Research ArticleAmyotrophic Lateral Sclerosis

Human genetics and neuropathology suggest a link between miR-218 and amyotrophic lateral sclerosis pathophysiology

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Science Translational Medicine  18 Dec 2019:
Vol. 11, Issue 523, eaav5264
DOI: 10.1126/scitranslmed.aav5264
  • Fig. 1 miR-218 is expressed in the human spinal motor neurons and is down-regulated in human ALS.

    (A to F) Three orthogonal miRNA quantification studies in human motor neurons from 20 ALS cases and 14 non-neurodegeneration controls. (A) miR-218 chromogenic in situ hybridization depicting broad expression along the cervical, thoracic, and lumbar regions of the adult human spinal cord. (B) qPCR analysis of miR-218, ISL1, HB9, and CHAT in human iPSCs and differentiated motor neurons. miR-218 normalized to U6 expression. mRNAs normalized to average of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β-actin expression, presented on a log scale; n = 3 independent wells per time point. (C) miR-218 expression in laser capture microdissection–enriched motor neurons from non-neurodegeneration controls (n = 7 human lumbar spinal cords) relative to surrounding non–motor neuron anterior horn tissue (n = 10), to Clarke’s column proprioceptive neurons (n = 4), or to ALS motor neurons (n = 9 sporadic and 2 familial nervous systems carrying the SOD1 A4V mutation).TaqMan qPCR analysis of miR-218 normalized to the average of RNU48/SNORD48, RNU44/SNORD44, and U6 in the same sample and to the average miR-218 expression in the anterior horn. One-way ANOVA, followed by Newman-Keuls multiple comparisons test, was performed on log-transformed data (means ± SD). (D) Volcano plot of relative miRNA expression in ALS lumbar ventral horns (n = 5) versus non-neurodegeneration controls (n = 2; x-axis log2 scale), screened by NanoString nCounter platform. Y axis depicts the differential expression P values (−log10 scale). Black dots indicate P < 0.05; light gray dots are nonsignificant. miR-218 is the most down-regulated miRNA in ALS nervous systems. Data normalized to the average of five control mRNAs (ACTB, B2M, GAPDH, RPL19, and RPLP0). (E) Reduced miR-218+ cell numbers in sALS patient anterior horns (n = 4) relative to non-neurodegeneration controls (n = 5) and representative miRNA in situ hybridization micrographs. Two-way ANOVA followed by Bonferroni’s multiple comparisons test (means ± SEM). (F) Chromogenic miR-218 in situ hybridization signal densitometry in motor neurons at different spinal cord levels (non-neurodegeneration control/ALS cases: cervical: n = 151 control cells, 85 ALS cells; thoracic: n = 54 control cells, 75 ALS cells; lumbar: n = 189 control cells, 92 ALS cells). One-tailed Mann-Whitney test (means ± SEM) was performed. OD, optical density. (G) Cumulative distribution function (CDF) plot of top 100 predicted miR-218-5p targets [TargetScan (20)], or all expressed mRNAs, in laser capture microdissection–enriched surviving motor neurons from lumbar spinal cords of patients with sALS with rostral onset and caudal progression (n = 13) relative to non-neurodegeneration controls [n = 6; (21)] and box plot (inset) depicting median, upper and lower quartiles, and extreme points. P value was calculated using Kolmogorov-Smirnov test comparing miR-218-5p target subset distribution to all genes. *P < 0.05; ***P < 0.001; ****P < 0.0001.

  • Fig. 2 miR-218 controls motor neuron network activity.

    (A) Seven most enriched gene ontology terms (22) of predicted miR-218 targets (20). P value of term enrichment (−log10; dashed orange line indicates P = 0.05). RISC, RNA-induced silencing complex. (B) CDF plot of miR-218 predicted targets, relative to all expressed mRNAs, after OE of miR-218 and box plot (insets), depicting median, upper and lower quartiles, and extreme points. P value was calculated using Kolmogorov-Smirnov test comparing miR-218-5p subset distribution to all genes. ****P < 0.0001. (C and D) Binding site enrichment of all known miRNAs, in ~10,000 expressed mRNAs, was tested after (C) miR-218 OE or (D) miR-218 KD relative to control virus. Significant enrichment for two miR-218-5p seed matches (blue and red) and lack of enrichment for any other miRNA (gray) via a Sylamer study (24). (E) Diagram of calcium transient imaging in embryonic rat spinal motor neurons, transduced with lentiviruses encoding control vector, miR-218 OE, or a miR-218 KD. E14, embryonic day 14. (F to H) Neuron time lapse micrographs (F), representative traces (G), and quantification (H) of spontaneous calcium spike frequencies (∆F/F > 0.5) from Fluo2 HighAff AM study after 12 days in vitro. Recorded from 58/76/41 control/OE/KD cells, respectively. Box plot depicting median, upper and lower quartiles, and extreme points. ***P < 0.001, Kruskal-Wallis test followed by Dunn’s multiple comparisons test. This experiment was repeated three independent times with similar results.

  • Fig. 3 The potassium channel Kv10.1 acts downstream of miR-218.

    (A) qPCR measuring the expression of mRNA targets after miR-218 OE (n = 15). Data normalized to control virus (n = 12) and to average expression of HPRT and β-actin; two technical duplicates, two-sided Student’s t test (means ± SEM). (B) Representative traces of individual motor neurons and (C to H) quantification of spontaneous calcium spike frequencies (∆F/F > 0.5) of embryonic rat spinal motor neurons, transduced with lentiviruses encoding a control vector, or miR-218 KD and further transfected with siRNA for specific target KD, or a nontargeting siRNA control (minus sign). Cells (≥55) were recorded per experimental condition; n ≥ 2 independent experimental repeats with similar results, Kruskal-Wallis test followed by Dunn’s multiple comparisons test. ns, nonsignificant. (I) Relative Renilla luminescence upstream of a WT Kv10.1 3′UTR or a mutated 3′UTR that is insensitive to miR-218, normalized to coexpressed firefly luciferase and to a negative control miRNA vector. n = 3 independent wells per experimental condition, one-way ANOVA followed by Bonferroni’s multiple comparisons test (means ± SEM). (J) miR-218:Kv10.1 3′UTR chimera from an AGO2 CLEAR-CLIP experiment in mouse cortex (29). (K) miR-218 expression (qPCR: n = 3, normalized to U6) and (L) Kv10.1 protein expression (Western blot: n = 5), upon miR-218 lentiviral KD or OE, in primary rat motor neurons and a representative blot detected with anti-Kv10.1 and anti–tubulin β-III (TUBB3) antibodies. Box plots depict median, upper and lower quartiles, and extreme points; one-way ANOVA followed by Newman-Keuls multiple comparisons test. a.u., arbitrary units. (M) Kv10.1 mRNA expression, as log2-normalized counts, from NGS study of induced ALS motor neurons (n = 4 different donors in duplicates) or non-neurodegeneration controls [n = 3 different donors in duplicates; (30)]. Box plots depict median, upper and lower quartiles, and extreme points (DESeq analysis). (N) Kv10.1 mRNA expression, as reads per kilobase million (RPKM) from NGS study of laser capture microdissection–enriched surviving motor neurons from lumbar spinal cords of patients with sALS with rostral onset and caudal progression (n = 12) and non-neurodegeneration controls [n = 8; (21); GSE76220]. Box plots depict median, upper and lower quartiles, and extreme points. *P < 0.05; **P < 0.01; ***P < 0.001, two-sided Student’s t test.

  • Fig. 4 Rare genetic miR-218 variants disrupt its ability to regulate neuronal activity.

    (A) Diagrams of hsa-miR-218-2 pri-miRNA (top) and the pre-miRNA hairpin (bottom), with demarcation of DROSHA, DGCR8, and DICER binding and arrows, revealing variant nucleotides (V1 to V6). Guide RNA is shown in red. (B) Table and forest plot depicting odds ratio (OR) estimates with 95% confidence intervals (CIs), across study cohorts and P values, calculated with SKAT-O or χ2 test with Yates’ correction. Vertical dotted line denotes OR = 3. (C) Representative motor neuron traces and (D) quantification of spontaneous calcium spike frequencies (∆F/F > 0.5) in embryonic rat spinal motor neurons, transduced with lentiviruses encoding WT or mutated human miR-218-2. Number of cells recorded in a single experiment: control: n = 131; WT miR-218-2: n = 114; single variant V2: n = 137; single variant V5: n = 119; multiple variant Vall: n = 118; unprocessable miR-218-2 Vdead: n = 111. n = 4 independent times with similar results. ***P < 0.001, Kruskal-Wallis test followed by Dunn’s multiple comparisons test.

  • Fig. 5 Rare genetic variants in miR-218 inhibit biogenesis.

    (A) Diagram of experimental design. HEPG2 cells transfected with WT miR-218-2 or miR-218-2 genetic variants and RNA processing for NGS and qPCR studies. (B) Pie chart of relative representation of different RNA families in NGS data [percentage of reads aligned to miRNA: 56%; transfer RNA (tRNA): 20%; ribosomal RNA (rRNA): 13%; other RNA types: 11%]. (C) The number of expressed miRNAs was comparable across samples (means ± SEM). (D) MA plot of miRNA expression in HEPG2 cells transfected with WT miR-218-2 relative to control vector. Abundance (x axis; presented on a log scale) against ratio of miRNA in cells overexpressing WT miR-218 versus a control vector (log2 fold change). (E) Histogram of number of reads per base for WT miR-218-2 sequences, aligned over the genomic sequence. (F) Bar graph of miR-218-5p isotypes (isomiR-218-5p, sequence denoted) in HEPG2 transfected with WT miR-218-2 or V2/V5 variants. (G and H) Relative expression of mature miR-218-5p from NGS (G) or TaqMan qPCR studies (H), normalized to miR-214-3p spike-in mimics. (I) Pre-miR-218-2 expression from NGS. (J) Ratio of pre-miR-218-2 (substrate) to mature miR-218-5p (product), defined as “inhibition score.” Inhibition score approximates a value of 1 in the WT condition, whereas a value of >1 reflects reduced DICER activity. Control: n = 3; WT miR-218-2: n = 5; single variant V2: n = 4; single variant V5: n = 4; multiple variant Vall: n = 5; unprocessable miR-218-2; Vdead: n = 3. Box plots depict median, upper and lower quartiles, and extreme points. *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA followed by Bonferroni’s multiple comparisons test performed on data (I) or log-transformed data (G, H, and J).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/523/eaav5264/DC1

    Materials and Methods

    Fig. S1. miR-218 is highly and specifically expressed in human and murine spinal motor neurons.

    Fig. S2. High content analysis of neuronal morphology after miR-218 perturbation.

    Fig. S3. miR-218 regulates intrinsic excitability.

    Fig. S4. qPCR validation of miR-218 target KD.

    Fig. S5. Evaluation of miR-218 upstream of the mRNA encoding for the potassium channel Kv4.2 (Kcnd2).

    Fig. S6. Kv10.1 (KCNH1) protein quantification by Western blot after miR-218 KD.

    Fig. S7. A summary diagram of key observations.

    Table S1. Identified hsa-miR-218-2 variants.

    Table S2. DsiRNA sequences used in the study.

    Table S3. Synthetic miR-218 sequences used for cloning into pMA-T vectors.

    Table S4. Primers used for quantitative real-time PCR.

    Data file S1. Individual-level data for miR-218 expression.

    Data file S2. NanoString nCounter data for miRNAs measured in lumbar ventral horns.

    Data file S3. Source data for Kv10.1 (KCNH1) Western blot studies.

    Data file S4. Source data for Kv4.2 (KCND2) Western blot studies.

    References (6172)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. miR-218 is highly and specifically expressed in human and murine spinal motor neurons.
    • Fig. S2. High content analysis of neuronal morphology after miR-218 perturbation.
    • Fig. S3. miR-218 regulates intrinsic excitability.
    • Fig. S4. qPCR validation of miR-218 target KD.
    • Fig. S5. Evaluation of miR-218 upstream of the mRNA encoding for the potassium channel Kv4.2 (Kcnd2).
    • Fig. S6. Kv10.1 (KCNH1) protein quantification by Western blot after miR-218 KD.
    • Fig. S7. A summary diagram of key observations.Table S1. Identified hsa-miR-218-2 variants.
    • Table S2. DsiRNA sequences used in the study.
    • Table S3. Synthetic miR-218 sequences used for cloning into pMA-T vectors.
    • Table S4. Primers used for quantitative real-time PCR.
    • Legends for data files S1 to S4
    • References (6172)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual-level data for miR-218 expression.
    • Data file S2 (Microsoft Excel format). NanoString nCounter data for miRNAs measured in lumbar ventral horns.
    • Data file S3 (Microsoft Excel format). Source data for Kv10.1 (KCNH1) Western blot studies.
    • Data file S4 (Microsoft Excel format). Source data for Kv4.2 (KCND2) Western blot studies.

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