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We report the first total syntheses of simonsol F (3), simonsinol (5), fargenin (4), and macranthol (6) in addition to syntheses of simonsol C (2), simonsol G (1), and honokiol (14). The syntheses are based upon a phosphonium ylide-mediated cascade reaction and upon natural product isomerization reactions which proceed through Cope rearrangements of putative biosynthetic dienone intermediates. As a corollary of the natural product isomerization reactions, we propose an alternative biosynthesis of honokiol (14), simonsinol (5), and macranthol (6) which unites the natural products in this family under a single common precursor, chavicol (7). Finally, we demonstrate that simonsol C (2) and simonsol F (3) promote axonal growth in primary mouse cortical neurons.
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Extracellular vesicles (EVs) are secreted by all cells in the CNS, including neurons and astrocytes. EVs are lipid membrane enclosed particles loaded with various bioactive cargoes reflecting the dynamic activities of cells of origin. In contrast to neurons, the specific role of EVs released by astrocytes is less well understood, partly due to the difficulty in maintaining primary astrocyte cultures in a quiescent state. The aim of this study was to establish a human serum-free astrocyte culture system that maintains primary astrocytes in a quiescent state to study the morphology, function, and protein cargoes of astrocyte-derived EVs. Serum-free medium with G5 supplement and serum-supplemented medium with 2% FBS were compared for the culture of commercially available human primary fetal astrocytes. Serum-free astrocytes displayed morphologies similar to in vivo astrocytes, and surprisingly, higher levels of astrocyte markers compared to astrocytes chronically cultured in FBS. In contrast, astrocyte and inflammatory markers in serum-free astrocytes were upregulated 24 h after either acute 2% FBS or cytokine exposure, confirming their capacity to become reactive. Importantly, this suggests that distinct signaling pathways are involved in acute and chronic astrocyte reactivity. Despite having a similar morphology, chronically serum-cultured astrocyte-derived EVs (ADEVs) were smaller in size compared to serum-free ADEVs and could reactivate serum-free astrocytes. Proteomic analysis identified distinct protein datasets for both types of ADEVs with enrichment of complement and coagulation cascades for chronically serum-cultured astrocyte-derived EVs, offering insights into their roles in the CNS. Collectively, these results suggest that human primary astrocytes cultured in serum-free medium bear similarities with in vivo quiescent astrocytes and the addition of serum induces multiple morphological and transcriptional changes that are specific to human reactive astrocytes and their ADEVs. Thus, more emphasis should be made on using multiple structural, molecular, and functional parameters when evaluating ADEVs as biomarkers of astrocyte health.
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Alzheimer's disease has been considered mostly as a neuronal pathology, although increasing evidence suggests that glial cells might play a key role in the disease onset and progression. In this sense, astrocytes, with their central role in neuronal metabolism and function, are of great interest for increasing our understanding of the disease. Thus, exploring the morphological and functional changes suffered by astrocytes along the course of this disorder has great therapeutic and diagnostic potential. In this work we isolated and cultivated astrocytes from symptomatic 9-10-months-old adult 3xTg-AD mice, with the aim of characterizing their phenotype and exploring their pathogenic potential. These "old" astrocytes occurring in the 3xTg-AD mouse model of Alzheimer's Disease presented high proliferation rate and differential expression of astrocytic markers compared with controls. They were neurotoxic to primary neuronal cultures both, in neuronal-astrocyte co-cultures and when their conditioned media (ACM) was added into neuronal cultures. ACM caused neuronal GSK3ß activation, changes in cytochrome c pattern, and increased caspase 3 activity, suggesting intrinsic apoptotic pathway activation. Exposure of neurons to ACM caused different subcellular responses. ACM application to the somato-dendritic domain in compartmentalised microfluidic chambers caused degeneration both locally in soma/dendrites and distally in axons. However, exposure of axons to ACM did not affect somato-dendritic nor axonal integrity. We propose that this newly described old 3xTg-AD neurotoxic astrocytic population can contribute towards the mechanistic understanding of the disease and shed light on new therapeutical opportunities.
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Enfermedad de Alzheimer , Síndromes de Neurotoxicidad , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Astrocitos/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Síndromes de Neurotoxicidad/metabolismoRESUMEN
Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored.We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons invivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5'-tRHs compose the great majority of tRNA-derived fragments observed invitro, a shift to 3'-tRNAs is observed in mature axons invivo.The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication.
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Axones/metabolismo , Comunicación Celular , Vesículas Extracelulares/metabolismo , Neuronas/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Transporte Biológico , Fraccionamiento Celular/métodos , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Anotación de Secuencia Molecular , Proyección Neuronal , Conformación de Ácido Nucleico , ARN Pequeño no Traducido/química , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Fracciones SubcelularesRESUMEN
Programmed cell death 4 (PDCD4) protein is a tumor suppressor that inhibits translation through the mTOR-dependent initiation factor EIF4A, but its functional role and mRNA targets in neurons remain largely unknown. Our work identified that PDCD4 is highly expressed in axons and dendrites of CNS and PNS neurons. Using loss- and gain-of-function experiments in cortical and dorsal root ganglia primary neurons, we demonstrated the capacity of PDCD4 to negatively control axonal growth. To explore PDCD4 transcriptome and translatome targets, we used Ribo-seq and uncovered a list of potential targets with known functions as axon/neurite outgrowth regulators. In addition, we observed that PDCD4 can be locally synthesized in adult axons in vivo, and its levels decrease at the site of peripheral nerve injury and before nerve regeneration. Overall, our findings demonstrate that PDCD4 can act as a new regulator of axonal growth via the selective control of translation, providing a target mechanism for axon regeneration and neuronal plasticity processes in neurons.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Axones/metabolismo , Dendritas/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Células Cultivadas , Mutación con Ganancia de Función , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Mutación con Pérdida de Función , Masculino , Ratones , Células PC12 , Cultivo Primario de Células , Biosíntesis de Proteínas , Proteínas de Unión al ARN/genética , Ratas , Regulación hacia ArribaRESUMEN
Both the establishment of neuronal polarity and axonal growth are crucial steps in the development of the nervous system. The local translation of mRNAs in the axon provides precise regulation of protein expression, and is now known to participate in axon development, pathfinding and synaptic formation and function. We have investigated the role of miR-26a in early stage mouse primary cortical neuron development. We show that micro-RNA-26a-5p (miR-26a) is highly expressed in neuronal cultures, and regulates both neuronal polarity and axon growth. Using compartmentalised microfluidic neuronal cultures, we identified a local role for miR-26a in the axon, where the repression of local synthesis of GSK3ß controls axon development and growth. Removal of this repression in the axon triggers local translation of GSK3ß protein and subsequent transport to the soma, where it can impact axonal growth. These results demonstrate how the axonal miR-26a can regulate local protein translation in the axon to facilitate retrograde communication to the soma and amplify neuronal responses, in a mechanism that influences axon development.
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Axones/metabolismo , Corteza Cerebral/citología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , MicroARNs/metabolismo , Neuronas/metabolismo , Animales , Línea Celular Tumoral , Polaridad Celular/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Neurogénesis/genética , Biosíntesis de Proteínas , Transporte de Proteínas/genética , TransfecciónRESUMEN
MicroRNAs (miRNAs) represent potential biomarkers for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). However, whether expression changes of individual miRNAs are simply an indication of cellular dysfunction and degeneration, or actually promote functional changes in target gene expression relevant to disease pathogenesis, is unclear. Here we used bioinformatics to test the hypothesis that ALS-associated miRNAs exert their effects through targeting genes implicated in disease etiology. We documented deregulated miRNAs identified in studies of ALS patients, noting variations in participants, tissue samples, miRNA detection or quantification methods used, and functional or bioinformatic assessments (if performed). Despite lack of experimental standardization, overlap of many deregulated miRNAs between studies was noted; however, direction of reported expression changes did not always concur. The use of in silico predictions of target genes for the most commonly deregulated miRNAs, cross-referenced to a selection of previously identified ALS genes, did not support our hypothesis. Specifically, although deregulated miRNAs were predicted to commonly target ALS genes, random miRNAs gave similar predictions. To further investigate biological patterns in the deregulated miRNAs, we grouped them by tissue source in which they were identified, indicating that for a core of frequently detected miRNAs, blood/plasma/serum may be useful for future profiling experiments. We conclude that in silico predictions of gene targets of deregulated ALS miRNAs, at least using currently available algorithms, are unlikely to be sufficient in informing disease pathomechanisms. We advocate experimental functional testing of candidate miRNAs and their predicted targets, propose miRNAs to prioritise, and suggest a concerted move towards protocol standardization for biomarker identification.
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Mutations in SWI/SNF genes are amongst the most common across all human cancers, but efficient therapeutic approaches that exploit vulnerabilities caused by SWI/SNF mutations are currently lacking. Here, we show that the SWI/SNF ATPases BRM/SMARCA2 and BRG1/SMARCA4 promote the expression of p62/GTF2H1, a core subunit of the transcription factor IIH (TFIIH) complex. Inactivation of either ATPase subunit downregulates GTF2H1 and therefore compromises TFIIH stability and function in transcription and nucleotide excision repair (NER). We also demonstrate that cells with permanent BRM or BRG1 depletion have the ability to restore GTF2H1 expression. As a consequence, the sensitivity of SWI/SNF-deficient cells to DNA damage induced by UV irradiation and cisplatin treatment depends on GTF2H1 levels. Together, our results expose GTF2H1 as a potential novel predictive marker of platinum drug sensitivity in SWI/SNF-deficient cancer cells.