EDITORIAL article Front. Mol. Neurosci., 30 May 2023Sec. Neuroplasticity and Development Volume 16 - 2023 | https://doi.org/10.3389/fnmol.2023.1214853

Despite their latent neurogenic potential, most normal parenchymal astrocytes fail to dedifferentiate to neural stem cells in response to injury. In contrast, aberrant lineage plasticity is a hallmark of gliomas, and this suggests that tumor suppressors may constrain astrocyte dedifferentiation. Here, we show that p53, one of the most commonly inactivated tumor suppressors in glioma, is a gatekeeper of astrocyte fate. In the context of stab-wound injury, p53 loss destabilized the identity of astrocytes, priming them to dedifferentiate in later life. This resulted from persistent and age-exacerbated neuroinflammation at the injury site and EGFR activation in periwound astrocytes. Mechanistically, dedifferentiation was driven by the synergistic upregulation of mTOR signaling downstream of p53 loss and EGFR, which reinstates stemness programs via increased translation of neurodevelopmental transcription factors. Thus, our findings suggest that first-hit mutations remove the barriers to injury-induced dedifferentiation by sensitizing somatic cells to inflammatory signals, with implications for tumorigenesis.

Neurons are morphologically complex cells that rely on the compartmentalization of protein expression to develop and maintain their cytoarchitecture. The targeting of RNA transcripts to axons is one of the mechanisms that allows rapid local translation of proteins in response to extracellular signals. 3′ Untranslated regions (UTRs) of mRNA are noncoding sequences that play a critical role in determining transcript localization and translation by interacting with specific RNA-binding proteins (RBPs). However, how 3′ UTRs contribute to mRNA metabolism and the nature of RBP complexes responsible for these functions remains elusive. We performed 3′ end sequencing of RNA isolated from cell bodies and axons of sympathetic neurons exposed to either nerve growth factor (NGF) or neurotrophin 3 (NTF3, also known as NT-3). NGF and NTF3 are growth factors essential for sympathetic neuron development through distinct signaling mechanisms. Whereas NTF3 acts mostly locally, NGF signal is retrogradely transported from axons to cell bodies. We discovered that both NGF and NTF3 affect transcription and alternative polyadenylation in the nucleus and induce the localization of specific 3′ UTR isoforms to axons, including short 3′ UTR isoforms found exclusively in axons. The integration of our data with CLIP sequencing data supports a model whereby long 3′ UTR isoforms associate with RBP complexes in the nucleus and, upon reaching the axons, are remodeled locally into shorter isoforms. Our findings shed new light into the complex relationship between nuclear polyadenylation, mRNA localization, and local 3′ UTR remodeling in developing neurons.

ABSTRACT Background Neurons are morphologically complex cells that rely on the compartmentalization of protein expression to develop and maintain their cytoarchitecture. Targeting of RNA transcripts to axons is one of the mechanisms that allows rapid local translation of proteins in response to extracellular signals. 3’ untranslated regions (UTRs) of mRNA are non-coding sequences that play a critical role in determining transcript localisation and translation by interacting with specific RNA binding proteins (RBPs). However, how 3’UTRs contribute to mRNA metabolism and the nature of RBP complexes responsible for these functions remain elusive. Results We performed 3’ end sequencing of RNA isolated from axons and cell bodies of sympathetic neurons exposed to either Nerve Growth factor (NGF) or Neurotrophin 3 (NT3). NGF and NT3 are growth factors essential for sympathetic neuron development that act through distinct signalling mechanisms. Whereas NT3 is thought to act only locally, NGF signals back from axons to the cell bodies. We discovered that both NGF and NT3 affect transcription and alternative polyadenylation and induce the localisation of specific 3’UTR isoforms to axons. The finding that many transcripts with short 3’UTR were detected only in axons suggest that these may undergo local post-transcriptional remodelling. The integration of our data with CLIP-sequencing data revealed that long 3’UTR isoforms associate with RBP complexes in the nucleus, and once in axons, regulate cytoplasmic 3’ UTR isoform cleavage into shorter isoform. Conclusions Our findings shed new light on the complex interplay between nuclear polyadenylation, mRNA localisation and local 3’UTR remodelling in developing neurons. Graphical abstract

This protocol illustrates the use of an in vitro assay to study the cleavage of the IMPA1 30UTR by the endonuclease Ago2 in sympathetic neurons. The procedure includes the preparation of cytoplasmic protein extracts and also describes the synthesis and labeling of the RNA probe. The protocol can be applied to other cell systems, RNA transcripts, and endonucleases to confirm the role of known cleavage site(s) and cleavage proteins, or to investigate new ones.For complete details on the use and execution of this protocol, please refer to Andreassi et al. (2021).

The 3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localization and translation. Incorrect processing and delivery of mRNA cause severe developmental defects and have been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3' UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3' UTR and stable 3' UTR-derived fragments of unknown function. Cleavage of the long 3' UTR of Inositol Monophosphatase 1 (IMPA1) mediated by a protein complex containing the endonuclease argonaute 2 (Ago2) generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates an additional class of 3' UTR-derived RNAs.

The 3’ untranslated regions (3’UTRs) of messenger RNAs (mRNA) are non-coding sequences that regulate several aspects of mRNA metabolism, including intracellular localisation and translation. Here, we show that in sympathetic neuron axons, the 3’UTRs of many transcripts undergo cleavage, generating both translatable isoforms expressing a shorter 3’UTR, and 3’UTR fragments. 3’end RNA sequencing indicated that 3’UTR cleavage is a potentially widespread event in axons, which is mediated by a protein complex containing the endonuclease Ago2 and the RNA binding protein HuD. Analysis of the Inositol monophosphatase 1 ( Impa1 ) mRNA revealed that a stem loop structure within the 3’UTR is necessary for Ago2 cleavage. Thus, remodeling of the 3’UTR provides an alternative mechanism that simultaneously regulates local protein synthesis and generates a new class of 3’UTR RNAs with yet unknown functions.

Neurons are extraordinarily large and highly polarized cells that require rapid and efficient communication between cell bodies and axons over long distances. In peripheral neurons, transcripts are transported along axons to growth cones, where they are rapidly translated in response to extrinsic signals. While studying Tp53inp2, a transcript highly expressed and enriched in sympathetic neuron axons, we unexpectedly discovered that Tp53inp2 is not translated. Instead, the transcript supports axon growth in a coding-independent manner. Increasing evidence indicates that mRNAs may function independently of their coding capacity; for example, acting as a scaffold for functionally related proteins. The Tp53inp2 transcript interacts with the nerve growth factor (NGF) receptor TrkA, regulating TrkA endocytosis and signaling. Deletion of Tp53inp2 inhibits axon growth in vivo, and the defects are rescued by a non-translatable form of the transcript. Tp53inp2 is an atypical mRNA that regulates axon growth by enhancing NGF-TrkA signaling in a translation-independent manner.

Neurons modulate gene expression in response to extrinsic signals to enable brain development, cognition, and learning and to process stimuli that regulate systemic physiological functions. This signal-to-gene communication is facilitated by posttranslational modifications such as S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to cysteine thiols. In the cerebral cortex, S-nitrosylation of histone deacetylase 2 (HDAC2) is required for gene transcription during neuronal development, but few other nuclear targets of S-nitrosylation have been identified to date. We used S-nitrosothiol resin-assisted capture on NO donor-treated nuclear extracts from rat cortical neurons and identified 614 S-nitrosylated nuclear proteins. Of these, 131 proteins have not previously been shown to be S-nitrosylated in any system, and 555 are previously unidentified targets of S-nitrosylation in neurons. The sites of S-nitrosylation were identified for 59% of the targets, and motifs containing single lysines were found at 33% of these sites. In addition, lysine motifs were necessary for promoting the S-nitrosylation of HDAC2 and methyl-CpG binding protein 3 (MBD3). Moreover, S-nitrosylation of the histone-binding protein RBBP7 was necessary for dendritogenesis of cortical neurons in culture. Together, our findings characterize S-nitrosylated nuclear proteins in neurons and identify S-nitrosylation motifs that may be shared with other targets of NO signaling.

Neurons are morphologically complex cells that rely on the compartmentalization of protein expression to develop and maintain their extraordinary cytoarchitecture. This formidable task is achieved, at least in part, by targeting mRNA to subcellular compartments where they are rapidly translated. mRNA transcripts are the conveyor of genetic information from DNA to the translational machinery, however, they are also endowed with additional functions linked to both the coding sequence (open reading frame, or ORF) and the flanking 5' and 3' untranslated regions (UTRs), that may harbor coding-independent functions. In this review, we will highlight recent evidences supporting new coding-dependent and -independent functions of mRNA and discuss how nuclear and cytoplasmic post-transcriptional modifications of mRNA contribute to localization and translation in mammalian cells with specific emphasis on neurons. We also describe recently developed techniques that can be employed to study RNA dynamics at subcellular level in eukaryotic cells in developing and regenerating neurons.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease, characterized by degeneration of the anterior horn cells of the spinal cord. SMA presents with a highly variable phenotype ranging from very severe to mild (type No cure for SMA is available at present. All forms of SMA are caused by homozygous loss of the functional survival motor neuron (SMN1) gene. However, all patients have one or more copies of the SMN2 gene, nearly identical to SMN1. Both genes encode the SMN protein but the level produced by SMN2 is insufficient to protect from disease. Increasing SMN2 gene expression could be of considerable therapeutic importance. The aim of this study was to assess whether SMN2 gene expression can be increased by 4-phenylbutyrate (PBA). Fibroblast cell cultures from 16 SMA patients affected by different clinical severities were treated with PBA, and full-length SMN2 transcripts were measured by real-time PCR. In all cell cultures, except one, PBA treatment caused an increase in full-length SMN2 transcripts, ranging from 50 to 160% in type I and from 80 to 400% in type II and III cultures. PBA was found also effective in enhancing SMN protein levels and the number of SMN-containing nuclear structures (gems). These data show that SMN expression is considerably increased by PBA, and suggest that the compound, owing also to its favorable pharmacological properties, could be a good candidate for the treatment of SMA.

Epigenetic modifications of chromatin represent a fundamental mechanism by which eukaryotic cells adapt their transcriptional response to developmental and environmental cues. Although an increasing number of molecules have been linked to epigenetic changes, the intracellular pathways that lead to their activation/repression have just begun to be characterized. Here, we demonstrate that inositol hexakisphosphate kinase 1 (IP(6)K1), the enzyme responsible for the synthesis of the high-energy inositol pyrophosphates (IP7), is associated with chromatin and interacts with Jumonji domain containing 2C (JMJD2C), a recently identified histone lysine demethylase. Reducing IP(6)K1 levels by RNAi or using mouse embryonic fibroblasts derived from ip(6)k1(-/-) knockout mice results in a decreased IP7 concentration that epigenetically translates to reduced levels of trimethyl-histone H3 lysine 9 (H3K9me3) and increased levels of acetyl-H3K9. Conversely, expression of IP(6)K1 induces JMJD2C dissociation from chromatin and increases H3K9me3 levels, which depend on IP(6)K1 catalytic activity. Importantly, these effects lead to changes in JMJD2C-target gene transcription. Our findings demonstrate that inositol pyrophosphate signaling influences nuclear functions by regulating histone modifications.

Cell proliferation is associated with a high rate of aerobic glycolysis, which has been widely interpreted as a compensatory mechanism for suppressed mitochondrial function, despite reports of high respiration rates. The molecular mechanisms that link cell proliferation with mitochondrial metabolism, dynamics, and biogenesis remain obscure. Here, we show that proliferation is associated with an increase in both glycolysis and respiration, in conjunction with mitochondrial fusion and biogenesis. Changes in mitochondrial morphology and mass are due to accumulation of OPA1, MFN1, and TFAM, silencing any of which hinders cell proliferation. Moreover, the levels of OPA1, MFN1, and TFAM are regulated by the ubiquitin ligase APC/C-CDH1, which also controls proteasomal degradation of key glycolytic, glutaminolytic, and cell-cycle proteins. Thus, we have identified an important component of the molecular mechanism that coordinates cell proliferation with activation of the mitochondrial metabolic machinery that provides the necessary energy and biosynthetic substrates.

Cell proliferation is associated with a high rate of aerobic glycolysis, which has been widely interpreted as a compensatory mechanism for suppressed mitochondrial function, despite reports of high respiration rates. The molecular mechanisms that link cell proliferation with mitochondrial metabolism, dynamics, and biogenesis remain obscure. Here, we show that proliferation is associated with an increase in both glycolysis and respiration, in conjunction with mitochondrial fusion and biogenesis. Changes in mitochondrial morphology and mass are due to accumulation of OPA1, MFN1, and TFAM, silencing any of which hinders cell proliferation. Moreover, the levels of OPA1, MFN1, and TFAM are regulated by the ubiquitin ligase APC/C(CDH1), which also controls proteasomal degradation of key glycolytic, glutaminolytic, and cell-cycle proteins. Thus, we have identified an important component of the molecular mechanism that coordinates cell proliferation with activation of the mitochondrial metabolic machinery that provides the necessary energy and biosynthetic substrates.

Cell proliferation is associated with a high rate of aerobic glycolysis, which has been widely interpreted as a compensatory mechanism for suppressed mitochondrial function, despite reports of high respiration rates. The molecular mechanisms that link cell proliferation with mitochondrial metabolism, dynamics, and biogenesis remain obscure. Here, we show that proliferation is associated with an increase in both glycolysis and respiration, in conjunction with mitochondrial fusion and biogenesis. Changes in mitochondrial morphology and mass are due to accumulation of OPA1, MFN1, and TFAM, silencing any of which hinders cell proliferation. Moreover, the levels of OPA1, MFN1, and TFAM are regulated by the ubiquitin ligase APC/C(CDH1), which also controls proteasomal degradation of key glycolytic, glutaminolytic, and cell-cycle proteins. Thus, we have identified an important component of the molecular mechanism that coordinates cell proliferation with activation of the mitochondrial metabolic machinery that provides the necessary energy and biosynthetic substrates.

Nat. Neurosci. 13, 291–301 (2010); published online 31 January 2010; corrected after print 19 April 2010 In the version of this article initially published, the last name of the fifth author was misspelled. The correct name is Serena De Vita. The error has been corrected in the HTML and PDF versionsof the article.

Translation of localized mRNA is a fast and efficient way of reacting to extracellular stimuli with the added benefit of providing spatial resolution to the cellular response. The efficacy of this adaptive response ultimately relies on the ability to express a particular protein at the right time and in the right place. Although mRNA localization is a mechanism shared by most organisms, it is especially relevant in highly polarized cells, such as differentiated neurons. 3'-Untranslated regions (3'UTRs) of mRNAs are critical both for the targeting of transcripts to specific subcellular compartments and for translational control. Here we review recent studies that indicate how, in response to extracellular cues, nuclear and cytoplasmic remodeling of the 3'UTR contributes to mRNA localization and local protein synthesis.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease, characterized by degeneration of the anterior horn cells of the spinal cord. SMA presents with a highly variable phenotype ranging from very severe to mild (type I–III). No cure for SMA is available at present. All forms of SMA are caused by homozygous loss of the functional survival motor neuron (SMN1) gene. However, all patients have one or more copies of the SMN2 gene, nearly identical to SMN1. Both genes encode the SMN protein but the level produced by SMN2 is insufficient to protect from disease. Increasing SMN2 gene expression could be of considerable therapeutic importance. The aim of this study was to assess whether SMN2 gene expression can be increased by 4-phenylbutyrate (PBA). Fibroblast cell cultures from 16 SMA patients affected by different clinical severities were treated with PBA, and full-length SMN2 transcripts were measured by real-time PCR. In all cell cultures, except one, PBA treatment caused an increase in full-length SMN2 transcripts, ranging from 50 to 160% in type I and from 80 to 400% in type II and III cultures. PBA was found also effective in enhancing SMN protein levels and the number of SMN-containing nuclear structures (gems). These data show that SMN expression is considerably increased by PBA, and suggest that the compound, owing also to its favorable pharmacological properties, could be a good candidate for the treatment of SMA.