miRNA malfunction causes spinal motor neuron disease

Abstract

Defective RNA metabolism is an emerging mechanism involved in ALS pathogenesis and possibly in other neurodegenerative disorders. Here, we show that microRNA (miRNA) activity is essential for long-term survival of postmitotic spinal motor neurons (SMNs) in vivo. Thus, mice that do not process miRNA in SMNs exhibit hallmarks of spinal muscular atrophy (SMA), including sclerosis of the spinal cord ventral horns, aberrant end plate architecture, and myofiber atrophy with signs of denervation. Furthermore, a neurofilament heavy subunit previously implicated in motor neuron degeneration is specifically up-regulated in miRNA-deficient SMNs. We demonstrate that the heavy neurofilament subunit is a target of miR-9, a miRNA that is specifically down-regulated in a genetic model of SMA. These data provide evidence for miRNA function in SMN diseases and emphasize the potential role of miR-9-based regulatory mechanisms in adult neurons and neurodegenerative states.

Fig. 1.

Inferior survival and motor activity of the MNDicer^mut mice. (A) Kaplan–Meier survival curve for controls and conditional Dicer1 KO mice (control, n = 12; MNDCR^mut, n = 12). The median survival of MNDicer^mut mice is 29 wk, and it is >60 wk for controls. (B) Weight gain of controls and MNDicer^mut mice. (C) Time to complete a turn in the pole task for controls and MNDicer^mut mice. (D and E) Open-field measures at 8, 16, and 31 wk of age. (D) Ratio of distance traveled in the open-field arena compared with the mean of controls (control, n = 12; MNDCR^mut, n = 12). (E) Ratio of rearing events performed in the open-field arena compared with the mean of controls (control, n = 12; MNDCR^mut, n = 12). Home-cage locomotion of MNDicer^mut and controls at 11 (F), 21 (G), and 50 (H) wk of age (control, n = 12; MNDCR^mut, n = 12). (Inset) Average of activity throughout the measured period. *P < 0.05; **P < 0.01.

Fig. 2.

MNDCR^mut mice exhibit muscular atrophy with signs of denervation. Hind-limb interosseous and gastrocnemius muscle bipolar EMG recording. (A) Representative EMG traces of control (Upper) and MNDicer^mut (Lower) mice under anesthesia. Frequent fibrillation potentials are annotated by red arrows. (B) EMG Pathology Index was evaluated for individual controls (○; n = 5) and MNDicer^mut mice (●; n = 5). This scale (range: 0–7) reflects the intensity and frequency of fibrillation potentials in coded mice, noting that the electromyographer was blinded as to the genotype of the mouse tested. (C) Basic ATPase staining of transverse section through control and MNDicer^mut tibialis anterior muscles. Fiber grouping events were observed only in mutant muscles. (D) H&E staining of transverse section through the tibialis anterior MNDicer^mut muscle. Angular fibers are marked by arrows. (Scale bar: 100 μm.) *P < 0.05; **P < 0.01.

Fig. 3.

MNDicer^mut mice exhibit spinal cord ventral horn sclerosis and axonopathy. (A) (Left) Representative Nissl staining of lumbar (L4-L5) sections from a MNDicer^mut mouse and a control littermate. (Insets) Enlargements of a ventral horn area in each section. The dashed line represents the border under which large-diameter cells (>20 μm) were counted. (Right) Average number of MNs counted per ventral horn section in lumbar spinal cord of 4-mo-old MNDicer^mut mice and controls (average of 7 and 10.1 MNs per section, respectively; 15 lumbar sections per animal; n = 5 and n = 5, respectively). (B) (Left) Representative lumbar section from 4-mo-old MNDicer^mut mice and controls immunostained for GFAP. (Right) Quantification of the GFAP immunofluorescence (IF) signal (arbitrary units, 3 lumbar sections per animal; n = 5 and n = 5, respectively) and quantification of GFAP by Western blot analysis of lower spinal cord extracts from MNDicer^mut mice (“mut”) and controls, normalized to the expression of β-tubulin (arbitrary units; n = 3 and n = 4, respectively). A representative capture from the Western blot analysis is provided for two mutants and two controls. (C) (Left) Representative dorsal (sensory, Left) and ventral (motor, Right) roots used for axon number measurements, stained with anti-NEFM antibody. (Right) Average axon number in dorsal and ventral roots of MNDicer^mut mice and controls (n = 2 and n = 2, respectively). (D) (Left) Representative hind-limb tibialis anterior NMJ, demonstrating complete overlap (Upper) or partial overlap (Lower) between the postsynapse (red, rhodamine-labeled bungarotoxin) and presynapse (green, mixture of anti-neurofilament and synaptophysin antibodies; yellow, merged channels) components. (Right) Percentage of pathological end plates in MNDicer^mut mice and controls. These represent 17 aberrant NMJs of 651 NMJs that were individually screened in control mice and 43 aberrant NMJs of 760 NMJs in MNDicer^mut mice (n = 2 and n = 2, respectively). *P < 0.05; **P < 0.01.

Fig. 4.

miR-9 is specifically down-regulated in a model of SMA and is located upstream of coordinated expression of the neurofilament subunits. (A) Binned distribution of neurofilament subunit expression intensity. The percentage of axons at any intensity bin is mentioned on the y axis. NEFL (Left), NEFM (Center), and NEFH (Right). Black and gray lines represent the global mean intensity of control and MNDicer^mut axons, respectively. (B) Illustration of sequences cloned into luciferase reporter constructs used for functional evaluation of miR-9 interactions with neurofilament subunit mRNAs, wherein NEFH^mut stands for seed-mutated NEFH. Gray boxes represent miR-9–binding sites (C) Heterologous luciferase reporter assay reveals that miR-9 may function upstream of the NF subunits. Levels of luciferase activity in HEK293 cells transfected with either an empty vector or a vector overexpressing miR-9. Data are normalized to the activity of a cotransfected β-galactosidase reporter and presented as the percentage of luciferase activity in the absence of miR-9. OC-2 (a fragment of the Onecut2 3′UTR) is used as a positive control. (D and E) WT control mouse ES cells (mESCs) and SMN1^mut mESCs harboring a homozygous mSMN1 mutation and two copies of an hSMN2 transgene were differentiated in vitro into MNs. The cells were FACS-purified according to the expression of GFP transgene, driven by the Hlxb9 promoter. (D) Volcano plot exemplifying the log₂ ratio of SMN1^mut/WT miRNA expression on the x axis and the log₁₀ P value obtained by a two-tailed Student's t test on the y axis. (E) Quantitative PCR analysis of miR-9 and miR-9* expression in MNs derived from SMN1^mut mESCs (gray bars) and WT mESCs (empty bars). *P < 0.05; **P < 0.01.