Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis

Accumulating evidence indicates that abnormal conformation of mutant superoxide dismutase 1 (SOD1) is an essential feature underlying the pathogenesis of mutant SOD1-linked familial amyotrophic lateral sclerosis (ALS). Here we investigated the role of ubiquitin-proteasome pathway in the mutant SOD1-related cell death and the effect of oxidative stress on the misfolding of mutant SOD1. Transient overexpression of ubiquitin with human SOD1 (wild-type, ala4val, gly85arg, gly93ala) in Neuro2A cells decreased the amount of mutant SOD1, but not of wild-type, while only mutants were co-immunoprecipitated with poly-ubiquitin. Proteasome inhibition by lactacystin augmented accumulation of mutant SOD1 in the non-ionic detergent-insoluble fraction. The spinal cord lysates from mutant SOD1 transgenic mice showed multiple carbonylated proteins, including mutant SOD1 with SDS-resistant dimer formation. Furthermore, the treatment of hSOD1-expressing cells with hydrogen peroxide promoted the oligomerization, and detergent-insolubility of mutant SOD1 alone, and the oxidized mutant SOD1 proteins were more heavily poly-ubiquitinated. In Neuro2A cells stably expressing human SOD1 protein, the proteasome function measured by chymotrypsin-like activity, was decreased over time without a quantitative alteration of the 20S proteasomal component. Finally, primary motor neurons from the mouse embryonic spinal cord were more vulnerable to lactacystin than non-motor neurons. These results indicate that the sustained expression of mutant SOD1 leads to proteasomal inhibition and motor neuronal death, which in part explains the pathogenesis of mutant SOD1-linked ALS.     

Aggregation modulating elements in mutant human superoxide dismutase 1

Mutations in superoxide dismutase 1 (SOD1) cause some forms of familial amyotrophic lateral sclerosis (fALS). Affected tissues of patients and transgenic mouse models of the disease accumulate misfolded and aggregated forms of the mutant protein. In the present study we have identified specific sequences in human SOD1 that modulate the aggregation of fALS mutant proteins. From our study of a panel of mutant proteins, we identify two sequence elements in human SOD1 (residues 42-50 and 109-123) that are critical in modulating the aggregation of the protein. These sequences are components of the 4th and 7th β-strands of the protein, and in the native structure are normally juxtaposed as elements of the core β-barrel. Our data suggest that some type of intermolecular interaction between these elements may occur in promoting mutant SOD1 aggregation.     

Sequence and structural determinants of Cu, Zn superoxide dismutase aggregation

Diverse point mutations in the enzyme Cu, Zn superoxide dismutase (SOD1) are linked to its aggregation in the familial form of the disease amyotrophic lateral sclerosis. The disease-associated mutations are known to destabilize the protein, but the structural basis of the aggregation of the destabilized protein and the structure of aggregates are not well understood. Here, we investigate in silico the sequence and structural determinants of SOD1 aggregation: (1) We identify sequence fragments in SOD1 that have a high aggregation propensity, using only the sequence of SOD1, and (2) we perform molecular dynamics simulations of the SOD1 dimer folding and misfolding. In both cases, we identify identical regions of the protein as having high propensity to form intermolecular interactions. These regions correspond to the N- and C-termini, and two crossover loops and two beta-strands in the Greek-key native fold of SOD1. Our results suggest that the high aggregation propensity of mutant SOD1 may result from a synergy of two factors: the presence of highly amyloidogenic sequence fragments ("hot spots"), and the presence of these fragments in regions of the protein that are structurally most likely to form intermolecular contacts under destabilizing conditions. Therefore, we postulate that the balance between the self-association of aggregation-prone sequences and the specific structural context of these sequences in the native state determines the aggregation propensity of proteins.     

A novel variant of human superoxide dismutase 1 harboring amyotrophic lateral sclerosis-associated and experimental mutations in metal-binding residues and free cysteines lacks toxicity in vivo

Mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis. The Cu-binding capacity of SOD1 has spawned hypotheses that implicate metal-mediated production of reactive species as a potential mechanism of toxicity. In past experiments, we have tested such hypotheses by mutating residues in SOD1 that normally coordinate the binding of Cu, finding that such mutants retain the capacity to induce motor neuron disease. We now describe the lack of disease in mice that express a variant of human SOD1 in which residues that coordinate the binding of Cu and Zn have been mutated (SODMD). SODMD encodes three disease-causing and four experimental mutations that ultimately eliminate all histidines involved in the binding of metals; and includes one disease-causing and one experimental mutation that eliminate secondary metal binding at C6 and C111. We show that the combined effect of these mutations produces a protein that is unstable but does not aggregate on its own, is not toxic, and does not induce disease when co-expressed with high levels of wild-type SOD1. In cell culture models, we determine that the combined mutation of C6 and C111 to G and S, respectively, dramatically reduces the aggregation propensity of SODMD and may account for the lack of toxicity for this mutant.     

Prion-like activity of Cu/Zn superoxide dismutase

Neurodegenerative diseases belong to a larger group of protein misfolding disorders, known as proteinopathies. There is increasing experimental evidence implicating prion-like mechanisms in many common neurodegenerative disorders, including Alzheimer disease, Parkinson disease, the tauopathies, and amyotrophic lateral sclerosis (ALS), all of which feature the aberrant misfolding and aggregation of specific proteins. The prion paradigm provides a mechanism by which a mutant or wild-type protein can dominate pathogenesis through the initiation of self-propagating protein misfolding. ALS, a lethal disease characterized by progressive degeneration of motor neurons is understood as a classical proteinopathy; the disease is typified by the formation of inclusions consisting of aggregated protein within and around motor neurons that can contribute to neurotoxicity. It is well established that misfolded/oxidized SOD1 protein is highly toxic to motor neurons and plays a prominent role in the pathology of ALS. Recent work has identified propagated protein misfolding properties in both mutant and wild-type SOD1, which may provide the molecular basis for the clinically observed contiguous spread of the disease through the neuroaxis. In this review we examine the current state of knowledge regarding the prion-like properties of SOD1 and comment on its proposed mechanisms of intercellular transmission.     

Modulation of mutant superoxide dismutase 1 aggregation by co-expression of wild-type enzyme

Mutations in superoxide dismutase 1 (SOD1, EC 1.15.1.1) cause familial amyotrophic lateral sclerosis; with aggregated forms of mutant protein accumulating in spinal cord tissues of transgenic mouse models and human patients. Mice over-expressing wild-type human SOD1 (WT hSOD1) do not develop amyotrophic lateral sclerosis-like disease, but co-expression of WT enzyme at high levels with mutant SOD1 accelerates the onset of motor neuron disease compared with mice expressing mutant hSOD1 alone. Spinal cords of mice expressing both proteins contain aggregated forms of mutant protein and, in some cases, evidence of co-aggregation of WT hSOD1 enzyme. In the present study, we used a cell culture model of mutant SOD1 aggregation to examine how the presence of WT SOD1 affects mutant protein aggregation, finding that co-expression of WT SOD1, hSOD1 or mouse SOD1, delayed the formation of mutant hSOD1 aggregates; in essence appearing to slow the aggregation rate. In some combinations of WT and mutant hSOD1 co-expression, the aggregates that did eventually form appeared to contain WT hSOD1 protein. However, WT mouse SOD1 did not co-aggregate with mutant hSOD1 despite displaying a similar ability to slow mutant hSOD1 aggregation. Together, these studies indicate that WT SOD1 (human or mouse), when expressed at levels equivalent to the mutant protein, modulates the aggregation of mutant SOD1.     

Exosome-dependent and independent mechanisms are involved in prion-like transmission of propagated Cu/Zn superoxide dismutase misfolding

Amyotrophic lateral sclerosis (ALS), a fatal adult-onset degenerative neuromuscular disorder with a poorly defined etiology, progresses in an orderly spatiotemporal manner from one or more foci within the nervous system, reminiscent of prion disease pathology. We have previously shown that misfolded mutant Cu/Zn superoxide dismutase (SOD1), mutation of which is associated with a subset of ALS cases, can induce endogenous wild-type SOD1 misfolding in the intracellular environment in a templating fashion similar to that of misfolded prion protein. Our recent observations further extend the prion paradigm of pathological SOD1 to help explain the intercellular transmission of disease along the neuroaxis. It has been shown that both mutant and misfolded wild-type SOD1 can traverse cell-to-cell either as protein aggregates that are released from dying cells and taken up by neighboring cells via macropinocytosis, or released to the extracellular environment on the surface of exosomes secreted from living cells. Furthermore, once propagation of misfolded wild-type SOD1 has been initiated in human cell culture, it continues over multiple passages of transfer and cell growth. Propagation and transmission of misfolded wild-type SOD1 is therefore a potential mechanism in the systematic progression of ALS pathology.     

Solvent sensitivity of protein aggregation in Cu,Zn superoxide dismutase: a molecular dynamics simulation study

Misfolding and aggregation of Cu, Zn Superoxide Dismutase (SOD1) is often found in amyotrophic lateral sclerosis (ALS) patients. The central apo SOD1 barrel was involved in protein maturation and pathological aggregation in ALS. In this work, we employed atomistic molecular dynamics (MD) simulations to study the conformational dynamics of SOD1^barrel monomer in different concentrations of trifluoroethanol (TFE). We find concentration dependence unusual structural and dynamical features, characterized by the local unfolding of SOD1^barrel. This partially unfolded structure is characterized by the exposure of hydrophobic core, is highly dynamic in nature, and is the precursor of aggregation seen in SOD1^barrel. Our computational studies supports the hypothesis of the formation of aggregation ‘building blocks’ by means of local unfolding of apo monomer as the mechanism of SOD1 fibrillar aggregation. The non-monotonic TFE concentration dependence of protein conformational changes was explored through simulation studies. Our results suggest that altered protein conformation and dynamics within its structure may underlie the aggregation of SOD1 in ALS.     

Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values

This article utilized “protein charge ladders”—chemical derivatives of proteins with similar structure, but systematically altered net charge—to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn²⁺ stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude—by up to 7.4 units per dimer at lysosomal pH—than values predicted from standard pK_a values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to +2.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn²⁺ to mutant SOD1 lowered its net charge by an additional +2.33 ± 0.01 to +3.18 ± 0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn²⁺ (ΔZ < 0.44 ± 0.07 per additional Zn²⁺). Both metalated and apo-SOD1 regulated net charge across subcellular pH, without inverting from negative to positive at the theoretical pI. Differential scanning calorimetry, hydrogen-deuterium exchange, and inductively coupled plasma mass spectrometry confirmed that the structure, stability, and metal content of mutant proteins were not significantly affected by lysine acetylation. Measured values of net charge should be used when correlating the biophysical properties of a specific ALS-variant SOD1 protein with its observed aggregation propensity or clinical phenotype.     

Inducible superoxide dismutase 1 aggregation in transgenic amyotrophic lateral sclerosis mouse fibroblasts

High molecular weight detergent-insoluble complexes of superoxide dismutase 1 (SOD1) enzyme are a biochemical abnormality associated with mutant SOD1-linked familial amyotrophic lateral sclerosis (FALS). In the present study, SOD1 protein from spinal cords of transgenic FALS mice was fractionated according to solubility in saline, zwitterionic, non-ionic or anionic detergents. Both endogenous mouse SOD1 and mutant human SOD1 were least soluble in SDS, followed by NP-40 and CHAPS, with an eight-fold greater detergent resistance of mutant protein overall. Importantly, high molecular weight mutant SOD1 complexes were isolated with SDS-extraction only. To reproduce SOD1 aggregate pathology in vitro, primary fibroblasts were isolated and cultured from neonatal transgenic FALS mice. Fibroblasts expressed abundant mutant SOD1 without spontaneous aggregation over time with passage. Proteasomal inhibition of cultures using lactacystin induced dose-dependent aggregation and increased the SDS-insoluble fraction of mutant SOD1, but not endogenous SOD1. In contrast, paraquat-mediated superoxide stress in fibroblasts promoted aggregation of endogenous SOD1, but not mutant SOD1. Treatment of cultures with peroxynitrite or the copper chelator diethyldithiocarbamate (DDC) alone did not modulate aggregation. However, DDC inhibited lactacystin-induced mutant SOD1 aggregation in transgenic fibroblasts, while exogenous copper slightly augmented aggregation. These data suggest that SOD1 aggregates may derive from proteasomal or oxidation-mediated oligomerisation pathways from mutant and endogenous subunits respectively. Furthermore, these pathways may be affected by copper availability. We propose that non-neural cultures such as these transgenic fibroblasts with inducible SOD1 aggregation may be useful for rapid screening of compounds with anti-aggregation potential in FALS.     

Folding of Cu, Zn superoxide dismutase and familial amyotrophic lateral sclerosis

Cu, Zn superoxide dismutase (SOD1) has been implicated in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). It has been suggested that mutant mediated SOD1 misfolding/aggregation is an integral part of the pathology of ALS. We study the folding thermodynamics and kinetics of SOD1 using a hybrid molecular dynamics approach. We reproduce the experimentally observed SOD1 folding thermodynamics and find that the residues which contribute the most to SOD1 thermal stability are also crucial for apparent two-state folding kinetics. Surprisingly, we find that these residues are located on the surface of the protein and not in the hydrophobic core. Mutations in some of the identified residues are found in patients with the disease. We argue that the identified residues may play an important role in aggregation. To further characterize the folding of SOD1, we study the role of cysteine residues in folding and find that non-native disulfide bond formation may significantly alter SOD1 folding dynamics and aggregation propensity.     

Phosphorylation state of the native high-molecular-weight neurofilament subunit protein from cervical spinal cord in sporadic amyotrophic lateral sclerosis

The intraneuronal aggregation of phosphorylated high-molecular-weight neurofilament protein (NFH) in spinal cord motor neurons is considered to be a key pathological marker of amyotrophic lateral sclerosis (ALS). In order to determine whether this observation is due to the aberrant or hyper-phosphorylation of NFH, we have purified and characterized NFH from the cervical spinal cords of ALS patients and controls. We observed no differences between ALS and normal controls in the physicochemical properties of NFH in Triton X-100 insoluble protein fractions, with respect to migration patterns on 2D-iso electrofocusing (IEF) gels, the rate of Escherichia coli alkaline phosphatase mediated dephosphorylation, or the rate of calpain-mediated proteolysis. The rate of calpain-mediated proteolysis was unaffected by either exhaustive NFH dephosphorylation or by the addition of calmodulin to the reaction. Phosphopeptides and the phosphorylated motifs characterized by liquid chromatography tandem mass spectroscopy (LC/MS/MS) analysis demonstrated that all the phosphorylated residues found in ALS NFH were also found to be phosphorylated in normal human NFH samples. Hence, we have observed no difference in the physicochemical properties of normal and ALS NFH extracted from cervical spinal cords, suggesting that the perikaryal aggregation of highly phosphorylated NF in ALS neurons reflects the aberrant somatotopic localization of normally phosphorylated NFH.     

Transglutaminase 2 accelerates neuroinflammation in amyotrophic lateral sclerosis through interaction with misfolded superoxide dismutase 1

Although the aberrant assembly of mutant superoxide dismutase 1 (mSOD1) is implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS), the molecular basis of superoxide dismutase 1 (SOD1) oligomerization remains undetermined. We investigated the roles of transglutaminase 2 (TG2), an endogenous cross‐linker in mSOD1‐linked ALS. TG2 interacted preferentially with mSOD1 and promoted its oligomerization in transfected cells. Purified TG2 directly oligomerized recombinant mutant SOD1 and the apo‐form of the wild‐type SOD1 proteins in a calcium‐dependent manner, indicating that misfolded SOD1 is a substrate of TG2. Moreover, the non‐cell‐autonomous effect of extracellular TG2 on the neuroinflammation was suggested, since the TG2‐mediated soluble SOD1 oligomers induced tumor necrosis factor‐α, interleukin‐1β, and nitric oxide in microglial BV2 cells. TG2 was up‐regulated in the spinal cord of pre‐symptomatic G93A SOD1 transgenic mice and in the hypoglossal nuclei of mice suffering nerve ligation. Furthermore, inhibition of spinal TG2 by cystamine significantly delayed the progression and reduced SOD1 oligomers and microglial activation. These results indicate a novel role of TG2 in SOD1 oligomer‐mediated neuroinflammation, as well as in the involvement in the intracellular aggregation of misfolded SOD1 in ALS.

     

Misfolded superoxide dismutase-1 in CSF from amyotrophic lateral sclerosis patients

Several of the superoxide dismutase-1 (SOD1) mutations linked to amyotrophic lateral sclerosis (ALS) lead to synthesis of structurally defective molecules, suggesting that any cytotoxic conformational species common for all mutations should be misfolded. SOD1 can be secreted and evidence from ALS model systems suggests that extracellular SOD1 may be involved in cytotoxicity. Three ELISAs specifically reacting with different sequence segments in misfolded SOD1 species were used for analysis of CSF from 38 neurological controls and from 96 ALS patients, 57 of whom were sporadic cases and 39 familial, including 22 patients carrying SOD1 mutations. Misfolded SOD1 was found in all samples. There were, however, no significant differences between patients with and without mutations, and between all the ALS patients and the controls. The estimated concentration of misfolded SOD1 in the interstitium of the CNS is a 1000 times lower than that required for appreciable cytotoxicity in model systems. The results argue against a direct cytotoxic role of extracellular misfolded SOD1 in ALS. Misfolded SOD1 in CSF cannot be used as a biomarker of ALS in patients with and without mutations in the enzyme.     

Novel behavioural characteristics of the superoxide dismutase 1 G93A (SOD1G93A) mouse model of amyotrophic lateral sclerosis include sex‐dependent phenotypes

Amyotrophic lateral sclerosis (ALS) involves the rapid degeneration of upper and lower motor neurons leading to weakening and paralysis of voluntary movements. Mutations in copper‐zinc superoxide dismutase 1 (SOD1) are a known genetic cause of ALS, and the SOD1 ^G93A mouse has been used extensively to investigate molecular mechanisms in ALS. In recent years, evidence suggests that ALS and frontotemporal dementia form a spectrum disorder ranging from motor to cognitive dysfunctions. Thus, we tested male and female SOD1 ^G93A mice for the first time before the onset of debilitating motor impairments in behavioural domains relevant to both ALS and frontotemporal dementia. SOD1 ^G93A males displayed reduced locomotion, exploration and increased anxiety‐like behaviours compared with control males. Intermediate‐term spatial memory was impaired in SOD1 ^G93A females, whereas long‐term spatial memory deficits as well as lower acoustic startle response, and prepulse inhibition were identified in SOD1 ^G93A mice of both sexes compared with respective controls. Interestingly, SOD1 ^G93A males exhibited an increased conditioned cue freezing response. Nosing behaviours were also elevated in both male and female SOD1 ^G93A when assessed in social paradigms. In conclusion, SOD1 ^G93A mice exhibit a variety of sex‐specific behavioural deficits beyond motor impairments supporting the notion of an ALS‐frontotemporal spectrum disorder. Thus, SOD1 ^G93A mice may represent a useful model to test the efficacy of therapeutic interventions on clinical symptoms in addition to declining motor abilities.     

Heat-shock protein 105 interacts with and suppresses aggregation of mutant Cu/Zn superoxide dismutase: clues to a possible strategy for treating ALS

A dominant mutation in the gene for copper-zinc superoxide dismutase (SOD1) is the most frequent cause of the inherited form of amyotrophic lateral sclerosis. Mutant SOD1 provokes progressive degeneration of motor neurons by an unidentified acquired toxicity. Exploiting both affinity purification and mass spectrometry, we identified a novel interaction between heat-shock protein 105 (Hsp105) and mutant SOD1. We detected this interaction both in spinal cord extracts of mutant SOD1(G93A) transgenic mice and in cultured neuroblastoma cells. Expression of Hsp105, which is found in mouse motor neurons, was depressed in the spinal cords of SOD1(G93A) mice as disease progressed, while levels of expression of two other heat-shock proteins, Hsp70 and Hsp27, were elevated. Moreover, Hsp105 suppressed the formation of mutant SOD1-containing aggregates in cultured cells. These results suggest that techniques that raise levels of Hsp105 might be promising tools for alleviation of the mutant SOD1 toxicity.     

Metabolomic analysis and signatures in motor neuron disease

Motor neuron diseases (MND) are a heterogeneous group of disorders that includes amyotrophic lateral sclerosis (ALS) and result in death of motor neurons. These diseases may produce characteristic perturbations of the metabolome, the collection of small-molecules (metabolites) present in a cell, tissue, or organism. To test this hypothesis, we used high performance liquid chromatography followed by electrochemical detection to profile blood plasma from 28 patients with MND and 30 healthy controls. Of 317 metabolites, 50 were elevated in MND patients and more than 70 were decreased (p<0.05). Among the compounds elevated, 12 were associated with the drug Riluzole. In a subsequent study of 19 subjects with MND who were not taking Riluzole and 33 healthy control subjects, six compounds were significantly elevated in MND, while the number of compounds with decreased concentration was similar to study 1. Our data also revealed a distinctive signature of highly correlated metabolites in a set of four patients, three of whom had lower motor neuron (LMN) disease. In both datasets we were able to separate MND patients from controls using multivariate regression techniques. These results suggest that metabolomic studies can be used to ascertain metabolic signatures of disease in a non-invasive fashion. Elucidation of the structures of signature molecules in ALS and other forms of MND should provide insight into aberrant biochemical pathways and may provide diagnostic markers and targets for drug design.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. ^†S.R., M.E.C. and M.B. contributed equally to this work. W.R.M. and B.S.K. contributed equally to this work. S.R., M.B., W.R.M., B.S.K., C.B., S.H., P.V., M.F.B., and R.K-D. have financial interests in Metabolon Inc., a company engaged in metabolic profiling. ^††Electronic supporting figures, tables and datasets are available at the Journal’s website.*To whom corrsepondence should be addressed.E-mail: kaddu001@mc.duke.eduCurrent address: Duke University Medical Center, Department of Psychiatry, P.O. Box 3950, Durham, NC 27710.     

Overexpression of manganese superoxide dismutase attenuates neuronal death in human cells expressing mutant (G37R) Cu/Zn-superoxide dismutase

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor function and eventual death as a result of degeneration of motor neurons in the spinal cord and brain. The discovery of mutations in SOD1, the gene encoding the antioxidant enzyme Cu/Zn-superoxide dismutase (CuZnSOD), in a subset of ALS patients has led to new insight into the pathophysiology of ALS. Utilizing a novel adenovirus gene delivery system, our laboratory has developed a human cell culture model using chemically differentiated neuroblastoma cells to investigate how mutations in SOD1 lead to neuronal death. Expression of mutant SOD1 (G37R) resulted in a time and dose-related death of differentiated neuroblastoma cells. This cell death was inhibited by overexpression of the antioxidant enzyme manganese superoxide dismutase (MnSOD). These observations support the hypothesis that mutant SOD1-associated neuronal death is associated with alterations in oxidative stress, and since MnSOD is a mitochondrial enzyme, suggest that mitochondria play a key role in disease pathogenesis. Our findings in this model of inhibition of mutant SOD1-associated death by MnSOD represent an unique approach to explore the underlying mechanisms of mutant SOD1 cytotoxicity and can be used to identify potential therapeutic agents for further testing.     

Selective loss of neurofilament expression in Cu/Zn superoxide dismutase (SOD1) linked amyotrophic lateral sclerosis

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 microdissected 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.