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Introduction: Germinal Matrix-Intraventricular Haemorrhage (GM-IVH) is one of the most common neurological complications in preterm infants, which can lead to accumulation of cerebrospinal fluid (CSF) and is a major cause of severe neurodevelopmental impairment in preterm infants. However, the pathophysiological mechanisms triggered by GM-IVH are poorly understood. Analyzing the CSF that accumulates following IVH may allow the molecular signaling and intracellular communication that contributes to pathogenesis to be elucidated. Growing evidence suggests that miRs, due to their key role in gene expression, have a significant utility as new therapeutics and biomarkers. Methods: The levels of 2,083 microRNAs (miRs) in 15 CSF samples from 10 infants with IVH were measured using miRNA whole transcriptome sequencing. Gene ontology (GO) and miR family analysis were used to uncover dysregulated signalling which were then validated in vitro in human foetal neural progenitor cells treated with IVH-CSF. Results: Five hundred eighty-seven miRs were differentially expressed in the CSF extracted at least 2 months after injury, compared to CSF extracted within the first month of injury. GO uncovered key pathways targeted by differentially expressed miRs including the MAPK cascade and the JAK/STAT pathway. Astrogliosis is known to occur in preterm infants, and we hypothesized that this could be due to abnormal CSF-miR signaling resulting in dysregulation of the JAK/STAT pathway - a key controller of astrocyte differentiation. We then confirmed that treatment with IVH-CSF promotes astrocyte differentiation from human fetal NPCs and that this effect could be prevented by JAK/STAT inhibition. Taken together, our results provide novel insights into the CSF/NPCs crosstalk following perinatal brain injury and reveal novel targets to improve neurodevelopmental outcomes in preterm infants.
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We previously reported that kenpaullone, which inhibits GSK-3a/b and CDKs inhibited CCCP mediated mitochondrial depolarisation and augments the mitochondrial network. To investigate the actions of this class of drug further, we compared the ability of kenpaullone, alsterpaullone, 1-azakenapaullone, AZD5438, AT7519 (CDK and GSK-3a/b inhibitors) and dexpramipexole and olesoxime (mitochondrial permeability transition pore inhibitors) to prevent CCCP mediated mitochondrial depolarisation and found that AZD5438 and AT7519, were the most effective. Furthermore, treatment with AZD5438 alone increased the complexity of the mitochondrial network. We also found that AZD5438 prevented the rotenone induced decrease in PGC-1alpha and TOM20 levels and that it mediated powerful anti-apoptotic effects and promoted glycolytic respiration. Importantly, experiments in human iPSC derived cortical and midbrain neurons showed AZD5438 mediated significant protective effects, preventing the neuronal cell death, and collapse in the neurite and mitochondrial network associated with rotenone treatment. These results suggest drugs that target GSK-3a/b and CDKs should be developed and assessed further as they may have significant therapeutic potential.
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Neurônios , Rotenona , Humanos , Carbonil Cianeto m-Clorofenil Hidrazona , Imidazóis , Inibidores de Proteínas Quinases , Quinases Ciclina-DependentesRESUMO
SAFB1 is a DNA and RNA binding protein that is highly expressed in the cerebellum and hippocampus and is involved in the processing of coding and non-coding RNAs, splicing and dendritic function. We analyzed SAFB1 expression in the post-mortem brain tissue of spinocerebellar ataxia (SCA), Huntington's disease (HD), Multiple sclerosis (MS), Parkinson's disease patients and controls. In SCA cases, the expression of SAFB1 in the nucleus was increased and there was abnormal and extensive expression in the cytoplasm where it co-localized with the markers of Purkinje cell injury. Significantly, no SAFB1 expression was found in the cerebellar neurons of the dentate nucleus in control or MS patients; however, in SCA patients, SAFB1 expression was increased significantly in both the nucleus and cytoplasm of dentate neurons. In HD, we found that SAFB1 expression was increased in the nucleus and cytoplasm of striatal neurons; however, there was no SAFB1 staining in the striatal neurons of controls. In PD substantia nigra, we did not see any changes in neuronal SAFB1 expression. iCLIP analysis found that SAFB1 crosslink sites within ATXN1 RNA were adjacent to the start and within the glutamine repeat sequence. Further investigation found increased binding of SAFB1 to pathogenic ATXN1-85Q mRNA. These novel data strongly suggest SAFB1 contributes to the etiology of SCA and Huntington's chorea and that it may be a pathological marker of polyglutamine repeat expansion diseases.
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Encéfalo/metabolismo , Doença de Huntington/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Neurônios/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores de Estrogênio/metabolismo , Ataxias Espinocerebelares/metabolismo , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Feminino , Humanos , Doença de Huntington/patologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Neurônios/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Ataxias Espinocerebelares/patologiaRESUMO
Genetic and biochemical evidence points to an association between mitochondrial dysfunction and Parkinson's disease (PD). PD-associated mutations in several genes have been identified and include those encoding PTEN-induced putative kinase 1 (PINK1) and parkin. To identify genes, pathways, and pharmacological targets that modulate the clearance of damaged or old mitochondria (mitophagy), here we developed a high-content imaging-based assay of parkin recruitment to mitochondria and screened both a druggable genome-wide siRNA library and a small neuroactive compound library. We used a multiparameter principal component analysis and an unbiased parameter-agnostic machine-learning approach to analyze the siRNA-based screening data. The hits identified in this analysis included specific genes of the ubiquitin proteasome system, and inhibition of ubiquitin-conjugating enzyme 2 N (UBE2N) with a specific antagonist, Bay 11-7082, indicated that UBE2N modulates parkin recruitment and downstream events in the mitophagy pathway. Screening of the compound library identified kenpaullone, an inhibitor of cyclin-dependent kinases and glycogen synthase kinase 3, as a modulator of parkin recruitment. Validation studies revealed that kenpaullone augments the mitochondrial network and protects against the complex I inhibitor MPP+. Finally, we used a microfluidics platform to assess the timing of parkin recruitment to depolarized mitochondria and its modulation by kenpaullone in real time and with single-cell resolution. We demonstrate that the high-content imaging-based assay presented here is suitable for both genetic and pharmacological screening approaches, and we also provide evidence that pharmacological compounds modulate PINK1-dependent parkin recruitment.
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Mitocôndrias/metabolismo , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Benzazepinas/química , Benzazepinas/metabolismo , Benzazepinas/farmacologia , Células HeLa , Humanos , Hidrazonas/química , Hidrazonas/metabolismo , Hidrazonas/farmacologia , Indóis/química , Indóis/metabolismo , Indóis/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Análise de Componente Principal , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Interferência de RNA , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genéticaRESUMO
The medial prefrontal cortex (mPFC) is known to be critical for specific forms of long-term recognition memory, however the cellular mechanisms in the mPFC that underpin memory maintenance have not been well characterized. This study examined the importance of phosphorylation of cAMP responsive element binding protein (CREB) in the mPFC for different forms of long-term recognition memory in the rat. Adenoviral transduction of the mPFC with a dominant-negative inhibitor of CREB impaired object-in-place memory following a 6 or 24 h retention delay, but no impairment was observed following delays of 5 min or 3 h. Long-term object temporal order memory and spatial temporal order memory was also impaired. In contrast, there were no impairments in novel object recognition or object location memory. These results establish, for the first time, the importance of CREB phosphorylation within the mPFC for memory of associative and temporal information crucial to recognition.
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Associação , Proteína de Ligação a CREB/fisiologia , Memória de Longo Prazo/fisiologia , Córtex Pré-Frontal/metabolismo , Reconhecimento Psicológico/fisiologia , Memória Espacial/fisiologia , Transcrição Gênica/genética , Animais , Comportamento Animal/fisiologia , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Dependovirus , Masculino , Fosforilação/fisiologia , RatosRESUMO
BACKGROUND: Preterm infants are at risk of germinal matrix haemorrhage-intraventricular haemorrhage (GMH-IVH) which leads to post-haemorrhagic hydrocephalus (PHH) in 30% of infants; this is associated with moderate-severe neurodevelopmental impairment and confers significant risk of cerebral palsy. There are however no predictive indicators of the severity or long-term outcome after GMH-IVH. In recent years, endosome-derived extracellular vesicles (EVs) or exosomes have been isolated from biofluids and shown to mediate intercellular communication via selective enrichment in proteins and micro-RNAs. METHODS: This study aimed to isolate and characterise EVs from the cerebrospinal fluid (CSF) of 3 preterm infants with PHH using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) with immunogold protein labelling, and micro-RNA analysis. RESULTS: NTA of unaltered CSF revealed a heterogeneous and dynamic population of EVs. Exosomal-sized EVs were isolated by differential ultracentrifugation and TEM confirmed the presence of CD63+ and CD81+ exosomes. The micro-RNAs miR-9, miR-17, miR-26a, miR-124 and miR-1911 were detected within the exosome-enriched fraction and profiled over time. CONCLUSION: This is the first reported characterisation of exosomes from the CSF of preterm infants with post-haemorrhagic hydrocephalus.
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Hemorragia Cerebral/líquido cefalorraquidiano , Exossomos/metabolismo , Hidrocefalia/líquido cefalorraquidiano , MicroRNAs/líquido cefalorraquidiano , Hemorragia Cerebral/complicações , Humanos , Hidrocefalia/etiologia , Recém-Nascido , Recém-Nascido PrematuroRESUMO
The overall survival for patients with primary glioblastoma is very poor. Glioblastoma contains a subpopulation of glioma stem cells (GSC) that are responsible for tumour initiation, treatment resistance and recurrence. PPARα is a transcription factor involved in the control of lipid, carbohydrate and amino acid metabolism. We have recently shown that PPARα gene and protein expression is increased in glioblastoma and has independent clinical prognostic significance in multivariate analyses. In this work, we report that PPARα is overexpressed in GSC compared to foetal neural stem cells. To investigate the role of PPARα in GSC, we knocked down its expression using lentiviral transduction with short hairpin RNA (shRNA). Transduced GSC were tagged with luciferase and stereotactically xenografted into the striatum of NOD-SCID mice. Bioluminescent and magnetic resonance imaging showed that knockdown (KD) of PPARα reduced the tumourigenicity of GSC in vivo. PPARα-expressing control GSC xenografts formed invasive histological phenocopies of human glioblastoma, whereas PPARα KD GSC xenografts failed to establish viable intracranial tumours. PPARα KD GSC showed significantly reduced proliferative capacity and clonogenic potential in vitro with an increase in cellular senescence. In addition, PPARα KD resulted in significant downregulation of the stem cell factors c-Myc, nestin and SOX2. This was accompanied by downregulation of the PPARα-target genes and key regulators of fatty acid oxygenation ACOX1 and CPT1A, with no compensatory increase in glycolytic flux. These data establish the aberrant overexpression of PPARα in GSC and demonstrate that this expression functions as an important regulator of tumourigenesis, linking self-renewal and the malignant phenotype in this aggressive cancer stem cell subpopulation. We conclude that targeting GSC PPARα expression may be a therapeutically beneficial strategy with translational potential as an adjuvant treatment. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Neoplasias Encefálicas/patologia , Glioblastoma/patologia , PPAR alfa/metabolismo , RNA Interferente Pequeno/farmacologia , Animais , Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes/métodos , Humanos , Lentivirus , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/patologia , Fenótipo , Transdução de Sinais/fisiologia , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Olfactory ensheathing cell (OEC) transplantation is a promising strategy for treating spinal cord injury (SCI), as has been demonstrated in experimental SCI models and naturally occurring SCI in dogs. However, the presence of chondroitin sulphate proteoglycans within the extracellular matrix of the glial scar can inhibit efficient axonal repair and limit the therapeutic potential of OECs. Here we have used lentiviral vectors to genetically modify canine OECs to continuously deliver mammalian chondroitinase ABC at the lesion site in order to degrade the inhibitory chondroitin sulphate proteoglycans in a rodent model of spinal cord injury. We demonstrate that these chondroitinase producing canine OECs survived at 4 weeks following transplantation into the spinal cord lesion and effectively digested chondroitin sulphate proteoglycans at the site of injury. There was evidence of sprouting within the corticospinal tract rostral to the lesion and an increase in the number of corticospinal axons caudal to the lesion, suggestive of axonal regeneration. Our results indicate that delivery of the chondroitinase enzyme can be achieved with the genetically modified OECs to increase axon growth following SCI. The combination of these two promising approaches is a potential strategy for promoting neural regeneration following SCI in veterinary practice and human patients.
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Axônios , Condroitina ABC Liase/biossíntese , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Doenças do Cão/metabolismo , Mucosa Olfatória/transplante , Traumatismos da Medula Espinal/veterinária , Animais , Doenças do Cão/patologia , Cães , Mucosa Olfatória/citologia , Mucosa Olfatória/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Abnormal alpha-synuclein (α-synuclein) expression and aggregation is a key characteristic of Parkinson's disease (PD). However, the exact mechanism(s) linking α-synuclein to the other central feature of PD, dopaminergic neuron loss, remains unclear. Therefore, improved cell and in vivo models are needed to investigate the role of α-synuclein in dopaminergic neuron loss. MicroRNA-7 (miR-7) regulates α-synuclein expression by binding to the 3' UTR of the Synuclein Alpha Non A4 Component of Amyloid Precursor (SNCA) gene and inhibiting its translation. We show that miR-7 is decreased in the substantia nigra of patients with PD and, therefore, may play an essential role in the regulation of α-synuclein expression. Furthermore, we have found that lentiviral-mediated expression of miR-7 complementary binding sites to stably induce a loss of miR-7 function results in an increase in α-synuclein expression in vitro and in vivo. We have also shown that depletion of miR-7 using a miR-decoy produces a loss of nigral dopaminergic neurons accompanied by a reduction of striatal dopamine content. These data suggest that miR-7 has an important role in the regulation of α-synuclein and dopamine physiology and may provide a new paradigm to study the pathology of PD.
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Neurônios Dopaminérgicos/metabolismo , MicroRNAs/metabolismo , Substância Negra/metabolismo , alfa-Sinucleína/metabolismo , Animais , Humanos , Lentivirus/genética , Locomoção/genética , Locomoção/fisiologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/genéticaRESUMO
Recognition memory enables us to judge whether we have encountered a stimulus before and to recall associated information, including where the stimulus was encountered. The perirhinal cortex (PRh) is required for judgment of stimulus familiarity, while hippocampus (HPC) and medial prefrontal cortex (mPFC) are additionally involved when spatial information associated with a stimulus needs to be remembered. While gene expression is known to be essential for the consolidation of long-term recognition memory, the underlying regulatory mechanisms are not fully understood. Here we investigated the roles of two epigenetic mechanisms, DNA methylation and histone deacetylation, in recognition memory. Infusion of DNA methyltransferase inhibitors into PRh impaired performance in novel object recognition and object-in-place tasks while infusions into HPC or mPFC impaired object-in-place performance only. In contrast, inhibition of histone deacetylases in PRh, but not mPFC, enhanced recognition memory. These results support the emerging role of epigenetic processes in learning and memory.
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We have used transcriptome analysis to identify genes and pathways that are activated during recognition memory formation in the perirhinal cortex. Rats were exposed to objects either repeatedly, so that the objects become familiar, or to novel objects in a bow-tie maze over six consecutive days. On the final day, one hour after the last exposure to the series of objects, RNA from the perirhinal cortex was sequenced to compare the transcriptome of naïve control rats and rats exposed to either novel or familiar stimuli. Differentially expressed genes were identified between group Novel and group Familiar rats. These included genes coding for transcription factors, GDNF receptors and extracellular matrix-related proteins. Moreover, differences in alternative splicing were also detected between the two groups, which suggests that this post-transcriptional mechanism may play a role in the consolidation of object recognition memory. To conclude, this study shows that RNA sequencing can be used as a tool to identify differences in gene expression in behaving animals undergoing the same task but encountering different exposures.
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Córtex Perirrinal/metabolismo , Reconhecimento Psicológico/fisiologia , Transcriptoma , Processamento Alternativo , Animais , Expressão Gênica , Ontologia Genética , Masculino , Aprendizagem em Labirinto/fisiologia , RatosRESUMO
Episodic memory formation depends on information about a stimulus being integrated within a precise spatial and temporal context, a process dependent on the hippocampus and prefrontal cortex. Investigations of putative functional interactions between these regions are complicated by multiple direct and indirect hippocampal-prefrontal connections. Here application of a pharmacogenetic deactivation technique enabled us to investigate the mnemonic contributions of two direct hippocampal-medial prefrontal cortex (mPFC) pathways, one arising in the dorsal CA1 (dCA1) and the other in the intermediate CA1 (iCA1). While deactivation of either pathway impaired episodic memory, the resulting pattern of mnemonic deficits was different: deactivation of the dCA1âmPFC pathway selectively disrupted temporal order judgments while iCA1âmPFC pathway deactivation disrupted spatial memory. These findings reveal a previously unsuspected division of function among CA1 neurons that project directly to the mPFC. Such subnetworks may enable the distinctiveness of contextual information to be maintained in an episodic memory circuit.
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Hipocampo/fisiologia , Memória Episódica , Vias Neurais/fisiologia , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Masculino , Rede Nervosa/fisiologia , Ratos , Memória Espacial/fisiologiaRESUMO
RNA-binding proteins play a central role in cellular metabolism by orchestrating the complex interactions of coding, structural and regulatory RNA species. The SAFB (scaffold attachment factor B) proteins (SAFB1, SAFB2 and SAFB-like transcriptional modulator, SLTM), which are highly conserved evolutionarily, were first identified on the basis of their ability to bind scaffold attachment region DNA elements, but attention has subsequently shifted to their RNA-binding and protein-protein interactions. Initial studies identified the involvement of these proteins in the cellular stress response and other aspects of gene regulation. More recently, the multifunctional capabilities of SAFB proteins have shown that they play crucial roles in DNA repair, processing of mRNA and regulatory RNA, as well as in interaction with chromatin-modifying complexes. With the advent of new techniques for identifying RNA-binding sites, enumeration of individual RNA targets has now begun. This review aims to summarise what is currently known about the functions of SAFB proteins.
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Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Reparo do DNA/genética , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismoRESUMO
We explored the response of a panel of selected microRNAs (miRNAs) in neuroprotection produced by ischemic preconditioning. Hippocampal neuronal cultures were exposed to a 30-min oxygen-glucose deprivation (OGD). In our hands, this duration of OGD does not result in neuronal loss in vitro but significantly reduces neuronal death from a subsequent 'lethal' OGD insult. RT-qPCR was used to determine the expression of 16 miRNAs of interest at 1 and 24-h post-OGD. One miRNA (miR-98) was significantly decreased at 1-h post-OGD. Ten miRNAs (miR-9, miR-21, miR-29b, miR-30e, miR-101a, miR-101b, miR-124a, miR-132, miR-153, miR-204) were increased significantly at 24-h post-OGD. No miRNAs were decreased at 24-h. The increases observed in the 24-h group suggested that these miRNAs might play a role in preconditioning-induced neuroprotection. We selected the widely studied miR-132, a brain enriched, CREB regulated miRNA, to explore its role in simulated ischemic insults. We found that hippocampal neurons transduced with lentiviral vectors expressing miR-132 were protected from OGD and NMDA treatment, but not hydrogen peroxide. These findings add to the growing literature that targeting neuroprotective pathways controlled by miRNAs may represent a therapeutic strategy for the treatment of ischemic brain injury.
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Glucose/deficiência , MicroRNAs/genética , Neurônios/metabolismo , Oxigênio/metabolismo , Regulação para Cima , Animais , Hipóxia Celular , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Glucose/metabolismo , Hipocampo/irrigação sanguínea , Hipocampo/citologia , Peróxido de Hidrogênio/toxicidade , Precondicionamento Isquêmico , MicroRNAs/metabolismo , N-Metilaspartato/toxicidade , Neurônios/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
BACKGROUND: SAFB1 is a RNA binding protein implicated in the regulation of multiple cellular processes such as the regulation of transcription, stress response, DNA repair and RNA processing. To gain further insight into SAFB1 function we used iCLIP and mapped its interaction with RNA on a genome wide level. RESULTS: iCLIP analysis found SAFB1 binding was enriched, specifically in exons, ncRNAs, 3' and 5' untranslated regions. SAFB1 was found to recognise a purine-rich GAAGA motif with the highest frequency and it is therefore likely to bind core AGA, GAA, or AAG motifs. Confirmatory RT-PCR experiments showed that the expression of coding and non-coding genes with SAFB1 cross-link sites was altered by SAFB1 knockdown. For example, we found that the isoform-specific expression of neural cell adhesion molecule (NCAM1) and ASTN2 was influenced by SAFB1 and that the processing of miR-19a from the miR-17-92 cluster was regulated by SAFB1. These data suggest SAFB1 may influence alternative splicing and, using an NCAM1 minigene, we showed that SAFB1 knockdown altered the expression of two of the three NCAM1 alternative spliced isoforms. However, when the AGA, GAA, and AAG motifs were mutated, SAFB1 knockdown no longer mediated a decrease in the NCAM1 9-10 alternative spliced form. To further investigate the association of SAFB1 with splicing we used exon array analysis and found SAFB1 knockdown mediated the statistically significant up- and downregulation of alternative exons. Further analysis using RNAmotifs to investigate the frequency of association between the motif pairs (AGA followed by AGA, GAA or AAG) and alternative spliced exons found there was a highly significant correlation with downregulated exons. Together, our data suggest SAFB1 will play an important physiological role in the central nervous system regulating synaptic function. We found that SAFB1 regulates dendritic spine density in hippocampal neurons and hence provide empirical evidence supporting this conclusion. CONCLUSIONS: iCLIP showed that SAFB1 has previously uncharacterised specific RNA binding properties that help coordinate the isoform-specific expression of coding and non-coding genes. These genes regulate splicing, axonal and synaptic function, and are associated with neuropsychiatric disease, suggesting that SAFB1 is an important regulator of key neuronal processes.
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Antígeno CD56/genética , Expressão Gênica , Glicoproteínas/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas do Tecido Nervoso/genética , Proteínas Associadas à Matriz Nuclear/genética , Splicing de RNA , Receptores de Estrogênio/genética , Processamento Alternativo , Antígeno CD56/metabolismo , Regulação para Baixo , Glicoproteínas/metabolismo , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Receptores de Estrogênio/metabolismo , Regulação para CimaRESUMO
Cerebral Dopamine Neurotrophic Factor (CDNF) and Mesencephalic Astrocyte-derived Neurotrophic factor (MANF) are members of a recently discovered family of neurotrophic factors (NTFs). Here, we used intranigral or intrastriatal lentiviral vector-mediated expression to evaluate their efficacy at protecting dopaminergic function in the 6-OHDA model of Parkinson's disease (PD). In contrast to the well-studied Glial-Derived Neurotrophic Factor (GDNF), no beneficial effects were demonstrated by striatal overexpression of either protein. Interestingly, nigral overexpression of CDNF decreased amphetamine-induced rotations and increased tyroxine hydroxylase (TH) striatal fiber density but had no effect on numbers of TH(+) cells in the SN. Nigral MANF overexpression had no effect on amphetamine-induced rotations or TH striatal fiber density but resulted in a significant preservation of TH(+) cells. Combined nigral overexpression of both factors led to a robust reduction in amphetamine-induced rotations, greater increase in striatal TH-fiber density and significant protection of TH(+) cells in the SN. We conclude that nigral CDNF and MANF delivery is more efficacious than striatal delivery. This is also the first study to demonstrate that combined NTF can have synergistic effects that result in enhanced neuroprotection, suggesting that multiple NTF delivery may be more efficacious for the treatment of PD than the single NTF approaches attempted so far.
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Expressão Gênica , Fatores de Crescimento Neural/genética , Doença de Parkinson/genética , Substância Negra/metabolismo , Animais , Comportamento Animal , Linhagem Celular , Modelos Animais de Doenças , Ordem dos Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Lentivirus/genética , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Oxidopamina/efeitos adversos , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Ratos , Proteínas Recombinantes de Fusão , Substância Negra/patologia , Transdução Genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The complex process of skeletal muscle differentiation is organized by the myogenic regulatory factors (MRFs), Myf5, MyoD, Myf6, and myogenin, where myogenin plays a critical role in the regulation of the final stage of muscle differentiation. In an effort to investigate the role microRNAs (miRNAs) play in regulating myogenin, a bioinformatics approach was used and six miRNAs (miR-182, miR-186, miR-135, miR-491, miR-329, and miR-96) were predicted to bind the myogenin 3'-untranslated region (UTR). However, luciferase assays showed only miR-186 inhibited translation and 3'-UTR mutagenesis analysis confirmed this interaction was specific. Interestingly, the expression of miR-186 mirrored that of its host gene, ZRANB2, during development. Functional studies demonstrated that miR-186 overexpression inhibited the differentiation of C2C12 and primary muscle cells. Our findings therefore identify miR-186 as a novel regulator of myogenic differentiation.
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Regiões 3' não Traduzidas/fisiologia , Diferenciação Celular/fisiologia , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Miogenina/biossíntese , Animais , Linhagem Celular , Camundongos , MicroRNAs/genética , Músculo Esquelético/citologia , Miogenina/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Some higher vertebrates can display unique muscle regenerative abilities through dedifferentiation. Research evidence suggests that induced dedifferentiation can be achieved in mammalian cells. TWIST is a bHLH (basic helix-loop-helix) transcription factor that is expressed during embryonic development and plays critical roles in diverse developmental systems including myogenesis. Several experiments demonstrated its role in inhibition of muscle cell differentiation. We have previously shown that overexpression of TWIST can reverse muscle cell differentiation in the presence of growth factors. Here we show that TWIST reverses muscle cell differentiation through binding and down-regulation of myogenin. Moreover, it can reverse cellular morphology in the absence of growth factors.
Assuntos
Diferenciação Celular , Inativação Gênica , Mioblastos/fisiologia , Miogenina/genética , Proteínas Nucleares/fisiologia , Proteína 1 Relacionada a Twist/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Forma Celular , Meios de Cultura Livres de Soro , Regulação para Baixo , Camundongos , Dados de Sequência Molecular , Desenvolvimento Muscular , Miogenina/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição GênicaRESUMO
Although tumour suppressor gene hypermethylation is a universal feature of cancer cells, little is known about the necessary molecular triggers. Here, we show that Wilms' tumour 1 (WT1), a developmental master regulator that can also act as a tumour suppressor or oncoprotein, transcriptionally regulates the de novo DNA methyltransferase 3A (DNMT3A) and that cellular WT1 levels can influence DNA methylation of gene promoters genome-wide. Specifically, we demonstrate that depletion of WT1 by short-interfering RNAs leads to reduced DNMT3A in Wilms' tumour cells and human embryonal kidney-derived cell lines. Chromatin immunoprecipitation assays demonstrate WT1 recruitment to the DNMT3A promoter region and reporter assays confirm that WT1 directly transactivates DNMT3A expression. Consistent with this regulatory role, immunohistochemical analysis shows co-expression of WT1 and DNMT3A proteins in nuclei of blastemal cells in human fetal kidney and Wilms' tumours. Using genome-wide promoter methylation arrays, we show that human embryonal kidney cells over-expressing WT1 acquire DNA methylation changes at specific gene promoters where DNMT3A recruitment is increased, with hypermethylation being associated with silencing of gene expression. Elevated DNMT3A is also demonstrated at hypermethylated genes in Wilms' tumour cells, including a region of long-range epigenetic silencing. Finally, we show that depletion of WT1 in Wilms' tumour cells can lead to reactivation of gene expression from methylated promoters, such as TGFB2, a key modulator of epithelial-mesenchymal transitions. Collectively, our work defines a new regulatory modality for WT1 involving elicitation of epigenetic alterations which is most likely crucial to its functions in development and disease.