RNG105 deficiency impairs the dendritic localization of mRNAs for Na+/K+ ATPase subunit isoforms and leads to the degeneration of neuronal networks

Abstract

mRNA transport and local translation in dendrites play key roles in use-dependent synaptic modification and in higher-order brain functions. RNG105, an RNA-binding protein, has previously been identified as a component of RNA granules that mediate dendritic mRNA localization and local translation. Here, we demonstrate that RNG105 knock-out in mice reduces the dendritic localization of mRNAs for Na+/K+ ATPase (NKA) subunit isoforms (i.e., α3, FXYD1, FXYD6, and FXYD7). The loss of dendritic mRNA localization is accompanied by the loss of function of NKA in dendrites without affecting the NKA function in the soma. Furthermore, we show that RNG105 deficiency affects the formation and maintenance of synapses and neuronal networks. These phenotypes are partly explained by an inhibition of NKA, which is known to influence synaptic functions as well as susceptibility to neurotoxicity. The present study first demonstrates the in vivo role of RNG105 in the dendritic localization of mRNAs and uncovers a novel link between dendritic mRNA localization and the development and maintenance of functional networks.

Figure 1.

Generation of RNG105-deficient mice. A, Gene structure of the Rng105 locus (R, Rng105 exon 1 including the start codon), targeting construct and after homologous recombination. PCR products for genotyping (arrows) are indicated. H, HindIII; A, AvrII; B, BamHI; M, MluI; N, NotI. B, PCR genotyping and Western blotting of one E17.5 litter. Cerebral cortex extracts (30 μg) were loaded in each lane and probed with an anti-RNG105 antibody. C, Littermates at E17.5. The weight (mean ± SEM) of the litter was as follows: Rng105^+/+, 1.09 ± 0.03 g (n = 5); Rng105^+/−, 1.09 ± 0.05 g (n = 3); and Rng105^−/−, 0.85 ± 0.05 g (n = 2).

Figure 2.

RNG105-dependent dendritic localization of mRNAs for NKA subunit isoforms α3 and FXYD1, 6, and 7. A, Visualization of mRNAs with MS2-GFP (green) and of RNG105-mRFP1 (red) in dendrites of primary cultured neurons from E17.5 mouse cerebral cortex. 3′-UTRs of NKA α3 and FXYD1, 6, and 7 mRNAs are recruited to RNG105-localizing RNA granules. A magnified image of a dendrite is shown in each panel. B, Live imaging of RNG105-mRFP1 with FXYD1 (top panels) or FXYD7 (bottom panels) 3′-UTR-MS2-GFP in dendrites of primary cultured neurons. A cell soma is located on the left side in each panel. Images were taken at time points 0 and 380 s. Between the two time points, time-lapse imaging of RNG105-mRFP1 was performed (30–320 s) (supplemental Movies 1–3, available at www.jneurosci.org as supplemental material). The arrowheads denote individual granules. Arrowheads “c” indicate a granule transported from the soma to dendrite during the time-lapse imaging. The granule denoted by arrowheads “b” moved distally after the end of the time-lapse imaging (320–380 s). The GFP-tagged 3′-UTRs were colocalized with RNG105-mRFP1, but sometimes slightly shifted because there was a time lag of 3 s between mRFP1 and GFP data acquisition. The insets are nonenhanced images of the granule “c” at the same size, which show that the granule is relatively small one (see also supplemental Movie 2, available at www.jneurosci.org as supplemental material). C, Cultured neurons transfected with both RNG105-mRFP1 and FXYD1 3′-UTR-MS2-GFP were immunostained with anti-ribosomal protein S6 (RPS6) antibody. The arrowheads denote an RNG105-localizing RNA granule. Scale bars, 10 μm. D, Distribution of FXYD1 3′-UTR-MS2-GFP in wild-type (+/+), Rng105^−/− (−/−), and RNG105-mRFP1-overexpressing cultured neurons. The inset shows a nonenhanced image of the cell soma where small clusters of FXYD1 3′-UTR-MS2-GFP are observed. Scale bar, 10 μm. The bottom graph shows number of GFP clusters in the soma and dendrites (>20 μm from the soma). n = 8; *p < 0.05, **p < 0.01; error bars are SEM. E, Distribution pattern of mRNA-localizing clusters in the dendrites of wild-type, Rng105^−/−, and RNG105-mRFP1-overexpressing neurons in D. a.u., Arbitrary units. Error bars are SEM.

Figure 3.

RNA granule proteins except G3BP are localized normally to dendrites of Rng105^−/− neurons. A, Immunostaining for staufen 1 and RPS6 in cultured neurons (10 DIV) from wild-type and Rng105^−/− E17.5 cortexes. The insets show magnified images of the boxed areas. B, Immunostaining for G3BP in cultured neurons (10 DIV) from wild-type and Rng105^−/− E17.5 cortexes. The arrowheads denote a dendrite emanating from the soma located on the left side in each panel. Scale bars, 10 μm. C, Distribution patterns of staufen 1, RPS6, CPEB1, FMRP, Pur α, and G3BP in dendrites of wild-type and Rng105^−/− neurons. Mean fluorescence intensity in the dendrite is normalized to that in the soma. n = 5; *p < 0.05, t test at each distance from the soma. Error bars are SEM.

Figure 4.

Dendritic localization of NKA subunit isoforms is decreased in Rng105^−/− neurons. A, Immunostaining for NKA isoforms and PSD-95 in cultured neurons (10 DIV) from E17.5 wild-type and Rng105^−/− cortexes. A cell soma is located on the left side in each panel. The arrowheads denote a dendrite that emanates from the cell soma. B, C, Magnified images of dendritic areas in wild-type cultured neurons. FXYD6 and FXYD7 show significant colocalization with PSD-95 (B) but little colocalization with gephyrin (C). D, Immunostaining for synapsin I and PSD-95 in cultured neurons (10 DIV) from E17.5 wild-type cortex. The insets show magnified images of a dendritic area. PSD-95 shows significant colocalization with synapsin I. In A–D, neurons were cultured at low density to separate neurites from each other. Scale bars, 10 μm. E, Distribution patterns of NKA isoforms and PSD-95 in the dendrites of wild-type and Rng105^−/− neurons in A. Mean fluorescence intensity in the dendrite is normalized to that in the soma. The bottom right panel shows the ratio of FXYD7/PSD-95 mean fluorescence intensity. F, Quantification of colocalization of FXYD proteins with PSD-95 or gephyrin in dendrites in A–C. Shown are percentages of PSD-95 or gephyrin cluster areas that overlap with the FXYD proteins. n = 10 neurons. Black asterisks, Tukey–Kramer test among wild-type neurons; red asterisks, t test between wild-type and Rng105^−/− neurons. *p < 0.05, **p < 0.01. Error bars are SEM.

Figure 5.

K⁺-induced Na⁺ efflux is decreased in Rng105^−/− neuronal dendrites and by RNAi of NKA subunit isoforms. A, Fluorescence images of Sodium Green in cultured neurons (10 DIV) from wild-type and Rng105^−/− E17.5 cortexes. Neurons were cultured at low density to separate neurites from each other. Scale bar, 10 μm. B, Time courses of Sodium Green fluorescence intensity in the cell soma and dendrites (>30 μm from the cell soma) of wild-type and Rng105^−/− neurons during time-lapse experiments. KCl (50 mm) was added at time 0. The same experiments were also performed in the continuous presence of 50 μm ouabain. Mean fluorescence intensity is normalized to that at t = −25 s (F₀). Shown are the mean ± SEM (top panels) and the mean (bottom panels). C, Mean duration of K⁺-induced Na⁺ efflux in the cell soma and dendrites of wild-type and Rng105^−/− neurons (left panels). The same experiments were performed on neurons cotransfected with a red fluorescent protein (DsRed2) reporter and siRNA for the indicated NKA subunit isoforms (middle panels). The right panels show rescue experiments: gray bars, only siRNAs were introduced; white bars, both siRNAs and target genes that were mutated at the siRNA target sequences were introduced. n = 4 independent experiments per group. *p < 0.05, **p < 0.01, significant difference compared with +/+ or control siRNA, Tukey-Kramer test. Error bars are SEM.

Figure 6.

RNG105-deficient neurons form poor dendrites and networks. A, Neurons from E16.5 cortexes were cultured at low density so that they did not come into contact with each other. Shown are phase contrast images of primary cultures (11 DIV) from wild-type and Rng105^−/− cortexes. B, The axons and dendrites in A were traced and their lengths were measured at 4, 6, 8, and 11 DIV. There was no significant difference in the length of axons or dendrites between wild-type and Rng105^−/− neurons except that dendrites of Rng105^−/− neurons were significantly shorter than those of wild-type neurons at 11 DIV. n = 20 for axon lengths; n = 179, 59, 64, and 26 (wild type) and 142, 79, 47, and 35 (Rng105^−/−) for dendrite lengths at 4, 6, 8, and 11 DIV, respectively. C, Neurons from wild-type and Rng105^−/− cerebral cortexes were transfected with a GFP reporter to visualize the neuronal morphology and cultured for 9 d. GFP images of the transfectants at 9 DIV are shown. D, Dendrites in C were quantified using Sholl analysis, which counts the number of dendrites crossing circles of various radiuses from the soma. n = 10. E, Phase contrast images of primary cultured neurons (22 DIV) from wild-type and Rng105^−/− E17.5 cortexes. Scale bars: A, C, E, 100 μm. F, Quantification of neuronal networks in E. n = 8 fields (2.0 mm² each), four independent cultures from two littermates per group. In B, D, and F, *p < 0.05, **p < 0.01, ***p < 0.001, t test at each DIV or each distance from the soma. Error bars are SEM.

Figure 7.

RNG105 deficiency affects the distribution pattern of synaptic proteins. A, Primary cultured neurons (10 DIV) from wild-type and Rng105^−/− E17.5 cortexes were immunostained for synapsin I. The arrowheads denote clusters of neuronal somata. In wild-type neurons, synapsin I-positive clusters are mainly distributed along neuronal networks. In contrast, in Rng105^−/− neurons, synapsin I-positive clusters are preferentially localized to the somata. B, Quantification of the immunostaining for synapsin I in A. Shown are the ratio of the mean pixel intensity of synapsin I clusters in the network area to that in the soma. C, Immunostaining for synapsin I and GAD65 in coronal slices of wild-type and Rng105^−/− E17.5 cortexes. Synapsin I clusters were decreased in the network area of Rng105^−/− brains. Synapsin I clusters were barely detected on the soma in both wild-type and Rng105^−/− brains, which was different from the results obtained in cultured neurons, although the reasons are not known. D, Quantification of the immunostaining for synapsin I and GAD65 clusters in C. Mean fluorescence intensity and cluster density are shown. a.u., Arbitrary units. E, Immunostaining for an AMPAR subunit (GluR1) and PSD-95 in slices of wild-type and Rng105^−/− E17.5 cortexes. Cell somata are masked with blue in the right panels. Unmasked regions are network areas. Scale bars: A, C, E, 10 μm. F, The ratio of the overlap between GluR1 and PSD-95 in the network area to that in total area in E. In B, D, and F, numbers inside the bars are the number of fluorescent clusters or the number of fields (4.5 × 10⁴ μm² each) analyzed. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars are SEM.

Figure 8.

Half-width of mEPSCs is decreased in Rng105^−/− cultured neurons. A, B, Representative sample traces of mEPSCs, passing through AMPAR channels, from wild-type and Rng105^−/− neurons. B shows expanded mEPSCs. C, Representative sample traces of mIPSCs from wild-type and Rng105^−/− neurons. D, Cumulative amplitude and half-width histograms of mEPSCs and mIPSCs. mEPSC half-width is significantly decreased in Rng105^−/− neurons. **p < 0.01 using Kolmogorov–Smirnov test. E, Average frequency, amplitude, and half-width of mEPSCs and mIPSCs from wild-type and Rng105^−/− neurons. **p < 0.01 using t test. Error bars are SEM.

Figure 9.

Inhibition of NKA reduces neuronal networks and synapsin I staining. A, Phase contrast images of primary neurons (10 DIV) from E17.5 cortex cultured in the absence or presence of ouabain (10 μm). Scale bar, 100 μm. B, Quantification of the neuronal networks in A. n = 6 fields (2.0 mm² each) per group. *p < 0.05, **p < 0.01, t test at each DIV. Error bars are SEM. C, Immunostaining for synapsin I and GAD65 in primary neurons (11 DIV) cultured in the absence or presence of ouabain (10 μm). A cluster of cell somata is located on the left side, and neurites elongate toward the right side. Scale bar, 10 μm. D, Quantification of synapsin I and GAD65 fluorescence intensity in C.

Figure 10.

RNAi of NKA affects the termination pattern of axons from normal neurons. A, Immunostaining for synapsin I and GAD65 in cultured neurons (10 DIV) cotransfected with a GFP reporter and siRNA for the indicated NKA isoform or for RNG105. Magnified image of a GFP-positive dendritic area is shown in each panel. Synapsin I and GAD65 clusters correspond to axon terminals from other GFP-negative neurons. The arrowheads denote spines. The insets show magnified images of representative spines. Scale bar, 10 μm. B, Spine size in the RNAi neurons in A. The bottom panel shows rescue experiments: −, only siRNAs were introduced into neurons; +, both siRNAs and target genes that were mutated at the siRNA target sequences were introduced. C, Fluorescence intensity of synapsin I and GAD65 clusters on the GFP-positive spines in A. The fluorescence intensity on spines is normalized to that in GFP-negative areas in the same field and plotted. Each dot in the graph shows fluorescence intensity on each spine. D, Mean fluorescence intensity of synapsin I and GAD65 in C. The right panel shows rescue experiments. In B and D, expression of rescue constructs significantly suppressed the effects of siRNAs except that the suppression of the effect of FXYD7 siRNA on spine size was partial. The numbers indicate the number of spines analyzed. *p < 0.05, **p < 0.01, significant difference compared with control, Tukey–Kramer test. Error bars are SEM.