Image_3_The 3’UTRs of Myelin Basic Protein mRNAs Regulate Transport, Local Translation and Sensitivity to Neuronal Activity in Zebrafish.tif

<p>Formation of functional myelin sheaths within the central nervous system depends on expression of myelin basic protein (MBP). Following process extension and wrapping around axonal segments, this highly basic protein is required for compaction of the multi-layered membrane sheath produced by oligodendrocytes. MBP is hypothesized to be targeted to the membrane sheath by mRNA transport and local translation, which ensures that its expression is temporally and spatially restricted. The mechanistic details of how this might be regulated are still largely unknown, in particular because a model system that allows this process to be studied in vivo is lacking. We here show that the expression of the zebrafish MBP orthologs, mbpa and mbpb, is developmentally regulated, and that expression of specific mbpa isoforms is restricted to the peripheral nervous system. By analysis of transgenic zebrafish, which express a fluorescent reporter protein specifically in myelinating oligodendrocytes, we demonstrate that both mbpa and mbpb include a 3’UTR sequence, by which mRNA transport and translation is regulated in vivo. Further functional analysis suggests that: (1) the 3’UTRs delay the onset of protein expression; and that (2) several regulatory elements contribute to targeting of the mbp mRNA to the myelin sheath. Finally, we show that a pharmacological compound known to enhance neuronal activity stimulates the translation of Mbp in zebrafish in a 3’UTR-dependent manner. A similar effect was obtained following stimulation with a TrkB receptor agonist, and cell-based assays further confirmed that the receptor ligand, BDNF, in combination with other signals reversed the inhibitory effect of the 3’UTR on translation.</p

Image_2_The 3’UTRs of Myelin Basic Protein mRNAs Regulate Transport, Local Translation and Sensitivity to Neuronal Activity in Zebrafish.tif

<p>Formation of functional myelin sheaths within the central nervous system depends on expression of myelin basic protein (MBP). Following process extension and wrapping around axonal segments, this highly basic protein is required for compaction of the multi-layered membrane sheath produced by oligodendrocytes. MBP is hypothesized to be targeted to the membrane sheath by mRNA transport and local translation, which ensures that its expression is temporally and spatially restricted. The mechanistic details of how this might be regulated are still largely unknown, in particular because a model system that allows this process to be studied in vivo is lacking. We here show that the expression of the zebrafish MBP orthologs, mbpa and mbpb, is developmentally regulated, and that expression of specific mbpa isoforms is restricted to the peripheral nervous system. By analysis of transgenic zebrafish, which express a fluorescent reporter protein specifically in myelinating oligodendrocytes, we demonstrate that both mbpa and mbpb include a 3’UTR sequence, by which mRNA transport and translation is regulated in vivo. Further functional analysis suggests that: (1) the 3’UTRs delay the onset of protein expression; and that (2) several regulatory elements contribute to targeting of the mbp mRNA to the myelin sheath. Finally, we show that a pharmacological compound known to enhance neuronal activity stimulates the translation of Mbp in zebrafish in a 3’UTR-dependent manner. A similar effect was obtained following stimulation with a TrkB receptor agonist, and cell-based assays further confirmed that the receptor ligand, BDNF, in combination with other signals reversed the inhibitory effect of the 3’UTR on translation.</p

Image_1_The 3’UTRs of Myelin Basic Protein mRNAs Regulate Transport, Local Translation and Sensitivity to Neuronal Activity in Zebrafish.TIF

<p>Formation of functional myelin sheaths within the central nervous system depends on expression of myelin basic protein (MBP). Following process extension and wrapping around axonal segments, this highly basic protein is required for compaction of the multi-layered membrane sheath produced by oligodendrocytes. MBP is hypothesized to be targeted to the membrane sheath by mRNA transport and local translation, which ensures that its expression is temporally and spatially restricted. The mechanistic details of how this might be regulated are still largely unknown, in particular because a model system that allows this process to be studied in vivo is lacking. We here show that the expression of the zebrafish MBP orthologs, mbpa and mbpb, is developmentally regulated, and that expression of specific mbpa isoforms is restricted to the peripheral nervous system. By analysis of transgenic zebrafish, which express a fluorescent reporter protein specifically in myelinating oligodendrocytes, we demonstrate that both mbpa and mbpb include a 3’UTR sequence, by which mRNA transport and translation is regulated in vivo. Further functional analysis suggests that: (1) the 3’UTRs delay the onset of protein expression; and that (2) several regulatory elements contribute to targeting of the mbp mRNA to the myelin sheath. Finally, we show that a pharmacological compound known to enhance neuronal activity stimulates the translation of Mbp in zebrafish in a 3’UTR-dependent manner. A similar effect was obtained following stimulation with a TrkB receptor agonist, and cell-based assays further confirmed that the receptor ligand, BDNF, in combination with other signals reversed the inhibitory effect of the 3’UTR on translation.</p

Molecular Cloning and Characterization of Porcine Na⁺/K⁺-ATPase Isoforms α1, α2, α3 and the ATP1A3 Promoter

<div><p>Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1–α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1–α3 genes, <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i>, their chromosomal localization, and expression patterns. Our <i>ATP1A1</i> sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine <i>ATP1A1</i> sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i> mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i> transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine <i>ATP1A3</i> promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine <i>ATP1A3</i> promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the <i>ATP1A3</i> promoter in attempts to generate transgenic porcine models of neurological diseases caused by <i>ATP1A3</i> mutations.</p></div

Comparative expression levels of porcine <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i> mRNA in different organs and tissues from adult pigs.

<p><i>β-actin</i> (b_ACT) is used as endogenous reference. Each column represents the mean expression of a triplicate from three different pigs. The considerable biological variation between the animals represented in each column is indicated by error bars showing the standard deviation. FCO: frontal cortex, CBE: cerebellum, HIP: hippocampus, BST: brain stem, HEA: heart, LDO: longissimus dorsi, BFE: biceps femoris, KID: kidney.</p

Relative expression pattern of porcine <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i> mRNA in different organs and tissues from adult pigs and from brain tissues at different stages of embryonic development.

<p><i>GAPDH</i> is used as endogenous reference. Each column represents the mean expression of a triplicate from three different pigs. The considerable biological variation between the animals represented in each column is indicated by error bars showing the standard deviation. KID: kidney, LUN: lung, LIV: liver, HEA: heart, THG: thyroid gland, LDO: longissimus dorsi, PGL: pituitary gland, SPC: spinal cord, FCO: frontal cortex, CBE: cerebellum; BST: brain stem, HIP: hippocampus, BSG: basal ganglia, D60: embryo of day 60, D80: embryo of day 80, D100: embryo of day 100, D115: embryo of day 115.</p

Functional importance of Arg841.

<p>The rat α1 Na⁺/K⁺-ATPase Arg843 homologous to pig Arg841 was replaced by alanine (“mutant”) and the functional consequences analyzed (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079127#s3" target="_blank">Methods</a>). Wild type, <i>closed circles</i>; mutant, <i>open circles</i>. The standard errors are indicated as error bars (seen only when larger than the size of the symbols). <b>A.</b> Na⁺ dependence of phosphorylation. Phosphorylation was carried out for 10 s at 0°C in the presence of 2 µM [γ-³²P]ATP in P-medium with oligomycin and the indicated concentrations of Na⁺. Each <i>line</i> shows the best fit of the Hill equation, giving <i>K</i>_0.5(Na⁺) values of 0.50±0.01 mM for wild type and 1.07±0.04 mM for the mutant. <b>B.</b> K⁺ dependence of Na⁺/K⁺-ATPase activity. The ATPase activity was measured at 37°C in A-medium with 40 mM Na⁺, 3 mM ATP, and the indicated concentrations of K⁺. Each <i>line</i> shows the best fit of the Hill equation, giving <i>K</i>_0.5(K⁺) values of 0.67±0.01 mM for wild type and 0.50±0.02 mM for the mutant. <b>C.</b> ATP dependence of Na⁺/K⁺-ATPase activity. The ATPase activity was measured at 37°C in A-medium with 130 mM Na⁺, 20 mM K⁺, and the indicated concentrations of ATP. Each <i>line</i> shows the best fit of the Hill equation, giving <i>K</i>_0.5(ATP) values of 0.50±0.03 mM for wild type and 0.43±0.04 mM for the mutant. <b>D.</b> Vanadate dependence of Na⁺/K⁺-ATPase activity. The ATPase activity was measured at 37°C in A-medium with 130 mM Na⁺, 20 mM K⁺, 3 mM ATP, and the indicated concentrations of vanadate. Each <i>line</i> shows the best fit of the Hill equation for inhibition, giving <i>K</i>_0.5(vanadate) values of 2.2±0.1 µM for wild type and 2.4±0.1 µM for the mutant. <b>E.</b> Distribution of phosphoenzyme intermediates between E1P and E2P. Phosphorylation was carried out for 10 s at 0°C in the presence of 2 µM [γ-³²P]ATP in P-medium with 20 mM Na⁺. Dephosphorylation was initiated by addition of 1 mM non-radioactive ATP and 2.5 mM ADP and terminated by acid quenching at the indicated times. Each <i>line</i> shows the best fit of a bi-exponential decay function giving amplitudes (corresponding to E2P) for the slow phase of 63±4% for wild type and 84±8% for the mutant. <b>F.</b> Rate of E1P→E2P interconversion. Phosphorylation was carried out for 15 s at 0°C in the presence of 2 µM [γ-³²P]ATP in P-medium with 600 mM Na⁺. Dephosphorylation was initiated by addition of a chase solution producing final concentrations of 600 mM Na⁺, 20 mM K⁺, and 1 mM non-radioactive ATP in addition to the components in the P-medium, and terminated by acid quenching at the indicated times. Each <i>line</i> shows the best fit by a bi-exponential decay function giving rate constants for the slow phase (corresponding to the E1P→E2P interconversion) of 0.14±0.05 s⁻¹ for wild type and 0.43±0.18 s⁻¹ for the mutant.</p

Specificity of <i>ATP1A3</i> promoter driven expression of GFP in zebrafish embryos.

<p><b>A.</b> and <b>B.</b> Weak GFP expression in the central nervous system and cells of the pronephros in F1 embryo 54 hours post fertilization. <b>C.</b> Mosaic expression in individual cells of the neural tube driven by the <i>ATP1A3</i> promoter in a representative embryo of the injected generation.</p

Schematic representation of elements in the minimal <i>Tol2</i>-vector construct, Tol2-pATP1A3:GFP, used for transgenesis in zebrafish.

<p>This construct is modified from the pT2AL200R150G plasmid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079127#pone.0079127-Urasaki1" target="_blank">[70]</a>. Tol2: the left and right terminal regions of the full-length Tol2, ATP1A3p: the porcine <i>ATP1A3</i> promoter sequence, GFP: Green Fluorescence Protein, Intron: the rabbit β-globin intron, polyA: SV40 polyA signal.</p

Methylation status of the <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i> genes in pig brain and liver (Sus scrofa 10.2).

<p>A selected promoter region of the <i>ATP1A3</i> (pATP1A3) gene was also included in this study.</p