Translational profiling identifies a cascade of damageinitiated in motor neurons and spreading to glia inmutant SOD1-mediated ALSShuying Suna,b, Ying Suna,b, Shuo-Chien Linga,b,1, Laura Ferraiuoloc,2, Melissa McAlonis-Downesa,b, Yiyang Zoub,Kevin Drennera,b, Yin Wanga,b, Dara Ditswortha,b, Seiya Tokunagaa,b, Alex Kopelevichb, Brian K. Kasparc,Clotilde Lagier-Tourennea,d,3, and Don W. Clevelanda,b,d,4

aLudwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA 92093; bDepartment of Cellular and Molecular Medicine, Universityof California at San Diego, La Jolla, CA 92093; cThe Research Institute at Nationwide Children’s Hospital, Department of Neuroscience, The Ohio StateUniversity, Columbus, OH 43205; and dDepartment of Neurosciences, University of California at San Diego, La Jolla, CA 92093

Contributed by Don W. Cleveland, October 26, 2015 (sent for review September 24, 2015)

Ubiquitous expression of amyotrophic lateral sclerosis (ALS)-causing mutations in superoxide dismutase 1 (SOD1) provokesnoncell autonomous paralytic disease. By combining ribosomeaffinity purification and high-throughput sequencing, a cascade ofmutant SOD1-dependent, cell type-specific changes are now iden-tified. Initial mutant-dependent damage is restricted to motorneurons and includes synapse and metabolic abnormalities, endo-plasmic reticulum (ER) stress, and selective activation of the PRKR-like ER kinase (PERK) arm of the unfolded protein response. PERKactivation correlates with what we identify as a naturally low levelof ER chaperones in motor neurons. Early changes in astrocytesoccur in genes that are involved in inflammation and metabolismand are targets of the peroxisome proliferator-activated receptor andliver X receptor transcription factors. Dysregulation of myelinationand lipid signaling pathways and activation of ETS transcription fac-tors occur in oligodendrocytes only after disease initiation. Thus, path-ogenesis involves a temporal cascade of cell type-selective damageinitiating in motor neurons, with subsequent damage within gliadriving disease propagation.

ALS | SOD1 | cell type selective toxicity | bacTRAP | RNA profiling

Amyotrophic lateral sclerosis (ALS) is an adult-onset neuro-degenerative disease with loss of upper and lower motor

neurons that leads to fatal paralysis, with a typical disease courseof 1–5 y (1). Dominant mutations in the Cu/Zn superoxide dis-mutase (SOD1) gene (2) account for 20% of familial ALS. Anal-ysis of chimeric mice comprised of mixtures of wild type andmutant-expressing cells (3, 4) and use of cell type-selective ex-cision of ubiquitously expressed SOD1 mutant transgenes (5–9)have established that disease pathogenesis is noncell autono-mous, a mechanistic feature that is likely to be common to manyneurological disorders (10). Mutant SOD1 in motor neuronsaccelerates disease onset, but does not affect the rate of diseaseprogression (5, 7, 8). Mutant synthesis by neighboring glial cells,especially astrocytes (8) and microglia (5), has been shown to ac-celerate disease progression (5, 8). Mutant SOD1 gene inactivationin NG2+ oligodendrocyte progenitors, but not in already maturedoligodendroctyes, of adult mice has been reported to delay the ageof disease onset (11). Mutant synthesis in as-yet unidentified celltypes beyond motor neurons and oligodendrocytes also drives theonset of disease in ALS mice, as demonstrated by delayed initiationof disease in mice in which all motor neurons and oligodendrocytesare mutant-expressing but variable proportions of other cell typesexpress mutant SOD1 (4).Two key questions in understanding the pathogenic mecha-

nisms of ALS are what causes the selective degeneration ofmotor neurons from a widely expressed mutant gene and whatgenetic regulators of aging influence late-onset disease. Multiplepathways for toxicity of mutant SOD1 have been implicated,including misfolded protein triggering abnormal mitochondrial

function, endoplasmic reticulum (ER) stress, axonal transportdefects, excessive production of extracellular superoxide, and ox-idative damage from aberrantly secreted mutant SOD1 (reviewedin ref. 12). What damage occurring during the course of diseaseis accumulated within motor neurons, astrocytes, or oligoden-drocytes remains unknown, however.Previous attempts to analyze gene expression changes caused

by mutant SOD1 within defined cell populations in the centralnervous system (CNS) have relied on the physical enrichment oftarget cell populations, with either laser-capture microdissection(13–17) or fluorescence-activated cell sorting (18). These ap-proaches have clear disadvantages, however, including cross-contamination from neighboring cells/environments; isolation ofRNAs only from neuronal cell bodies but not dendrites, axons,or synapses; and artifacts introduced during cellular isolationprocedures. In addition, becaue of technical limitations, most

Significance

Amyotrophic lateral sclerosis can be caused by a mutation insuperoxide dismutase. Ubiquitously expressed, disease mech-anism involves damage within motor neurons (whose de-generation is responsible for progressive paralysis) and glia. Bycombining ribosome affinity purification from each of three celltypes, a temporal cascade of damage is identified that initiateswithin motor neurons, with subsequent damage within gliadriving disease propagation. Mutant-dependent damage tomotor neurons, which are shown to express very low levels ofendoplasmic reticulum chaperones, includes synapse and met-abolic abnormalities and selective activation of the PERK armof the unfolded protein response. Early changes in astrocytesare to genes involved in inflammation and metabolism, whiledysregulation of myelination and lipid signaling pathways inoligodendrocytes occurs only after disease initiation.

Author contributions: S.S. and D.W.C. designed research; S.S., S.-C.L., L.F., M.M.-D., Y.Z.,K.D., Y.W., D.D., S.T., and A.K. performed research; B.K.K. and C.L.-T. contributed newreagents/analytic tools; S.S. and Y.S. analyzed data; and S.S. and D.W.C. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Data deposition: The raw RNA-seq data have been deposited in the Gene ExpressionOmnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE74724).1Present address: Department of Physiology, National University of Singapore, Singapore117549.

2Present address: Sheffield Institute for Translational Neuroscience, University of Sheffield,Sheffield S10 2TN, United Kingdom.3Present address: MassGeneral Institute for Neurodegenerative Diseases, Department ofNeurology, Massachusetts General Hospital, Charlestown, MA 02129.

4To whom correspondence should be addressed. Email: dcleveland@ucsd.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1520639112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1520639112 PNAS | Published online November 30, 2015 | E6993–E7002

NEU

ROSC

IENCE

PNASPL

US

previous studies have focused on motor neurons alone or amixture of cells from white matter rather than on individual glialcell types. Furthermore, most previous work used transgenicmice with a highly accelerated disease course from a very highdegree of overexpression of mutant SOD1, which in the mostfrequently studied mouse (19) yields >20 times the normal en-dogenous level by end-stage disease, raising concern about howclosely the mechanism of toxicity in this accelerated model re-flects the human situation.Non–cell-autonomous toxicity has been demonstrated in cell

cultures as well, with astrocytes (20–25) or microglia (26) car-rying SOD1 mutations generating toxicity to cocultured embry-onic motor neurons. Although gene expression changes inducedby the SOD1G93A mutant in such cocultures have been reported(27), the extent to which this reflects age-dependent diseasecourse has not been established.Here we coupled high-throughput RNA sequencing with the

translating ribosome affinity purification (TRAP) methodology(28, 29) to evaluate damage within motor neurons, astrocytes,and oligodendrocytes during the course of disease in mice thatdevelop fatal ALS-like paralysis from ubiquitous expression of amoderate level of the familial ALS-causing mutation SOD1G37R

(5). BacTRAP reporter transgenes that encode an EGFP-taggedribosomal protein L10a (Rpl10a) and driven by a cell type-specifictransgene promoter allow isolation of actively translating, poly-ribosome-associated mRNAs from specific cell types in the CNS.Ribosome subunits and the bound mRNAs can be maintainedintact and isolated by EGFP immunoprecipitation from an un-stressed, in vivo cellular environment with intact cell–cell con-nections at any chosen time point during aging. With thisapproach, we have identified a cascade of damage, including

selective activation of the protein kinase RNA-like endoplasmicreticulum kinase or PRKR-like ER kinase (PERK) arm of theunfolded protein response (UPR), to start within motor neurons,followed by dysregulation of metabolic and inflammatory genesin astrocytes and membrane proteins and lipid signaling path-ways in oligodendrocytes.

ResultsIsolation of Translated mRNAs from Motor Neurons, Astrocytes, orOligodendrocytes. To determine the damage caused by ALS-linked mutant SOD1 within spinal motor neurons, astrocytes, oroligodendrocytes during the disease course, we mated a mouseline (LoxSOD1G37R) that develops age-dependent, fatal paralyticmotor neuron disease from ubiquitous expression of a moderatelevel of an ALS-linked point mutation in SOD1 (5) to bacTRAPreporter mouse lines (28, 29) (Fig. 1A). The LoxSOD1G37R linewas chosen because of its wide use in identifying cell types whosemutant SOD1 synthesis contributes to a non–cell-autonomousdisease mechanism (5, 8, 11, 30, 31) and in which overt diseaseonset initiates at approximately 8 mo of age (Fig. 1B). The diseasecourse after initiation includes nearly complete denervation-inducedmuscle atrophy and accompanying weight loss, motor neuron death,and progressive paralysis (5).Cohorts of LoxSOD1G37R mice expressing EGFP-tagged ribo-

some protein Rpl10a only within motor neurons (Chat-bacTRAP),astrocytes (Aldh1l1-bacTRAP), or oligodendrocytes (Cnp1-bacTRAP)were obtained (28, 29) (Fig. 1A). All mice were in a commonC57BL/6 genetic background. The predicted EGFP-Rpl10a ex-pression patterns in spinal cords of the three reporter mouselines were confirmed by immunofluorescence (Fig. 1C). Chatpromoter-driven EGFP-Rpl10a was expressed in the same cell

Chat-bacTRAPMotor Neuron

Aldh1l1-bacTRAPAstrocyte

Cnp1-bacTRAPOligodendrocyte

× LoxSOD1 G37R

High-throughput RNA sequencing

AAA

7MeG

7MeG

EGFP immunoprecipitation

mRNA purification

AAA

AAA7MeG

Ribosome

EGFP-labeled ribosome (by EGFP-Rpl10a)

mRNA

Anti-EGFP-coated magnetic bead

B Age dependent disease in LoxSOD1 miceG37R

8 10.5Age

(month)

presymptomatic onset symptomatic end stage

fullyinnervated

initiate denervation

continuingdenervation,

muscle atrophy,and weight loss

paralysis

Diseasestage

A Chat-EGFP-Rpl10a CHAT

Cnp1-EGFP-Rpl10a CC1

Aldh1l1-EGFP-Rpl10a GFAP

Ast

rocy

tes

Mot

orne

uron

sO

li god

end r

o cyt

es

100 μm

10 μm

100 μm

C

0

100

200

300

400

500

Aldh1l1-GFP

Cnp1-GFP

Chat-GFP

no GFPG

FP- im

mun

opre

cip i

tat e

dR

NA

per m

ouse

(ng)

D

28S18S

Bio

anal

yze r

no GFPChat-G

FP

Aldh1l1-GFP

Cnp1-GFPE

Motor neuronMarkers

AstrocyteMarkers

OligodendrocyteMarkers

Astrocytes

Nor

mal

ized

fold

chan

g e

Chat Scl18a3 Aldh1l1 Gfap Cnp1 Mbp0

0.5

1

Motor neurons Oligodendrocytes1.5F

Fig. 1. The bacTRAP methodology for isolating cell type-specific translational mRNAs. (A) The experimental design to identify cell type-specific damages inmotor neurons, astrocytes, and oligodendrocytes caused by the SOD1G37R mutant, coupling the bacTRAP methodology and high-throughput RNA sequencing.The polyribosome-associated translational mRNAs were isolated by EGFP immunoprecipitation from specifically labeled cell types in the spinal cord ofbacTRAP reporter mice. (B) The disease course of the SOD1G37R transgenic mouse. To identify early changes, RNA from all three cell types was first isolated atdisease onset (∼8 mo; red). RNA from oligodendrocytes was also examined at an early symptomatic stage (∼10.5 mo; purple). (C) Direct fluorescence of GFP(for motor neuron reporter) or double immunostaining of mouse spinal cord with an anti-GFP antibody (green, for astrocyte and oligodendrocyte reporters)and antibodies for motor neuron marker Chat (red), astrocyte marker GFAP (red), and marker for oligodendrocytes CC1 (red). (D) Average amount of GFP-immunoprecipitated RNA from each spinal cord of the three EGFP-Rpl10a reporter mice and mice without the GFP transgene. (E) The quality of RNA afterimmunoprecipitation was measured with a Bioanalyzer. The intact 18S and 28S bands demonstrate that the RNA was not degraded. (F) qRT-PCR for motorneuron markers Chat and Slcl18a3(VAChT), astrocyte markers Aldh1 and Gfap, and oligodendrocyte markers Cnp1 and Mbp.

E6994 | www.pnas.org/cgi/doi/10.1073/pnas.1520639112 Sun et al.

population that expressed the motor neuron-specific proteincholine acetyltransferase (Chat). The Aldh1l1 promoter-drivenEGFP-tagged ribosome-labeled cells exhibited the morphologyexpected for astrocytes with many fine processes (32, 33) thatpartially overlap the astrocyte marker GFAP, whose localizationis restricted to a subset of astrocytic cytoplasm (33). The Cnp1promoter-driven EGFP-RPl10a was specifically expressed in ol-igodendrocytes, as indicated by coaccumulation with CC1. In allthree cases, EGFP-tagged Rpl10a was found diffusely within thecorresponding cytoplasm and bound to nucleoli (Fig. 1C), asexpected for a ribosomal protein.To identify early changes that might trigger or contribute di-

rectly to pathogenesis rather than reflect consequences of initialmotor neuron degeneration and death, we harvested spinal cordsfrom each of the three EGFP-Rpl10a mouse lines, with orwithout the LoxSOD1G37R transgene, from 8-mo-old mice, anage at which muscle denervation in most mutant animals hadstarted but before overt phenotypic symptoms had developed(30) (Fig. 1B). GFP immunoprecipitation of extracts from mousespinal cords successfully recovered polyribosome-associatedmRNAs from each EGFP-Rpl10a reporter mouse, with verylittle background RNA from mice without the GFP-containingtransgene (Fig. 1D). mRNAs purified by EGFP immunoprecip-

itation from motor neurons, astrocytes, and oligodendrocytesremained intact (Fig. 1E) and were highly enriched for corre-sponding marker genes, including Chat and Slc18a3 (VAChT)for motor neurons, Aldh1l1 and Gfap for astrocytes, and Cnp1and Mbp for oligodendrocytes, as demonstrated by quantitativeRT-PCR (qRT-PCR) (Fig. 1F).

Cell Type-Specific Translational mRNA Changes Induced by SOD1G37R.Cell type-specific mRNAs recovered from spinal cords of micewith or without the LoxSOD1G37R transgene were converted tocDNA libraries and then sequenced. Each biological groupcontained between three and six sex-matched animals. For eachsample, an average of ∼40 million 50-bp reads were uniquelymapped to the annotated mouse (mm9) genome (SI Appendix,Table S1). Expression levels for each annotated protein-codinggene were determined by the number of fragments per kilobaseof transcript per million mapped reads (FPKM). Genome-widecomparison using unbiased (Spearman) hierarchical clustering ofexpression levels of mRNAs purified from the three cell types ofnontransgenic mice revealed a high correlation between bi-ological replicates of each condition and a clear distinction ofmRNAs from motor neurons, astrocytes, oligodendrocytes, andwhole spinal cord (SI Appendix, Fig. S1). Inspection of scatterplots

A30

25

20

15

10

5

00 5 10 15 20 25

Ast

rocy

te

Motor neuron

Gfap

Aqp4

Aldh1l1Fgfr3

ChatSlc18a3

Mnx1Isl1

mean FPKM

0 5 10 15 20 25Motor neuron

30

25

20

15

10

5

0

MbpCnp1MagMog

Sox10Slc18a3

ChatIsl2Mnx1Isl1

0 5 10 15 20 25Oligodendrocyte

30

30

25

20

15

10

5

0

Ast

rocy

te

MbpCnp

MagMog

Sox10

Gfap

Aqp4

Aldh1l1Fgfr3

mean FPKM

Log2 Log10

3

1

2

0

456

7

89

Log2 Log10

3

12

0

456

7

89

mean FPKMLog2 Log10

3

12

0

456

7

89

0

0.2

0.4

0.6

0.8

1

1.2

Non-TgG37R

Non-Tg G37R non-Tg G37RMotor neuron Astrocyte Oligodendrocyte

Rel

ativ

e m

ouse

SO

D1

RN

A(n

orm

aliz

ed to

Dna

ja2)

B

SOD1 SOD1 SOD1

0

5

10

15

20

CTRL G37R CTRL G37R CTRL G37RMotor neuron Astrocyte Oligodendrocyte

Rel

ativ

e hu

man

SO

D1

RN

A(n

orm

aliz

ed to

Dna

ja2)

C

SOD1 SOD1 SOD1

-100 0 100 200 300

Motor neuron

Astrocyte

Oligodendrocyte

Number of gene expression changes

up

down

SOD1 compared to Non-Tg at onsetD G37R

Acsbg1

SOD1G37R

SOD1G37R

SOD1G37R

Non-Tg

Non-Tg

Non-Tg

Olig

oden

droc

yte

Ast

rocy

teM

otor

neu

ron

E Overlaps of gene expression changesamong the three cell types

Motorneuron

Astrocyte

Oligodendrocyte

239 8216

23 711

F

Ol ig

oden

droc

yte

220

220

0

0

0

0

3000

3000

0

0

200

200

Fig. 2. Cell type-specific transcriptome changes induced by SOD1G37R mutation. (A) Scatterplots of average gene expression from multiple biological rep-licates of immunoprecipitated mRNA from motor neurons versus astrocytes (Top), oligodendrocytes versus motor neurons (Middle), and astrocytes versusoligodendrocytes (Bottom). The dots representing the cell type marker genes are labeled in the figures. (B) qRT-PCR of mouse SOD1 in motor neurons, as-trocytes, and oligodendrocytes. The relative level is normalized to internal control Dnaja2. Error bars represent SD in three or four biological replicates.(C) qRT-PCR of human SOD1 in motor neurons, astrocytes, and oligodendrocytes. The relative level is normalized to internal control Dnaja2. Error barsrepresent SD in three or four biological replicates. (D) Number of gene expression changes in the three cell types, comparing SOD1G37R transgenic mice andnontransgenic mice. (E) Number of overlapped gene changes induced by SOD1G37R in motor neurons, astrocytes, and oligodendrocytes. (F) RNA-seq readsfrom SOD1G37R transgenic mice and nontransgenic controls in the three cell types, showing the specific up-regulation of Acsbg1 mRNA in motor neurons.

Sun et al. PNAS | Published online November 30, 2015 | E6995

NEU

ROSC

IENCE

PNASPL

US

of average gene expression from multiple biological replicates ofimmunoprecipitated mRNA frommotor neurons versus astrocytesshowed distinct gene expression patterns (Fig. 2A, Top). A robustenrichment for motor neuron marker genes (Chat, Slc18a3, Isl1,Isl2, andMnx1), astrocyte markers (Aldh1l1,Gfap, Fgfr3, and Aqp4),or oligodendrocyte markers (Cnp1,Mbp,Mog,Mag, and Sox10) wasfound, respectively, in the immunoprecipitated mRNAs from thethree cell types (Fig. 2A).Endogenous and mutant SOD1 were expressed at high levels

in all three cell types. The endogenous mouse SOD1 mRNAcontent was highest in motor neurons, with 70% of the motorneuron level found in astrocytes and 40% in oligodendrocytes(Fig. 2B). Human SOD1G37R RNA matched the correspondingratios for mouse SOD1 in motor neurons and oligodendrocytes(100% and 40%, respectively), with mutant SOD1 expressed atonly approximately one-half the corresponding endogenous levelin astrocytes (30% of the level in motor neurons) (Fig. 2C).We next tested whether gene expression changes were induced

in each cell type from expression of mutant SOD1G37R. A genome-wide comparison was performed on RNAs isolated just beforedisease onset from motor neurons, astrocytes, oligodendrocytes,and whole spinal cord (Fig. 1B). An initial unbiased (Spearman)hierarchical clustering of all gene expression values from motorneuron samples was used to determine that all four nontransgeniccontrols clustered closely together, as expected (SI Appendix,Fig. S2A). Two of the four SOD1G37R motor neuron sampleswere well separated from the controls, whereas two othersdeveloped more moderated differences with those controls (SIAppendix, Fig. S2A). Given the variability in age of diseaseonset across a 100-d window between the mice with the earliestand latest disease onset in this SOD1G37R line (34), we in-terpret the variability in affected gene expression in the mutantmice to reflect animals with earlier and later disease initiation.Indeed, a heat map of genes with altered expression clearlyshows that the two more widely separated mutant animals exhibitedmore dramatic changes, with more modest changes in the samegenes and in the same directions in the remaining two mutantmice (SI Appendix, Fig. S2B).We next performed statistical comparisons to identify mRNAs

that were changed significantly, using all annotated protein-coding genes (with Cuffdiff FPKM value at least 0.1 in onecondition; q < 0.05). Near disease onset, the gene expressionchanges were most dramatic in motor neurons (Fig. 2D), with260 significant changes, ranging from a 50-fold increase in aserotonin transporter (Slc6a4) mRNA to a 35% decrease inRunx2 mRNA SI Appendix, Table S2). Fewer changes were seenin astrocyte mRNAs (108 mRNAs altered) and almost nochanges were detected in translating oligodendrocyte mRNAs(23 mRNAs altered; a maximum sixfold change). Of note, mostof these changes (85% in motor neurons, 62% in astrocytes, and61% in oligodendrocytes) were up-regulations in expression(with a mean up-regulation of 2.5-fold). Most gene changes werecell type-specific; for example, 239 genes changed only in motorneurons, despite the expression of almost all of them in all threecell types (Fig. 2E and SI Appendix, Table S9), indicating aunique response by each cell type to mutant SOD1 (synthesizedwithin either it or its cell partners). Furthermore, very few ofthese changes were identified from an analysis of whole spinalcord RNAs (SI Appendix, Fig. S2C), reinforcing the value of thebacTRAP method in identifying translational mRNA changeswithin individual cell types.

Activation of ER Stress in Motor Neurons Expressing Mutant SOD1.Examination of the 260 mRNA changes found in LoxSOD1G37R

motor neurons revealed a strong enrichment for genes involvedin synapses and cell junctions (SI Appendix, Table S3). Approx-imately 10% of all of the gene changes are in this category,consistent with synapse dysfunction accompanying the known

denervation that occurs as one of the earliest detected changes inthis mouse line (34). Up-regulated gene changes were enrichedin genes involved in metabolism, with notable perturbation inthe alanine, aspartate, and glutamate metabolism pathways(marked with red stars and red shade in SI Appendix, Fig. S3).Down-regulated genes were highly enriched in those encodingribosomes and components of the translation machinery(SI Appendix, Table S3), consistent with disruption of normaloverall protein synthesis on ER stress activation.Analysis of potential transcription factor-binding motifs and

coregulatory elements from the total 260 changes in motorneurons revealed two heat-shock proteins, HSF2 and HSF1, andan ER stress responsive transcription factor, CHOP (Fig. 3A),strongly indicating UPR activation. The UPR is a major intra-cellular pathway activated in response to an accumulation ofunfolded or misfolded proteins (35, 36). The three branchesof the UPR are dependent on cAMP-dependent transcriptionfactor ATF-6 alpha (ATF6), PERK, and IRE1α (a serine/threonine-protein kinase/endoribonuclease). The three branchesof the UPR function to sense protein misfolding in the ER andtransduce the initial misfolding signal for induction of compo-nents of each of the three branches (35, 36). Activation of PERKphosphorylates eIF2α, which enhances the translation of cAMP-dependent transcription factor 4 (ATF4) mRNA (37). Analysisof the mRNAs accumulated within motor neurons at diseaseonset revealed ER stress, including induction of the ER chap-erone PDI and the autophagy component SQSTM1 (p62), andactivation of the PERK arm of the UPR pathway, including el-evation of ATF4, whose activity in turn induces CHOP (alsoknown as DNA damage-inducible transcript 3 protein), a proa-poptotic transcription factor (35). qRT-PCR analysis confirmedelevated levels of CHOP and ATF4 in SOD1G37R motor neurons(Fig. 3B). PERK activation was further supported by the ∼2.2-fold enhanced phosphorylation of eIF2α in SOD1G37R motorneurons within lumbar sections of mouse spinal cord at diseaseonset (Fig. 3 F and G).Despite activation of the PERK pathway, neither the ATF6 nor

IRE1α pathway of the UPR was similarly activated in motorneurons, as demosnrtated by the absence of changes identified inBip (also known as Grp78), an ATF6-activated ER chaperoneprotein, or in X-box-binding protein 1 (XBP1), a key downstreamcomponent of the IRE1α pathway (Fig. 3B). Furthermore, acti-vation of the IRE1α pathway triggers the RNase activity of IRE1α,which excises a 26-nt intron of the unspliced XBP1 mRNA (XBP1u)to generate an active transcription factor, termed spliced XBP1(XBP1s) (35). At disease onset, no XBP1s isoform was generated inthe motor neurons of the SOD1G37R mice (Fig. 3B).ER stress and activation of the PERK branch of the UPR was

found in motor neurons, but not in astrocytes or oligodendrocytes(Fig. 3 C and D). Analysis of the FPKM values of RNAs encodingER chaperone proteins in normal motor neurons, astrocytes, andoligodendrocytes revealed much higher expression levels in the twoglial cell types than in motor neurons in ER chaperones PDI andFKBP9 (Fig. 3E). For example, PDI mRNA levels were approxi-mately 17-fold higher in astrocytes and 12-fold higher in oligo-dendrocytes compared with motor neurons. Even after a threefoldinduction in the SOD1G37R motor neurons relative to non-transgenic neurons (Fig. 3B), PDI levels remained sixfold belowthe age-matched levels in normal astrocytes and oligodendroctyes(Fig. 3E), consistent with motor neurons being intrinsically morevulnerable to unfolded protein accumulation. FKBP9 levels aresimilarly elevated by 12-fold in astrocytes and sixfold in oligoden-drocytes relative to normal motor neurons (Fig. 3E).To determine whether the selective activation of the PERK

arm of the UPR was common to disease-linked SOD1 mutantsof different biochemical characters, we examined ER stresscomponents in mice (38) that develop fatal disease from anintrinsically misfolded, dismutase-inactive mutation SOD1G85R

E6996 | www.pnas.org/cgi/doi/10.1073/pnas.1520639112 Sun et al.

(39), which develop slowly progressing disease with modest mutantSOD1 accumulation, similar to the SOD1G37R mouse line exceptthat the mutation is dismutase-inactive. Laser capture micro-dissection was used to isolate RNAs from motor neurons at anearly symptomatic stage (age 10.5 mo). qRT-PCR analysis revealeddramatic elevations of ATF4 expression (40-fold) and CHOPexpression (12-fold) (SI Appendix, Fig. S2D), indicative of acti-vation of the PERK branch of ER stress as a common responsein motor neurons induced by dismutase-active and -inactiveSOD1 mutations.

Dysfunction of Nuclear Receptors Peroxisome Proliferator-ActivatedReceptor and Liver X Receptor in Astrocytes Expressing Mutant SOD1.We initially analyzed SOD1-mutant dependent changes in ribo-some-bound RNAs recovered from astroctyes using unsuper-vised Spearman hierarchical clustering of all gene expressionvalues. This analysis revealed that the six nontransgenic controlsclustered tightly together, as did three of the four SOD1G37R

samples (SI Appendix, Fig. S4A). As seen in the motor neurons,the majority of the significant RNA changes in astrocytes (67 of108) were increases, ranging from a 1.8-fold increase for crem(cAMP-responsive element modulator) to a 30-fold increase forCcl6 (C-C motif chemokine 6) (Fig. 4A). There were moremodest reductions in 42 RNAs, with the maximum reduction to33% of the initial level for Kif20a (SI Appendix, Table S4). Geneontology analysis revealed enrichment of genes linked to im-mune responses and with extracellular functions (SI Appendix,Table S5), including the chemokine CXCL10 (C-X-C motifchemokine 10) and an insulin-like growth factor-binding protein

(IGFBP7), both of which have been shown to have toxic orproapoptotic effects on neurons and in other tissues (40–42).qRT-PCR analysis of RNAs purified from the Aldh1l1-promotedEGFP-Rpl10a ribosomes validated fourfold and twofold in-creases, respectively, in the RNAs encoding CXCL10 andIGFBP7 in the astrocytes from the dismutase-active (SOD1G37R)mutant SOD1 mice (SI Appendix, Fig. S4B). Similar analyses ofRNAs isolated at onset stage from astrocytes of mice that willdevelop fatal motor paralysis from expressing dismutase-inactive(SOD1G85R) mutant mice showed similar (twofold) increases inCXCL10 and IGFBP7 (SI Appendix, Fig. S4B).Although the mRNA levels of the two candidates were un-

changed, two families of nuclear receptors, peroxisome pro-liferator-activated receptor (PPAR) and liver X receptor (LXR),were predicted to be the major transcription coactivators in-volved in the 108 gene changes in SOD1 mutant-expressingastroctyes (Fig. 4B). Both of these proteins can be activated byfatty acid and cholesterol derivatives, and they control the ex-pression of genes involved in metabolism and inflammation (43).On ligand binding, both receptors undergo a conformationalchange, followed by changes in interacting proteins, and theyaffect transcription through multiple modes, including directactivation, repression, and transrepression (43, 44). In general,PPAR and LXR activate metabolism and play important roles inenergy and lipid homeostasis, and also repress inflammatorygene expression (43, 45) (Fig. 4C). In SOD1G37R astrocytes,there was extensive up-regulation of inflammatory gene expres-sion (Fig. 4D) and a trend toward a reduction in metabolic genes

00.2

0.6

1.0

1.4

ATF4 CHOP PDI

00.2

0.6

1

1.4

onset sympt onset sympt onset symptATF4 CHOP PDI

0

5

10

15

20

25

PDI FKBP9 ERp72

motor neuronastrocyteoligodendrocyte

A C

D

E

Rel

ativ

eFP

KM

in o

ligod

endr

ocyt

es

SOD1

Rel

ativ

eFP

KM

inas

trocy

tes

SOD1G37R

ER

chap

eron

eR

NA

leve

l(r

elat

ive

FPK

M)

0

0.050.1

0.150.2

0.25

0

4

8

12

Top 6 candidate transcription factors implicated in SOD1-mediated gene upregulation

Impo

rtanc

e

Occ

urre

nce

(%)

Non-TgG37R

Non-TgER stress

CHOP

0

1

2

3

4

Non-Tg

Non-Tg SOD1

SOD1G37R

ATF4 CHOP Bip XBP1 PDI p62Nor

mal

ized

fold

cha

nge

inm

otor

neu

rons

at d

i sea

se o

nset

B

** ***

*

Factors involved in unfolded protein response

G37R

Non-Tg SOD1G37R

CH

AT-G

FP-R

PL 1

0Aph

osph

o-eI

F2a

ME

RG

E

F

0

0.5

1

1.5

2

2.5

3G

G37R

Pho

spho

-eIF

2αr e

lativ

ein

tens

ity

Non-Tg SOD1

**

Fig. 3. Activation of ER stress specifically in motor neurons at disease onset. (A) Candidate transcription factors of coregulated genes in SOD1 G37R motorneurons, as predicted by the web server DiRE. (B) qRT-PCR of genes involved in the UPR-activated ER stress pathway in SOD1 G37R-expressed motor neuronscompared with nontransgenic controls. Error bars represent SEM in three or four biological replicates. *P < 0.05, **P < 0.005, Student t test. (Top) Schematicdiagram of the three branches activated by UPR-induced ER stress. (Top Right) RT-PCR of unspliced and spliced isoforms of XBP1 in motor neurons with orwithout the G37R transgene. (C) Relative expression levels of ATF4, CHOP, and PDI in astrocytes at disease onset. The FPKM values from RNA-seq werenormalized to those in nontransgenic samples of each gene. Error bars represent SD in four to six biological replicates. (D) Relative expression levels (FPKMfrom RNA-seq) of ATF4, CHOP, and PDI in oligodendrocytes at both onset and an early symptomatic stage. Error bars represent SD in four to six biologicalreplicates. (E) Relative expression levels (FPKM) of ER chaperones in the three cell types of nontransgenic mice. The expression levels of each gene werenormalized to the value in motor neurons. (F) Immunofluorescence of phospho-eIF2α in spinal cords of Chat-EGFP–labeled motor neuron reporter mice, withor without the SOD1G37R transgene. (G) Quantification of the intensity of red fluorescence normalized to green fluorescence as in F. There were three mice ineach group, and approximately 100 motor neurons were quantified in each mouse. Error bars represent SD. **P < 0.005, Student t test.

Sun et al. PNAS | Published online November 30, 2015 | E6997

NEU

ROSC

IENCE

PNASPL

US

(Fig. 4E), several of which have been shown to be targets ofPPAR/LXR (46–48).Along with the apparently increased PPAR/LXR activity,

peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC1α) was first down-regulated in mutant SOD1G37R

astrocytes, but not in motor neurons or oligodendrocytes (Fig.4E and SI Appendix, Fig. S4C). PGC1α is a transcriptionalcoactivator of nuclear receptors and other transcription factorsthat play key roles in mitochondria biogenesis and oxidative me-tabolism (49, 50). Its expression can be regulated by PPAR, and italso cooperates with PPAR in transcriptional control of nucleargenes encoding mitochondrial fatty acid metabolism (51). A de-crease in PGC1α level is not a typical gene expression changeaccompanying astrogliosis induced by neuroinflammation orstroke (52), suggesting that it may be an intrinsic toxic effect ofmutant SOD1 in astrocytes rather than an inflammatory responseto the motor neuron dysfunction. Thus, dysfunction of mito-chondria and/or oxidative metabolism might account in part forthe noncell autonomous toxicity of astrocytes to motor neurons.

Gene Expression Changes in Oligodendrocytes at an Early SymptomaticStage. The Cnp1-Rpl10a bacTrap approach identified very fewchanges (only 14 up-regulated and 9 down-regulated RNAs) inSOD1G37R mutant oligodendrocytes at disease onset. Moreover,these changes were smaller in magnitude relative to the changes inmotor neurons, ranging from a maximal 2.4-fold increase forPla2g4e (cytosolic phospholipase A2 epsilon) to repression to 37%of the initial level for Ccne2 (G1/S-specific cyclin-E2) (SI Appendix,Table S6).To test whether changes appeared in oligodendrocytes later in

the disease course, we used the Cnp1-Rpl10a bacTrap approachto purify translating RNAs from oligodendrocytes at an earlysymptomatic disease stage (age 10.5 mo) (Fig. 1B). As before,unsupervised hierarchical clustering of all gene expression valuesrevealed that the five nontransgenic controls clustered together,whereas the three SOD1G37R samples were largely distinct fromthe controls (SI Appendix, Fig. S5A). A total of 750 gene ex-pression changes were identified in annotated protein-codinggenes (with a cuffdiff FPKM value of at least 0.1 in one condi-tion; q < 0.05; Fig. 5A), 628 of which were up-regulated (between1.7- and 33-fold) (SI Appendix, Table S7). A total of 439 geneswere up-regulated at least twofold, 92 genes were up-regulatedmore than fivefold, and 12 genes were up-regulated more than

10-fold (Fig. 5B). The use of qRT-PCR to provide an in-dependent assessment of RNA levels validated the changes inRNA levels (Fig. 5C). Fourteen of the 23 RNA changes seen atdisease onset showed enhanced dysregulation at an early symp-tomatic stage (SI Appendix, Table S7).Gene ontology analysis revealed that membrane protein-encod-

ing genes were disproportionately misregulated, composing ∼25%of the up-regulated RNAs and 45% of the down-regulated RNAs(SI Appendix, Table S8). Furthermore, all three main proteinscomposing the myelin sheath—myelin basic protein (MBP), myelinoligodendrocyte glycoprotein (MOG), and proteolipid protein(Plp1)—were down-regulated in the SOD1G37R oligodendrocytes(Fig. 5D). In addition, RNAs encoding components of three majorsignaling pathways—phosphatidylinositol signaling (P = 0.0008,Benjamini test), FcγR-mediated phagocytosis (P = 0.0007, Benja-mini test) and a calcium signaling pathway (P = 0.0009, Benjaminitest)— were perturbed (SI Appendix, Figs. 6 and 7). Members ofphosphoinositide phospholipase C (PLC) and Ca2+/calmodulin-dependent protein kinase (CaMK) protein families were up-regu-lated. PLCs participate in phosphatidylinositol 4,5-bisphosphate(PIP2) metabolism and lipid signaling pathways in a calcium-dependent manner, and are essential for intracellular calcium ho-meostasis (53). CaMK is a serine/threonine-specific protein kinaseregulated by the Ca2+/calmodulin complex that has been shown toregulate oligodendrocyte maturation and myelination (54). Strik-ingly, nine of the 15 members of the PLC family and six CaMKswere up-regulated in oligodendrocytes in SOD1G37R mice at thisearly symptomatic stage (Fig. 5 E and F). Similar analysis of RNAsfrom oligodendrocytes of SOD1G85R mice isolated using the Cnp1-Rpl10a bacTrap approach at early symptomatic stage revealed asimilar trend of changes (albeit of lower magnitude) in the two genefamilies (SI Appendix, Fig. 5 B and C), indicating common defectsfrom dismutase-active and -inactive SOD1 mutants.It was previously reported that the main lactate transporter,

monocarboxylate transporter 1 (MCT1), which provides metabolicsupport to axons, was suppressed in oligodendrocytes of SOD1G93A

mice (55). Surprisingly, however, despite 750 altered RNAs,changes in MCT1 mRNAs were not seen in oligodendrocytes ateither disease onset or the early symptomatic stage in eitherSOD1G37R or SOD1G85R mutant mice (SI Appendix, Fig. S5D).Thus, altered levels of MCT1 synthesis (e.g., translating mRNAs)cannot be an early damaging factor that drives initiation of non–cell-autonomous toxicity from oligodendrocytes to motor neurons.

0

4

8

12

0

0.1

0.2

0.3

Top 5 candidate transcription factors implicated in SOD1-mediated gene changes

Impo

rtanc

e

Occ

urre

nce

(%)

-50 0 50 100

all

> 2 fold

> 5 fold

> 10 foldupdown

Number of gene expression changes

A

PPAR,LXR (nuclear receptors)

Activation of inflammation

Enhanced metabolism

Fatty acid and cholesterol ligands

SOD1

CG37R

012345678

Junb Cebpd Cebpb

D G37R

of in

flam

mat

ion

g ene

sN

orm

a liz

e d fo

ld c

han g

e

* *

*

00.20.40.60.8

11.21.4

Sertad2 Prrx1 PGC1a

Nor

mal

izae

d fo

ld c

hang

e

E

of m

e ta b

oli s

m g

ene s *

Non-Tg SOD1 G37RNon-Tg SOD1

B

Fig. 4. Gene expression changes in astrocytes at disease onset. (A) Number of gene expression changes above 2-, 5-, and 10-fold in SOD1G37R astrocytes atdisease onset. (B) Candidate transcription factors of coregulated genes in SOD1G37R astrocytes, as predicted by the web server DiRE. (C) Diagram of PPAR andLXR functions and how they are altered by the SOD1G37R mutant in astrocytes (red). (D) qRT-PCR of inflammatory genes up-regulated in SOD1G37R astrocytes.Error bars represent SEM in three or four biological replicates. *P < 0.05, Student t test. (E) qRT-PCR of genes involved in metabolism down-regulated in G37Rastrocytes. Error bars represent SEM in three or four biological replicates. *P < 0.05, Student t test.

E6998 | www.pnas.org/cgi/doi/10.1073/pnas.1520639112 Sun et al.

Our analysis of potential binding motifs and coregulatoryelements identified four subfamilies of the ETS transcriptionfactors—ETS, PEA3, PU1 (SPI), and ELF—as candidate tran-scription factors whose elevated activity could underlie increasedexpression of 427 of the 622 genes with greater than twofoldchanges in mutant-expressing oligodendrocytes (Fig. 5G). TheETS (E26 transformation-specific or E-twenty-six) family is oneof the largest families of transcription factors in mice, with 12subfamilies and 28 total members (56, 57). RNAs encoding sevenof these factors were significantly up-regulated in SOD1G37R

oligodendrocytes, with two (Elf4 and Fli1) increased by morethan fivefold (Fig. 5H). Elevated levels of these RNAs (elevatedto a smaller extent) were also identified at early symptomaticstage in SOD1G85R oligodendrocytes (SI Appendix, Fig. S5E).Functional gene ontology analysis with candidate ETS targets

in the SOD1 mutant-mediated expression-changed genes revealedsignificant elevations in genes in the phosphatidylinositol signalingsystem (P = 0.005, Benjamini test) and FcγR-mediated phagocytosis(P = 0.000008, Benjamini test) (SI Appendix, Fig. S6 A and B).Phagocytosis is a major mechanism for removing pathogens and celldebris. It was once thought to be carried out mainly by microglia inthe CNS (58), but recently was found to be performed by astrocytesas well, for synapse and neuronal debris elimination (59, 60).

As a further test to confirm that the changes identified intranslating RNAs from oligodendrocytes are not likely to beremaining RNA contaminants from large changes in microglia orastroctyes, we examined the fold changes of genes in the FcγR-mediated phagocytosis pathway in whole spinal cord. Up-regulationwas either not observed or observed at a much smaller magnitudecompared with that in oligodendrocyte-derived RNAs (SI Ap-pendix, Fig. S5F), consistent with increased synthesis in phago-cytosis components within oligodendrocytes. Although we areunaware of any reported evidence of phagocytosis in oligo-dendrocytes, Fc receptor mediated-signaling has been im-plicated in the induction of oligodendrocyte differentiation andmyelination (61).

DiscussionMotor neuron degeneration caused by mutant SOD1 is non–cell-autonomous (14), with contributions from neighboring glia cells(3–5, 7–9, 11), but no consensus has been reached on the precisemechanisms involved. To examine this question, we combined ri-bosome tagging in mice (bacTRAP) to enable isolation of cell type-specific polyribosome-associated mRNAs in the context of intactneuronal-glial networks with high-throughput sequencing (RNA-seq). Starting with RNAs isolated from mice ubiquitously expressing

OligodendrocyteA

-200 0 200 400 600 800

sympt

onset

Number of gene expression changes

updown

(23 total)

(750 total)

Oligodendrocyte at early symptomatic stageB

-200 0 200 400 600 800

> 2 fold

> 5 fold

> 10 fold

Number of gene expression changes

updown

(12)

(92)

(439)

(750)All(>1.5 fold)0

0.5

1

1.5

2

2.5

3

Cadps Rusc1Tmeff1 Nkain2

Nor

mal

ized

fold

cha

nge

C

Olig

ode n

droc

yte * *

**

Non-TgG37RSOD1

0

0.2

0.4

0.6

0.8

1

1.2

Mbp Mog Mag Plp1

D** ** **

Rel

ativ

eex

pres

sion

in o

ligod

endr

ocyt

e **

SOD1G37R

Non-Tg

0

2

4

6

8

10

Rel

ativ

e FP

KM

Non-TgSOD1G37R

Pho

spho

lipas

e C

var

iant

s** ** ** **

** *****

*

00.5

11.5

22.5

3

Rel

ativ

e FP

KM

G37R

00.050.1

0.150.2

0.250.3

05

10152025G

Top 5 candidate transcription factorsimplicated in SOD1-mediated gene changes

Occ

urre

nce

(%)

Impo

rtanc

e

ETS family ETS family

02468

1012141618

Elf4 Fli1 Ets1 Ets2 Etv4 Etv5 Elk3

OligodendrocytesWhole spinal cord

no changeSO

D1

ver

sus

Non

-Tg

G37

RF o

ld c

h ang

e of

ge n

es in

** ** ** ****

**

E F Non-Tg SOD1

H

Fig. 5. Gene expression changes in oligodendrocytes. (A) Number of gene expression changes in oligodendrocytes at disease onset and early symptomatic stage,comparing SOD1G37R transgenic mice with nontransgenic mice. (B) Number of gene expression changes above 2-, 5-, and 10-fold in SOD1G37R oligodendrocytes at anearly symptomatic stage. (C) qRT-PCR validation of gene changes in SOD1G37R oligodendrocytes at early symptomatic stage. Error bars represent SEM in three or fourbiological replicates. *P < 0.05, **P < 0.005, Student t test. (D–F) Relative expression levels of myelin proteins (D), PLC family genes (E), and CaMK genes (F) inoligodendrocytes at early symptomatic stage. The FPKM values were normalized to values in nontransgene samples for each gene. Error bars represent SD in threeto five biological replicates. *P < 0.05, **P < 0.005, Student t test. (G) Candidate transcription factors of coregulated genes in SOD1G37R olidodendrocytes, aspredicted by the web server DiRE. (H) Fold change of ETS transcription factors in SOD1G37R oligodendrocytes or whole spinal cord compared with those in non-transgenic controls. The FPKM values were normalized to values in nontransgene samples for each gene. Error bars represent SD in three to five biological replicates.

Sun et al. PNAS | Published online November 30, 2015 | E6999

NEU

ROSC

IENCE

PNASPL

US

dismutase-active or -inactive mutant SOD1 at or just before diseaseonset, we identified early events within motor neurons, followed bylater RNA dysregulation in astrocytes and oligodendrocytes.Early RNA expression changes identified in mutant SOD1-

expressing motor neurons include ER stress, synapse, and met-abolic abnormalities (Fig. 6). Few RNA changes were initiallyfound in astrocytes, with many accumulating later, including ingenes involved in inflammation and metabolism, enriched fortargets of PPAR and LXR nuclear receptors (Fig. 6). Thisfinding is consistent with earlier efforts using genetic deletion ofmutant SOD1 from motor neurons to delay disease onset (5, 7),whereas similar deletion from astrocytes slowed disease pro-gression with no strong effect on onset (8, 9). Curiously, althoughmutant SOD1 gene inactivation in NG2+ progenitor oligoden-drocytes reportedly delays the age of disease onset (11), withonly a modest effect in slowing progression after onset inSOD1G37R mice, our genomic data show hardly any changes inoligodendrocytes at this age, with dramatic changes arising by anearly symptomatic stage, consisting mainly of dysregulation ofmyelination and lipid signaling pathways coupled with activationof ETS transcription factors (Fig. 6). The strong effect on diseaseonset from removal of mutant SOD1 from NG2+ cells impliesthat they may have additional functional roles or are progenitorsof additional cell populations beyond a role as the in vivo pro-genitors of mature Cnp1-expressing oligodendrocytes.Activation of ER stress has been implicated in the pathogenesis

of ALS (62–66). Up-regulation of Bip (downstream of the ATF6branch) was initially reported in cell cultures transfected withmutant SOD1 and spinal cords of transgenic SOD1H46R andSOD1L84V mice (64). Another study at disease onset in SOD1G93A

mice reported increased activity of CHOP, a downstream targetindicating activation of the PERK branch of the UPR (66). Ac-tivation of all three arms of the UPR with activated ATF6, ATF4,and XBP1 found in spinal cords, but not cerebellum, has beenreported in end-stage SOD1G93A and SOD1G85R mice (63).Adding to this knowledge, we have established that ER stressstarts within motor neurons with selective activation of the PERK-ATF4-eIF2α pathway, leading to up-regulation of CHOP and itstranscriptional targets, and have further confirmed the specificactivation of its upstream regulators at disease onset. This findingadds an in vivo example during disease pathogenesis to previouscell culture evidence establishing that the three UPR sensors canhave fundamental differences in the timing of their signaling and

responses to particular ER stress stimuli (35, 67). Our data es-tablish that chronic accumulation of misfolded SOD1 initiatesactivation of the PERK branch of ER stress selectively in post-mitotic motor neurons, but not in astrocytes or oligodendrocyteseven at symptomatic disease stages, supporting a special vulner-ability of motor neurons to misfolded SOD1 accumulation. Onesimple explanation for this vulnerability is our identification ofhighly divergent levels of ER chaperones, with much higher levelsin astrocytes and oligodendrocytes than in motor neurons.Furthermore, immunostaining of phospho-eIF2α within indi-

vidual motor neurons has revealed that some motor neuronsdevelop ER stress activation earlier than others, consistent withreport of ER stress activation in selectively vulnerable motorneuron subtypes (68). A recent cell culture study also reportedER stress in both IREα and PERK branches induced by mutantSOD1 in immature induced pluripotent cells (iPSC)-derivedmotor neurons, with the basal level of ER stress (reported byspliced Xbp1) inherently higher in healthy motor neurons com-pared with other neurons and nonneuronal cell types in in vitrocultures (69). The differences between our in vivo findings ofselective PERK pathway activation compared with broader ERstress activation in cell cultures (spliced Xbp1) (Fig. 3B) likelyreflect the additive effects of those from mutant SOD1 and thestressed environment facing cells in cell cultures. ER stress isusually triggered by the accumulation of misfolded proteinswithin the ER lumen. Although SOD1 is not a secreted proteinand is not synthesized inside the ER lumen, mutant SOD1 hasbeen shown to directly interact with Derlin-1, a component of theER-associated degradation (ERAD) machinery, and activates ERstress through dysfunction of ERAD (70). It also has been shownthat misfolded SOD1 has increased association with mitochondriaand ER membranes (71–75). Furthermore, the conformation ofmembrane-associated mutant SOD1 has been solved by NMR,revealing that the association is mediated by interfacial amphi-philic helices (76). Based on the foregoing findings, we proposethat the abnormal insertion of misfolded SOD1 onto/into ERmembranes in motor neurons interferes with the normal functionof other membrane proteins, including protein folding, modifica-tion, and degradation, with activation of the PERK pathway of ERstress from direct SOD1 mutant damage to Derlin or indirectdamage from disrupted protein homeostasis.Whether manipulation of the ER stress pathways can provide a

protective effect remains controversial. Breeding with ATF4−/−

mice (77) or nervous system deletion of XBP-1 (78) has beenreported to modestly delay disease onset and/or prolong the lifespan, but only in one-half of SOD1G86R mice studied; however,crossing with PERK+/−mice has been reported to accelerate diseaseonset and shorten life span in SOD1G85R mice (79). The ER stress-protective agent salubrinal, an inhibitor of eIF2α dephosphory-lation, has been shown to mitigate disease progression (68).Furthermore, eIF2α phosphorylation was found to be up-regulatedby TDP-43 toxicity in flies, and a PERK inhibitor could attenuate itsdamage in flies and mammalian neurons (80). Therefore, distur-bance of ER proteostasis might be a more general phenomenonunderlying the selective motor neuron degeneration caused by dif-ferent mutant genes in ALS. The divergent findings produce acomplex scenario regarding therapy development, with interventionat different steps of the ER stress pathways, at different timings andunder different circumstances, or on different cell types apparentlyresulting in opposite effects on the neurodegeneration process.Finally, our bac-Trap approach has identified a temporal

cascade of different cellular pathways activated by mutant SOD1in motor neurons, astrocytes, and oligodendrocytes. ER stressactivated in motor neurons is an initiating event, presumablymediated by activation of a UPR sensor on ER membranes.Altered lipid signaling is seen in all three cell types. PPAR andLXR nuclear receptors that are normally activated by fatty acidand cholesterol derivatives were predicted to be dysfunctional in

ER stress

Synapse and metabolism responses

Abnormal metabolism and

inflamma�on

Membrane and lipid signaling

defects

PERK-ATF4-CHOP

PPAR nuclear receptorsac�vated by lipid ligand

ETS family of transcrip�on factors

Motor neuron Astrocyte

Oligodendrocyte

❶ ❷

❸

Fig. 6. Cell type-specific toxicity of mutant SOD1 in motor neurons, astro-cytes, and oligodendrocytes. (1) Initial damage occurs in motor neurons atdisease onset, including synapse and metabolism responses and activation ofER stress. (2) Damage in astrocytes includes abnormal metabolism and in-flammation, owing in part to the dysfunction of PPAR and LXR nuclear re-ceptors that are activated by lipid ligands. (3) Damage in oligodendrocytesoccurs in an early symptomatic stage, later than that in motor neurons andastrocytes. The toxicity includes mainly myelination and lipid signaling de-fects, coupled with activation of the ETS family of transcription factors.

E7000 | www.pnas.org/cgi/doi/10.1073/pnas.1520639112 Sun et al.

astrocytes. Extensive membrane proteins and lipid signalingpathways were subsequently dysregulated in oligodendrocytes.Taken together, the collective evidence supports the idea thatthe toxicity of misfolded SOD1 originates from its abnormalmembrane association. With aging, a decreasing ability to de-grade unfolded proteins leads to increasing associations of mu-tant SOD1 with different membranes and eventually initiates acascade of cellular responses and gene expression changes thatresult in motor neuron dysfunction and death. Owing to thedistinctive intrinsic properties of the different cell types, thiscommon toxic feature provokes cell type-specific responses thatcontribute to disease initiation and progression in a coordinatedmanner. Motor neurons are most vulnerable to the accumulatedmutant SOD1 and abnormal cellular pathways because theysynthesize high levels of SOD1 and have very low levels of ERchaperones. Subsequent damage developed from mutant SOD1synthesis within astrocytes and oligodendrocytes is essential foramplifying the initial damage within motor neurons.

Materials and MethodsAnimals. ALS mouse lines SOD1G37R (5), SOD1G85R (38), and SOD1G93A (19) areall heterozygous for a 12-kb genomic DNA fragment encoding the humanmutant SOD1 transgene, under its endogenous promoter. The bacTRAPtransgenic mouse line Chat-bacTRAP line expresses an EGFP-tagged ribo-some protein Rpl10a only within motor neurons. The Aldh1l1-bacTRAP lineexpresses the same EGFP-tagged Rpl10a in astrocytes, whereas the Cnp1-bacTRAP expresses it in oligodendrocytes (28, 29). All animal experimentsused in this work were approved by the University of California at San DiegoInstitutional Animal Care and Use Committee.

Purification of Cell Type-Specific mRNA from bacTRAP Mice. Dissected mousespinal cord was immediately homogenized in ice-cold polysome extractionbuffer (20 mM Hepes pH 7.4, 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT,100 μg/mL cycloheximide, protease inhibitors, and RNase inhibitors). Homoge-nates were centrifuged at 2,000 × g for 10 min at 4 °C, after which NonidetP-40 and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids)were added to the supernatant at final concentrations of 1% and 30 mM.The lysates were centrifuged at 13,000 × g for 15 min at 4 °C after incubationon ice for 5 min. Two monoclonal GFP antibody (Htz-GFP19C8 and Htz-GFP19F7; Memorial Sloan Kettering Cancer Center Monoclonal AntibodyCore Facility)-coated magnetic beads (Dynabeads Protein G; Invitrogen)were added to the supernatant, followed by incubation at 4 °C with rotationovernight. Beads were subsequently washed five times with high-salt poly-some wash buffer (20 mM Hepes pH 7.4, 350 mM KCl, 5 mM MgCl2, 0.5 mMDTT, 1% Nonidet P-40, and 100 μg/mL cycloheximide). The RNA bound onthe beads were extracted with an Absolutely RNA Nanoprep Kit (Stratagene)and quantified by the RiboGreen RNA assay (Invitrogen).

RNA-seq Library Preparation and Transcriptome Analysis. RNA quality wasmeasured using the Agilent Bioanalyzer system according to the manufac-turer’s recommendations. RNA-seq libraries were prepared from RNAsextracted from specific cell types or whole spinal cord, using an IlluminaTruSeq RNA Sample Preparation Kit. RNA-seq libraries were sequenced onan Illumina HiSEq 2000 sequencer for 50 cycles from a single end.

The reads were aligned to a reference mouse genome obtained from theUniversity of California Santa Cruz (mm9; NCBI build 37) using Tophat (de-

fault parameters, with the exception of reporting reads mapping to uniquelocations on the reference) (81). RNA-seq results were visualized in the UCSCGenome Browser (genome.ucsc.edu/) by generating custom tracks using big-wig files. The relative abundance of transcripts was measured by FPKM usingCufflinks (82). Expression variations of protein-coding genes between non-trangenic control and mutant SOD1 groups were quantified and compared bycuffdiff (83). Scatterplots, heat maps, and hierarchical clustering were gener-ated by R software using FPKM values from each sample. Gene ontologyanalysis was performed using DAVID (https://david.ncifcrf.gov/). Distant regu-latory elements of coregulated genes were analyzed by DiRE (dire.dcode.org/).

Laser Capture Microdissection. Mice were perfused with sterile ice-cold PBS.Spinal cords were dissected, incubated in 20% (wt/vol) sucrose (in PBS) at 4 °Covernight, and subsequently embedded in optimal cutting temperature (OCT)compound (Sakura) and frozen in isopentane cooled to −40 °C on dry ice.Tissues were cut into 14-μm sections and stained with Cresyl violet. Approxi-mately 700 motor neurons were laser capture microdissected from eachmouse, and RNA was extracted using the RNAqueous-Micro Kit (Ambion) andamplified using the MessageAmp II aRNA amplification kit (Ambion).

qRT-PCR. For first-strand cDNA synthesis, random hexamers were used with ahigh-capacity cDNA reverse transcription kit (Applied Biosystems). qRT-PCRreactions were performed with three or four biological replicates for eachgroup and two technical replicates using iQ SYBR Green Supermix (Bio-Rad)on the iQ5 Multicolor Real-Time PCR system (Bio-Rad). The data were ana-lyzed using iQ5 optical system software, version 2.1 (Bio-Rad). Expressionvalues were normalized to the control genes Rpl23 and Dnaja2. Intergroupdifferences were assessed using the two-tailed Student t test. Primer se-quences are presented in SI Appendix, Table S10.

Immunofluorescence. Mice were perfused intracardially with 4% (vol/vol)paraformaldehyde in 0.1 M Sorenson’s phosphate buffer, pH 7.2. The entirespinal cord was dissected, postfixed for 2 h in fixative, and the transferred in a30% sucrose phosphate buffer for at least 2 d. The lumbar spinal cord wasembedded in OCT compound and snap-frozen in isopentane cooled to −40 °Con dry ice. Floating lumbar spinal cord cryosections (30 μm) were incubated ina blocking solution containing PBS, 0.5% Tween 20, and 1.5% (wt/vol) BSA for1 h, 30 min at room temperature and then in PBS and 0.3% Triton X-100overnight at room temperature, with the following primary polyclonalantibodies against ChAT (Millipore), GFAP (Dako), CC1 (Calbiochem), GFP(a gift from the Oegema/Desai laboratory), and phosphor-eIF2α (Cell SignalingTechnology). Primary antibodies were washed with PBS and then detectedusing FITC or Cy3-coupled secondary antibodies (Jackson ImmunoResearch).The sections were washed with PBS and mounted. Analysis was performedusing a Nikon Eclipse laser scanning confocal microscope. Fluorescence in-tensity from unsaturated images captured with identical confocal settings(a minimum of six spinal cord sections imaged per animal) was quantifiedusing NIS-Elements software (Nikon).

ACKNOWLEDGMENTS. We thank Drs. Nathaniel Heintz and Jeffery Rothsteinfor the bacTRAP reporter mice; Dr. Myriam Heiman for the detailed protocolfor the bacTRAP RNA purification method; members of B. Ren’s laboratory,especially Z. Ye, S. Kuan, and B. Li, for technical help with the Illuminasequencing; and all members of the D.W.C. laboratory for critical commentsand helpful suggestions. This work was supported by National Institutes ofHealth Grant R01 NS27036 (to D.W.C.). S.S. was a recipient of the MiltonSafenowitz Postdoctoral Fellowship from the ALS Association, a Target ALSSpringboard Fellowship, and a National Institutes of Health K99/R00 Award(NS091538-01).

1. Boillée S, Vande Velde C, Cleveland DW (2006) ALS: A disease of motor neurons andtheir nonneuronal neighbors. Neuron 52(1):39–59.

2. Rosen DR (1993) Mutations in Cu/Zn superoxide dismutase gene are associated withfamilial amyotrophic lateral sclerosis. Nature 362(6415):59–62.

3. Clement AM, et al. (2003) Wild-type nonneuronal cells extend survival of SOD1 mu-tant motor neurons in ALS mice. Science 302(5642):113–117.

4. Yamanaka K, et al. (2008) Mutant SOD1 in cell types other than motor neurons andoligodendrocytes accelerates onset of disease in ALS mice. Proc Natl Acad Sci USA105(21):7594–7599.

5. Boillée S, et al. (2006) Onset and progression in inherited ALS determined by motorneurons and microglia. Science 312(5778):1389–1392.

6. Kang SH, Fukaya M, Yang JK, Rothstein JD, Bergles DE (2010) NG2+ CNS glial pro-genitors remain committed to the oligodendrocyte lineage in postnatal life andfollowing neurodegeneration. Neuron 68(4):668–681.

7. Wang L, Sharma K, Grisotti G, Roos RP (2009) The effect of mutant SOD1 dismutaseactivity on non-cell autonomous degeneration in familial amyotrophic lateral scle-rosis. Neurobiol Dis 35(2):234–240.

8. Yamanaka K, et al. (2008) Astrocytes as determinants of disease progression in in-herited amyotrophic lateral sclerosis. Nat Neurosci 11(3):251–253.

9. Wang L, Gutmann DH, Roos RP (2011) Astrocyte loss of mutant SOD1 delays ALSdisease onset and progression in G85R transgenic mice. Hum Mol Genet 20(2):286–293.

10. Lobsiger CS, Cleveland DW (2007) Glial cells as intrinsic components of non–cell-autonomous neurodegenerative disease. Nat Neurosci 10(11):1355–1360.

11. Kang SH, et al. (2013) Degeneration and impaired regeneration of gray matter oli-godendrocytes in amyotrophic lateral sclerosis. Nat Neurosci 16(5):571–579.

12. Ilieva H, Polymenidou M, Cleveland DW (2009) Non-cell autonomous toxicity inneurodegenerative disorders: ALS and beyond. J Cell Biol 187(6):761–772.

13. Perrin FE, et al. (2005) No widespread induction of cell death genes occurs in puremotoneurons in an amyotrophic lateral sclerosis mouse model. Hum Mol Genet14(21):3309–3320.

14. Lobsiger CS, Boillée S, Cleveland DW (2007) Toxicity from different SOD1 mutantsdysregulates the complement system and the neuronal regenerative response in ALSmotor neurons. Proc Natl Acad Sci USA 104(18):7319–7326.

Sun et al. PNAS | Published online November 30, 2015 | E7001

NEU

ROSC

IENCE

PNASPL

US

15. Ferraiuolo L, et al. (2007) Microarray analysis of the cellular pathways involved in theadaptation to and progression of motor neuron injury in the SOD1 G93A mousemodel of familial ALS. J Neurosci 27(34):9201–9219.

16. Ferraiuolo L, et al. (2011) Dysregulation of astrocyte-motoneuron cross-talk in mutantsuperoxide dismutase 1-related amyotrophic lateral sclerosis. Brain 134(Pt 9):2627–2641.

17. Bandyopadhyay U, et al. (2013) RNA-seq profiling of spinal cord motor neurons froma presymptomatic SOD1 ALS mouse. PLoS One 8(1):e53575.

18. Chiu IM, et al. (2013) A neurodegeneration-specific gene-expression signature ofacutely isolated microglia from an amyotrophic lateral sclerosis mouse model. CellReports 4(2):385–401.

19. Gurney ME, et al. (1994) Motor neuron degeneration in mice that express a humanCu,Zn superoxide dismutase mutation. Science 264(5166):1772–1775.

20. Di Giorgio FP, Carrasco MA, Siao MC, Maniatis T, Eggan K (2007) Non-cell autonomouseffect of glia on motor neurons in an embryonic stem cell-based ALS model. NatNeurosci 10(5):608–614.

21. Di Giorgio FP, Boulting GL, Bobrowicz S, Eggan KC (2008) Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation. Cell Stem Cell 3(6):637–648.

22. Haidet-Phillips AM, et al. (2011) Astrocytes from familial and sporadic ALS patientsare toxic to motor neurons. Nat Biotechnol 29(9):824–828.

23. Marchetto MC, et al. (2008) Non–cell-autonomous effect of human SOD1 G37R as-trocytes on motor neurons derived from human embryonic stem cells. Cell Stem Cell3(6):649–657.

24. Nagai M, et al. (2007) Astrocytes expressing ALS-linked mutated SOD1 release factorsselectively toxic to motor neurons. Nat Neurosci 10(5):615–622.

25. Meyer K, et al. (2014) Direct conversion of patient fibroblasts demonstrates non-cellautonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS. ProcNatl Acad Sci USA 111(2):829–832.

26. Frakes AE, et al. (2014) Microglia induce motor neuron death via the classical NF-κBpathway in amyotrophic lateral sclerosis. Neuron 81(5):1009–1023.

27. Phatnani HP, et al. (2013) Intricate interplay between astrocytes and motor neurons inALS. Proc Natl Acad Sci USA 110(8):E756–E765.

28. Heiman M, et al. (2008) A translational profiling approach for the molecular char-acterization of CNS cell types. Cell 135(4):738–748.

29. Doyle JP, et al. (2008) Application of a translational profiling approach for thecomparative analysis of CNS cell types. Cell 135(4):749–762.

30. Da Cruz S, et al. (2012) Elevated PGC-1α activity sustains mitochondrial biogenesis andmuscle function without extending survival in a mouse model of inherited ALS. CellMetab 15(5):778–786.

31. Zhong Z, et al. (2009) Activated protein C therapy slows ALS-like disease in mice bytranscriptionally inhibiting SOD1 in motor neurons and microglia cells. J Clin Invest119(11):3437–3449.

32. Bushong EA, Martone ME, Ellisman MH (2004) Maturation of astrocyte morphologyand the establishment of astrocyte domains during postnatal hippocampal devel-opment. Int J Dev Neurosci 22(2):73–86.

33. Molofsky AV, et al. (2012) Astrocytes and disease: A neurodevelopmental perspective.Genes Dev 26(9):891–907.

34. Parone PA, et al. (2013) Enhancing mitochondrial calcium buffering capacity reducesaggregation of misfolded SOD1 and motor neuron cell death without extendingsurvival in mouse models of inherited amyotrophic lateral sclerosis. J Neurosci 33(11):4657–4671.

35. Hetz C (2012) The unfolded protein response: Controlling cell fate decisions under ERstress and beyond. Nat Rev Mol Cell Biol 13(2):89–102.

36. Walter P, Ron D (2011) The unfolded protein response: From stress pathway to ho-meostatic regulation. Science 334(6059):1081–1086.

37. Harding HP, et al. (2000) Regulated translation initiation controls stress-induced geneexpression in mammalian cells. Mol Cell 6(5):1099–1108.

38. Bruijn LI, et al. (1997) ALS-linked SOD1 mutant G85R mediates damage to astrocytesand promotes rapidly progressive disease with SOD1-containing inclusions. Neuron18(2):327–338.

39. Borchelt DR, et al. (1994) Superoxide dismutase 1 with mutations linked to familialamyotrophic lateral sclerosis possesses significant activity. Proc Natl Acad Sci USA91(17):8292–8296.

40. van Weering HR, et al. (2011) CXCL10/CXCR3 signaling in glia cells differentially af-fects NMDA-induced cell death in CA and DG neurons of the mouse hippocampus.Hippocampus 21(2):220–232.

41. Sui Y, et al. (2006) CXCL10-induced cell death in neurons: Role of calcium dysregu-lation. Eur J Neurosci 23(4):957–964.

42. Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR (2008) Oncogenic BRAFinduces senescence and apoptosis through pathways mediated by the secreted pro-tein IGFBP7. Cell 132(3):363–374.

43. Hong C, Tontonoz P (2008) Coordination of inflammation and metabolism by PPARand LXR nuclear receptors. Curr Opin Genet Dev 18(5):461–467.

44. Glass CK, Rosenfeld MG (2000) The coregulator exchange in transcriptional functionsof nuclear receptors. Genes Dev 14(2):121–141.

45. Li AC, Glass CK (2004) PPAR- and LXR-dependent pathways controlling lipid metab-olism and the development of atherosclerosis. J Lipid Res 45(12):2161–2173.

46. Lemay DG, Hwang DH (2006) Genome-wide identification of peroxisome proliferatorresponse elements using integrated computational genomics. J Lipid Res 47(7):1583–1587.

47. Pehkonen P, et al. (2012) Genome-wide landscape of liver X receptor chromatinbinding and gene regulation in human macrophages. BMC Genomics 13:50–70.

48. Boergesen M, et al. (2012) Genome-wide profiling of liver X receptor, retinoid X re-ceptor, and peroxisome proliferator-activated receptor α in mouse liver reveals ex-tensive sharing of binding sites. Mol Cell Biol 32(4):852–867.

49. Finck BN, Kelly DP (2006) PGC-1 coactivators: inducible regulators of energy metab-olism in health and disease. J Clin Invest 116(3):615–622.

50. St-Pierre J, et al. (2006) Suppression of reactive oxygen species and neurodegenerationby the PGC-1 transcriptional coactivators. Cell 127(2):397–408.

51. Vega RB, Huss JM, Kelly DP (2000) The coactivator PGC-1 cooperates with peroxisomeproliferator-activated receptor alpha in transcriptional control of nuclear genes en-coding mitochondrial fatty acid oxidation enzymes. Mol Cell Biol 20(5):1868–1876.

52. Zamanian JL, et al. (2012) Genomic analysis of reactive astrogliosis. J Neurosci 32(18):6391–6410.

53. Meldrum E, Parker PJ, Carozzi A (1991) The PtdIns-PLC superfamily and signal trans-duction. Biochim Biophys Acta 1092(1):49–71.

54. Waggener CT, Dupree JL, Elgersma Y, Fuss B (2013) CaMKIIβ regulates oligodendro-cyte maturation and CNS myelination. J Neurosci 33(25):10453–10458.

55. Lee Y, et al. (2012) Oligodendroglia metabolically support axons and contribute toneurodegeneration. Nature 487(7408):443–448.

56. Gutierrez-Hartmann A, Duval DL, Bradford AP (2007) ETS transcription factors inendocrine systems. Trends Endocrinol Metab 18(4):150–158.

57. Hollenhorst PC, McIntosh LP, Graves BJ (2011) Genomic and biochemical insights intothe specificity of ETS transcription factors. Annu Rev Biochem 80:437–471.

58. Brown GC, Neher JJ (2014) Microglial phagocytosis of live neurons. Nat Rev Neurosci15(4):209–216.

59. Chung WS, et al. (2013) Astrocytes mediate synapse elimination through MEGF10 andMERTK pathways. Nature 504(7480):394–400.

60. Tasdemir-Yilmaz OE, Freeman MR (2014) Astrocytes engage unique molecular pro-grams to engulf pruned neuronal debris from distinct subsets of neurons. Genes Dev28(1):20–33.

61. Nakahara J, et al. (2003) Signaling via immunoglobulin Fc receptors induces oligo-dendrocyte precursor cell differentiation. Dev Cell 4(6):841–852.

62. Atkin JD, et al. (2006) Induction of the unfolded protein response in familial amyo-trophic lateral sclerosis and association of protein-disulfide isomerase with superox-ide dismutase 1. J Biol Chem 281(40):30152–30165.

63. Kikuchi H, et al. (2006) Spinal cord endoplasmic reticulum stress associated with amicrosomal accumulation of mutant superoxide dismutase-1 in an ALS model. ProcNatl Acad Sci USA 103(15):6025–6030.

64. Tobisawa S, et al. (2003) Mutant SOD1 linked to familial amyotrophic lateral sclerosis,but not wild-type SOD1, induces ER stress in COS7 cells and transgenic mice. BiochemBiophys Res Commun 303(2):496–503.

65. Wate R, et al. (2005) Expression of an endoplasmic reticulum-resident chaperone,glucose-regulated stress protein 78, in the spinal cord of a mouse model of amyo-trophic lateral sclerosis. Acta Neuropathol 110(6):557–562.

66. Vlug AS, et al. (2005) ATF3 expression precedes death of spinal motoneurons inamyotrophic lateral sclerosis-SOD1 transgenic mice and correlates with c-Jun phos-phorylation, CHOP expression, somato-dendritic ubiquitination and Golgi fragmen-tation. Eur J Neurosci 22(8):1881–1894.

67. DuRose JB, Tam AB, Niwa M (2006) Intrinsic capacities of molecular sensors of theunfolded protein response to sense alternate forms of endoplasmic reticulum stress.Mol Biol Cell 17(7):3095–3107.

68. Saxena S, Cabuy E, Caroni P (2009) A role for motoneuron subtype-selective ER stressin disease manifestations of FALS mice. Nat Neurosci 12(5):627–636.

69. Kiskinis E, et al. (2014) Pathways disrupted in human ALS motor neurons identifiedthrough genetic correction of mutant SOD1. Cell Stem Cell 14(6):781–795.

70. Nishitoh H, et al. (2008) ALS-linked mutant SOD1 induces ER stress- and ASK1-dependent motor neuron death by targeting Derlin-1. Genes Dev 22(11):1451–1464.

71. Vande Velde C, Miller TM, Cashman NR, Cleveland DW (2008) Selective association ofmisfolded ALS-linked mutant SOD1 with the cytoplasmic face of mitochondria. ProcNatl Acad Sci USA 105(10):4022–4027.

72. Liu J, et al. (2004) Toxicity of familial ALS-linked SOD1 mutants from selective re-cruitment to spinal mitochondria. Neuron 43(1):5–17.

73. Israelson A, et al. (2010) Misfolded mutant SOD1 directly inhibits VDAC1 conductancein a mouse model of inherited ALS. Neuron 67(4):575–587.

74. Homma K, et al. (2013) SOD1 as a molecular switch for initiating the homeostatic ERstress response under zinc deficiency. Mol Cell 52(1):75–86.

75. Israelson A, et al. (2015) Macrophage migration inhibitory factor as a chaperone in-hibiting accumulation of misfolded SOD1. Neuron 86(1):218–232.

76. Lim L, Lee X, Song J (2015) Mechanism for transforming cytosolic SOD1 into integralmembrane proteins of organelles by ALS-causing mutations. Biochim Biophys Acta1848(1 Pt A):1–7.

77. Matus S, Lopez E, Valenzuela V, Nassif M, Hetz C (2013) Functional contribution of thetranscription factor ATF4 to the pathogenesis of amyotrophic lateral sclerosis. PLoSOne 8(7):e66672.

78. Hetz C, et al. (2009) XBP-1 deficiency in the nervous system protects against amyo-trophic lateral sclerosis by increasing autophagy. Genes Dev 23(19):2294–2306.

79. Wang L, Popko B, Roos RP (2011) The unfolded protein response in familial amyo-trophic lateral sclerosis. Hum Mol Genet 20(5):1008–1015.

80. Kim HJ, et al. (2014) Therapeutic modulation of eIF2α phosphorylation rescues TDP-43toxicity in amyotrophic lateral sclerosis disease models. Nat Genet 46(2):152–160.

81. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: Discovering splice junctions withRNA-Seq. Bioinformatics 25(9):1105–1111.

82. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping andquantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5(7):621–628.

83. Trapnell C, et al. (2010) Transcript assembly and quantification by RNA-Seq reveals un-annotated transcripts and isoform switching during cell differentiation. Nat Biotechnol28(5):511–515.

E7002 | www.pnas.org/cgi/doi/10.1073/pnas.1520639112 Sun et al.

SUPPORTING INFORMATION (SI) APPENDIX

Translational profiling identifies a cascade of damage initiated in motor

neurons and spreading to glia in mutant SOD1-mediated ALS

Shuying Sun, Ying Sun, Shuo-Chien Ling, Laura Ferraiuolo, Melissa McAlonis-Downes,

Yiyang Zou, Kevin Drenner, Yin Wang, Dara Ditsworth, Seiya Tokunaga, Alex Kopelevich,

Brian K. Kaspar, Clotilde Lagier-Tourenne, and Don W. Cleveland

Supplementary Figures:

SI Figure 1. High-throughput RNA sequencing of translational mRNAs purified from motor

neurons, astrocytes and oligodendrocytes.

SI Figure 2. Gene expression changes in mutant SOD1 motor neurons.

SI Figure 3. Signaling pathways enriched in SOD1G37R

induced gene expression changes in

motor neurons at disease onset.

SI Figure 4. Gene expression changes in astrocytes at disease onset.

SI Figure 5. Gene expression changes in oligodendrocytes with mutant SOD1 transgene.

SI Figure 6. Signaling pathways enriched in SOD1G37R

induced gene expression changes in

oligodendrocytes at symptomatic stage.

SI Figure 7. The Calcium Signaling pathway enriched in SOD1G37R

induced gene expression

changes in oligodendrocytes at symptomatic stage.

Supplementary Tables:

SI Table 1. Number of uniquely mapped reads obtained by RNA‐seq of the different biological

replicates in each condition as marked.

SI Table 2. Gene expression changes in SOD1G37R

motor neurons compared to non-transgenic

controls at disease onset.

SI Table 3. Main functional categories enriched in SOD1G37R

induced gene changes in motor

neurons.

SI Table 4. Gene expression changes in SOD1G37R

astrocytes compared to non-transgenic

controls at disease onset.

SI Table 5. Main functional categories enriched in SOD1G37

induced gene changes in astrocytes.

SI Table 6. Gene expression changes in SOD1G37R

oligodendrocytes compared to non-transgenic

controls at disease onset.

SI Table 7. Gene expression changes in SOD1G37R

oligodendrocytes compared to non-transgenic

controls at early symptomatic stage.

SI Table 8. Main functional categories enriched in SOD1G37R

induced gene changes in

oligodendrocytes at early symptomatic stage.

SI Table 9. Overlaps of gene expression changes in SOD1G37R

motor neurons, astrocytes and

oligodendrocytes at disease onset.

SI Table 10. Primer sequences.

1.0

0.9

0.8

0.7

Ald

h1l1

-3

Ald

h1l1

-5

Ald

h1l1

-4

Ald

h1l1

-6

Ald

h1l1

-1

Ald

h1l1

-2

Cnp

1-1

Cnp

1-3

Cnp

1-4

Cnp

1-2

Cnp

1-5

Tota

l-2

Tota

l-1

Tota

l-3

Tota

l-4

Cha

t-3

Cha

t-5

Cha

t-6

Cha

t-1

Cha

t-2

Cha

t-4

Oligodendrocyte Motor neuronAstrocyte Spinal cord

Supplementary Figure 1: High-throughput RNA sequencing of translational mRNAs purified from motor neurons, astrocytes and oligodendrocytes. Hierarchical clustering of all expressed genes in motor neurons, astrocytes, oligodendrocytes and whole spinal cord tissue of non-transgenic animal, with 4-6 biological replicates in each group.

A

G37

R 1

G37

R 2

G37

R 3

G37

R 4

Non

-Tg

4

Non

-Tg

1

Non

-Tg

2

Non

-Tg

3

Motor neuron(onset)

B

Non-Tg G37R

Up-

regu

late

d D

own-

regu

late

d G

37R

Motor neuron

Whole spinal cord

Astrocyte

205 27355

61 28147

C

Oligodendrocyte

14 3199

G37R Non-Tg

D

0

10

20

30

40

50

60

70

ATF4 CHOP

Nor

mal

ized

fold

cha

nge

Non-TgSOD1

G85R

Uns

uper

vise

d cl

uste

ring

0.99

50.

990

0.98

50.

980

0.97

5

in S

OD

1

ve

rsus

non

-tran

sgen

tic m

otor

neu

rons

SOD1 spinal cord(10.5 months)

G85R Laser capture

microdissectionmotor

neuronsRNA extraction

and amplification

qRT-PCR to measure RNAsencoding UPR components

SOD1

SOD1

Supplementary Figure 2: Gene expression changes in mutant SOD1 motor neurons. (A) Hierarchical clustering of all expressed genes in motor neurons with or without SOD1G37R transgene. (B) Heat map with hierarchical clustering of all the 260 genes differentially expressed in non-transgenic and SOD1G37R motor neurons. (C) The number of overlapped gene changes induced by SOD1 G37R in motor neurons, astrocytes and oligodendrocytes, compared to ones identified in whole spinal cord. (D) qRT-PCR of ATF4 and CHOP in SOD1G85R expressed motor neurons compared to the non-transgenic control. Error bars represent s.e.m in 2 biological and 4 technical replicates.

Aspa

Adssl1

AbatGad1,2 Glul

Supplementary Figure 3: Signaling pathways enriched in SOD1G37R induced gene expression changes in motor neurons at disease onset. The Alanine, Aspartate and Glutamate Metabolism pathway annotated by KEGG (Kyoto Encyclopedia of Genes and Genomes) was found to be significant using DAVID Functional Annotation Bioinformatics Analysis. Red box-shaded genes and red star-labeled reactions are altered in SOD1G37R mutant motor neurons.

A Astrocyte(onset)

G37

R 3

G37

R 2G

37R

1

G37

R 4

Non

-Tg

1

Non

-Tg

3

Non

-Tg

4Non

-Tg

5

Non

-Tg

6

0

1

2

3

4

5

Cxcl10 Igfbp7

Nor

mal

ized

fold

cha

nge

Non-TgG37RSOD1

SOD1G85R

B******

***

Non

-Tg

2

Non-TgG37R

00.20.40.60.8

11.21.4

Non-TgSOD1

C

G37RRel

ativ

e P

GC

1α le

vel

(FP

KM

)

**

Uns

uper

vise

d cl

uste

ring

0.99

50.

985

0.98

00.

975

0.99

00.

970

0.96

5

SOD1

Supplementary Figure 4: Gene expression changes in astrocytes at disease onset. (A) Hierarchical clustering of all expressed genes in astrocytes with or without SOD1G37R transgene. (B) qRT-PCR validation of up-regulated genes encoding secreted proteins with potential toxicity on neurons in SOD1G37R and SOD1G85R astrocytes. Error bars represent s.e.m. in 3-4 biological replicates. *p<0.05, **p<0.005, ***p<0.0005, Student’s t-test. (C) The relative expression levels of PGC1α in motor neurons, astrocytes, oligodendrocytes and whole spinal cord, comparing G37R with non-transgene mice at disease onset. The FPKM values in each group were normalized to that in non-transgenic astrocytes. Error bars represent s.e.m. in 3-6 biological replicates. **p<0.005, Student’s t-test.

0

2

4

6

8

10

12

14

16

18

Elf4 Fli1 Ets1 Ets2 Etv4 Etv5 Elk3

0

2

4

6

8

10

Rel

ativ

e FP

KM

Non-TgSOD1

Oligodendrocytes(early symptomatic)

Non

-Tg

1

Non

-Tg

2

Non

-Tg

3

Non

-Tg

4

Non

-Tg

5

G37

R 1

G37

R 2

G37

R 3

0

0.2

0.4

0.6

0.8

1

1.2

onset symptomatic

Rel

ativ

e FP

KM

of M

CT1

non-Tg

SOD1SOD1

D

B

C

A

0

2

4

6

8

10

12 OligodendrocytesWhole spinal cord

E

G37R

G85R

G37

R

G85R

Pho

spho

lipas

e C

var

iant

s

0

0.5

1

1.5

2

2.5

3

Rel

ativ

e FP

KM

Non-TgSOD1G85R

Ca2

+/ca

lmod

ulin

-de

pend

ent k

inas

es

SOD1SOD1

G37R

G85R

Fold

cha

nge

of g

enes

in m

utan

t SO

D1

no change

vers

us N

on-T

g ol

igod

endr

ocyt

es

F*

** *

*

* * * * **

G37RNon-Tg

Uns

uper

vise

d cl

uste

ring

0.98

00.

975

0.97

00.

965

0.96

00.

955

0.95

0

SOD1

SO

D1

ver

sus

Non

-Tg

Fold

cha

nge

of g

enes

in

G37

R

Supplementary Figure 5: Gene expression changes in oligodendrocytes with mutant SOD1 transgene. (A) Hierarchical clustering of all expressed genes in astrocytes with or without SOD1G37R transgene. (B) Relative expression levels of PLC family genes in SOD1G85R oligodendrocytes at symptomatic stage. The FPKM values were normalized to ones in non-transgene samples for each gene. Error bars represent s.d. in 3-5 biological replicates. *p<0.05, Student’s t-test. (C) Relative expression levels (FPKM values from RNA-seq) of CaMK genes in G85R oligodendrocytes at symptomatic stage. Error bars represent s.d. in 3-5 biological replicates. *p<0.05, Student’s t-test. (D) Relative expression levels (FPKM) of MCT1 in SOD1G37R and SOD1G85R oligdendrocytes compared to non-transgenic samples at both disease onset and symptomatic stage. (E) Fold change of ETS transcription factors in SOD1G37R or SOD1G85R oligodendrocytes compared to ones in non-transgenic controls. The FPKM values were normalized to ones in non-transgene samples for each gene. Error bars represent s.d. in 3-5 biological replicates. (F) Fold change of genes in the FcγR-mediated phagocytosis pathway in G37R oligodendrocytes or whole spinal cord compared to non-transgenic controls. The FPKM values were normalized to ones in non-transgene samples for each gene. Error bars represent s.d. in 3-5 biological replicates.

Synj1

Pik3cg

PLC

PLCPLC

Inpp4a

Inpp5d

Inpp4a

Dgkb

Fcgr2b

Ptprc

Inpp5d

Src Pik3cg

Dnm1

Myo10

Plcg2Prkcd

Marcks

Itpkb

Ncf1

Was

Rac2Vav1

Dock2

A

B

Supplementary Figure 6: Signaling pathways enriched in SOD1G37R induced gene expression changes in oligodendrocytes at symptomatic stage. The phosphatidylinositol signaling pathway (A) and FcγR-mediated Phagocytosis (B) annotated by KEGG (Kyoto Encyclopedia of Genes and Genomes) were found to be significant using DAVID Functional Annotation Bioinformatics Analysis. Red box-shaded genes are altered (up-regulated) in SOD1G37R mutant oligodendrocytes. Darker red represents relatively higher fold change than lighter red. Purple star-labeled genes are potential targets of ETS transcription factors.

Slc8a2

Cacna1i

Bdkrb2 Gq

Adcy8

PLCδ

PLCβ

PLCγ

PLCε

Ryr2

Mylk

Phkg

CAMK

Adcy8

Pde1b

Ptk2b

Itpkb

Slc8a2

Supplementary Figure 7: The Calcium Signaling pathway enriched in SOD1G37R induced gene expression changes in oligodendrocytes at symptomatic stage. The Calcium Signaling pathway annotated by KEGG (Kyoto Encyclopedia of Genes and Genomes) were found to be significant using DAVID Functional Annotation Bioinformatics Analysis. Red box-shaded genes are up-regulated in SOD1G37R oligodendrocytes. Darker red represents relatively higher fold change than lighter red. Blue box-shaded genes are down-regulated in SOD1G37R oligodendrocytes.

Supplementary Table 1: Number of uniquely mapped reads obtained by RNA‐seq of the different biological replicates in each condition as marked

Non‐Tg G37R Non‐Tg      G37R Non‐Tg G37R Non‐Tg  G37R Non‐Tg  G37R G85R Non‐Tg  G37RSample 1 46,560,673 44,811,418 40,417,656 39,321,273 33,874,841 38,870,700 58,534,768 55,787,566 48,354,420 46,481,238 41,514,393 21,838,068 30,256,872Sample 2 40,613,401 35,384,166 47,044,682 41,926,905 32,257,716 37,733,359 60,334,899 53,600,581 43,917,893 45,196,376 44,308,212 29,239,200 33,006,700Sample 3 36,678,375 37,955,944 42,117,355 38,124,359 37,603,930 30,199,733 47,506,358 54,209,556 44,857,846 39,094,208 38,529,849 29,222,841 35,308,474Sample 4 34,961,748 41,693,935 45,533,830 37,242,469 35,255,915 38,972,192 56,022,231 44,736,327 41,026,823Sample 5 40,005,888 37,710,915 47,685,360Sample 6 39,588,673

Whole spinal cordOligodendrocyte11 month8 month

Motor neuron Astrocyte Oligodendrocyte Whole spinal cord

Supplementary Table 2: Gene expression changes in SOD1 G37R motor neurons compared to non-transgenic controls at disease onset.gene locus FPKM value for non-transgenic control FPKM value for SOD1 G37R ln(fold_change) q_value

Slc6a4 chr11:76812098-76845845 0.01 0.65 3.77 7.18E-04Sprr1a chr3:92287875-92289803 1.96 54.74 3.33 0.00E+00Phox2b chr5:67485635-67490365 0.20 3.64 2.89 0.00E+00Fgf21 chr7:52869259-52870860 0.55 8.67 2.75 9.75E-14Tbx20 chr9:24525255-24578747 0.07 0.67 2.29 1.29E-07Atf3 chr1:192994175-193007212 0.93 9.14 2.29 0.00E+00Rtdr1 chr10:74420221-74495331 0.21 2.04 2.28 4.51E-05Myo1f chr17:33692651-33744709 0.09 0.74 2.13 3.46E-05Shox2 chr3:66777190-66785693 0.14 1.12 2.10 6.44E-03Lpl chr8:71404453-71430831 0.09 0.63 1.96 8.01E-05Slc17a7 chr7:52419290-52431509 0.27 1.81 1.89 1.24E-07Crabp2 chr3:87752614-87757294 0.21 1.39 1.87 3.14E-02Cckar chr5:54089723-54098943 0.07 0.45 1.86 2.13E-02Fcrls chr3:87054886-87067446 0.11 0.67 1.85 1.92E-02Rasal3 chr17:32527605-32541753 0.06 0.36 1.75 2.49E-02Fmod chr1:135934091-135944854 0.07 0.41 1.75 3.47E-02Lyz2 chr10:116714596-116719328 0.85 4.81 1.73 5.42E-07Inpp5d chr1:89516886-89617083 0.19 1.06 1.73 4.04E-10Aspg chr12:113344893-113365784 0.09 0.49 1.72 2.41E-02Thpo chr16:20724526-20742457 0.15 0.82 1.70 2.55E-03Trh chr6:92192055-92194642 0.22 1.17 1.69 1.43E-02Clec7a chr6:129411608-129422795 0.11 0.58 1.68 2.78E-02Vav1 chr17:57418522-57468659 0.09 0.43 1.61 5.72E-03Ptpn6 chr6:124670735-124688727 0.24 1.17 1.60 1.67E-03Tbx3 chr5:120120677-120134610 0.06 0.29 1.57 1.51E-02Tyrobp chr7:31198806-31202598 1.32 6.10 1.53 4.23E-04Capg chr6:72494432-72512974 0.52 2.35 1.51 2.79E-04Trem2 chr17:48485725-48491598 0.40 1.75 1.48 1.52E-02Gbp2 chr3:142283626-142300972 0.12 0.54 1.48 4.46E-02Gpnmb chr6:48986516-49008181 0.10 0.42 1.46 2.78E-02Anxa3 chr5:97222403-97274987 0.65 2.61 1.40 1.24E-04Hmha1 chr10:79479416-79494216 0.14 0.55 1.39 5.70E-03Hk2 chr6:82675020-82724448 0.11 0.43 1.38 5.83E-03Hk3 chr13:55050792-55122746 0.37 1.39 1.33 3.13E-06C1qa chr4:136451830-136454759 1.87 6.93 1.31 5.35E-06Irf8 chr8:123260275-123280592 0.30 1.09 1.30 2.60E-03Mpeg1 chr19:12535268-12539775 0.21 0.76 1.29 1.49E-03C1qb chr4:136436060-136442092 0.99 3.55 1.28 1.44E-03Hpgd chr8:58773348-58799843 1.23 4.37 1.27 5.33E-06Nupr1 chr7:133766759-133768984 4.60 16.04 1.25 5.61E-07Timp1 chrX:20425668-20498044 0.54 1.89 1.24 4.21E-02Lgals3 chr14:47993534-48005842 1.43 4.95 1.24 7.58E-07Crh chr3:19593400-19595396 0.56 1.90 1.23 2.20E-02Cyth4 chr15:78427476-78452449 0.42 1.42 1.21 1.15E-03Ncf1 chr5:134696129-134705495 0.49 1.60 1.18 9.80E-04Gfap chr11:102748649-102758514 40.07 129.75 1.18 0.00E+00Nr4a2 chr2:56959636-56976414 0.35 1.11 1.16 1.02E-03Gbp7 chr3:142193301-142213047 0.11 0.34 1.16 2.01E-02Pde5a chr3:122432075-122562292 0.11 0.34 1.16 3.13E-02C4b chr17:34865325-34880842 0.79 2.47 1.15 2.00E-07Fermt3 chr19:7070620-7093959 0.43 1.33 1.13 5.63E-03En1 chr1:122499063-122504568 0.67 2.03 1.11 1.10E-03Ctss chr3:95330707-95360325 2.03 6.08 1.10 8.90E-05C1qc chr4:136445716-136448829 1.20 3.55 1.09 1.13E-02Adhfe1 chr1:9538126-9621173 1.08 3.12 1.06 6.91E-07Dach2 chrX:110411864-110949995 0.26 0.75 1.05 1.31E-03Phgdh chr3:98117093-98143892 2.18 6.14 1.03 1.79E-05Slc14a1 chr18:78296829-78338858 0.32 0.90 1.02 1.55E-02Myo1b chr1:51806609-51972818 0.17 0.46 1.02 3.20E-02Aldh1l1 chr6:90500841-90549165 2.57 6.99 1.00 1.67E-07Naprt1 chr15:75721393-75724911 0.71 1.93 1.00 2.06E-02Vim chr2:13495937-13504453 6.35 17.17 0.99 5.75E-09Cd9 chr6:125410283-125444773 2.40 6.48 0.99 6.28E-04Col16a1 chr4:129725083-129776521 0.25 0.67 0.97 2.57E-02Acsbg1 chr9:54452803-54509692 12.02 31.52 0.96 4.64E-12Chrnb4 chr9:54875962-54896351 0.32 0.85 0.96 4.39E-02Neu4 chr1:95917069-95924907 0.33 0.84 0.93 2.38E-02Ngfr chr11:95430139-95449012 0.55 1.37 0.91 1.67E-02

Genes upregulated in SOD1 G37R motor neurons

Pdk4 chr6:5433350-5446278 0.98 2.37 0.89 1.59E-03Pgm5 chr19:24748497-24936332 0.33 0.78 0.87 6.11E-03Npy chr6:49772727-49779504 3.46 8.26 0.87 4.25E-02Tagln2 chr1:174430376-174437506 7.56 18.00 0.87 5.20E-06Kit chr5:75971011-76052746 0.41 0.98 0.86 6.25E-03Chrna3 chr9:54859149-54874366 0.58 1.36 0.85 4.08E-02Padi2 chr4:140462274-140513817 4.52 10.54 0.85 5.56E-08Acss1 chr2:150443846-150494668 0.55 1.29 0.84 2.83E-02Rfx4 chr10:84218792-84369283 0.69 1.59 0.84 3.59E-03Csf1r chr18:61265225-61290793 0.53 1.20 0.83 3.83E-02Dock1 chr7:141862369-142365330 1.05 2.36 0.81 1.72E-04Ddc chr11:11714103-11798147 2.69 6.06 0.81 1.32E-03Papss2 chr19:32670204-32741677 0.56 1.25 0.81 2.17E-02C3 chr17:57343395-57367559 0.85 1.86 0.79 3.72E-03Phkg1 chr5:130322865-130354954 1.30 2.83 0.78 2.16E-02Gpr88 chr3:115952571-115956402 0.66 1.45 0.78 4.93E-02Arap2 chr5:62993684-63157416 1.43 3.10 0.78 5.58E-05Dock10 chr1:80497647-80755128 1.20 2.55 0.76 3.00E-04Cbs chr17:31749588-31774086 2.18 4.62 0.75 5.17E-04Il33 chr19:29999603-30035205 8.10 17.02 0.74 3.54E-06Sgk1 chr10:21601989-21719708 23.44 49.10 0.74 0.00E+00Epas1 chr17:87153203-87232750 1.47 3.07 0.73 1.07E-03Fam59b chr5:30430123-30481520 1.10 2.28 0.73 1.36E-02Acsf2 chr11:94418415-94463100 0.99 2.06 0.73 4.46E-02Cacna1i chr15:80117667-80228722 0.55 1.14 0.72 7.22E-03Caskin2 chr11:115660665-115674906 0.92 1.88 0.72 1.83E-02Nfe2l2 chr2:75513575-75542698 1.32 2.71 0.72 3.57E-02Stbd1 chr5:93032076-93035605 9.28 18.97 0.72 2.38E-05Gpd1 chr15:99548023-99555438 10.59 21.65 0.72 2.08E-06Chl1 chr6:103460869-103683029 1.49 3.03 0.71 2.67E-04Aldh1l2 chr10:82950191-82996885 0.78 1.58 0.71 1.21E-02Spon1 chr7:120909511-121186889 0.70 1.43 0.71 1.76E-02Tns3 chr11:8331654-8564538 1.82 3.71 0.71 1.05E-04Rhpn2 chr7:36119255-36181786 1.49 3.04 0.71 9.81E-03Stxbp3a chr3:108596097-108643420 3.46 6.98 0.70 2.18E-03Cnp chr11:100436252-100443053 81.60 164.78 0.70 1.94E-13Arhgef2 chr3:88420128-88451974 2.01 4.01 0.69 1.30E-05A2m chr6:121586190-121629256 0.95 1.90 0.69 2.30E-02Epb4.1l2 chr10:25079603-25243324 1.87 3.73 0.69 7.35E-04Nbr1 chr11:101413420-101443265 76.73 152.66 0.69 8.30E-09Slc25a18 chr6:120723785-120744000 6.71 13.30 0.68 2.78E-03Daam2 chr17:49595346-49703662 3.04 6.01 0.68 7.18E-05Prex2 chr1:10983545-11293763 0.49 0.96 0.68 2.06E-02Clic4 chr4:134769884-134828675 8.65 17.02 0.68 6.25E-06Mlc1 chr15:88786313-88808983 3.88 7.54 0.66 4.32E-03Txnip chr3:96359690-96370724 1.35 2.61 0.66 1.99E-02Pygm chr19:6384428-6398459 4.49 8.68 0.66 1.02E-03Plin3 chr17:56418384-56429934 3.01 5.79 0.66 1.46E-02Lhfpl2 chr13:94827750-94965364 1.01 1.95 0.65 4.48E-02Arap1 chr7:108496582-108561100 0.83 1.58 0.65 1.69E-02Gad1 chr2:70327996-70440069 14.68 28.17 0.65 9.59E-06Prex1 chr2:166391844-166539332 2.90 5.57 0.65 2.79E-04Rftn1 chr17:50132631-50329822 2.88 5.53 0.65 7.08E-03Maob chrX:16286406-16394492 2.53 4.82 0.64 2.36E-02Rock1 chr18:10064398-10181790 0.74 1.41 0.64 4.45E-02Psat1 chr19:15979612-15999549 39.79 75.60 0.64 8.22E-07Nmral1 chr16:4711317-4719059 5.59 10.60 0.64 2.38E-02Agxt2l1 chr3:130320365-130338668 1.86 3.51 0.63 1.32E-02Prkd3 chr17:79348744-79420156 0.52 0.98 0.63 3.44E-02Enc1 chr13:98011059-98022995 3.22 6.02 0.63 1.32E-03Galnt6 chr15:100520345-100559807 0.83 1.55 0.62 3.73E-02Phldb1 chr9:44494390-44543281 2.90 5.38 0.62 1.94E-03Ndrg1 chr15:66760879-66801203 29.57 54.77 0.62 1.42E-05Efhd1 chr1:89160938-89207366 3.61 6.68 0.62 3.52E-02Arhgef10 chr8:14911716-15001085 2.59 4.78 0.62 8.59E-05Phlpp1 chr1:108068445-108290822 4.95 9.10 0.61 2.20E-04Aspa chr11:73118489-73138136 5.97 10.87 0.60 2.16E-02Inf2 chr12:113826994-113853768 2.06 3.75 0.60 1.58E-02Arid5a chr1:36364577-36380874 0.54 0.98 0.59 4.85E-02Apoe chr7:20281592-20284515 27.74 50.02 0.59 5.29E-04Dnm2 chr9:21229351-21314630 5.99 10.77 0.59 1.26E-07

Rassf2 chr2:131818585-131855724 3.15 5.67 0.59 4.47E-03Gpd2 chr2:57090088-57223130 3.11 5.57 0.58 2.62E-04Rasgrp3 chr17:75835244-75928393 1.07 1.91 0.58 4.93E-02Serpinb1b chr13:33175971-33186249 15.47 27.66 0.58 7.53E-04Gal chr19:3409916-3414457 27.55 49.19 0.58 3.27E-03Acap2 chr16:31092498-31201324 2.30 4.09 0.58 5.28E-03Gad2 chr2:22477846-22549397 4.83 8.61 0.58 7.64E-04Lgals9 chr11:78776480-78798426 4.50 8.02 0.58 3.13E-02Dock4 chr12:41172639-41573075 1.46 2.61 0.58 1.42E-02Serpinb1a chr13:32933960-32943054 4.40 7.83 0.58 3.25E-02Car2 chr3:14886425-14900770 57.30 101.93 0.58 7.93E-05Rasa3 chr8:13567217-13677587 5.82 10.34 0.58 1.38E-03Klk6 chr7:51079913-51087397 6.12 10.88 0.58 1.02E-02Plcl1 chr1:55462789-55811129 3.21 5.70 0.57 1.78E-03Cdkn1a chr17:29227930-29237667 4.69 8.32 0.57 2.77E-02Rapgef3 chr15:97575200-97598097 1.69 2.99 0.57 3.09E-02Clmn chr12:106001323-106103286 0.76 1.33 0.56 6.10E-03Camk2a chr18:61085285-61147806 5.18 9.04 0.56 4.46E-05Carhsp1 chr16:8658679-8672246 9.66 16.83 0.55 1.26E-03Erbb2ip chr13:104608865-104710594 2.38 4.12 0.55 5.14E-03Sox10 chr15:78985342-78994920 3.38 5.81 0.54 4.82E-02Myo6 chr9:80012840-80159536 1.53 2.63 0.54 1.99E-02Fam40b chr6:29867012-29909680 1.18 2.03 0.54 3.54E-02Trf chr9:103111205-103132616 65.17 111.79 0.54 2.71E-04Ddit3 chr10:126727848-126748842 30.43 51.54 0.53 4.25E-03Aldoc chr11:78137699-78140262 328.25 553.91 0.52 3.32E-03Slc32a1 chr2:158436493-158441483 16.43 27.68 0.52 1.98E-03Mag chr7:31684201-31699851 26.43 44.25 0.52 1.08E-03Eno1 chr4:149611305-149622982 40.44 67.50 0.51 1.26E-03Fmnl2 chr2:52716901-52993236 2.27 3.76 0.51 3.60E-02Acot11 chr4:106406519-106472436 2.52 4.18 0.51 3.35E-02Gab1 chr8:83288332-83404378 2.93 4.85 0.50 3.75E-02Ermn chr2:57897525-57905163 6.70 11.07 0.50 1.03E-02Baiap3 chr17:25377114-25393309 5.49 9.02 0.50 1.00E-02Gm98 chr19:10282760-10315238 3.36 5.52 0.50 2.39E-02Pdxk chr10:77899491-77927693 10.43 17.06 0.49 2.79E-03Ppp2r3a chr9:101007319-101154162 1.68 2.75 0.49 2.22E-02Ifrd1 chr12:40929715-40949776 12.17 19.74 0.48 2.04E-02Tubb2b chr13:34218876-34222223 16.70 27.09 0.48 1.37E-02Mllt4 chr17:13853442-14042801 2.76 4.48 0.48 2.68E-02Trim66 chr7:116592514-116651648 1.12 1.81 0.48 3.80E-03Slc17a6 chr7:58877199-58926496 11.61 18.69 0.48 5.57E-03Rasgrf2 chr13:92020011-92901449 5.84 9.40 0.48 9.70E-03Add3 chr19:53214934-53321889 6.19 9.93 0.47 2.78E-06Marcks chr10:36853048-36858732 6.90 11.04 0.47 1.65E-02Gabra1 chr11:41944981-41996432 4.61 7.38 0.47 4.52E-02Adssl1 chr12:113858257-113879566 14.58 23.29 0.47 2.49E-02Glul chr1:155747074-155756844 163.46 259.56 0.46 1.12E-02Camkv chr9:107838250-107852022 10.60 16.82 0.46 1.83E-02Anln chr9:22135657-22193650 5.51 8.70 0.46 1.94E-02Cldn11 chr3:31048841-31063248 23.92 37.22 0.44 1.66E-02Slc7a5 chr8:124405045-124431586 10.39 16.11 0.44 2.49E-02Hsd17b4 chr18:50287854-50355924 11.22 17.33 0.43 3.62E-02Zzz3 chr3:152058973-152344396 2.26 3.49 0.43 2.39E-02Rnh1 chr7:148346224-148358750 26.03 40.16 0.43 7.01E-03Nptx1 chr11:119400032-119409134 7.33 11.30 0.43 3.01E-02Efr3a chr15:65618602-65705374 17.45 26.88 0.43 9.48E-03Mat2a chr6:72382792-72390609 20.93 32.21 0.43 1.60E-02Ppp6r3 chr19:3454927-3575749 4.58 7.05 0.43 3.81E-02Fscn1 chr5:143722033-143734868 20.83 31.85 0.42 2.20E-02Ahi1 chr10:20672352-20800235 36.20 55.14 0.42 3.11E-04Vgf chr5:137506164-137509221 72.30 109.73 0.42 1.12E-02Ebf3 chr7:144385353-144506128 3.85 5.76 0.40 9.23E-03Sept2 chr1:95375569-95406309 6.66 9.97 0.40 2.54E-02Wars chr12:110098239-110132384 49.14 73.22 0.40 2.43E-04Gap43 chr16:42248673-42340764 138.60 205.99 0.40 2.49E-02Spg20 chr3:54916029-54941254 13.40 19.86 0.39 3.72E-04Enpp2 chr15:54670452-54751701 17.06 25.25 0.39 1.76E-02Abat chr16:8513521-8621660 27.94 41.08 0.39 1.84E-03Ank3 chr10:68996455-69490184 6.44 9.40 0.38 1.53E-04Anp32e chr3:95733179-95751310 16.82 24.50 0.38 1.26E-02

Grb10 chr11:11830501-11937423 8.99 12.93 0.36 1.24E-02Mbp chr18:82644514-82755029 104.67 150.50 0.36 1.27E-05Mon2 chr10:122429116-122513561 2.85 4.09 0.36 5.75E-03Ssrp1 chr2:84877357-84887268 16.34 23.43 0.36 1.55E-02Llgl1 chr11:60513191-60540723 8.50 12.19 0.36 3.14E-03Gtf2i chr5:134713703-134790616 19.60 27.28 0.33 1.81E-04Ralgds chr2:28368686-28408602 9.85 13.63 0.32 1.92E-02Ndrg2 chr14:52524945-52533163 123.08 166.49 0.30 1.39E-02Ddx17 chr15:79346837-79377171 26.14 34.48 0.28 4.46E-02Sept8 chr11:53333237-53357598 25.37 32.93 0.26 1.54E-02Rap1gap chr4:137220640-137285776 36.21 46.01 0.24 2.25E-02Add1 chr5:34916362-34974954 56.05 69.52 0.22 3.62E-02

Ppp2r2b chr18:42804922-43219125 404.29 298.21 -0.30 8.74E-03Fam78b chr1:168931547-169021433 20.66 15.12 -0.31 2.07E-02Lin7a chr10:106708886-106862199 13.96 10.19 -0.32 2.68E-02Lrrcc1 chr3:14533787-14572658 8.96 6.46 -0.33 2.16E-02Rpl38 chr11:114529856-114533645 570.51 408.73 -0.33 1.50E-02Caln1 chr5:130845327-131316515 45.09 32.22 -0.34 1.20E-02Cd59a chr2:103935957-103955508 93.01 64.59 -0.36 3.46E-02Atox1 chr11:55260144-55274640 568.68 394.62 -0.37 4.42E-02Rpl30 chr15:34370260-34373031 106.09 73.35 -0.37 3.86E-02Rps24 chr14:25309902-25315368 226.14 154.87 -0.38 7.62E-04Pcp4 chr16:96689212-96747400 475.32 324.65 -0.38 3.03E-02Rpl14 chr9:120480633-120483770 315.69 215.36 -0.38 2.52E-02Cryab chr9:50560862-50564738 343.68 231.40 -0.40 1.99E-02Hddc2 chr10:31033210-31047892 100.39 67.50 -0.40 3.83E-02Rpl35a chr16:33056538-33060274 60.75 40.70 -0.40 2.81E-02Mctp1 chr13:76522408-77171071 18.70 12.44 -0.41 2.81E-02Rab3b chr4:108551674-108644682 63.67 42.32 -0.41 1.15E-02Rxrg chr1:169528492-169569754 14.18 9.39 -0.41 4.32E-02Rpl37 chr15:5066612-5069140 174.41 115.05 -0.42 3.78E-02Rps8 chr4:116826440-116828737 80.04 52.39 -0.42 3.40E-02Rgs11 chr17:26339906-26348269 21.44 14.00 -0.43 4.69E-02Lrrc49 chr9:60435043-60535941 194.06 125.00 -0.44 4.00E-04Rgs7bp chr13:105737232-105845010 57.86 37.14 -0.44 4.27E-03Polr3g chr13:81812835-81850012 31.60 20.01 -0.46 5.15E-03Prss12 chr3:123149830-123209520 42.11 26.60 -0.46 3.41E-03Chrm2 chr6:36283137-36474774 10.87 6.58 -0.50 4.46E-02Itm2a chrX:104592533-104598699 60.85 36.10 -0.52 5.08E-04Mrvi1 chr7:118011780-118125609 15.18 8.98 -0.53 4.42E-04Uhmk1 chr1:172129386-172145524 26.21 15.42 -0.53 3.31E-03Tcp11l1 chr2:104520136-104552319 5.91 3.30 -0.58 1.04E-02Zfp672 chr11:58128615-58143841 27.53 15.31 -0.59 7.42E-04Hsd17b7 chr1:171879667-171899336 11.61 6.36 -0.60 2.33E-04Gpr146 chr5:139835692-139936488 2.02 1.06 -0.64 2.71E-02Rbm3 chrX:7716100-7725089 10.16 5.28 -0.65 1.60E-02Akap9 chr5:3928185-4080204 2.53 1.32 -0.65 2.02E-03Zfp605 chr5:110539110-110558813 2.98 1.51 -0.68 3.72E-02Ppp1r1b chr11:98210051-98219597 23.27 11.33 -0.72 2.16E-05Runx2 chr17:44740949-45256233 0.44 0.15 -1.12 1.12E-02

Genes downregulated in SOD1 G37R motor neurons

Supplementary Table 3: Main functional categories enriched in SOD1G37R induced gene changes in motor neurons.

GO categories GO term Benjamini

All genes Cellular

component GO:0045202 synapse 0.002025175 GO:0030054 cell junction 0.00762269

Upregulated genes

Molecular function

GO:0030170 pyridoxal phosphate binding 0.002590255 GO:0070279 vitamin B6 binding 0.002590255 GO:0003779 actin binding 0.012795866 GO:0005516 calmodulin binding 0.014144813

GO:0005085 guanyl-nucleotide exchange

factor activity 0.01536069

Biological process

GO:0006006 glucose metabolic process 0.01030087 GO:0019318 hexose metabolic process 0.026219843

GO:0005996 monosaccharide metabolic

process 0.034273322 GO:0002443 leukocyte mediated immunity 0.009494858

GO:0002449 lymphocyte mediated

immunity 0.030247327

GO:0006958 complement activation,

classical pathway 0.030150948 GO:0002252 immune effector process 0.034709754 GO:0009611 response to wounding 0.03495991

Downregulated genes Cellular

component GO:0005840 ribosome 0.004876113 Molecular function GO:0003735

structural constituent of ribosome 3.55E-04

Biological process GO:0006412 translation 0.003114477

Supplementary Table 4: Gene expression changes in SOD1 G37R astrocytes compared to non-transgenic controls at disease onset.gene locus FPKM value for non-transgenic control FPKM value for SOD1 G37R ln(fold_change) q_value

Ccl6 chr11:83395562-83437195 0.04 1.03 3.38 6.66E-03Htr1b chr9:81524998-81538763 0.01 0.14 2.40 2.02E-03Tgm1 chr14:56318845-56332329 0.12 1.12 2.20 1.14E-05Hspb1 chr5:136363788-136365433 28.29 193.42 1.92 0.00E+00Fkbp5 chr17:28536039-28623057 7.78 50.13 1.86 0.00E+00Lamc2 chr1:154969885-155033577 0.05 0.29 1.72 4.42E-02Timp1 chrX:20425668-20498044 2.68 14.36 1.68 9.85E-11Arl4d chr11:101526854-101529146 9.02 46.21 1.63 2.13E-111810011O10Rik chr8:25548087-25549418 6.80 34.14 1.61 2.04E-11Wisp2 chr2:163646569-163658883 0.88 4.37 1.61 2.34E-05Ecm1 chr3:95538070-95543492 0.50 2.47 1.61 2.92E-05Serpinf2 chr11:75245237-75253003 0.20 0.93 1.53 4.33E-02Crym chr7:127329897-127345502 0.85 3.58 1.44 3.40E-03Lyz2 chr10:116714596-116719328 1.67 6.78 1.40 5.23E-04Pla2g4e chr2:119992147-120071071 0.21 0.81 1.36 8.92E-03Adamtsl2 chr2:26934900-26964133 0.42 1.57 1.32 3.49E-03S100a4 chr3:90407691-90409967 25.45 92.93 1.30 2.53E-08Tekt4 chr17:25608534-25613539 7.81 26.56 1.22 5.17E-07Maff chr15:79178107-79189506 1.96 6.64 1.22 2.92E-04Fcgr2b chr1:172890688-172906202 0.84 2.75 1.18 4.33E-02Cebpd chr16:15887378-16146926 6.22 20.22 1.18 4.20E-07Tcf23 chr5:31271049-31279391 0.40 1.25 1.13 2.05E-02Socs3 chr11:117827400-117830680 0.98 3.04 1.13 4.42E-03Lgals3 chr14:47993534-48005842 34.84 107.19 1.12 3.32E-14Map3k6 chr4:132796732-132808843 0.38 1.16 1.11 2.59E-02Cebpb chr2:167514414-167515918 10.45 31.69 1.11 4.14E-06Anxa3 chr5:97222403-97274987 3.15 9.46 1.10 1.06E-03Rasl11a chr5:147656646-147659302 4.62 13.82 1.10 7.65E-04Trem2 chr17:48485725-48491598 1.48 4.40 1.09 4.71E-02Tyrobp chr7:31198806-31202598 6.56 19.49 1.09 5.04E-03Tagln chr9:45671773-45744141 15.12 44.20 1.07 9.73E-07Lrrc33 chr16:32142910-32165562 2.47 7.17 1.07 1.89E-04C1qa chr4:136451830-136454759 6.52 18.92 1.07 4.75E-04C1qb chr4:136436060-136442092 6.09 17.63 1.06 7.17E-04Junb chr8:87500807-87502647 4.02 11.62 1.06 3.67E-04Serpina3n chr12:105644917-105652539 25.39 72.28 1.05 1.44E-06Cxcl10 chr5:92760866-92843653 3.11 8.78 1.04 1.13E-02C1qc chr4:136445716-136448829 5.21 14.56 1.03 2.50E-03Prss23 chr7:96656294-96666096 0.98 2.72 1.02 1.80E-02Slc37a1 chr17:31432427-31487643 0.57 1.55 1.00 2.31E-02Nupr1 chr7:133766759-133768984 6.45 17.03 0.97 1.98E-02Mthfd2 chr6:83255697-83267598 34.16 88.44 0.95 1.03E-05Plin4 chr17:56219079-56249225 0.87 2.25 0.95 9.04E-03Tagln2 chr1:174430376-174437506 24.54 63.11 0.94 2.80E-05Tm4sf1 chr3:57090985-57105841 6.90 17.11 0.91 1.17E-03Sult1a1 chr7:133816383-133819871 5.99 14.38 0.88 1.04E-02Sgk1 chr10:21601989-21719708 27.25 62.68 0.83 1.31E-06Iigp1 chr18:60535682-60552283 1.04 2.38 0.83 4.72E-02Vim chr2:13495937-13504453 136.47 312.10 0.83 1.25E-03S100a11 chr3:93324417-93330210 32.62 73.78 0.82 6.62E-03Ctss chr3:95330707-95360325 7.26 16.10 0.80 3.50E-02Arrdc2 chr8:73359036-73363619 12.83 28.28 0.79 1.69E-03Zbtb16 chr9:48462401-48644050 2.80 6.01 0.76 2.11E-02Hr chr14:70953862-70973349 28.73 61.59 0.76 3.88E-03Cdkn1a chr17:29227930-29237667 139.85 298.90 0.76 5.10E-03Gadd45g chr13:51942043-51943843 160.40 342.24 0.76 4.96E-03Igfbp7 chr5:77739508-77837070 10.79 22.95 0.75 2.83E-02Inpp5d chr1:89516886-89617083 0.48 1.03 0.75 3.32E-02Tmem52 chr4:154843222-154844967 18.15 37.23 0.72 4.67E-03S100a6 chr3:90416815-90418336 300.99 614.31 0.71 8.93E-032010002N04Rik chr18:60633844-60661637 62.68 126.75 0.70 1.43E-02Emd chrX:71500177-71503086 64.90 124.94 0.65 2.57E-02Nfkbia chr12:56590395-56593634 37.87 71.44 0.63 3.24E-02Mylk chr16:34785035-35002520 6.18 11.40 0.61 4.83E-02Txnip chr3:96359690-96370724 18.95 34.79 0.61 1.09E-03Anxa2 chr9:69301489-69339592 42.12 77.12 0.60 4.31E-02Crem chr18:3266351-3366861 12.34 21.35 0.55 3.76E-05

Genes upregulated in SOD1 G37R astrocytes

Tcf12 chr9:71692058-71959626 25.94 18.71 -0.33 2.49E-02Fnbp1 chr2:30881725-30997528 58.51 41.93 -0.33 6.28E-03Mbnl1 chr3:60276751-60433670 23.21 14.98 -0.44 5.74E-05Prkd3 chr17:79348744-79420156 10.49 6.69 -0.45 2.69E-02Sema6d chr2:124436031-124493506 15.04 9.34 -0.48 6.39E-04Kazn chr4:141658304-141795316 37.89 23.26 -0.49 2.93E-04Nfix chr8:87231497-87324239 45.31 27.21 -0.51 1.50E-02Prr5l chr2:101554441-101679137 37.67 22.35 -0.52 4.64E-02Atl2 chr17:80247731-80295463 38.50 22.70 -0.53 3.85E-02Anks1b chr10:89336253-90435729 10.10 5.77 -0.56 4.07E-04Sertad2 chr11:20443255-20553026 12.40 7.01 -0.57 1.14E-02D16Ertd472e chr16:78540580-78576933 6.00 3.37 -0.58 1.38E-03Insig1 chr5:28397951-28405202 64.65 35.06 -0.61 4.72E-02Cpeb2 chr5:43542924-43680963 11.76 6.37 -0.61 1.13E-03Fam53b chr7:139903766-140004879 12.77 6.90 -0.62 2.53E-03Aven chr2:112333120-112471410 69.40 37.24 -0.62 1.50E-03Mob3b chr4:34896322-35104733 14.49 7.74 -0.63 4.31E-02Ppargc1a chr5:51845487-51945160 10.75 5.67 -0.64 6.87E-03Fam123a chr14:60997122-60999840 31.61 16.65 -0.64 1.28E-03Rasgrp3 chr17:75835244-75928393 13.20 6.90 -0.65 2.19E-02Prrx1 chr1:165175249-165243781 8.16 4.27 -0.65 3.76E-04Heyl chr4:122910798-122927113 32.27 16.81 -0.65 1.55E-02Gtf2ird1 chr5:134833530-134932586 6.17 3.22 -0.65 2.16E-07Cdc42ep1 chr15:78673076-78685959 78.05 39.96 -0.67 1.13E-022810459M11Rik chr1:87942437-87952031 19.54 9.97 -0.67 1.22E-03Olig2 chr16:91225794-91228922 45.59 22.69 -0.70 8.93E-03Fbxo32 chr15:58007433-58046447 4.54 2.25 -0.70 4.69E-02Trib2 chr12:15798532-15823591 28.60 14.13 -0.71 5.81E-03Foxn2 chr17:88840051-88889873 8.91 4.36 -0.71 1.80E-02Plekho2 chr9:65402190-65427841 91.66 44.63 -0.72 3.49E-03Bcl9l chr9:44307218-44318495 4.95 2.37 -0.74 4.83E-02Map6d1 chr16:20233381-20241431 19.53 9.28 -0.74 6.11E-03Wnt7a chr6:91313976-91361363 12.38 5.86 -0.75 1.81E-02Trim36 chr18:46324953-46372261 9.80 4.42 -0.80 3.24E-03Glmn chr5:107963729-108090857 5.08 2.25 -0.81 2.66E-02Ddit4l chr3:137286635-137291296 52.06 22.57 -0.84 2.82E-04Trim59 chr3:68839215-68848664 11.89 4.88 -0.89 1.97E-03Smtnl2 chr11:72203615-72225215 5.67 2.32 -0.89 2.24E-02Fam84b chr15:60650550-60662955 2.93 1.09 -0.99 3.40E-03Fam181a chr12:104553168-104555275 21.55 7.32 -1.08 1.25E-04Kif20a chr18:34758268-34811390 1.19 0.39 -1.11 5.96E-03

Genes downregulated in SOD1 G37R astrocytes

Supplementary Table 5: Main functional categories enriched in SOD1G37R induced gene changes in astrocytes.

GO categories GO term Benjamini

All genes Cellular

component GO:0030528 transcription regulator activity 0.033649388 Upregulated genes

Cellular component GO:0005576 extracellular region 9.91E-04

Biological process

GO:0002250 adaptive immune response 0.006877283 GO:0002526 acute inflammatory response 0.003692776

GO:0016064 immunoglobulin mediated immune response 0.011421196

GO:0019724 B cell mediated immunity 0.010011371

GO:0002449 lymphocyte mediated immunity 0.015928235

GO:0006954 inflammatory response 0.022856364 GO:0002443 leukocyte mediated immunity 0.021361855

 

Supplementary Table 6: Gene expression changes in SOD1 G37R oligodendrocytes compared to non-transgenic controls at disease onset.gene locus FPKM value for non-transgenic control FPKM value for SOD1 G37R ln(fold_change) q_value

Pla2g4e chr2:119992147‐120071071 0.40 2.35 1.77 6.11E‐12Socs3 chr11:117827400‐117830680 0.33 1.35 1.39 6.80E‐04Bcl3 chr7:20393810‐20408104 0.76 2.91 1.35 4.25E‐05Trib1 chr15:59480208‐59488654 0.97 2.24 0.84 2.58E‐03Lgals3 chr14:47993534‐48005842 1.56 3.40 0.78 3.66E‐02Fmn1 chr2:113167892‐113556924 0.62 1.29 0.73 8.13E‐04Serpina3n chr12:105644917‐105652539 38.84 77.03 0.68 3.98E‐06Vim chr2:13495937‐13504453 9.00 16.34 0.60 4.60E‐03C4b chr17:34865325‐34880842 13.29 22.30 0.52 3.22E‐03Spna2 chr2:29821079‐29886970 9.65 14.68 0.42 1.10E‐04Plec chr15:76001403‐76061808 0.99 1.46 0.39 1.55E‐02Arhgef40 chr14:52604507‐52640408 3.79 5.56 0.38 2.33E‐02Madd chr2:90977516‐91023204 10.48 14.42 0.32 1.41E‐02Rap1gap chr4:137220640‐137285776 32.52 42.19 0.26 1.65E‐02

Fam123a chr14:60997122‐60999840 25.61 15.39 ‐0.51 2.59E‐04D4Wsu53e chr4:134479539‐134483285 122.26 73.27 ‐0.51 4.97E‐03Nfkbia chr12:56590395‐56593634 45.45 26.88 ‐0.53 1.60E‐02Hspa1a chr17:35106303‐35109101 10.27 5.98 ‐0.54 1.89E‐02Aven chr2:112333120‐112471410 65.64 37.67 ‐0.56 3.79E‐05Arl4d chr11:101526854‐101529146 176.53 100.28 ‐0.57 8.93E‐03Dnajb1 chr8:86132073‐86135802 64.55 35.48 ‐0.60 1.66E‐04Sgk1 chr10:21601989‐21719708 728.50 378.12 ‐0.66 7.99E‐04Ccne2 chr4:11118500‐11181406 1.06 0.39 ‐1.01 1.05E‐02

Genes upregulated in SOD1 G37R oligodendrocytes

Genes downregulated in SOD1 G37R oligodendrocytes

Supplementary Table 7: Gene expression changes in SOD1 G37R oligodendrocytes compared to non-transgenic controls at early symptomatic stage.gene locus FPKM value for non-transgenic control FPKM value for SOD1 G37R ln(fold_change) q_value Changed at onset

AI607873 chr1:175653558‐175671940 0.01 0.46 3.50 2.88E‐03Trib3 chr2:152163160‐152169796 0.12 3.57 3.42 2.65E‐10Tgm1 chr14:56318845‐56332329 0.07 2.21 3.39 4.80E‐08Cst7 chr2:150396150‐150404680 0.13 3.03 3.18 6.54E‐05Mmp12 chr9:7347373‐7360461 0.02 0.40 3.18 2.85E‐03Lilrb4 chr10:51210780‐51216417 0.03 0.72 3.16 2.27E‐02Clec7a chr6:129411608‐129422795 0.15 2.87 2.93 3.86E‐10Cybb chrX:9012377‐9046450 0.03 0.51 2.91 1.26E‐04Gpnmb chr6:48986516‐49008181 0.19 2.69 2.66 1.76E‐12Lyz2 chr10:116714596‐116719328 2.66 37.91 2.66 0.00E+00Lyz1 chr10:116724850‐116729924 0.23 3.24 2.64 1.92E‐06Serpina3m chr12:105625373‐105632467 0.08 0.82 2.37 6.71E‐03Pbk chr14:66424747‐66436659 0.09 0.84 2.29 9.59E‐03Wisp2 chr2:163646569‐163658883 0.11 1.05 2.27 1.86E‐03Arhgap25 chr6:87409378‐87483229 0.19 1.75 2.23 1.79E‐05Cd22 chr7:31650422‐31665361 0.04 0.36 2.23 4.87E‐03Ccl3 chr11:83461344‐83462880 0.15 1.34 2.18 4.10E‐02Lgals3 chr14:47993534‐48005842 2.59 22.07 2.14 5.27E‐10 YesRgs1 chr1:146091798‐146096234 0.09 0.76 2.11 3.44E‐02Fcrls chr3:87054886‐87067446 0.78 6.39 2.10 8.08E‐11Top2a chr11:98854260‐98885503 0.06 0.48 2.10 3.34E‐04Klhl6 chr16:19946585‐19983122 0.35 2.89 2.10 3.26E‐08Atf3 chr1:192994175‐193007212 0.38 3.08 2.09 2.71E‐07Ly9 chr1:173518743‐173537491 0.07 0.56 2.09 9.66E‐03Pla2g4e chr2:119992147‐120071071 0.51 4.15 2.09 0.00E+00 YesMyo1f chr17:33692651‐33744709 0.61 4.91 2.08 0.00E+00Bcl3 chr7:20393810‐20408104 0.81 6.44 2.07 2.05E‐10 YesSocs3 chr11:117827400‐117830680 0.40 3.10 2.04 9.08E‐09 YesOsmr chr15:6763576‐6824313 0.07 0.52 2.02 3.83E‐04Crym chr7:127329897‐127345502 0.33 2.49 2.01 2.36E‐04Fam46c chr3:100275458‐100293115 0.31 2.27 2.00 1.36E‐05Irf8 chr8:123260275‐123280592 0.88 6.52 2.00 7.21E‐13Vav1 chr17:57418522‐57468659 0.47 3.44 2.00 5.49E‐06Cd48 chr1:173612185‐173635388 0.15 1.07 1.99 3.17E‐02Apobec1 chr6:122527809‐122552462 0.21 1.57 1.99 1.11E‐04Slfn8 chr11:82815659‐82834312 0.07 0.51 1.99 9.93E‐03Itgax chr7:135273081‐135294171 0.14 0.99 1.98 2.66E‐05Bcl2a1b chr9:89094110‐89102676 0.46 3.31 1.98 1.98E‐03Capg chr6:72494432‐72512974 1.78 12.93 1.98 2.45E‐10Ch25h chr19:34548273‐34549625 0.12 0.88 1.97 2.96E‐02Slfn2 chr11:82878613‐82884180 0.23 1.63 1.97 4.18E‐04Slfn5 chr11:82724754‐82778352 0.09 0.63 1.96 1.25E‐04Ptpn6 chr6:124670735‐124688727 1.25 8.84 1.96 1.76E‐12Tnfsf11 chr14:78677252‐78707850 0.17 1.18 1.94 8.99E‐04Cd68 chr11:69477872‐69479564 1.09 7.60 1.94 3.85E‐07Dock2 chr11:34126863‐34597325 0.42 2.90 1.94 5.91E‐08Col5a3 chr9:20574493‐20619478 0.12 0.87 1.94 1.92E‐06Mpeg1 chr19:12535268‐12539775 0.91 6.27 1.93 0.00E+00Aspg chr12:113344893‐113365784 0.43 2.99 1.93 5.93E‐08Anxa3 chr5:97222403‐97274987 1.86 12.83 1.93 7.21E‐13Rasal3 chr17:32527605‐32541753 0.39 2.61 1.90 2.47E‐08Trim30a chr7:111557539‐111613707 0.15 0.97 1.89 6.75E‐05Samsn1 chr16:75859038‐75909511 0.24 1.59 1.88 3.98E‐04Cd180 chr13:103483637‐103496711 0.15 0.95 1.86 2.63E‐03Hlx chr1:186551023‐186556372 0.11 0.69 1.86 1.87E‐02Atp6v0d2 chr4:19803984‐19849713 0.11 0.69 1.85 7.38E‐03Myzap chr9:71352153‐71440167 0.68 4.28 1.84 2.56E‐08Serpina3n chr12:105644917‐105652539 56.92 357.60 1.84 0.00E+00 YesArhgap30 chr1:173319090‐173340370 0.20 1.27 1.83 4.29E‐06Vim chr2:13495937‐13504453 13.63 84.31 1.82 0.00E+00 YesGbp2 chr3:142283626‐142300972 0.63 3.79 1.80 5.93E‐08Blnk chr19:41003416‐41069025 0.27 1.60 1.79 3.59E‐04Nckap1l chr15:103284255‐103329231 1.13 6.74 1.79 0.00E+00Ifi204 chr1:175677424‐175697050 0.16 0.93 1.77 3.76E‐03Fgr chr4:132530009‐132557797 0.10 0.60 1.76 4.85E‐03Ifi44 chr3:151393886‐151412923 0.06 0.37 1.76 4.33E‐02Gbp5 chr3:142159897‐142185308 0.17 0.98 1.75 6.61E‐04Tagln2 chr1:174430376‐174437506 4.44 25.22 1.74 0.00E+00Naip2 chr13:100914017‐100972047 0.13 0.73 1.73 3.47E‐04Cd52 chr4:133649452‐133650988 1.40 7.91 1.73 1.72E‐03Cyth4 chr15:78427476‐78452449 1.39 7.75 1.72 3.01E‐11Psd4 chr2:24240916‐24264249 0.14 0.80 1.72 1.67E‐04Pik3ap1 chr19:41348707‐41459560 0.35 1.95 1.71 4.08E‐05Rhoh chr5:66254807‐66287939 0.06 0.34 1.71 9.84E‐03Klk8 chr7:51052946‐51059192 0.56 3.09 1.71 2.59E‐04Casp12 chr9:5345475‐5373034 0.34 1.89 1.71 3.46E‐05Hpgd chr8:58773348‐58799843 3.11 17.08 1.71 7.21E‐13Lpl chr8:71404453‐71430831 0.67 3.67 1.70 1.81E‐09Arl11 chr14:61928589‐61930773 0.26 1.40 1.69 7.17E‐03

Genes upregulated in SOD1 G37R oligodendrocytes

Sh3tc1 chr5:36039828‐36071925 0.11 0.58 1.69 2.13E‐03Dock8 chr19:25074018‐25276922 0.26 1.39 1.68 3.71E‐08Fyb chr15:6529870‐6613312 0.18 0.96 1.68 2.03E‐03Icam1 chr9:20820403‐20833240 0.10 0.54 1.66 2.90E‐02Tlr7 chrX:163742862‐163768460 0.09 0.48 1.66 1.08E‐02BC013712 chr4:132338271‐132352279 0.18 0.97 1.65 3.68E‐03Slc4a11 chr2:130509843‐130523255 0.29 1.50 1.65 8.68E‐05Hcls1 chr16:36935068‐36963300 0.53 2.75 1.65 3.82E‐05Sfpi1 chr2:90936953‐90955913 1.24 6.43 1.65 2.85E‐06Lpxn chr19:12873098‐12908298 0.36 1.85 1.64 1.43E‐03Fcgr2b chr1:172890688‐172906202 0.39 1.97 1.62 3.29E‐03Ncf4 chr15:78075240‐78093010 0.29 1.46 1.62 9.90E‐03Myo1g chr11:6406550‐6420961 0.10 0.50 1.61 2.29E‐02Sash3 chrX:45499703‐45514740 0.17 0.82 1.58 7.83E‐03Wdfy4 chr14:33772732‐33998252 0.09 0.43 1.58 6.40E‐04Egr2 chr10:67000616‐67004936 0.12 0.58 1.57 1.97E‐02Nes chr3:87775014‐87784373 0.18 0.86 1.57 5.89E‐05Lrrk1 chr7:73403632‐73533227 0.15 0.71 1.56 8.75E‐05C3ar1 chr6:122797157‐122806175 0.11 0.51 1.56 7.25E‐03Uba7 chr9:107877897‐107886387 0.20 0.96 1.56 2.41E‐03Sprr1a chr3:92287875‐92289803 0.44 2.08 1.55 4.45E‐02Plcb2 chr2:118533252‐118554174 0.11 0.52 1.55 3.82E‐03Psmb8 chr17:34335139‐34338399 2.14 9.98 1.54 6.25E‐07Ecm1 chr3:95538070‐95543492 0.20 0.92 1.54 3.25E‐02Apobec3 chr15:79719961‐79738859 0.41 1.88 1.53 5.60E‐04Gadl1 chr9:115818572‐115985294 0.09 0.43 1.52 2.52E‐02Itgb2 chr10:76993092‐77028419 0.58 2.64 1.52 1.92E‐05C1qa chr4:136451830‐136454759 12.10 54.63 1.51 1.34E‐12Pik3cg chr12:32858261‐32893514 0.27 1.20 1.49 8.99E‐04Nlrc5 chr8:96996662‐97051172 0.09 0.40 1.49 4.76E‐03Tmem106a chr11:101443555‐101453099 0.14 0.63 1.48 4.30E‐02Lyn chr4:3605267‐3718759 0.56 2.45 1.48 1.01E‐02Fmod chr1:135934091‐135944854 0.58 2.54 1.48 3.59E‐05Inpp5d chr1:89516886‐89617083 1.15 5.05 1.48 1.62E‐06Eif4ebp1 chr8:28370798‐28386128 0.51 2.24 1.47 2.12E‐02Arap3 chr18:38132276‐38158623 0.23 1.00 1.47 5.61E‐03Rasl11a chr5:147656646‐147659302 0.69 2.97 1.46 5.85E‐03Parp14 chr16:35832963‐35871468 0.16 0.71 1.46 2.02E‐04Hmha1 chr10:79479416‐79494216 0.78 3.31 1.45 4.27E‐05Parvg chr15:84155149‐84173408 0.35 1.50 1.45 1.77E‐03Pion chr5:20692084‐20797519 0.26 1.08 1.44 1.05E‐03Mdfic chr6:15670660‐15752169 0.14 0.59 1.44 1.73E‐02Cd109 chr9:78463352‐78564067 0.15 0.62 1.43 1.66E‐03Trem2 chr17:48485725‐48491598 2.19 9.14 1.43 1.94E‐05Fes chr7:87522643‐87532832 0.44 1.82 1.43 5.24E‐04Cd84 chr1:173769827‐173820849 0.18 0.74 1.43 1.21E‐02Ptprc chr1:139959437‐140071882 0.07 0.30 1.42 4.40E‐02Arhgdib chr6:136872229‐136890238 1.63 6.74 1.42 8.68E‐05Ccl6 chr11:83395562‐83437195 0.34 1.39 1.42 3.35E‐02Parp9 chr16:35926600‐35972707 0.36 1.46 1.41 1.63E‐02Mlxipl chr5:135582760‐135614252 0.13 0.54 1.41 2.25E‐02Tlr13 chrX:103338613‐103355832 0.10 0.43 1.41 3.79E‐02Akr1b8 chr6:34304163‐34318454 0.56 2.27 1.40 8.12E‐03Alox5 chr6:116360088‐116411196 0.20 0.79 1.40 2.18E‐02Aif1 chr17:35307936‐35312946 1.74 6.98 1.39 6.58E‐04Ifit1 chr19:34715378‐34724499 0.84 3.37 1.39 2.21E‐05Rin3 chr12:103521283‐103629064 0.18 0.73 1.39 8.69E‐03Nlrp3 chr11:59356187‐59380458 0.10 0.41 1.39 4.28E‐02Fermt3 chr19:7070620‐7093959 1.38 5.52 1.38 4.91E‐03Tyrobp chr7:31198806‐31202598 6.76 26.91 1.38 1.18E‐05Gna15 chr10:80965058‐80986970 0.63 2.50 1.38 1.21E‐03Cxcl16 chr11:70267735‐70279701 0.23 0.89 1.37 3.23E‐02Ang chr14:51710751‐51725826 2.51 9.79 1.36 5.38E‐04Lag3 chr6:124854376‐124861723 0.73 2.82 1.35 7.01E‐04Casp1 chr9:5298516‐5307281 0.77 2.97 1.35 1.98E‐03Trib1 chr15:59480208‐59488654 1.09 4.17 1.34 5.12E‐07 YesAdamtsl3 chr7:89484203‐89762958 0.12 0.46 1.34 4.76E‐03Itpripl2 chr7:125628625‐125635489 0.07 0.27 1.34 3.97E‐02Snx20 chr8:91150726‐91160027 0.68 2.58 1.34 7.00E‐03Hspb1 chr5:136363788‐136365433 6.99 26.72 1.34 2.73E‐07Ncf1 chr5:134696129‐134705495 1.83 6.98 1.34 3.23E‐07Gfap chr11:102748649‐102758514 154.40 587.08 1.34 1.57E‐08Hpgds chr6:65067286‐65094724 0.42 1.59 1.34 6.89E‐04Ctss chr3:95330707‐95360325 11.72 44.40 1.33 1.86E‐10Irf5 chr6:29476624‐29487320 0.77 2.88 1.32 4.36E‐04Hck chr2:152934203‐152977177 0.47 1.76 1.32 5.43E‐03Was chrX:7658591‐7667617 0.26 0.96 1.32 4.08E‐02Plscr2 chr9:92170439‐92192590 1.17 4.32 1.31 2.02E‐04Tnfaip2 chr12:112680871‐112693229 0.32 1.19 1.31 3.29E‐03C1qb chr4:136436060‐136442092 7.29 26.82 1.30 9.09E‐08Gsdmd chr15:75692768‐75697834 0.43 1.57 1.30 1.61E‐02Fblim1 chr4:141131976‐141161967 0.45 1.64 1.30 1.51E‐03

Tgm2 chr2:157942140‐157972128 1.15 4.22 1.30 4.29E‐06Fgd2 chr17:29497858‐29516480 0.56 2.02 1.29 6.85E‐03Serpina3g chr12:105452753‐105480144 0.93 3.39 1.29 6.67E‐04Clic1 chr17:35187187‐35195664 5.57 20.17 1.29 3.79E‐07Emp1 chr6:135312948‐135333191 0.18 0.65 1.29 4.97E‐02Cd14 chr18:36884720‐36886308 0.70 2.55 1.29 7.78E‐03Tlr2 chr3:83640193‐83645530 0.38 1.35 1.28 6.48E‐03Cfh chr1:141982431‐142079988 0.17 0.62 1.28 1.47E‐02C1qc chr4:136445716‐136448829 6.74 23.86 1.26 6.37E‐07Tnc chr4:63620818‐63708049 0.09 0.33 1.26 2.42E‐02Gbp7 chr3:142193301‐142213047 0.36 1.25 1.26 4.36E‐04Sla chr15:66502331‐66682282 0.41 1.44 1.25 7.33E‐03Flnc chr6:29383152‐29411888 0.57 2.00 1.25 1.98E‐06Ehd2 chr7:16534335‐16552884 1.14 3.97 1.24 3.45E‐05C3 chr17:57343395‐57367559 3.59 12.37 1.24 1.42E‐09A2m chr6:121586190‐121629256 2.97 10.25 1.24 6.72E‐09Tbxas1 chr6:38868984‐39034578 0.54 1.86 1.23 1.43E‐02Rbp1 chr9:98323379‐98346969 0.58 1.96 1.22 2.88E‐03Rac2 chr15:78389598‐78403213 0.43 1.47 1.22 4.76E‐03Rasa4 chr5:136559785‐136587730 0.95 3.20 1.22 2.45E‐04C4a chr17:34946036‐34960399 1.32 4.45 1.22 5.81E‐07Plek chr11:16871208‐16908721 1.40 4.69 1.21 2.45E‐06Pycard chr7:135134886‐135137381 0.34 1.15 1.21 3.50E‐02Fn1 chr1:71632096‐71699745 0.08 0.27 1.21 4.34E‐02Rrm2 chr12:25393118‐25399011 0.49 1.63 1.21 1.29E‐02Fcer1g chr1:173159702‐173164480 2.65 8.88 1.21 7.03E‐03Akna chr4:63028161‐63064479 0.21 0.70 1.21 7.90E‐03Myof chr19:37973525‐38118067 0.37 1.24 1.21 2.52E‐04Ctsc chr7:95426602‐95459385 0.44 1.47 1.21 1.10E‐02Mylk chr16:34785035‐35002520 0.21 0.71 1.20 2.18E‐03Fmnl3 chr15:99147653‐99200897 0.77 2.57 1.20 8.81E‐05Psmb9 chr17:34319043‐34324275 1.24 4.12 1.20 3.01E‐02Stat6 chr10:127080041‐127103759 0.74 2.44 1.20 1.87E‐02Ly86 chr13:37437213‐37510905 2.20 7.26 1.20 1.46E‐031700084C01Rchr1:171859069‐171864784 3.16 10.44 1.20 2.82E‐05Samd9l chr6:3322256‐3349571 0.26 0.85 1.20 4.06E‐03Gfpt2 chr11:49607656‐49652122 0.58 1.91 1.19 2.57E‐03Gbp3 chr3:142223015‐142236176 0.60 1.96 1.19 3.39E‐03Zfp831 chr2:174469034‐174536331 0.08 0.26 1.18 3.23E‐02C4b chr17:34865325‐34880842 17.61 57.33 1.18 3.28E‐10 YesCebpa chr7:35904311‐35906951 1.86 6.02 1.17 2.87E‐05Prss23 chr7:96656294‐96666096 0.37 1.20 1.17 1.03E‐02Lsp1 chr7:149646774‐149701914 0.95 3.07 1.17 1.29E‐02Ikzf1 chr11:11586215‐11672929 0.21 0.68 1.16 3.98E‐02Igfbp3 chr11:7106095‐7113926 1.49 4.71 1.15 2.32E‐04Oasl2 chr5:115346942‐115362254 1.11 3.50 1.15 1.94E‐04Fos chr12:86814850‐86818219 6.11 19.21 1.15 3.38E‐07Fam111a chr19:12648014‐12671056 0.39 1.22 1.14 2.43E‐02Fcgr3 chr1:172981299‐172989534 1.70 5.34 1.14 1.98E‐03Ccnd1 chr7:152115835‐152125830 7.15 22.34 1.14 8.78E‐09Cyba chr8:124948670‐124956840 2.52 7.80 1.13 9.90E‐03Junb chr8:87500807‐87502647 3.77 11.68 1.13 1.64E‐05Ip6k3 chr17:27280915‐27304709 0.70 2.14 1.11 9.79E‐03Rab32 chr10:10264836‐10278005 1.06 3.21 1.10 3.88E‐03Ccdc88b chr19:6919112‐6932701 0.71 2.15 1.10 5.48E‐04Mocos chr18:24812191‐24860057 0.49 1.47 1.10 1.70E‐02Mafb chr2:160189412‐160192801 1.94 5.74 1.08 4.10E‐05Pgm5 chr19:24748497‐24936332 0.92 2.70 1.08 2.00E‐05Tnfaip3 chr10:18720716‐18735216 0.37 1.10 1.08 2.20E‐02Dbx2 chr15:95453993‐95485202 4.01 11.71 1.07 1.07E‐05Ier5 chr1:156943496‐156946766 1.91 5.58 1.07 6.55E‐05Kank1 chr19:25311691‐25508986 1.90 5.52 1.07 5.70E‐06Renbp chrX:71167459‐71176189 2.98 8.65 1.07 8.55E‐03Nfatc1 chr18:80802943‐80909810 0.59 1.70 1.06 4.31E‐02Afap1l2 chr19:56986843‐57083065 0.75 2.17 1.06 1.98E‐03Plce1 chr19:38598686‐38859590 1.05 3.03 1.06 8.77E‐06Lxn chr3:67234036‐67278990 24.89 71.69 1.06 4.23E‐04Cd44 chr2:102651299‐102741822 0.74 2.12 1.05 1.38E‐03Pros1 chr16:62854159‐62929166 2.13 6.07 1.05 5.73E‐05Ldha chr7:54101173‐54110997 31.21 88.97 1.05 3.63E‐08Cebpb chr2:167514414‐167515918 5.30 14.95 1.04 3.28E‐04Glipr2 chr4:43970573‐43991990 1.30 3.67 1.04 4.94E‐03Naprt1 chr15:75721393‐75724911 3.56 9.90 1.02 1.94E‐04Irgm2 chr11:58028478‐58036285 0.56 1.56 1.02 1.20E‐02Tubb6 chr18:67550384‐67562403 3.93 10.87 1.02 2.26E‐04Pld4 chr12:113998865‐114007197 0.96 2.66 1.02 1.92E‐02Prkcd chr14:31408539‐31439394 4.48 12.38 1.02 1.70E‐05Hk2 chr6:82675020‐82724448 0.95 2.60 1.01 7.48E‐04Cchcr1 chr17:35654060‐35667960 0.92 2.52 1.00 9.68E‐03Lgals3bp chr11:118254065‐118263245 4.08 11.08 1.00 1.03E‐04Olfml3 chr3:103539316‐103541924 1.74 4.71 1.00 5.77E‐03Pdlim4 chr11:53868429‐53882519 3.86 10.39 0.99 2.37E‐03

Fabp7 chr10:57504728‐57508256 11.30 30.32 0.99 2.52E‐04Xdh chr17:74233247‐74299522 0.35 0.93 0.98 3.58E‐02Pygl chr12:71291801‐71328670 1.18 3.17 0.98 4.85E‐03Tmem176a chr6:48791481‐48795363 2.82 7.53 0.98 1.18E‐02Sardh chr2:27043912‐27102823 1.48 3.94 0.98 3.25E‐04Sox6 chr7:122614857‐123182258 0.36 0.97 0.98 4.91E‐03Ampd3 chr7:117916117‐117955909 3.00 7.95 0.97 3.83E‐05Plcd1 chr9:118980645‐119002614 1.66 4.37 0.97 2.45E‐03P4ha3 chr7:107434029‐107468209 1.35 3.56 0.97 6.25E‐03Dock11 chrX:33428826‐33616557 0.77 2.03 0.97 1.08E‐03Steap3 chr1:122122992‐122167659 0.83 2.16 0.96 3.35E‐02Bcar3 chr3:122122697‐122233100 1.25 3.24 0.95 3.44E‐03Papss2 chr19:32670204‐32741677 2.05 5.33 0.95 9.32E‐03Myc chr15:61816895‐61821916 1.17 3.04 0.95 4.59E‐02Aldh1b1 chr4:45811893‐45817480 1.80 4.67 0.95 3.93E‐03Athl1 chr7:148127479‐148133557 1.02 2.61 0.94 9.84E‐03Laptm5 chr4:130469248‐130492063 2.13 5.46 0.94 2.46E‐03Itpkb chr1:182257099‐182353790 3.93 10.08 0.94 4.43E‐05Stard8 chrX:96237919‐96270067 0.36 0.92 0.94 4.00E‐02Bsn chr9:107998352‐108092714 0.67 1.72 0.93 1.35E‐04Slc14a1 chr18:78296829‐78338858 1.54 3.90 0.93 1.99E‐02Bdh2 chr3:134944184‐134967389 2.31 5.84 0.93 4.43E‐02Th chr7:150078680‐150085871 1.68 4.26 0.93 1.54E‐02Zfp36l1 chr12:81208746‐81214000 1.51 3.82 0.93 3.73E‐03Dtx3l chr16:35926600‐35972707 0.54 1.37 0.92 4.06E‐02Mcc chr18:44584713‐44971836 0.32 0.81 0.92 2.30E‐02Tmem176b chr6:48783810‐48791373 8.99 22.41 0.91 2.43E‐03Igsf10 chr3:58810899‐59148178 0.78 1.95 0.91 3.25E‐02Gmip chr8:72332585‐72345769 0.64 1.61 0.91 2.38E‐02Rgs20 chr1:4899656‐5060366 3.83 9.53 0.91 2.73E‐03Anxa2 chr9:69301489‐69339592 18.09 44.87 0.91 2.50E‐05Myo10 chr15:25552304‐25743426 0.94 2.32 0.90 9.53E‐04Tnfrsf1a chr6:125299740‐125312501 1.64 4.02 0.90 1.39E‐02Aldh1l2 chr10:82950191‐82996885 4.72 11.59 0.90 1.57E‐05Prex2 chr1:10983545‐11293763 1.16 2.85 0.90 2.83E‐03Aldh1l1 chr6:90500841‐90549165 11.61 28.47 0.90 1.57E‐05Plcg2 chr8:120022190‐120159042 1.05 2.57 0.90 6.74E‐03Ccnd2 chr6:127075726‐127162437 1.14 2.79 0.89 2.15E‐03Flna chrX:71468799‐71491873 1.08 2.63 0.89 6.78E‐04Hspa1b chr17:35093373‐35096183 21.93 53.42 0.89 9.32E‐03Lrrc16a chr13:24104352‐24372659 1.60 3.88 0.89 1.07E‐03Ifit3 chr19:34658018‐34663472 2.95 7.12 0.88 3.49E‐03Tspo chr15:83394002‐83404633 7.90 19.07 0.88 4.94E‐03Axl chr7:26541518‐26573752 1.27 3.05 0.87 3.41E‐02Coro1a chr7:133843287‐133848268 20.03 48.04 0.87 3.22E‐05I830012O16R chr19:34682446‐34687891 1.82 4.36 0.87 1.43E‐021200009I06Richr12:112655640‐112669394 0.83 1.99 0.87 4.54E‐02Sorbs2 chr8:46593141‐46913260 1.50 3.56 0.86 3.36E‐03Lamb2 chr9:108382192‐108392861 0.81 1.92 0.86 1.06E‐02Ngef chr1:89373403‐89470445 10.17 24.01 0.86 5.89E‐05Baiap3 chr17:25377114‐25393309 10.94 25.81 0.86 3.84E‐04Rusc1 chr3:88887900‐88897285 48.48 114.27 0.86 1.08E‐04Aplnr chr2:84976516‐84980080 0.96 2.24 0.85 2.39E‐02Serpinb1b chr13:33175971‐33186249 37.68 88.27 0.85 2.11E‐05Arhgef26 chr3:62142698‐62266143 1.95 4.52 0.84 1.28E‐03Sbno2 chr10:79519758‐79565447 3.76 8.73 0.84 3.26E‐04Plcb3 chr19:7028202‐7044242 2.94 6.79 0.84 9.26E‐04Mapk8ip2 chr15:89284341‐89292878 12.05 27.89 0.84 5.65E‐05Serpinb9 chr13:33096409‐33109824 1.79 4.13 0.84 5.72E‐03Ddo chr10:40349816‐40369737 1.69 3.90 0.84 1.01E‐02Aldh18a1 chr19:40624746‐40662953 5.67 13.07 0.84 3.75E‐03C77370 chrX:101272774‐101396456 0.32 0.74 0.84 2.12E‐02Ppm1e chr11:87040407‐87172496 7.33 16.79 0.83 6.26E‐05Sox9 chr11:112643523‐112649071 1.16 2.66 0.83 1.41E‐02Dpysl4 chr7:146271899‐146287690 2.08 4.76 0.83 1.06E‐02Ank1 chr8:24085353‐24260968 15.81 35.92 0.82 9.66E‐03Phkg1 chr5:130322865‐130354954 4.10 9.29 0.82 3.67E‐02Fosl2 chr5:32438844‐32460212 0.60 1.36 0.82 3.13E‐02Parp3 chr9:106372683‐106378982 2.63 5.96 0.82 6.57E‐03Acsbg1 chr9:54452803‐54509692 60.01 135.75 0.82 1.42E‐04Marcks chr10:36853048‐36858732 7.63 17.25 0.82 1.27E‐04Ryr2 chr13:11645369‐12199212 1.30 2.93 0.81 4.11E‐04Dapk1 chr13:60703307‐60864547 2.91 6.58 0.81 7.83E‐03Rfx4 chr10:84218792‐84369283 2.32 5.22 0.81 3.01E‐03Acss1 chr2:150443846‐150494668 2.82 6.35 0.81 2.91E‐03Tgfbr2 chr9:115996812‐116084481 1.07 2.40 0.81 2.48E‐02Spna2 chr2:29821079‐29886970 9.69 21.79 0.81 2.10E‐03 YesKctd12 chr14:103375797‐103381854 2.62 5.89 0.81 8.16E‐04Heatr7a chr15:76210942‐76307699 9.67 21.73 0.81 1.66E‐02Chi3l1 chr1:136078980‐136086608 5.76 12.93 0.81 3.82E‐03Acaa2 chr18:74938865‐74965861 8.05 18.04 0.81 2.22E‐03Unc13a chr8:74150611‐74195656 3.98 8.92 0.81 2.06E‐04

Ephx2 chr14:66703213‐66743337 3.54 7.93 0.81 7.31E‐03Gad1 chr2:70327996‐70440069 38.71 86.61 0.81 1.35E‐04Camkv chr9:107838250‐107852022 21.08 47.09 0.80 8.75E‐05Dmxl2 chr9:54212964‐54349433 6.67 14.90 0.80 1.07E‐04Arhgef17 chr7:108018259‐108080675 8.40 18.75 0.80 1.02E‐04Pfkm chr15:97869174‐97962878 127.75 284.88 0.80 5.72E‐03Camk2b chr11:5869668‐5965751 50.72 112.70 0.80 2.15E‐03Sep6 chrX:34450828‐34529690 16.83 37.37 0.80 1.64E‐04Ccdc141 chr2:76847962‐77008692 0.68 1.51 0.80 2.48E‐02Mtap1b chr13:100191418‐100286557 33.15 73.54 0.80 1.26E‐03Fmnl1 chr11:103032451‐103060214 4.13 9.15 0.80 1.58E‐03Ophn1 chrX:95752853‐96086324 1.86 4.11 0.79 7.17E‐03Lrrk2 chr15:91503654‐91646555 0.93 2.05 0.79 7.34E‐03Gna14 chr19:16510156‐16685308 2.07 4.57 0.79 8.45E‐03Plcd3 chr11:102931609‐102962972 1.19 2.62 0.79 4.63E‐02Trim66 chr7:116592514‐116651648 1.38 3.05 0.79 1.99E‐02Cx3cr1 chr9:119957810‐119977400 1.67 3.68 0.79 1.26E‐02Irgm1 chr11:48678750‐48684848 4.13 9.10 0.79 4.91E‐03Sphkap chr1:83252355‐83404775 3.75 8.27 0.79 5.40E‐04Pdzd2 chr15:12286808‐12522311 0.47 1.03 0.79 1.06E‐02Id3 chr4:135699736‐135701307 6.13 13.46 0.79 2.48E‐02Gadd45g chr13:51942043‐51943843 13.34 29.24 0.78 3.28E‐03Chac1 chr2:119176977‐119180062 4.72 10.34 0.78 1.08E‐02Nek6 chr2:38367216‐38443010 4.28 9.38 0.78 5.08E‐03AW551984 chr9:39394980‐39411709 3.99 8.74 0.78 1.80E‐03Csf1r chr18:61265225‐61290793 3.41 7.45 0.78 2.86E‐03Amotl1 chr9:14346410‐14419444 1.68 3.68 0.78 1.46E‐03Kank4 chr4:98421582‐98484228 2.01 4.39 0.78 4.72E‐03Nynrin chr14:56472951‐56493573 1.21 2.64 0.78 5.72E‐03Ass1 chr2:31325789‐31376190 13.62 29.71 0.78 9.02E‐04Spnb1 chr12:77681474‐77811534 1.97 4.28 0.78 8.99E‐04Dock7 chr4:98603355‐98787606 1.87 4.07 0.78 2.96E‐03Plcl2 chr17:50648871‐50827819 4.85 10.57 0.78 8.99E‐04Galm chr17:80526810‐80584372 1.77 3.85 0.77 4.35E‐02Ripk1 chr13:34094742‐34127039 0.97 2.10 0.77 3.17E‐02Pck2 chr14:56159102‐56168854 4.87 10.53 0.77 1.97E‐03Grb10 chr11:11830501‐11937423 6.85 14.79 0.77 2.64E‐03Tbc1d1 chr5:64551449‐64742725 1.78 3.83 0.77 6.06E‐03Myo5a chr9:74919012‐75071494 8.39 18.06 0.77 2.95E‐04Pfkp chr13:6547402‐6647970 54.30 116.91 0.77 1.97E‐03Isyna1 chr8:73118379‐73121189 10.24 22.05 0.77 1.98E‐03Akr1b10 chr6:34334246‐34346949 16.88 36.19 0.76 1.25E‐03Ehd4 chr2:119915222‐119980311 6.45 13.81 0.76 1.16E‐03Egr1 chr18:35020860‐35024610 3.91 8.36 0.76 5.43E‐03Frmpd4 chrX:163909240‐165015165 1.41 3.01 0.76 4.07E‐03Rap1gap chr4:137220640‐137285776 37.77 80.71 0.76 5.29E‐04 YesAldoc chr11:78137699‐78140262 769.71 1643.46 0.76 1.97E‐02Magee2 chrX:102050290‐102052606 2.40 5.12 0.76 2.54E‐02Klhl1 chr14:96504483‐96918253 2.88 6.14 0.76 4.87E‐03Ddah1 chr3:145421655‐145557241 8.06 17.20 0.76 6.40E‐04Frmpd1 chr4:45197777‐45298808 2.14 4.56 0.76 6.64E‐03Drp2 chrX:130939318‐130991112 1.80 3.82 0.75 4.26E‐03Dusp6 chr10:98725864‐98730123 3.02 6.42 0.75 1.07E‐02Rasgrf2 chr13:92020011‐92901449 7.51 15.93 0.75 4.60E‐04Nrp1 chr8:130882972‐131029375 1.17 2.48 0.75 1.49E‐02Asns chr6:7625170‐7643182 35.20 74.60 0.75 4.18E‐04Camk2a chr18:61085285‐61147806 10.88 23.05 0.75 1.72E‐03Mpp3 chr11:101860966‐101888269 3.07 6.51 0.75 1.08E‐02Nefh chr11:4838758‐4942797 105.59 223.50 0.75 1.88E‐02Hecw1 chr13:14318704‐14615493 2.84 5.97 0.74 1.11E‐03Ppp4r4 chr12:104770774‐104852042 4.09 8.57 0.74 3.76E‐03Akap13 chr7:82600419‐82899495 0.48 1.01 0.74 2.38E‐02Pamr1 chr2:102390177‐102483197 1.66 3.48 0.74 4.55E‐02Camsap3 chr8:3587449‐3609075 2.61 5.46 0.74 1.20E‐02Slc25a18 chr6:120723785‐120744000 23.10 48.22 0.74 1.28E‐03Wdr6 chr9:108472222‐108481001 31.71 66.18 0.74 4.76E‐03Plcb1 chr2:134611899‐135300994 5.93 12.36 0.73 1.64E‐02Rimklb chr6:122403626‐122436323 2.53 5.26 0.73 9.22E‐03Cebpd chr16:15887378‐16146926 10.68 22.23 0.73 1.20E‐02Plvap chr8:74021651‐74035668 2.81 5.84 0.73 4.21E‐02Gprin1 chr13:54838033‐54851030 2.49 5.18 0.73 9.90E‐03Arhgap26 chr18:39152798‐39535939 2.37 4.92 0.73 2.58E‐03Usp29 chr7:6683451‐6929373 3.04 6.31 0.73 5.38E‐03Mlc1 chr15:88786313‐88808983 17.19 35.59 0.73 1.05E‐03Cpne5 chr17:29293465‐29374735 2.16 4.47 0.73 1.22E‐02Ampd2 chr3:107876979‐107889545 15.62 32.26 0.72 7.48E‐04Chd5 chr4:151712759‐151764303 6.20 12.80 0.72 9.66E‐03Lgals9 chr11:78776480‐78798426 11.51 23.74 0.72 9.30E‐03Pfkl chr10:77449693‐77472541 36.65 75.56 0.72 8.99E‐04Madd chr2:90977516‐91023204 12.48 25.69 0.72 2.69E‐03 YesSep11 chr5:93522482‐93603984 2.62 5.38 0.72 6.78E‐03Cadps2 chr6:23212773‐23789421 4.02 8.24 0.72 3.07E‐02

Cadps chr14:13205076‐13655593 39.33 80.67 0.72 1.38E‐03Myh9 chr15:77591018‐77672545 3.58 7.34 0.72 1.98E‐03Stxbp5l chr16:37107395‐37385044 2.16 4.43 0.72 7.64E‐03Aqp4 chr18:15547902‐15562193 12.36 25.30 0.72 1.10E‐03Gpr101 chrX:54749844‐54756934 1.16 2.36 0.71 3.40E‐02Abr chr11:76230235‐76391229 29.79 60.59 0.71 1.89E‐03Dzip3 chr16:48924340‐48994225 4.00 8.13 0.71 6.42E‐03Asap3 chr4:135762279‐135802488 1.39 2.83 0.71 2.39E‐02Gad2 chr2:22477846‐22549397 8.38 17.03 0.71 1.34E‐03Wnk2 chr13:49131670‐49243383 4.32 8.78 0.71 2.57E‐03Fndc3b chr3:27315083‐27609361 1.36 2.77 0.71 1.49E‐02Camk2g chr14:21554096‐21613310 76.83 156.04 0.71 2.87E‐02Akap6 chr12:53800369‐54252002 7.61 15.44 0.71 9.48E‐04Nmnat2 chr1:154802230‐154966391 18.00 36.51 0.71 9.45E‐04Rgs12 chr5:35292096‐35376242 2.35 4.76 0.71 2.41E‐02Dlgap1 chr17:70774131‐71170753 4.29 8.68 0.71 1.66E‐02Gm10336 chr13:12274983‐12278755 1.80 3.63 0.70 3.10E‐02Oplah chr15:76124863‐76137675 4.59 9.26 0.70 6.17E‐03Fmn1 chr2:113167892‐113556924 0.56 1.12 0.70 2.82E‐02 YesCkmt1 chr2:121184376‐121206676 206.82 416.62 0.70 3.92E‐03Inpp4a chr1:37356737‐37467580 9.28 18.67 0.70 9.58E‐03Gm15800 chr5:121670227‐121818586 3.79 7.60 0.70 1.31E‐03Alox8 chr11:68997386‐69011341 2.75 5.52 0.70 2.25E‐02Pwwp2b chr7:146434380‐146453152 4.27 8.57 0.70 1.79E‐02Camk1g chr1:195172539‐195196476 11.30 22.66 0.70 3.01E‐03Vgf chr5:137506164‐137509221 18.16 36.37 0.69 2.04E‐03Dnm1 chr2:32163990‐32208824 188.95 378.25 0.69 1.97E‐02Abat chr16:8513521‐8621660 51.77 103.55 0.69 9.66E‐03Fbxo41 chr6:85419571‐85452880 3.51 7.01 0.69 4.32E‐03Lrrc48 chr11:60166881‐60207835 3.72 7.43 0.69 4.63E‐02Btbd11 chr10:84849558‐85123037 8.23 16.44 0.69 3.05E‐031300001I01Richr11:74462996‐74484349 12.84 25.60 0.69 1.58E‐03Fry chr5:151062504‐151300328 2.75 5.48 0.69 3.05E‐03Lrp1 chr10:126975213‐127058204 3.69 7.33 0.69 2.19E‐03Vat1l chr8:116729539‐116897970 41.37 82.22 0.69 2.27E‐03Ap3b2 chr7:88605284‐88638811 28.00 55.65 0.69 1.72E‐03Sgsm1 chr5:113672239‐113739806 10.73 21.31 0.69 9.58E‐03Arhgef40 chr14:52604507‐52640408 4.80 9.52 0.69 1.18E‐02 YesSyn2 chr6:115084919‐115232644 39.17 77.65 0.68 1.99E‐02Dcaf12l1 chrX:42139743‐42143374 7.28 14.44 0.68 6.49E‐03Trrap chr5:145529660‐145620642 1.68 3.33 0.68 5.72E‐03Arhgap20 chr9:51573456‐51683990 2.83 5.61 0.68 7.83E‐03Tbc1d9 chr8:85689250‐85796839 8.71 17.23 0.68 8.96E‐03Klk6 chr7:51079913‐51087397 260.30 514.54 0.68 1.64E‐02Gprasp2 chrX:132373572‐132379269 18.18 35.93 0.68 4.70E‐03Trim9 chr12:71345523‐71448601 9.44 18.65 0.68 1.12E‐02Tspyl3 chr2:153048105‐153051177 2.65 5.23 0.68 3.34E‐02Klhl23 chr2:69660426‐69674708 2.39 4.70 0.68 1.78E‐02Plcb4 chr2:135567565‐135838804 7.25 14.28 0.68 7.67E‐03Pde2a chr7:108570204‐108661343 4.34 8.54 0.68 4.08E‐02Nxn chr11:76070727‐76212643 4.51 8.88 0.68 1.76E‐02Ina chr19:46961320‐47098836 65.82 129.48 0.68 1.36E‐02Cryl1 chr14:57893870‐58017320 5.85 11.49 0.68 4.26E‐02Spon1 chr7:120909511‐121186889 2.99 5.87 0.68 8.55E‐03Plxnb2 chr15:88985978‐89011218 1.96 3.86 0.67 1.27E‐02Pygb chr2:150612531‐150657484 63.36 124.29 0.67 5.13E‐03Lgi4 chr7:31844953‐31861716 5.32 10.44 0.67 1.32E‐02Phlpp2 chr8:112392502‐112468571 3.15 6.17 0.67 5.38E‐03Syne1 chr10:5151833‐5326342 3.65 7.14 0.67 3.06E‐02Lmtk3 chr7:53039316‐53059500 3.88 7.59 0.67 9.58E‐03Ezr chr17:6942479‐6987129 2.77 5.43 0.67 3.59E‐02Reln chr5:21390271‐21850523 1.59 3.11 0.67 9.23E‐03Plec chr15:76001403‐76061808 1.39 2.71 0.67 1.64E‐02 YesUnc80 chr1:66515020‐66745722 6.35 12.38 0.67 3.05E‐03Fam65a chr8:108129128‐108146118 6.03 11.75 0.67 6.30E‐03Nap1l2 chrX:100379397‐100382003 23.78 46.27 0.67 2.46E‐03Pacs1 chr19:5133684‐5273119 6.89 13.37 0.66 5.14E‐03Cap2 chr13:46597271‐46745650 6.43 12.48 0.66 7.19E‐03Snap47 chr11:59220651‐59263458 219.06 423.68 0.66 9.90E‐03Slc8a2 chr7:16715648‐16745860 2.33 4.50 0.66 3.44E‐02Lpin2 chr17:71533317‐71599158 6.67 12.90 0.66 4.18E‐03Chl1 chr6:103460869‐103683029 3.37 6.49 0.66 7.17E‐03Tmem22 chr9:100452606‐100471504 9.59 18.49 0.66 1.79E‐02Efr3a chr15:65618602‐65705374 30.98 59.70 0.66 5.11E‐03Hk1 chr10:61731602‐61842656 47.00 90.51 0.66 6.34E‐03D430041D05Rchr2:103983231‐104250491 1.29 2.48 0.65 2.08E‐02Fxyd7 chr7:31827533‐31836473 71.37 137.02 0.65 5.03E‐03Plin2 chr4:86302468‐86315963 6.42 12.31 0.65 2.82E‐02Ccrn4l chr3:51028368‐51055576 10.18 19.47 0.65 6.37E‐03Ubr4 chr4:138936573‐139045447 5.14 9.82 0.65 4.18E‐03Mcf2 chrX:57309132‐57400820 2.56 4.88 0.65 4.09E‐02Raph1 chr1:60540028‐60623609 1.38 2.63 0.65 2.36E‐02

Ecel1 chr1:89044229‐89051602 11.86 22.61 0.65 7.34E‐03Gucy1a3 chr3:81896348‐81949799 5.29 10.09 0.65 9.21E‐03Unc79 chr12:104187068‐104422207 1.08 2.05 0.64 4.09E‐02Capn1 chr19:5988544‐6018459 11.84 22.54 0.64 6.30E‐03Klhl13 chrX:22796396‐22942179 13.77 26.21 0.64 4.95E‐03Tanc2 chr11:105451299‐105790617 3.01 5.72 0.64 5.73E‐03Anxa6 chr11:54792463‐54846973 57.16 108.70 0.64 3.92E‐03Midn chr10:79611034‐79621112 4.78 9.08 0.64 1.75E‐02Trio chr15:27660403‐27955603 2.05 3.89 0.64 1.07E‐02Col16a1 chr4:129725083‐129776521 1.83 3.47 0.64 4.44E‐02Cpne4 chr9:104472117‐104936874 3.36 6.37 0.64 2.94E‐02Iqsec3 chr6:121322950‐121423696 8.43 15.98 0.64 4.76E‐03Ralgapa1 chr12:56703885‐56922154 7.06 13.35 0.64 2.94E‐02Jak1 chr4:100741642‐100937887 27.10 51.22 0.64 7.64E‐03Pnck chrX:70901330‐70905409 41.18 77.53 0.63 2.20E‐02Heatr1 chr13:12487641‐12531160 1.49 2.81 0.63 4.30E‐02Ptk2b chr14:66772093‐66899889 3.77 7.09 0.63 3.38E‐02Dfna5 chr6:50157401‐50211768 5.49 10.30 0.63 3.97E‐02Stk10 chr11:32433265‐32524595 2.20 4.13 0.63 4.03E‐02Nkrf chrX:34427536‐34442894 11.65 21.87 0.63 7.31E‐03Pde1b chr15:103333728‐103360483 30.86 57.88 0.63 5.38E‐03Tubb2a chr13:34166146‐34169877 147.93 277.48 0.63 9.28E‐03Pygm chr19:6384428‐6398459 19.99 37.47 0.63 6.58E‐03Ahi1 chr10:20672352‐20800235 56.07 105.06 0.63 4.37E‐02Hif1a chr12:75008853‐75048517 15.44 28.94 0.63 6.10E‐03Hexb chr13:97907891‐97968312 25.68 48.11 0.63 2.64E‐02Sall2 chr14:52930851‐52948345 7.48 13.99 0.63 9.63E‐03Arhgef19 chr4:140798798‐140813477 4.21 7.86 0.62 3.29E‐02Wdr7 chr18:63868348‐64149413 21.08 39.25 0.62 8.73E‐03Ttc3 chr16:94580369‐94690828 62.20 115.78 0.62 3.63E‐02Arhgef6 chrX:54484661‐54591906 2.97 5.53 0.62 3.35E‐02Mast1 chr8:87435751‐87461252 21.41 39.83 0.62 7.03E‐03Wdr35 chr12:8980806‐9035653 7.78 14.47 0.62 3.34E‐02Camta1 chr4:150433631‐151235877 24.15 44.91 0.62 4.33E‐02Htt chr5:35104388‐35255170 2.70 5.02 0.62 9.30E‐03Plk2 chr13:111185251‐111191051 5.67 10.51 0.62 2.82E‐02Dock3 chr9:106795155‐107134240 7.67 14.22 0.62 7.17E‐03Lasp1 chr11:97660985‐97700078 6.27 11.62 0.62 1.86E‐02Astn1 chr1:160292434‐160621917 5.72 10.58 0.62 1.64E‐02Dync1i1 chr6:5675638‐5978039 41.49 76.79 0.62 2.91E‐02Magee1 chrX:102315734‐102319250 41.82 77.36 0.62 8.07E‐03Grip2 chr6:91711502‐91757387 2.97 5.49 0.61 3.17E‐02Cacna1i chr15:80117667‐80228722 1.85 3.42 0.61 2.50E‐02Tbc1d4 chr14:101841576‐102008408 3.01 5.56 0.61 2.26E‐02Nbea chr3:55429119‐55987623 5.37 9.91 0.61 1.48E‐02Mapk4 chr18:74088140‐74224603 4.06 7.50 0.61 2.42E‐02Rap1gap2 chr11:74196984‐74403660 8.48 15.66 0.61 9.03E‐03Arrb1 chr7:106683995‐106755281 13.44 24.80 0.61 9.32E‐03Flot1 chr17:35960301‐35969732 83.49 153.98 0.61 7.82E‐03Bhlhb9 chrX:132420389‐132425620 5.87 10.82 0.61 2.82E‐02Kcnab3 chr11:69139759‐69146543 35.22 64.89 0.61 7.25E‐03Gmpr chr13:45602837‐45641750 42.37 77.98 0.61 7.67E‐03Tuba4a chr1:75207403‐75215828 210.81 387.95 0.61 4.40E‐02Ica1 chr6:8580526‐8728484 14.14 26.01 0.61 3.55E‐02Ets2 chr16:95924013‐95942656 5.25 9.66 0.61 2.66E‐02Adcy8 chr15:64530596‐64753858 2.91 5.34 0.61 3.92E‐02Zcchc12 chrX:33735898‐33739153 16.68 30.53 0.60 1.23E‐02Ctnnd2 chr15:30102347‐30959098 7.12 13.03 0.60 1.06E‐02Mtus2 chr5:148768895‐149127641 2.29 4.19 0.60 2.97E‐02Ttc39b chr4:82866204‐82970093 2.94 5.37 0.60 1.87E‐02Gria1 chr11:56825119‐57143746 9.15 16.69 0.60 9.28E‐03Gucy1b3 chr3:81835925‐81878633 14.82 27.03 0.60 9.21E‐03Cpne9 chr6:113232300‐113255565 5.38 9.81 0.60 4.08E‐02Ctsd chr7:149561820‐149573775 229.54 417.76 0.60 3.38E‐02Apba2 chr7:71646591‐71898762 21.12 38.43 0.60 1.06E‐02Bbs4 chr9:59169772‐59201315 19.25 35.01 0.60 1.08E‐02Plxna4 chr6:32094558‐32538192 2.10 3.82 0.60 2.07E‐02Impdh1 chr6:29150439‐29162271 19.00 34.51 0.60 1.21E‐02Myh10 chr11:68505416‐68630126 13.23 24.01 0.60 1.24E‐02Pgm2l1 chr7:107376116‐107427382 6.89 12.48 0.59 1.31E‐02Zfyve28 chr5:34537542‐34630973 5.23 9.46 0.59 2.96E‐02Cgnl1 chr9:71474315‐71619409 6.28 11.35 0.59 1.40E‐02Hspa4l chr3:40549534‐40594287 37.75 68.17 0.59 1.46E‐02Ipo4 chr14:56244465‐56254515 28.54 51.51 0.59 1.19E‐02Lcp1 chr14:75530929‐75630649 7.91 14.27 0.59 4.56E‐02Sarm1 chr11:78285831‐78311256 3.12 5.63 0.59 4.97E‐02Fam40a chr3:107415449‐107434628 22.57 40.66 0.59 1.09E‐02Efr3b chr12:3962553‐4038915 20.47 36.82 0.59 1.42E‐026330439K17Rchr2:144296292‐144353134 22.90 41.19 0.59 1.54E‐02Spnb3 chr19:4711222‐4752352 2.18 3.91 0.59 3.81E‐02Rundc3b chr5:8490335‐8622952 5.71 10.28 0.59 3.05E‐02Rab3c chr13:110844395‐111070414 10.94 19.66 0.59 1.32E‐02

Cad chr5:31357183‐31380852 2.30 4.13 0.59 4.62E‐023830431G21Rchr12:81793587‐81825203 4.88 8.76 0.59 4.08E‐02Synj1 chr16:90936341‐91044624 25.17 45.18 0.58 2.94E‐02Dgkb chr12:38607291‐39359997 3.12 5.59 0.58 4.35E‐02Pnmal2 chr7:17530030‐17534177 54.00 96.74 0.58 2.07E‐02Apeh chr9:107987744‐107996811 13.73 24.60 0.58 2.32E‐02Ube2o chr11:116399066‐116442761 24.76 44.32 0.58 1.49E‐02Rab3b chr4:108551674‐108644682 9.72 17.39 0.58 2.09E‐02Crmp1 chr5:37633318‐37683372 23.97 42.88 0.58 1.36E‐02Pitpnm2 chr5:124568698‐124666427 3.38 6.05 0.58 3.34E‐02Abcd2 chr15:90976301‐91022238 4.77 8.53 0.58 2.55E‐02Atp6v1a chr16:44085516‐44139132 94.46 168.90 0.58 3.86E‐02Pde4d chr13:109444370‐110746177 2.84 5.08 0.58 3.40E‐02Tbc1d30 chr10:120700875‐120748245 7.61 13.60 0.58 1.78E‐02Ppfia3 chr7:52594495‐52625934 10.32 18.42 0.58 4.03E‐02Hspa12a chr19:58870240‐58935474 32.06 57.20 0.58 2.04E‐02Phyhip chr14:70857323‐70868631 28.76 51.28 0.58 1.34E‐02Gbf1 chr19:46227047‐46361000 15.27 27.19 0.58 1.49E‐02Actl6b chr5:137994782‐138010801 13.28 23.61 0.58 4.24E‐02Ranbp6 chr19:29882597‐29887464 10.50 18.67 0.58 1.75E‐02Kif3a chr11:53380880‐53417746 30.30 53.80 0.57 2.00E‐02Hspb8 chr5:116858503‐116872873 12.73 22.57 0.57 3.61E‐02Dlg3 chrX:97963061‐98013749 7.49 13.28 0.57 4.36E‐02Plekha6 chr1:135142673‐135200012 8.21 14.55 0.57 3.23E‐02Ppp1r3c chr19:36806220‐36811094 7.06 12.50 0.57 4.43E‐02Eno1 chr4:149611305‐149622982 74.70 132.17 0.57 2.94E‐02Luzp1 chr4:136025675‐136105233 4.38 7.74 0.57 2.36E‐02Uhrf1bp1 chr17:27993451‐28036985 2.82 4.98 0.57 3.41E‐02Klhl22 chr16:17759713‐17793475 16.39 28.94 0.57 2.25E‐02Herc1 chr9:66198256‐66362400 2.01 3.55 0.57 3.73E‐02Cyfip2 chr11:46007350‐46126361 48.37 85.37 0.57 4.33E‐02Spnb4 chr7:28141401‐28231608 5.72 10.10 0.57 3.57E‐02Pitpnm1 chr19:4100116‐4113965 17.21 30.38 0.57 1.70E‐02Snph chr2:151416285‐151458269 18.92 33.31 0.57 1.70E‐02Rgl1 chr1:154364659‐154472241 7.82 13.76 0.56 3.13E‐02Lonrf2 chr1:38851353‐38878060 14.63 25.67 0.56 2.40E‐02Stmn3 chr2:181041163‐181049205 346.39 607.23 0.56 3.86E‐02Hivep2 chr10:13686184‐13871184 2.21 3.86 0.56 4.38E‐02Ogfod1 chr8:96561097‐96591822 13.60 23.73 0.56 3.17E‐02L1cam chrX:71099118‐71126173 12.90 22.49 0.56 2.39E‐02Nckipsd chr9:108710710‐108720697 32.82 57.16 0.55 2.10E‐02Plxna2 chr1:196446022‐196643062 2.15 3.75 0.55 4.43E‐02Stxbp5 chr10:9475344‐9620838 5.31 9.24 0.55 2.52E‐02Megf8 chr7:26102182‐26150936 7.04 12.21 0.55 2.48E‐02Hpcal4 chr4:122860746‐122871942 5.63 9.74 0.55 4.54E‐02Gdap1l1 chr2:163264202‐163281060 16.97 29.37 0.55 2.97E‐02Slc25a22 chr7:148615647‐148623773 33.40 57.76 0.55 3.34E‐02BC037034 chr5:138700885‐138713982 18.50 31.98 0.55 3.73E‐02Camkk1 chr11:72832509‐72855567 33.31 57.46 0.55 2.55E‐02Plxna1 chr6:89266307‐89312607 3.68 6.35 0.54 3.87E‐02Abcg4 chr9:44081272‐44096327 8.68 14.95 0.54 4.33E‐02Pank4 chr4:154338241‐154355047 12.08 20.78 0.54 4.54E‐02Rab3gap1 chr1:129765349‐129840453 18.28 31.27 0.54 3.08E‐02Herc2 chr7:63305524‐63487168 4.08 6.96 0.53 3.23E‐02Mtor chr4:147822690‐147931794 5.83 9.94 0.53 3.60E‐02Clu chr14:66587319‐66600382 122.25 207.76 0.53 4.57E‐02Ndn chr7:69493162‐69494813 114.72 194.11 0.53 4.15E‐02

Cnih4 chr1:183081061‐183099125 17.46 10.33 ‐0.52 4.46E‐02Golga7 chr8:24351797‐24367552 149.78 88.33 ‐0.53 3.58E‐02Nxt2 chrX:138662479‐138674235 66.98 39.49 ‐0.53 2.97E‐021190002N15Rchr9:94418282‐94438500 13.38 7.89 ‐0.53 4.54E‐02Nipal4 chr11:45961656‐45979861 35.59 20.96 ‐0.53 3.23E‐02Dixdc1 chr9:50470857‐50536089 51.85 30.47 ‐0.53 4.15E‐021810037I17Richr3:122627314‐122629112 123.66 72.66 ‐0.53 3.65E‐02Tmem125 chr4:118213545‐118216331 154.95 90.97 ‐0.53 3.67E‐02St6galnac3 chr3:152865472‐153388097 23.98 14.06 ‐0.53 3.06E‐02H2afv chr11:6327228‐6344446 40.92 23.91 ‐0.54 3.93E‐02Sft2d1 chr17:8503967‐8520307 56.56 33.04 ‐0.54 4.38E‐02Grm3 chr5:9485235‐9725352 33.42 19.48 ‐0.54 2.57E‐02Aven chr2:112333120‐112471410 75.18 43.75 ‐0.54 3.78E‐02 YesRnf130 chr11:49838832‐49918235 245.83 143.03 ‐0.54 3.97E‐02Tmeff2 chr1:50984366‐51244114 106.66 62.03 ‐0.54 3.92E‐02BC005537 chr13:24893525‐24904768 48.88 28.42 ‐0.54 2.48E‐02Tnfaip6 chr2:51893632‐51912201 81.38 47.24 ‐0.54 2.65E‐02Ttyh2 chr11:114536781‐114582298 148.86 86.19 ‐0.55 4.88E‐02Gramd3 chr18:56591785‐56663446 28.49 16.48 ‐0.55 2.94E‐02Vma21 chrX:69062117‐69070045 15.56 9.00 ‐0.55 2.96E‐02Adi1 chr12:29360071‐29367039 240.13 138.82 ‐0.55 4.08E‐02Map6d1 chr16:20233381‐20241431 89.46 51.71 ‐0.55 2.95E‐02Tmem98 chr11:80623916‐80635535 122.51 70.79 ‐0.55 2.41E‐02Tspan14 chr14:41719732‐41780096 52.81 30.51 ‐0.55 2.54E‐02

Genes downregulated in SOD1 G37R oligodendrocytes

Lhfpl2 chr13:94827750‐94965364 15.68 9.06 ‐0.55 3.67E‐024732418C07Rchr4:115410677‐115449932 75.58 43.62 ‐0.55 3.29E‐02Usp30 chr5:114550341‐114572933 59.46 34.29 ‐0.55 2.30E‐022810407C02Rchr3:58380579‐58397466 70.81 40.83 ‐0.55 2.62E‐022810468N07Rchr17:25707755‐25711988 336.95 194.06 ‐0.55 3.92E‐02Cab39l chr14:60059817‐60167740 47.20 27.18 ‐0.55 2.88E‐022900064A13Rchr2:112295181‐112307593 140.65 80.90 ‐0.55 2.23E‐02Ninj2 chr6:120043397‐120150356 66.80 38.41 ‐0.55 4.33E‐02Ptpra chr2:130276013‐130389485 64.68 37.18 ‐0.55 3.34E‐022610001J05Richr6:13819073‐13821483 44.15 25.36 ‐0.55 2.48E‐02Ube2d1 chr10:70717727‐70748010 230.50 132.33 ‐0.55 2.98E‐02Tmem159 chr7:127245939‐127264500 37.21 21.36 ‐0.56 3.65E‐02H47 chr7:73224534‐73234291 73.99 42.36 ‐0.56 2.40E‐02Sys1 chr2:164286470‐164291010 113.39 64.86 ‐0.56 2.36E‐02Hhip chr8:82489749‐82581907 5.65 3.23 ‐0.56 2.64E‐02Cisd2 chr3:135069375‐135086397 23.29 13.27 ‐0.56 2.15E‐02Fbxo7 chr10:85484673‐85511073 176.76 100.71 ‐0.56 2.52E‐02Pde8a chr7:88358689‐88478508 52.45 29.75 ‐0.57 1.55E‐02Rnf7 chr9:96371375‐96379014 303.98 172.31 ‐0.57 2.62E‐02Slc25a27 chr17:43778848‐43803964 53.57 30.36 ‐0.57 1.84E‐02Ldlrad3 chr2:101790357‐102026534 18.90 10.68 ‐0.57 1.97E‐02Cyp20a1 chr1:60400215‐60444231 26.62 15.03 ‐0.57 3.34E‐02Ptpn13 chr5:103854210‐104027380 4.20 2.37 ‐0.57 3.53E‐02Ddc chr11:11714103‐11798147 65.41 36.88 ‐0.57 1.50E‐02Slc38a2 chr15:96517822‐96530129 139.04 78.25 ‐0.57 4.85E‐02Ctnnbip1 chr4:148892349‐148940546 35.22 19.78 ‐0.58 3.57E‐02Nkd1 chr8:91045242‐91118786 34.73 19.49 ‐0.58 1.61E‐02Leprel4 chr11:100270063‐100276133 57.81 32.44 ‐0.58 1.54E‐02Snapc5 chr9:64027103‐64030495 82.62 46.33 ‐0.58 2.44E‐02Tgfa chr6:86145244‐86225443 49.56 27.73 ‐0.58 1.38E‐02Olfml1 chr7:114710946‐114734879 46.12 25.79 ‐0.58 1.40E‐02Sypl chr12:33638809‐33664367 91.73 51.15 ‐0.58 1.78E‐02Tspan15 chr10:61648143‐61693966 49.48 27.56 ‐0.59 1.26E‐02Serinc5 chr13:93381092‐93481901 64.66 35.97 ‐0.59 1.66E‐02Trim59 chr3:68839215‐68848664 77.21 42.92 ‐0.59 1.54E‐02Gm410 chr3:57471343‐57496459 25.33 14.06 ‐0.59 2.43E‐02Hopx chr5:77516010‐77544148 388.22 214.38 ‐0.59 3.06E‐02Nipa1 chr7:63217900‐63274943 116.81 64.50 ‐0.59 1.54E‐02Klf9 chr19:23215715‐23241401 45.95 25.36 ‐0.59 1.27E‐02Gm15663 chr10:105011606‐105020926 8.37 4.62 ‐0.59 3.81E‐02Tmem123 chr9:7764076‐7794332 28.71 15.82 ‐0.60 1.09E‐02Cyp2j12 chr4:95766008‐95807843 29.52 16.26 ‐0.60 1.43E‐02Reep3 chr10:66471936‐66559736 25.90 14.26 ‐0.60 9.79E‐03Sox2ot chr3:34459302‐34576915 168.98 93.02 ‐0.60 3.34E‐02Dpy19l1 chr9:24216224‐24307584 113.79 62.59 ‐0.60 2.25E‐02Tmem144 chr3:79617074‐79646584 25.87 14.23 ‐0.60 1.34E‐02Nkain2 chr10:31409124‐32609721 74.98 41.14 ‐0.60 2.39E‐02Snhg11 chr2:158201373‐158211881 9.18 5.02 ‐0.60 1.49E‐02Ptch1 chr13:63612840‐63666828 5.36 2.93 ‐0.60 4.67E‐02Ctsk chr3:95303207‐95313284 27.68 15.12 ‐0.60 2.39E‐02Gstm7 chr3:107729251‐107734663 557.23 304.21 ‐0.61 2.54E‐02Palm2 chr4:57581119‐57730000 8.91 4.86 ‐0.61 1.10E‐02Piga chrX:160857718‐160871847 16.18 8.80 ‐0.61 1.06E‐02Mfsd2a chr4:122624093‐122638431 17.87 9.67 ‐0.61 2.02E‐02Lpar1 chr4:58448123‐58566363 72.25 38.85 ‐0.62 6.18E‐03Etv1 chr12:39506844‐39594802 76.97 41.13 ‐0.63 1.23E‐02Mar1 chr8:68141938‐68995536 9.85 5.23 ‐0.63 1.20E‐02Sspn chr6:145882666‐145913745 11.95 6.34 ‐0.63 7.64E‐03Zcchc24 chr14:26531125‐26588342 94.02 49.87 ‐0.63 9.82E‐03Fam57a chr11:76015557‐76021759 39.13 20.73 ‐0.64 1.88E‐02Apln chrX:45378322‐45388029 36.07 19.06 ‐0.64 4.91E‐03Rab31 chr17:66001065‐66122092 275.88 145.51 ‐0.64 3.10E‐02Pcolce2 chr9:95538046‐95595970 83.93 44.13 ‐0.64 5.10E‐03Fktn chr4:53727053‐53776143 9.10 4.73 ‐0.66 1.37E‐02Insig1 chr5:28397951‐28405202 55.09 28.58 ‐0.66 3.85E‐03Dnajc24 chr2:105806864‐105843706 45.62 23.62 ‐0.66 7.17E‐03Zfp536 chr7:38264127‐38554771 5.10 2.64 ‐0.66 3.23E‐02Gm13375 chr2:20890500‐20891975 40.36 20.83 ‐0.66 7.89E‐039330117O12 chr18:54771397‐54799524 13.05 6.72 ‐0.66 1.70E‐02Phactr3 chr2:177853679‐178073197 33.67 17.27 ‐0.67 1.41E‐02Thap2 chr10:114807021‐114821491 4.91 2.51 ‐0.67 2.94E‐02Tmeff1 chr4:48598064‐48676003 205.85 105.29 ‐0.67 6.18E‐03C630043F03Rchr4:71862277‐71864964 9.44 4.82 ‐0.67 1.54E‐02Shisa4 chr1:137268032‐137271640 387.95 196.85 ‐0.68 3.76E‐03Ptn chr6:36665662‐36761361 87.34 44.03 ‐0.69 1.72E‐03Pon2 chr6:5214623‐5248373 63.03 31.58 ‐0.69 1.38E‐03Crip1 chr12:114390222‐114392090 65.88 32.42 ‐0.71 2.21E‐02C030030A07Rchr6:136777363‐136789075 83.17 39.77 ‐0.74 1.15E‐03Itgad chr7:135317459‐135349880 3.60 1.71 ‐0.74 3.44E‐02Bdkrb2 chr12:106801381‐106831281 5.87 2.72 ‐0.77 4.63E‐02Prrg1 chrX:75694951‐75829235 12.89 5.97 ‐0.77 4.63E‐04Rnf141 chr7:117960048‐117987895 43.95 20.30 ‐0.77 1.24E‐03

Scd3 chr19:44277777‐44318506 13.19 6.09 ‐0.77 8.29E‐04Lefty1 chr1:182865169‐182868532 9.99 4.58 ‐0.78 1.46E‐02Fam171b chr2:83652884‐83721515 33.04 15.09 ‐0.78 2.28E‐04Sgk3 chr1:9788210‐9892649 28.38 12.95 ‐0.78 1.38E‐03Gpr98 chr13:81234066‐81772143 1.26 0.57 ‐0.79 2.45E‐03Gjc3 chr5:138395036‐138404187 131.50 58.56 ‐0.81 1.99E‐04Miat chr5:112642247‐112657968 2.01 0.89 ‐0.81 3.99E‐03Scd1 chr19:44468939‐44482199 101.25 43.67 ‐0.84 1.11E‐04Expi chr11:83522505‐83524862 50.19 21.34 ‐0.86 3.29E‐03A230001M10 chr3:102066327‐102092694 38.30 15.58 ‐0.90 1.30E‐05Arid5b chr10:67558340‐67741474 8.50 3.42 ‐0.91 9.51E‐05Pla2g3 chr11:3388229‐3394169 15.57 6.01 ‐0.95 3.39E‐05Meg3 chr12:110779205‐110809939 9.96 3.76 ‐0.97 2.30E‐021500015L24R chr19:20479777‐20497275 10.58 3.89 ‐1.00 9.79E‐03Gabrr2 chr4:33150086‐33182840 6.56 1.78 ‐1.30 3.59E‐05Ubtf chr11:102165873‐102180410 119.03 27.50 ‐1.47 3.36E‐03

Supplementary Table 8: Main functional categories enriched in SOD1G37R induced gene changes in oligodendrocytes at early symptomatic stage.

GO categories GO term Benjamini

Upregulated genes Cellular

component GO:0005886 plasma membrane 5.28E-07

Molecular function

GO:0030695 GTPase regulator activity 9.79E-12

GO:0046578 regulation of Ras protein signal transduction 1.29E-05

GO:0005089 Rho guanyl-nucleotide exchange factor activity 3.14E-04

GO:0004435 phosphoinositide phospholipase C activity 1.23E-06

GO:0006955 Immune response 2.11E-06 GO:0032555 purine ribonucleotide binding 3.32E-08 GO:0003779 actin binding 1.44E-04

Downregulated genes Cellular

component GO:0031224 intrinsic to membrane 0.00285104  

Supplementary Table 9: Overlaps of gene expression changes in SOD1 G37R motor neurons, astrocytes and oligodendrocytes at disease onset.Overlaps between motor neurons and astrocytes Overlaps between motor neurons and oligodendrocytes Overlaps between motor neurons and oligodendrocytes Overlaps among all three cell types

Anxa3 C4b Arl4d Lgals3C1qa Lgals3 Aven Sgk1C1qb Rap1gap Fam123a VimC1qc Sgk1 Lgals3

Cdkn1a Vim NfkbiaCtss Pla2g4e

Inpp5d Serpina3nLgals3 Sgk1Lyz2 Socs3Nupr1 VimPrkd3

Rasgrp3Sgk1

Tagln2Timp1Trem2Txnip

TyrobpVim

Supplementary Table 10: Primer sequencesGene name Forward primer Reverse primerhuSOD1 GTAATGGACCAGTGAAGGTGTGG AGTGAGGACCTGCACTGGTACAmSOD1 GCAAGCGGTGAACCAGTTGTGT ATGAGGTCCTGCACTGGTACAGDnaja2 TGTGCTCCGACTGTAATGGAG CTTCCCCAGTGAACGTAATCCRpl23 ACAGACTTCCTGCTGCTGGTG GACCCCTGCGTTATCTTCAAAGATF4 TGGATGATGGCTTGGCCAGTG GAGCTCATCTGGCATGGTTTC Chop CTGCCTTTCACCTTGGAGACG CTTTGGGATGTGCGTGTGACCBip/Grp78 GTGCAGCAGGACATCAAGTTC TACGCCTCAGCAGTCTCCTTCXbp1 (qPCR) AAGCGCTGCGGAGGAAACTG GCCGTGAGTTTTCTCCCGTAAXbp1 (splicing) GGGAATGGACACGCTGGATCC GTCCATGGGAAGATGTTCTGGPDI GTTGCCCAAGGAGGACTGTAG TATAGCGCTGGCCAAAGAACGp62 AGGGAACACAGCAAGCTCATC TGACTCAGCTGTAGGGCAAGGCxcl10 TGGGTCTGAGTGGGACTCAAG CGTGGCAATGATCTCAACACGIgfbp7 GGAAAATCTGGCCATTCAGACC TTCATGGAGGGCATCAACCACCadps CTGCAGCTCCACATTTACCA GACGCTGTAGCTTCCTCCACRusc1 CCCCCAACAGTAAGTGTGCT GCAGTGTGGTCACAGAGAGCTmeff1 GCAGCCATTATTGGAGCAGT ATATAAAAGTCCGCGGCTCANkain2 TATGGATACCAAGGGCCTCA GAGTCCATGGGCTGACAGAEGFP CCTACGGCGTGCAGTGCTTC CGAGCTGCACGCTGCCGTCCβ-actin AGAGGGAAATCGTGCGTGAC CAATAGTGATGACCTGGCCGT