Translational profiling identifies a cascade of damage PNAS PLUS initiated in motor neurons and spreading to glia in mutant SOD1-mediated ALS

Shuying 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, University of California at San Diego, La Jolla, CA 92093; cThe Research Institute at Nationwide Children’s Hospital, Department of Neuroscience, The Ohio State University, 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)- function, endoplasmic reticulum (ER) stress, axonal transport causing mutations in dismutase 1 (SOD1) provokes defects, excessive production of extracellular superoxide, and ox- noncell autonomous paralytic . By combining ribosome idative damage from aberrantly secreted mutant SOD1 (reviewed affinity purification and high-throughput sequencing, a cascade of in ref. 12). What damage occurring during the course of disease mutant SOD1-dependent, cell type-specific changes are now iden- is accumulated within motor neurons, astrocytes, or oligoden- tified. Initial mutant-dependent damage is restricted to motor drocytes remains unknown, however. neurons and includes synapse and metabolic abnormalities, endo- Previous attempts to analyze expression changes caused plasmic reticulum (ER) stress, and selective activation of the PRKR- by mutant SOD1 within defined cell populations in the central like ER kinase (PERK) arm of the unfolded response. PERK nervous system (CNS) have relied on the physical enrichment of activation correlates with what we identify as a naturally low level target cell populations, with either laser-capture microdissection of ER chaperones in motor neurons. Early changes in astrocytes (13–17) or fluorescence-activated cell sorting (18). These ap- occur in that are involved in inflammation and metabolism and are targets of the peroxisome proliferator-activated and proaches have clear disadvantages, however, including cross- X receptor transcription factors. Dysregulation of myelination contamination from neighboring cells/environments; isolation of and lipid signaling pathways and activation of ETS transcription fac- RNAs only from neuronal cell bodies but not dendrites, axons, tors occur in oligodendrocytes only after disease initiation. Thus, path- or synapses; and artifacts introduced during cellular isolation ogenesis involves a temporal cascade of cell type-selective damage procedures. In addition, becaue of technical limitations, most initiating in motor neurons, with subsequent damage within glia driving disease propagation. Significance

ALS | SOD1 | cell type selective toxicity | bacTRAP | RNA profiling Amyotrophic lateral sclerosis can be caused by a mutation in . Ubiquitously expressed, disease mech- myotrophic lateral sclerosis (ALS) is an adult-onset neuro- anism involves damage within motor neurons (whose de- Adegenerative disease with loss of upper and lower motor generation is responsible for progressive paralysis) and glia. By neurons that leads to fatal paralysis, with a typical disease course combining ribosome affinity purification from each of three cell of 1–5 y (1). Dominant mutations in the Cu/Zn superoxide dis- types, a temporal cascade of damage is identified that initiates mutase (SOD1) gene (2) account for 20% of familial ALS. Anal- within motor neurons, with subsequent damage within glia NEUROSCIENCE ysis of chimeric mice comprised of mixtures of wild type and driving disease propagation. Mutant-dependent damage to mutant-expressing cells (3, 4) and use of cell type-selective ex- motor neurons, which are shown to express very low levels of cision of ubiquitously expressed SOD1 mutant (5–9) endoplasmic reticulum chaperones, includes synapse and met- have established that disease pathogenesis is noncell autono- abolic abnormalities and selective activation of the PERK arm mous, a mechanistic feature that is likely to be common to many of the unfolded protein response. Early changes in astrocytes neurological disorders (10). Mutant SOD1 in motor neurons are to genes involved in inflammation and metabolism, while accelerates disease onset, but does not affect the rate of disease dysregulation of myelination and lipid signaling pathways in progression (5, 7, 8). Mutant synthesis by neighboring glial cells, oligodendrocytes occurs only after disease initiation. especially astrocytes (8) and microglia (5), has been shown to ac- celerate disease progression (5, 8). Mutant SOD1 gene inactivation Author contributions: S.S. and D.W.C. designed research; S.S., S.-C.L., L.F., M.M.-D., Y.Z., + in NG2 oligodendrocyte progenitors, but not in already matured K.D., Y.W., D.D., S.T., and A.K. performed research; B.K.K. and C.L.-T. contributed new reagents/analytic tools; S.S. and Y.S. analyzed data; and S.S. and D.W.C. wrote the paper. oligodendroctyes, of adult mice has been reported to delay the age of disease onset (11). Mutant synthesis in as-yet unidentified cell The authors declare no conflict of interest. types beyond motor neurons and oligodendrocytes also drives the Freely available online through the PNAS open access option. onset of disease in ALS mice, as demonstrated by delayed initiation Data deposition: The raw RNA-seq data have been deposited in the of disease in mice in which all motor neurons and oligodendrocytes Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE74724). 1 are mutant-expressing but variable proportions of other cell types Present address: Department of Physiology, National University of Singapore, Singapore 117549. express mutant SOD1 (4). 2Present address: Sheffield Institute for Translational Neuroscience, University of Sheffield, Two key questions in understanding the pathogenic mecha- Sheffield S10 2TN, United Kingdom. nisms of ALS are what causes the selective degeneration of 3Present address: MassGeneral Institute for Neurodegenerative , Department of motor neurons from a widely expressed mutant gene and what Neurology, Massachusetts General Hospital, Charlestown, MA 02129. genetic regulators of aging influence late-onset disease. Multiple 4To whom correspondence should be addressed. Email: [email protected]. pathways for toxicity of mutant SOD1 have been implicated, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. including misfolded protein triggering abnormal mitochondrial 1073/pnas.1520639112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1520639112 PNAS | Published online November 30, 2015 | E6993–E7002 previous studies have focused on motor neurons alone or a selective activation of the protein kinase RNA-like endoplasmic mixture of cells from white matter rather than on individual glial reticulum kinase or PRKR-like ER kinase (PERK) arm of the cell types. Furthermore, most previous work used transgenic unfolded protein response (UPR), to start within motor neurons, mice with a highly accelerated disease course from a very high followed by dysregulation of metabolic and inflammatory genes degree of overexpression of mutant SOD1, which in the most in astrocytes and membrane and lipid signaling path- frequently studied mouse (19) yields >20 times the normal en- ways in oligodendrocytes. dogenous level by end-stage disease, raising concern about how closely the mechanism of toxicity in this accelerated model re- Results flects the human situation. Isolation of Translated mRNAs from Motor Neurons, Astrocytes, or Non–cell-autonomous toxicity has been demonstrated in cell Oligodendrocytes. To determine the damage caused by ALS- cultures as well, with astrocytes (20–25) or microglia (26) car- linked mutant SOD1 within spinal motor neurons, astrocytes, or rying SOD1 mutations generating toxicity to cocultured embry- oligodendrocytes during the disease course, we mated a mouse onic motor neurons. Although gene expression changes induced line (LoxSOD1G37R) that develops age-dependent, fatal paralytic by the SOD1G93A mutant in such cocultures have been reported from ubiquitous expression of a moderate (27), the extent to which this reflects age-dependent disease level of an ALS-linked point mutation in SOD1 (5) to bacTRAP course has not been established. reporter mouse lines (28, 29) (Fig. 1A). The LoxSOD1G37R line Here we coupled high-throughput RNA sequencing with the was chosen because of its wide use in identifying cell types whose translating ribosome affinity purification (TRAP) methodology mutant SOD1 synthesis contributes to a non–cell-autonomous (28, 29) to evaluate damage within motor neurons, astrocytes, disease mechanism (5, 8, 11, 30, 31) and in which overt disease and oligodendrocytes during the course of disease in mice that onset initiates at approximately 8 mo of age (Fig. 1B). The disease develop fatal ALS-like paralysis from ubiquitous expression of a course after initiation includes nearly complete denervation-induced moderate level of the familial ALS-causing mutation SOD1G37R muscle atrophy and accompanying weight loss, motor neuron death, (5). BacTRAP reporter transgenes that encode an EGFP-tagged and progressive paralysis (5). ribosomal protein L10a (Rpl10a) and driven by a cell type-specific Cohorts of LoxSOD1G37R mice expressing EGFP-tagged ribo- promoter allow isolation of actively translating, poly- some protein Rpl10a only within motor neurons (Chat-bacTRAP), ribosome-associated mRNAs from specific cell types in the CNS. astrocytes (Aldh1l1-bacTRAP), or oligodendrocytes (Cnp1-bacTRAP) Ribosome subunits and the bound mRNAs can be maintained were obtained (28, 29) (Fig. 1A). All mice were in a common intact and isolated by EGFP immunoprecipitation from an un- C57BL/6 genetic background. The predicted EGFP-Rpl10a ex- stressed, in vivo cellular environment with intact cell–cell con- pression patterns in spinal cords of the three reporter mouse nections at any chosen time point during aging. With this lines were confirmed by immunofluorescence (Fig. 1C). Chat approach, we have identified a cascade of damage, including promoter-driven EGFP-Rpl10a was expressed in the same cell

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Fig. 1. The bacTRAP methodology for isolating cell type-specific translational mRNAs. (A) The experimental design to identify cell type-specific damages in motor 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 cordof bacTRAP reporter mice. (B) The disease course of the SOD1G37R transgenic mouse. To identify early changes, RNA from all three cell types was first isolated at disease 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 after immunoprecipitation was measured with a Bioanalyzer. The intact 18S and 28S bands demonstrate that the RNA was not degraded. (F) qRT-PCR for motor neuron 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 protein itation from motor neurons, astrocytes, and oligodendrocytes PNAS PLUS choline acetyltransferase (Chat). The Aldh1l1 promoter-driven remained intact (Fig. 1E) and were highly enriched for corre- EGFP-tagged ribosome-labeled cells exhibited the morphology sponding marker genes, including Chat and Slc18a3 (VAChT) expected for astrocytes with many fine processes (32, 33) that for motor neurons, Aldh1l1 and Gfap for astrocytes, and Cnp1 partially overlap the astrocyte marker GFAP, whose localization and Mbp for oligodendrocytes, as demonstrated by quantitative is restricted to a subset of astrocytic cytoplasm (33). The Cnp1 RT-PCR (qRT-PCR) (Fig. 1F). promoter-driven EGFP-RPl10a was specifically expressed in ol- igodendrocytes, as indicated by coaccumulation with CC1. In all Cell Type-Specific Translational mRNA Changes Induced by SOD1G37R. three cases, EGFP-tagged Rpl10a was found diffusely within the Cell type-specific mRNAs recovered from spinal cords of mice G37R corresponding cytoplasm and bound to nucleoli (Fig. 1C), as with or without the LoxSOD1 transgene were converted to expected for a ribosomal protein. cDNA libraries and then sequenced. Each biological group To identify early changes that might trigger or contribute di- contained between three and six sex-matched animals. For each rectly to pathogenesis rather than reflect consequences of initial sample, an average of ∼40 million 50-bp reads were uniquely motor neuron degeneration and death, we harvested spinal cords mapped to the annotated mouse (mm9) genome (SI Appendix, from each of the three EGFP-Rpl10a mouse lines, with or Table S1). Expression levels for each annotated protein-coding without the LoxSOD1G37R transgene, from 8-mo-old mice, an gene were determined by the number of fragments per kilobase age at which muscle denervation in most mutant animals had of transcript per million mapped reads (FPKM). Genome-wide started but before overt phenotypic symptoms had developed comparison using unbiased (Spearman) hierarchical clustering of (30) (Fig. 1B). GFP immunoprecipitation of extracts from mouse expression levels of mRNAs purified from the three cell types of spinal cords successfully recovered polyribosome-associated nontransgenic mice revealed a high correlation between bi- mRNAs from each EGFP-Rpl10a reporter mouse, with very ological replicates of each condition and a clear distinction of little background RNA from mice without the GFP-containing mRNAs from motor neurons, astrocytes, oligodendrocytes, and transgene (Fig. 1D). mRNAs purified by EGFP immunoprecip- whole spinal cord (SI Appendix,Fig.S1). Inspection of scatterplots

A B 1.2 E Log2mean FPKM Log10 Overlaps of gene expression changes 30 9 1 among the three cell types Gfap 8 25 0.8 Aqp4 7 Fgfr3 0.6 20 Aldh1l1 6 Slc18a3 Motor 5 0.4 23916 82 Astrocyte 15 Chat neuron Mnx1 4 Isl1 0.2 3 7

Astrocyte 10 3 2 2 (normalized to Dnaja2) 0 11 5 SOD1 RNA mouse Relative Non-Tg SOD1Non-Tg SOD1non-Tg SOD1 1 G37R G37R G37R Oligodendrocyte 0 0 Motor neuron Astrocyte Oligodendrocyte 0 5 10 15 20 25 Motor neuron C 20 F Acsbg1 Log2mean FPKM Log10 220 30 9 15 SOD1 Cnp1 Mbp 8 G37R Mag yte 25 Mog

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Ol 2 G37R G37R G37R 5 Motor neuron Astrocyte Oligodendrocyte 1 SOD1 0 0 G37R 0 5 10 15 20 25 0 Motor neuron 3000 D SOD1G37R compared to Non-Tg at onset

mean FPKM Log10 Astrocyte Log2 Non-Tg 30 9 Gfap Mbp 8 up 25 Oligodendrocyte 0 Aqp4 Cnp 7 200 Fgfr3 Mag down 20 Aldh1l1 Mog 6 Sox10 SOD1 5 Astrocyte G37R 15 4 0 200 Astrocyte 10 3 2 Motor neuron 5 Non-Tg 1

0 0 -100 0 100 200 300 Oligodendrocyte 0 0 51015 20 25 30 Number of gene expression changes Oligodendrocyte

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 versus oligodendrocytes (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 bars represent SD in three or four biological replicates. (D) Number of gene expression changes in the three cell types, comparing SOD1G37R transgenic mice and nontransgenic mice. (E) Number of overlapped gene changes induced by SOD1G37R in motor neurons, astrocytes, and oligodendrocytes. (F) RNA-seq reads from 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 of average gene expression from multiple biological replicates of denervation that occurs as one of the earliest detected changes in immunoprecipitated mRNA from motor neurons versus astrocytes this mouse line (34). Up-regulated gene changes were enriched showed distinct gene expression patterns (Fig. 2A, Top). A robust in genes involved in metabolism, with notable perturbation in enrichment for motor neuron marker genes (Chat, Slc18a3, Isl1, the , aspartate, and glutamate metabolism pathways Isl2,andMnx1), astrocyte markers (Aldh1l1, Gfap, Fgfr3,andAqp4), (marked with red stars and red shade in SI Appendix, Fig. S3). or oligodendrocyte markers (Cnp1, Mbp, Mog, Mag,andSox10)was Down-regulated genes were highly enriched in those encoding found, respectively, in the immunoprecipitated mRNAs from the ribosomes and components of the translation machinery three cell types (Fig. 2A). (SI Appendix, Table S3), consistent with disruption of normal Endogenous and mutant SOD1 were expressed at high levels overall protein synthesis on ER stress activation. in all three cell types. The endogenous mouse SOD1 mRNA Analysis of potential -binding motifs and content was highest in motor neurons, with 70% of the motor coregulatory elements from the total 260 changes in motor neuron level found in astrocytes and 40% in oligodendrocytes neurons revealed two heat-shock proteins, HSF2 and HSF1, and (Fig. 2B). Human SOD1G37R RNA matched the corresponding an ER stress responsive transcription factor, CHOP (Fig. 3A), ratios for mouse SOD1 in motor neurons and oligodendrocytes strongly indicating UPR activation. The UPR is a major intra- (100% and 40%, respectively), with mutant SOD1 expressed at cellular pathway activated in response to an accumulation of only approximately one-half the corresponding endogenous level unfolded or misfolded proteins (35, 36). The three branches in astrocytes (30% of the level in motor neurons) (Fig. 2C). of the UPR are dependent on cAMP-dependent transcription We next tested whether gene expression changes were induced factor ATF-6 alpha (ATF6), PERK, and IRE1α (a serine/ in each cell type from expression of mutant SOD1G37R. A genome- threonine-protein kinase/endoribonuclease). The three branches wide comparison was performed on RNAs isolated just before of the UPR function to sense protein misfolding in the ER and disease onset from motor neurons, astrocytes, oligodendrocytes, transduce the initial misfolding signal for induction of compo- and whole spinal cord (Fig. 1B). An initial unbiased (Spearman) nents of each of the three branches (35, 36). Activation of PERK hierarchical clustering of all gene expression values from motor phosphorylates eIF2α, which enhances the translation of cAMP- neuron samples was used to determine that all four nontransgenic dependent transcription factor 4 (ATF4) mRNA (37). Analysis controls clustered closely together, as expected (SI Appendix, of the mRNAs accumulated within motor neurons at disease Fig. S2A). Two of the four SOD1G37R motor neuron samples onset revealed ER stress, including induction of the ER chap- were well separated from the controls, whereas two others erone PDI and the autophagy component SQSTM1 (p62), and developed more moderated differences with those controls (SI activation of the PERK arm of the UPR pathway, including el- Appendix,Fig.S2A). Given the variability in age of disease evation of ATF4, whose activity in turn induces CHOP (also onset across a 100-d window between the mice with the earliest known as DNA damage-inducible transcript 3 protein), a proa- and latest disease onset in this SOD1G37R line (34), we in- poptotic transcription factor (35). qRT-PCR analysis confirmed terpret the variability in affected gene expression in the mutant elevated levels of CHOP and ATF4 in SOD1G37R motor neurons mice to reflect animals with earlier and later disease initiation. (Fig. 3B). PERK activation was further supported by the ∼2.2- Indeed, a heat map of genes with altered expression clearly fold enhanced phosphorylation of eIF2α in SOD1G37R motor shows that the two more widely separated mutant animals exhibited neurons within lumbar sections of mouse spinal cord at disease more dramatic changes, with more modest changes in the same onset (Fig. 3 F and G). genes and in the same directions in the remaining two mutant Despite activation of the PERK pathway, neither the ATF6 nor mice (SI Appendix,Fig.S2B). IRE1α pathway of the UPR was similarly activated in motor We next performed statistical comparisons to identify mRNAs neurons, as demosnrtated by the absence of changes identified in that were changed significantly, using all annotated protein- Bip (also known as Grp78), an ATF6-activated ER chaperone coding genes (with Cuffdiff FPKM value at least 0.1 in one protein, or in X-box-binding protein 1 (XBP1), a key downstream condition; q < 0.05). Near disease onset, the gene expression component of the IRE1α pathway (Fig. 3B). Furthermore, acti- changes were most dramatic in motor neurons (Fig. 2D), with vation of the IRE1α pathway triggers the RNase activity of IRE1α, 260 significant changes, ranging from a 50-fold increase in a which excises a 26-nt intron of the unspliced XBP1 mRNA (XBP1u) serotonin transporter (Slc6a4) mRNA to a 35% decrease in to generate an active transcription factor, termed spliced XBP1 Runx2 mRNA SI Appendix, Table S2). Fewer changes were seen (XBP1s) (35). At disease onset, no XBP1s isoform was generated in in astrocyte mRNAs (108 mRNAs altered) and almost no the motor neurons of the SOD1G37R mice (Fig. 3B). changes were detected in translating oligodendrocyte mRNAs ER stress and activation of the PERK branch of the UPR was (23 mRNAs altered; a maximum sixfold change). Of note, most found in motor neurons, but not in astrocytes or oligodendrocytes of these changes (85% in motor neurons, 62% in astrocytes, and (Fig. 3 C and D). Analysis of the FPKM values of RNAs encoding 61% in oligodendrocytes) were up-regulations in expression ER chaperone proteins in normal motor neurons, astrocytes, and (with a mean up-regulation of 2.5-fold). Most gene changes were oligodendrocytes revealed much higher expression levels in the two cell type-specific; for example, 239 genes changed only in motor glial cell types than in motor neurons in ER chaperones PDI and neurons, despite the expression of almost all of them in all three FKBP9 (Fig. 3E). For example, PDI mRNA levels were approxi- cell types (Fig. 2E and SI Appendix, Table S9), indicating a mately 17-fold higher in astrocytes and 12-fold higher in oligo- unique response by each cell type to mutant SOD1 (synthesized dendrocytes compared with motor neurons. Even after a threefold within either it or its cell partners). Furthermore, very few of induction in the SOD1G37R motor neurons relative to non- these changes were identified from an analysis of whole spinal transgenic neurons (Fig. 3B), PDI levels remained sixfold below cord RNAs (SI Appendix, Fig. S2C), reinforcing the value of the the age-matched levels in normal astrocytes and oligodendroctyes bacTRAP method in identifying translational mRNA changes (Fig. 3E), consistent with motor neurons being intrinsically more within individual cell types. vulnerable to unfolded protein accumulation. FKBP9 levels are similarly elevated by 12-fold in astrocytes and sixfold in oligoden- Activation of ER Stress in Motor Neurons Expressing Mutant SOD1. drocytes relative to normal motor neurons (Fig. 3E). Examination of the 260 mRNA changes found in LoxSOD1G37R To determine whether the selective activation of the PERK motor neurons revealed a strong enrichment for genes involved arm of the UPR was common to disease-linked SOD1 mutants in synapses and cell junctions (SI Appendix, Table S3). Approx- of different biochemical characters, we examined ER stress imately 10% of all of the gene changes are in this category, components in mice (38) that develop fatal disease from an consistent with synapse dysfunction accompanying the known intrinsically misfolded, dismutase-inactive mutation SOD1G85R

E6996 | www.pnas.org/cgi/doi/10.1073/pnas.1520639112 Sun et al. PNAS PLUS Top 6 candidate transcription factors G37R

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Fig. 3. Activation of ER stress specifically in motor neurons at disease onset. (A) Candidate transcription factors of coregulated genes in SOD1 G37R motor neurons, 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 neurons compared with nontransgenic controls. Error bars represent SEM in three or four biological replicates. *P < 0.05, **P < 0.005, Student t test. (Top) Schematic diagram 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 or without the G37R transgene. (C) Relative expression levels of ATF4, CHOP, and PDI in astrocytes at disease onset. The FPKM values from RNA-seq were normalized to those in nontransgenic samples of each gene. Error bars represent SD in four to six biological replicates. (D) Relative expression levels (FPKM from 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 biological replicates. (E) Relative expression levels (FPKM) of ER chaperones in the three cell types of nontransgenic mice. The expression levels of each gene were normalized to the value in motor neurons. (F) Immunofluorescence of phospho-eIF2α in spinal cords of Chat-EGFP–labeled motor neuron reporter mice, with or without the SOD1G37R transgene. (G) Quantification of the intensity of red fluorescence normalized to green fluorescence as in F. There were three mice in each group, and approximately 100 motor neurons were quantified in each mouse. Error bars represent SD. **P < 0.005, Student t test.

(39), which develop slowly progressing disease with modest mutant (IGFBP7), both of which have been shown to have toxic or SOD1 accumulation, similar to the SOD1G37R mouse line except proapoptotic effects on neurons and in other tissues (40–42). that the mutation is dismutase-inactive. Laser capture micro- qRT-PCR analysis of RNAs purified from the Aldh1l1-promoted dissection was used to isolate RNAs from motor neurons at an EGFP-Rpl10a ribosomes validated fourfold and twofold in- NEUROSCIENCE early symptomatic stage (age 10.5 mo). qRT-PCR analysis revealed creases, respectively, in the RNAs encoding CXCL10 and dramatic elevations of ATF4 expression (40-fold) and CHOP IGFBP7 in the astrocytes from the dismutase-active (SOD1G37R) expression (12-fold) (SI Appendix, Fig. S2D), indicative of acti- mutant SOD1 mice (SI Appendix, Fig. S4B). Similar analyses of vation of the PERK branch of ER stress as a common response RNAs isolated at onset stage from astrocytes of mice that will in motor neurons induced by dismutase-active and -inactive develop fatal motor paralysis from expressing dismutase-inactive SOD1 mutations. (SOD1G85R) mutant mice showed similar (twofold) increases in CXCL10 and IGFBP7 (SI Appendix,Fig.S4B). Dysfunction of Nuclear Receptors Peroxisome Proliferator-Activated Although the mRNA levels of the two candidates were un- Receptor and in Astrocytes Expressing Mutant SOD1. changed, two families of nuclear receptors, peroxisome pro- We initially analyzed SOD1-mutant dependent changes in ribo- liferator-activated receptor (PPAR) and liver X receptor (LXR), some-bound RNAs recovered from astroctyes using unsuper- vised Spearman hierarchical clustering of all gene expression were predicted to be the major transcription coactivators in- values. This analysis revealed that the six nontransgenic controls volved in the 108 gene changes in SOD1 mutant-expressing clustered tightly together, as did three of the four SOD1G37R astroctyes (Fig. 4B). Both of these proteins can be activated by samples (SI Appendix, Fig. S4A). As seen in the motor neurons, fatty acid and cholesterol derivatives, and they control the ex- the majority of the significant RNA changes in astrocytes (67 of pression of genes involved in metabolism and inflammation (43). 108) were increases, ranging from a 1.8-fold increase for crem On ligand binding, both receptors undergo a conformational (cAMP-responsive element modulator) to a 30-fold increase for change, followed by changes in interacting proteins, and they Ccl6 (C-C motif chemokine 6) (Fig. 4A). There were more affect transcription through multiple modes, including direct modest reductions in 42 RNAs, with the maximum reduction to activation, repression, and transrepression (43, 44). In general, 33% of the initial level for Kif20a (SI Appendix, Table S4). Gene PPAR and LXR activate metabolism and play important roles in ontology analysis revealed enrichment of genes linked to im- energy and lipid homeostasis, and also repress inflammatory mune responses and with extracellular functions (SI Appendix, gene expression (43, 45) (Fig. 4C). In SOD1G37R astrocytes, Table S5), including the chemokine CXCL10 (C-X-C motif there was extensive up-regulation of inflammatory gene expres- chemokine 10) and an insulin-like growth factor-binding protein sion (Fig. 4D) and a trend toward a reduction in metabolic genes

Sun et al. PNAS | Published online November 30, 2015 | E6997 A C Fatty acid and cholesterol ligands > 10 fold up SOD1G37R > 5 fold down PPAR,LXR (nuclear receptors) > 2 fold

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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 at disease onset. (B) Candidate transcription factors of coregulated genes in SOD1G37R astrocytes, as predicted by the web server DiRE. (C) Diagram of PPAR and LXR 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 G37R astrocytes. Error bars represent SEM in three or four biological replicates. *P < 0.05, Student t test.

(Fig. 4E), several of which have been shown to be targets of 10-fold (Fig. 5B). The use of qRT-PCR to provide an in- PPAR/LXR (46–48). dependent assessment of RNA levels validated the changes in Along with the apparently increased PPAR/LXR activity, RNA levels (Fig. 5C). Fourteen of the 23 RNA changes seen at peroxisome proliferator-activated receptor gamma coactivator disease onset showed enhanced dysregulation at an early symp- 1-alpha (PGC1α) was first down-regulated in mutant SOD1G37R tomatic stage (SI Appendix, Table S7). astrocytes, but not in motor neurons or oligodendrocytes (Fig. analysis revealed that membrane protein-encod- 4E and SI Appendix,Fig.S4C). PGC1α is a transcriptional ing genes were disproportionately misregulated, composing ∼25% coactivator of nuclear receptors and other transcription factors of the up-regulated RNAs and 45% of the down-regulated RNAs that play key roles in mitochondria biogenesis and oxidative me- (SI Appendix,TableS8). Furthermore, all three main proteins tabolism (49, 50). Its expression can be regulated by PPAR, and it composing the myelin sheath—myelin basic protein (MBP), myelin also cooperates with PPAR in transcriptional control of nuclear oligodendrocyte glycoprotein (MOG), and proteolipid protein genes encoding mitochondrial fatty acid metabolism (51). A de- (Plp1)—were down-regulated in the SOD1G37R oligodendrocytes crease in PGC1α level is not a typical gene expression change (Fig. 5D). In addition, RNAs encoding components of three major accompanying astrogliosis induced by neuroinflammation or signaling pathways—phosphatidylinositol signaling (P = 0.0008, stroke (52), suggesting that it may be an intrinsic toxic effect of Benjamini test), FcγR-mediated phagocytosis (P = 0.0007, Benja- mutant SOD1 in astrocytes rather than an inflammatory response mini test) and a calcium signaling pathway (P = 0.0009, Benjamini to the motor neuron dysfunction. Thus, dysfunction of mito- test)— were perturbed (SI Appendix,Figs.6and7). Members of + chondria and/or oxidative metabolism might account in part for phosphoinositide phospholipase C (PLC) and Ca2 /calmodulin- the noncell autonomous toxicity of astrocytes to motor neurons. dependent protein kinase (CaMK) protein families were up-regu- lated. PLCs participate in phosphatidylinositol 4,5-bisphosphate Gene Expression Changes in Oligodendrocytes at an Early Symptomatic (PIP2) metabolism and lipid signaling pathways in a calcium- Stage. The Cnp1-Rpl10a bacTrap approach identified very few dependent manner, and are essential for intracellular calcium ho- changes (only 14 up-regulated and 9 down-regulated RNAs) in meostasis (53). CaMK is a serine/threonine-specific protein kinase + SOD1G37R mutant oligodendrocytes at disease onset. Moreover, regulated by the Ca2 /calmodulin complex that has been shown to these changes were smaller in magnitude relative to the changes in regulate oligodendrocyte maturation and myelination (54). Strik- motor neurons, ranging from a maximal 2.4-fold increase for ingly, nine of the 15 members of the PLC family and six CaMKs Pla2g4e (cytosolic phospholipase A2 epsilon) to repression to 37% were up-regulated in oligodendrocytes in SOD1G37R mice at this of the initial level for Ccne2 (G1/S-specific cyclin-E2) (SI Appendix, early symptomatic stage (Fig. 5 E and F). Similar analysis of RNAs Table S6). from oligodendrocytes of SOD1G85R mice isolated using the Cnp1- To test whether changes appeared in oligodendrocytes later in Rpl10a bacTrap approach at early symptomatic stage revealed a the disease course, we used the Cnp1-Rpl10a bacTrap approach similar trend of changes (albeit of lower magnitude) in the two gene to purify translating RNAs from oligodendrocytes at an early families (SI Appendix,Fig.5B and C), indicating common defects symptomatic disease stage (age 10.5 mo) (Fig. 1B). As before, from dismutase-active and -inactive SOD1 mutants. unsupervised hierarchical clustering of all gene expression values It was previously reported that the main lactate transporter, revealed that the five nontransgenic controls clustered together, monocarboxylate transporter 1 (MCT1), which provides metabolic whereas the three SOD1G37R samples were largely distinct from support to axons, was suppressed in oligodendrocytes of SOD1G93A the controls (SI Appendix, Fig. S5A). A total of 750 gene ex- mice (55). Surprisingly, however, despite 750 altered RNAs, pression changes were identified in annotated protein-coding changes in MCT1 mRNAs were not seen in oligodendrocytes at genes (with a cuffdiff FPKM value of at least 0.1 in one condi- either disease onset or the early symptomatic stage in either tion; q < 0.05; Fig. 5A), 628 of which were up-regulated (between SOD1G37R or SOD1G85R mutant mice (SI Appendix, Fig. S5D). 1.7- and 33-fold) (SI Appendix, Table S7). A total of 439 genes Thus, altered levels of MCT1 synthesis (e.g., translating mRNAs) were up-regulated at least twofold, 92 genes were up-regulated cannot be an early damaging factor that drives initiation of non– more than fivefold, and 12 genes were up-regulated more than cell-autonomous toxicity from oligodendrocytes to motor neurons.

E6998 | www.pnas.org/cgi/doi/10.1073/pnas.1520639112 Sun et al. PNAS PLUS A Oligodendrocyte B Oligodendrocyte at early symptomatic stage C 3 Non-Tg * SOD1G37R > 10 fold (12) e 2.5 * up up onset (23 total) down 2 down > 5 fold (92) rocyt

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G37R D EF10 Non-Tg SOD1 1.2 ** * ** ** ** 3 8 Non-Tg ** sion 1 ** ** ** ** SOD1G37R 2.5 ** 0.8 6 2 Non-Tg 0.6 * ** * 1.5 4 ** ** ** ** 0.4 SOD1G37R * ** 1 Relative FPKM Relative 2 0.2 0.5 in oligodendrocyte Relative FPKM Relative expres 0 0 0

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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 an early symptomatic stage. (C) qRT-PCR validation of gene changes in SOD1G37R oligodendrocytes at early symptomatic stage. Error bars represent SEM in three or four biological 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)in oligodendrocytes at early symptomatic stage. The FPKM values were normalized to values in nontransgene samples for each gene. Error bars represent SDinthree to five biological replicates. *P < 0.05, **P < 0.005, Student t test. (G) Candidate transcription factors of coregulated genes in SOD1G37R olidodendrocytes, as predicted 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. NEUROSCIENCE Our analysis of potential binding motifs and coregulatory As a further test to confirm that the changes identified in elements identified four subfamilies of the ETS transcription translating RNAs from oligodendrocytes are not likely to be factors—ETS, PEA3, PU1 (SPI), and ELF—as candidate tran- remaining RNA contaminants from large changes in microglia or scription factors whose elevated activity could underlie increased astroctyes, we examined the fold changes of genes in the FcγR- expression of 427 of the 622 genes with greater than twofold mediated phagocytosis pathway in whole spinal cord. Up-regulation changes in mutant-expressing oligodendrocytes (Fig. 5G). The was either not observed or observed at a much smaller magnitude ETS (E26 transformation-specific or E-twenty-six) family is one compared with that in oligodendrocyte-derived RNAs (SI Ap- of the largest families of transcription factors in mice, with 12 pendix, Fig. S5F), consistent with increased synthesis in phago- subfamilies and 28 total members (56, 57). RNAs encoding seven cytosis components within oligodendrocytes. Although we are of these factors were significantly up-regulated in SOD1G37R unaware of any reported evidence of phagocytosis in oligo- oligodendrocytes, with two (Elf4 and Fli1) increased by more dendrocytes, Fc receptor mediated-signaling has been im- than fivefold (Fig. 5H). Elevated levels of these RNAs (elevated plicated in the induction of oligodendrocyte differentiation and to a smaller extent) were also identified at early symptomatic myelination (61). stage in SOD1G85R oligodendrocytes (SI Appendix, Fig. S5E). Functional gene ontology analysis with candidate ETS targets Discussion in the SOD1 mutant-mediated expression-changed genes revealed Motor neuron degeneration caused by mutant SOD1 is non–cell- significant elevations in genes in the phosphatidylinositol signaling autonomous (14), with contributions from neighboring glia cells system (P = 0.005, Benjamini test) and FcγR-mediated phagocytosis (3–5, 7–9, 11), but no consensus has been reached on the precise (P = 0.000008, Benjamini test) (SI Appendix,Fig.S6A and B). mechanisms involved. To examine this question, we combined ri- Phagocytosis is a major mechanism for removing pathogens and cell bosome tagging in mice (bacTRAP) to enable isolation of cell type- debris. It was once thought to be carried out mainly by microglia in specific polyribosome-associated mRNAs in the context of intact the CNS (58), but recently was found to be performed by astrocytes neuronal-glial networks with high-throughput sequencing (RNA- as well, for synapse and neuronal debris elimination (59, 60). seq). Starting with RNAs isolated from mice ubiquitously expressing

Sun et al. PNAS | Published online November 30, 2015 | E6999 Abnormal responses to particular ER stress stimuli (35, 67). Our data es- ❶ ❷ metabolism and tablish that chronic accumulation of misfolded SOD1 initiates Synapse and activation of the PERK branch of ER stress selectively in post- Motor neuron Astrocyte inflammaon metabolism mitotic motor neurons, but not in astrocytes or oligodendrocytes responses PPAR nuclear receptors even at symptomatic disease stages, supporting a special vulner- acvated by lipid ligand ability of motor neurons to misfolded SOD1 accumulation. One ER stress simple explanation for this vulnerability is our identification of highly divergent levels of ER chaperones, with much higher levels Membrane and in astrocytes and oligodendrocytes than in motor neurons. PERK-ATF4-CHOP α lipid signaling Furthermore, immunostaining of phospho-eIF2 within indi- Oligodendrocyte defects vidual motor neurons has revealed that some motor neurons develop ER stress activation earlier than others, consistent with ❸ ETS family of report of ER stress activation in selectively vulnerable motor transcripon factors neuron subtypes (68). A recent cell culture study also reported ER stress in both IREα and PERK branches induced by mutant Fig. 6. Cell type-specific toxicity of mutant SOD1 in motor neurons, astro- SOD1 in immature induced pluripotent cells (iPSC)-derived cytes, and oligodendrocytes. (1) Initial damage occurs in motor neurons at motor neurons, with the basal level of ER stress (reported by disease onset, including synapse and metabolism responses and activation of ER stress. (2) Damage in astrocytes includes abnormal metabolism and in- spliced Xbp1) inherently higher in healthy motor neurons com- flammation, owing in part to the dysfunction of PPAR and LXR nuclear re- pared with other neurons and nonneuronal cell types in in vitro ceptors that are activated by lipid ligands. (3) Damage in oligodendrocytes cultures (69). The differences between our in vivo findings of occurs in an early symptomatic stage, later than that in motor neurons and selective PERK pathway activation compared with broader ER astrocytes. The toxicity includes mainly myelination and lipid signaling de- stress activation in cell cultures (spliced Xbp1) (Fig. 3B) likely fects, coupled with activation of the ETS family of transcription factors. reflect the additive effects of those from mutant SOD1 and the stressed environment facing cells in cell cultures. ER stress is usually triggered by the accumulation of misfolded proteins dismutase-active or -inactive mutantSOD1atorjustbeforedisease within the ER lumen. Although SOD1 is not a secreted protein onset, we identified early events within motor neurons, followed by and is not synthesized inside the ER lumen, mutant SOD1 has later RNA dysregulation in astrocytes and oligodendrocytes. been shown to directly interact with Derlin-1, a component of the Early RNA expression changes identified in mutant SOD1- ER-associated degradation (ERAD) machinery, and activates ER expressing motor neurons include ER stress, synapse, and met- stress through dysfunction of ERAD (70). It also has been shown abolic abnormalities (Fig. 6). Few RNA changes were initially that misfolded SOD1 has increased association with mitochondria found in astrocytes, with many accumulating later, including in and ER membranes (71–75). Furthermore, the conformation of genes involved in inflammation and metabolism, enriched for membrane-associated mutant SOD1 has been solved by NMR, targets of PPAR and LXR nuclear receptors (Fig. 6). This revealing that the association is mediated by interfacial amphi- finding is consistent with earlier efforts using genetic deletion of philic helices (76). Based on the foregoing findings, we propose mutant SOD1 from motor neurons to delay disease onset (5, 7), that the abnormal insertion of misfolded SOD1 onto/into ER whereas similar deletion from astrocytes slowed disease pro- membranes in motor neurons interferes with the normal function gression with no strong effect on onset (8, 9). Curiously, although + of other membrane proteins, including , modifica- mutant SOD1 gene inactivation in NG2 progenitor oligoden- tion, and degradation, with activation of the PERK pathway of ER drocytes reportedly delays the age of disease onset (11), with stress from direct SOD1 mutant damage to Derlin or indirect only a modest effect in slowing progression after onset in damage from disrupted protein homeostasis. G37R SOD1 mice, our genomic data show hardly any changes in Whether manipulation of the ER stress pathways can provide a − − oligodendrocytes at this age, with dramatic changes arising by an protective effect remains controversial. Breeding with ATF4 / early symptomatic stage, consisting mainly of dysregulation of mice (77) or nervous system deletion of XBP-1 (78) has been myelination and lipid signaling pathways coupled with activation reported to modestly delay disease onset and/or prolong the life of ETS transcription factors (Fig. 6). The strong effect on disease span, but only in one-half of SOD1G86R mice studied; however, + + − onset from removal of mutant SOD1 from NG2 cells implies crossing with PERK / mice has been reported to accelerate disease that they may have additional functional roles or are progenitors onset and shorten life span in SOD1G85R mice (79). The ER stress- of additional cell populations beyond a role as the in vivo pro- protective agent salubrinal, an inhibitor of eIF2α dephosphory- genitors of mature Cnp1-expressing oligodendrocytes. lation, has been shown to mitigatediseaseprogression(68). Activation of ER stress has been implicated in the pathogenesis Furthermore, eIF2α phosphorylation was found to be up-regulated of ALS (62–66). Up-regulation of Bip (downstream of the ATF6 by TDP-43 toxicity in flies, and a PERK inhibitor could attenuate its branch) was initially reported in cell cultures transfected with damage in flies and mammalian neurons (80). Therefore, distur- mutant SOD1 and spinal cords of transgenic SOD1H46R and bance of ER proteostasis might be a more general phenomenon SOD1L84V mice (64). Another study at disease onset in SOD1G93A underlying the selective motor neuron degeneration caused by dif- mice reported increased activity of CHOP, a downstream target ferent mutant genes in ALS. The divergent findings produce a indicating activation of the PERK branch of the UPR (66). Ac- complex scenario regarding therapy development, with intervention tivation of all three arms of the UPR with activated ATF6, ATF4, at different steps of the ER stress pathways, at different timings and and XBP1 found in spinal cords, but not cerebellum, has been under different circumstances, or on different cell types apparently reported in end-stage SOD1G93A and SOD1G85R mice (63). resulting in opposite effects on the process. Adding to this knowledge, we have established that ER stress Finally, our bac-Trap approach has identified a temporal starts within motor neurons with selective activation of the PERK- cascade of different cellular pathways activated by mutant SOD1 ATF4-eIF2α pathway, leading to up-regulation of CHOP and its in motor neurons, astrocytes, and oligodendrocytes. ER stress transcriptional targets, and have further confirmed the specific activated in motor neurons is an initiating event, presumably activation of its upstream regulators at disease onset. This finding mediated by activation of a UPR sensor on ER membranes. adds an in vivo example during disease pathogenesis to previous Altered lipid signaling is seen in all three cell types. PPAR and cell culture evidence establishing that the three UPR sensors can LXR nuclear receptors that are normally activated by fatty acid have fundamental differences in the timing of their signaling and and cholesterol derivatives were predicted to be dysfunctional in

E7000 | www.pnas.org/cgi/doi/10.1073/pnas.1520639112 Sun et al. astrocytes. Extensive membrane proteins and lipid signaling fault parameters, with the exception of reporting reads mapping to unique PNAS PLUS pathways were subsequently dysregulated in oligodendrocytes. locations on the reference) (81). RNA-seq results were visualized in the UCSC Taken together, the collective evidence supports the idea that Genome Browser (genome.ucsc.edu/) by generating custom tracks using big- the toxicity of misfolded SOD1 originates from its abnormal wig files. The relative abundance of transcripts was measured by FPKM using Cufflinks (82). Expression variations of protein-coding genes between non- membrane association. With aging, a decreasing ability to de- trangenic control and mutant SOD1 groups were quantified and compared by grade unfolded proteins leads to increasing associations of mu- cuffdiff (83). Scatterplots, heat maps, and hierarchical clustering were gener- tant SOD1 with different membranes and eventually initiates a ated by R software using FPKM values from each sample. Gene ontology cascade of cellular responses and gene expression changes that analysis was performed using DAVID (https://david.ncifcrf.gov/). Distant regu- result in motor neuron dysfunction and death. Owing to the latory elements of coregulated genes were analyzed by DiRE (dire.dcode.org/). distinctive intrinsic properties of the different cell types, this common toxic feature provokes cell type-specific responses that Laser Capture Microdissection. Mice were perfused with sterile ice-cold PBS. contribute to disease initiation and progression in a coordinated Spinal cords were dissected, incubated in 20% (wt/vol) sucrose (in PBS) at 4 °C manner. Motor neurons are most vulnerable to the accumulated overnight, and subsequently embedded in optimal cutting temperature (OCT) − mutant SOD1 and abnormal cellular pathways because they 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- synthesize high levels of SOD1 and have very low levels of ER mately 700 motor neurons were laser capture microdissected from each chaperones. Subsequent damage developed from mutant SOD1 mouse, and RNA was extracted using the RNAqueous-Micro Kit (Ambion) and synthesis within astrocytes and oligodendrocytes is essential for amplified using the MessageAmp II aRNA amplification kit (Ambion). amplifying the initial damage within motor neurons. qRT-PCR. For first-strand cDNA synthesis, random hexamers were used with a Materials and Methods high-capacity cDNA reverse transcription kit (Applied Biosystems). qRT-PCR Animals. ALS mouse lines SOD1G37R (5), SOD1G85R (38), and SOD1G93A (19) are reactions were performed with three or four biological replicates for each all heterozygous for a 12-kb genomic DNA fragment encoding the human group and two technical replicates using iQ SYBR Green Supermix (Bio-Rad) mutant SOD1 transgene, under its endogenous promoter. The bacTRAP on the iQ5 Multicolor Real-Time PCR system (Bio-Rad). The data were ana- transgenic mouse line Chat-bacTRAP line expresses an EGFP-tagged ribo- lyzed using iQ5 optical system software, version 2.1 (Bio-Rad). Expression some protein Rpl10a only within motor neurons. The Aldh1l1-bacTRAP line values were normalized to the control genes Rpl23 and Dnaja2. Intergroup expresses the same EGFP-tagged Rpl10a in astrocytes, whereas the Cnp1- differences were assessed using the two-tailed Student t test. Primer se- bacTRAP expresses it in oligodendrocytes (28, 29). All animal experiments quences are presented in SI Appendix, Table S10. used in this work were approved by the University of California at San Diego Institutional Animal Care and Use Committee. Immunofluorescence. Mice were perfused intracardially with 4% (vol/vol) paraformaldehyde in 0.1 M Sorenson’s phosphate buffer, pH 7.2. The entire Purification of Cell Type-Specific mRNA from bacTRAP Mice. Dissected mouse spinal cord was dissected, postfixed for 2 h in fixative, and the transferred in a spinal cord was immediately homogenized in ice-cold polysome extraction 30% sucrose phosphate buffer for at least 2 d. The lumbar spinal cord was − buffer (20 mM Hepes pH 7.4, 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT, embedded in OCT compound and snap-frozen in isopentane cooled to 40 °C 100 μg/mL cycloheximide, protease inhibitors, and RNase inhibitors). Homoge- on dry ice. Floating lumbar spinal cord cryosections (30 μm) were incubated in nates were centrifuged at 2,000 × g for 10 min at 4 °C, after which Nonidet a blocking solution containing PBS, 0.5% Tween 20, and 1.5% (wt/vol) BSA for P-40 and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids) 1 h, 30 min at room temperature and then in PBS and 0.3% Triton X-100 were added to the supernatant at final concentrations of 1% and 30 mM. overnight at room temperature, with the following primary polyclonal The lysates were centrifuged at 13,000 × g for 15 min at 4 °C after incubation antibodies against ChAT (Millipore), GFAP (Dako), CC1 (Calbiochem), GFP on ice for 5 min. Two monoclonal GFP antibody (Htz-GFP19C8 and Htz- (a gift from the Oegema/Desai laboratory), and phosphor-eIF2α (Cell Signaling GFP19F7; Memorial Sloan Kettering Cancer Center Monoclonal Antibody Technology). Primary antibodies were washed with PBS and then detected Core Facility)-coated magnetic beads (Dynabeads Protein G; Invitrogen) using FITC or Cy3-coupled secondary antibodies (Jackson ImmunoResearch). were added to the supernatant, followed by incubation at 4 °C with rotation The sections were washed with PBS and mounted. Analysis was performed overnight. Beads were subsequently washed five times with high-salt poly- using a Nikon Eclipse laser scanning confocal microscope. Fluorescence in- some wash buffer (20 mM Hepes pH 7.4, 350 mM KCl, 5 mM MgCl2,0.5mM tensity from unsaturated images captured with identical confocal settings DTT, 1% Nonidet P-40, and 100 μg/mL cycloheximide). The RNA bound on (a minimum of six spinal cord sections imaged per animal) was quantified

the beads were extracted with an Absolutely RNA Nanoprep Kit (Stratagene) using NIS-Elements software (Nikon). NEUROSCIENCE and quantified by the RiboGreen RNA assay (Invitrogen). ACKNOWLEDGMENTS. We thank Drs. Nathaniel Heintz and Jeffery Rothstein RNA-seq Library Preparation and Transcriptome Analysis. RNA quality was for the bacTRAP reporter mice; Dr. Myriam Heiman for the detailed protocol ’ measured using the Agilent Bioanalyzer system according to the manufac- for the bacTRAP RNA purification method; members of B. Ren s laboratory, turer’s recommendations. RNA-seq libraries were prepared from RNAs especially Z. Ye, S. Kuan, and B. Li, for technical help with the Illumina sequencing; and all members of the D.W.C. laboratory for critical comments extracted from specific cell types or whole spinal cord, using an Illumina and helpful suggestions. This work was supported by National Institutes of TruSeq RNA Sample Preparation Kit. RNA-seq libraries were sequenced on Health Grant R01 NS27036 (to D.W.C.). S.S. was a recipient of the Milton an Illumina HiSEq 2000 sequencer for 50 cycles from a single end. Safenowitz Postdoctoral Fellowship from the ALS Association, a Target ALS The reads were aligned to a reference mouse genome obtained from the Springboard Fellowship, and a National Institutes of Health K99/R00 Award University of California Santa Cruz (mm9; NCBI build 37) using Tophat (de- (NS091538-01).

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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.

0.7

0.8

0.9

1.0 3 5 4 6 1 2 5 2 4 3 1 4 3 5 6 1 2 4 3 1 2 ------1 - 1 - 1 - 1 - 1 - 1 - l l l l l l a l - a l - a l - a l - t t t t 1 1 1 1 1 1 h a t h a t h a t h a t h a t h a t n p 1 n p 1 n p 1 n p 1 n p 1 o o o o h h h h h h T C C C C C C T T T C C C C C d d d d d d l l l l l l A A A A A A Astrocyte Oligodendrocyte Spinal cord Motor neuron

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 Motor neuron B (onset) 5 7 9 . 0 g n i 0 8 e r 9 t

. 0 s e d

u G37R Non-Tg SOD1 t s a l c l n

u o 5 g r 8 e e d 9 u . - r 0 n n e v i s

w r r o e o D t 0 9 s u p m o 9 .

n 0 c i t U 4 4 3 g 5 T g e n R R 1 2 1 - 9 3 s 7 7 g 2 n 9 g 3 3 . g R R T o - 0 T 7 7 T G G

- N n - r a n 3 3 t d n o n - e G G o o t N n N a N l o u n

G37R g s

SOD1 e r

Non-Tg u - p r s U v e

R

7 3 C G Whole spinal cord O D 1 S

n Motor i neuron 55 273 G85R 205 D SOD1 Laser capture motor RNA extraction spinal cord microdissection neurons and amplification (10.5 months)

qRT-PCR to measure RNAs Astrocyte 70 encoding UPR components 61 47 281 60 h a n g e

c 50 d 40 Non-Tg

e d f o l 30 G85R z Oligodendrocyte i SOD1

a l 20 14 9 319 m 10 N o r 0 ATF4 CHOP

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

Abat Gad1,2 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. Astrocyte A B ***

5 (onset) *** 6 e 5 9 g . 0 a n 0

h 4 * 7 c 9 ** . g d 0 n l i o

5 3 f

e r Non-Tg 7

t 9 d s . G37R 0 e u SOD1

z 2 i c l 0

4

8 G85R a l

9 SOD1 . e d R m 0 1 r 7 o 5 3 v i s 8 r N G 9 e 3 . 0 0 g 1 T 0

- s u p Cxcl10 Igfbp7 9 n n R 9 . o 7 0 U 5 3 N g 5 2 3 G T

9 - 9 R R . n 0 7 7 o 3 3 2 1 N 6 4 g g G G g g T T T T - - - - n n n n o o o o N N N N SOD1G37R Non-Tg

C l 1.4 ** e v e

l 1.2

α

) 1 C 1

G 0.8 K M P P

F 0.6 e ( v i t 0.4

a Non-Tg l

e 0.2 G37R

R SOD1 0

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. A Oligodendrocytes D (early symptomatic) 1.2 0

5 1 9 . 0 0.8 5

M of MCT1 non-Tg 5 K 9 0.6 . P 0 G37R

g SOD1 F

n 0.4 i e 0 G85R v 6

i SOD1 e r t 9 t

. 0.2 a s 0 l u e c l

5 0 R

6 9 . e d

0 onset symptomatic v i s 1 r 0

e 7 R 9 . 7 0 3 s u p n G

5 E U 7 1 9 3 2 . g

0 T R R D 1 - 18 s 7 7 n O 0 3 2 3 3 o t e S 5 4 8 g g 16 9 t G G N g g G37R . T T n SOD1 0 T T - - o c y a - - t n n 14 G85R n n u o o SOD1 n d r o o m N N e

12 N N d n i o g s 10 G37R i Non-Tg SOD1 l n e o 8 g g e - T s f t

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d R K a i n 2 * *

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* G m

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1.5 1 d v 7 a l l i e t 3 D c

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a 0.5 0 C d e p 0

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. A

Pik3cg

PLC PLC Synj1

Inpp4a Itpkb

PLC Inpp5d

Inpp4a Dgkb

B

Dnm1 Fcgr2b Inpp5d

Ptprc Myo10

Src Pik3cg

Marcks Plcg2 Prkcd

Ncf1

Was

Vav1 Rac2

Dock2

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 Slc8a2

Adcy8

Mylk Phkg Cacna1i

Ryr2

CAMK

Bdkrb2 Gq

PLCδ PLCβ Adcy8 PLCγ Pde1b PLCε

Ptk2b

Itpkb

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 8 month 11 month Motor neuron Astrocyte Oligodendrocyte Whole spinal cord Oligodendrocyte Whole spinal cord Non‐Tg G37R Non‐Tg G37R Non‐Tg G37R Non‐Tg G37R Non‐Tg G37R G85R Non‐Tg G37R Sample 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 30,256,872 Sample 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 33,006,700 Sample 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 35,308,474 Sample 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 41,026,823 Sample 5 40,005,888 37,710,915 47,685,360 Sample 6 39,588,673 Supplementary Table 2: Gene expression changes in SOD1 G37R motor neurons compared to non-transgenic controls at disease onset. gene FPKM value for non-transgenic control FPKM value for SOD1 G37R ln(fold_change) q_value Genes upregulated in SOD1 G37R motor neurons Slc6a4 chr11:76812098-76845845 0.01 0.65 3.77 7.18E-04 Sprr1a chr3:92287875-92289803 1.96 54.74 3.33 0.00E+00 Phox2b chr5:67485635-67490365 0.20 3.64 2.89 0.00E+00 Fgf21 chr7:52869259-52870860 0.55 8.67 2.75 9.75E-14 Tbx20 chr9:24525255-24578747 0.07 0.67 2.29 1.29E-07 Atf3 chr1:192994175-193007212 0.93 9.14 2.29 0.00E+00 Rtdr1 chr10:74420221-74495331 0.21 2.04 2.28 4.51E-05 Myo1f chr17:33692651-33744709 0.09 0.74 2.13 3.46E-05 Shox2 chr3:66777190-66785693 0.14 1.12 2.10 6.44E-03 Lpl chr8:71404453-71430831 0.09 0.63 1.96 8.01E-05 Slc17a7 chr7:52419290-52431509 0.27 1.81 1.89 1.24E-07 Crabp2 chr3:87752614-87757294 0.21 1.39 1.87 3.14E-02 Cckar chr5:54089723-54098943 0.07 0.45 1.86 2.13E-02 Fcrls chr3:87054886-87067446 0.11 0.67 1.85 1.92E-02 Rasal3 chr17:32527605-32541753 0.06 0.36 1.75 2.49E-02 Fmod chr1:135934091-135944854 0.07 0.41 1.75 3.47E-02 Lyz2 chr10:116714596-116719328 0.85 4.81 1.73 5.42E-07 Inpp5d chr1:89516886-89617083 0.19 1.06 1.73 4.04E-10 Aspg chr12:113344893-113365784 0.09 0.49 1.72 2.41E-02 Thpo chr16:20724526-20742457 0.15 0.82 1.70 2.55E-03 Trh chr6:92192055-92194642 0.22 1.17 1.69 1.43E-02 Clec7a chr6:129411608-129422795 0.11 0.58 1.68 2.78E-02 Vav1 chr17:57418522-57468659 0.09 0.43 1.61 5.72E-03 Ptpn6 chr6:124670735-124688727 0.24 1.17 1.60 1.67E-03 Tbx3 chr5:120120677-120134610 0.06 0.29 1.57 1.51E-02 Tyrobp chr7:31198806-31202598 1.32 6.10 1.53 4.23E-04 Capg chr6:72494432-72512974 0.52 2.35 1.51 2.79E-04 Trem2 chr17:48485725-48491598 0.40 1.75 1.48 1.52E-02 Gbp2 chr3:142283626-142300972 0.12 0.54 1.48 4.46E-02 Gpnmb chr6:48986516-49008181 0.10 0.42 1.46 2.78E-02 Anxa3 chr5:97222403-97274987 0.65 2.61 1.40 1.24E-04 Hmha1 chr10:79479416-79494216 0.14 0.55 1.39 5.70E-03 Hk2 chr6:82675020-82724448 0.11 0.43 1.38 5.83E-03 Hk3 chr13:55050792-55122746 0.37 1.39 1.33 3.13E-06 C1qa chr4:136451830-136454759 1.87 6.93 1.31 5.35E-06 Irf8 chr8:123260275-123280592 0.30 1.09 1.30 2.60E-03 Mpeg1 chr19:12535268-12539775 0.21 0.76 1.29 1.49E-03 C1qb chr4:136436060-136442092 0.99 3.55 1.28 1.44E-03 Hpgd chr8:58773348-58799843 1.23 4.37 1.27 5.33E-06 Nupr1 chr7:133766759-133768984 4.60 16.04 1.25 5.61E-07 Timp1 chrX:20425668-20498044 0.54 1.89 1.24 4.21E-02 Lgals3 chr14:47993534-48005842 1.43 4.95 1.24 7.58E-07 Crh chr3:19593400-19595396 0.56 1.90 1.23 2.20E-02 Cyth4 chr15:78427476-78452449 0.42 1.42 1.21 1.15E-03 Ncf1 chr5:134696129-134705495 0.49 1.60 1.18 9.80E-04 Gfap chr11:102748649-102758514 40.07 129.75 1.18 0.00E+00 Nr4a2 chr2:56959636-56976414 0.35 1.11 1.16 1.02E-03 Gbp7 chr3:142193301-142213047 0.11 0.34 1.16 2.01E-02 Pde5a chr3:122432075-122562292 0.11 0.34 1.16 3.13E-02 C4b chr17:34865325-34880842 0.79 2.47 1.15 2.00E-07 Fermt3 chr19:7070620-7093959 0.43 1.33 1.13 5.63E-03 En1 chr1:122499063-122504568 0.67 2.03 1.11 1.10E-03 Ctss chr3:95330707-95360325 2.03 6.08 1.10 8.90E-05 C1qc chr4:136445716-136448829 1.20 3.55 1.09 1.13E-02 Adhfe1 chr1:9538126-9621173 1.08 3.12 1.06 6.91E-07 Dach2 chrX:110411864-110949995 0.26 0.75 1.05 1.31E-03 Phgdh chr3:98117093-98143892 2.18 6.14 1.03 1.79E-05 Slc14a1 chr18:78296829-78338858 0.32 0.90 1.02 1.55E-02 Myo1b chr1:51806609-51972818 0.17 0.46 1.02 3.20E-02 Aldh1l1 chr6:90500841-90549165 2.57 6.99 1.00 1.67E-07 Naprt1 chr15:75721393-75724911 0.71 1.93 1.00 2.06E-02 Vim chr2:13495937-13504453 6.35 17.17 0.99 5.75E-09 Cd9 chr6:125410283-125444773 2.40 6.48 0.99 6.28E-04 Col16a1 chr4:129725083-129776521 0.25 0.67 0.97 2.57E-02 Acsbg1 chr9:54452803-54509692 12.02 31.52 0.96 4.64E-12 Chrnb4 chr9:54875962-54896351 0.32 0.85 0.96 4.39E-02 Neu4 chr1:95917069-95924907 0.33 0.84 0.93 2.38E-02 Ngfr chr11:95430139-95449012 0.55 1.37 0.91 1.67E-02 Pdk4 chr6:5433350-5446278 0.98 2.37 0.89 1.59E-03 Pgm5 chr19:24748497-24936332 0.33 0.78 0.87 6.11E-03 Npy chr6:49772727-49779504 3.46 8.26 0.87 4.25E-02 Tagln2 chr1:174430376-174437506 7.56 18.00 0.87 5.20E-06 Kit chr5:75971011-76052746 0.41 0.98 0.86 6.25E-03 Chrna3 chr9:54859149-54874366 0.58 1.36 0.85 4.08E-02 Padi2 chr4:140462274-140513817 4.52 10.54 0.85 5.56E-08 Acss1 chr2:150443846-150494668 0.55 1.29 0.84 2.83E-02 Rfx4 chr10:84218792-84369283 0.69 1.59 0.84 3.59E-03 Csf1r chr18:61265225-61290793 0.53 1.20 0.83 3.83E-02 Dock1 chr7:141862369-142365330 1.05 2.36 0.81 1.72E-04 Ddc chr11:11714103-11798147 2.69 6.06 0.81 1.32E-03 Papss2 chr19:32670204-32741677 0.56 1.25 0.81 2.17E-02 C3 chr17:57343395-57367559 0.85 1.86 0.79 3.72E-03 Phkg1 chr5:130322865-130354954 1.30 2.83 0.78 2.16E-02 Gpr88 chr3:115952571-115956402 0.66 1.45 0.78 4.93E-02 Arap2 chr5:62993684-63157416 1.43 3.10 0.78 5.58E-05 Dock10 chr1:80497647-80755128 1.20 2.55 0.76 3.00E-04 Cbs chr17:31749588-31774086 2.18 4.62 0.75 5.17E-04 Il33 chr19:29999603-30035205 8.10 17.02 0.74 3.54E-06 Sgk1 chr10:21601989-21719708 23.44 49.10 0.74 0.00E+00 Epas1 chr17:87153203-87232750 1.47 3.07 0.73 1.07E-03 Fam59b chr5:30430123-30481520 1.10 2.28 0.73 1.36E-02 Acsf2 chr11:94418415-94463100 0.99 2.06 0.73 4.46E-02 Cacna1i chr15:80117667-80228722 0.55 1.14 0.72 7.22E-03 Caskin2 chr11:115660665-115674906 0.92 1.88 0.72 1.83E-02 Nfe2l2 chr2:75513575-75542698 1.32 2.71 0.72 3.57E-02 Stbd1 chr5:93032076-93035605 9.28 18.97 0.72 2.38E-05 Gpd1 chr15:99548023-99555438 10.59 21.65 0.72 2.08E-06 Chl1 chr6:103460869-103683029 1.49 3.03 0.71 2.67E-04 Aldh1l2 chr10:82950191-82996885 0.78 1.58 0.71 1.21E-02 Spon1 chr7:120909511-121186889 0.70 1.43 0.71 1.76E-02 Tns3 chr11:8331654-8564538 1.82 3.71 0.71 1.05E-04 Rhpn2 chr7:36119255-36181786 1.49 3.04 0.71 9.81E-03 Stxbp3a chr3:108596097-108643420 3.46 6.98 0.70 2.18E-03 Cnp chr11:100436252-100443053 81.60 164.78 0.70 1.94E-13 Arhgef2 chr3:88420128-88451974 2.01 4.01 0.69 1.30E-05 A2m chr6:121586190-121629256 0.95 1.90 0.69 2.30E-02 Epb4.1l2 chr10:25079603-25243324 1.87 3.73 0.69 7.35E-04 Nbr1 chr11:101413420-101443265 76.73 152.66 0.69 8.30E-09 Slc25a18 chr6:120723785-120744000 6.71 13.30 0.68 2.78E-03 Daam2 chr17:49595346-49703662 3.04 6.01 0.68 7.18E-05 Prex2 chr1:10983545-11293763 0.49 0.96 0.68 2.06E-02 Clic4 chr4:134769884-134828675 8.65 17.02 0.68 6.25E-06 Mlc1 chr15:88786313-88808983 3.88 7.54 0.66 4.32E-03 Txnip chr3:96359690-96370724 1.35 2.61 0.66 1.99E-02 Pygm chr19:6384428-6398459 4.49 8.68 0.66 1.02E-03 Plin3 chr17:56418384-56429934 3.01 5.79 0.66 1.46E-02 Lhfpl2 chr13:94827750-94965364 1.01 1.95 0.65 4.48E-02 Arap1 chr7:108496582-108561100 0.83 1.58 0.65 1.69E-02 Gad1 chr2:70327996-70440069 14.68 28.17 0.65 9.59E-06 Prex1 chr2:166391844-166539332 2.90 5.57 0.65 2.79E-04 Rftn1 chr17:50132631-50329822 2.88 5.53 0.65 7.08E-03 Maob chrX:16286406-16394492 2.53 4.82 0.64 2.36E-02 Rock1 chr18:10064398-10181790 0.74 1.41 0.64 4.45E-02 Psat1 chr19:15979612-15999549 39.79 75.60 0.64 8.22E-07 Nmral1 chr16:4711317-4719059 5.59 10.60 0.64 2.38E-02 Agxt2l1 chr3:130320365-130338668 1.86 3.51 0.63 1.32E-02 Prkd3 chr17:79348744-79420156 0.52 0.98 0.63 3.44E-02 Enc1 chr13:98011059-98022995 3.22 6.02 0.63 1.32E-03 Galnt6 chr15:100520345-100559807 0.83 1.55 0.62 3.73E-02 Phldb1 chr9:44494390-44543281 2.90 5.38 0.62 1.94E-03 Ndrg1 chr15:66760879-66801203 29.57 54.77 0.62 1.42E-05 Efhd1 chr1:89160938-89207366 3.61 6.68 0.62 3.52E-02 Arhgef10 chr8:14911716-15001085 2.59 4.78 0.62 8.59E-05 Phlpp1 chr1:108068445-108290822 4.95 9.10 0.61 2.20E-04 Aspa chr11:73118489-73138136 5.97 10.87 0.60 2.16E-02 Inf2 chr12:113826994-113853768 2.06 3.75 0.60 1.58E-02 Arid5a chr1:36364577-36380874 0.54 0.98 0.59 4.85E-02 Apoe chr7:20281592-20284515 27.74 50.02 0.59 5.29E-04 Dnm2 chr9:21229351-21314630 5.99 10.77 0.59 1.26E-07 Rassf2 chr2:131818585-131855724 3.15 5.67 0.59 4.47E-03 Gpd2 chr2:57090088-57223130 3.11 5.57 0.58 2.62E-04 Rasgrp3 chr17:75835244-75928393 1.07 1.91 0.58 4.93E-02 Serpinb1b chr13:33175971-33186249 15.47 27.66 0.58 7.53E-04 Gal chr19:3409916-3414457 27.55 49.19 0.58 3.27E-03 Acap2 chr16:31092498-31201324 2.30 4.09 0.58 5.28E-03 Gad2 chr2:22477846-22549397 4.83 8.61 0.58 7.64E-04 Lgals9 chr11:78776480-78798426 4.50 8.02 0.58 3.13E-02 Dock4 chr12:41172639-41573075 1.46 2.61 0.58 1.42E-02 Serpinb1a chr13:32933960-32943054 4.40 7.83 0.58 3.25E-02 Car2 chr3:14886425-14900770 57.30 101.93 0.58 7.93E-05 Rasa3 chr8:13567217-13677587 5.82 10.34 0.58 1.38E-03 Klk6 chr7:51079913-51087397 6.12 10.88 0.58 1.02E-02 Plcl1 chr1:55462789-55811129 3.21 5.70 0.57 1.78E-03 Cdkn1a chr17:29227930-29237667 4.69 8.32 0.57 2.77E-02 Rapgef3 chr15:97575200-97598097 1.69 2.99 0.57 3.09E-02 Clmn chr12:106001323-106103286 0.76 1.33 0.56 6.10E-03 Camk2a chr18:61085285-61147806 5.18 9.04 0.56 4.46E-05 Carhsp1 chr16:8658679-8672246 9.66 16.83 0.55 1.26E-03 Erbb2ip chr13:104608865-104710594 2.38 4.12 0.55 5.14E-03 Sox10 chr15:78985342-78994920 3.38 5.81 0.54 4.82E-02 Myo6 chr9:80012840-80159536 1.53 2.63 0.54 1.99E-02 Fam40b chr6:29867012-29909680 1.18 2.03 0.54 3.54E-02 Trf chr9:103111205-103132616 65.17 111.79 0.54 2.71E-04 Ddit3 chr10:126727848-126748842 30.43 51.54 0.53 4.25E-03 Aldoc chr11:78137699-78140262 328.25 553.91 0.52 3.32E-03 Slc32a1 chr2:158436493-158441483 16.43 27.68 0.52 1.98E-03 Mag chr7:31684201-31699851 26.43 44.25 0.52 1.08E-03 Eno1 chr4:149611305-149622982 40.44 67.50 0.51 1.26E-03 Fmnl2 chr2:52716901-52993236 2.27 3.76 0.51 3.60E-02 Acot11 chr4:106406519-106472436 2.52 4.18 0.51 3.35E-02 Gab1 chr8:83288332-83404378 2.93 4.85 0.50 3.75E-02 Ermn chr2:57897525-57905163 6.70 11.07 0.50 1.03E-02 Baiap3 chr17:25377114-25393309 5.49 9.02 0.50 1.00E-02 Gm98 chr19:10282760-10315238 3.36 5.52 0.50 2.39E-02 Pdxk chr10:77899491-77927693 10.43 17.06 0.49 2.79E-03 Ppp2r3a chr9:101007319-101154162 1.68 2.75 0.49 2.22E-02 Ifrd1 chr12:40929715-40949776 12.17 19.74 0.48 2.04E-02 Tubb2b chr13:34218876-34222223 16.70 27.09 0.48 1.37E-02 Mllt4 chr17:13853442-14042801 2.76 4.48 0.48 2.68E-02 Trim66 chr7:116592514-116651648 1.12 1.81 0.48 3.80E-03 Slc17a6 chr7:58877199-58926496 11.61 18.69 0.48 5.57E-03 Rasgrf2 chr13:92020011-92901449 5.84 9.40 0.48 9.70E-03 Add3 chr19:53214934-53321889 6.19 9.93 0.47 2.78E-06 Marcks chr10:36853048-36858732 6.90 11.04 0.47 1.65E-02 Gabra1 chr11:41944981-41996432 4.61 7.38 0.47 4.52E-02 Adssl1 chr12:113858257-113879566 14.58 23.29 0.47 2.49E-02 Glul chr1:155747074-155756844 163.46 259.56 0.46 1.12E-02 Camkv chr9:107838250-107852022 10.60 16.82 0.46 1.83E-02 Anln chr9:22135657-22193650 5.51 8.70 0.46 1.94E-02 Cldn11 chr3:31048841-31063248 23.92 37.22 0.44 1.66E-02 Slc7a5 chr8:124405045-124431586 10.39 16.11 0.44 2.49E-02 Hsd17b4 chr18:50287854-50355924 11.22 17.33 0.43 3.62E-02 Zzz3 chr3:152058973-152344396 2.26 3.49 0.43 2.39E-02 Rnh1 chr7:148346224-148358750 26.03 40.16 0.43 7.01E-03 Nptx1 chr11:119400032-119409134 7.33 11.30 0.43 3.01E-02 Efr3a chr15:65618602-65705374 17.45 26.88 0.43 9.48E-03 Mat2a chr6:72382792-72390609 20.93 32.21 0.43 1.60E-02 Ppp6r3 chr19:3454927-3575749 4.58 7.05 0.43 3.81E-02 Fscn1 chr5:143722033-143734868 20.83 31.85 0.42 2.20E-02 Ahi1 chr10:20672352-20800235 36.20 55.14 0.42 3.11E-04 Vgf chr5:137506164-137509221 72.30 109.73 0.42 1.12E-02 Ebf3 chr7:144385353-144506128 3.85 5.76 0.40 9.23E-03 Sept2 chr1:95375569-95406309 6.66 9.97 0.40 2.54E-02 Wars chr12:110098239-110132384 49.14 73.22 0.40 2.43E-04 Gap43 chr16:42248673-42340764 138.60 205.99 0.40 2.49E-02 Spg20 chr3:54916029-54941254 13.40 19.86 0.39 3.72E-04 Enpp2 chr15:54670452-54751701 17.06 25.25 0.39 1.76E-02 Abat chr16:8513521-8621660 27.94 41.08 0.39 1.84E-03 Ank3 chr10:68996455-69490184 6.44 9.40 0.38 1.53E-04 Anp32e chr3:95733179-95751310 16.82 24.50 0.38 1.26E-02 Grb10 chr11:11830501-11937423 8.99 12.93 0.36 1.24E-02 Mbp chr18:82644514-82755029 104.67 150.50 0.36 1.27E-05 Mon2 chr10:122429116-122513561 2.85 4.09 0.36 5.75E-03 Ssrp1 chr2:84877357-84887268 16.34 23.43 0.36 1.55E-02 Llgl1 chr11:60513191-60540723 8.50 12.19 0.36 3.14E-03 Gtf2i chr5:134713703-134790616 19.60 27.28 0.33 1.81E-04 Ralgds chr2:28368686-28408602 9.85 13.63 0.32 1.92E-02 Ndrg2 chr14:52524945-52533163 123.08 166.49 0.30 1.39E-02 Ddx17 chr15:79346837-79377171 26.14 34.48 0.28 4.46E-02 Sept8 chr11:53333237-53357598 25.37 32.93 0.26 1.54E-02 Rap1gap chr4:137220640-137285776 36.21 46.01 0.24 2.25E-02 Add1 chr5:34916362-34974954 56.05 69.52 0.22 3.62E-02 Genes downregulated in SOD1 G37R motor neurons Ppp2r2b chr18:42804922-43219125 404.29 298.21 -0.30 8.74E-03 Fam78b chr1:168931547-169021433 20.66 15.12 -0.31 2.07E-02 Lin7a chr10:106708886-106862199 13.96 10.19 -0.32 2.68E-02 Lrrcc1 chr3:14533787-14572658 8.96 6.46 -0.33 2.16E-02 Rpl38 chr11:114529856-114533645 570.51 408.73 -0.33 1.50E-02 Caln1 chr5:130845327-131316515 45.09 32.22 -0.34 1.20E-02 Cd59a chr2:103935957-103955508 93.01 64.59 -0.36 3.46E-02 Atox1 chr11:55260144-55274640 568.68 394.62 -0.37 4.42E-02 Rpl30 chr15:34370260-34373031 106.09 73.35 -0.37 3.86E-02 Rps24 chr14:25309902-25315368 226.14 154.87 -0.38 7.62E-04 Pcp4 chr16:96689212-96747400 475.32 324.65 -0.38 3.03E-02 Rpl14 chr9:120480633-120483770 315.69 215.36 -0.38 2.52E-02 Cryab chr9:50560862-50564738 343.68 231.40 -0.40 1.99E-02 Hddc2 chr10:31033210-31047892 100.39 67.50 -0.40 3.83E-02 Rpl35a chr16:33056538-33060274 60.75 40.70 -0.40 2.81E-02 Mctp1 chr13:76522408-77171071 18.70 12.44 -0.41 2.81E-02 Rab3b chr4:108551674-108644682 63.67 42.32 -0.41 1.15E-02 Rxrg chr1:169528492-169569754 14.18 9.39 -0.41 4.32E-02 Rpl37 chr15:5066612-5069140 174.41 115.05 -0.42 3.78E-02 Rps8 chr4:116826440-116828737 80.04 52.39 -0.42 3.40E-02 Rgs11 chr17:26339906-26348269 21.44 14.00 -0.43 4.69E-02 Lrrc49 chr9:60435043-60535941 194.06 125.00 -0.44 4.00E-04 Rgs7bp chr13:105737232-105845010 57.86 37.14 -0.44 4.27E-03 Polr3g chr13:81812835-81850012 31.60 20.01 -0.46 5.15E-03 Prss12 chr3:123149830-123209520 42.11 26.60 -0.46 3.41E-03 Chrm2 chr6:36283137-36474774 10.87 6.58 -0.50 4.46E-02 Itm2a chrX:104592533-104598699 60.85 36.10 -0.52 5.08E-04 Mrvi1 chr7:118011780-118125609 15.18 8.98 -0.53 4.42E-04 Uhmk1 chr1:172129386-172145524 26.21 15.42 -0.53 3.31E-03 Tcp11l1 chr2:104520136-104552319 5.91 3.30 -0.58 1.04E-02 Zfp672 chr11:58128615-58143841 27.53 15.31 -0.59 7.42E-04 Hsd17b7 chr1:171879667-171899336 11.61 6.36 -0.60 2.33E-04 Gpr146 chr5:139835692-139936488 2.02 1.06 -0.64 2.71E-02 Rbm3 chrX:7716100-7725089 10.16 5.28 -0.65 1.60E-02 Akap9 chr5:3928185-4080204 2.53 1.32 -0.65 2.02E-03 Zfp605 chr5:110539110-110558813 2.98 1.51 -0.68 3.72E-02 Ppp1r1b chr11:98210051-98219597 23.27 11.33 -0.72 2.16E-05 Runx2 chr17:44740949-45256233 0.44 0.15 -1.12 1.12E-02 Supplementary Table 3: Main functional categories enriched in SOD1G37R induced gene changes in motor neurons. GO categories GO term Benjamini All genes Cellular GO:0045202 synapse 0.002025175 component GO:0030054 cell junction 0.00762269 Upregulated genes GO:0030170 pyridoxal phosphate binding 0.002590255 GO:0070279 vitamin B6 binding 0.002590255 Molecular GO:0003779 binding 0.012795866 function GO:0005516 calmodulin binding 0.014144813 guanyl-nucleotide exchange GO:0005085 factor activity 0.01536069 GO:0006006 glucose metabolic process 0.01030087 GO:0019318 hexose metabolic process 0.026219843 monosaccharide metabolic GO:0005996 process 0.034273322 GO:0002443 leukocyte mediated immunity 0.009494858 Biological lymphocyte mediated process GO:0002449 immunity 0.030247327 complement activation, GO:0006958 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 structural constituent of function GO:0003735 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 Genes upregulated in SOD1 G37R astrocytes Ccl6 chr11:83395562-83437195 0.04 1.03 3.38 6.66E-03 Htr1b chr9:81524998-81538763 0.01 0.14 2.40 2.02E-03 Tgm1 chr14:56318845-56332329 0.12 1.12 2.20 1.14E-05 Hspb1 chr5:136363788-136365433 28.29 193.42 1.92 0.00E+00 Fkbp5 chr17:28536039-28623057 7.78 50.13 1.86 0.00E+00 Lamc2 chr1:154969885-155033577 0.05 0.29 1.72 4.42E-02 Timp1 chrX:20425668-20498044 2.68 14.36 1.68 9.85E-11 Arl4d chr11:101526854-101529146 9.02 46.21 1.63 2.13E-11 1810011O10Rik chr8:25548087-25549418 6.80 34.14 1.61 2.04E-11 Wisp2 chr2:163646569-163658883 0.88 4.37 1.61 2.34E-05 Ecm1 chr3:95538070-95543492 0.50 2.47 1.61 2.92E-05 Serpinf2 chr11:75245237-75253003 0.20 0.93 1.53 4.33E-02 Crym chr7:127329897-127345502 0.85 3.58 1.44 3.40E-03 Lyz2 chr10:116714596-116719328 1.67 6.78 1.40 5.23E-04 Pla2g4e chr2:119992147-120071071 0.21 0.81 1.36 8.92E-03 Adamtsl2 chr2:26934900-26964133 0.42 1.57 1.32 3.49E-03 S100a4 chr3:90407691-90409967 25.45 92.93 1.30 2.53E-08 Tekt4 chr17:25608534-25613539 7.81 26.56 1.22 5.17E-07 Maff chr15:79178107-79189506 1.96 6.64 1.22 2.92E-04 Fcgr2b chr1:172890688-172906202 0.84 2.75 1.18 4.33E-02 Cebpd chr16:15887378-16146926 6.22 20.22 1.18 4.20E-07 Tcf23 chr5:31271049-31279391 0.40 1.25 1.13 2.05E-02 Socs3 chr11:117827400-117830680 0.98 3.04 1.13 4.42E-03 Lgals3 chr14:47993534-48005842 34.84 107.19 1.12 3.32E-14 Map3k6 chr4:132796732-132808843 0.38 1.16 1.11 2.59E-02 Cebpb chr2:167514414-167515918 10.45 31.69 1.11 4.14E-06 Anxa3 chr5:97222403-97274987 3.15 9.46 1.10 1.06E-03 Rasl11a chr5:147656646-147659302 4.62 13.82 1.10 7.65E-04 Trem2 chr17:48485725-48491598 1.48 4.40 1.09 4.71E-02 Tyrobp chr7:31198806-31202598 6.56 19.49 1.09 5.04E-03 Tagln chr9:45671773-45744141 15.12 44.20 1.07 9.73E-07 Lrrc33 chr16:32142910-32165562 2.47 7.17 1.07 1.89E-04 C1qa chr4:136451830-136454759 6.52 18.92 1.07 4.75E-04 C1qb chr4:136436060-136442092 6.09 17.63 1.06 7.17E-04 Junb chr8:87500807-87502647 4.02 11.62 1.06 3.67E-04 Serpina3n chr12:105644917-105652539 25.39 72.28 1.05 1.44E-06 Cxcl10 chr5:92760866-92843653 3.11 8.78 1.04 1.13E-02 C1qc chr4:136445716-136448829 5.21 14.56 1.03 2.50E-03 Prss23 chr7:96656294-96666096 0.98 2.72 1.02 1.80E-02 Slc37a1 chr17:31432427-31487643 0.57 1.55 1.00 2.31E-02 Nupr1 chr7:133766759-133768984 6.45 17.03 0.97 1.98E-02 Mthfd2 chr6:83255697-83267598 34.16 88.44 0.95 1.03E-05 Plin4 chr17:56219079-56249225 0.87 2.25 0.95 9.04E-03 Tagln2 chr1:174430376-174437506 24.54 63.11 0.94 2.80E-05 Tm4sf1 chr3:57090985-57105841 6.90 17.11 0.91 1.17E-03 Sult1a1 chr7:133816383-133819871 5.99 14.38 0.88 1.04E-02 Sgk1 chr10:21601989-21719708 27.25 62.68 0.83 1.31E-06 Iigp1 chr18:60535682-60552283 1.04 2.38 0.83 4.72E-02 Vim chr2:13495937-13504453 136.47 312.10 0.83 1.25E-03 S100a11 chr3:93324417-93330210 32.62 73.78 0.82 6.62E-03 Ctss chr3:95330707-95360325 7.26 16.10 0.80 3.50E-02 Arrdc2 chr8:73359036-73363619 12.83 28.28 0.79 1.69E-03 Zbtb16 chr9:48462401-48644050 2.80 6.01 0.76 2.11E-02 Hr chr14:70953862-70973349 28.73 61.59 0.76 3.88E-03 Cdkn1a chr17:29227930-29237667 139.85 298.90 0.76 5.10E-03 Gadd45g chr13:51942043-51943843 160.40 342.24 0.76 4.96E-03 Igfbp7 chr5:77739508-77837070 10.79 22.95 0.75 2.83E-02 Inpp5d chr1:89516886-89617083 0.48 1.03 0.75 3.32E-02 Tmem52 chr4:154843222-154844967 18.15 37.23 0.72 4.67E-03 S100a6 chr3:90416815-90418336 300.99 614.31 0.71 8.93E-03 2010002N04Rik chr18:60633844-60661637 62.68 126.75 0.70 1.43E-02 Emd chrX:71500177-71503086 64.90 124.94 0.65 2.57E-02 Nfkbia chr12:56590395-56593634 37.87 71.44 0.63 3.24E-02 Mylk chr16:34785035-35002520 6.18 11.40 0.61 4.83E-02 Txnip chr3:96359690-96370724 18.95 34.79 0.61 1.09E-03 Anxa2 chr9:69301489-69339592 42.12 77.12 0.60 4.31E-02 Crem chr18:3266351-3366861 12.34 21.35 0.55 3.76E-05 Genes downregulated in SOD1 G37R astrocytes Tcf12 chr9:71692058-71959626 25.94 18.71 -0.33 2.49E-02 Fnbp1 chr2:30881725-30997528 58.51 41.93 -0.33 6.28E-03 Mbnl1 chr3:60276751-60433670 23.21 14.98 -0.44 5.74E-05 Prkd3 chr17:79348744-79420156 10.49 6.69 -0.45 2.69E-02 Sema6d chr2:124436031-124493506 15.04 9.34 -0.48 6.39E-04 Kazn chr4:141658304-141795316 37.89 23.26 -0.49 2.93E-04 Nfix chr8:87231497-87324239 45.31 27.21 -0.51 1.50E-02 Prr5l chr2:101554441-101679137 37.67 22.35 -0.52 4.64E-02 Atl2 chr17:80247731-80295463 38.50 22.70 -0.53 3.85E-02 Anks1b chr10:89336253-90435729 10.10 5.77 -0.56 4.07E-04 Sertad2 chr11:20443255-20553026 12.40 7.01 -0.57 1.14E-02 D16Ertd472e chr16:78540580-78576933 6.00 3.37 -0.58 1.38E-03 Insig1 chr5:28397951-28405202 64.65 35.06 -0.61 4.72E-02 Cpeb2 chr5:43542924-43680963 11.76 6.37 -0.61 1.13E-03 Fam53b chr7:139903766-140004879 12.77 6.90 -0.62 2.53E-03 Aven chr2:112333120-112471410 69.40 37.24 -0.62 1.50E-03 Mob3b chr4:34896322-35104733 14.49 7.74 -0.63 4.31E-02 Ppargc1a chr5:51845487-51945160 10.75 5.67 -0.64 6.87E-03 Fam123a chr14:60997122-60999840 31.61 16.65 -0.64 1.28E-03 Rasgrp3 chr17:75835244-75928393 13.20 6.90 -0.65 2.19E-02 Prrx1 chr1:165175249-165243781 8.16 4.27 -0.65 3.76E-04 Heyl chr4:122910798-122927113 32.27 16.81 -0.65 1.55E-02 Gtf2ird1 chr5:134833530-134932586 6.17 3.22 -0.65 2.16E-07 Cdc42ep1 chr15:78673076-78685959 78.05 39.96 -0.67 1.13E-02 2810459M11Rik chr1:87942437-87952031 19.54 9.97 -0.67 1.22E-03 Olig2 chr16:91225794-91228922 45.59 22.69 -0.70 8.93E-03 Fbxo32 chr15:58007433-58046447 4.54 2.25 -0.70 4.69E-02 Trib2 chr12:15798532-15823591 28.60 14.13 -0.71 5.81E-03 Foxn2 chr17:88840051-88889873 8.91 4.36 -0.71 1.80E-02 Plekho2 chr9:65402190-65427841 91.66 44.63 -0.72 3.49E-03 Bcl9l chr9:44307218-44318495 4.95 2.37 -0.74 4.83E-02 Map6d1 chr16:20233381-20241431 19.53 9.28 -0.74 6.11E-03 Wnt7a chr6:91313976-91361363 12.38 5.86 -0.75 1.81E-02 Trim36 chr18:46324953-46372261 9.80 4.42 -0.80 3.24E-03 Glmn chr5:107963729-108090857 5.08 2.25 -0.81 2.66E-02 Ddit4l chr3:137286635-137291296 52.06 22.57 -0.84 2.82E-04 Trim59 chr3:68839215-68848664 11.89 4.88 -0.89 1.97E-03 Smtnl2 chr11:72203615-72225215 5.67 2.32 -0.89 2.24E-02 Fam84b chr15:60650550-60662955 2.93 1.09 -0.99 3.40E-03 Fam181a chr12:104553168-104555275 21.55 7.32 -1.08 1.25E-04 Kif20a chr18:34758268-34811390 1.19 0.39 -1.11 5.96E-03 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 GO:0002250 adaptive immune response 0.006877283 GO:0002526 acute inflammatory response 0.003692776 immunoglobulin mediated GO:0016064 immune response 0.011421196 Biological GO:0019724 B cell mediated immunity 0.010011371 process lymphocyte mediated GO:0002449 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 Genes upregulated in SOD1 G37R oligodendrocytes Pla2g4e chr2:119992147‐120071071 0.40 2.35 1.77 6.11E‐12 Socs3 chr11:117827400‐117830680 0.33 1.35 1.39 6.80E‐04 Bcl3 chr7:20393810‐20408104 0.76 2.91 1.35 4.25E‐05 Trib1 chr15:59480208‐59488654 0.97 2.24 0.84 2.58E‐03 Lgals3 chr14:47993534‐48005842 1.56 3.40 0.78 3.66E‐02 Fmn1 chr2:113167892‐113556924 0.62 1.29 0.73 8.13E‐04 Serpina3n chr12:105644917‐105652539 38.84 77.03 0.68 3.98E‐06 Vim chr2:13495937‐13504453 9.00 16.34 0.60 4.60E‐03 C4b chr17:34865325‐34880842 13.29 22.30 0.52 3.22E‐03 Spna2 chr2:29821079‐29886970 9.65 14.68 0.42 1.10E‐04 Plec chr15:76001403‐76061808 0.99 1.46 0.39 1.55E‐02 Arhgef40 chr14:52604507‐52640408 3.79 5.56 0.38 2.33E‐02 Madd chr2:90977516‐91023204 10.48 14.42 0.32 1.41E‐02 Rap1gap chr4:137220640‐137285776 32.52 42.19 0.26 1.65E‐02 Genes downregulated in SOD1 G37R oligodendrocytes Fam123a chr14:60997122‐60999840 25.61 15.39 ‐0.51 2.59E‐04 D4Wsu53e chr4:134479539‐134483285 122.26 73.27 ‐0.51 4.97E‐03 Nfkbia chr12:56590395‐56593634 45.45 26.88 ‐0.53 1.60E‐02 Hspa1a chr17:35106303‐35109101 10.27 5.98 ‐0.54 1.89E‐02 Aven chr2:112333120‐112471410 65.64 37.67 ‐0.56 3.79E‐05 Arl4d chr11:101526854‐101529146 176.53 100.28 ‐0.57 8.93E‐03 Dnajb1 chr8:86132073‐86135802 64.55 35.48 ‐0.60 1.66E‐04 Sgk1 chr10:21601989‐21719708 728.50 378.12 ‐0.66 7.99E‐04 Ccne2 chr4:11118500‐11181406 1.06 0.39 ‐1.01 1.05E‐02 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 Genes upregulated in SOD1 G37R oligodendrocytes AI607873 chr1:175653558‐175671940 0.01 0.46 3.50 2.88E‐03 Trib3 chr2:152163160‐152169796 0.12 3.57 3.42 2.65E‐10 Tgm1 chr14:56318845‐56332329 0.07 2.21 3.39 4.80E‐08 Cst7 chr2:150396150‐150404680 0.13 3.03 3.18 6.54E‐05 Mmp12 chr9:7347373‐7360461 0.02 0.40 3.18 2.85E‐03 Lilrb4 chr10:51210780‐51216417 0.03 0.72 3.16 2.27E‐02 Clec7a chr6:129411608‐129422795 0.15 2.87 2.93 3.86E‐10 Cybb chrX:9012377‐9046450 0.03 0.51 2.91 1.26E‐04 Gpnmb chr6:48986516‐49008181 0.19 2.69 2.66 1.76E‐12 Lyz2 chr10:116714596‐116719328 2.66 37.91 2.66 0.00E+00 Lyz1 chr10:116724850‐116729924 0.23 3.24 2.64 1.92E‐06 Serpina3m chr12:105625373‐105632467 0.08 0.82 2.37 6.71E‐03 Pbk chr14:66424747‐66436659 0.09 0.84 2.29 9.59E‐03 Wisp2 chr2:163646569‐163658883 0.11 1.05 2.27 1.86E‐03 Arhgap25 chr6:87409378‐87483229 0.19 1.75 2.23 1.79E‐05 Cd22 chr7:31650422‐31665361 0.04 0.36 2.23 4.87E‐03 Ccl3 chr11:83461344‐83462880 0.15 1.34 2.18 4.10E‐02 Lgals3 chr14:47993534‐48005842 2.59 22.07 2.14 5.27E‐10 Yes Rgs1 chr1:146091798‐146096234 0.09 0.76 2.11 3.44E‐02 Fcrls chr3:87054886‐87067446 0.78 6.39 2.10 8.08E‐11 Top2a chr11:98854260‐98885503 0.06 0.48 2.10 3.34E‐04 Klhl6 chr16:19946585‐19983122 0.35 2.89 2.10 3.26E‐08 Atf3 chr1:192994175‐193007212 0.38 3.08 2.09 2.71E‐07 Ly9 chr1:173518743‐173537491 0.07 0.56 2.09 9.66E‐03 Pla2g4e chr2:119992147‐120071071 0.51 4.15 2.09 0.00E+00 Yes Myo1f chr17:33692651‐33744709 0.61 4.91 2.08 0.00E+00 Bcl3 chr7:20393810‐20408104 0.81 6.44 2.07 2.05E‐10 Yes Socs3 chr11:117827400‐117830680 0.40 3.10 2.04 9.08E‐09 Yes Osmr chr15:6763576‐6824313 0.07 0.52 2.02 3.83E‐04 Crym chr7:127329897‐127345502 0.33 2.49 2.01 2.36E‐04 Fam46c chr3:100275458‐100293115 0.31 2.27 2.00 1.36E‐05 Irf8 chr8:123260275‐123280592 0.88 6.52 2.00 7.21E‐13 Vav1 chr17:57418522‐57468659 0.47 3.44 2.00 5.49E‐06 Cd48 chr1:173612185‐173635388 0.15 1.07 1.99 3.17E‐02 Apobec1 chr6:122527809‐122552462 0.21 1.57 1.99 1.11E‐04 Slfn8 chr11:82815659‐82834312 0.07 0.51 1.99 9.93E‐03 Itgax chr7:135273081‐135294171 0.14 0.99 1.98 2.66E‐05 Bcl2a1b chr9:89094110‐89102676 0.46 3.31 1.98 1.98E‐03 Capg chr6:72494432‐72512974 1.78 12.93 1.98 2.45E‐10 Ch25h chr19:34548273‐34549625 0.12 0.88 1.97 2.96E‐02 Slfn2 chr11:82878613‐82884180 0.23 1.63 1.97 4.18E‐04 Slfn5 chr11:82724754‐82778352 0.09 0.63 1.96 1.25E‐04 Ptpn6 chr6:124670735‐124688727 1.25 8.84 1.96 1.76E‐12 Tnfsf11 chr14:78677252‐78707850 0.17 1.18 1.94 8.99E‐04 Cd68 chr11:69477872‐69479564 1.09 7.60 1.94 3.85E‐07 Dock2 chr11:34126863‐34597325 0.42 2.90 1.94 5.91E‐08 Col5a3 chr9:20574493‐20619478 0.12 0.87 1.94 1.92E‐06 Mpeg1 chr19:12535268‐12539775 0.91 6.27 1.93 0.00E+00 Aspg chr12:113344893‐113365784 0.43 2.99 1.93 5.93E‐08 Anxa3 chr5:97222403‐97274987 1.86 12.83 1.93 7.21E‐13 Rasal3 chr17:32527605‐32541753 0.39 2.61 1.90 2.47E‐08 Trim30a chr7:111557539‐111613707 0.15 0.97 1.89 6.75E‐05 Samsn1 chr16:75859038‐75909511 0.24 1.59 1.88 3.98E‐04 Cd180 chr13:103483637‐103496711 0.15 0.95 1.86 2.63E‐03 Hlx chr1:186551023‐186556372 0.11 0.69 1.86 1.87E‐02 Atp6v0d2 chr4:19803984‐19849713 0.11 0.69 1.85 7.38E‐03 Myzap chr9:71352153‐71440167 0.68 4.28 1.84 2.56E‐08 Serpina3n chr12:105644917‐105652539 56.92 357.60 1.84 0.00E+00 Yes Arhgap30 chr1:173319090‐173340370 0.20 1.27 1.83 4.29E‐06 Vim chr2:13495937‐13504453 13.63 84.31 1.82 0.00E+00 Yes Gbp2 chr3:142283626‐142300972 0.63 3.79 1.80 5.93E‐08 Blnk chr19:41003416‐41069025 0.27 1.60 1.79 3.59E‐04 Nckap1l chr15:103284255‐103329231 1.13 6.74 1.79 0.00E+00 Ifi204 chr1:175677424‐175697050 0.16 0.93 1.77 3.76E‐03 Fgr chr4:132530009‐132557797 0.10 0.60 1.76 4.85E‐03 Ifi44 chr3:151393886‐151412923 0.06 0.37 1.76 4.33E‐02 Gbp5 chr3:142159897‐142185308 0.17 0.98 1.75 6.61E‐04 Tagln2 chr1:174430376‐174437506 4.44 25.22 1.74 0.00E+00 Naip2 chr13:100914017‐100972047 0.13 0.73 1.73 3.47E‐04 Cd52 chr4:133649452‐133650988 1.40 7.91 1.73 1.72E‐03 Cyth4 chr15:78427476‐78452449 1.39 7.75 1.72 3.01E‐11 Psd4 chr2:24240916‐24264249 0.14 0.80 1.72 1.67E‐04 Pik3ap1 chr19:41348707‐41459560 0.35 1.95 1.71 4.08E‐05 Rhoh chr5:66254807‐66287939 0.06 0.34 1.71 9.84E‐03 Klk8 chr7:51052946‐51059192 0.56 3.09 1.71 2.59E‐04 Casp12 chr9:5345475‐5373034 0.34 1.89 1.71 3.46E‐05 Hpgd chr8:58773348‐58799843 3.11 17.08 1.71 7.21E‐13 Lpl chr8:71404453‐71430831 0.67 3.67 1.70 1.81E‐09 Arl11 chr14:61928589‐61930773 0.26 1.40 1.69 7.17E‐03 Sh3tc1 chr5:36039828‐36071925 0.11 0.58 1.69 2.13E‐03 Dock8 chr19:25074018‐25276922 0.26 1.39 1.68 3.71E‐08 Fyb chr15:6529870‐6613312 0.18 0.96 1.68 2.03E‐03 Icam1 chr9:20820403‐20833240 0.10 0.54 1.66 2.90E‐02 Tlr7 chrX:163742862‐163768460 0.09 0.48 1.66 1.08E‐02 BC013712 chr4:132338271‐132352279 0.18 0.97 1.65 3.68E‐03 Slc4a11 chr2:130509843‐130523255 0.29 1.50 1.65 8.68E‐05 Hcls1 chr16:36935068‐36963300 0.53 2.75 1.65 3.82E‐05 Sfpi1 chr2:90936953‐90955913 1.24 6.43 1.65 2.85E‐06 Lpxn chr19:12873098‐12908298 0.36 1.85 1.64 1.43E‐03 Fcgr2b chr1:172890688‐172906202 0.39 1.97 1.62 3.29E‐03 Ncf4 chr15:78075240‐78093010 0.29 1.46 1.62 9.90E‐03 Myo1g chr11:6406550‐6420961 0.10 0.50 1.61 2.29E‐02 Sash3 chrX:45499703‐45514740 0.17 0.82 1.58 7.83E‐03 Wdfy4 chr14:33772732‐33998252 0.09 0.43 1.58 6.40E‐04 Egr2 chr10:67000616‐67004936 0.12 0.58 1.57 1.97E‐02 Nes chr3:87775014‐87784373 0.18 0.86 1.57 5.89E‐05 Lrrk1 chr7:73403632‐73533227 0.15 0.71 1.56 8.75E‐05 C3ar1 chr6:122797157‐122806175 0.11 0.51 1.56 7.25E‐03 Uba7 chr9:107877897‐107886387 0.20 0.96 1.56 2.41E‐03 Sprr1a chr3:92287875‐92289803 0.44 2.08 1.55 4.45E‐02 Plcb2 chr2:118533252‐118554174 0.11 0.52 1.55 3.82E‐03 Psmb8 chr17:34335139‐34338399 2.14 9.98 1.54 6.25E‐07 Ecm1 chr3:95538070‐95543492 0.20 0.92 1.54 3.25E‐02 Apobec3 chr15:79719961‐79738859 0.41 1.88 1.53 5.60E‐04 Gadl1 chr9:115818572‐115985294 0.09 0.43 1.52 2.52E‐02 Itgb2 chr10:76993092‐77028419 0.58 2.64 1.52 1.92E‐05 C1qa chr4:136451830‐136454759 12.10 54.63 1.51 1.34E‐12 Pik3cg chr12:32858261‐32893514 0.27 1.20 1.49 8.99E‐04 Nlrc5 chr8:96996662‐97051172 0.09 0.40 1.49 4.76E‐03 Tmem106a chr11:101443555‐101453099 0.14 0.63 1.48 4.30E‐02 Lyn chr4:3605267‐3718759 0.56 2.45 1.48 1.01E‐02 Fmod chr1:135934091‐135944854 0.58 2.54 1.48 3.59E‐05 Inpp5d chr1:89516886‐89617083 1.15 5.05 1.48 1.62E‐06 Eif4ebp1 chr8:28370798‐28386128 0.51 2.24 1.47 2.12E‐02 Arap3 chr18:38132276‐38158623 0.23 1.00 1.47 5.61E‐03 Rasl11a chr5:147656646‐147659302 0.69 2.97 1.46 5.85E‐03 Parp14 chr16:35832963‐35871468 0.16 0.71 1.46 2.02E‐04 Hmha1 chr10:79479416‐79494216 0.78 3.31 1.45 4.27E‐05 Parvg chr15:84155149‐84173408 0.35 1.50 1.45 1.77E‐03 Pion chr5:20692084‐20797519 0.26 1.08 1.44 1.05E‐03 Mdfic chr6:15670660‐15752169 0.14 0.59 1.44 1.73E‐02 Cd109 chr9:78463352‐78564067 0.15 0.62 1.43 1.66E‐03 Trem2 chr17:48485725‐48491598 2.19 9.14 1.43 1.94E‐05 Fes chr7:87522643‐87532832 0.44 1.82 1.43 5.24E‐04 Cd84 chr1:173769827‐173820849 0.18 0.74 1.43 1.21E‐02 Ptprc chr1:139959437‐140071882 0.07 0.30 1.42 4.40E‐02 Arhgdib chr6:136872229‐136890238 1.63 6.74 1.42 8.68E‐05 Ccl6 chr11:83395562‐83437195 0.34 1.39 1.42 3.35E‐02 Parp9 chr16:35926600‐35972707 0.36 1.46 1.41 1.63E‐02 Mlxipl chr5:135582760‐135614252 0.13 0.54 1.41 2.25E‐02 Tlr13 chrX:103338613‐103355832 0.10 0.43 1.41 3.79E‐02 Akr1b8 chr6:34304163‐34318454 0.56 2.27 1.40 8.12E‐03 Alox5 chr6:116360088‐116411196 0.20 0.79 1.40 2.18E‐02 Aif1 chr17:35307936‐35312946 1.74 6.98 1.39 6.58E‐04 Ifit1 chr19:34715378‐34724499 0.84 3.37 1.39 2.21E‐05 Rin3 chr12:103521283‐103629064 0.18 0.73 1.39 8.69E‐03 Nlrp3 chr11:59356187‐59380458 0.10 0.41 1.39 4.28E‐02 Fermt3 chr19:7070620‐7093959 1.38 5.52 1.38 4.91E‐03 Tyrobp chr7:31198806‐31202598 6.76 26.91 1.38 1.18E‐05 Gna15 chr10:80965058‐80986970 0.63 2.50 1.38 1.21E‐03 Cxcl16 chr11:70267735‐70279701 0.23 0.89 1.37 3.23E‐02 Ang chr14:51710751‐51725826 2.51 9.79 1.36 5.38E‐04 Lag3 chr6:124854376‐124861723 0.73 2.82 1.35 7.01E‐04 Casp1 chr9:5298516‐5307281 0.77 2.97 1.35 1.98E‐03 Trib1 chr15:59480208‐59488654 1.09 4.17 1.34 5.12E‐07 Yes Adamtsl3 chr7:89484203‐89762958 0.12 0.46 1.34 4.76E‐03 Itpripl2 chr7:125628625‐125635489 0.07 0.27 1.34 3.97E‐02 Snx20 chr8:91150726‐91160027 0.68 2.58 1.34 7.00E‐03 Hspb1 chr5:136363788‐136365433 6.99 26.72 1.34 2.73E‐07 Ncf1 chr5:134696129‐134705495 1.83 6.98 1.34 3.23E‐07 Gfap chr11:102748649‐102758514 154.40 587.08 1.34 1.57E‐08 Hpgds chr6:65067286‐65094724 0.42 1.59 1.34 6.89E‐04 Ctss chr3:95330707‐95360325 11.72 44.40 1.33 1.86E‐10 Irf5 chr6:29476624‐29487320 0.77 2.88 1.32 4.36E‐04 Hck chr2:152934203‐152977177 0.47 1.76 1.32 5.43E‐03 Was chrX:7658591‐7667617 0.26 0.96 1.32 4.08E‐02 Plscr2 chr9:92170439‐92192590 1.17 4.32 1.31 2.02E‐04 Tnfaip2 chr12:112680871‐112693229 0.32 1.19 1.31 3.29E‐03 C1qb chr4:136436060‐136442092 7.29 26.82 1.30 9.09E‐08 Gsdmd chr15:75692768‐75697834 0.43 1.57 1.30 1.61E‐02 Fblim1 chr4:141131976‐141161967 0.45 1.64 1.30 1.51E‐03 Tgm2 chr2:157942140‐157972128 1.15 4.22 1.30 4.29E‐06 Fgd2 chr17:29497858‐29516480 0.56 2.02 1.29 6.85E‐03 Serpina3g chr12:105452753‐105480144 0.93 3.39 1.29 6.67E‐04 Clic1 chr17:35187187‐35195664 5.57 20.17 1.29 3.79E‐07 Emp1 chr6:135312948‐135333191 0.18 0.65 1.29 4.97E‐02 Cd14 chr18:36884720‐36886308 0.70 2.55 1.29 7.78E‐03 Tlr2 chr3:83640193‐83645530 0.38 1.35 1.28 6.48E‐03 Cfh chr1:141982431‐142079988 0.17 0.62 1.28 1.47E‐02 C1qc chr4:136445716‐136448829 6.74 23.86 1.26 6.37E‐07 Tnc chr4:63620818‐63708049 0.09 0.33 1.26 2.42E‐02 Gbp7 chr3:142193301‐142213047 0.36 1.25 1.26 4.36E‐04 Sla chr15:66502331‐66682282 0.41 1.44 1.25 7.33E‐03 Flnc chr6:29383152‐29411888 0.57 2.00 1.25 1.98E‐06 Ehd2 chr7:16534335‐16552884 1.14 3.97 1.24 3.45E‐05 C3 chr17:57343395‐57367559 3.59 12.37 1.24 1.42E‐09 A2m chr6:121586190‐121629256 2.97 10.25 1.24 6.72E‐09 Tbxas1 chr6:38868984‐39034578 0.54 1.86 1.23 1.43E‐02 Rbp1 chr9:98323379‐98346969 0.58 1.96 1.22 2.88E‐03 Rac2 chr15:78389598‐78403213 0.43 1.47 1.22 4.76E‐03 Rasa4 chr5:136559785‐136587730 0.95 3.20 1.22 2.45E‐04 C4a chr17:34946036‐34960399 1.32 4.45 1.22 5.81E‐07 Plek chr11:16871208‐16908721 1.40 4.69 1.21 2.45E‐06 Pycard chr7:135134886‐135137381 0.34 1.15 1.21 3.50E‐02 Fn1 chr1:71632096‐71699745 0.08 0.27 1.21 4.34E‐02 Rrm2 chr12:25393118‐25399011 0.49 1.63 1.21 1.29E‐02 Fcer1g chr1:173159702‐173164480 2.65 8.88 1.21 7.03E‐03 Akna chr4:63028161‐63064479 0.21 0.70 1.21 7.90E‐03 Myof chr19:37973525‐38118067 0.37 1.24 1.21 2.52E‐04 Ctsc chr7:95426602‐95459385 0.44 1.47 1.21 1.10E‐02 Mylk chr16:34785035‐35002520 0.21 0.71 1.20 2.18E‐03 Fmnl3 chr15:99147653‐99200897 0.77 2.57 1.20 8.81E‐05 Psmb9 chr17:34319043‐34324275 1.24 4.12 1.20 3.01E‐02 Stat6 chr10:127080041‐127103759 0.74 2.44 1.20 1.87E‐02 Ly86 chr13:37437213‐37510905 2.20 7.26 1.20 1.46E‐03 1700084C01Rchr1:171859069‐171864784 3.16 10.44 1.20 2.82E‐05 Samd9l chr6:3322256‐3349571 0.26 0.85 1.20 4.06E‐03 Gfpt2 chr11:49607656‐49652122 0.58 1.91 1.19 2.57E‐03 Gbp3 chr3:142223015‐142236176 0.60 1.96 1.19 3.39E‐03 Zfp831 chr2:174469034‐174536331 0.08 0.26 1.18 3.23E‐02 C4b chr17:34865325‐34880842 17.61 57.33 1.18 3.28E‐10 Yes Cebpa chr7:35904311‐35906951 1.86 6.02 1.17 2.87E‐05 Prss23 chr7:96656294‐96666096 0.37 1.20 1.17 1.03E‐02 Lsp1 chr7:149646774‐149701914 0.95 3.07 1.17 1.29E‐02 Ikzf1 chr11:11586215‐11672929 0.21 0.68 1.16 3.98E‐02 Igfbp3 chr11:7106095‐7113926 1.49 4.71 1.15 2.32E‐04 Oasl2 chr5:115346942‐115362254 1.11 3.50 1.15 1.94E‐04 Fos chr12:86814850‐86818219 6.11 19.21 1.15 3.38E‐07 Fam111a chr19:12648014‐12671056 0.39 1.22 1.14 2.43E‐02 Fcgr3 chr1:172981299‐172989534 1.70 5.34 1.14 1.98E‐03 Ccnd1 chr7:152115835‐152125830 7.15 22.34 1.14 8.78E‐09 Cyba chr8:124948670‐124956840 2.52 7.80 1.13 9.90E‐03 Junb chr8:87500807‐87502647 3.77 11.68 1.13 1.64E‐05 Ip6k3 chr17:27280915‐27304709 0.70 2.14 1.11 9.79E‐03 Rab32 chr10:10264836‐10278005 1.06 3.21 1.10 3.88E‐03 Ccdc88b chr19:6919112‐6932701 0.71 2.15 1.10 5.48E‐04 Mocos chr18:24812191‐24860057 0.49 1.47 1.10 1.70E‐02 Mafb chr2:160189412‐160192801 1.94 5.74 1.08 4.10E‐05 Pgm5 chr19:24748497‐24936332 0.92 2.70 1.08 2.00E‐05 Tnfaip3 chr10:18720716‐18735216 0.37 1.10 1.08 2.20E‐02 Dbx2 chr15:95453993‐95485202 4.01 11.71 1.07 1.07E‐05 Ier5 chr1:156943496‐156946766 1.91 5.58 1.07 6.55E‐05 Kank1 chr19:25311691‐25508986 1.90 5.52 1.07 5.70E‐06 Renbp chrX:71167459‐71176189 2.98 8.65 1.07 8.55E‐03 Nfatc1 chr18:80802943‐80909810 0.59 1.70 1.06 4.31E‐02 Afap1l2 chr19:56986843‐57083065 0.75 2.17 1.06 1.98E‐03 Plce1 chr19:38598686‐38859590 1.05 3.03 1.06 8.77E‐06 Lxn chr3:67234036‐67278990 24.89 71.69 1.06 4.23E‐04 Cd44 chr2:102651299‐102741822 0.74 2.12 1.05 1.38E‐03 Pros1 chr16:62854159‐62929166 2.13 6.07 1.05 5.73E‐05 Ldha chr7:54101173‐54110997 31.21 88.97 1.05 3.63E‐08 Cebpb chr2:167514414‐167515918 5.30 14.95 1.04 3.28E‐04 Glipr2 chr4:43970573‐43991990 1.30 3.67 1.04 4.94E‐03 Naprt1 chr15:75721393‐75724911 3.56 9.90 1.02 1.94E‐04 Irgm2 chr11:58028478‐58036285 0.56 1.56 1.02 1.20E‐02 Tubb6 chr18:67550384‐67562403 3.93 10.87 1.02 2.26E‐04 Pld4 chr12:113998865‐114007197 0.96 2.66 1.02 1.92E‐02 Prkcd chr14:31408539‐31439394 4.48 12.38 1.02 1.70E‐05 Hk2 chr6:82675020‐82724448 0.95 2.60 1.01 7.48E‐04 Cchcr1 chr17:35654060‐35667960 0.92 2.52 1.00 9.68E‐03 Lgals3bp chr11:118254065‐118263245 4.08 11.08 1.00 1.03E‐04 Olfml3 chr3:103539316‐103541924 1.74 4.71 1.00 5.77E‐03 Pdlim4 chr11:53868429‐53882519 3.86 10.39 0.99 2.37E‐03 Fabp7 chr10:57504728‐57508256 11.30 30.32 0.99 2.52E‐04 Xdh chr17:74233247‐74299522 0.35 0.93 0.98 3.58E‐02 Pygl chr12:71291801‐71328670 1.18 3.17 0.98 4.85E‐03 Tmem176a chr6:48791481‐48795363 2.82 7.53 0.98 1.18E‐02 Sardh chr2:27043912‐27102823 1.48 3.94 0.98 3.25E‐04 Sox6 chr7:122614857‐123182258 0.36 0.97 0.98 4.91E‐03 Ampd3 chr7:117916117‐117955909 3.00 7.95 0.97 3.83E‐05 Plcd1 chr9:118980645‐119002614 1.66 4.37 0.97 2.45E‐03 P4ha3 chr7:107434029‐107468209 1.35 3.56 0.97 6.25E‐03 Dock11 chrX:33428826‐33616557 0.77 2.03 0.97 1.08E‐03 Steap3 chr1:122122992‐122167659 0.83 2.16 0.96 3.35E‐02 Bcar3 chr3:122122697‐122233100 1.25 3.24 0.95 3.44E‐03 Papss2 chr19:32670204‐32741677 2.05 5.33 0.95 9.32E‐03 chr15:61816895‐61821916 1.17 3.04 0.95 4.59E‐02 Aldh1b1 chr4:45811893‐45817480 1.80 4.67 0.95 3.93E‐03 Athl1 chr7:148127479‐148133557 1.02 2.61 0.94 9.84E‐03 Laptm5 chr4:130469248‐130492063 2.13 5.46 0.94 2.46E‐03 Itpkb chr1:182257099‐182353790 3.93 10.08 0.94 4.43E‐05 Stard8 chrX:96237919‐96270067 0.36 0.92 0.94 4.00E‐02 Bsn chr9:107998352‐108092714 0.67 1.72 0.93 1.35E‐04 Slc14a1 chr18:78296829‐78338858 1.54 3.90 0.93 1.99E‐02 Bdh2 chr3:134944184‐134967389 2.31 5.84 0.93 4.43E‐02 Th chr7:150078680‐150085871 1.68 4.26 0.93 1.54E‐02 Zfp36l1 chr12:81208746‐81214000 1.51 3.82 0.93 3.73E‐03 Dtx3l chr16:35926600‐35972707 0.54 1.37 0.92 4.06E‐02 Mcc chr18:44584713‐44971836 0.32 0.81 0.92 2.30E‐02 Tmem176b chr6:48783810‐48791373 8.99 22.41 0.91 2.43E‐03 Igsf10 chr3:58810899‐59148178 0.78 1.95 0.91 3.25E‐02 Gmip chr8:72332585‐72345769 0.64 1.61 0.91 2.38E‐02 Rgs20 chr1:4899656‐5060366 3.83 9.53 0.91 2.73E‐03 Anxa2 chr9:69301489‐69339592 18.09 44.87 0.91 2.50E‐05 Myo10 chr15:25552304‐25743426 0.94 2.32 0.90 9.53E‐04 Tnfrsf1a chr6:125299740‐125312501 1.64 4.02 0.90 1.39E‐02 Aldh1l2 chr10:82950191‐82996885 4.72 11.59 0.90 1.57E‐05 Prex2 chr1:10983545‐11293763 1.16 2.85 0.90 2.83E‐03 Aldh1l1 chr6:90500841‐90549165 11.61 28.47 0.90 1.57E‐05 Plcg2 chr8:120022190‐120159042 1.05 2.57 0.90 6.74E‐03 Ccnd2 chr6:127075726‐127162437 1.14 2.79 0.89 2.15E‐03 Flna chrX:71468799‐71491873 1.08 2.63 0.89 6.78E‐04 Hspa1b chr17:35093373‐35096183 21.93 53.42 0.89 9.32E‐03 Lrrc16a chr13:24104352‐24372659 1.60 3.88 0.89 1.07E‐03 Ifit3 chr19:34658018‐34663472 2.95 7.12 0.88 3.49E‐03 Tspo chr15:83394002‐83404633 7.90 19.07 0.88 4.94E‐03 Axl chr7:26541518‐26573752 1.27 3.05 0.87 3.41E‐02 Coro1a chr7:133843287‐133848268 20.03 48.04 0.87 3.22E‐05 I830012O16R chr19:34682446‐34687891 1.82 4.36 0.87 1.43E‐02 1200009I06Richr12:112655640‐112669394 0.83 1.99 0.87 4.54E‐02 Sorbs2 chr8:46593141‐46913260 1.50 3.56 0.86 3.36E‐03 Lamb2 chr9:108382192‐108392861 0.81 1.92 0.86 1.06E‐02 Ngef chr1:89373403‐89470445 10.17 24.01 0.86 5.89E‐05 Baiap3 chr17:25377114‐25393309 10.94 25.81 0.86 3.84E‐04 Rusc1 chr3:88887900‐88897285 48.48 114.27 0.86 1.08E‐04 Aplnr chr2:84976516‐84980080 0.96 2.24 0.85 2.39E‐02 Serpinb1b chr13:33175971‐33186249 37.68 88.27 0.85 2.11E‐05 Arhgef26 chr3:62142698‐62266143 1.95 4.52 0.84 1.28E‐03 Sbno2 chr10:79519758‐79565447 3.76 8.73 0.84 3.26E‐04 Plcb3 chr19:7028202‐7044242 2.94 6.79 0.84 9.26E‐04 Mapk8ip2 chr15:89284341‐89292878 12.05 27.89 0.84 5.65E‐05 Serpinb9 chr13:33096409‐33109824 1.79 4.13 0.84 5.72E‐03 Ddo chr10:40349816‐40369737 1.69 3.90 0.84 1.01E‐02 Aldh18a1 chr19:40624746‐40662953 5.67 13.07 0.84 3.75E‐03 C77370 chrX:101272774‐101396456 0.32 0.74 0.84 2.12E‐02 Ppm1e chr11:87040407‐87172496 7.33 16.79 0.83 6.26E‐05 Sox9 chr11:112643523‐112649071 1.16 2.66 0.83 1.41E‐02 Dpysl4 chr7:146271899‐146287690 2.08 4.76 0.83 1.06E‐02 Ank1 chr8:24085353‐24260968 15.81 35.92 0.82 9.66E‐03 Phkg1 chr5:130322865‐130354954 4.10 9.29 0.82 3.67E‐02 Fosl2 chr5:32438844‐32460212 0.60 1.36 0.82 3.13E‐02 Parp3 chr9:106372683‐106378982 2.63 5.96 0.82 6.57E‐03 Acsbg1 chr9:54452803‐54509692 60.01 135.75 0.82 1.42E‐04 Marcks chr10:36853048‐36858732 7.63 17.25 0.82 1.27E‐04 Ryr2 chr13:11645369‐12199212 1.30 2.93 0.81 4.11E‐04 Dapk1 chr13:60703307‐60864547 2.91 6.58 0.81 7.83E‐03 Rfx4 chr10:84218792‐84369283 2.32 5.22 0.81 3.01E‐03 Acss1 chr2:150443846‐150494668 2.82 6.35 0.81 2.91E‐03 Tgfbr2 chr9:115996812‐116084481 1.07 2.40 0.81 2.48E‐02 Spna2 chr2:29821079‐29886970 9.69 21.79 0.81 2.10E‐03 Yes Kctd12 chr14:103375797‐103381854 2.62 5.89 0.81 8.16E‐04 Heatr7a chr15:76210942‐76307699 9.67 21.73 0.81 1.66E‐02 Chi3l1 chr1:136078980‐136086608 5.76 12.93 0.81 3.82E‐03 Acaa2 chr18:74938865‐74965861 8.05 18.04 0.81 2.22E‐03 Unc13a chr8:74150611‐74195656 3.98 8.92 0.81 2.06E‐04 Ephx2 chr14:66703213‐66743337 3.54 7.93 0.81 7.31E‐03 Gad1 chr2:70327996‐70440069 38.71 86.61 0.81 1.35E‐04 Camkv chr9:107838250‐107852022 21.08 47.09 0.80 8.75E‐05 Dmxl2 chr9:54212964‐54349433 6.67 14.90 0.80 1.07E‐04 Arhgef17 chr7:108018259‐108080675 8.40 18.75 0.80 1.02E‐04 Pfkm chr15:97869174‐97962878 127.75 284.88 0.80 5.72E‐03 Camk2b chr11:5869668‐5965751 50.72 112.70 0.80 2.15E‐03 Sep6 chrX:34450828‐34529690 16.83 37.37 0.80 1.64E‐04 Ccdc141 chr2:76847962‐77008692 0.68 1.51 0.80 2.48E‐02 Mtap1b chr13:100191418‐100286557 33.15 73.54 0.80 1.26E‐03 Fmnl1 chr11:103032451‐103060214 4.13 9.15 0.80 1.58E‐03 Ophn1 chrX:95752853‐96086324 1.86 4.11 0.79 7.17E‐03 Lrrk2 chr15:91503654‐91646555 0.93 2.05 0.79 7.34E‐03 Gna14 chr19:16510156‐16685308 2.07 4.57 0.79 8.45E‐03 Plcd3 chr11:102931609‐102962972 1.19 2.62 0.79 4.63E‐02 Trim66 chr7:116592514‐116651648 1.38 3.05 0.79 1.99E‐02 Cx3cr1 chr9:119957810‐119977400 1.67 3.68 0.79 1.26E‐02 Irgm1 chr11:48678750‐48684848 4.13 9.10 0.79 4.91E‐03 Sphkap chr1:83252355‐83404775 3.75 8.27 0.79 5.40E‐04 Pdzd2 chr15:12286808‐12522311 0.47 1.03 0.79 1.06E‐02 Id3 chr4:135699736‐135701307 6.13 13.46 0.79 2.48E‐02 Gadd45g chr13:51942043‐51943843 13.34 29.24 0.78 3.28E‐03 Chac1 chr2:119176977‐119180062 4.72 10.34 0.78 1.08E‐02 Nek6 chr2:38367216‐38443010 4.28 9.38 0.78 5.08E‐03 AW551984 chr9:39394980‐39411709 3.99 8.74 0.78 1.80E‐03 Csf1r chr18:61265225‐61290793 3.41 7.45 0.78 2.86E‐03 Amotl1 chr9:14346410‐14419444 1.68 3.68 0.78 1.46E‐03 Kank4 chr4:98421582‐98484228 2.01 4.39 0.78 4.72E‐03 Nynrin chr14:56472951‐56493573 1.21 2.64 0.78 5.72E‐03 Ass1 chr2:31325789‐31376190 13.62 29.71 0.78 9.02E‐04 Spnb1 chr12:77681474‐77811534 1.97 4.28 0.78 8.99E‐04 Dock7 chr4:98603355‐98787606 1.87 4.07 0.78 2.96E‐03 Plcl2 chr17:50648871‐50827819 4.85 10.57 0.78 8.99E‐04 Galm chr17:80526810‐80584372 1.77 3.85 0.77 4.35E‐02 Ripk1 chr13:34094742‐34127039 0.97 2.10 0.77 3.17E‐02 Pck2 chr14:56159102‐56168854 4.87 10.53 0.77 1.97E‐03 Grb10 chr11:11830501‐11937423 6.85 14.79 0.77 2.64E‐03 Tbc1d1 chr5:64551449‐64742725 1.78 3.83 0.77 6.06E‐03 Myo5a chr9:74919012‐75071494 8.39 18.06 0.77 2.95E‐04 Pfkp chr13:6547402‐6647970 54.30 116.91 0.77 1.97E‐03 Isyna1 chr8:73118379‐73121189 10.24 22.05 0.77 1.98E‐03 Akr1b10 chr6:34334246‐34346949 16.88 36.19 0.76 1.25E‐03 Ehd4 chr2:119915222‐119980311 6.45 13.81 0.76 1.16E‐03 Egr1 chr18:35020860‐35024610 3.91 8.36 0.76 5.43E‐03 Frmpd4 chrX:163909240‐165015165 1.41 3.01 0.76 4.07E‐03 Rap1gap chr4:137220640‐137285776 37.77 80.71 0.76 5.29E‐04 Yes Aldoc chr11:78137699‐78140262 769.71 1643.46 0.76 1.97E‐02 Magee2 chrX:102050290‐102052606 2.40 5.12 0.76 2.54E‐02 Klhl1 chr14:96504483‐96918253 2.88 6.14 0.76 4.87E‐03 Ddah1 chr3:145421655‐145557241 8.06 17.20 0.76 6.40E‐04 Frmpd1 chr4:45197777‐45298808 2.14 4.56 0.76 6.64E‐03 Drp2 chrX:130939318‐130991112 1.80 3.82 0.75 4.26E‐03 Dusp6 chr10:98725864‐98730123 3.02 6.42 0.75 1.07E‐02 Rasgrf2 chr13:92020011‐92901449 7.51 15.93 0.75 4.60E‐04 Nrp1 chr8:130882972‐131029375 1.17 2.48 0.75 1.49E‐02 Asns chr6:7625170‐7643182 35.20 74.60 0.75 4.18E‐04 Camk2a chr18:61085285‐61147806 10.88 23.05 0.75 1.72E‐03 Mpp3 chr11:101860966‐101888269 3.07 6.51 0.75 1.08E‐02 Nefh chr11:4838758‐4942797 105.59 223.50 0.75 1.88E‐02 Hecw1 chr13:14318704‐14615493 2.84 5.97 0.74 1.11E‐03 Ppp4r4 chr12:104770774‐104852042 4.09 8.57 0.74 3.76E‐03 Akap13 chr7:82600419‐82899495 0.48 1.01 0.74 2.38E‐02 Pamr1 chr2:102390177‐102483197 1.66 3.48 0.74 4.55E‐02 Camsap3 chr8:3587449‐3609075 2.61 5.46 0.74 1.20E‐02 Slc25a18 chr6:120723785‐120744000 23.10 48.22 0.74 1.28E‐03 Wdr6 chr9:108472222‐108481001 31.71 66.18 0.74 4.76E‐03 Plcb1 chr2:134611899‐135300994 5.93 12.36 0.73 1.64E‐02 Rimklb chr6:122403626‐122436323 2.53 5.26 0.73 9.22E‐03 Cebpd chr16:15887378‐16146926 10.68 22.23 0.73 1.20E‐02 Plvap chr8:74021651‐74035668 2.81 5.84 0.73 4.21E‐02 Gprin1 chr13:54838033‐54851030 2.49 5.18 0.73 9.90E‐03 Arhgap26 chr18:39152798‐39535939 2.37 4.92 0.73 2.58E‐03 Usp29 chr7:6683451‐6929373 3.04 6.31 0.73 5.38E‐03 Mlc1 chr15:88786313‐88808983 17.19 35.59 0.73 1.05E‐03 Cpne5 chr17:29293465‐29374735 2.16 4.47 0.73 1.22E‐02 Ampd2 chr3:107876979‐107889545 15.62 32.26 0.72 7.48E‐04 Chd5 chr4:151712759‐151764303 6.20 12.80 0.72 9.66E‐03 Lgals9 chr11:78776480‐78798426 11.51 23.74 0.72 9.30E‐03 Pfkl chr10:77449693‐77472541 36.65 75.56 0.72 8.99E‐04 Madd chr2:90977516‐91023204 12.48 25.69 0.72 2.69E‐03 Yes Sep11 chr5:93522482‐93603984 2.62 5.38 0.72 6.78E‐03 Cadps2 chr6:23212773‐23789421 4.02 8.24 0.72 3.07E‐02 Cadps chr14:13205076‐13655593 39.33 80.67 0.72 1.38E‐03 Myh9 chr15:77591018‐77672545 3.58 7.34 0.72 1.98E‐03 Stxbp5l chr16:37107395‐37385044 2.16 4.43 0.72 7.64E‐03 Aqp4 chr18:15547902‐15562193 12.36 25.30 0.72 1.10E‐03 Gpr101 chrX:54749844‐54756934 1.16 2.36 0.71 3.40E‐02 Abr chr11:76230235‐76391229 29.79 60.59 0.71 1.89E‐03 Dzip3 chr16:48924340‐48994225 4.00 8.13 0.71 6.42E‐03 Asap3 chr4:135762279‐135802488 1.39 2.83 0.71 2.39E‐02 Gad2 chr2:22477846‐22549397 8.38 17.03 0.71 1.34E‐03 Wnk2 chr13:49131670‐49243383 4.32 8.78 0.71 2.57E‐03 Fndc3b chr3:27315083‐27609361 1.36 2.77 0.71 1.49E‐02 Camk2g chr14:21554096‐21613310 76.83 156.04 0.71 2.87E‐02 Akap6 chr12:53800369‐54252002 7.61 15.44 0.71 9.48E‐04 Nmnat2 chr1:154802230‐154966391 18.00 36.51 0.71 9.45E‐04 Rgs12 chr5:35292096‐35376242 2.35 4.76 0.71 2.41E‐02 Dlgap1 chr17:70774131‐71170753 4.29 8.68 0.71 1.66E‐02 Gm10336 chr13:12274983‐12278755 1.80 3.63 0.70 3.10E‐02 Oplah chr15:76124863‐76137675 4.59 9.26 0.70 6.17E‐03 Fmn1 chr2:113167892‐113556924 0.56 1.12 0.70 2.82E‐02 Yes Ckmt1 chr2:121184376‐121206676 206.82 416.62 0.70 3.92E‐03 Inpp4a chr1:37356737‐37467580 9.28 18.67 0.70 9.58E‐03 Gm15800 chr5:121670227‐121818586 3.79 7.60 0.70 1.31E‐03 Alox8 chr11:68997386‐69011341 2.75 5.52 0.70 2.25E‐02 Pwwp2b chr7:146434380‐146453152 4.27 8.57 0.70 1.79E‐02 Camk1g chr1:195172539‐195196476 11.30 22.66 0.70 3.01E‐03 Vgf chr5:137506164‐137509221 18.16 36.37 0.69 2.04E‐03 Dnm1 chr2:32163990‐32208824 188.95 378.25 0.69 1.97E‐02 Abat chr16:8513521‐8621660 51.77 103.55 0.69 9.66E‐03 Fbxo41 chr6:85419571‐85452880 3.51 7.01 0.69 4.32E‐03 Lrrc48 chr11:60166881‐60207835 3.72 7.43 0.69 4.63E‐02 Btbd11 chr10:84849558‐85123037 8.23 16.44 0.69 3.05E‐03 1300001I01Richr11:74462996‐74484349 12.84 25.60 0.69 1.58E‐03 Fry chr5:151062504‐151300328 2.75 5.48 0.69 3.05E‐03 Lrp1 chr10:126975213‐127058204 3.69 7.33 0.69 2.19E‐03 Vat1l chr8:116729539‐116897970 41.37 82.22 0.69 2.27E‐03 Ap3b2 chr7:88605284‐88638811 28.00 55.65 0.69 1.72E‐03 Sgsm1 chr5:113672239‐113739806 10.73 21.31 0.69 9.58E‐03 Arhgef40 chr14:52604507‐52640408 4.80 9.52 0.69 1.18E‐02 Yes Syn2 chr6:115084919‐115232644 39.17 77.65 0.68 1.99E‐02 Dcaf12l1 chrX:42139743‐42143374 7.28 14.44 0.68 6.49E‐03 Trrap chr5:145529660‐145620642 1.68 3.33 0.68 5.72E‐03 Arhgap20 chr9:51573456‐51683990 2.83 5.61 0.68 7.83E‐03 Tbc1d9 chr8:85689250‐85796839 8.71 17.23 0.68 8.96E‐03 Klk6 chr7:51079913‐51087397 260.30 514.54 0.68 1.64E‐02 Gprasp2 chrX:132373572‐132379269 18.18 35.93 0.68 4.70E‐03 Trim9 chr12:71345523‐71448601 9.44 18.65 0.68 1.12E‐02 Tspyl3 chr2:153048105‐153051177 2.65 5.23 0.68 3.34E‐02 Klhl23 chr2:69660426‐69674708 2.39 4.70 0.68 1.78E‐02 Plcb4 chr2:135567565‐135838804 7.25 14.28 0.68 7.67E‐03 Pde2a chr7:108570204‐108661343 4.34 8.54 0.68 4.08E‐02 Nxn chr11:76070727‐76212643 4.51 8.88 0.68 1.76E‐02 Ina chr19:46961320‐47098836 65.82 129.48 0.68 1.36E‐02 Cryl1 chr14:57893870‐58017320 5.85 11.49 0.68 4.26E‐02 Spon1 chr7:120909511‐121186889 2.99 5.87 0.68 8.55E‐03 Plxnb2 chr15:88985978‐89011218 1.96 3.86 0.67 1.27E‐02 Pygb chr2:150612531‐150657484 63.36 124.29 0.67 5.13E‐03 Lgi4 chr7:31844953‐31861716 5.32 10.44 0.67 1.32E‐02 Phlpp2 chr8:112392502‐112468571 3.15 6.17 0.67 5.38E‐03 Syne1 chr10:5151833‐5326342 3.65 7.14 0.67 3.06E‐02 Lmtk3 chr7:53039316‐53059500 3.88 7.59 0.67 9.58E‐03 Ezr chr17:6942479‐6987129 2.77 5.43 0.67 3.59E‐02 Reln chr5:21390271‐21850523 1.59 3.11 0.67 9.23E‐03 Plec chr15:76001403‐76061808 1.39 2.71 0.67 1.64E‐02 Yes Unc80 chr1:66515020‐66745722 6.35 12.38 0.67 3.05E‐03 Fam65a chr8:108129128‐108146118 6.03 11.75 0.67 6.30E‐03 Nap1l2 chrX:100379397‐100382003 23.78 46.27 0.67 2.46E‐03 Pacs1 chr19:5133684‐5273119 6.89 13.37 0.66 5.14E‐03 Cap2 chr13:46597271‐46745650 6.43 12.48 0.66 7.19E‐03 Snap47 chr11:59220651‐59263458 219.06 423.68 0.66 9.90E‐03 Slc8a2 chr7:16715648‐16745860 2.33 4.50 0.66 3.44E‐02 Lpin2 chr17:71533317‐71599158 6.67 12.90 0.66 4.18E‐03 Chl1 chr6:103460869‐103683029 3.37 6.49 0.66 7.17E‐03 Tmem22 chr9:100452606‐100471504 9.59 18.49 0.66 1.79E‐02 Efr3a chr15:65618602‐65705374 30.98 59.70 0.66 5.11E‐03 Hk1 chr10:61731602‐61842656 47.00 90.51 0.66 6.34E‐03 D430041D05Rchr2:103983231‐104250491 1.29 2.48 0.65 2.08E‐02 Fxyd7 chr7:31827533‐31836473 71.37 137.02 0.65 5.03E‐03 Plin2 chr4:86302468‐86315963 6.42 12.31 0.65 2.82E‐02 Ccrn4l chr3:51028368‐51055576 10.18 19.47 0.65 6.37E‐03 Ubr4 chr4:138936573‐139045447 5.14 9.82 0.65 4.18E‐03 Mcf2 chrX:57309132‐57400820 2.56 4.88 0.65 4.09E‐02 Raph1 chr1:60540028‐60623609 1.38 2.63 0.65 2.36E‐02 Ecel1 chr1:89044229‐89051602 11.86 22.61 0.65 7.34E‐03 Gucy1a3 chr3:81896348‐81949799 5.29 10.09 0.65 9.21E‐03 Unc79 chr12:104187068‐104422207 1.08 2.05 0.64 4.09E‐02 Capn1 chr19:5988544‐6018459 11.84 22.54 0.64 6.30E‐03 Klhl13 chrX:22796396‐22942179 13.77 26.21 0.64 4.95E‐03 Tanc2 chr11:105451299‐105790617 3.01 5.72 0.64 5.73E‐03 Anxa6 chr11:54792463‐54846973 57.16 108.70 0.64 3.92E‐03 Midn chr10:79611034‐79621112 4.78 9.08 0.64 1.75E‐02 Trio chr15:27660403‐27955603 2.05 3.89 0.64 1.07E‐02 Col16a1 chr4:129725083‐129776521 1.83 3.47 0.64 4.44E‐02 Cpne4 chr9:104472117‐104936874 3.36 6.37 0.64 2.94E‐02 Iqsec3 chr6:121322950‐121423696 8.43 15.98 0.64 4.76E‐03 Ralgapa1 chr12:56703885‐56922154 7.06 13.35 0.64 2.94E‐02 Jak1 chr4:100741642‐100937887 27.10 51.22 0.64 7.64E‐03 Pnck chrX:70901330‐70905409 41.18 77.53 0.63 2.20E‐02 Heatr1 chr13:12487641‐12531160 1.49 2.81 0.63 4.30E‐02 Ptk2b chr14:66772093‐66899889 3.77 7.09 0.63 3.38E‐02 Dfna5 chr6:50157401‐50211768 5.49 10.30 0.63 3.97E‐02 Stk10 chr11:32433265‐32524595 2.20 4.13 0.63 4.03E‐02 Nkrf chrX:34427536‐34442894 11.65 21.87 0.63 7.31E‐03 Pde1b chr15:103333728‐103360483 30.86 57.88 0.63 5.38E‐03 Tubb2a chr13:34166146‐34169877 147.93 277.48 0.63 9.28E‐03 Pygm chr19:6384428‐6398459 19.99 37.47 0.63 6.58E‐03 Ahi1 chr10:20672352‐20800235 56.07 105.06 0.63 4.37E‐02 Hif1a chr12:75008853‐75048517 15.44 28.94 0.63 6.10E‐03 Hexb chr13:97907891‐97968312 25.68 48.11 0.63 2.64E‐02 Sall2 chr14:52930851‐52948345 7.48 13.99 0.63 9.63E‐03 Arhgef19 chr4:140798798‐140813477 4.21 7.86 0.62 3.29E‐02 Wdr7 chr18:63868348‐64149413 21.08 39.25 0.62 8.73E‐03 Ttc3 chr16:94580369‐94690828 62.20 115.78 0.62 3.63E‐02 Arhgef6 chrX:54484661‐54591906 2.97 5.53 0.62 3.35E‐02 Mast1 chr8:87435751‐87461252 21.41 39.83 0.62 7.03E‐03 Wdr35 chr12:8980806‐9035653 7.78 14.47 0.62 3.34E‐02 Camta1 chr4:150433631‐151235877 24.15 44.91 0.62 4.33E‐02 Htt chr5:35104388‐35255170 2.70 5.02 0.62 9.30E‐03 Plk2 chr13:111185251‐111191051 5.67 10.51 0.62 2.82E‐02 Dock3 chr9:106795155‐107134240 7.67 14.22 0.62 7.17E‐03 Lasp1 chr11:97660985‐97700078 6.27 11.62 0.62 1.86E‐02 Astn1 chr1:160292434‐160621917 5.72 10.58 0.62 1.64E‐02 Dync1i1 chr6:5675638‐5978039 41.49 76.79 0.62 2.91E‐02 Magee1 chrX:102315734‐102319250 41.82 77.36 0.62 8.07E‐03 Grip2 chr6:91711502‐91757387 2.97 5.49 0.61 3.17E‐02 Cacna1i chr15:80117667‐80228722 1.85 3.42 0.61 2.50E‐02 Tbc1d4 chr14:101841576‐102008408 3.01 5.56 0.61 2.26E‐02 Nbea chr3:55429119‐55987623 5.37 9.91 0.61 1.48E‐02 Mapk4 chr18:74088140‐74224603 4.06 7.50 0.61 2.42E‐02 Rap1gap2 chr11:74196984‐74403660 8.48 15.66 0.61 9.03E‐03 Arrb1 chr7:106683995‐106755281 13.44 24.80 0.61 9.32E‐03 Flot1 chr17:35960301‐35969732 83.49 153.98 0.61 7.82E‐03 Bhlhb9 chrX:132420389‐132425620 5.87 10.82 0.61 2.82E‐02 Kcnab3 chr11:69139759‐69146543 35.22 64.89 0.61 7.25E‐03 Gmpr chr13:45602837‐45641750 42.37 77.98 0.61 7.67E‐03 Tuba4a chr1:75207403‐75215828 210.81 387.95 0.61 4.40E‐02 Ica1 chr6:8580526‐8728484 14.14 26.01 0.61 3.55E‐02 Ets2 chr16:95924013‐95942656 5.25 9.66 0.61 2.66E‐02 Adcy8 chr15:64530596‐64753858 2.91 5.34 0.61 3.92E‐02 Zcchc12 chrX:33735898‐33739153 16.68 30.53 0.60 1.23E‐02 Ctnnd2 chr15:30102347‐30959098 7.12 13.03 0.60 1.06E‐02 Mtus2 chr5:148768895‐149127641 2.29 4.19 0.60 2.97E‐02 Ttc39b chr4:82866204‐82970093 2.94 5.37 0.60 1.87E‐02 Gria1 chr11:56825119‐57143746 9.15 16.69 0.60 9.28E‐03 Gucy1b3 chr3:81835925‐81878633 14.82 27.03 0.60 9.21E‐03 Cpne9 chr6:113232300‐113255565 5.38 9.81 0.60 4.08E‐02 Ctsd chr7:149561820‐149573775 229.54 417.76 0.60 3.38E‐02 Apba2 chr7:71646591‐71898762 21.12 38.43 0.60 1.06E‐02 Bbs4 chr9:59169772‐59201315 19.25 35.01 0.60 1.08E‐02 Plxna4 chr6:32094558‐32538192 2.10 3.82 0.60 2.07E‐02 Impdh1 chr6:29150439‐29162271 19.00 34.51 0.60 1.21E‐02 Myh10 chr11:68505416‐68630126 13.23 24.01 0.60 1.24E‐02 Pgm2l1 chr7:107376116‐107427382 6.89 12.48 0.59 1.31E‐02 Zfyve28 chr5:34537542‐34630973 5.23 9.46 0.59 2.96E‐02 Cgnl1 chr9:71474315‐71619409 6.28 11.35 0.59 1.40E‐02 Hspa4l chr3:40549534‐40594287 37.75 68.17 0.59 1.46E‐02 Ipo4 chr14:56244465‐56254515 28.54 51.51 0.59 1.19E‐02 Lcp1 chr14:75530929‐75630649 7.91 14.27 0.59 4.56E‐02 Sarm1 chr11:78285831‐78311256 3.12 5.63 0.59 4.97E‐02 Fam40a chr3:107415449‐107434628 22.57 40.66 0.59 1.09E‐02 Efr3b chr12:3962553‐4038915 20.47 36.82 0.59 1.42E‐02 6330439K17Rchr2:144296292‐144353134 22.90 41.19 0.59 1.54E‐02 Spnb3 chr19:4711222‐4752352 2.18 3.91 0.59 3.81E‐02 Rundc3b chr5:8490335‐8622952 5.71 10.28 0.59 3.05E‐02 Rab3c chr13:110844395‐111070414 10.94 19.66 0.59 1.32E‐02 Cad chr5:31357183‐31380852 2.30 4.13 0.59 4.62E‐02 3830431G21Rchr12:81793587‐81825203 4.88 8.76 0.59 4.08E‐02 Synj1 chr16:90936341‐91044624 25.17 45.18 0.58 2.94E‐02 Dgkb chr12:38607291‐39359997 3.12 5.59 0.58 4.35E‐02 Pnmal2 chr7:17530030‐17534177 54.00 96.74 0.58 2.07E‐02 Apeh chr9:107987744‐107996811 13.73 24.60 0.58 2.32E‐02 Ube2o chr11:116399066‐116442761 24.76 44.32 0.58 1.49E‐02 Rab3b chr4:108551674‐108644682 9.72 17.39 0.58 2.09E‐02 Crmp1 chr5:37633318‐37683372 23.97 42.88 0.58 1.36E‐02 Pitpnm2 chr5:124568698‐124666427 3.38 6.05 0.58 3.34E‐02 Abcd2 chr15:90976301‐91022238 4.77 8.53 0.58 2.55E‐02 Atp6v1a chr16:44085516‐44139132 94.46 168.90 0.58 3.86E‐02 Pde4d chr13:109444370‐110746177 2.84 5.08 0.58 3.40E‐02 Tbc1d30 chr10:120700875‐120748245 7.61 13.60 0.58 1.78E‐02 Ppfia3 chr7:52594495‐52625934 10.32 18.42 0.58 4.03E‐02 Hspa12a chr19:58870240‐58935474 32.06 57.20 0.58 2.04E‐02 Phyhip chr14:70857323‐70868631 28.76 51.28 0.58 1.34E‐02 Gbf1 chr19:46227047‐46361000 15.27 27.19 0.58 1.49E‐02 Actl6b chr5:137994782‐138010801 13.28 23.61 0.58 4.24E‐02 Ranbp6 chr19:29882597‐29887464 10.50 18.67 0.58 1.75E‐02 Kif3a chr11:53380880‐53417746 30.30 53.80 0.57 2.00E‐02 Hspb8 chr5:116858503‐116872873 12.73 22.57 0.57 3.61E‐02 Dlg3 chrX:97963061‐98013749 7.49 13.28 0.57 4.36E‐02 Plekha6 chr1:135142673‐135200012 8.21 14.55 0.57 3.23E‐02 Ppp1r3c chr19:36806220‐36811094 7.06 12.50 0.57 4.43E‐02 Eno1 chr4:149611305‐149622982 74.70 132.17 0.57 2.94E‐02 Luzp1 chr4:136025675‐136105233 4.38 7.74 0.57 2.36E‐02 Uhrf1bp1 chr17:27993451‐28036985 2.82 4.98 0.57 3.41E‐02 Klhl22 chr16:17759713‐17793475 16.39 28.94 0.57 2.25E‐02 Herc1 chr9:66198256‐66362400 2.01 3.55 0.57 3.73E‐02 Cyfip2 chr11:46007350‐46126361 48.37 85.37 0.57 4.33E‐02 Spnb4 chr7:28141401‐28231608 5.72 10.10 0.57 3.57E‐02 Pitpnm1 chr19:4100116‐4113965 17.21 30.38 0.57 1.70E‐02 Snph chr2:151416285‐151458269 18.92 33.31 0.57 1.70E‐02 Rgl1 chr1:154364659‐154472241 7.82 13.76 0.56 3.13E‐02 Lonrf2 chr1:38851353‐38878060 14.63 25.67 0.56 2.40E‐02 Stmn3 chr2:181041163‐181049205 346.39 607.23 0.56 3.86E‐02 Hivep2 chr10:13686184‐13871184 2.21 3.86 0.56 4.38E‐02 Ogfod1 chr8:96561097‐96591822 13.60 23.73 0.56 3.17E‐02 L1cam chrX:71099118‐71126173 12.90 22.49 0.56 2.39E‐02 Nckipsd chr9:108710710‐108720697 32.82 57.16 0.55 2.10E‐02 Plxna2 chr1:196446022‐196643062 2.15 3.75 0.55 4.43E‐02 Stxbp5 chr10:9475344‐9620838 5.31 9.24 0.55 2.52E‐02 Megf8 chr7:26102182‐26150936 7.04 12.21 0.55 2.48E‐02 Hpcal4 chr4:122860746‐122871942 5.63 9.74 0.55 4.54E‐02 Gdap1l1 chr2:163264202‐163281060 16.97 29.37 0.55 2.97E‐02 Slc25a22 chr7:148615647‐148623773 33.40 57.76 0.55 3.34E‐02 BC037034 chr5:138700885‐138713982 18.50 31.98 0.55 3.73E‐02 Camkk1 chr11:72832509‐72855567 33.31 57.46 0.55 2.55E‐02 Plxna1 chr6:89266307‐89312607 3.68 6.35 0.54 3.87E‐02 Abcg4 chr9:44081272‐44096327 8.68 14.95 0.54 4.33E‐02 Pank4 chr4:154338241‐154355047 12.08 20.78 0.54 4.54E‐02 Rab3gap1 chr1:129765349‐129840453 18.28 31.27 0.54 3.08E‐02 Herc2 chr7:63305524‐63487168 4.08 6.96 0.53 3.23E‐02 Mtor chr4:147822690‐147931794 5.83 9.94 0.53 3.60E‐02 Clu chr14:66587319‐66600382 122.25 207.76 0.53 4.57E‐02 Ndn chr7:69493162‐69494813 114.72 194.11 0.53 4.15E‐02 Genes downregulated in SOD1 G37R oligodendrocytes Cnih4 chr1:183081061‐183099125 17.46 10.33 ‐0.52 4.46E‐02 Golga7 chr8:24351797‐24367552 149.78 88.33 ‐0.53 3.58E‐02 Nxt2 chrX:138662479‐138674235 66.98 39.49 ‐0.53 2.97E‐02 1190002N15Rchr9:94418282‐94438500 13.38 7.89 ‐0.53 4.54E‐02 Nipal4 chr11:45961656‐45979861 35.59 20.96 ‐0.53 3.23E‐02 Dixdc1 chr9:50470857‐50536089 51.85 30.47 ‐0.53 4.15E‐02 1810037I17Richr3:122627314‐122629112 123.66 72.66 ‐0.53 3.65E‐02 Tmem125 chr4:118213545‐118216331 154.95 90.97 ‐0.53 3.67E‐02 St6galnac3 chr3:152865472‐153388097 23.98 14.06 ‐0.53 3.06E‐02 H2afv chr11:6327228‐6344446 40.92 23.91 ‐0.54 3.93E‐02 Sft2d1 chr17:8503967‐8520307 56.56 33.04 ‐0.54 4.38E‐02 Grm3 chr5:9485235‐9725352 33.42 19.48 ‐0.54 2.57E‐02 Aven chr2:112333120‐112471410 75.18 43.75 ‐0.54 3.78E‐02 Yes Rnf130 chr11:49838832‐49918235 245.83 143.03 ‐0.54 3.97E‐02 Tmeff2 chr1:50984366‐51244114 106.66 62.03 ‐0.54 3.92E‐02 BC005537 chr13:24893525‐24904768 48.88 28.42 ‐0.54 2.48E‐02 Tnfaip6 chr2:51893632‐51912201 81.38 47.24 ‐0.54 2.65E‐02 Ttyh2 chr11:114536781‐114582298 148.86 86.19 ‐0.55 4.88E‐02 Gramd3 chr18:56591785‐56663446 28.49 16.48 ‐0.55 2.94E‐02 Vma21 chrX:69062117‐69070045 15.56 9.00 ‐0.55 2.96E‐02 Adi1 chr12:29360071‐29367039 240.13 138.82 ‐0.55 4.08E‐02 Map6d1 chr16:20233381‐20241431 89.46 51.71 ‐0.55 2.95E‐02 Tmem98 chr11:80623916‐80635535 122.51 70.79 ‐0.55 2.41E‐02 Tspan14 chr14:41719732‐41780096 52.81 30.51 ‐0.55 2.54E‐02 Lhfpl2 chr13:94827750‐94965364 15.68 9.06 ‐0.55 3.67E‐02 4732418C07Rchr4:115410677‐115449932 75.58 43.62 ‐0.55 3.29E‐02 Usp30 chr5:114550341‐114572933 59.46 34.29 ‐0.55 2.30E‐02 2810407C02Rchr3:58380579‐58397466 70.81 40.83 ‐0.55 2.62E‐02 2810468N07Rchr17:25707755‐25711988 336.95 194.06 ‐0.55 3.92E‐02 Cab39l chr14:60059817‐60167740 47.20 27.18 ‐0.55 2.88E‐02 2900064A13Rchr2:112295181‐112307593 140.65 80.90 ‐0.55 2.23E‐02 Ninj2 chr6:120043397‐120150356 66.80 38.41 ‐0.55 4.33E‐02 Ptpra chr2:130276013‐130389485 64.68 37.18 ‐0.55 3.34E‐02 2610001J05Richr6:13819073‐13821483 44.15 25.36 ‐0.55 2.48E‐02 Ube2d1 chr10:70717727‐70748010 230.50 132.33 ‐0.55 2.98E‐02 Tmem159 chr7:127245939‐127264500 37.21 21.36 ‐0.56 3.65E‐02 H47 chr7:73224534‐73234291 73.99 42.36 ‐0.56 2.40E‐02 Sys1 chr2:164286470‐164291010 113.39 64.86 ‐0.56 2.36E‐02 Hhip chr8:82489749‐82581907 5.65 3.23 ‐0.56 2.64E‐02 Cisd2 chr3:135069375‐135086397 23.29 13.27 ‐0.56 2.15E‐02 Fbxo7 chr10:85484673‐85511073 176.76 100.71 ‐0.56 2.52E‐02 Pde8a chr7:88358689‐88478508 52.45 29.75 ‐0.57 1.55E‐02 Rnf7 chr9:96371375‐96379014 303.98 172.31 ‐0.57 2.62E‐02 Slc25a27 chr17:43778848‐43803964 53.57 30.36 ‐0.57 1.84E‐02 Ldlrad3 chr2:101790357‐102026534 18.90 10.68 ‐0.57 1.97E‐02 Cyp20a1 chr1:60400215‐60444231 26.62 15.03 ‐0.57 3.34E‐02 Ptpn13 chr5:103854210‐104027380 4.20 2.37 ‐0.57 3.53E‐02 Ddc chr11:11714103‐11798147 65.41 36.88 ‐0.57 1.50E‐02 Slc38a2 chr15:96517822‐96530129 139.04 78.25 ‐0.57 4.85E‐02 Ctnnbip1 chr4:148892349‐148940546 35.22 19.78 ‐0.58 3.57E‐02 Nkd1 chr8:91045242‐91118786 34.73 19.49 ‐0.58 1.61E‐02 Leprel4 chr11:100270063‐100276133 57.81 32.44 ‐0.58 1.54E‐02 Snapc5 chr9:64027103‐64030495 82.62 46.33 ‐0.58 2.44E‐02 Tgfa chr6:86145244‐86225443 49.56 27.73 ‐0.58 1.38E‐02 Olfml1 chr7:114710946‐114734879 46.12 25.79 ‐0.58 1.40E‐02 Sypl chr12:33638809‐33664367 91.73 51.15 ‐0.58 1.78E‐02 Tspan15 chr10:61648143‐61693966 49.48 27.56 ‐0.59 1.26E‐02 Serinc5 chr13:93381092‐93481901 64.66 35.97 ‐0.59 1.66E‐02 Trim59 chr3:68839215‐68848664 77.21 42.92 ‐0.59 1.54E‐02 Gm410 chr3:57471343‐57496459 25.33 14.06 ‐0.59 2.43E‐02 Hopx chr5:77516010‐77544148 388.22 214.38 ‐0.59 3.06E‐02 Nipa1 chr7:63217900‐63274943 116.81 64.50 ‐0.59 1.54E‐02 Klf9 chr19:23215715‐23241401 45.95 25.36 ‐0.59 1.27E‐02 Gm15663 chr10:105011606‐105020926 8.37 4.62 ‐0.59 3.81E‐02 Tmem123 chr9:7764076‐7794332 28.71 15.82 ‐0.60 1.09E‐02 Cyp2j12 chr4:95766008‐95807843 29.52 16.26 ‐0.60 1.43E‐02 Reep3 chr10:66471936‐66559736 25.90 14.26 ‐0.60 9.79E‐03 Sox2ot chr3:34459302‐34576915 168.98 93.02 ‐0.60 3.34E‐02 Dpy19l1 chr9:24216224‐24307584 113.79 62.59 ‐0.60 2.25E‐02 Tmem144 chr3:79617074‐79646584 25.87 14.23 ‐0.60 1.34E‐02 Nkain2 chr10:31409124‐32609721 74.98 41.14 ‐0.60 2.39E‐02 Snhg11 chr2:158201373‐158211881 9.18 5.02 ‐0.60 1.49E‐02 Ptch1 chr13:63612840‐63666828 5.36 2.93 ‐0.60 4.67E‐02 Ctsk chr3:95303207‐95313284 27.68 15.12 ‐0.60 2.39E‐02 Gstm7 chr3:107729251‐107734663 557.23 304.21 ‐0.61 2.54E‐02 Palm2 chr4:57581119‐57730000 8.91 4.86 ‐0.61 1.10E‐02 Piga chrX:160857718‐160871847 16.18 8.80 ‐0.61 1.06E‐02 Mfsd2a chr4:122624093‐122638431 17.87 9.67 ‐0.61 2.02E‐02 Lpar1 chr4:58448123‐58566363 72.25 38.85 ‐0.62 6.18E‐03 Etv1 chr12:39506844‐39594802 76.97 41.13 ‐0.63 1.23E‐02 Mar1 chr8:68141938‐68995536 9.85 5.23 ‐0.63 1.20E‐02 Sspn chr6:145882666‐145913745 11.95 6.34 ‐0.63 7.64E‐03 Zcchc24 chr14:26531125‐26588342 94.02 49.87 ‐0.63 9.82E‐03 Fam57a chr11:76015557‐76021759 39.13 20.73 ‐0.64 1.88E‐02 Apln chrX:45378322‐45388029 36.07 19.06 ‐0.64 4.91E‐03 Rab31 chr17:66001065‐66122092 275.88 145.51 ‐0.64 3.10E‐02 Pcolce2 chr9:95538046‐95595970 83.93 44.13 ‐0.64 5.10E‐03 Fktn chr4:53727053‐53776143 9.10 4.73 ‐0.66 1.37E‐02 Insig1 chr5:28397951‐28405202 55.09 28.58 ‐0.66 3.85E‐03 Dnajc24 chr2:105806864‐105843706 45.62 23.62 ‐0.66 7.17E‐03 Zfp536 chr7:38264127‐38554771 5.10 2.64 ‐0.66 3.23E‐02 Gm13375 chr2:20890500‐20891975 40.36 20.83 ‐0.66 7.89E‐03 9330117O12 chr18:54771397‐54799524 13.05 6.72 ‐0.66 1.70E‐02 Phactr3 chr2:177853679‐178073197 33.67 17.27 ‐0.67 1.41E‐02 Thap2 chr10:114807021‐114821491 4.91 2.51 ‐0.67 2.94E‐02 Tmeff1 chr4:48598064‐48676003 205.85 105.29 ‐0.67 6.18E‐03 C630043F03Rchr4:71862277‐71864964 9.44 4.82 ‐0.67 1.54E‐02 Shisa4 chr1:137268032‐137271640 387.95 196.85 ‐0.68 3.76E‐03 Ptn chr6:36665662‐36761361 87.34 44.03 ‐0.69 1.72E‐03 Pon2 chr6:5214623‐5248373 63.03 31.58 ‐0.69 1.38E‐03 Crip1 chr12:114390222‐114392090 65.88 32.42 ‐0.71 2.21E‐02 C030030A07Rchr6:136777363‐136789075 83.17 39.77 ‐0.74 1.15E‐03 Itgad chr7:135317459‐135349880 3.60 1.71 ‐0.74 3.44E‐02 Bdkrb2 chr12:106801381‐106831281 5.87 2.72 ‐0.77 4.63E‐02 Prrg1 chrX:75694951‐75829235 12.89 5.97 ‐0.77 4.63E‐04 Rnf141 chr7:117960048‐117987895 43.95 20.30 ‐0.77 1.24E‐03 Scd3 chr19:44277777‐44318506 13.19 6.09 ‐0.77 8.29E‐04 Lefty1 chr1:182865169‐182868532 9.99 4.58 ‐0.78 1.46E‐02 Fam171b chr2:83652884‐83721515 33.04 15.09 ‐0.78 2.28E‐04 Sgk3 chr1:9788210‐9892649 28.38 12.95 ‐0.78 1.38E‐03 Gpr98 chr13:81234066‐81772143 1.26 0.57 ‐0.79 2.45E‐03 Gjc3 chr5:138395036‐138404187 131.50 58.56 ‐0.81 1.99E‐04 Miat chr5:112642247‐112657968 2.01 0.89 ‐0.81 3.99E‐03 Scd1 chr19:44468939‐44482199 101.25 43.67 ‐0.84 1.11E‐04 Expi chr11:83522505‐83524862 50.19 21.34 ‐0.86 3.29E‐03 A230001M10 chr3:102066327‐102092694 38.30 15.58 ‐0.90 1.30E‐05 Arid5b chr10:67558340‐67741474 8.50 3.42 ‐0.91 9.51E‐05 Pla2g3 chr11:3388229‐3394169 15.57 6.01 ‐0.95 3.39E‐05 Meg3 chr12:110779205‐110809939 9.96 3.76 ‐0.97 2.30E‐02 1500015L24R chr19:20479777‐20497275 10.58 3.89 ‐1.00 9.79E‐03 Gabrr2 chr4:33150086‐33182840 6.56 1.78 ‐1.30 3.59E‐05 Ubtf 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 GO:0030695 GTPase regulator activity 9.79E-12 regulation of Ras protein signal GO:0046578 transduction 1.29E-05 Rho guanyl-nucleotide Molecular GO:0005089 exchange factor activity 3.14E-04 function phosphoinositide GO:0004435 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 Lgals3 C1qa Lgals3 Aven Sgk1 C1qb Rap1gap Fam123a Vim C1qc Sgk1 Lgals3 Cdkn1a Vim Nfkbia Ctss Pla2g4e Inpp5d Serpina3n Lgals3 Sgk1 Lyz2 Socs3 Nupr1 Vim Prkd3 Rasgrp3 Sgk1 Tagln2 Timp1 Trem2 Txnip Tyrobp Vim Supplementary Table 10: Primer sequences Gene name Forward primer Reverse primer huSOD1 GTAATGGACCAGTGAAGGTGTGG AGTGAGGACCTGCACTGGTACA mSOD1 GCAAGCGGTGAACCAGTTGTGT ATGAGGTCCTGCACTGGTACAG Dnaja2 TGTGCTCCGACTGTAATGGAG CTTCCCCAGTGAACGTAATCC Rpl23 ACAGACTTCCTGCTGCTGGTG GACCCCTGCGTTATCTTCAAAG ATF4 TGGATGATGGCTTGGCCAGTG GAGCTCATCTGGCATGGTTTC Chop CTGCCTTTCACCTTGGAGACG CTTTGGGATGTGCGTGTGACC Bip/Grp78 GTGCAGCAGGACATCAAGTTC TACGCCTCAGCAGTCTCCTTC Xbp1 (qPCR) AAGCGCTGCGGAGGAAACTG GCCGTGAGTTTTCTCCCGTAA Xbp1 (splicing) GGGAATGGACACGCTGGATCC GTCCATGGGAAGATGTTCTGG PDI GTTGCCCAAGGAGGACTGTAG TATAGCGCTGGCCAAAGAACG p62 AGGGAACACAGCAAGCTCATC TGACTCAGCTGTAGGGCAAGG Cxcl10 TGGGTCTGAGTGGGACTCAAG CGTGGCAATGATCTCAACACG Igfbp7 GGAAAATCTGGCCATTCAGACC TTCATGGAGGGCATCAACCAC Cadps CTGCAGCTCCACATTTACCA GACGCTGTAGCTTCCTCCAC Rusc1 CCCCCAACAGTAAGTGTGCT GCAGTGTGGTCACAGAGAGC Tmeff1 GCAGCCATTATTGGAGCAGT ATATAAAAGTCCGCGGCTCA Nkain2 TATGGATACCAAGGGCCTCA GAGTCCATGGGCTGACAGA EGFP CCTACGGCGTGCAGTGCTTC CGAGCTGCACGCTGCCGTCC β-actin AGAGGGAAATCGTGCGTGAC CAATAGTGATGACCTGGCCGT