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

Abstract

Motor neuron-specific microRNA-218 (miR-218) has recently received attention because of its roles in mouse development. However, miR-218 relevance to human motor neuron disease was not yet explored. Here, we demonstrate by neuropathology that miR-218 is abundant in healthy human motor neurons. However, in amyotrophic lateral sclerosis (ALS) motor neurons, miR-218 is down-regulated and its mRNA targets are reciprocally up-regulated (derepressed). We further identify the potassium channel Kv10.1 as a new miR-218 direct target that controls neuronal activity. In addition, we screened thousands of ALS genomes and identified six rare variants in the human miR-218-2 sequence. miR-218 gene variants fail to regulate neuron activity, suggesting the importance of this small endogenous RNA for neuronal robustness. The underlying mechanisms involve inhibition of miR-218 biogenesis and reduced processing by DICER. Therefore, miR-218 activity in motor neurons may be susceptible to failure in human ALS, suggesting that miR-218 may be a potential therapeutic target in motor neuron disease.

Fig. 1.. miR-218 is expressed in the human spinal motor neurons and is downregulated in human ALS.

(A-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, Hb9, Isl1 and ChAT in human iPSCs and differentiated motor neurons. miR-218 normalized to U6 expression. mRNAs normalized to average of HPRT and β–actin expression, presented on a log scale; n=3 independent wells per time point. (C) miR-218 expression in laser-capture micro-dissected human lumbar motor neurons. miR-218 expression in non-neurodegeneration motor neurons (n=7 human 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 comparison test 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 non-significant. miR-218 is the most downregulated miRNA in ALS nervous systems. Data normalized to the average of five control mRNAs (ACTB, B2M, GAPDH, RPL19, 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 comparison test, Means ± SEM, and (F) chromogenic miR-218 in situ hybridization signal densitometry in motor neurons at different spinal cord levels (non-neurodegeneration control/ALS cases: Cervical n=151/85 cells; Thoracic n=54/75; Lumbar 189/92). One-tailed Mann-Whitney test, Means ± SEM. (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 calculated using Kolmogorov–Smirnov test comparing miR-218-5p targets 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). (B) CDF plot of miR-218 predicted targets, relative to all expressed mRNAs, following OE of miR-218 and box-plot (insets), depicting median, upper and lower quartiles and extreme points. P-value calculated using Kolmogorov–Smirnov test comparing miR-218-5p subset distribution to all genes. **** P<0.0001. 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, 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. Neuron time lapse micrographs (F), representative traces (G) and (H) quantification of spontaneous calcium spike frequencies (ΔF/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 the median, upper and lower quartiles and extreme points. Kruskal-Wallis test followed by Dunn’s multiple comparison test, *** P<0.001. This experiment was repeated 3 independent times with similar results.

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

(A) qPCR measuring the expression of mRNAs targets, following 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-H) quantification of spontaneous calcium spike frequencies (ΔF/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 non-targeting siRNA control (minus sign). ≥55 cells recorded per each experimental condition; N≥2 independent experimental repeats with similar results. Kruskal-Wallis test followed by Dunn’s multiple comparison test. (I) Relative Renilla luminescence upstream of a wild-type Kv10.1 3′UTR or a mutated 3′UTR that is insensitive to miR-218, normalized to co-expressed firefly luciferase and to a negative control miRNA vector. n=3 independent wells per experimental condition. One-way ANOVA followed by Bonferronie’s multiple comparison 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 Beta-III (TUBB3) antibodies. Box-Plots depict median, upper / lower quartiles & extreme points, one-way ANOVA followed by Newman-Keuls multiple comparison test. (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 / lower quartiles & 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 / lower quartiles & extreme points, two-sided student’s t-test. * P<0.05; ** P<0.01; *** P<0.001; ns – non-significant.

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

(A) Diagrams of miR-218-2 pri-miRNA (upper) and the pre-miRNA hairpin (lower), with demarcation of DROSHA, DGCR8 and DICER binding and arrows, revealing variant nucleotides (V1-V6). Guide RNA in red. (B) Table and forest plot depicting odds ratio (OR) estimates with 95% confidence intervals (CI), across study cohorts and p-values, calculated with SKAT-O or Chi-squared test with Yate’s correction. Vertical dotted line denotes OR=3. (C) Representative motor neuron traces and (D) quantification of spontaneous calcium spike frequencies (ΔF/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 V_all, n=118; Unprocessable miR-218-2 V_dead, n=111. N=4 independent times with similar results. Kruskal-Wallis test followed by Dunn’s multiple comparison test. *** P<0.001; ns – non-significant.

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 processing of RNA 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%; tRNA −20%; 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 wild-type 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 vs a control vector (log 2 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-218-2-5p isotypes (isomiR-218-2-5p, sequence denoted) in HEPG2 transfected with WT miR-218-2, or V₂ / V₅ variants. Relative expression of mature miR-218-2 from (G) NGS or (H) TaqMan qPCR studies, normalized to miR-214-3p spike-in mimics. (I) Pre-miR-218-2 expression from NGS. (J) The ratio of pre-miR-218-2 (substrate) to mature miR-218 (product), defined as “inhibition score”. Inhibition score approximates a value of 1 in the WT condition, whereas a value > 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 V_all, n=5; Unprocessable miR-218-2; V_dead, n=3. Box-Plots depict median, upper / lower quartiles & extreme points, One-way ANOVA followed by Bonferroni’s multiple comparison test performed on data (I) or log-transformed data (G, H, J), * P<0.05; ** P<0.01; *** P<0.001; ns – non-significant.