Wei-Yuan Chow

Identifications, Classification, and Evolution of the Vertebrate α-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid (AMPA) Receptor Subunit Genes

The AMPA receptor (AMPAR), a pharmacologically defined ionotropic glutamate receptor, mediates fast excitatory synaptic transmission in the vertebrate central nervous system. Mammalian and avian AMPARs are assembled from the products of four genes (GRIA1–GRIA4) conserved in their translated sequences and gene organizations. Teleost fish also express AMPAR subunits; however, the AMPAR genes have not been extensively investigated in lower vertebrates. To elucidate the evolution of vertebrate AMPAR genes, reverse-transcriptase PCR-based surveys of subunits expressed in the brains of eight nonmammalian vertebrates were performed. The newly cloned vertebrate AMPAR subunits were classified by their sequence identities to the mammalian AMPAR subunits. The results of molecular and phylogenetic analyses indicated that the members of the AMPAR gene family increased from two in the jawless hagfish to four in the tetrapods and the shark and to more than four in the teleost fish. The sizes of AMPAR gene families correlate well with those of many multigene families observed in various vertebrates. Moreover, all vertebrates expressed at least one AMPAR subunit bearing an arginine (R) at the Q/R site, at which no invertebrate glutamate receptor subunit has been found to have an R residue, suggesting that the low calcium-permeable AMPARs appeared at early evolutionary stages of vertebrate central nervous systems. Uniquely, the loop 1 (L1) regions between hydrophobic domain 1 and hydrophobic domain 2 of the hagfish putative GRIA2 and all the teleost GRIA1 subunits were much longer than those of the remaining known ionotropic glutamate receptor subunits. The length and sequence of the L1 of teleost GRIA1 subunits were heterogeneous, suggesting that the amino acid residues in L1 were not highly selected.

Characterization of granular particles isolated from postsynaptic densities

Abstract: We describe here the isolation and biochemical characterization of a population of protein aggregates from the postsynaptic density (PSD) prepared from pig cerebral cortex. The protein constituents of these aggregates are linked together primarily by disulfide bonds. Negative staining electron microscopy revealed that the isolated protein aggregates were granular objects with an average outside diameter of ∼21 nm and with small protrusions on their surface. The major constituents of the isolated granular aggregates consist of tubulin and an unidentified protein of 70 kDa in size. Small amounts of the α subunit of calcium/calmodulin‐dependent protein kinase II and subunits of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid and NMDA subtypes of glutamate receptors were also detected by immunoblotting. Actin, however, was not found in these granular aggregates. We propose that these granular protein aggregates correspond to the ∼20‐nm‐diameter granular particles of the PSD on the basis of their biochemical and morphological characteristics. The spatial arrangement of these granular aggregates relative to other components of the postsynaptic terminal is also postulated here.

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°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, α,β‐tubulins, calcium, calmodulin‐dependent protein kinase IIα, 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 Gd3+‐sensitive manner. The cold‐induced exit of tubulins from dendritic spines was further found to be an energy‐dependent process involving the activation of Gd3+‐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.

Embryonic expression of zebrafish AMPA receptor genes: Zygotic gria2α 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.

Effects of disulfide bonds formed during isolation process on the structure of the postsynaptic density

The biochemical, morphological and structural properties of rat postsynaptic densities (PSDs) isolated under conditions where disulfide bond formation was allowed or curtailed were studied here. Biochemical analyses revealed that the isolated PSDs were composed by a similar set of proteins regardless of the differences in their isolation processes. The PSDs isolated under the conditions where disulfide bond formation was curtailed were more easily dissociated by treatments with urea, guanidine hydrochloride and deoxycholate than the PSDs isolated under conditions where disulfide bond formation was allowed. Consistently, the structure of the PSDs isolated under the former condition appeared to be more fragmented than those isolated under the latter condition, as revealed by electron microscopy. The results indicate that the disulfide bonds formed during the isolation process significantly tighten the PSD structure and further suggest that the PSD in vivo is a protein aggregate whose constituent proteins be held together primarily by non-covalent interactions.

Molecular and electrophysiological characterizations of fGluR3α, an ionotropic glutamate receptor subunit of a teleost fish

Here we report the cloning and functional analysis of a cDNA encoding a functional glutamate receptor subunit of Oreochromis sp., a freshwater teleost fish. The deduced amino acid sequence of this cDNA clone, fGluR3 alpha, displays the highest sequence identity to that of the mammalian GluR3 subunit. Results of quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis indicated that the expression level of fGluR3 alpha in the cerebellum was much less than that in the telencephalon and optical lobe. Similar to its mammalian counterpart, variants of fGluR3 alpha were created by alternative splicing and RNA editing at the R/G site. The channel properties of homomeric fGluR3 alpha expressed in Xenopus oocytes were similar to those of the mammalian alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-preferring receptors. The rank order of agonist potency of the expressed fGluR3 alpha is AMPA > or = glutamate > or = quisqualate > domoate > or = kainate. This is the first functional glutamate receptor of teleost fish being demonstrated to be sensitive to AMPA. Furthermore, this study suggested a strong functional conservation of AMPA-preferring receptors in vertebrates.

Solubilization and characterization of glutamate binding sites from porcine brain.

The majority of l-glutamate binding sites in the synaptic membranes isolated from porcine brain were solubilized by a mixture containing Triton X-100 and digitonin. The solubilized l-glutamate binding sites bind l-glutamate reversibly and with a K(d) value of 0.67 ?M. The solubilized sites also bind l-aspartate, cysteate, and homocysteate, but not N- methyl- d -aspartate , kainate or quisqualate. Various salts, sodium chloride, sodium acetate, potassium chloride, calcium chloride, and magnesium acetate, enhance the l-glutamate binding activity of the solubilized preparation. When solubilized preparations were fractionated by gel-filtration chromatography, no binding activity was detected in the resulting fractions, whereas activity could be partially recovered in the combinations of different fractions. l-Glutamate binding activity of the glutaraldehyde-treated solubilized preparation was eluted out of the gel-filtration column as a single peak. The results suggest that this l-glutamate binding site consists of at least two components and that phospholipids may play important roles in the receptor function.

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.

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.