Fig 1: Arundic acid preserves motor functions and electrophysiological properties of late-onset SMA mice in an SMN-independent manner. a Immunostaining of SMN (green) in the spinal cord ventral horns of wt or SMA mice treated with vehicle or AA at P44. Nucleic DNA was stained with Dapi (blue). The relative SMN protein level in the ventral horns of SMA mice treated with vehicle or AA was reduced compared to wt vehicle mice (p < 0.001), confirming that the effects of AA were SMN-independent. b Western blot analysis of SMN protein levels in spinal cord tissue of vehicle-treated wt, or vehicle- or AA-treated SMA mice at P44. Beta-actin was used as a loading control. SMA mice showed reduced SMN protein levels (p < 0.01) compared to wt mice. c Rotarod assessment of wt or SMA mice treated with vehicle or AA at P33 and P44. Vehicle-treated SMA mice showed a reduced running duration on the rotarod compared to wt mice at P33 (p < 0.01) and P44 (p < 0.001). When SMA mice were treated with AA, the running duration was enhanced compared to the vehicle-treated SMA mice at P33 (p < 0.05) and P44 (p < 0.001). d Grip strength measurements of wt or SMA mice treated with vehicle or AA at P33 and P44. Vehicle-treated SMA mice showed reduced grip strength at P33 (p < 0.05) and P44 (p < 0.05) compared to wt mice. The grip strength of AA-treated SMA mice was enhanced at P33 (p < 0.01) and P44 (p < 0.05) compared to vehicle-treated SMA mice. e MPA measurements of wt or SMA mice treated with vehicle or AA at P33 and P44. At P44, MPA was reduced in vehicle-treated SMA mice compared to AA-treated SMA (p < 0.05) and vehicle-treated wt mice (p < 0.01). f At P44, motor conduction velocity was slightly reduced in vehicle-treated SMA mice compared to wt mice (p < 0.01). g The body weight of SMA mice was not altered by AA. h The phenotype of SMA mice was not affected by AA treatment. N = 6 animals per condition for immunostaining. Three slices per lumbar spinal cord were investigated. Each data point reflects the mean of three spinal cord slices per animal. N = 3 animals per condition for Western blot analysis and glutamate measurements. N = 6 animals per condition for motor behavior and electrophysiological experiments. Scale bar: 20 µm. AA arundic acid, MPA motor potential amplitude, P postnatal day, SMA spinal muscular atrophy, SMN,survival of motor neuron, wt,wild type. P values: *p < 0.05, **p < 0.01, or ***p < 0.001
Fig 2: Late-onset SMA mice show early reduced EAAT1 expression and increased glutamate levels. a Immunostaining of EAAT1 (green) in the ventral horn of lumbar spinal cord slices from wt or SMA mice at P15, P20, and P42. Nucleic DNA was stained with Dapi (blue). The relative EAAT1 protein level in the ventral horn of SMA mice was reduced at P20, before motor neuron loss, and P42 (p < 0.001) compared to wt mice. b Western blot analysis of EAAT1 protein levels in spinal cord tissue of wt or SMA mice at P15, P20, and P42. Beta-actin was used as a loading control. SMA mice showed a reduced EAAT1 protein level at P20 (p < 0.01) and P42 (p < 0.001) compared to wt mice. c No change of EAAT1 mRNA expression was observed at any timepoint (p > 0.05). d Measurement of glutamate level in the spinal cord of wt or SMA mice at P15, P20, and P42 using a glutamate assay kit. The glutamate level in the spinal cord tissue of SMA mice was elevated at P20 (p < 0.001) and P42 (p < 0.001) compared to wt mice. n = 6 animals per condition for immunostaining. Three slices per lumbar spinal cord were investigated. Each data point reflects the mean of three spinal cord slices per animal. n = 3 animals per condition for Western blot analysis, qPCR analysis, and glutamate measurements. Scale bar: 20 µm. Abbreviations: EAAT1, excitatory amino acid transporter 1; mRNA, messenger ribonucleic acid; P, postnatal day; qPCR, real-time polymerase chain reaction; SMA, spinal muscular atrophy; wt, wild type. P values: *p < 0.05, **p < 0.01, or ***p < 0.001
Fig 3: Late-onset SMA mice show phenotypical changes, reduced body weights, and SMN protein levels. a SMA mice showed reduced grip strength at P33 (p < 0.05) and P44 (p < 0.05). b Body weight in SMA mice was reduced at P20 and P42 compared to wt mice (p < 0.001). c Photographs of wt and SMA mice at P42 showing necrotic tail tissue under SMA conditions at P42. At P15, no difference was observed (p > 0.05). d Immunostaining of SMN (green) in the ventral horn of lumbar spinal cord slices from wt or SMN mice at P15, P20, and P42. Nucleic DNA was stained with Dapi (blue). The relative SMN level in the ventral horn of SMA mice spinal cords was reduced at all timepoints compared to wt mice (p < 0.001). e Western blot analysis of SMN protein levels in spinal cord tissue of wt or SMA mice at P15, P20, and P42. Beta-actin was used as a loading control. Analysis revealed reduced SMN protein levels in SMA mice at all timepoints compared to wt control mice (p < 0.01 to p < 0.001). n = 6 animals per condition for immunostaining. Three slices per lumbar spinal cord were investigated. Each data point reflects the mean of three spinal cord slices per animal. n = 3 animals per condition for Western blot analysis. n = 12 animals per condition for body weight analysis. Scale bar: 20 µm. Abbreviations: P, postnatal day; SMA, spinal muscular atrophy; SMN, survival of motor neuron; wt, wild type. P values: *p < 0.05, **p < 0.01, or ***p < 0.001
Fig 4: Spinal astrocyte activation precedes the loss of spinal MN. a Immunostaining of spinal MN (SMI-32, green) in the ventral horn of lumbar spinal cord slices from wt or SMA mice at P15, P20, and P42. Nucleic DNA was stained with Dapi (blue). The number of spinal MNs in SMA mice was reduced at P42 (p < 0.001). b Immunostaining of GFAP (magenta) as a marker of astrocyte reactivity in the ventral horn of lumbar spinal cord slices from wt or SMA mice at P15, P20, and P42. Nucleic DNA was stained with Dapi (blue). SMA mice showed an elevated relative GFAP protein level at P20, before MN loss, and P42 compared to wt mice (p < 0.001). c Western blot analysis of GFAP protein levels in spinal cord tissue of wt or SMA mice at P15, P20, and P42. Beta-actin was used as a loading control. SMA mice showed an enhanced GFAP protein level at P20 and P42 compared to wt mice (p < 0.001). n = 6 animals per condition for immunostaining. Three slices per lumbar spinal cord were investigated. Each data point reflects the mean of three spinal cord slices per animal. n = 3 animals per condition for Western blot analysis. Scale bar: 20 µm. Abbreviations: GFAP, glial fibrillary acid protein; MN, motor neuron; P, postnatal day; SMA, spinal muscular atrophy; wt, wild type; #, number. P values: *p < 0.05, **p < 0.01, or ***p < 0.001
Supplier Page from OriGene Technologies for Actb Mouse qPCR Primer Pair (NM_007393)