Short- and Long-Term Regulation of HuD: A Molecular Switch Mediated by Folic Acid?

The RNA-binding protein HuD has been shown to play a crucial role in gene regulation in the nervous system and is involved in various neurological and psychiatric diseases. In this study, through the creation of an interaction network on HuD and its potential targets, we identified a strong association between HuD and several diseases of the nervous system. Specifically, we focused on the relationship between HuD and the brain-derived neurotrophic factor (BDNF), whose protein is implicated in several neuronal diseases and is involved in the regulation of neuronal development, survival, and function. To better investigate this relationship and given that we previously demonstrated that folic acid (FA) is able to directly bind HuD itself, we performed in vitro experiments in neuron-like human SH-SY5Y cells in the presence of FA, also known to be a pivotal environmental factor influencing the nervous system development. Our findings show that FA exposure results in a significant increase in both HuD and BDNF transcripts and proteins after 2 and 4 h of treatment, respectively. Similar data were obtained after 2 h of FA incubation followed by 2 h of washout. This increase was no longer detected upon 24 h of FA exposure, probably due to a signaling shutdown mechanism. Indeed, we observed that following 24 h of FA exposure HuD is methylated. These findings indicate that FA regulates BDNF expression via HuD and suggest that FA can behave as an epigenetic modulator of HuD in the nervous system acting via short- and long-term mechanisms. Finally, the present results also highlight the potential of BDNF as a therapeutic target for specific neurological and psychiatric diseases.

The formation of HuR/YB1 complex is required for the stabilization of target mRNA to promote myogenesis.

mRNA stability is the mechanism by which cells protect transcripts allowing their expression to execute various functions that affect cell metabolism and fate. It is well-established that RNA binding proteins (RBPs) such as HuR use their ability to stabilize mRNA targets to modulate vital processes such as muscle fiber formation (myogenesis). However, the machinery and the mechanisms regulating mRNA stabilization are still elusive. Here, we identified Y-Box binding protein 1 (YB1) as an indispensable HuR binding partner for mRNA stabilization and promotion of myogenesis. Both HuR and YB1 bind to 409 common mRNA targets, 147 of which contain a U-rich consensus motif in their 3' untranslated region (3'UTR) that can also be found in mRNA targets in other cell systems. YB1 and HuR form a heterodimer that associates with the U-rich consensus motif to stabilize key promyogenic mRNAs. The formation of this complex involves a small domain in HuR (227-234) that if mutated prevents HuR from reestablishing myogenesis in siHuR-treated muscle cells. Together our data uncover that YB1 is a key player in HuR-mediated stabilization of pro-myogenic mRNAs and provide the first indication that the mRNA stability mechanism is as complex as other key cellular processes such as mRNA decay and translation.

Posttranscriptional Regulation of Gene Expression Participates in the Myelin Restoration in Mouse Models of Multiple Sclerosis: Antisense Modulation of HuR and HuD ELAV RNA Binding Protein.

Neuropathic pain is the most difficult-to-treat pain syndrome in multiple sclerosis. Evidence relates neuropathic pain to demyelination, which often originates from unresolved neuroinflammation or altered immune response. Posttranscriptional regulation of gene expression might play a fundamental role in the regulation of these processes. The ELAV RNA-binding proteins HuR and HuD are involved in the promotion of inflammatory phenomena and in neuronal development and maintenance, respectively. Thus, the aim of this study was to investigate the role of HuR and HuD in demyelination-associated neuropathic pain in the mouse experimental autoimmune encephalomyelitis (EAE) model. HuR resulted overexpressed in the spinal cord of MOG35-55-EAE and PLP139-151-EAE mice and was detected in CD11b + cells. Conversely, HuD was largely downregulated in the MOG-EAE spinal cord, along with GAP43 and neurofilament H, while in PLP-EAE mice, HuD and neuronal markers remained unaltered. Intranasal antisense oligonucleotide (ASO) delivery to knockdown HuR, increased myelin basic protein expression, and Luxol Fast Blue staining in both EAE models, an indication of increased myelin content. These effects temporally coincided with attenuation of pain hypersensitivity. Anti-HuR ASO increased the expression of HuD in GAP43-expressing cells and promoted a HuD-mediated neuroprotective activity in MOG-EAE mice, while in PLP-EAE mice, HuR silencing dampened pro-inflammatory responses mediated by spinal microglia activation. In conclusion, anti-HuR ASO showed myelin protection at analgesic doses with multitarget mechanisms, and it deserves further consideration as an innovative agent to counteract demyelination in neuropathic pain states.

Regulation of neuronal RNA signatures by ELAV/Hu proteins.

The RNA-binding proteins encoded by the highly conserved elav/Hu gene family, found in all metazoans, regulate the expression of a wide range of genes, at both the co-transcriptional and posttranscriptional level. Nervous-system-specific ELAV/Hu proteins are prominent for their essential role in neuron differentiation, and mutations have been associated with human neurodevelopmental and neurodegenerative diseases. Drosophila ELAV, the founding member of the protein family, mediates the synthesis of neuronal RNA signatures by promoting alternative splicing and alternative polyadenylation of hundreds of genes. The recent identification of ELAV's direct RNA targets revealed the protein's central role in shaping the neuronal transcriptome, and highlighted the importance of neuronal transcript signatures for neuron maintenance and organism survival. Animals have evolved multiple cellular mechanisms to ensure robustness of ELAV/Hu function. In Drosophila, elav autoregulates in a 3'UTR-dependent manner to maintain optimal protein levels. A complete absence of ELAV causes the activation and nuclear localization of the normally cytoplasmic paralogue FNE, in a process termed EXon-Activated functional Rescue (EXAR). Other species, including mammals, seem to utilize different strategies, such as protein redundancy, to maintain ELAV protein function and effectively safeguard the identity of the neuronal transcriptome. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Development RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Molecular insights into binding dynamics of tandem RNA recognition motifs (tRRMs) of human antigen R (HuR) with mRNA and the effect of point mutations in impaired HuR-mRNA recognition.

Human antigen R (HuR) is a key regulatory protein with prominent roles in RNA metabolism and post-transcriptional gene regulation. Many studies have shown the involvement of HuR in plethora of human diseases, which are often manifestations of impaired HuR-RNA interactions. However, the inherent complexities of highly flexible protein-RNA interactions have limited our understanding of the structural basis of HuR-RNA recognition. In this study, we dissect the underlying molecular mechanism of interaction between N-terminal tandem RNA-recognition motifs (tRRMs) of HuR and mRNA using molecular dynamics simulation. We have also explored the effect of point mutations (T90A, R97A and R136A) of three reported critical residues in HuR-mRNA binding specificity. Our findings show that N-terminal tRRMs exhibit conformational stability upon RNA binding. We further show that R136A and R97A mutants significantly lose their binding affinity owing to the loss of critical interactions with mRNA. This may be attributed to the larger domain rearrangements in the mutant complexes, especially the ß2ß3 loops in both the tRRMs, leading to unfavourable conformations and loss of binding affinity. We have identified critical binding residues in tRRMs of HuR, contributing favourable binding energy in mRNA recognition. This study contributes significantly to understand the molecular mechanism of RNA recognition by tandem RRMs and provides a platform to modulate binding affinities through mutations. This may further guide in future structure-based drug-therapies targeting impaired HuR-RNA interactions.Communicated by Ramaswamy H. Sarma.

Inhibition of the DAPKs-L13a axis prevents a GAIT-like motif-mediated HuR insufficiency in melanoma cells.

We previously showed that the adaptive response of BRAFV600-mutated melanoma cells to BRAF inhibition emerges from a subpopulation of cells expressing an intermittent lower level of the mRNA-binding protein HuR. In this study, following initial overexpression experiments in which we confirm our previous results, we use wild-type and mutants HuR full-length mRNA constructs and in vivo-interacting assays and demonstrate that a highly conserved interferon-γ-activated inhibitor of translation (GAIT)-like motif located upstream of the GU-rich elements of HuR major polyadenylation site (PAS2), interacts with constituents of the GAIT complex and affects HuR post-transcriptional expression regulation. Knockdown of the ribosomal protein L13a or the inhibition of the DAPK1-ZIPK axis involved in L13a phosphorylation, reduces the proportion of HuRLow cells at steady-state and attenuates the adaptive response of the whole melanoma-cell population to BRAF inhibition. These results have further potential therapeutic implications for disease conditions associated with HuR insufficient expression.

Cell-Penetrating Peptide TAT-HuR-HNS3 Suppresses Proinflammatory Gene Expression via Competitively Blocking Interaction of HuR with Its Partners.

Proinflammatory cytokines/chemokines are commonly regulated by RNA-binding proteins at posttranscriptional levels. Human Ag R (HuR)/embryonic lethal abnormal vision-like 1 (ELAVL1) is one of the well-characterized RNA-binding proteins that increases the stability of short-lived mRNAs, which encode proinflammatory mediators. HuR employs its nucleocytoplasmic shuttling sequence (HNS) domain, interacting with poly(ADP-ribose) polymerase 1 (PARP1), which accounts for the enhanced poly-ADP-ribosylation and cytoplasmic shuttling of HuR. Also by using its HNS domain, HuR undergoes dimerization/oligomerization, underlying the increased binding of HuR with proinflammatory cytokine/chemokine mRNAs and the disassociation of the miRNA-induced silencing complex from the targets. Therefore, competitively blocking the interactions of HuR with its partners may suppress proinflammatory mediator production. In this study, peptides derived from the sequence of the HuR-HNS domain were synthesized, and their effects on interfering HuR interacting with PARP1 and HuR itself were analyzed. Moreover, cell-penetrating TAT-HuR-HNS3 was delivered into human and mouse cells or administered into mouse lungs with or without exposure of TNF-α or LPS. mRNA levels of proinflammatory mediators as well as neutrophil infiltration were evaluated. We showed that TAT-HuR-HNS3 interrupts HuR-PARP1 interaction and therefore results in a lowered poly-ADP-ribosylation level and decreased cytoplasmic distribution of HuR. TAT-HuR-HNS3 also blocks HuR dimerization and promotes Argonaute 2-based miRNA-induced silencing complex binding to the targets. Moreover, TAT-HuR-HNS3 lowers mRNA stability of proinflammatory mediators in TNF-α-treated epithelial cells and macrophages, and it decreases TNF-α-induced inflammatory responses in lungs of experimental animals. Thus, TAT-HuR-HNS3 is a promising lead peptide for the development of inhibitors to treat inflammation-related diseases.

A genome-wide CRISPR screen identifies HuR as a regulator of apoptosis induced by dsRNA and virus.

We performed an unbiased whole-genome CRISPR/Cas9 screen in A549 cells to identify potential regulators involved in cell death triggered by double-stranded RNA (dsRNA). Of several top candidate genes, we identified the RNA-binding gene ELAV like protein 1 (256529), which encodes the protein Hu antigen R (HuR). Depletion of HuR led to less cell death induced by dsRNA. HuR is mainly involved in apoptosis, and all of its RNA recognition motifs are essential for its pro-apoptotic function. We further showed that the HuR depletion had no influence on the mRNA level of the anti-apoptotic gene BCL2, but instead that HuR downregulates BCL2 translation in a cap-independent way. Polysome fractionation studies showed that HuR retarded the BCL2 mRNA in the non-translating pool of polysomes. Moreover, protection from dsRNA-induced apoptosis by HuR depletion required the presence of BCL2, indicating that the pro-apoptotic function of HuR is executed by suppressing BCL2. Consistent with this, HuR regulated apoptosis induced by infection of encephalomyocarditis or Semliki Forest virus. Collectively, our work identified a suite of proteins that regulate dsRNA-induced cell death, and elucidated the mechanism by which HuR acts as a pro-apoptotic factor.

SRI-42127, a novel small molecule inhibitor of the RNA regulator HuR, potently attenuates glial activation in a model of lipopolysaccharide-induced neuroinflammation.

Glial activation with the production of pro-inflammatory mediators is a major driver of disease progression in neurological processes ranging from acute traumatic injury to chronic neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. Posttranscriptional regulation is a major gateway for glial activation as many mRNAs encoding pro-inflammatory mediators contain adenine- and uridine-rich elements (ARE) in the 3' untranslated region which govern their expression. We have previously shown that HuR, an RNA regulator that binds to AREs, plays a major positive role in regulating inflammatory cytokine production in glia. HuR is predominantly nuclear in localization but translocates to the cytoplasm to exert a positive regulatory effect on RNA stability and translational efficiency. Homodimerization of HuR is necessary for translocation and we have developed a small molecule inhibitor, SRI-42127, that blocks this process. Here we show that SRI-42127 suppressed HuR translocation in LPS-activated glia in vitro and in vivo and significantly attenuated the production of pro-inflammatory mediators including IL1ß, IL-6, TNF-α, iNOS, CXCL1, and CCL2. Cytokines typically associated with anti-inflammatory effects including TGF-ß1, IL-10, YM1, and Arg1 were either unaffected or minimally affected. SRI-42127 suppressed microglial activation in vivo and attenuated the recruitment/chemotaxis of neutrophils and monocytes. RNA kinetic studies and luciferase studies indicated that SRI-42127 has inhibitory effects both on mRNA stability and gene promoter activation. In summary, our findings underscore HuR's critical role in promoting glial activation and the potential for SRI-42127 and other HuR inhibitors for treating neurological diseases driven by this activation.

ELAVL1a is an immunocompetent protein that protects zebrafish embryos from bacterial infection.

Previous studies have shown that ELAVL1 plays multiple roles, but its overall biological function remains ill-defined. Here we clearly demonstrated that zebrafish ELAVL1a was a lipoteichoic acid (LTA)- and LPS-binding protein abundantly stored in the eggs/embryos of zebrafish. ELAVL1a acted not only as a pattern recognition receptor, capable of identifying LTA and LPS, as well as bacteria, but also as an effector molecule, capable of inhibiting the growth of Gram-positive and -negative bacteria. Furthermore, we reveal that the C-terminal 62 residues of ELAVL1a positioned at 181-242 were indispensable for ELAVL1a antibacterial activity. Additionally, site-directed mutagenesis revealed that the hydrophobic residues Val192/Ile193, as well as the positively charged residues Arg203/Arg204, were the functional determinants contributing to the antimicrobial activity of rELAVL1a. Importantly, microinjection of rELAVL1a into embryos markedly promoted their resistance against pathogenic Aeromonas hydrophila challenge, and this pathogen-resistant activity was considerably reduced by co-injection of anti-ELAVL1a antibody or by knockdown with morpholino for elavl1a. Collectively, our results indicate that ELAVL1a is a maternal immune factor that can protect zebrafish embryos from bacterial infection. This work also provides another angle for understanding the biological roles of ELAVL1a.

ELAV Proteins Bind and Stabilize C/EBP mRNA in the Induction of Long-Term Memory in Aplysia.

Long-term memory (LTM) formation is a critical survival process by which an animal retains information about prior experiences to guide future behavior. In the experimentally advantageous marine mollusk Aplysia, LTM for sensitization can be induced by the presentation of two aversive shocks to the animal's tail. Each of these training trials recruits distinct growth factor signaling systems that promote LTM formation. Specifically, whereas intact TrkB signaling during Trial 1 promotes an initial and transient increase of the immediate early gene apc/ebp mRNA, a prolonged increase in apc/ebp gene expression required for LTM formation requires the addition of TGFß signaling during Trial 2. Here we explored the molecular mechanisms by which Trial 2 achieves the essential prolonged gene expression of apc/ebp We find that this prolonged gene expression is not dependent on de novo transcription, but that apc/ebp mRNA synthesized by Trial 1 is post-transcriptionally stabilized by interacting with the RNA-binding protein ApELAV. This interaction is promoted by p38 MAPK activation initiated by TGFß. We further demonstrate that blocking the interaction of ApELAV with its target mRNA during Trial 2 blocks both the prolonged increase in apc/ebp gene expression and the behavioral induction of LTM. Collectively, our findings elucidate both when and how ELAV proteins are recruited for the stabilization of mRNA in LTM formation. Stabilization of a transiently expressed immediate early gene mRNA by a repeated training trial may therefore serve as a "filter" for learning, permitting only specific events to cause lasting transcriptional changes and behavioral LTM.SIGNIFICANCE STATEMENT: In the present paper, we significantly extend the general field of molecular processing in long-term memory (LTM) by describing a novel form of pretranslational processing required for LTM, which relies on the stabilization of a newly synthesized mRNA by a class of RNA binding proteins (ELAVs). There are now compelling data showing that important processing can occur after transcription of a gene, but before translation of the message into protein. Although the potential importance of ELAV proteins in LTM formation has previously been reported, the specific actions of ELAV proteins during LTM formation remained to be understood. Our new findings thus complement and extend this literature by demonstrating when and how this post-transcriptional gene regulation is mediated in the induction of LTM.

Overlapping Activities of ELAV/Hu Family RNA Binding Proteins Specify the Extended Neuronal 3' UTR Landscape in Drosophila.

The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.

ELAV and FNE Determine Neuronal Transcript Signatures through EXon-Activated Rescue.

The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. Here, we report that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. We uncover an endogenous strategy of functional gene rescue that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. We propose that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.

RNA structural dynamics regulate early embryogenesis through controlling transcriptome fate and function.

BACKGROUND: Vertebrate early embryogenesis is initially directed by a set of maternal RNAs and proteins, yet the mechanisms controlling this program remain largely unknown. Recent transcriptome-wide studies on RNA structure have revealed its pervasive and crucial roles in RNA processing and functions, but whether and how RNA structure regulates the fate of the maternal transcriptome have yet to be determined. RESULTS: Here we establish the global map of four nucleotide-based mRNA structures by icSHAPE during zebrafish early embryogenesis. Strikingly, we observe that RNA structurally variable regions are enriched in the 3' UTR and contain cis-regulatory elements important for maternal-to-zygotic transition (MZT). We find that the RNA-binding protein Elavl1a stabilizes maternal mRNAs by binding to the cis-elements. Conversely, RNA structure formation suppresses Elavl1a's binding leading to the decay of its maternal targets. CONCLUSIONS: Our study finds that RNA structurally variable regions are enriched in mRNA 3' UTRs and contain cis-regulatory elements during zebrafish early embryogenesis. We reveal that Elavl1a regulates maternal RNA stability in an RNA structure-dependent fashion. Overall, our findings reveal a broad and fundamental role of RNA structure-based regulation in vertebrate early embryogenesis.

Elav-Mediated Exon Skipping and Alternative Polyadenylation of the Dscam1 Gene Are Required for Axon Outgrowth.

Many metazoan genes express alternative long 3' UTR isoforms in the nervous system, but their functions remain largely unclear. In Drosophila melanogaster, the Dscam1 gene generates short and long (Dscam1-L) 3' UTR isoforms because of alternative polyadenylation (APA). Here, we found that the RNA-binding protein Embryonic Lethal Abnormal Visual System (Elav) impacts Dscam1 biogenesis at two levels, including regulation of long 3' UTR biogenesis and skipping of an upstream exon (exon 19). MinION long-read sequencing confirmed the connectivity of this alternative splicing event to the long 3' UTR. Knockdown or CRISPR deletion of Dscam1-L impaired axon outgrowth in Drosophila. The Dscam1 long 3' UTR was found to be required for correct Elav-mediated skipping of exon 19. Elav thus co-regulates APA and alternative splicing to generate specific Dscam1 transcripts that are essential for neural development. This coupling of APA to alternative splicing might represent a new class of regulated RNA processing.

Neural stem/progenitor cells are activated during tail regeneration in the leopard gecko (Eublepharis macularius).

As for many lizards, the leopard gecko (Eublepharis macularius) can self-detach its tail to avoid predation and then regenerate a replacement. The replacement tail includes a regenerated spinal cord with a simple morphology: an ependymal layer surrounded by nerve tracts. We hypothesized that cells within the ependymal layer of the original spinal cord include populations of neural stem/progenitor cells (NSPCs) that contribute to the regenerated spinal cord. Prior to tail loss, we performed a bromodeoxyuridine pulse-chase experiment and found that a subset of ependymal layer cells (ELCs) were label-retaining after a 140-day chase period. Next, we conducted a detailed spatiotemporal characterization of these cells before, during, and after tail regeneration. Our findings show that SOX2, a hallmark protein of NSPCs, is constitutively expressed by virtually all ELCs before, during, and after regeneration. We also found that during regeneration, ELCs express an expanded panel of NSPC and lineage-restricted progenitor cell markers, including MSI-1, SOX9, and TUJ1. Using electron microscopy, we determined that multiciliated, uniciliated, and biciliated cells are present, although the latter was only observed in regenerated spinal cords. Our results demonstrate that cells within the ependymal layer of the original, regenerating and fully regenerate spinal cord represent a heterogeneous population. These include radial glia comparable to Type E and Type B cells, and a neuronal-like population of cerebrospinal fluid-contacting cells. We propose that spinal cord regeneration in geckos represents a truncation of the restorative trajectory observed in some urodeles and teleosts, resulting in the formation of a structurally distinct replacement.

Transient vimentin expression during the embryonic development of the chicken cerebellum.

Complex morphogenetic events, critical for the development of normal cerebellum foliation and layering, are known to involve type III intermediate filament protein such as vimentin expressed by Bergmann glia. The present study aimed to determine aspects of intermediate and late embryonic pattern of vimentin expression during the corticogenesis of chicken cerebellum at embryonic days 10-19 (E10-E19), using single and double immunohistochemistry/immunofluorescence. Vimentin expression showed partial co-localization with the glial markers GFAP and BLBP. Within cerebellar cortex, vimentin+ fibers were first found within lobules I and X (E10) and gradually extended to all folia (E15-E17), located within the external granule (EGL) the molecular cell layer, showing a radial orientation towards the inner granular layer and the cerebellar white matter oriented longitudinally. Interestingly, within the immature fissures base of most lobules, vimentin+ fibers radiate in a fan shape. Short-term BrdU experiments revealed that EGL cell proliferation was higher in the fissure base compared to folia apex. In addition, following 24-h survival, BrdU+ cells were found in close association to vimentin+ fibers in the EGL pre-migratory zone and within immature molecular layer. Paralleling cerebellum development, vimentin expression gradually extended to all folia sub-regions (base, wall, apex), but, at day E19, it was mainly confined to the folia apex and secondary fissure base. Taken together our data further support the possible role of vimentin+ fibers in the structural events of cerebellum corticogenesis, suggesting the participation of radial/Bergmann glia in chicken cerebellum foliation, similarly to that described for mammalian cerebellum morphogenesis.

3'UTR Length-Dependent Control of SynGAP Isoform α2 mRNA by FUS and ELAV-like Proteins Promotes Dendritic Spine Maturation and Cognitive Function.

FUS is an RNA-binding protein associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Previous reports have demonstrated intrinsic roles of FUS in synaptic function. However, the mechanism underlying FUS's regulation of synaptic morphology has remained unclear. We found that reduced mature spines after FUS depletion were associated with the internalization of PSD-95 within the dendritic shaft. Mass spectrometry of PSD-95-interacting proteins identified SynGAP, whose expression decreased after FUS depletion. Moreover, FUS and the ELAV-like proteins ELAVL4 and ELAVL1 control SynGAP mRNA stability in a 3'UTR length-dependent manner, resulting in the stable expression of the alternatively spliced SynGAP isoform α2. Finally, abnormal spine maturation and FTLD-like behavioral deficits in FUS-knockout mice were ameliorated by SynGAP α2. Our findings establish an important link between FUS and ELAVL proteins for mRNA stability control and indicate that this mechanism is crucial for the maintenance of synaptic morphology and cognitive function.

Additive neurogenesis supported by multiple stem cell populations mediates adult spinal cord development: A spatiotemporal statistical mapping analysis in a teleost model of indeterminate growth.

The knifefish Apteronotus leptorhynchus exhibits indeterminate growth throughout adulthood. This phenomenon extends to the spinal cord, presumably through the continuous addition of new neurons and glial cells. However, little is known about the developmental dynamics of cells added during adult growth. The present work characterizes the structural and functional development of the adult spinal cord in this model organism through a comprehensive quantitative analysis of the spatial and temporal dynamics of new cells at various developmental stages. This analysis, based on a novel statistical mapping approach, revealed within the adult spinal cord a wide distribution of both mitotically active and quiescent Sox2-expressing stem/progenitor cells (SPCs). While such cells are particularly concentrated within the ependymal layer near the central canal, the majority of them reside in the parenchyma, resembling the distribution of SPCs observed in the mammalian spinal cord. The active SPCs in the adult knifefish spinal cord give rise to transit amplifying progenitor cells that undergo a few additional mitotic divisions before developing into Hu C/D+ neurons and S100+ glial cells. There is no evidence of long-distance migration of the newborn cells. The persistence of cell proliferation and differentiation, combined with low levels of apoptosis, leads to a continuous addition of cells to the existing tissue. Newly generated neurons have functional and behavioral relevance, as indicated by the integration of axons of new electromotor neurons into the electric organ of these weakly electric fish. This results in a gradual increase in the amplitude of the electric organ discharge during adult development. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1269-1307, 2017.

Comparative analysis of monoaminergic cerebrospinal fluid-contacting cells in Osteichthyes (bony vertebrates).

Cerebrospinal fluid-contacting (CSF-c) cells containing monoamines such as dopamine (DA) and serotonin (5-HT) occur in the periventricular zones of the hypothalamic region of most vertebrates except for placental mammals. Here we compare the organization of the CSF-c cells in chicken, Xenopus, and zebrafish, by analyzing the expression of synthetic enzymes of DA and 5-HT, respectively, tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), and draw an evolutionary scenario for this cell population. Due to the lack of TH immunoreactivity in this region, the hypothalamic CSF-c cells have been thought to take up DA from the ventricle instead of synthesizing it. We demonstrate that a second TH gene (TH2) is expressed in the CSF-c cells of all the three species, suggesting that these cells do indeed synthetize DA. Furthermore, we found that many CSF-c cells coexpress TH2 and TPH1 and contain both DA and 5-HT, a dual neurotransmitter phenotype hitherto undescribed in the brain of any vertebrate. The similarities of CSF-c cells in chicken, Xenopus, and zebrafish suggest that these characteristics are inherited from the common ancestor of the Osteichthyes. A significant difference between tetrapods and teleosts is that teleosts possess an additional CSF-c cell population around the posterior recess (PR) that has emerged in specific groups of Actinopterygii. Our comparative analysis reveals that the hypothalamus in mammals and teleosts has evolved in a divergent manner: placental mammals have lost the monoaminergic CSF-c cells, while teleosts have increased their relative number.