AAV9-mediated delivery of miR-23a reduces disease severity in Smn2B/-SMA model mice

Abstract

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in survival motor neuron 1 (SMN1). The molecular mechanisms underlying motor neuron degeneration in SMA remain elusive, as global cellular dysfunction obscures the identification and characterization of disease-relevant pathways and potential therapeutic targets. Recent reports have implicated microRNA (miRNA) dysregulation as a potential contributor to the pathological mechanism in SMA. To characterize miRNAs that are differentially regulated in SMA, we profiled miRNA levels in SMA induced pluripotent stem cell (iPSC)-derived motor neurons. From this array, miR-23a downregulation was identified selectively in SMA motor neurons, consistent with previous reports where miR-23a functioned in neuroprotective and muscle atrophy-antagonizing roles. Reintroduction of miR-23a expression in SMA patient iPSC-derived motor neurons protected against degeneration, suggesting a potential miR-23a-specific disease-modifying effect. To assess this activity in vivo, miR-23a was expressed using a self-complementary adeno-associated virus serotype 9 (scAAV9) viral vector in the Smn2B/- SMA mouse model. scAAV9-miR-23a significantly reduced the pathology in SMA mice, including increased motor neuron size, reduced neuromuscular junction pathology, increased muscle fiber area, and extended survival. These experiments demonstrate that miR-23a is a novel protective modifier of SMA, warranting further characterization of miRNA dysfunction in SMA.

Figure 1

miR-23a is downregulated in SMA patient iPSC-derived motor neurons. Motor neurons were differentiated from control and SMA patient-derived iPSCs and analyzed for miR expression. (A) Volcano plot of miRNA PCR array revealing 16 significantly downregulated miRNAs in SMA vs. control iPSC-derived motor neurons at 28 days of differentiation. Horizontal line denotes a P-value 0.05. Dashed vertical lines indicate 2-fold change. Arrow indicates miR-23a. (B) qRT-PCR comparing miR-23a levels in wild-type and SMA motor neurons, compared using Student's t-test. (C) Representative images of immunohistochemical staining of iPSC-derived SMA motor neurons cultured with control astrocyte-conditioned media (CTL ACM), SMA astrocyte-conditioned media (SMA ACM), SMA astrocyte-conditioned media and transfected with miR scramble (SMA ACM + miR scramble), or SMA astrocyte-conditioned media and transfected with synthetic miR-23a (SMA ACM + miR-23a). Cells were stained with ChAT (red) to label motor neurons, Tuj1 (green) to label neurons, and Hoescht (blue) to label nuclei. Images represent merged channels. Images were taken at 40× (scale bar = 100 μm). (D) Survival of iPSC-derived SMA motor neurons cultured with CTL ACM, SMA ACM, SMA ACM + scramble, or SMA ACM + miR-23a at 28 days of differentiation. Groups were compared using one-way ANOVA and Newman-Keuls multiple comparisons test. Scatter plot is shown as mean ± SEM (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, significance not indicated P>0.05).

Figure 2

scAAV9-miR-23a improves motor neuron soma size and perimeter. L3–L5 spinal cords were dissected from mice harvested at P21, cross sectioned, and stained (n = 3 mice). (A) Representative images of L3–L5 cross sections immunohistochemically labeled with NeuroTrace Nissl (green) to label Nissl substance and ChAT (red) to label motor neurons. Images were taken with 40× objective lens (scale bar = 50 μm). (B) Scatter plot representing number of ChAT-positive motor neuron soma in the L3–L5 spinal cord. (C) Scatter plot representing cross-sectional area of ChAT-positive motor neuron soma in the L3–L5 spinal cord. (D) Scatter plot representing perimeter of ChAT-positive motor neuron soma in the L3–L5 spinal cord. (E) Representative images of L3–L5 cross sections immunohistochemically labeled with VGLUT1 (green) to label proprioceptive inputs and ChAT (red). Images were taken with 40× objective lens (scale bar = 50 μm). (F) Scatter plot representing quantification of VGLUT1⁺ proprioceptive synaptic inputs on motor neuron soma. (G) Western blot of SMN in spinal cord whole tissue lysates. Experimental groups were compared using one-way ANOVA with Newman-Keuls multiple comparisons test. Scatter plots are shown as mean ± SEM (^**P < 0.01, ^***P < 0.001, ^nsP > 0.05).

Figure 3

scAAV9-miR-23a reduces neuromuscular junction defects in Smn^2B/− mice. Transverse abdominus muscles were dissected from mice harvested on P17, fixed, and stained (n = 5 mice). (A) Representative images of neuromuscular junctions stained with neurofilament (green) to label motor axons, synaptophysin (green) to label presynaptic terminals, and α-bungarotoxin (red) to label endplates. Arrows point toward fully denervated endplates. Images were taken with 40× objective lens (scale bar = 20 μm). (B) Bar graph representing average percentages of fully innervated, partially innervated, and denervated motor endplates. Statistical analysis was performed comparing degree of fully innervated endplates, represented by grey dots. (C) Scatter plot representing quantification of motor endplate area. Experimental groups were compared using one-way ANOVA with Newman-Keuls multiple comparisons test. Scatter plots are shown as mean ± SEM (^***P < 0.001).

Figure 4

scAAV9-miR-23a decreases atrogene transcript levels and increases muscle fiber area and in Smn^2B/− mice. Histological and molecular analyses were conducted on muscle harvested from mice at P17. (A) Quantification of relative transcript levels of miR-23a-3p in hindlimb skeletal muscle via qRT-PCR (n = 3 mice per group). (B) Quantification of relative transcript levels of MuRF1 and Atrogin1 in hindlimb skeletal muscle via qRT-PCR (n = 3 mice per group). (C) Representative images of cross sections of TA and SO. Sections were stained with laminin (green) to label muscle fiber boundaries. Images were taken with 40× objective lens (scale bar = 50 μm). (D) Scatter plot representing cross-sectional area of TA and SO muscle fibers (n = 4 mice per group; n > 350 fibers per TA; n > 200 fibers per SO). Experimental groups were compared using one-way ANOVA with Newman-Keuls multiple comparisons test. Scatter plots are shown as mean ± SEM (^**P < 0.0 1, ^***P < 0.001, ^nsP > 0.05).

Figure 5

scAAV9-miR-23a extends survival in Smn^2B/− mice. (A) Kaplan-Meier curve of Smn^2B/− mice treated with scAAV9-miR-23a via IV or ICV injection. Difference in survival between treatment groups and untreated Smn^2B/− controls was calculated using the log-rank Mantel-Cox test. (B) Weight gain of Smn^2B/− mice treated with scAAV9-miR-23a via IV or ICV injection (^***P < 0.001).