BDNF elevates the axonal levels of hnRNPs Q and R in cultured rat cortical neurons.

Local translation plays important roles in the maintenance and various functions of axons, and dysfunctions of local translation in axons are implicated in various neurological diseases. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA binding proteins with multiple functions in RNA metabolism. Here, we identified 20 hnRNPs in the axons of cultured rat cortical neurons by interrogating published axon mass spectrometric databases with rat protein databases. Among those identified in axons are highly related hnRNPs Q and R. RT-PCR analysis indicated that axons also contained low levels of hnRNPs Q and R mRNAs. We further found that BDNF treatments raised the levels of hnRNPs Q and R proteins in whole neurons and axons. BDNF also increased the level of poly(A) RNA as well as the proportion of poly(A) RNA granules containing hnRNPs Q and R in the axon. However, following severing the connection between the cell bodies and axons, BDNF did not affect the levels of hnRNPs Q and R, the content of poly(A) RNA, or the colocalization of poly(A) RNA and hnRNPs Q and R in the axon any more, although BDNF still stimulated the local translation in severed axons as it did in intact axons. The results are consistent with that BDNF enhances the axonal transport of RNA granules. The results further suggest that hnRNPs Q and R play a role in the mechanism underlying the enhancement of axonal RNA transport by BDNF.

In vitro growth conditions and development affect differential distributions of RNA in axonal growth cones and shafts of cultured rat hippocampal neurons.

Local synthesis of proteins in the axons participates in axonogenesis and axon guidance to establish appropriate synaptic connections and confer plasticity. To study the transcripts present in the growth cones and axonal shafts of cultured rat hippocampal neurons, two chip devices, differing in their abilities to support axonal growth and branching, are designed and employed here to isolate large quantities of axonal materials. Cone-, shaft- and axon-residing transcripts with amounts higher than that of a somatodendritic transcript, Actg1 (γ-actin), are selected and classified. Since the chips are optically transparent, distribution of transcripts over axons can be studied by fluorescence in situ hybridization. Three transcripts, Cadm1 (cell adhesion molecule 1), Nefl (neurofilament light polypeptide), and Cfl1 (non-muscle cofilin) are confirmed to be preferentially localized to the growth cones, while Pfn2 (profilin2) is preferentially localized to the shafts of those axons growing on the chip that restricts axonal growth. The different growing conditions of axons on chips and on conventional coverslips do not affect the cone-preferred localization of Cadm1 and shaft-preferred localization of Pfn2, but affect the distributions of Nefl and Cfl1 over the axons at 14th day in vitro. Furthermore, the distributions of Cadm1 and Nefl over the axons growing on conventional coverslips undergo changes during in vitro development. Our results suggest a dynamic nature of the mechanisms regulating the distributions of transcripts in axonal substructures in a manner dependent upon both growth conditions and neuronal maturation.

Zebrafish Adar2 Edits the Q/R site of AMPA receptor Subunit gria2α transcript to ensure normal development of nervous system and cranial neural crest cells.

BACKGROUND: Adar2 deaminates selective adenosines to inosines (A-to-I RNA editing) in the double-stranded region of nuclear transcripts. Although the functions of mouse Adar2 and its biologically most important substrate gria2, encoding the GluA2 subunit of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor, have been extensively studied, the substrates and functions of zebrafish Adar2 remain elusive. METHODS/PRINCIPAL FINDINGS: Expression of Adar2 was perturbed in the adar2 morphant (adar2MO), generated by antisense morpholio oligonucleotides. The Q/R editing of gria2α was reduced in the adar2MO and was enhanced by overexpression of Adar2, demonstrating an evolutionarily conserved activity between zebrafish and mammalian Adar2 in editing the Q/R site of gria2. To delineate the role of Q/R editing of gria2α in the developmental defects observed in the adar2MO, the Q/R editing of gria2α was specifically perturbed in the gria2αQRMO, generated by a morpholio oligonucleotide complementary to the exon complementary sequence (ECS) required for the Q/R editing. Analogous to the adar2-deficient and Q/R-editing deficient mice displaying identical neurological defects, the gria2αQRMO and adar2MO displayed identical developmental defects in the nervous system and cranial cartilages. Knockdown p53 abolished apoptosis and partially suppressed the loss of spinal cord motor neurons in these morphants. However, reducing p53 activity neither replenished the brain neuronal populations nor rescued the developmental defects. The expressions of crestin and sox9b in the neural crest cells were reduced in the adar2MO and gria2αQRMO. Overexpressing the edited GluA2αR in the adar2MO restored normal expressions of cresting and sox9b. Moreover, overexpressing the unedited GluA2αQ in the wild type embryos resulted in reduction of crestin and sox9b expressions. These results argue that an elevated GluA2αQ level is sufficient for generating the cranial neural crest defects observed in the adar2MO. Our results present a link between dysfunction of AMPA receptors and defective development of the nervous system and cranial neural crest in the zebrafish.

A lab-on-a-chip platform for studying the subcellular functional proteome of neuronal axons.

Axons are long, slender processes extending from the cell bodies of neurons and play diverse and crucial roles in the development and function of nervous systems. Here, we describe the development of a chip device that can be used to produce large quantities of axons for proteomic and RNA analyses. On the chip surface, bundles of axons of rat hippocampal neurons in culture are guided to grow in areas distinct and distant from where their cell bodies reside. Fluorescence immunocytochemical studies have confirmed that the areas where these axons are guided to grow are occupied exclusively by axons and not by neuronal somatodendrites or astroglial cells. These axon-occupied parts are easily separated from the remainder of the chip and collected by breaking the chip along the well-positioned linear grooves made on the backside. One- and two-dimensional gel electrophoresis and Western blotting analyses reveal that the axons and whole cells differ in their protein compositions. RT-PCR analyses also indicate that the axons contain only a subset of neuronal RNAs. Furthermore, the chip device could be easily modified to address other issues concerning neuronal axons, such as the molecular composition of the axon substructure, the growth cone and shaft, the degeneration and regeneration processes associated with injured axons and the effects of extrinsic molecules, such as axon guidance cues and cell adhesion molecules, on the axon. With these diverse applications, the chip device described here will serve as a powerful platform for studying the functional proteome of neuronal axons.

Cold-induced exodus of postsynaptic proteins from dendritic spines.

Dendritic spines are small protrusions on neuronal dendrites and the major target of the excitatory inputs in mammalian brains. Cultured neurons and brain slices are important tools in studying the biochemical and cellular properties of dendritic spines. During the processes of immunocytochemical studies of neurons and the preparation of brain slices, neurons were often kept at temperatures lower than 37 degrees C for varied lengths of time. This study sought to investigate whether and how cold treatment would affect the protein composition of dendritic spines. The results indicated that upon cold treatment four postsynaptic proteins, namely, alpha,beta-tubulins, calcium, calmodulin-dependent protein kinase IIalpha, and cytoplasmic dynein heavy chain and microtubule-associated protein 2, but not PSD-95 or AMPA receptors, exited from the majority of dendritic spines of cultured rat hippocampal neurons in a Gd(3+)-sensitive manner. The cold-induced exit of tubulins from dendritic spines was further found to be an energy-dependent process involving the activation of Gd(3+)-sensitive calcium channels and ryanodine receptors. The results thus indicate that changes in temperature, calcium concentration, and energy supply of the medium surrounding neurons would affect the protein composition of the dendritic spines and conceivably the protein composition of the subcellular organizations, such as the postsynaptic density, in the cytoplasm of dendritic spines.

A real-time PCR method for the quantitative analysis of RNA editing at specific sites.

In this study, a quantitative PCR (qPCR) method was developed to determine the A-to-I RNA editing frequencies at specific sites. The A-to-I RNA editing of nuclear transcripts exerts profound effects on the biological activities of gene products. RNA editing of nuclear gene transcripts have been shown to be developmentally regulated and tissue specific, and alternations of RNA editing activities have been observed under pathological conditions. Two sites of ionotropic glutamate receptor subunits, the Q/R site of zebrafish gria2alpha and the Y/C site of grik2alpha, were chosen in this study to demonstrate the applicability of the SYBR Green detection-based real-time PCR method to measure RNA editing activities during zebrafish development. The results obtained by qPCR were consistent with those obtained by the limited primer extension. However, the qPCR method has the advantages of easy handling and low cost.

Molecular and functional studies of tilapia (Oreochromis mossambicus) NMDA receptor NR1 subunits.

NMDA (N-methyl-d-aspartate) receptor, a subclass of the ionotropic glutamate receptors, participates in synaptic transmission and plays important roles in various higher brain functions in the vertebrate central nervous system. Here, we report the cloning of two NR1 subunits of tilapia (Oreochromis mossambicus). Phylogenetic analysis strongly supports that the two tilapia NR1 genes are paralogous, resulting from a gene duplication event in the teleost lineage. The electrophysiological and pharmacological properties of the tilapia NR1.2 subunit coexpressed with rat NR2B in the Xenopus oocytes are similar to that of the recombinant rat NR1/NR2B. Both tilapia NR1 transcripts are alternatively spliced at the N and C terminal coding regions. The C terminal exons, C1' and C1", originally discovered in the knifefish NR1 gene, are present in the tNR1.1 but not in the tNR1.2. Majorities of the NR1 transcripts expressed in the tilapia and zebrafish brains do not include these alternative splice exons. The splicing patterns of NR1 transcripts differ in various brain subregions. The regional expression patterns of splice variants are not fully preserved between tilapia and zebrafish. Nevertheless, tectum opticum regions of teleost and rat express high levels of NR1 splicing variant with N1 cassette.

Identification of degenerate motifs using position restricted selection and hybrid ranking combination.

The identification of regulatory elements recognized by transcription factors and chromatin remodeling factors is essential to studying the regulation of gene expression. When no auxiliary data, such as orthologous sequences or expression profiles, are used, the accuracy of most tools for motif discovery is strongly influenced by the motif degeneracy and the lengths of sequence. Since suitable auxiliary data may not always be available, more work must be conducted to enhance tool performance to identify transcription elements in the metazoan. A non-alignment-based algorithm, MotifSeeker, is proposed to enhance the accuracy of discovering degenerate motifs. MotifSeeker utilizes the property that variable sites of transcription elements are usually position-specific to reduce exposure to noise. Consequently, the efficiency and accuracy of motif identification are improved. Using data fusion, the ranking process integrates two measures of motif significance, resulting in a more robust significance measure. Testing results for the synthetic data reveal that the accuracy of MotifSeeker is less sensitive to the motif degeneracy and the length of input sequences. Furthermore, MotifSeeker has been tested on a well-known benchmark [M. Tompa, N. Li, T.L. Bailey, G.M. Church, B. De Moor, E. Eskin, A.V. Favorov, M.C. Frith, Y. Fu, W.J. Kent, et al. (2005) Nat. Biotechnol., 23, 137-144], yielding a correlation coefficient of 0.262, which compares favorably with those of other tools. The high applicability of MotifSeeker to biological data is further demonstrated experimentally on regulons of Saccharomyces cerevisiae and liver-specific genes with experimentally verified regulatory elements.

Embryonic expression of zebrafish AMPA receptor genes: zygotic gria2alpha expression initiates at the midblastula transition.

The AMPA-preferring receptors (AMPARs) mediate rapid excitatory synaptic transmission in the central nervous system of vertebrates. Expression profiles of 8 AMPAR genes were studied by RT-PCR analyses to elucidate the properties of AMPARs during early zebrafish development. Transcripts of all AMPAR genes are detected at the time of fertilization, suggesting maternal transcriptions of zebrafish AMPAR genes. The amounts of gria1 and gria2 transcripts are several-fold higher than that of gria3 and gria4 between 10 and 72 hpf (hour postfertilization). The edited gria2alpha transcript decreases during gastrulation period, suggesting that zygotic expression of gria2alpha begins around the time of midblastula transition. Relative to the amount of beta-actin, the amounts of AMPAR transcripts increase significantly after the completion of neurulation. The amounts of gria2 transcripts exceed the total amounts of the remaining AMPAR transcripts after 36 hpf, suggesting increases in the representation of low Ca2+ permeable AMPARs during neuronal maturation. Many but not all of the known mammalian protein-protein interaction motifs are preserved in the C-terminal domains (CTD) of zebrafish AMPARs. Before 16 hpf, the embryos express predominantly the alternative splice forms encoding longer CTD. Representations of the short CTD splice forms of gria2 and gria4alpha increase after 24 hpf, when neurulation is nearly completed.

GeneAlign: a coding exon prediction tool based on phylogenetical comparisons.

GeneAlign is a coding exon prediction tool for predicting protein coding genes by measuring the homologies between a sequence of a genome and related sequences, which have been annotated, of other genomes. Identifying protein coding genes is one of most important tasks in newly sequenced genomes. With increasing numbers of gene annotations verified by experiments, it is feasible to identify genes in the newly sequenced genomes by comparing to annotated genes of phylogenetically close organisms. GeneAlign applies CORAL, a heuristic linear time alignment tool, to determine if regions flanked by the candidate signals (initiation codon-GT, AG-GT and AG-STOP codon) are similar to annotated coding exons. Employing the conservation of gene structures and sequence homologies between protein coding regions increases the prediction accuracy. GeneAlign was tested on Projector dataset of 491 human-mouse homologous sequence pairs. At the gene level, both the average sensitivity and the average specificity of GeneAlign are 81%, and they are larger than 96% at the exon level. The rates of missing exons and wrong exons are smaller than 1%. GeneAlign is a free tool available at http://genealign.hccvs.hc.edu.tw.

The mutually exclusive flip and flop exons of AMPA receptor genes were derived from an intragenic duplication in the vertebrate lineage.

The incomplete correlation between the organismal complexities and the number of genes among eukaryotic organisms can be partially explained by multiple protein products of a gene created by alternative splicing. One type of alternative splicing involves alternative selection of mutually exclusive exons and creates protein products with substitution of one segment of the amino acid sequence for another. To elucidate the evolution of the mutually exclusive 115-bp exons, designated flip and flop, of vertebrate AMPA receptor genes, the gene structures of chordate (tunicate, cephalochordate, and vertebrate) and protostome (Drosophila and Caenorhabditis elegans) AMPA receptor subunits were compared. Phylogenetic analysis supports that the vertebrate flip and flop exons evolved from a common sequence. Flip and flop exons exist in all vertebrate AMPA receptor genes but only one 115-bp exon is present in the genes of tunicates and cephalochordates, suggesting that the exon duplication event occurred at the ancestral vertebrate AMPA receptor gene after the separation of vertebrates from primitive chordates. The structures of animal AMPA receptor genes also suggest that an intron insertion to separate the primordial flip/flop exon from the M4-coding exon occurred before the exon duplication event and probably at the chordate lineage.