HuD mRNA Research Articles

ALS-related FUS mutations alter axon growth in motoneurons and affect HuD/ELAVL4 and FMRP activity

Mutations in the RNA-binding protein (RBP) FUS have been genetically associated with the motoneuron disease amyotrophic lateral sclerosis (ALS). Using both human induced pluripotent stem cells and mouse models, we found that FUS-ALS causative mutations affect the activity of two relevant RBPs with important roles in neuronal RNA metabolism: HuD/ELAVL4 and FMRP. Mechanistically, mutant FUS leads to upregulation of HuD protein levels through competition with FMRP for HuD mRNA 3’UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show increased axon branching and growth upon injury, which could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, increased axonal growth and branching might represent broad early events in the pathogenesis of ALS.

Neuronal RNA-binding protein HuD regulates addiction-related gene expression and behavior.

The neuronal RNA-binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD-mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuDOE ). When this set was further limited to genes in the knowledgebase of addiction-related genes database (KARG) that contains predicted HuD-binding sites in their 3' untranslated regions (3'UTRs), we found that HuD regulates networks that have been associated with addiction-like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction-related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild-type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuDOE and wild-type mice in CPP and found that HuDOE mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction-related behaviors such as cocaine conditioning and seeking, through increased plasticity-related gene expression.

Characterization of Multiple Exon 1 Variants in Mammalian HuD mRNA and Neuron-Specific Transcriptional Control via Neurogenin 2

The RBP (RNA-binding protein) and Hu/ELAV family member HuD regulates mRNA metabolism of genes directly or indirectly involved in neuronal differentiation, learning and memory, and several neurological diseases. Given the important functions of HuD in a variety of processes, we set out to determine the mechanisms that promote HuD mRNA expression in neurons using a mouse model. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in developing neurons. Bioinformatic and 5'RACE (rapid amplification of cDNA ends) analyses of the 5' genomic flanking region identified eight conserved HuD leader exons (E1s), two of which are novel. Expression of all E1 variants was determined in mouse embryonic (E14.5) and adult brains. Sequential deletion of the 5' regulatory region upstream of the predominantly expressed E1c variant revealed a well conserved 400 bp DNA region that contains five E-boxes and is capable of directing HuD expression specifically in neurons. Using EMSA (electrophoretic mobility shift assay), ChIP (chromatin immunoprecipitation), and 5' regulatory region deletion and mutation analysis, we found that two of these E-boxes are targets of Neurogenin 2 (Ngn2) and that this mechanism is important for HuD mRNA induction. Together, our findings reveal that transcriptional regulation of HuD involves the use of alternate leader exons and Ngn2 mediates neuron-specific mRNA expression. To our knowledge, this is the first study to identify molecular events that positively regulate HuD mRNA expression.

MiR-375 Inhibits Differentiation of Neurites by Lowering HuD Levels

Neuronal development and plasticity are maintained by tightly regulated gene expression programs. Here, we report that the developmentally regulated microRNA miR-375 affects dendrite formation and maintenance. miR-375 overexpression in mouse hippocampus potently reduced dendrite density. We identified the predominantly neuronal RNA-binding protein HuD as a key effector of miR-375 influence on dendrite maintenance. Heterologous reporter analysis verified that miR-375 repressed HuD expression through a specific, evolutionarily conserved site on the HuD 3' untranslated region. miR-375 overexpression lowered both HuD mRNA stability and translation and recapitulated the effects of HuD silencing, which reduced the levels of target proteins with key functions in neuronal signaling and cytoskeleton organization (N-cadherin, PSD-95, RhoA, NCAM1, and integrin alpha1). Moreover, the increase in neurite outgrowth after brain-derived neurotrophic factor (BDNF) treatment was diminished by miR-375 overexpression; this effect was rescued by reexpression of miR-375-refractory HuD. Our findings indicate that miR-375 modulates neuronal HuD expression and function, in turn affecting dendrite abundance.

Increase of the RNA-binding protein HuD and posttranscriptional up-regulation of the GAP-43 gene during spatial memory.

Neuronal ELAV-like proteins (HuB, HuC, and HuD) are highly conserved RNA-binding proteins able to selectively associate with the 3' UTR of a subset of target mRNAs and increase their cytoplasmic stability and rate of translation. We previously demonstrated the involvement of these proteins in learning, reporting that they undergo a sustained up-regulation in the hippocampus of mice trained in a spatial discrimination task. Here, we extend this finding, showing that a similar up-regulation occurs in the hippocampus of rats trained in another spatial learning paradigm, the Morris water maze. HuD, a strictly neuron-specific ELAV-like protein, is shown to increase after learning, with a preferential binding to the cytoskeletal fraction. HuD up-regulation is associated with an enhancement of GAP-43 mRNA and protein levels, with an apparently increased HuD colocalization with the GAP-43 mRNA and an increased association of neuronal ELAV-like proteins with the GAP-43 mRNA. These learning-dependent biochemical events appear to be spatiotemporally controlled, because they do not occur in another brain region involved in learning, the retrosplenial cortex, and at the level of protein expression they show extinction 1 month after training despite memory retention. By contrast, HuD mRNA levels still remain increased after 1 month in the CA1 region. This persistence may have implications for long-term memory recall.

Expression of mRNA for the elav-like neural-specific RNA binding protein, HuD, during nervous system development

The expression of mRNA for the neuronal antigen HuD (Elavl4) associated with paraneoplastic encephalomyelitis and sensory neuronopathy was evaluated in the developing and adult rat nervous system. Using RNase protection assay and non-radioactive in situ hybridization histochemistry HuD expression was shown to be expressed at high levels at the earliest time point observed (E15), but declined significantly during the first postnatal week to levels which were maintained into adulthood. In the adult, HuD expression became restricted primarily to large pyramidal-like neurons. Exceptions of note were many smaller neurons within a variety of thalamic nuclei. Expression of HuD was observed to be coincident with terminal differentiation of all neuronal structures evaluated regardless of the timing of their development, providing correlative evidence for a role in neuronal differentiation or the maintenance of neuronal phenotype. The marked restriction of HuD mRNA expression with maturity suggests that its functional role in adult neurons varies significantly throughout the CNS.

Differential expression of the neuroendocrine genes HelN1 and HuD in small‐cell lung carcinoma: Evidence for down‐regulation of HuD in the variant phenotype

Hel-N1 and HuD belong to the elav gene family and have gained recent attention as potential neuroendocrine markers for small-cell lung carcinoma (SCLC). Members of this conserved family normally appear at different stages of neuronal maturation, raising the possibility that their expression patterns in SCLC reflect the degree of neuroendocrine differentiation. I have utilized a ribonuclease protection assay to analyze Hel-N1 and HuD expression in cultured SCLC cells with high (classic phenotype) and low (variant phenotype) levels of neuroendocrine differentiation. Hel-N1 was detected in both classic and variant SCLC. Although HuD was detected consistently in classic SCLC, it was low to absent in variant SCLC, indicating a significant down-regulation in that phenotype. The expression patterns of Hel-N1 and HuD also were analyzed in 9 primary SCLC and 10 non-SCLC lung-tumor samples. In the majority of SCLC samples, either Hel-N1 or HuD was detected exclusively or predominantly, indicating a pattern of variable gene expression similar to cultured SCLC. Neither transcript could be detected in the non-SCLC samples. These data indicate that (i) HuD mRNA expression is associated with a higher level of neuroendocrine differentiation in SCLC, (ii) Hel-N1and HuD expressions are variable in both primary and cultured SCLC and (iii) HuD and Hel-N1, in combination, are neurogenetic markers for SCLC. Int. J. Cancer 74:378–382, 1997. © 1997 Wiley-Liss, Inc.

The RNA binding protein HuD: rat cDNA and analysis of the alternative spliced mRNA in neuronal differentiating cell lines P19 and PC12.

HuD belongs to a family of neurospecific RNA binding proteins found in man, frog and fly [49]. To investigate whether this protein is involved in regulation of neuronal differentiation of rodent cells in vivo and in vitro, the cDNA of the rat homolog gene (r-HuD) was cloned, its expression was studied in rat brain and in neurogenic cell lines, and the splicing of its RNA was analyzed. Coding sequences of HuD from man and rat were found to be 99.5 and 95% identical at protein and DNA level, respectively. In rat brain r-HuD transcripts 3.7 and 4.2 kb in length were detected by Northern blot analysis. RT-PCR and in situ hybridization revealed that rodent homologues of HuD transcripts are present in P19 mouse embryo carcinoma and in PC12 rat pheochromocytoma cell lines both able to differentiate into neurons. In contrast, r-HuD transcripts were not detectable in the rat glioma cell line C6. In P19 cells a strong induction of HuD mRNA was observed after triggering neuronal differentiation by retinoic acid, whereas in PC12 cells the mRNA was present before and after nerve growth factor (NGF) induced neuronal differentiation. In both neuronal cell lines and in brain of adult rat and mouse HuD mRNA is alternatively spliced in a region which encodes a proline rich linker domain between the second and third RNA recognition motif. This RNA processing event seems to be differently regulated in PC12 cells on the one hand, and in P19 cells and brain of rat and mouse on the other.

Molecular analysis of the HuD gene encoding a paraneoplastic encephalomyelitis antigen in human lung cancer cell lines: Sekido Y, Bader SA, Carbone DP, Johnson BE, Minna JD. Simmons Cancer Center, Texas University SW Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8590. Cancer Res 1994;54:4988–4992

Small cell lung cancer (SCLC) is known to express the HuD protein, the neuronal antigen homologous to Drosophila Elav and Sxl genes involved in neuronal and sex development. HuD is the target of an immune response including high titered antibodies causing paraneoplastic encephalomyelitis and sensory neuropathy. Because the p53 recessive oncogene is mutated and anti-p53 antibodies frequently occur in cancer patients, we wondered if the development of anti-HuD antibodies signaled the presence of HuD mutations in lung cancer. The HuD gene was mapped to chromosome region 1p using a human-mouse hybrid cell panel. We confirmed that 26 of 46 cancer (43 lung cancer and 3 mesothelioma) cell lines expressed HuD mRNA and that this expression, as well as protein expression by Western blot, correlated strongly with the SCLC neuroendocrine phenotype. Southern blot and single-strand conformation polymorphism analyses showed that HuD was not mutated in 78 lung cancers, including patients with the severe paraneoplastic syndrome. Northern blot analysis showed that lung cancer cell lines expressed two major mRNAs (4.3 and 4.0 kilobases) of HuD. We found the three previously described alternative spliced mRNA forms (HuDpro, HuD, and HuDmex). In addition, we also found HuD mRNA had an alternative splicing form in its 5'-coding region. This alternative splice introduced 87 base pairs of sequence and a termination codon resulting in a predicted small, truncated protein (11 amino acids) reminiscent of the male-specific truncated protein in the Sex-lethal (Sxl) gene of Drosophila. However, mRNAs encoding both full-length and truncated proteins were expressed in all SCLCs. These results show that the HuD gene is not mutated in lung cancer, including tumors from patients producing anti-HuD antibodies, but HuD expression is an independent marker or determinant of the neuroendocrine differentiation seen in SCLC.

HuD, a paraneoplastic encephalomyelitis antigen, contains RNA-binding domains and is homologous to Elav and sex-lethal

A neuronal antigen (HuD) recognized by the sera of patients with antibody-associated paraneoplastic encephalomyelitis has been isolated by screening a lambda cerebellar expression library. The recombinant antigen provides an unambiguous assay for this rare condition associated with small cell lung cancer. The recombinant antigen has been used to identify specific infiltrating lymphocytes in tumors and affected brain tissues of patients with antibody-associated paraneoplastic encephalomyelitis and sensory neuronopathy. HuD mRNA is uniquely expressed in brain tissue. The HuD protein shows a remarkable homology to the Drosophila proteins Elav and Sex-lethal and is likely to play a role in neuron-specific RNA processing.