Fig 1: Ex vivo model of glutamate-mediated MN death. a Immunostaining of spinal MNs (SMI-32, green) in OTSCs from wt mice 48 h post-exposure (p.e.) to a 30 min pulse of 50 or 500 µM glutamate. Nucleic DNA was stained with Dapi (blue). Forty-eight hours after glutamate pulse, the number of spinal MNs was reduced (p < 0.001). b Immunostaining of spinal MNs (SMI-32, green) in OTSC from wt mice after exposure to the EAAT1 inhibitor PDC for 48 h. The inhibition of EAAT1 reduced spinal MNs after 48 h (P < 0.001). c PDC treatment enhanced glutamate level in OTSC (p > 0.01) d PDC treatment enhanced glutamate level in supernatant of OTSCs (p > 0.001) n = 10 slices per condition. Scale bar: 20 µm. Abbreviations: EAAT1, excitatory amino acid transporter 1; MN, motor neuron; OTSC, organotypic spinal cord slice cultures, PDC, L-trans-pyrrolidine-2,4-dicarboxylic acid; SMI-23, neurofilament heavy polypeptide; wt, wild type; #, number per slice. 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: SMA-like astrocytes generated by siRNA show similar protein alteration and dysfunctions in glutamate uptake as the late-onset SMA mouse model. a Immunostaining of SMN (green) in cultured wt spinal astrocytes transfected with scrambled or SMN1 siRNA at DIV 10. Nucleic DNA was stained with Dapi (blue). Astrocytes transfected with SMN siRNA showed reduced SMN protein levels (p < 0.01). b Immunostaining of GFAP (magenta) in cultured wt spinal astrocytes transfected with scrambled or SMN siRNA at DIV 10. Nucleic DNA was stained with Dapi (blue). Astrocytes transfected with SMN siRNA showed elevated GFAP protein levels compared to astrocytes transfected with scrambled siRNA (p < 0.01). c Immunostaining of EAAT1 (magenta) in cultured wt spinal astrocytes transfected with scrambled or SMN siRNA at DIV 10. Nucleic DNA was stained with Dapi (blue). Astrocytes transfected with SMN siRNA showed reduced EAAT1 protein levels compared to scrambled siRNA-transfected cells (p < 0.01). d In Western blot analysis the EAAT1 protein level in SMA-like astrocytes was reduced (p < 0.05) e No alteration in EAAT mRNA expression was detected (p > 0.05) in qPCR analysis. f When scrambled or SMN siRNA transfected astrocytes were exposed to 200 µM glutamate for 4 h, SMN-deficient astrocytes showed reduced glutamate uptake (p < 0.001). A similar effect was observed when scrambled siRNA-transfected astrocytes were exposed to the EAAT1 inhibitor PDC. Glutamate uptake was reduced compared to cells not exposed to PDC (p < 0.001). g Astrocytes transfected with SMN siRNA but not exposed to glutamate showed an increased release of glutamate compared to astrocytes transfected with scrambled siRNA (p < 0.001). n = 6 independent experiments for immunostaining. For each experiment, > 50 cells were analyzed per condition. N = 3 independent experiments for functional assays, qPCR, and Western blot analysis. Scale bar: 50 µm. Abbreviations: DIV, days in vitro; EAAT1, excitatory amino acid transporter 1; GFAP, glial fibrillary acid protein; mRNA, messenger ribonucleic acid; PDC, L-trans-pyrrolidine-2,4-dicarboxylic acid; qPCR, real-time polymerase chain reaction; siRNA, small interfering ribonucleic acid; SMN, survival of motor neuron; wt, wild type. P values: *p < 0.05, **p < 0.01, or ***p < 0.001
Fig 4: In vivo administration of AA inhibits astrocyte activation, enhances EAAT1 expression, and avoids MN loss. a Immunostaining of GFAP (magenta) 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). SMA mice treated with vehicle showed elevated protein levels of GFAP compared to wt mice (p < 0.001). When SMA mice were treated with AA, GFAP protein levels were reduced compared to vehicle-treated SMA animals (p < 0.001). b Western blot analysis of the GFAP protein level in the spinal of wt or SMA mice treated with vehicle or AA at P44. Similar effects as described for immunostaining were observed (p < 0.01). c Immunostaining of EAAT1 (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). SMA mice treated with vehicle showed reduced protein levels of EAAT1 compared to wt mice (p < 0.001). When SMA mice were treated with AA, EAAT1 protein levels were elevated compared to vehicle-treated SMA animals (p < 0.001). d Western blot analysis of EAAT1 protein level in the spinal cord tissue of wt or SMA mice treated with vehicle or AA at P44. Similar effects as described for immunostaining were observed (p < 0.01 to p < 0.001). e EAAT1 mRNA level was increased in AA-treated SMA mice (p < 0.05). In contrast, the mRNA level was not affected in vehicle-treated SMA mice (p > 0.05) f Measurement of glutamate levels in the spinal cord tissue of wt or SMA mice treated with vehicle or AA using a glutamate assay kit. The glutamate level in the spinal cord tissue of vehicle-treated SMA mice was elevated compared to wt mice (p < 0.001). When SMA mice were treated with AA, the glutamate level in their spinal cord tissue was reduced to the control level (p < 0.001). g Immunostaining of spinal MN (SMI-32, 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). Vehicle-treated SMA mice showed a reduced number of MNs compared to wt mice at P44 (p < 0.01). When SMA mice were treated with AA, the number of MNs stayed at the level of wt mice (p < 0.01 to vehicle SMA; p > 0.05 to wt). 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: AA, arundic acid; EAAT1, excitatory amino acid transporter 1; GFAP, glial fibrillary acid protein; MN, motor neuron; mRNA, messenger ribonucleic acid; P, postnatal day; qPCR, real-time polymerase chain reaction; SMA, spinal muscular atrophy; SMI-23, neurofilament heavy polypeptide; wt, wild type; #, number per slice. P values: *p < 0.05, **p < 0.01, or ***p < 0.001
Fig 5: Impaired glutamate homeostasis in induced human astrocytes and human CSF and serum samples. a Immunostaining of SMN (green) in induced human astrocytes transfected with scrambled or SMN siRNA at DIV 10. Nucleic DNA was stained with Dapi (blue). Astrocytes transfected with SMN siRNA showed reduced SMN protein levels (p < 0.001). b Immunostaining of EAAT1 (green) in induced human astrocytes transfected with scrambled or SMN siRNA at DIV 10. Nucleic DNA was stained with Dapi (blue). Astrocytes transfected with SMN siRNA showed reduced EAAT1 protein levels compared to scrambled siRNA-transfected cells (p < 0.001). c Induced human astrocytes transfected with SMN siRNA but not exposed to glutamate showed an increased release of glutamate compared to astrocytes transfected with scrambled siRNA (p < 0.001). d When scrambled or SMN siRNA-transfected induced human astrocytes were exposed to 200 µM glutamate for 4 h, SMN-deficient induced human astrocytes showed reduced uptake of glutamate (p < 0.01). e CSF samples of SMA type 2 and 3 patients showed elevated glutamate levels compared to controls (p < 0.01). f Serum samples of SMA type 2 and 3 patients showed elevated glutamate levels compared to controls (p < 0.001). g In CSF samples of SMA type 2 and type 3 patients EAAT1 level was reduced prior to treatment. Nusinersen-treatment did not affect EAAT1 level. n = 6 independent experiments for immunostaining. For each experiment, > 50 cells were analyzed per condition. n = 3 independent experiments for functional assays. n = 7 patients for glutamate assay of CSF or serum samples. Scale bar: 50 µm. DIV days in vitro; EAAT1, excitatory amino acid transporter 1, siRNA small interfering ribonucleic acid, SMA spinal muscular atrophy, SMN survival of motor neuron. P values: *p < 0.05, **p < 0.01, or ***p < 0.001
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