An α2-Na/K ATPase/α-adducin complex in astrocytes triggers non-cell autonomous neurodegeneration

Abstract

Perturbations of astrocytes trigger neurodegeneration in several diseases, but the glial cell-intrinsic mechanisms that induce neurodegeneration remain poorly understood. We found that a protein complex of α2-Na/K ATPase and α-adducin was enriched in astrocytes expressing mutant superoxide dismutase 1 (SOD1), which causes familial amyotrophic lateral sclerosis (ALS). Knockdown of α2-Na/K ATPase or α-adducin in mutant SOD1 astrocytes protected motor neurons from degeneration, including in mutant SOD1 mice in vivo. Heterozygous disruption of the α2-Na/K ATPase gene suppressed degeneration in vivo and increased the lifespan of mutant SOD1 mice. The pharmacological agent digoxin, which inhibits Na/K ATPase activity, protected motor neurons from mutant SOD1 astrocyte-induced degeneration. Notably, α2-Na/K ATPase and α-adducin were upregulated in spinal cord of sporadic and familial ALS patients. Collectively, our findings define chronic activation of the α2-Na/K ATPase/α-adducin complex as a critical glial cell-intrinsic mechanism of non-cell autonomous neurodegeneration, with implications for potential therapies for neurodegenerative diseases.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1

Upregulation of α-adducin in SOD1G93A astrocytes mediates non–cell autonomous degeneration of motor neurons. (a) Lysates of spinal cord from symptomatic SOD1G93A transgenic mice and control non-transgenic mice were subjected to immunoblotting using an antibody that recognizes phosphorylation events in cells following exposure to oxidative stress. Lysates in lanes 5–8 were incubated with λ-phosphatase, which largely eliminated the 105-kDa immunoreactive band. (b) Lysates of spinal cords from symptomatic SOD1G93A and control mice were subjected to immunoprecipitation using the α-adducin antibody followed by immunoblotting with our phospho-antibody. (c) Immunoblots from spinal cord lysates showed an increase in α-adducin and phosphorylated α-adducin relative to the internal control proteins ERK and 14-3-3β in symptomatic SOD1G93A mice as compared with control wild-type littermates (120 d). (d) Immunoblots revealed that α-adducin and phosphorylated α-adducin were predominately expressed in primary glial cultures enriched with the astrocyte marker GFAP relative to primary motor neuron cultures enriched with the neuron marker β-tubulin. HSP60 was used as an internal control (lower panel). Blots shown in a–d are cropped. Full-length blots are presented in Supplementary Figure 11. (e) Immunohistochemistry of symptomatic SOD1G93A lumbar spinal cord sections revealed that phosphorylated-Ser436 α-adducin (phospho-α-adducin) colocalized with the astrocyte protein GFAP. Dashed line indicates ventral horn. The boxed areas are shown at high magnification; scale bars represent 50 μm. (f) Co-cultured astrocytes and motor neurons were subjected to immunocytochemistry with antibodies recognizing the motor neuron nuclear protein Islet1 (red) and the dendrite protein Map2 (green); scale bar represents 50 μm. Wild-type astrocytes transfected with the control U6 or α-adducin RNAi plasmid had little or no effect on motor neuron morphology or survival (upper and lower left panels). Control U6 SOD1G93A astrocytes induced non–cell autonomous motor neuron cell death and dendrite abnormalities (upper right panel). α-adducin knockdown in SOD1G93A astrocytes protected motor neurons against the non–cell autonomous cell death and dendrite abnormality (lower right panel). (g,h) Quantification of motor neuron survival was derived from n ≥ 900 cells per condition and values presented are the average of three independent experiments; two-tailed unpaired t test, P = 0.0095. Quantification of dendrite length was derived from n ≥ 240 images per condition and values presented are the average of three independent experiments; two-tailed unpaired t test, P = 0.0001. Data are presented as mean ± s.e.m. **P < 0.01, ***P < 0.001.

Figure 2

Knockdown of α-adducin in SOD1G93A mice suppresses motor neuron degeneration in vivo. (a,c) Spinal cords from SOD1G93A mice injected intraspinally were subjected to immunohistochemistry at end stage. End stage was defined as a time point at which the animal was unable to upright itself within 30 s of placement on its side. Immunohistochemistry with GFP in sections of the spinal cord in SOD1G93A mice lumbar revealed delivery of control virus (LV-U6; top, a) or α-adducin RNAi virus (LV-Addi; top, c) into the ventral horn; scale bars represent 100 μm. Alternating GFP-positive sections were subjected to immunohistochemistry using the GFP antibody and the neurofilament-SMi32 antibody (red), a motor neuron marker, or Nissl stained (bottom) for quantification of surviving motor neurons in GFP-labeled injected ventral horn and contralateral non-injected ventral horn (n ≥ 20 sections per animal); scale bars represent 50 μm. (b) Control LV-U6 SOD1G93A mice (n = 5) displayed equivalent degeneration of motor neurons in injected GFP-labeled ventral horn and non-injected contralateral ventral horn. (d) α-adducin knockdown in SOD1G93A mice (LV-Addi SOD1G93A; n = 5) increased motor neuron survival in GFP-labeled injected ventral horn as compared with non-injected contralateral ventral horn. Two-tailed unpaired t test: LV-U6, P = 0.340; LV-Addi, P = 0.0100. Data are presented as mean ± s.e.m. **P < 0.01.

Figure 3

Enrichment of the α2-Na/K ATPase/α-adducin complex in SOD1G93A astrocytes triggers motor neuron degeneration. (a) Immunoblots from spinal cord lysates showed an increase in the protein levels of α2-Na/K ATPase relative to the internal control proteins β2-ATPase and 14-3-3β in symptomatic SOD1G93A mice as compared with control wild-type littermates (120 d). (b) Immunoblots revealed that α2-Na/K ATPase was upregulated in SOD1G93A astrocytes as compared with non-transgenic controls. Knockdown of α-adducin in SOD1G93A astrocytes attenuated α2-Na/K ATPase protein levels. Protein levels are relative to ERK and 14-3-3β. Blots shown in a and b are cropped. Full-length blots are presented in Supplementary Figure 11. (c) Co-cultured astrocytes and motor neurons were subjected to immunocytochemistry with the motor neuron nuclear protein Islet1 (red) and the dendrite protein Map2 (green); scale bar represents 50 μm. Wild-type astrocytes transfected with the control U6 or α2-Na/K ATPase RNAi plasmid had little or no effect on motor neuron morphology or survival (upper and lower left panels). Control U6 SOD1G93A astrocytes induced non–cell autonomous motor neuron cell death and dendrite abnormalities (upper right panel). α2-Na/K ATPase knockdown in SOD1G93A astrocytes protected motor neurons against the non–cell autonomous cell death and dendrite abnormalities (lower right panel). (d,e) Quantification of motor neuron survival was derived from n ≥ 900 cells per condition and values presented are the average of three independent experiments; two-tailed unpaired t test, P = 0.0080. Quantification of dendrite length was derived from n ≥ 240 images per condition and values presented are the average of three independent experiments; two-tailed unpaired t test, P = 0.0001. Data are presented as mean ± s.e.m. **P < 0.01, ***P < 0.001. (f,g) Alternating GFP-positive sections from SOD1G93A mice injected intraspinally with lentivirus expressing α2-Na/K ATPase RNAi were subjected to immunohistochemistry using the GFP and neurofilament-SMi32 (red) antibodies or Nissl stained (bottom panels) for quantification of surviving motor neurons in GFP-labeled injected ventral horn and contralateral non-injected ventral horn at end stage (n ≥ 20 sections per animal); scale bars represent 100 (top) and 50 (middle, bottom) μm. End stage was defined as a time point at which the animal was unable to upright itself within 30 s of placement on its side. α2-Na/K ATPase knockdown in SOD1G93A mice (LV-ATPi SOD1G93A, n = 5) increased motor neuron survival in GFP-labeled injected ventral horn as compared with non-injected contralateral ventral horn. Arrowheads indicate surviving motor neurons (quantified in g). Two-tailed unpaired t test, P = 0.0005. Data are presented as mean ± s.e.m. ***P < 0.001.

Figure 4

Heterozygous disruption of Atp1a2 in SOD1G93A mice suppresses motor neuron degeneration and enhances mouse lifespan. (a) Downregulation of α2-Na/K ATPase in SOD1G93A astrocytes by crossing SOD1G93A mice with Atp1a2+/− (right) protected motor neurons from non–cell autonomous cell death and dendrite abnormalities induced by control SOD1G93A astrocytes (left). Scale bar represents 50 μm. (b,c) Quantification of motor neuron survival is derived from n ≥ 900 cells per condition and values presented are the average of three independent experiments; unpaired t test, P = 0.0003. Quantification of dendrite length is derived from n ≥ 240 images per condition and values presented are the average of three independent experiments performed in duplicates; two-tailed unpaired t test, P = 0.0001. Data are presented as mean ± s.e.m. ***P < 0.001. (d) Disease onset, that is, initial day of weight loss, was significantly delayed in α Atp1a2+/−; SOD1G93A mice (Atp1a2+/−, n = 14) as compared with control SOD1G93A littermates (Atp1a2+/+, n = 14); two-tailed unpaired t test, P = 0.0009. (e) Early disease process, that is, age at which 10% of weight loss is reached, was significantly delayed in Atp1a2+/−; SOD1G93A mice (Atp1a2+/−; SOD1G93A, red circles, n = 14) as compared with control SOD1G93A littermates (Atp1a2+/+; SOD1G93A, black squares, n = 14) (P = 0.0001). (f) Early phase disease progression, that is, days from onset to 10% weight loss, displayed no change between α Atp1a2+/−; SOD1G93A mice and control SOD1G93A littermates; two-tailed unpaired t test, P = 0.2023. (g) Late-phase disease progression, that is, from 10% weight loss to end stage, displayed significant delay in Atp1a2+/−; SOD1G93A mice (Atp1a2+/−, n = 14) as compared with control SOD1G93A littermates (Atp1a2+/+, n = 14); two-tailed unpaired t test, P = 0.0005. (h) Kaplan-Meier survival plots showed substantial and significant increase in lifespan for Atp1a2+/−; SOD1G93A mice (Atp1a2+/−; SOD1G93A, red circles, n = 14) as compared with control SOD1G93A littermates (Atp1a2+/+; SOD1G93A, black squares, n = 14) (P = 0.0001). (i) Control SOD1G93A mice displayed morbidity of reduced mobility and the inability to upright at endstage (Atp1a2+/+; SOD1G93A), whereas age-matched SOD1G93A littermates that harbor a heterozygous null Atp1a2 allele displayed increased mobility and health (Supplementary Movies 1 and 2). (j) Immunohistochemistry of NMJs from gastrocnemius muscle using the presynaptic marker synapsin (green) and postsynaptic marker α-bungarotoxin (red, Bu Tx) at 120 d showed that control SOD1G93A mice had denervated NMJs (left), whereas Atp1a2+/− displayed an increased NMJ integrity (right); scale bar represents 20 μm. (k) Quantification of NMJs from n = ~300 NMJs from three animals per group (two-tailed unpaired t test: complete denervated, P = 0.0196; partial denervated, P = 0.0203; completely innervated, P = 0.0370). Data are presented as mean ± s.e.m. *P < 0.05.

Figure 5

Na/K ATPase stimulates mitochondrial respiration and expression of inflammatory genes in SOD1G93A astrocytes. (a) Control SOD1G93A astrocytes (left) induced non–cell autonomous motor neuron cell death and dendrite abnormalities. Pharmacological inhibition of Na/K ATPase with ouabain (middle) and digoxin (right) was neuroprotective from the non–cell autonomous cell death and dendrite abnormalities induced by SOD1G93A astrocytes (left); scale bar represents 50 μm. Pharmacological inhibition of Na/K ATPase in control co-cultures non-transgenic astrocytes (Non-tg) and motor neurons with ouabain and digoxin did not alter motor neuron survival or morphology. (b,c) Quantification of motor neuron survival was derived from n ≥ 900 cells per condition and values presented are the average of three independent experiments (two-tailed unpaired t test: ouabain, P = 0.0024; digoxin, P = 0.0001). Quantification of dendrite lengths was derived from n ≥ 240 images per condition and values presented are the average of three independent experiments (two-tailed unpaired t test: ouabain, P = 0.0002; digoxin, P = 0.0001). Data are presented as mean ± s.e.m. **P < 0.01, ***P < 0.001. (d) A representative plot of oxygen consumption measured in astrocytes from control non-transgenic, SOD1G93A and heterozygous-null SOD1G93A mice using Seahorse Bioscience XF Analyzer. Arrow indicates time when the mitochondrial uncoupler FCCP was added. (e) Basal and maximum oxygen consumption were quantified in control non-transgenic (n = 3), SOD1G93A (n = 3) and heterozygous-null SOD1G93A mice (n = 3) astrocytes using Seahorse Bioscience XF Analyzer (two-tailed unpaired t test: basal consumption, P = 0.0001; maximum consumption, P = 0.0001). Data are presented as mean ± s.e.m. ***P < 0.001. (f) Total RNA of astrocytes from control non-transgenic (n = 3), SOD1G93A (n = 3) and heterozygous-null SOD1G93A mice (n = 3) were subjected to qRT-PCR analyses using primers to a panel of pro-inflammatory genes. Gene expression was normalized to GAPDH expression. The expression of 18 inflammatory genes was upregulated in SOD1G93A astrocytes. Downregulation of α2-Na/K ATPase in SOD1G93A astrocytes significantly decreased expression of half of the upregulated inflammatory genes (two-tailed unpaired t test: SPP1, P = 0.0253; LCN2, P = 0.0013; CLM1, P = 0.0052; WNT1, P = 0.0198; CCL11, P = 0.0387; CXCL1, P = 0.0246; CCR4, P = 0.0314; Il1b2, P = 0.0421; Itgb2, P = 0.0215; II1r1, P = 0.0092). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

The α2-Na/K ATPase/α-adducin complex is upregulated in spinal cord in familial and sporadic ALS patients. (a) Immunoblots from patients with familial ALS (n = 5) revealed elevated protein levels of α2-Na/K ATPase (top) and α-adducin (bottom) in spinal cord lysates as compared to control patients (n = 3). ERK served as a loading control. (b,c) Quantification of the relative densitometry protein levels for α2-Na/K ATPase and α-adducin relative to the internal control ERK; two-tailed unpaired t test: α2-Na/K ATPase, P = 0.0302; α-adducin, P = 0.0336. Blots shown in a and b are cropped. Full-length blots are presented in Supplementary Figure 11. (d) Immunoblots from patients with sporadic ALS (n = 5) showed elevated protein levels of α2-Na/K ATPase (top) and α-adducin (bottom) in spinal cord lysates as compared with control patients (n = 3). (e,f) Quantification of the relative densitometry protein levels for α2-Na/K ATPase and α-adducin relative to the internal control ERK; two-tailed unpaired t test: α2-Na/K ATPase, P = 0.0467; α-adducin, P = 0.0181. Data are presented as mean ± s.e.m. *P < 0.05.