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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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS OLIGODENDROCYTE-ASTROCYTE CROSSTALK IN PARKINSON’S DISEASE MEDIATES NEURONAL FERROPTOSIS VIA

THE FGF SIGNALING PATHWAY Article Open access 23 May 2025 PROTEOMIC ANALYSIS LINKS ALTERATIONS OF BIOENERGETICS, MITOCHONDRIA-ER INTERACTIONS AND PROTEOSTASIS IN HIPPOCAMPAL ASTROCYTES FROM

3XTG-AD MICE Article Open access 18 August 2020 FERROPTOSIS MEDIATES SELECTIVE MOTOR NEURON DEATH IN AMYOTROPHIC LATERAL SCLEROSIS Article 02 December 2021 REFERENCES * Allen, N.J. &

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423–428 (2005). Article  PubMed  Google Scholar  Download references 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. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Neurobiology, Harvard

Medical School, Boston, Massachusetts, USA Gilbert Gallardo, Jessica Barowski & Azad Bonni * Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis,

Missouri, USA Gilbert Gallardo & Azad Bonni * Department of Neurosciences, University of California, San Diego, La Jolla, California, USA., John Ravits * Department of Neurology,

Northwestern Feinberg School of Medicine, Chicago, Illinois, USA Teepu Siddique * Department of Molecular and Cellular Biology, Northwestern Feinberg School of Medicine, Chicago, Illinois,

USA Teepu Siddique * Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio, USA Jerry B Lingrel * Tanz Centre for

Research in Neurodegenerative Diseases and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Janice Robertson * Department of Pathology,

Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA Hanno Steen Authors * Gilbert Gallardo View author publications You can also search for this author

inPubMed Google Scholar * Jessica Barowski View author publications You can also search for this author inPubMed Google Scholar * John Ravits View author publications You can also search for

this author inPubMed Google Scholar * Teepu Siddique View author publications You can also search for this author inPubMed Google Scholar * Jerry B Lingrel View author publications You can

also search for this author inPubMed Google Scholar * Janice Robertson View author publications You can also search for this author inPubMed Google Scholar * Hanno Steen View author

publications You can also search for this author inPubMed Google Scholar * Azad Bonni View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS 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. CORRESPONDING AUTHOR Correspondence to Azad Bonni. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. INTEGRATED SUPPLEMENTARY INFORMATION

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 panel_s_. 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). SUPPLEMENTARY FIGURE 11 FULL SCANS OF KEY WESTERN BLOT DATA. IN MANY EXPERIMENTS, MEMBRANES WERE STRIPPED AND REBLOTTED WITH A SECOND

ANTIBODY. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–11 (PDF 11653 kb) SUPPLEMENTARY METHODS CHECKLIST (PDF 1629 kb) SOD1G93A MICE AT ENDSTAGE. (MPG

40096 KB) HETEROZYGOUS-NULL Α2-NA/K ATPASE+/-; SOD1G93A AGE MATCHED LITTERMATES (MPG 17088 KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 21 July 2014 * Accepted: 29 September 2014 * Published: 26 October 2014 * Issue Date: December 2014 * DOI:

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