Research ArticleNEUROPHYSIOLOGY

The long noncoding RNA neuroLNC regulates presynaptic activity by interacting with the neurodegeneration-associated protein TDP-43

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Science Advances  18 Dec 2019:
Vol. 5, no. 12, eaay2670
DOI: 10.1126/sciadv.aay2670
  • Fig. 1 An integrated screening strategy identifies NeuroLNC, a conserved lncRNA that regulates SV release.

    (A) Scheme depicting the experimental steps of the bioinformatic strategy and of the SV assay. gDNA, genomic DNA. (B) Quantification of the pHluorin experiments upon the pBI-driven overexpression of the lncRNAs following 60-AP field stimulation at 20 Hz. Mean values are plotted (± SEM). The tetanus toxin light chain (TetTxLC) was used as a negative control. Statistical test, one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test versus control (***P < 0.001). (C) Quantification and statistics as in (B), following a 600-AP field stimulation at 20 Hz. (D and E) Quantification of the SV exo-endocytosis experiments following either a 60- or 600-AP 20-Hz field stimulation, respectively, upon neuroLNC down-regulation. Statistical test, unpaired two-tailed Student’s t test (*P < 0.05 and **P < 0.01). (F) Schematic representation of the genomic locus of neuroLNC for rat, mouse, and human.

  • Fig. 2 NeuroLNC and miRNA 124-1 are two separate entities with distinct TSS.

    (A) Scatter plot showing the expression of neuroLNC and pri-miR-124-1. Since the two RNAs are transcribed from nearby genomic regions, we tested the correlation of the expression of the two RNAs (r2 = 0.81). (B) Experimental strategy used to test the presence of different RNA transcripts in the region between pri-miR-124-1 and neuroLNC. In the lower panel, the couple of primers used (light gray) and the fragments amplified (black) are represented to scale. (C) Results of the experiment exemplified in (B). The upper panel corresponds to the control PCR reaction on genomic DNA, and the lower panel shows the amplification of the retrotranscribed brain RNA. The lack of continuity between neuroLNC and mirR-124-1 (regions 2 and 3 and regions 3 and 4) indicates separate entities. For details concerning the primer sequences, refer to data S1J. bp, base pair; cDNA, complementary DNA. (D and E) Overexpression of neuroLNC in either primary hippocampal neurons (D) or differentiated PC-12 cells (E) does not influence the expression of pri-miR-124-1. The expression level values plotted are expressed as a percentage of the AAV-control or the control plasmid. NeuroLNC is significantly overexpressed, but there is no effect on the levels of pri-miR-124-1. Statistical tests, two-tailed Student’s t test (*P < 0.05 and **P < 0.01); n.s., not significant. (F) NeuroLNC overexpression does not influence the processing of miR-124-1 and the relative levels of its 3p and 5p forms. (G) TSS analysis. The scheme summarizes the observed starting sites, detailed in data S1C.

  • Fig. 3 NeuroLNC is neuron specific and nuclear, regulates presynaptic Ca2+ influx, and is modulated by neuronal activity.

    (A) Relative expression of neuroLNC in the rat central nervous system (CNS) and other tissues, as measured by quantitative real-time PCR (qRT-PCR). Statistical test, one-way ANOVA followed by Bonferroni’s multiple comparison test versus all non-CNS samples (***P < 0.001). (B) RNA fluorescence in situ hybridization (RNA-FISH) of neuroLNC on coronal brain slice of rat (P14). Note the localization to the nuclei of CA1 neurons in the zoom. Scale bars, 0.5 mm (left); 50 μm (right). DAPI, 4′,6-diamidino-2-phenylindole. (C) Combined RNA-FISH and immunofluorescence in primary mixed neuroglial cultures. Note that β-III tubulin–positive neurons are neuroLNC positive (left, white arrowhead), while cells positive for the glial marker glial fibrillary acidic protein (GFAP) are neuroLNC negative (right, white arrowhead). Scale bars, 10 μm. (D) Quantification of Ca2+ influx in primary hippocampal neurons upon neuroLNC overexpression following a 20-AP field stimulation at different frequencies. Mean values are plotted (± SEM). Insets indicate the difference in fluorescence measured at the maximum peak for each experiment. Statistical test, unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; and ***P < 0.001). See fig. S5 for details. (E) Quantification of Ca2+ influx as in (D), following shRNA-mediated neuroLNC down-regulation with a 600-AP stimulation at 20 Hz. Upon neuroLNC down-regulation, Ca2+ influx is significantly decreased. (F) Physiological (unstimulated) network activity measured in neurons following overexpression or down-regulation of neuroLNC. Overexpression increases spontaneous activity, while down-regulation has the opposite effect. Unpaired two-tailed Student’s t test (*P < 0.05). (G) The number of neuroLNC foci following activity modulation of neurons. Blocking the activity of neurons with TTX or AP5 + CNQX has no effect. The number of neuroLNC foci is promoted by increasing neuronal activity by applying bicuculline or 4-AP. In the upper panels, representative images are shown. Scale bar, 5 μm. Normalized averages are shown. One-way ANOVA followed by Bonferroni’s multiple comparison test versus control (**P < 0.01).

  • Fig. 4 NeuroLNC influences neurite elongation and branching in vitro and neuronal migration in vivo.

    (A) Exemplary traces of reconstructed primary hippocampal neurons transfected with either scramble shRNAs or a pool of three shRNAs against neuroLNC. White arrowheads indicate position of the cell body, and numbers designate different cells. Scale bar, 250 μm. (B) Summary of morphometric analysis on reconstructed neurons upon neuroLNC down-regulation. Box plots summarize the data distribution of ~700 neurites as explained in the methods. Unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; and ***P < 0.001). (C) Exemplary traces of reconstructed neurons as in (A), but upon neuroLNC overexpression. Scale bar, 250 μm. (D) Summary of the morphometric analysis of reconstructed neurons. Tracing and analysis of ~1000 neurites as in (B). (E) Exemplary confocal images of neuronal cells in E17.5 mouse cortices following E14.5 in utero electroporation (i.u.e.) with either scramble shRNAs or the shRNA pool against neuroLNC. Upper-left inset represents the portion of the coronal section imaged. Green fluorescent protein (GFP) was included for visualization of electroporated neurons (positive neurons are color-coded in magenta). Scale bar, 100 μm. (F) Frequency distribution and quantification of positive neurons upon neuroLNC down-regulation from the VZ (1st cortical segment) to the CP (10th cortical segment). Equal bins are used and data indicate mean ± SEM (experimental n = 5 in control and 7 in neuroLNC down-regulation). Two-way ANOVA with Bonferroni multiple comparison test for each single bin (*P < 0.05 and **P < 0.01). (G) Exemplary confocal images showing neuroLNC overexpression in an i.u.e. designed as in (E). A GFP version of the same plasmid was used as a negative control and coelectroporated for identifying positive neurons (color-coded in magenta). Scale bar, 100 μm. (H) Frequency distribution and quantification of positive neurons upon neuroLNC overexpression performed as in (F). Experimental n = 6 in both control neuroLNC overexpression. Two-way ANOVA with Bonferroni multiple comparison test for single bin pairs (**P < 0.01).

  • Fig. 5 NeuroLNC interacts in vivo with DNA and mRNA sequences of genes implicated in synaptic transmission, binds TDP-43, and requires its presence for modulating SV exocytosis.

    (A) Experimental workflow. For details and controls, see Materials and Methods and fig. S6. RBP, RNA binding protein. (B) Summary of DNA interactors of neuroLNC. All the hits are in data S1D. Among genes, preferentially bound top-ranking are shown in the gray inset. (C) Scatter plot of the false discovery rate (FDR, expressed as −log10) and enrichment ratio of the significant GO terms. GO analysis confirms the propensity of neuroLNC to be in proximity of genes related to chemical transmission, regulation of membrane potential, and synapse development. For an extensive list of GOs, see data S1E. (D) Summary of RNA targets found in the RNA interactome. (E) Summary of most abundant significantly enriched mRNA interactors. All mRNAs are significantly enriched versus the controls (P < 0.01), and >15 counts are represented in plot. For a comprehensive list, see data S1F. Among the most enriched mRNAs, three are also significantly enriched in STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis (gray inset, FDR < 0.01). (F) Scatter plot of the MS identification of the protein interactors of neuroLNC. TDP-43 is the only highly significant hit (P < 0.001). For MS data, refer to data S1H. (G) Enrichment of TDP-43 in MS experiments, detailing the negative controls (oligos against LacZ and no probe) and RNA purification performed with neuroLNC oligos upon ribonuclease (RNase) treatment. (H) Reverse experiment, where upon immunoprecipitating TDP-43, the presence of neuroLNC was tested by qRT-PCR, and the enrichment versus the housekeeping mRNA glyceraldehyde phosphate dehydrogenase (GAPDH) was calculated. Ab, antibody; IgG, immunoglobulin G. (I and J) Quantification of the SV exo-endocytosis experiments upon pBI-driven overexpression of either TDP-43, its antisense RNA (to decrease its levels), neuroLNC, a mutated form of neuroLNC that does not contain UG-repeats and does not bind TDP-43 (neuroLNCMUT), or several combinations of these constructs. SV exo-endocytosis as in Fig. 1 (B and C), following either a 60- or 600-AP field stimulation at 20 Hz. Down-regulation of TDP-43 blocks the effects of neuroLNC at both 60 and 600 AP. The neuroLNCMUT shows no effect on SV exo-endocytosis. In both schemes below, graph significances are summarized. One-way ANOVA followed by Bonferroni’s multiple comparison test (*P < 0.05; **P < 0.01; and ***P < 0.001). LFQ, label-free quantification.

  • Fig. 6 NeuroLNC overexpression increases the levels of SV protein and SV protein–associated mRNAs.

    Results of the qRT-PCR following overexpression of neuroLNC in primary hippocampal neurons with an AAV. Data were GAPDH-normalized and expressed as relative increase with respect to the control cultures (infected with a control AAV). Considering individual mRNA species, only SNAP25 and Stx1a are significantly increased across all tested SV and SV-associated mRNAs. Statistical test, one-way ANOVA followed by Bonferroni’s multiple comparison test versus controls (**P < 0.01). An overall analysis of the relative expression of all the SV and SV-associated mRNAs against their expression in the controls shows a highly significant increase of the whole class (8.2 ± 1.8% increase), suggesting that the effect on all these mRNAs has a limited amplitude but is overall consistent, and single significant differences are probably too small to emerge because of the modest sampling depth. Statistical test, two-tailed Student’s t test (***P < 0.001).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/12/eaay2670/DC1

    Fig. S1. Details of the screening strategy used for studying the role of lncRNAs in neuronal activity and down-regulation in culture.

    Fig. S2. NeuroLNC coding potential, conservation, and locus organization.

    Fig. S3. Specificity of the RNA-FISH approach and expression of neuroLNC in rodent cultures and in adult human brain.

    Fig. S4. Controls for the calcium dynamics experiments.

    Fig. S5. Controls and additional information concerning Fig. 5.

    Fig. S6. NeuroLNC overexpression does not affect the expression of the mRNAs that encode for calcium-related pathways, neuronal activity, or synaptic scaffolds.

    Fig. S7. Schematic representation summarizing the function of NeuroLNC in the nucleus of neurons.

    Data S1. Summary of data used here, see first sheet for details.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. Details of the screening strategy used for studying the role of lncRNAs in neuronal activity and down-regulation in culture.
    • Fig. S2. NeuroLNC coding potential, conservation, and locus organization.
    • Fig. S3. Specificity of the RNA-FISH approach and expression of neuroLNC in rodent cultures and in adult human brain.
    • Fig. S4. Controls for the calcium dynamics experiments.
    • Fig. S5. Controls and additional information concerning Fig. 5.
    • Fig. S6. NeuroLNC overexpression does not affect the expression of the mRNAs that encode for calcium-related pathways, neuronal activity, or synaptic scaffolds.
    • Fig. S7. Schematic representation summarizing the function of NeuroLNC in the nucleus of neurons.

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    Other Supplementary Material for this manuscript includes the following:

    • Data S1 (Microsoft Excel format). Summary of data used here, see first sheet for details.

    Files in this Data Supplement:

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