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.
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Acknowledgements
We thank members of the Bonni laboratory for helpful discussions. We thank L. Zinman (University of Toronto) for providing human patient tissue samples. This work was supported by a grant from the Edward R. and Anne G. Lefler Foundation (A.B.) and The Ruth L. Kirschstein National Research Service Awards T32 5T32AG00222 (G.G.). Human spinal cord material provided from Northwestern University autopsy program is partially funded from the Les Turner ALS Foundation. Additional human tissue samples were obtained from the Human Brain and Spinal Fluid Resource Center, which is sponsored by the National Institute of Neurological Disorders and Stroke and the US National Institutes of Health, National Multiple Sclerosis Society, and Department of Veterans Affairs.
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A.B. directed and coordinated the project. G.G. designed and performed or participated in all experiments. J.B. performed mouse husbandry and survival studies. J.B.L. provided α2-Na/K ATPase knockout mice. H.S. performed mass spectrometry analysis. J. Ravits, T.S. and J. Robertson provided human tissue samples. The manuscript was written by G.G. and A.B. and commented on by all authors.
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Supplementary Figure 1 α-Adducin in spinal cord is upregulated in symptomatic SOD1G93A spinal cord within astrocytes.
(a) Immunoblots for α-Adducin protein at 60 and 90 day old SOD1G93A mice show α-Adducin is upregulated at 90 days. (b) Immunohistochemistry with in sections of the lumbar spinal cord from symptomatic SOD1G93A mice displays Ser436-phosphorylated α-Adducin does not co-localize with the motor neuron marker (SMi32). Arrowheads indicate motor neurons; scale bar 50μm. (c) Immunohistochemistry from sections of control wild type lumbar spinal cord displays Ser436-phosphorylated α-Adducin does not co-localize with the motor neuron marker (SMi32); upper panels. Arrowheads indicate motor neurons. Immunohistochemistry from sections of control wild type lumbar spinal cord displays Ser436-phosphorylated α-Adducin co-localize with the astrocyte marker (GFAP) lower panels. Scale bar 50μm. (a) are cropped; full length images are presented in Supplementary Figure 11.
Supplementary Figure 2 Expression of a RNAi-resistant form of α-Adducin in SOD1G93A astrocytes restores the ability of SOD1G93A astrocytes to induce non-cell autonomous motor neuron cell death.
(a) Knockdown of α-Adducin relative to control U6 in astrocytes (b) 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 50μm. Wild type astrocytes transfected with the control U6 or α-Adducin RNAi plasmid had little or no effect on motor primary motor neurons cell death or dendrite abnormalities (upper left panel); quantified (c and d). Control U6 SOD1G93A astrocytes induced non-cell autonomous motor neuron cell death and dendrite abnormalities (upper right panel); quantified (c and d). Knockdown of α-Adducin in SOD1G93A astrocytes protected motor neurons against the non-cell autonomous cell death and dendrite abnormalities (lower left panel); quantified (c and d). Expressions of an RNAi-resistant form of α-Adducin (Add-Res) in the background of α-Adducin RNAi in SOD1G93A astrocytes restored the ability of the SOD1G93A astrocytes to induce non-cell autonomous cell death and dendrite abnormalities in motor neurons (lower right panel); quantified (d and e). All data in bar charts show mean ± s.e.m (***p<0.001; unpaired t-test). (a) are cropped; full length images are presented in Supplementary Figure 11.
Supplementary Figure 3 Lentivirial mediated knockdown in vivo predominately target astrocytes.
(a) Immunohistochemistry with GFP in sections of the lumbar spinal cord from SOD1G93A mice displaying percent GFP positive astrocytes (GFAP), microglia (Iba1) and motor neurons (SMi32) 30 days post-injection. Arrowheads indicate motor neurons; scale bar 50μm. (b) Quantifications of percent GFP positive cells revealed lentivirus predominately target astrocytes; n=~300 per cell type/three. All data in bar charts show ± s.e.m (***p<0.001; ANOVA). (c) Immunohistochemistry with GFP in sections of the lumbar spinal cord from wild type mice displaying percent GFP positive astrocytes (GFAP), microglia (Iba1) and motor neurons (SMi32) 30 days post-injection. Arrowheads indicate motor neurons; scale bar 50μm. (d) Quantifications of percent GFP positive cells revealed lentivirus predominately target astrocytes; n=~300 per cell type/three. All data in bar charts show ± s.e.m (***p<0.001; unpaired t-test).
Supplementary Figure 4 Knockdown of α-Adducin in SOD1G93A mice decreases immunoreactivity for phosphorylated Ser436-α-Adducin.
Spinal cord from SOD1G93A mice injected intraspinally with lentivirus expressing short hairpin RNAs targeting α-Adducin and encoding GFP (LV-Addi) or the corresponding control U6 (LV-U6) were subjected to immunohistochemistry using GFP and phospho-α-Adducin (red) antibodies. Knockdown of α-Adducin (LV-Addi) led to a decreased in immunoreactivity of phospho-α-Adducin within the GFP-labeled ventral horn as compared to control U6 (LV-U6) injected ventral horn; scale bar 50μm.
Supplementary Figure 5 Knockdown of α-Adducin or α2-Na/K ATPase in SOD1G93A mice do not alter gliosis in the spinal cord.
Spinal cord from SOD1G93A mice injected intraspinally with lentivirus expressing short hairpin RNAs targeting α-Adducin or α2-Na/K ATPase or the corresponding control U6 virus were subjected to immunohistochemistry using GFP and the GFAP (red) antibodies. Knockdown of α-Adducin (LV-Addi) or α2-Na/K ATPase (LV-ATPi) had little or no effect on the presence or abundance of astrocytes within the GFP-labeled ventral horn; scale bar 50μm.
Supplementary Figure 6 Knockdown of α-Adducin or α2-Na/K ATPase in SOD1G93A mice do not alter microgliosis in spinal cord.
Spinal cord from SOD1G93A mice injected intraspinally with lentivirus expressing short hairpin RNAs targeting α-Adducin or α2-Na/K ATPase or the corresponding control U6 virus were subjected to immunohistochemistry using GFP and the Iba1 (red) antibodies. Knockdown of α-Adducin (LV-Addi) or α2-Na/K ATPase (LV-ATPi) had little or no effect on the presence or abundance of microglia within the GFP-labeled ventral horn; scale bar 50μm.
Supplementary Figure 7 α2-Na/K ATPase co-immunoprecipitates with α-Adducin in spinal cord lysates and is specifically upregulated in astrocytes in symptomatic SOD1G93A mice.
(a) Immunoblots show immunoprecipitated α-Adducin from SOD1G93A and control wild type spinal cord lysates subjected to immunoblotting with the α-Adducin and α2-Na/K ATPase antibodies following glycine elution, confirming α2-Na/K ATPase as an interactor of α-Adducin (left panels). Immunoblots show α2-Na/K ATPase is predominately expressed in primary glial cultures relative to primary motor neuron cultures enriched with the neuron marker β-tubulin. 14-3-3β is used as an internal control (right panel). (b) Immunohistochemistry with astrocyte marker GFAP and α2-Na/K ATPase antibody in sections of the lumbar spinal cord from SOD1G93A mice at 60 days displays α2-Na/K ATPase expression within astrocytes; scale bar 50μm. (c) Immunohistochemistry with GFAP and α2-Na/K ATPase antibody in sections of the lumbar spinal cord from symptomatic SOD1G93A mice at 120 days displays upregulation of α2-Na/K ATPase expression within astrocytes; scale bar 50μm. (a) are cropped; full length images are presented in Supplementary Figure 11.
Supplementary Figure 8 Intraspinally injection of control lentivirus in SOD1G93A mice had no effect on motor neuron survival.
(a) Spinal cord from end stage SOD1G93A mice injected at age 90 days intraspinally with the control lentivirus encoding GFP (LV-U6 SOD1G93A) was subjected to immunohistochemistry at end stage. End stage was defined as a time point at which the animal was unable to upright itself within 30s of placement on its side. Immunohistochemistry with GFP in SOD1G93A lumbar sections revealed delivery of control injected virus (LV-U6) into the ventral horn; scale bar 100μm. Alternating GFP positive sections were subjected to immunohistochemistry using the GFP antibody and the neurofilment-SMi32 antibody (red), a motor neuron marker, or Nissl stained (lower panels) for quantification of surviving motor neurons within GFP-labeled injected ventral horn and contralateral non-injected ventral horn (n≥20 sections per animal); scale bar 50μm. Control LV-U6 SOD1G93A mice (n=3) displayed equivalent degeneration of motor neurons within injected GFP-labeled ventral horn and non-injected contralateral ventral horn. Arrowheads indicate surviving motor neurons; quantification shown in (b).
Supplementary Figure 9 Heterozygous disruption of the α2-Na/K ATPase gene in SOD1G93A mice delays motor neuron degeneration.
(a) Nissl stained sections from endstage control SOD1G93A mice (n=5) and aged-matched SOD1G93A littermates heterozygous-null for the α2-Na/K ATPase allele (n=5) displayed more than twice the number of motor neurons in ATPase+/-;SOD1G93A than control SOD1G93A mice. Arrow heads indicate surviving motor neurons; quantification shown in (b); scale bar 50μm (***p<0.001; unpaired t-test).
Supplementary Figure 10 Condition media from heterozygous-null from α2-Na/K ATPase SOD1G93A astrocytes is neuroprotective.
(a) Precondition media from wild type, SOD1G93A, and heterozygous-null α2-Na/K ATPase; SOD1G93A astrocytes were exposed to motor neurons and subjected to immunocytochemistry with antibodies recognizing the motor neuron nuclear protein Islet1 (red) and the dendrite protein MAP2 (green); scale bar 50μm. Precondition media from wild type astrocytes had little or no effect on motor neuron survival (upper panels); quantified (b). Preconditioned medium from SOD1G93A astrocytes induced non-cell autonomous motor neuron cell death (middle); quantified (b). Preconditioned medium from heterozygous-null α2-Na/K ATPase; SOD1G93A astrocytes protected motor neurons against the non-cell autonomous cell death (lower panel); All data in bar charts show mean ± s.e.m (***p<0.001; unpaired t-test).
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Gallardo, G., Barowski, J., Ravits, J. et al. An α2-Na/K ATPase/α-adducin complex in astrocytes triggers non–cell autonomous neurodegeneration. Nat Neurosci 17, 1710–1719 (2014). https://doi.org/10.1038/nn.3853
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DOI: https://doi.org/10.1038/nn.3853
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