Selective loss of neurofilament expression in Cu/Zn superoxide dismutase (SOD1) linked amyotrophic lateral sclerosis FionaM.Menzies,* ,1 AndrewJ.Grierson,*MarkR.Cookson,* ,1 PaulR.Heath,*JanineTomkins,* Denise A. Figlewicz,à PaulG.Ince andPamelaJ.Shaw* *Academic Neurology Unit and  Academic Unit of Pathology, The Medical School, University of Sheffield, Sheffield, South Yorkshire, UK àDepartments of Neurology, Neurobiology and Anatomy, University of Rochester, New York, USA Abstract Neurofilament pathology is a hallmark of sporadic and familial amyotrophic lateral sclerosis (SALS and FALS). The disease mechanisms underlying this pathology are presently unclear, but recent evidence in SALS patients suggest that reductions in neurofilament light subunit (NFL) mRNA may contribute to the death of motor neurones. Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) represent the best- studied cause of FALS, and a number of laboratory models of SOD1-mediated disease exist. Here we have used microdis- sected lumbar spinal cord motor neurones from human SOD1 FALS patients as well as G93A SOD1 transgenic mice and demonstrated that reduced NFL mRNA levels are seen in both. To probe the molecular mechanisms underpinning these observations, we generated NSC34 motor neurone-like cell lines expressing wild-type and mutant SOD1. NSC34 cells expressing G37R or G93A SOD1 showed selective reductions in NFL and NFM mRNA and protein. These data suggest that NFL mRNA reductions are common to SALS and FALS patients, and that cells and mice expressing mutant SOD1 may enable us to characterize the molecular mechanism(s) responsible for the loss of neurofilament mRNA. Keywords: amyotrophic lateral sclerosis, gene expression, neurofilaments, SOD1. J. Neurochem. (2002) 82, 1118–1128. Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disease characterized by the degeneration and cell death of motor neurones of the spinal cord, brain stem and motor cortex. Although 90% of cases are sporadic, the remainder are familial and, of these, 20% are due to mutations in the gene encoding Cu-Zn super- oxide dismutase (SOD1; Rosen et al. 1993). A common featureofthepathologyofbothsporadicandfamilialforms of the disease, including those with SOD1 mutations, is the presence of axonal and perikaryal neurofilament accumula- tions, suggesting a role for neurofilaments in the pathogen- esis of ALS (Carpenter 1968; Hirano et al. 1984; Rouleau et al. 1996; Ince et al. 1998). Neurofilament proteins are members of the predominant intermediate filaments expressed in large calibre neurones. The three subunits, neurofilament light (NFL), medium (NFM) and heavy (NFH)are68kDa,115kDaand160kDa,respectively.All three proteins are highly expressed in motor neurones, which is consistent with their potential to contribute to the development of ALS. Several laboratories have reported NFH mutations in sporadicALSpatients(Figlewicz et al. 1994;Tomkins et al. Received March 10, 2002; revised manuscript received May 8, 2002; acceptedMay15,2002. Address correspondence and reprint requests to Pamela J. Shaw, AcademicNeurologyUnit,TheMedicalSchool,UniversityofSheffield, BeechHillRoad,Sheffield,SouthYorkshireS102RX,UK. E-mail: pamela.shaw@sheffield.ac.uk 1 PresentaddressforF.M.MenziesandM.R.CooksonisNeurogenetics Branch, NINDS, NIH, Building 10, Room 3B12, 9000 Rockville Pike, Bethesda,MD20892,USA. Abbreviations used: ALS, amyotrophic lateral sclerosis; DAB, diam- inobenzidine; DEPC, diethyl pyrocarbonate; DMEM, Dulbecco’s mod- ified Eagle’s medium; ECL, enhanced chemiluminesence; FALS, familial amyotrophic lateral sclerosis; GAPDH, glyceraldehydephos- phatedehydrogenasemRNAmessengerRNA;NF,neurofilament;NFH, neurofilament heavy subunit; NFL, neurofilament light subunit; NFM, neurofilament medium subunit; nSOD1, normal Cu-Zn superoxide dismutase; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; RT, room temperature; SALS, sporadic amyotrophic lateral sclerosis; SDS, sodium dodecyl sulfate; SOD1, Cu/Zn superoxide dismutase; TBS, Tris-buffered saline. Journal of Neurochemistry ,2002, 82,1118–1128 1118 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry , 82,1118–1128