Motoneuron transplantation rescues the phenotype of SMARD1 (spinal muscular atrophy with respiratory distress type 1)

Abstract

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal form of infantile motoneuron disease. There is currently no effective treatment, although motor neuron replacement is a possible therapeutic strategy. We transplanted purified motor neurons into the spinal cord of nmd mice, an animal model of SMARD1. We also administered pharmacological treatment targeting the induction of axonal growth toward skeletal muscle target. At the end stage of the disease, donor-derived motor neurons were detected in the nmd anterior horns, extended axons into the ventral roots, and formed new neuromuscular junctions. These data correlated with improved neuromuscular function and increased life spans. The neuroprotective effect was associated with a reduction in proinflammatory molecules in treated spinal cords. This is the first report that functional restoration of motor units with transplanted motoneurons is feasible in an animal model of a human motoneuron disease, opening up new possibilities for therapeutic intervention.

Figure 1.

Phenotype of spinal cord-derived LeX+ cells. a, LeX+ cells were isolated from VZ cells of E10.5 mouse spinal cords by immunomagnetic selection and expanded in vitro. b, FACS analysis of negative control cells. c, FACS analysis showed that positive fractions were 70–98% pure for LeX+ cells. d–f, Isolated LeX+ cells formed a monolayer of cells in culture that were positive for both nestin (e, red) and LeX (d, green). g–i, Phase-contrast and immunofluorescence images of LeX+ cells expressing both SOX2 (red) and the proliferation marker PCNA (light blue). j, k, LeX+ cells coexpressed the stem cell markers SOX 2 (green) and Musashi-1 (red). Nuclei are labeled with DAPI (4′,6-diamidino-2-phenylindole; blue). f, i, and l show the merged images. Scale bar: d–f, 40 μm; g–l, 50 μm.

Figure 2.

LeX+ cells can acquire a motoneuron phenotype in vitro. a, b, After in vitro differentiation, LeX+ cells derived from HB9-GFP transgenic mice that express GFP protein (green) in motoneurons acquired a motoneuron phenotype as demonstrated by expression of GFP (a) and ChAT (b–d). c, e, Merged images. f, Images merged with phase-contrast image. g, Quantification of cells expressing HB9-GFP and ChAT after 48 h in culture (black bars) and after 7 d (green bars). Error bars represent SD. h, FACS analysis of HB9-GFP expression as an indicator of motoneuron purity after selection for p75NTR surface antigen by immunomagnetic selection. i, j, Cultured HB9-GFP cells after immunomagnetic selection for p75NTR. The culture was enriched for cells positive for both HB9-GFP (i, j) and ChAT (j; merged image of the green HB9-GFP signal with the red ChAT signal). Scale bar: a–c, 80 μm; d–f, 40 μm; i, j, 100 μm.

Figure 3.

Motoneuron transplantation increases survival, reduces weight loss, and improves the neuromuscular function of nmd mice. nmd mice received transplanted motoneurons (nmd-trans, group 1) or transplanted motoneurons and dbcAMP+rolipram+GDNF treatment (nmd-trans+drugs, group 3). Alternatively, nmd mice received vehicle only (nmd-veh, group 2) or vehicle plus dbcAMP+rolipram+GDNF (nmdveh+drugs, group 4). wt mice were vehicle treated. For each group, n = 24. The mice were assessed for the following: appearance, survival, weight, and neuromuscular function. a, Representative photographs of mice from the indicated treatment groups. nmd-veh mice clenched their hindlimbs compared with wt mice when picked up by the tail; nmd mice from the two treated groups (groups 1 and 3) had a greater capacity to open and extend their lower limbs. b, Time on the rotarod treadmill for treated nmd mice (groups 1 and 3), untreated mice (groups 2 and 4), and wt controls (n = 24). The time that mice could stay on the rotarod treadmill was statistically different between the treated and untreated nmd mice at age 8 weeks (p < 0.00001). Error bars show the SDs. c, Mean body weight of nmd treated mice (groups 1 and 3) or untreated mice (groups 2 and 4) and unaffected wt littermates (n = 24 for each group). The plot shows mean body weight for each week; error bars show the SDs. Both groups of treated nmd mice grew faster than untreated nmd mice (at 8 weeks; p < 0.00001). d, Kaplan-Meier survival curves of nmd-treated and untreated mice. Treated nmd mice (nmdtrans, group 1) survived significantly longer than vehicle-treated mice (nmd-veh, group 2) (group 1 vs group 2: log-rank test, χ² = 30.41, p < 0.00001). Indeed, group 3 mice (nmd-trans+drugs) survived longer than all other groups (group 3 vs group 1: log-rank test; χ² = 7.38, p = 0.0066; group 3 vs group 2: log-rank test; χ² = 45.77, p < 0.00001; group 3 vs group 4 (nmdveh+drugs): log-rank test, χ² = 38.26, p < 0.00001). nmd mice transplanted with primary murine fibroblasts (nmd-fibro) did not survive significantly longer than the untreated group (fibroblast treated vs group 2: log-rank test, χ² = 0.001691, p = 0.96719).

Figure 4.

Motoneurons transplanted into nmd mice engraft in the spinal cord anterior horns. Motoneurons derived from the LeX+ fraction of HB9-GFP spinal cord were transplanted by intraparenchymal injection. GFP+ motoneurons engrafted in the anterior horn of the spinal cord are shown in spinal cord coronal sections of treated nmd mice (group 1, nmd-trans). a, Merged image (composite image to present entire spinal cord) of red TuJ1 signal and green HB9-GFP signals. Immunohistochemistry of neuroectodermal markers confirmed that these cells displayed motoneuron characteristics. Confocal microscopy demonstrated that GFP motoneurons coexpressed neuron-specific proteins such as NeuN and MAP2. b, GFP (green). c, NeuN (red). d, Merged image. e, GFP (green). f, MAP2 (red). g, Merged image. h–j, GFP motoneurons had a cholinergic phenotype as shown by ChAT expression: h, merge of GFP (green) and NeuN (red); i, ChAT (light blue); j, merged image of h and i. Nuclei were stained with DAPI (blue). Scale bar: a, 250 μm; b–d, 75 μm; e–g, 100 μm; h–j, 50 μm.

Figure 5.

Transplanted motoneuron axons reach their skeletal muscle targets in nmd mice. a, Schematic diagram of a motoneuron with its axon forming a connection with muscle. b–d, Transplanted HB9-GFP motoneurons (green) in the nmd spinal cord (from group 3, nmd-trans+drugs mouse) were retrogradely labeled with Fluorogold (blue), showing their connection with the muscle. d, Merged image of b and c. e–g, Donor-derived GFP axons were detected in the anterior roots. e, GFP (green signal). f, NF (red signal). g, Merged image of e and f. h–j, HB9-GFP axons (h, green) reached the muscle and formed new neuromuscular junctions, identified by rhodamine-conjugated bungarotoxin (btx; i) with the host skeletal muscle. j, Merged image. Scale bar: b–d, h, i, 75 μm; e–g, 50 μm; j, 30 μm.

Figure 6.

Profiles of cytokines expressed in the spinal cords of treated and untreated nmd mice. The xMAP assay was used to simultaneously detect and quantify 26 cytokines in the spinal cord tissues from nmd mice in each of the four groups [group 1 (nmd-trans; for transplant) received transplanted motoneurons; group 2 (nmd-veh; for vehicle) received vehicle only (mock transplantation); group 3 (nmd-trans+drugs; for transplant plus drugs) received both transplanted motoneurons plus pharmacological agents to promote axonal extension (described below); and group 4 (nmd-veh-drugs; for vehicle plus drugs) received vehicle only plus the pharmacological agents] and wt mice. a, b, The chemokines MCP1 and MCP3 were expressed at significantly higher levels in the spinal cords of group 2 (nmd-veh) than in transplanted (groups 1 and 3) and wt mice. c, RANTES was expressed at a significantly higher levels (∼2-fold higher) in nmd-veh compared with the transplanted (groups 1 and 3) and wt groups. d, MIP1 was expressed at a significantly higher level (∼9-fold higher) in nmd-veh compared with group 1 (nmd-trans), whereas it was undetectable in group 3 (nmd-trans+drugs) mice and in the wt group. e, Spinal cords from nmd-veh mice expressed significantly higher levels of IL-1β than transplanted nmd mice (groups 1 and 3). f, IL-1α showed a significant increased level in nmd-veh mice with respect to wt mice and a higher level in nmd-veh mice with respect to transplanted mice, even if not significant. g, Other proinflammatory cytokines were either not expressed in measurable amounts or the differences between the groups of mice were nonsignificant. (**p < 0.01; *p < 0.05). Error bars represent SD.