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 superoxide dismutase 1 (SOD1) provokes defects, excessive production of extracellular superoxide, and ox- noncell autonomous paralytic disease. 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 gene 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 protein 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- NEUROSCIENCE occur in genes that are involved in inflammation and metabolism and are targets of the peroxisome proliferator-activated receptor and proaches have clear disadvantages, however, including cross- liver 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 superoxide dismutase. 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 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 transgenes (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 Gene Expression 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 Diseases, 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 Early Edition | 1of10 Downloaded by guest on September 24, 2021 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 proteins 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 motor neuron disease 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