MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of motor neurons, denervation of target muscles, muscle atrophy, and paralysis. Understanding ALS pathogenesis may require a fuller understanding of the bidirectional signaling between motor neurons and skeletal muscle fibers at neuromuscular synapses. Here, we show that a key regulator of this signaling is miR-206, a skeletal muscle-specific microRNA that is dramatically induced in a mouse model of ALS. Mice that are genetically deficient in miR-206 form normal neuromuscular synapses during development, but deficiency of miR-206 in the ALS mouse model accelerates disease progression. miR-206 is required for efficient regeneration of neuromuscular synapses after acute nerve injury, which probably accounts for its salutary effects in ALS. miR-206 mediates these effects at least in part through histone deacetylase 4 and fibroblast growth factor signaling pathways. Thus, miR-206 slows ALS progression by sensing motor neuron injury and promoting the compensatory regeneration of neuromuscular synapses.

Figure 1. Regulation of miR-206 in response to ALS and denervation

(A) Up-regulation of miR-206 during the progression of ALS in G93A-SOD1 mice as determined by Northern blot and quantified by densitometry. *p< 0.0001 by t test. n=2–4. (B) Transcripts of miR-206, miR-133b, miR-1, and miR-133a were detected by real time PCR in TA muscles following ten days of denervation (+). The contralateral muscle was used as a control (−). *p < 0.02, **p < 0.005 by t test. n=3–4. (C) Sequence alignment of the mouse miR-206 5’ flanking sequence from different species shows the conserved upstream region containing E-boxes. Position (0) denotes the start of pre-miR-206. Bracketed region represents the identified enhancer region. (D) β-galactosidase staining of (G/P) muscle isolated from denervated transgenic mice containing a lacZ transgene controlled by the WT Enhancer or the Mutant Enhancer (containing mutated E-boxes) (bracket in panel C). Contra-lateral muscle was used as a control. Lower panels show transverse section of muscle. Scale bar =200 µm. Values represent means ± SEM.

Figure 2. Regulation of ALS pathogenesis by miR-206

(A) Age of disease onset for G93A-SOD1 (black) (n=9) (188 days), miR-206^−/−; G93A-SOD1 (red) (n=10) (187 days), and miR-206 KO (blue) littermates. (B) Days of disease progression for G93A-SOD1 (78 days) and miR-206−/−; G93A-SOD1 littermates (56 days). p < 0.005 by log-rank test. (C) Survival curve for G93A-SOD1 (266 days), miR-206^−/−;G93A-SOD1 (244 days). p < 0.05 by log-rank test. (D) Survival of G93A-SOD1 and miR-206^−/−;G93A-SOD1 mice. *p < 0.02 by t test. (E) G93A-SOD1 and miR-206^−/−;G93A-SOD1 mice at approximately 7.5 months of age. (F) X-ray reveals kyphosis in miR-206^−/−;G93A-SOD1 mice. (G) Wheat-germ agglutinin (WGA) staining of transverse sections of muscle show accelerated muscle atrophy in miR-206^−/−;G93A-SOD1 mice compared to G93A-SOD1 littermates. Scale bar= 100 µm. Values represent means ± SEM.

Figure 3. Delayed NMJ reinnervation in miR-206 mutant mice

(A) Quantitative real time PCR reveals miR-206 expression is enriched in synaptic regions of muscle fibers. *p < 0.0002 by t test. n=3. (B) Following sciatic nerve transection (as indicated in weeks) a delay in reinnervation is seen in miR-206^−/− mice (KO) compared to WT mice as detected by the superimposition of anti-ZNP staining (green) with BTX (red). Scale bar=10 µm. (C) Time course and quantification of the number of reinnervated NMJs in WT and miR-206^−/− (KO) mice following sciatic nerve transection. n=2–6. (D) Immunohistochemistry shows a delay in pre-synaptic differentiation and partial re-occupancy of postsynaptic sites in miR-206^−/− NMJs 5 weeks after sciatic nerve transection. Scale bar =10 µm. (E) Postsynaptic area occupied by the reinnervating nerve (in %) 5 weeks after cutting the sciatic nerve in WT and miR-206^−/− (KO) mice. *p < 0.02 by t test. (F) Immunohistochemistry shows increased NMJ dysfunction and denervation in miR-206^−/−;G93A-SOD1 compared to G93A-SOD1 littermates. Scale bar= 10 µm. (G) Number of innervated NMJs in G93A-SOD1 and miR-206^−/−;G93A-SOD1 mice at 7 months of age. *p < 0.0005 by t test. (H) Number of fragmented NMJs in G93A-SOD1 and miR-206^−/−;G93A-SOD1 mice at 7 months of age. *p < 0.005 by t test. Values represent means ± SEM.

Figure 4. miR-206 targets HDAC4

(A) Luciferase activity of COS1 cells co-transfected with WT or mutant HDAC4 3’UTR-luciferase constructs with a miR-206 expression plasmid. (B) Quantitation of HDAC4 protein expression in muscle lysates isolated from wild-type (WT) and miR-206^−/− (miR-206 KO) mice following 3 weeks of denervation. *p < 0.02 by t test. n=3. (C) Immunohistochemistry shows an increase in reinnervation in HDAC4 mKO mutant mice compared to WT mice 7 days following nerve crush. Scale bar=10 µm. (D) Number of reinnervated NMJs in WT and HDAC4 mKO mice following sciatic nerve crush for 7 days. *p < 0.05 by t test. n=3–8. (E) Postsynaptic area occupied by the reinnervating nerve (in %) 7 days after crushing the sciatic nerve in WT and HDAC4 mKO mice. *p < 0.02. (F) Decrease in expression of Fgfbp1 transcripts in miR-206^−/− (KO) muscles 3 weeks after nerve transection. *p < 0.02 by t test. n=3–5. (G) Increase in expression of Fgfbp1 transcripts in HDAC4 mKO muscles 7 days after nerve crush. *p < 0.0005 by t test. n=3–5. (H) Immunohistochemistry shows an inhibition of synaptic-vesicle clustering in neonatal NMJs upon knock-down of FGFBP1. Scale bar= 10 µm. (I) NMJ size in muscle fibers expressing LacZ or FGFBP1 shRNAs. Mice were electroporated at P0 and analyzed at P8. *p < 0.02 by t test. n=4. Values represent means ± SEM (J) Schematic of miR-206 up-regulation and function after denervation. (K) Mechanism of miR-206-dependent reinnervation.