência Intelectual/genética , Proteínas do Tecido Nervoso/genética , Comportamento Social , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Aberrações Cromossômicas , Cromossomos Humanos Par 15/genética , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Distribuição Aleatória
12.
Clin Chim Acta ; 490: 6-11, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30552869

RESUMO

Reelin is a glycoprotein associated with synaptic plasticity and neurotransmission. The malfunctioning of reelin signaling in the brain is likely to contribute to the pathogenesis of Alzheimer's disease (AD). Reelin binding to Apolipoprotein E receptor 2 (ApoER2) activates downstream signaling and induces the proteolytic cleavage of ApoER2, resulting in the generation of soluble fragments. To evaluate the efficiency of reelin signaling in AD, we have quantified the levels of reelin and soluble ectodomain fragments of ApoER2 (ectoApoER2) in the cerebrospinal fluid (CSF). CSF from sporadic AD patients (sAD; n = 14, age 54-83 years) had lower levels of ecto-ApoER2 (~31% reduction; p = .005) compared to those in the age-matched controls (n = 10, age 61-80), and a higher reelin/ecto-ApoER2 ratio. In contrast, autosomal dominant AD patients, carriers of PSEN1 mutations (ADAD; n = 7, age 31-49 years) had higher ecto-ApoER2 levels (~109% increment; p = .001) and a lower reelin/ecto-ApoER2 ratio than the non-mutation carriers from the same families (n = 7, age 25-47 years). Our data suggest that the levels of ecto-ApoER2 in CSF could be a suitable read-out of an impaired reelin signaling in AD, but also indicate differences between sAD and ADAD.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/patologia , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/líquido cefalorraquidiano , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Presenilina-1/genética
13.
Neuroreport ; 30(1): 14-18, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30395007

RESUMO

Reelin is an extracellular matrix glycoprotein that modulates synaptic function and plasticity, with a crucial role in neuronal migration. Changes in the expression of this protein have been reported in neurodegenerative diseases, such as Alzheimer's disease (AD). This molecule is produced by Cajal-Retzius neurons during development and by inhibitory neurons in the adult nervous system. Individuals with Down syndrome (DS) present an early development of AD; therefore, we analyzed the alterations in this molecule and its receptors in the murine model for DS Ts65Dn as well as in human with DS. We performed immunofluorescence analysis for reelin and its receptors very-low-density lipoprotein receptor and apolipoprotein R receptor 2 in the temporal cortex of mice and humans and have quantified the density of reelin-expressing neurons and the intensity of expression of both receptors. We have observed an increment in the density of reelin immunoreactive neurons in both the temporal cortex of adult Ts65Dn mice and humans with DS. Moreover, these reelin immunoreactive neurons displayed a disorganized distribution when compared with wild-type mice. Regarding reelin receptors, very-low-density lipoprotein receptor expression remained unaltered in both Ts65Dn and humans with DS, whereas apolipoprotein R receptor 2 decreased in both individuals with DS and Ts65Dn mice. These alterations are similar to those observed in individuals with AD.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Síndrome de Down/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios , Receptores de Superfície Celular/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Lobo Temporal , Bancos de Tecidos , Adulto , Idoso , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Neurônios/citologia , Neurônios/metabolismo , Lobo Temporal/citologia , Lobo Temporal/metabolismo
14.
Int J Nanomedicine ; 13: 8297-8308, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30584301

RESUMO

Background: Nanoscale surface modifications are widely touted to improve the biocompatibility of medically relevant materials. Immune cells, such as macrophages, play a critical role in the initial healing events following implantation. Methods: To understand the response of macrophages to nanotopography better, we exposed U937-derived macrophages to a distinctive mesoporous titanium surface (TiNano) produced by a process of simple chemical nanocavitation, and to mechanically polished titanium (TiPolished) and glass coverslip (Glass) surfaces as controls. Cell numbers and morphology were evaluated. Osteopontin expression and that of the proinflammatory SPARC protein and its stabilin 1 receptor were analyzed. Release of inflammation-associated cytokines and chemokines was also measured. Results: Compared to the two control surfaces, there were fewer U937 cells on TiNano, and these exhibited a more rounded morphology with long filopodia. The cells showed areas of punctate actin distribution, indicating formation of podosomes. Of the three proteins examined, only osteopontin's immunofluorescence signal was clearly reduced. Irrespective of the substrate, the cytokine assay revealed important variations in expression levels of the multiple molecules analyzed and downregulation in a number of chemokines by the TiNano surface. Conclusion: These results indicate that macrophages sense and respond to the physicochemical cueing generated by the nanocavitated surface, triggering cellular and molecular changes consistent with lesser inflammatory propensity. Given the previously reported beneficial outcome of this mesoporous surface on osteogenic activity, it could be presumed that modulation of the macrophagic response it elicits may also contribute to initial bone-integration events.


Assuntos
Macrófagos/metabolismo , Nanopartículas/química , Titânio/farmacologia , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Nanopartículas/ultraestrutura , Osteonectina/metabolismo , Osteopontina/metabolismo , Fagocitose/efeitos dos fármacos , Receptores de Retorno de Linfócitos/metabolismo , Propriedades de Superfície , Titânio/química , Células U937
15.
J Toxicol Sci ; 43(11): 631-643, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30404997

RESUMO

Autism is a complex neurodevelopmental disorder characterized by impaired social communication and social interactions, and repetitive behaviors. The etiology of autism remains unknown and its molecular basis is not yet well understood. Pregnant Sprague-Dawley (SD) rats were administered 600 mg/kg of valproic acid (VPA) by intraperitoneal injection on day 12.5 of gestation. Both 11- to 13-week-old male and female rat models of VPA-induced autism showed impaired sociability and impaired preference for social novelty as compared to the corresponding control SD rats. Significantly reduced mRNA expressions of social behavior-related genes, such as those encoding the serotonin receptor, brain-derived neurotrophic factor and neuroligin3, and significantly increased expression levels of proinflammatory cytokines, such as interleukin-1 ß and tumor necrosis factor-α, were noted in the hippocampi of both male and female rats exposed to VPA in utero. The hippocampal expression level of gamma amino butyric acid (GABA) enzyme glutamic acid decarboxylase (GAD) 67 protein was reduced in both male and female VPA-exposed rats as compared to the corresponding control animals. Our results indicate that developmental exposure to VPA affects the social behavior in rats by modulating the expression levels of social behavior-related genes and inflammatory mediators accompanied with changes in GABA enzyme in the hippocampus.


Assuntos
Transtorno Autístico/metabolismo , Transtorno Autístico/psicologia , Glutamato Descarboxilase/metabolismo , Comportamento Social , Ácido Valproico/efeitos adversos , Animais , Transtorno Autístico/induzido quimicamente , Transtorno Autístico/genética , Comportamento Animal , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Hipocampo/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo
16.
Science ; 361(6404)2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30139844

RESUMO

The architecture of the neurovascular unit (NVU) is controlled by the communication of neurons, glia, and vascular cells. We found that the neuronal guidance cue reelin possesses proangiogenic activities that ensure the communication of endothelial cells (ECs) with the glia to control neuronal migration and the establishment of the blood-brain barrier in the mouse brain. Apolipoprotein E receptor 2 (ApoER2) and Disabled1 (Dab1) expressed in ECs are required for vascularization of the retina and the cerebral cortex. Deletion of Dab1 in ECs leads to a reduced secretion of laminin-α4 and decreased activation of integrin-ß1 in glial cells, which in turn control neuronal migration and barrier properties of the NVU. Thus, reelin signaling in the endothelium is an instructive and integrative cue essential for neuro-glia-vascular communication.


Assuntos
Comunicação Celular , Córtex Cerebral/irrigação sanguínea , Endotélio Vascular/fisiologia , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/fisiologia , Neurônios/fisiologia , Vasos Retinianos/fisiologia , Animais , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/fisiologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular , Endotélio Vascular/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Deleção de Genes , Integrina beta1/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Laminina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/metabolismo , Vasos Retinianos/citologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais
17.
Dev Cell ; 46(2): 189-203.e4, 2018 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-30016621

RESUMO

The ability to form specific cell-cell connections within complex cellular environments is critical for multicellular organisms. However, the underlying mechanisms of cell matching that instruct these connections remain elusive. Here, we quantitatively explored the dynamics and regulation of cell matching processes utilizing Drosophila cardiogenesis. We found that cell matching is highly robust at boundaries between cardioblast (CB) subtypes, and filopodia of different CB subtypes have distinct binding affinities. Cdc42 is involved in regulating this selective filopodia binding adhesion and influences CB matching. Further, we identified adhesion molecules Fasciclin III (Fas3) and Ten-m, both of which also regulate synaptic targeting, as having complementary differential expression in CBs. Altering Fas3 expression changes differential filopodia adhesion and leads to CB mismatch. Furthermore, only when both Fas3 and Ten-m are lost is CB alignment severely impaired. Our results show that differential adhesion mediated by selective filopodia binding efficiently regulates precise and robust cell matching.


Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Adesão Celular/fisiologia , Proteínas de Drosophila/fisiologia , Tenascina/fisiologia , Animais , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Coração/fisiologia , Pseudópodes/metabolismo , Pseudópodes/fisiologia , Sinapses/fisiologia , Tenascina/metabolismo
18.
Elife ; 72018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30039797

RESUMO

In neural circuits, individual neurons often make projections onto multiple postsynaptic partners. Here, we investigate molecular mechanisms by which these divergent connections are generated, using dyadic synapses in C. elegans as a model. We report that C. elegans nrx-1/neurexin directs divergent connectivity through differential actions at synapses with partnering neurons and muscles. We show that cholinergic outputs onto neurons are, unexpectedly, located at previously undefined spine-like protrusions from GABAergic dendrites. Both these spine-like features and cholinergic receptor clustering are strikingly disrupted in the absence of nrx-1. Excitatory transmission onto GABAergic neurons, but not neuromuscular transmission, is also disrupted. Our data indicate that NRX-1 located at presynaptic sites specifically directs postsynaptic development in GABAergic neurons. Our findings provide evidence that individual neurons can direct differential patterns of connectivity with their post-synaptic partners through partner-specific utilization of synaptic organizers, offering a novel view into molecular control of divergent connectivity.


Assuntos
Animais Geneticamente Modificados/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Neurônios GABAérgicos/fisiologia , Junção Neuromuscular/fisiologia , Transmissão Sináptica , Acetilcolina/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Moléculas de Adesão Celular Neuronais/genética , Neurônios GABAérgicos/citologia , Junção Neuromuscular/citologia , Receptores Colinérgicos , Receptores Nicotínicos/metabolismo , Sinapses
19.
Elife ; 72018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-30044218

RESUMO

The nanoscale organization of neurotransmitter receptors regarding pre-synaptic release sites is a fundamental determinant of the synaptic transmission amplitude and reliability. How modifications in the pre- and post-synaptic machinery alignments affects synaptic currents, has only been addressed with computer modelling. Using single molecule super-resolution microscopy, we found a strong spatial correlation between AMPA receptor (AMPAR) nanodomains and the post-synaptic adhesion protein neuroligin-1 (NLG1). Expression of a truncated form of NLG1 disrupted this correlation without affecting the intrinsic AMPAR organization, shifting the pre-synaptic release machinery away from AMPAR nanodomains. Electrophysiology in dissociated and organotypic hippocampal rodent cultures shows these treatments significantly decrease AMPAR-mediated miniature and EPSC amplitudes. Computer modelling predicts that ~100 nm lateral shift between AMPAR nanoclusters and glutamate release sites induces a significant reduction in AMPAR-mediated currents. Thus, our results suggest the synapses necessity to release glutamate precisely in front of AMPAR nanodomains, to maintain a high synaptic responses efficiency.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Sinapses/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores , Feminino , Hipocampo/citologia , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Neurônios/citologia , Ratos , Transmissão Sináptica
20.
Int J Mol Sci ; 19(6)2018 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-29857505

RESUMO

The Fasciclin 1 (FAS1) domain is an ancient structural motif in extracellular proteins present in all kingdoms of life and particularly abundant in plants. The FAS1 domain accommodates multiple interaction surfaces, enabling it to bind different ligands. The frequently observed tandem FAS1 arrangement might both positively and negatively regulate ligand binding. Additional protein domains and post-translational modifications are partially conserved between different evolutionary clades. Human FAS1 family members are associated with multiple aspects of health and disease. At the cellular level, mammalian FAS1 proteins are implicated in extracellular matrix structure, cell to extracellular matrix and cell to cell adhesion, paracrine signaling, intracellular trafficking and endocytosis. Mammalian FAS1 proteins bind to the integrin family of receptors and to protein and carbohydrate components of the extracellular matrix. FAS1 protein encoding plant genes exert effects on cellulosic and non-cellulosic cell wall structure and cellular signaling but to establish the modes of action for any plant FAS1 protein still requires biochemical experimentation. In fungi, eubacteria and archaea, the differential presence of FAS1 proteins in closely related organisms and isolated biochemical data suggest functions in pathogenicity and symbiosis. The inter-kingdom comparison of FAS1 proteins suggests that molecular mechanisms mediating interactions between cells and their environment may have evolved at the earliest known stages of evolution.


Assuntos
Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Adesão Celular , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Humanos , Família Multigênica , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade
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