RESUMEN
HuD protein (also known as ELAVL4) has been shown to stabilize mRNAs with AU-rich elements (ARE) in their 3' untranslated regions (UTRs), including Gap43, which has been linked to axon growth. HuD also binds to neuritin (Nrn1) mRNA, whose 3'UTR contains ARE sequences. Although the Nrn1 3'UTR has been shown to mediate its axonal localization in embryonic hippocampal neurons, it is not active in adult dorsal root ganglion (DRG) neurons. Here, we asked why the 3'UTR is not sufficient to mediate the axonal localization of Nrn1 mRNA in DRG neurons. HuD overexpression increases the ability of the Nrn1 3'UTR to mediate axonal localizing in DRG neurons. HuD binds directly to the Nrn1 ARE with about a two-fold higher affinity than to the Gap43 ARE. Although the Nrn1 ARE can displace the Gap43 ARE from HuD binding, HuD binds to the full 3'UTR of Gap43 with higher affinity, such that higher levels of Nrn1 are needed to displace the Gap43 3'UTR. The Nrn1 3'UTR can mediate a higher level of axonal localization when endogenous Gap43 is depleted from DRG neurons. Taken together, our data indicate that endogenous Nrn1 and Gap43 mRNAs compete for binding to HuD for their axonal localization and activity of the Nrn1 3'UTR.
Asunto(s)
Regiones no Traducidas 3' , Axones/metabolismo , Proteína 4 Similar a ELAV/metabolismo , Proteína GAP-43/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Animales , Axones/ultraestructura , Secuencia de Bases , Unión Competitiva , Proteína 4 Similar a ELAV/genética , Proteína GAP-43/genética , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Hipocampo/metabolismo , Hipocampo/ultraestructura , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/ultraestructura , Neuropéptidos/genética , Cultivo Primario de Células , Unión Proteica , Ratas , Ratas Sprague-Dawley , Elementos de Respuesta , Transducción de SeñalRESUMEN
Although intra-axonal protein synthesis is well recognized in cultured neurons and during development in vivo, there have been few reports of mRNA localization and/or intra-axonal translation in mature CNS axons. Indeed, previous work indicated that mature CNS axons contain much lower quantities of translational machinery than PNS axons, leading to the conclusion that the capacity for intra-axonal protein synthesis is linked to the intrinsic capacity of a neuron for regeneration, with mature CNS neurons showing much less growth after injury than PNS neurons. However, when regeneration by CNS axons is facilitated, it is not known whether the intra-axonal content of translational machinery changes or whether mRNAs localize into these axons. Here, we have used a peripheral nerve segment grafted into the transected spinal cord of adult rats as a supportive environment for regeneration by ascending spinal axons. By quantitative fluorescent in situ hybridization combined with immunofluorescence to unambiguously distinguish intra-axonal mRNAs, we show that regenerating spinal cord axons contain ß-actin, GAP-43, Neuritin, Reg3a, Hamp, and Importin ß1 mRNAs. These axons also contain 5S rRNA, phosphorylated S6 ribosomal protein, eIF2α translation factor, and 4EBP1 translation factor inhibitory protein. Different levels of these mRNAs in CNS axons from regenerating PNS axons may relate to differences in the growth capacity of these neurons, although the presence of mRNA transport and likely local translation in both CNS and PNS neurons suggests an active role in the regenerative process. SIGNIFICANCE STATEMENT: Although peripheral nerve axons retain the capacity to locally synthesize proteins into adulthood, previous studies have argued that mature brain and spinal cord axons cannot synthesize proteins. Protein synthesis in peripheral nerve axons is increased during regeneration, and intra-axonally synthesized proteins have been shown to contribute to nerve regeneration. Here, we show that mRNAs and translational machinery are transported into axons regenerating from the spinal cord into the permissive environment of a peripheral nerve graft. Our data raise the possibility that spinal cord axons may make use of localized protein synthesis for regeneration.
Asunto(s)
Axones/metabolismo , Regeneración Nerviosa/fisiología , Iniciación de la Cadena Peptídica Traduccional/fisiología , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , Médula Espinal/fisiopatología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Ganglios Espinales/citología , Hepcidinas/genética , Hepcidinas/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Masculino , Proteínas Asociadas a Pancreatitis , Iniciación de la Cadena Peptídica Traduccional/genética , Traumatismos de los Nervios Periféricos/patología , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/patología , Traumatismos de la Médula Espinal/patologíaRESUMEN
High mobility group (HMG) proteins concentrate in the nucleus, interacting with chromatin. Amphoterin is an HMG protein (HMGB1) that has been shown to have extranuclear functions and can be secreted from some cell types. Exogenous amphoterin can increase neurite growth, suggesting that the secreted protein may have growth promoting activities in neurons. Consistent with this, we show that depletion of amphoterin mRNA from cultured adult rat DRG neurons attenuates neurite outgrowth, pointing to autocrine or paracrine mechanisms for its growth-promoting effects. The mRNA encoding amphoterin localizes to axonal processes and we showed recently that its 3'-UTR is sufficient for axonal localization of heterologous transcripts (Donnelly et al., 2013). Here, we show that amphoterin mRNA is transported constitutively into axons of adult DRG neurons. A preconditioning nerve injury increases the levels of amphoterin protein in axons without a corresponding increase in amphoterin mRNA in the axons. A 60 nucleotide region of the amphoterin mRNA 3'-UTR is necessary and sufficient for its localization into axons of cultured sensory neurons. Amphoterin mRNA 3'-UTR is also sufficient for axonal localization in distal axons of DRG neurons in vivo. Overexpression of axonally targeted amphoterin mRNA increases axon outgrowth in cultured sensory neurons, but axon growth is not affected when the overexpressed mRNA is restricted to the cell body.
Asunto(s)
Axones/metabolismo , Regulación de la Expresión Génica/genética , Proteína HMGB1/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/metabolismo , Células Receptoras Sensoriales/citología , Regiones no Traducidas 3'/genética , Animales , Axones/efectos de los fármacos , Transporte Biológico/genética , Células Cultivadas , Ganglios Espinales/citología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteína HMGB1/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fotoblanqueo , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/efectos de los fármacos , Transducción GenéticaRESUMEN
Many neuronal mRNAs are actively transported into distal axons. The 3' untranslated regions (UTRs) of axonal mRNAs often contain cues for their localization. The 3' UTR of neuritin mRNA was shown to be sufficient for localization into axons of hippocampal neurons. Here, we show that neuritin mRNA localizes into axons of rat sensory neurons, but this is predominantly driven by the 5' rather than 3' UTR. Neuritin mRNA shifts from cell body to axon predominantly after nerve crush injury, suggesting that it encodes a growth-associated protein. Consistent with this, overexpression of neuritin increases axon growth but only when its mRNA localizes into the axons.
Asunto(s)
Regiones no Traducidas 3'/fisiología , Regiones no Traducidas 5'/fisiología , Axones/metabolismo , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Neuronas/citología , ARN Mensajero/metabolismo , Análisis de Varianza , Animales , Células Cultivadas , Ganglios Espinales/citología , Regulación de la Expresión Génica/fisiología , Hipocampo/citología , Masculino , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Fotoblanqueo , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Transfección , Proteínas tau/metabolismoRESUMEN
Increasing evidence points to the importance of local protein synthesis for axonal growth and responses to axotomy, yet there is little insight into the functions of individual locally synthesized proteins. We recently showed that expression of a reporter mRNA with the axonally localizing ß-actin mRNA 3'UTR competes with endogenous ß-actin and GAP-43 mRNAs for binding to ZBP1 and axonal localization in adult sensory neurons (Donnelly et al., 2011). Here, we show that the 3'UTR of GAP-43 mRNA can deplete axons of endogenous ß-actin mRNA. We took advantage of this 3'UTR competition to address the functions of axonally synthesized ß-actin and GAP-43 proteins. In cultured rat neurons, increasing axonal synthesis of ß-actin protein while decreasing axonal synthesis of GAP-43 protein resulted in short highly branched axons. Decreasing axonal synthesis of ß-actin protein while increasing axonal synthesis of GAP-43 protein resulted in long axons with few branches. siRNA-mediated depletion of overall GAP-43 mRNA from dorsal root ganglia (DRGs) decreased the length of axons, while overall depletion of ß-actin mRNA from DRGs decreased the number of axon branches. These deficits in axon growth could be rescued by transfecting with siRNA-resistant constructs encoding ß-actin or GAP-43 proteins, but only if the mRNAs were targeted for axonal transport. Finally, in ovo electroporation of axonally targeted GAP-43 mRNA increased length and axonally targeted ß-actin mRNA increased branching of sensory axons growing into the chick spinal cord. These studies indicate that axonal translation of ß-actin mRNA supports axon branching and axonal translation of GAP-43 mRNA supports elongating growth.
Asunto(s)
Actinas/biosíntesis , Axones/metabolismo , Proteína GAP-43/fisiología , Ganglios Espinales/crecimiento & desarrollo , Ganglios Espinales/metabolismo , Actinas/fisiología , Animales , Axones/fisiología , Células Cultivadas , Embrión de Pollo , Proteína GAP-43/biosíntesis , Masculino , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Spinal cord injury (SCI) is a debilitating disease resulting in an estimated 18,000 new cases in the United States on an annual basis. Significant behavioral research on animal models has led to a large amount of data, some of which has been catalogued in the Open Data Commons for Spinal Cord Injury (ODC-SCI). More recently, high throughput sequencing experiments have been utilized to understand molecular mechanisms associated with SCI, with nearly 6,000 samples from over 90 studies available in the Sequence Read Archive. However, to date, no resource is available for efficiently mining high throughput sequencing data from SCI experiments. Therefore, we have developed a protocol for processing RNA-Seq samples from high-throughput sequencing experiments related to SCI resulting in both raw and normalized data that can be efficiently mined for comparisons across studies as well as homologous discovery across species. We have processed 1,196 publicly available RNA-seq samples from 50 bulk RNA-Seq studies across nine different species, resulting in an SQLite database that can be used by the SCI research community for further discovery. We provide both the database as well as a web-based front-end that can be used to query the database for genes of interest, differential gene expression, genes with high variance, and gene set enrichments.
RESUMEN
Spinal cord injury (SCI) is a debilitating condition with an estimated 18,000 new cases annually in the United States. The field has accepted and adopted standardized databases such as the Open Data Commons for Spinal Cord Injury (ODC-SCI) to aid in broader analyses, but these currently lack high-throughput data despite the availability of nearly 6000 samples from over 90 studies available in the Sequence Read Archive. This limits the potential for large datasets to enhance our understanding of SCI-related mechanisms at the molecular and cellular level. Therefore, we have developed a protocol for processing RNA-Seq samples from high-throughput sequencing experiments related to SCI resulting in both raw and normalized data that can be efficiently mined for comparisons across studies, as well as homologous discovery across species. We have processed 1196 publicly available RNA-Seq samples from 50 bulk RNA-Seq studies across nine different species, resulting in an SQLite database that can be used by the SCI research community for further discovery. We provide both the database as well as a web-based front-end that can be used to query the database for genes of interest, differential gene expression, genes with high variance, and gene set enrichments.
RESUMEN
Neurite outgrowth is essential for development of the nervous system. Neurotrophins including BDNF are among extracellular signals that regulate neurite outgrowth. The ERK1/2 pathway contributes to intracellular signaling networks transducing the pro-neuritic effects of BDNF. In the nucleolus, RNA polymerase-1 (Pol1)-mediated transcription regulates ribosomal biogenesis, enabling cellular protein synthesis and growth. Hence, we tested the possibility that Pol1 is an effector for pro-neuritic signals such as BDNF. We report that Pol1-mediated nucleolar transcription was increased by BDNF in an ERK1/2-dependent manner in rat forebrain neurons. Conversely, in cultured hippocampal neurons, knockdown of a Pol1 coactivator, transcription initiation factor 1A (TIF1A), attenuated BDNF- or ERK1/2-induced neurite outgrowth. Also, upon overexpression, a constitutively active mutant of TIF1A strongly promoted neurite outgrowth, including increases in total neurite length and branching. Finally, overexpression of wild-type TIF1A enhanced the pro-neuritic effects of ERK1/2 activation. These observations indicate that the Pol1-mediated nucleolar transcription regulates neurite outgrowth and serves as a major pro-neuritic effector of the BDNF-activated ERK1/2 pathway. Thus, development of the nervous system appears critically dependent on the nucleolus.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Nucléolo Celular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Neuritas/metabolismo , ARN Polimerasa I/metabolismo , Transcripción Genética/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Nucléolo Celular/genética , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Hipocampo/citología , Hipocampo/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prosencéfalo/citología , Prosencéfalo/metabolismo , ARN Polimerasa I/genética , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Although DNA damaging topoisomerase inhibitors induce apoptosis in developing neurons, their effects on adult neurons have not yet been characterized. We report a blockage of RNA-Polymerase-1-driven transcription and nucleolar stress in neocortical neurons of adult rats after intracarotid injection of the DNA-topoisomerase-2 inhibitor, etoposide. Intracerebroventricular injection of etoposide induced a similar response in neonatal rats. In contrast, etoposide triggered neuronal apoptosis in the neonates, but not the adults. Nucleolar disruption and apoptosis were also observed in etoposide-challenged cultured cortical neurons from newborn rats. In that system, activation of the DNA double strand break signaling kinase ataxia telangiectasia-mutated protein kinase, p53 and p53-dependent apoptosis required lower etoposide concentrations than did the p53-independent induction of nucleolar stress. These distinct responses may be coupled to different forms of etoposide-induced DNA damage. Indeed, double strand breaks by the over-expressed endonuclease I-Ppo1 were sufficient to induce p53-dependent apoptosis. Moreover, nucleolar transcription was insensitive to such damage implying single strand breaks and/or topoisomerase-2-DNA adducts as triggers of nucleolar stress. Because nucleolar stress is not age-restricted, it may underlie non-apoptotic neurotoxicity of chemotherapy- or neurodegeneration-associated DNA damage by reducing ribosomal biogenesis in adult brain. Conversely, nucleolar insensitivity to double strand breaks likely contributes to mature neuron tolerance of such lesions.
Asunto(s)
Antineoplásicos/toxicidad , Nucléolo Celular/efectos de los fármacos , Daño del ADN , Etopósido/toxicidad , Neuronas/efectos de los fármacos , Inhibidores de Topoisomerasa II/toxicidad , Factores de Edad , Animales , Animales Recién Nacidos , Antineoplásicos/administración & dosificación , Proteínas de la Ataxia Telangiectasia Mutada , Arterias Carótidas , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/ultraestructura , Células Cultivadas , Corteza Cerebral/citología , Cromosomas de los Mamíferos/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Etopósido/administración & dosificación , Inyecciones Intraarteriales , Inyecciones Intraventriculares , Masculino , Ratones , Neuronas/ultraestructura , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa I/fisiología , Ratas , Ratas Sprague-Dawley , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Reduced muscle mass and increased fatiguability are major complications after spinal cord injury (SCI), and often hinder the rehabilitation efforts of patients. Such detriments to the musculoskeletal system, and the concomitant reduction in level of activity, contribute to secondary complications such as cardiovascular disease, diabetes, bladder dysfunction and liver damage. As a result of decreased weight-bearing capacity after SCI, muscles undergo morphological, metabolic, and contractile changes. Recent studies have shown that exercise after SCI decreases muscle wasting and reduces the burden of secondary complications. Here, we describe RNA sequencing data for detecting chronic transcriptomic changes in the rat soleus after SCI at two levels of injury severity, under conditions of restricted in-cage activity and two methods of applied exercise, swimming or shallow water walking. We demonstrate that the sequenced data are of good quality and show a high alignment rate to the Rattus norvegicus reference assembly (Rn6). The raw data, along with UCSC Genome Browser tracks created to facilitate exploration of gene expression, are available in the NCBI Gene Expression Omnibus (GEO; GSE129694).
RESUMEN
The length of untranslated regions at the 3' end of transcripts (3'UTRs) is regulated by alternate polyadenylation (APA). 3'UTRs contain regions that harbor binding motifs for regulatory molecules. However, the mechanisms that coordinate the 3'UTR length of specific groups of transcripts are not well-understood. We therefore developed a method, CSI-UTR, that models 3'UTR structure as tandem segments between functional alternative-polyadenylation sites (termed cleavage site intervals-CSIs). This approach facilitated (1) profiling of 3'UTR isoform expression changes and (2) statistical enrichment of putative regulatory motifs. CSI-UTR analysis is UTR-annotation independent and can interrogate legacy data generated from standard RNA-Seq libraries. CSI-UTR identified a set of CSIs in human and rodent transcriptomes. Analysis of RNA-Seq datasets from neural tissue identified differential expression events within 3'UTRs not detected by standard gene-based differential expression analyses. Further, in many instances 3'UTR and CDS from the same gene were regulated differently. This modulation of motifs for RNA-interacting molecules with potential condition-dependent and tissue-specific RNA binding partners near the polyA signal and CSI junction may play a mechanistic role in the specificity of alternative polyadenylation. Source code, CSI BED files and example datasets are available at: https://github.com/UofLBioinformatics/CSI-UTR.
RESUMEN
Multi-organ dysfunction is a major complication after spinal cord injury (SCI). In addition to local injury within the spinal cord, SCI causes major disruption to the peripheral organ innervation and regulation. The liver contains sympathetic, parasympathetic, and small sensory axons. The bi-directional signaling of sensory dorsal root ganglion (DRG) neurons that provide both efferent and afferent information is of key importance as it allows sensory neurons and peripheral organs to affect each other. SCI-induced liver inflammation precedes and may exacerbate intraspinal inflammation and pathology after SCI, which may be modulated by activity and exercise. In this study, we collected comprehensive gene expression data through RNA sequencing of liver tissue from rats with chronic SCI to determine the effects of activity and exercise on those expression patterns. The sequenced data are of high quality and show a high alignment rate to the Rn6 genome. Gene expression is demonstrated for genes associated with known liver pathologies. UCSC Genome Browser expression tracks are provided with the data to facilitate exploration of the samples.
Asunto(s)
Hígado/metabolismo , Traumatismos de la Médula Espinal , Transcriptoma , Animales , Enfermedad Crónica , Actividad Motora , Condicionamiento Físico Animal , Ratas , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Spinal cord injury (SCI) is a devastating clinical condition resulting in significant disabilities. Apart from local injury within the spinal cord, SCI patients develop a myriad of complications including multi-organ dysfunction. Some of the dysfunctions may be directly or indirectly related to the sensory neurons of the dorsal root ganglia (DRG), which signal to both the spinal cord and the peripheral organs. After SCI, some classes of DRG neurons exhibit sensitization and undergo axonal sprouting both peripherally and centrally. Such physiological and anatomical re-organization after SCI contributes to both adaptive and maladaptive plasticity processes, which may be modulated by activity and exercise. In this study, we collected comprehensive gene expression data in whole DRG below the levels of the injury to compare the effects of SCI with and without two different forms of exercise in rats.
Asunto(s)
Ganglios Espinales/metabolismo , Traumatismos de la Médula Espinal , Transcriptoma , Animales , Conducta Animal , Plasticidad Neuronal , Neuronas Aferentes/metabolismo , Condicionamiento Físico Animal , Ratas , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
In post-mitotic neurons, the mechanisms of the apoptotic checkpoint that is activated by DNA damage remain unclear. Here we show that in cultured cortical neurons, the DNA damaging agent camptothecin (CPT) reduced transcription of rRNA and disrupted nucleolar staining for B23/nucleophosmin suggesting DNA damage-induced nucleolar stress. Although CPT activated the pro-apoptotic protein p53, the CPT-induced nucleolar stress was unaffected by p53 inhibition. In addition, brain-derived neurotrophic factor-mediated protection from CPT-induced apoptosis prevented neither nucleolar stress nor p53 activation. Therefore, inhibition of rRNA transcription might be upstream of the pro-apoptotic p53 activity. Indeed, short hairpin RNA-mediated inhibition of a RNA-Polymerase-I co-factor, transcription initiation factor IA, attenuated rRNA transcription causing nucleolar stress and p53-dependent neuronal apoptosis. The protein synthesis inhibitor cycloheximide blocked apoptosis that was induced by over-expressed shTIF-IA or active form of p53. Also, the general transcription inhibitor actinomycin D triggered nucleolar stress and activated p53. However, it did not induce apoptosis except at the low concentration of 0.05 microg/mL with stronger inhibitory activity against nucleolar than extranucleolar transcription. Hence, nucleolar stress-activated apoptosis requires extranucleolar transcription. This study identifies the nucleoli of post-mitotic neurons as sensors of DNA damage coupling reduced rRNA transcription to p53-mediated apoptosis that requires de novo expression of protein-coding genes. Thus, rDNA selectivity of DNA damage may determine its ability to induce neuronal apoptosis.
Asunto(s)
Apoptosis/fisiología , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Neuronas/metabolismo , Transcripción Genética/fisiología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Secuencia de Bases , Camptotecina/toxicidad , Nucléolo Celular/efectos de los fármacos , Células Cultivadas , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Daño del ADN/fisiología , Ratones , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/fisiología , Ratas , Ratas Sprague-Dawley , Transcripción Genética/efectos de los fármacosRESUMEN
Over the last decade there have been increasing efforts to develop three-dimensional (3D) scaffolds for bone tissue engineering from bioactive ceramics with 3D printing emerging as a promising technology. The overall objective of the present study was to generate a tissue engineered synthetic bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro, thereby mimicking the advantageous properties of autogenous bone grafts and facilitating usage for reconstructing segmental discontinuity defects in vivo. To this end, 3D scaffolds were developed from a silica-containing calcium alkali orthophosphate, using, first, a replica technique - the Schwartzwalder-Somers method - and, second, 3D printing, (i.e. rapid prototyping). The mechanical and physical scaffold properties and their potential to facilitate homogenous colonization by osteogenic cells and extracellular bone matrix formation throughout the porous scaffold architecture were examined. Osteoblastic cells were dynamically cultured for 7 days on both scaffold types with two different concentrations of 1.5 and 3 × 109 cells/l. The amount of cells and bone matrix formed and osteogenic marker expression were evaluated using hard tissue histology, immunohistochemical and histomorphometric analysis. 3D-printed scaffolds (RPS) exhibited more micropores, greater compressive strength and silica release. RPS seeded with 3 × 109 cells/l displayed greatest cell and extracellular matrix formation, mineralization and osteocalcin expression. In conclusion, RPS displayed superior mechanical and biological properties and facilitated generating a tissue engineered synthetic bone graft in vitro, which mimics the advantageous properties of autogenous bone grafts, by containing homogenously distributed terminally differentiated osteoblasts and mineralizing bone matrix and therefore is suitable for subsequent in vivo implantation for regenerating segmental discontinuity bone defects. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Matriz Ósea/efectos de los fármacos , Trasplante Óseo , Calcificación Fisiológica/efectos de los fármacos , Cerámica/farmacología , Osteoblastos/citología , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Línea Celular , Fuerza Compresiva , Iones , Porosidad , Solubilidad , Andamios del Tejido/químicaRESUMEN
How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size-regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin ß1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3' untranslated region (3'UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3'UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling.
Asunto(s)
Axones/metabolismo , Ganglios Espinales/lesiones , Biosíntesis de Proteínas , Nervio Ciático/lesiones , Serina-Treonina Quinasas TOR/biosíntesis , Regiones no Traducidas 3' , Animales , Tamaño de la Célula , Ratones , Ratones Endogámicos , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Endogámicas BB , Ratas Sprague-Dawley , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , NucleolinaRESUMEN
Mammalian spermatogenesis occurs in a precise and coordinated manner in the seminiferous tubules. One of the attempts to understand the detailed biological process during mammalian spermatogenesis at the molecular level has been to identify the testis specific genes followed by study of the testicular expression pattern of the genes. From the subtracted cDNA library of rat testis prepared using representational difference analysis (RDA) method, a complimentary DNA clone encoding type III member of a DnaJ family protein, DnaJC18, was cloned (GenBank Accession No. DQ158861). The full-length DnaJC18 cDNA has the longest open reading frame of 357 amino acids. Tissue and developmental Northern blot analysis revealed that the DnaJC18 gene was expressed specifically in testis and began to express from postnatal week 4 testis, respectively. In situ hybridization studies showed that DnaJC18 mRNA was expressed only during the maturation stages of late pachy- tene, round and elongated spermatids of adult rat testis. Western blot analysis with DnaJC18 antibody revealed that 41.2 kDa DnaJC18 protein was detected only in adult testis. Immunohistochemistry study further confirmed that DnaJC18 protein, was expressed in developing germ cells and the result was in concert with the in situ hybridization result. Confocal microscopy with GFP tagged DnaJC18 protein revealed that it was localized in the cytoplasm of cells. Taken together, these results suggested that testis specific DnaJC18, a member of the type III DnaJ protein family, might play a role during germ cell maturation in adult rat testis.
RESUMEN
Ryanodine receptors (RyRs) are highly conductive intracellular Ca2+ release channels and are widely expressed in many tissues, including the central nervous system. RyRs have been implicated in intracellular Ca2+ overload which can drive secondary damage following traumatic injury to the spinal cord (SCI), but the spatiotemporal expression of the three isoforms of RyRs (RyR1-3) after SCI remains unknown. Here, we analyzed the gene and protein expression of RyR isoforms in the murine lumbar dorsal root ganglion (DRG) and the spinal cord lesion site at 1, 2 and 7 d after a mild contusion SCI. Quantitative RT PCR analysis revealed that RyR3 was significantly increased in lumbar DRGs and at the lesion site at 1 and 2 d post contusion compared to sham (laminectomy only) controls. Additionally, RyR2 expression was increased at 1 d post injury within the lesion site. RyR2 and -3 protein expression was localized to lumbar DRG neurons and their spinal projections within the lesion site acutely after SCI. In contrast, RyR1 expression within the DRG and lesion site remained unaltered following trauma. Our study shows that SCI initiates acute differential expression of RyR isoforms in DRG and spinal cord.
Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina/genética , Traumatismos de la Médula Espinal/metabolismo , Animales , Ganglios Espinales/metabolismo , Expresión Génica , Ratones , Ratones Transgénicos , Isoformas de Proteínas/genéticaRESUMEN
How can cells sense their own size to coordinate biosynthesis and metabolism with their growth needs? We recently proposed a motor-dependent bidirectional transport mechanism for axon length and cell size sensing, but the nature of the motor-transported size signals remained elusive. Here, we show that motor-dependent mRNA localization regulates neuronal growth and cycling cell size. We found that the RNA-binding protein nucleolin is associated with importin ß1 mRNA in axons. Perturbation of nucleolin association with kinesins reduces its levels in axons, with a concomitant reduction in axonal importin ß1 mRNA and protein levels. Strikingly, subcellular sequestration of nucleolin or importin ß1 enhances axonal growth and causes a subcellular shift in protein synthesis. Similar findings were obtained in fibroblasts. Thus, subcellular mRNA localization regulates size and growth in both neurons and cycling cells.
Asunto(s)
Tamaño de la Célula , Neuronas Motoras/metabolismo , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Axones/metabolismo , Ratones Transgénicos , Neurogénesis , Biosíntesis de Proteínas/fisiología , NucleolinaRESUMEN
Cadmium is a widely used heavy metal in industry and affects the male reproductive system of animals, including humans, as a result of occupational and environmental exposures. However, the molecular mechanism underlying its effect on steroidogenesis in gonads remains unclear. In this study, we demonstrated that exposure of K28 mouse testicular Leydig tumor cells to cadmium led to a significant increase in the mRNA level, promoter activity and protein level of the steroidogenic acute regulatory protein (StAR), an essential factor for steroid biosynthesis. It has been well documented that StAR gene transcription is regulated by multiple transcription factors, including cAMP-responsive element binding protein (CREB) family members and SF-1. Cadmium treatment caused an increase in CREB phosphorylation but did not alter the CREB protein level in the nucleus. EMSA studies revealed that cadmium-induced phosphorylated CREB formed specific complexes with the proximal region of the StAR gene promoter. Furthermore, co-transfection with a CREB expression plasmid significantly increased cadmium-induced StAR promoter activity. However, the nuclear level and the affinity of SF-1 protein for the StAR proximal promoter were dramatically decreased upon exposure to cadmium. Taken together, these results suggest that cadmium up-regulates StAR gene expression through phosphorylated CREB rather than through SF-1 in mouse testicular Leydig cells.