Long-term synaptic alterations such as long-term potentiation or long-term depression require the selective delivery and local translation of specific types of mRNAs in the neuronal dendrites [1–3]. Recent studies have shown that the dendrite-targeted mRNAs are transported by large mRNA–protein complex granules (mRNPs) [2,4,5], which also contain multiple RNA-binding proteins including Purα, heterogeneous RNPs, TLS, staufen, and FMRP [4–6].
Earlier, we have reported that RNA-binding protein, TLS , is observed in the dendritic spines of mature hippocampal neurons [7,8] and is associated with Nd1-L mRNA that encodes an actin-stabilizing protein . Although Nd1-L mRNA is expressed in TLS-null mouse (TLS-KO) neurons, the amount of Nd1-L mRNA in the dendrites of TLS-KO neurons is significantly reduced, whereas the amount of other mRNAs such as β-actin mRNA remains unchanged . In addition, we have shown that hippocampal neurons of TLS-KO  exhibit abnormal spine morphology , which may be attributed to an improper supply of specific dendritic mRNAs because of the TLS deficiency. The dendritic localization of Nd1-L mRNA was restored by the exogenous expression of TLS in TLS-KO neurons ; however, the abnormal spine phenotypes of TLS-KO did not seem to be rescued by the transient expression of TLS. Therefore, it is still elusive as to whether the abnormal spine phenotypes of TLS-KO neurons are solely induced by a failure to transport TLS-target mRNA to the dendrites.
Materials and methods
Wild-type (WT) and TLS-KO mouse hippocampal neurons were prepared as described earlier . All animal experiments in this study were regulated by the National Advisory Committee on Laboratory Animal Research in Singapore and Osaka Bioscience Institute in Japan.
Fluorescent cell in-situ hybridization
After the infection of adenovirus carrying TLS-GFP, the neurons were fixed with 4% paraformaldehyde/PBS containing 4% sucrose for 30 min followed by the incubation in RNase-free 70% ethanol for 10 min. After washing in 2 mM MgCl2/PBS, the neurons were incubated at 42°C for 60 min in hybridization buffer and then at 37°C for 60 min with digoxigenin-labeled complementary RNA probe complementary to a short nucleotide sequence in 3′-UTR of Nd1-L mRNA. The bound probes were indirectly detected by AP-anti-digoxigenin antibody and HNPP (2-hydroxy-3-naphtonic acid-2′-phenylalanine phosphate) fluorescent detection kit (Roche, Penzberg, Germany). HNPP was coupled with Fast Red TR (Invitrogen, Carlsbad, California, USA) to optimize the binding of the dephosphorylated product HNPP (HNP) onto the probe. When necessary, GFP signals were enhanced by immunostaining using anti-GFP antibody (Invitrogen) and Alexa Fluor 488-conjugated secondary IgG (Invitrogen).
Fluorescent cell in-situ hybridization (FISH) images were acquired with Olympus FV1000 confocal laser-scanning microscope equipped with IX81 microscope (Olympus, Japan). For visualizing whole spine morphology, multiple optical images (15 slices, z-spacing of 0.4–0.6 μm) spanning 4–6 μm along the z-axis were combined using Metamorph (Universal Imaging Corporation, Pennsylvania, USA) in the maximum brightness mode. Fluorescent images were taken by excitation at 488 and 546 nm to detect GFP (Em 510 nm) and HNP/Fast Red TR (Em 562 nm), respectively.
Colocalization of pixels in FISH images was quantitated using CoLocalizer Pro 2.0 software (CoLocalization Research Software, http://homepage.mac.com/colocalizerpro/) . The values of the following coefficients were calculated: Pearson's correlation coefficient as a standard measure for the correlation between the different signal distributions, an overlap coefficient according to Manders' , and overlap coefficients k1–k2 . Manders' overlap coefficient indicates an overlap of the signals, which is less sensitive to the limitations of typical fluorescence imaging such as efficiency of hybridization, sample photobleaching, and camera quantum efficiency. The k1–k2 overlap coefficients can split the value of colocalization into the two separate parameters where each k1–k2 coefficients depend on the sum of the products of the intensities by two different channels. Colocalization data were statistically analyzed using analysis of variance and Student's unpaired t-test.
Quantitative analysis of colocalization between TLS and Nd1-L mRNA
We first determined that Nd1-L mRNA specifically binds to TLS in hippocampal neuronal cultures by immunoprecipitation reverse transcription-PCR (our unpublished data). We also confirmed that the average expression level of Nd1-L mRNA was not decreased in TLS-KO neurons (1.923±0.697) than in WT neurons (1.259±0.324) by real time reverse transcription-PCR (data not shown). Exogenously expressed TLS-GFP is recruited to mRNPs without interfering with endogenous TLS . Therefore, we overexpressed TLS-GFP in both WT and TLS-KO hippocampal neurons at 21 DIV, thereby evaluating colocalization of TLS-GFP and endogenous Nd1-L mRNA in the dendrites. FISH images displayed punctuate localization of TLS-GFP and Nd1-L mRNA as fluorescent clusters in the dendrites (WT, Fig. 1a). In WT neurons, both TLS-GFP and Nd1-L mRNA were mainly found in the spines (Fig. 1b). In TLS-KO neurons, TLS-GFP clusters containing Nd1-L mRNA were barely observed in the dendrites (KO, Fig. 1a). Moreover, these TLS-GFP clusters were hardly found in the existing spines of TLS-KO neurons (KO, Fig. 1a). To confirm this observation, we calculated Pearson's correlation coefficient to determine the degree of colocalization between the two fluorescent signals derived by TLS-GFP and Nd1-L mRNA in the FISH images. Pearson's correlation coefficient was indeed lower in KO neurons (0.52±0.04) than that calculated for WT neurons (0.73±0.02), indicating that the colocalization was significantly decreased in TLS-KO neurons (Fig. 1c). Manders' overlap coefficient also revealed that the colocalization was significantly reduced in TLS-KO neurons compared with that of WT neurons (Fig. 1d; 0.81±0.01 and 0.53±0.03 in WT and TLS-KO, respectively). We further confirmed the above results by calculating Manders' overlap coefficients in the dendritic spines. Colocalization between TLS-GFP and Nd1-L mRNA was significantly decreased in KO dendritic spines (Fig. 1e, 0.517±0.009 and 0.402±0.037 in WT and TLS-KO, respectively). The values of overlap coefficients k1–k2 of TLS-GFP and Nd1-L mRNA also proved the reduced colocalization in KO dendritic spines (Fig. 1f, 0.556±0.043 and 0.934±0.043 in WT and TLS-KO, respectively). These results suggest that the formation of TLS–Nd1-L mRNP in the dendrites, a presumable local mRNA pool near the spines [7,10], was significantly impaired in TLS-KO neurons although Nd1-L mRNA was transported by exogenous TLS-GFP to the dendrites in TLS-KO neurons .
TLS-mRNP in distal dendrites
Next, we addressed a question of whether there is any difference between WT and TLS-KO neurons in the distribution of TLS-GFP and Nd1-L mRNA along the dendrites. We examined the degree of colocalization in the dendrites within a distance of 10 μm from the cell body (‘proximal’) and that between 50 and 60 μm from the cell body (‘distal’) (Fig. 2a). Both Manders' overlap coefficients (Fig. 2b) and k1–k2 coefficients (Fig. 2d) indicated that the colocalization was significantly higher in the distal part of the dendrites than in the proximal part. Higher colocalization in the distal dendrites was also detected in TLS-KO neurons, as indicated by both Manders' (Fig. 2c) and the k1–k2 coefficients (Fig. 2e).
Exogenous TLS does not rescue the spine phenotypes in TLS-KO neurons
Finally, we evaluated the density and shape of the spines by their representative morphology (Fig. 3a) in both WT and KO neurons as well as those expressing TLS-GFP. Mouse hippocampal primary cultures (WT, Fig. 3b) at 21 DIV typically display the numerous mature mushroom-shaped spines (M, Fig. 3a) whereas in KO neurons (KO, Fig. 3b), the immature spines, such as filopodia, thin or stubby short spines, are predominantly observed (I, Fig. 3a). We calculated the spine density according to their morphological category. The statistical analyses revealed that the exogenous expression of TLS-GFP neither increase the population of mature spines in WT neurons (Fig. 3c) nor changed the number and shape of the spines in KO neurons (Fig. 3d).
Our results confirmed that the stable complex of TLS-GFP with Nd1-L mRNA in TLS-KO neurons was significantly disrupted (Fig. 1), both in the individual dendrites (Fig. 1d and Fig. 2) and spines (Fig. 1e). We cannot exclude a possibility that the GFP-tag may interfere with the correct mRNP formation near the spines. It is, however, least possible because both myc/His-tagged and GFP-tagged TLS were able to bind Nd1-L mRNA in in-vitro binding assay with MyoVa and other polysomal proteins (our unpublished data). The decreased colocalization in the TLS-KO dendrites could not be because of a decreased neuronal activation (or glutamate sensitivity) through mGluR5 because mGluR5 proteins are expressed normally in TLS-KO neurons . The association of TLS with Nd1-L mRNA was largely independent on mGluR5 activation (our unpublished data). In addition, the stability of Nd1-L mRNA is not changed by TLS deficiency . Quantitative colocalization analysis in this study proved to be applicable for an AP-based FISH method as all different coefficient values were within the range of their standard values (Figs 1 and 2).
We have consistently observed that TLS-containing mRNPs (TLS-mRNP) in the dendrites of mature neurons form a large stationary cluster in the vicinity of the spines , from which the smaller TLS granules were translocated to the spines . The stationary TLS-mRNP cluster may be a ‘storage pool of mRNAs’ that supplies certain mRNAs required for spine maintenance in response to specific synaptic signals (e.g. mGluR5 activation). The higher colocalization of TLS-GFP and Nd1-L mRNA in the distal dendrites (Fig. 3) supports a view that the large TLS-mRNP cluster may render ‘mRNA pool’ in the distal dendrites.
Taken together with our present data, we propose that important factors for the formation of the stationary TLS-mRNP cluster in the dendrites may be impaired in TLS-KO neurons. Thus, TLS functions during the neuronal maturation may be critical for the recruitment of TLS and Nd1-L mRNA into the stationary ‘mRNA pool’ near the spines. It would be interesting to investigate the possible roles of TLS in transcriptional control and formation of mRNP complex in the context of acquisition of neuronal activity at the early developmental stage because TLS is implicated in nucleocytoplasmic shuttling as a component of RNA-splicing complex [15,16].
We determined that Nd-1L mRNA is a specific binding partner for TLS in mouse hippocampal neuronal dendrites. The colocalization of TLS and Nd1-L mRNA was significantly decreased in both the dendrites and the spines of TLS-KO neurons. Moreover, the exogenous expression of TLS in TLS-KO neurons at the later stage could not compensate for the aberrant spine development. The results suggest that a timely TLS expression is required for proper expression of Nd1-L in the neuronal dendrites and spine maturation.
The authors thank G. Hicks for providing heterozygous TLS-deficient mice and Y. Shima and M. Nakano for the excellent technical assistance. This study was supported in part by grants from Ministry of Education, Culture, Sports, Science and Technology (MEXT) and Academic Frontier Project for Private Universities, and Grant-in-Aid for Scientific Research on Priority Areas-Research on Regulation of Nano-systems in Cells from the MEXT and by research grant from the Life Science Foundation of Japan to T.T.
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