Recent advances in research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 gene mutations: neuronal Lewy body-like hyaline inclusions and astrocytic hyaline inclusions.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily involves the motor neuron system. Of all patients with ALS, approximately 5%-10% of them are familial and most of the others are sporadic. Superoxide dismutase 1 (SOD1) gene mutations are shown to be associated with about 20% of familial ALS (FALS) patients. FALS is neuropathologically classified into two subtypes: classical FALS in which degeneration is restricted to only motor neurons and FALS which is characterized by the degeneration of the posterior column in addition to the lesion of the motor neuron system. The neuronal Lewy body-like hyaline inclusion (LBHI) is a characteristic neuropathological marker of mutant SOD1-linked FALS with posterior column involvement. Inclusions similar to the neuronal LBHIs have been discovered in astrocytes in certain patients with FALS exhibiting SOD1 gene mutations. The purpose of this review is to discuss the novel neuropathological significance of the astrocytic hyaline inclusions (Ast-HIs) and neuronal LBHIs in brain tissues from individuals with the posterior-column-involvement-type FALS with SOD1 gene mutations. In hematoxylin and eosin preparations, both Ast-HIs and neuronal LBHIs are eosinophilic inclusions and sometimes show eosinophilic cores with paler peripheral halos. Immunohistochemically, both inclusions are intensely positive for SOD1. At the ultrastructural level, both inclusions consist of approximately 15-25 nm-sized granule-coated fibrils and granular materials. Immunoelectron microscopically, these abnormal granule-coated fibrils and granular materials are positive for SOD1. Therefore, the FALS disease process originating from SOD1 gene mutations occurs in astrocytes as well as neurons and is involved in the formation of both inclusions.     

Evidence of Increased Oxidative Damage in Both Sporadic and Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal dominant familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), suggesting that oxidative damage may play a role in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined markers of oxidative damage to protein, lipids, and DNA in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Protein carbonyl and nuclear DNA 8-hydroxy-2'-deoxyguanosine (OH⁸dG) levels were increased in SALS motor cortex but not in FALS patients. Malondialdehyde levels showed no significant changes. Immunohistochemical studies showed increased neuronal staining for hemeoxygenase-1, malondialdehyde-modified protein, and OH⁸dG in both SALS and FALS spinal cord. These studies therefore provide further evidence that oxidative damage may play a role in the pathogenesis of neuronal degeneration in both SALS and FALS.     

Metabolic Dysfunction in Familial, but Not Sporadic, Amyotrophic Lateral Sclerosis

Abstract: Autosomal dominant familial amyotrophic lateral sclerosis (FALS) is associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Previous studies have implicated the involvement of metabolic dysfunction in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined SOD activity and mitochondrial oxidative phosphorylation enzyme activities in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Cytosolic SOD activity, predominantly Cu/Zn SOD, was decreased ∼50% in all regions in FALS patients with SOD mutations but was not significantly altered in other patient groups. Marked increases in complex I and II–III activities were seen in FALS patients with SOD mutations but not in SALS patients. We also measured electron transport chain enzyme activities in a transgenic mouse model of FALS. Complex I activity was significantly increased in the forebrain of 60-day-old G93A transgenic mice overexpressing human mutant SOD1, relative to levels in transgenic wild-type animals, supporting the hypothesis that the motor neuron disorder associated with SOD1 mutations involves a defect in mitochondrial energy metabolism.     

Integrative role of cPLA with COX-2 and the effect of non-steriodal anti-inflammatory drugs in a transgenic mouse model of amyotrophic lateral sclerosis

Cyclooxygenase-2 (COX-2) is a key molecule in the inflammatory pathway in amyotrophic lateral sclerosis (ALS). Cytosolic phospholipase A (cPLA2) is an important enzyme providing substrate for cyclooxygenases. We therefore examined cPLA2 expression in human ALS and mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice and its relation to COX-2. Immunohistochemistry and real-time RT-PCR revealed elevated cPLA2 protein and its mRNA levels in the lumbar spinal cord of mutant SOD1 mice. COX-2 immunoreactivity was increased in lumbar spinal cord sections from both familial ALS (FALS) and sporadic ALS (SALS) as compared to controls, and cPLA2 immunoreactivity was increased in a patient with FALS. Oral administration of the non-selective cyclooxygenase (COX) inhibitor, sulindac, extended the survival (by 10%) of G93A SOD1 mice as compared to littermate controls. Sulindac, as well as the selective COX-2 inhibitors, rofecoxib and celecoxib reduced cPLA2 immunoreactivity in the lumbar spinal cord of G93A transgenic mice. Sulindac treatment preserved motor neurons, and reduced microglial activation and astrocytosis, in the spinal cord of G93A SOD1 transgenic mice. These results suggest that cPLA2 plays an important role in supplying arachidonic acid to the COX-2 driven inflammatory pathway in ALS associated with SOD1 mutations.     

Unique characteristics of the genetics epidemiology of amyotrophic lateral sclerosis in China

Continual discoveries of new genes and unraveling the genetic etiology in amyotrophic lateral sclerosis(ALS) have provided greater insight into the underlying pathogenesis in motor neuron degeneration, as well as facilitating the disease modeling and the testing of targeted therapeutics. While, the genetic etiology accounted for two-thirds of FALS and approximately 11% of SALS in Caucasians. However, the contributions of these causative genes to ALS vary among different populations. Furthermore, the prominent difference between Chinese population and other ethnics remains a source of ongoing debate. We systemically reviewed genetics literature of Chinese ALS populations and updated the mutation frequencies of the main ALS-implicated genes aiming to determine the genetic features of ALS in Chinese population. We also reviewed the associations between ALSimplicated single nucleotide polymorphisms(SNPs) and the risk of ALS in Chinese population. A total of 116 studies were included in this analysis(86 gene mutation study articles and 30 SNPs study articles). The results showed that the overall gene mutation rates of ALS-related causative genes were 55.0% in familial ALS(FALS) and 11.7% in sporadic ALS(SALS) in Chinese population. In Chinese FALS, the highest mutation frequency was found in SOD1 gene(25.6%), followed by FUS(5.8%), TARDBP(5.8%), DCTN1(3.6%) and C9 orf 72(3.5%). In Chinese SALS, the highest mutation frequency was also identified in SOD1 gene(1.6%), followed by ANXA11(1.4%), FUS(1.3%), SQSTM1(1.0%), OPTN(0.9%) and CCNF(0.8%).The associations between several SNPs and risk of ALS were also reported in Chinese population. The genetic features of ALS in Chinese population are significantly different from those in Caucasian population, indicating an association between genetic susceptibility and origin of population. Further explorations are required to understand the gene complexity of ALS, including the contribution of most minor genes and the molecular mechanisms in ALS pathologies.     

Superoxide Dismutase Activity, Oxidative Damage, and Mitochondrial Energy Metabolism in Familial and Sporadic Amyotrophic Lateral Sclerosis

The cause of neuronal death in amyotrophic lateral sclerosis (ALS) is unknown. Recently, it was found that some patients with autosomal-dominant familial ALS (FALS) have point mutations in the gene that encodes Cu/Zn superoxide dismutase (SOD1). In this study of postmortem brain tissue, we examined SOD activity and quantified protein carbonyl groups, a marker of oxidative damage, in samples of frontal cortex (Brodmann area 6) from 10 control patients, three FALS patients with known SOD1 mutations (FALS-1), one autosomal-dominant FALS patient with no identifiable SOD1 mutations (FALS-0), and 11 sporadic ALS (SALS) patients. Also, we determined the activities of components of the electron transport chain (complexes I, II-III, and IV) in these samples. The cytosolic SOD activity, which is primarily SOD1 activity, was reduced by 38.8% (p < 0.05) in the FALS-1 patients and not significantly altered in the SALS patients or the FALS-0 patient relative to the control patients. The mitochondrial SOD activity, which is primarily SOD2 activity, was not significantly altered in the FALS-1, FALS-0, or SALS patients. The protein carbonyl content was elevated by 84.8% (p < 0.01) in the SALS patients relative to the control patients. Finally, the complex I activity was increased by 55.3% (p < 0.001) in the FALS-1 patients relative to the control patients. These results from cortical tissue demonstrate that SOD1 activity is reduced and complex I activity is increased in FALS-1 patients and that oxidative damage to proteins is increased in SALS patients.     

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.     

Patient‐derived olfactory mucosa for study of the non‐neuronal contribution to amyotrophic lateral sclerosis pathology

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease which currently has no cure. Research using rodent ALS models transgenic for mutant superoxide dismutase 1 (SOD1) has implicated that glial–neuronal interactions play a major role in the destruction of motor neurons, but the generality of this mechanism is not clear as SOD1 mutations only account for less than 2% of all ALS cases. Recently, this hypothesis was backed up by observation of similar effects using astrocytes derived from post‐mortem spinal cord tissue of ALS patients which did not carry SOD1 mutations. However, such necropsy samples may not be easy to obtain and may not always yield viable cell cultures. Here, we have analysed olfactory mucosa (OM) cells, which can be easily isolated from living ALS patients. Disease‐specific changes observed when ALS OM cells were co‐cultured with human spinal cord neurons included decreased neuronal viability, aberrant neuronal morphology and altered glial inflammatory responses. Our results show the potential of OM cells as new cell models for ALS.     

Identification of two novel loci for dominantly inherited familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, adult-onset motor neuron disease that arises as a dominantly inherited trait in approximately 10% of ALS cases. Mutations in one gene, cytosolic Cu/Zn superoxide dismutase (SOD1), account for approximately 25% of familial ALS (FALS) cases. We have performed a genetic linkage screen in 16 pedigrees with FALS with no evidence for mutations in the SOD1 gene and have identified novel ALS loci on chromosomes 16 and 20. The analysis of these genes will delineate pathways implicated as determinants of motor-neuron viability and provide insights into possible therapies for ALS.     

Rapid Communication: Cu/Zn Superoxide Dismutase Activity in Familial and Sporadic Amyotrophic Lateral Sclerosis

Abstract: Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that is inherited as an autosomal dominant trait in ~ 10% of cases. Recently we and others identified several single-base mutations in the Cu/Zn superoxide dismutase (SOD1) gene in patients with familial ALS (FALS). Using single-strand conformational polymorphism, we studied the C to G mutation in exon 2 of the SOD1 gene (resulting in a leucine to valine substitution in position 38) in affected and unaffected members of a large Belgian family with FALS. We measured the SOD1 activity in red blood cell lysates in 14 members of this family, including the only surviving clinically affected patient. SOD1 activity of the family members carrying the mutation was less than half that of members without the mutation. In addition, in 11 patients with sporadic ALS and 11 age- and sex-matched controls, red blood cell SOD1 activity was normal. These studies indicate that SOD1 activity is reduced in these FALS patients but not in sporadic ALS patients. Moreover, this SOD1 enzyme abnormality is detectable years before onset of clinical ALS in carriers of this FALS mutation.     

A specific inhibitor of janus kinase-3 increases survival in a transgenic mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disorder involving the motor neurons of cortex, brain stem, and spinal cord. About 10% of all ALS patients are familial cases (FALS), of which 20% have mutations in the Cu, Zn-superoxide dismutase (SOD1) gene. The murine model for FALS, which overexpresses a FALS variant of the SOD1 gene, exhibits progressive limbic paralysis followed by death. Treatment of FALS mice with WHI-P131, a specific inhibitor of Janus kinase 3 (JAK3), increased survival by more than two months, suggesting that specific inhibitors of JAK3 may be useful in the treatment of human ALS. These results uniquely establish JAK3 as a novel molecular target for the treatment of FALS.     

Astroglial inhibition of NF-κB does not ameliorate disease onset and progression in a mouse model for amyotrophic lateral sclerosis (ALS)

Motor neuron death in amyotrophic lateral sclerosis (ALS) is considered a "non-cell autonomous" process, with astrocytes playing a critical role in disease progression. Glial cells are activated early in transgenic mice expressing mutant SOD1, suggesting that neuroinflammation has a relevant role in the cascade of events that trigger the death of motor neurons. An inflammatory cascade including COX2 expression, secretion of cytokines and release of NO from astrocytes may descend from activation of a NF-κB-mediated pathway observed in astrocytes from ALS patients and in experimental models. We have attempted rescue of transgenic mutant SOD1 mice through the inhibition of the NF-κB pathway selectively in astrocytes. Here we show that despite efficient inhibition of this major pathway, double transgenic mice expressing the mutant SOD1(G93A) ubiquitously and the dominant negative form of IκBα (IκBαAA) in astrocytes under control of the GFAP promoter show no benefit in terms of onset and progression of disease. Our data indicate that motor neuron death in ALS cannot be prevented by inhibition of a single inflammatory pathway because alternative pathways are activated in the presence of a persistent toxic stimulus.     

DREAM-Dependent Activation of Astrocytes in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin and characterized by a relentless loss of motor neurons that causes a progressive muscle weakness until death. Among the several pathogenic mechanisms that have been related to ALS, a dysregulation of calcium-buffering proteins in motor neurons of the brain and spinal cord can make these neurons more vulnerable to disease progression. Downstream regulatory element antagonist modulator (DREAM) is a neuronal calcium-binding protein that plays multiple roles in the nucleus and cytosol. The main aim of this study was focused on the characterization of DREAM and glial fibrillary acid protein (GFAP) in the brain and spinal cord tissues from transgenic SOD1^G93A mice and ALS patients to unravel its potential role under neurodegenerative conditions. The DREAM and GFAP levels in the spinal cord and different brain areas from transgenic SOD1^G93A mice and ALS patients were analyzed by Western blot and immunohistochemistry. Our findings suggest that the calcium-dependent excitotoxicity progressively enhanced in the CNS in ALS could modulate the multifunctional nature of DREAM, strengthening its apoptotic way of action in both motor neurons and astrocytes, which could act as an additional factor to increase neuronal damage. The direct crosstalk between astrocytes and motor neurons can become vulnerable under neurodegenerative conditions, and DREAM could act as an additional switch to enhance motor neuron loss. Together, these findings could pave the way to further study the molecular targets of DREAM to find novel therapeutic strategies to fight ALS.     

Aberrant localization of FUS and TDP43 is associated with misfolding of SOD1 in amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is incurable and characterized by progressive paralysis of the muscles of the limbs, speech and swallowing, and respiration due to the progressive degeneration of voluntary motor neurons. Clinically indistinguishable ALS can be caused by genetic mutations of Cu/Zn superoxide dismutase (SOD1), TAR-DNA binding protein 43 (TDP43), or fused in sarcoma/translocated in liposarcoma (FUS/TLS), or can occur in the absence of known mutation as sporadic disease. In this study, we tested the hypothesis that FUS/TLS and TDP43 gain new pathogenic functions upon aberrant accumulation in the cytosol that directly or indirectly include misfolding of SOD1.

Methodology/Principal Findings

Patient spinal cord necropsy immunohistochemistry with SOD1 misfolding-specific antibodies revealed misfolded SOD1 in perikarya and motor axons of SOD1-familial ALS (SOD1-FALS), and in motor axons of R521C-FUS FALS and sporadic ALS (SALS) with cytoplasmic TDP43 inclusions. SOD1 misfolding and oxidation was also detected using immunocytochemistry and quantitative immunoprecipitation of human neuroblastoma SH-SY5Y cells as well as cultured murine spinal neural cells transgenic for human wtSOD1, which were transiently transfected with human cytosolic mutant FUS or TDP43, or wtTDP43.

Conclusion/Significance

We conclude that cytosolic mislocalization of FUS or TDP43 in vitro and ALS in vivo may kindle wtSOD1 misfolding in non-SOD1 FALS and SALS. The lack of immunohistochemical compartmental co-localization of misfolded SOD1 with cytosolic TDP43 or FUS suggests an indirect induction of SOD1 misfolding followed by propagation through template directed misfolding beyond its site of inception. The identification of a final common pathway in the molecular pathogenesis of ALS provides a treatment target for this devastating disease.     

Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.     

Identification of new mutations in the Cu/Zn superoxide dismutase gene of patients with familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. Although most cases of ALS are sporadic, approximately 10% are inherited as an autosomal dominant trait. Mutations in the Cu/Zn superoxide dismutase gene (SOD 1) are responsible for a fraction of familial ALS (FALS). Screening our FALS kindreds by SSCP, we have identified mutations in 15 families, of which 9 have not been previously reported. Two of the new mutations alter amino acids that have never been implicated in FALS. One of them affects a highly conserved amino acid involved in dimer contact, and the other one affects the active-site loop of the enzyme. These two mutations reduce significantly SOD 1 enzyme activity in lymphoblasts. Our results suggest that SOD 1 mutations are responsible for > or = 13% of FALS cases.     

Mitochondria-Targeted Catalase Reverts the Neurotoxicity of hSOD1G93A Astrocytes without Extending the Survival of ALS-Linked Mutant hSOD1 Mice

Dominant mutations in the Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. The molecular mechanism underlying the toxic gain-of-function of mutant hSOD1s remains uncertain. Several lines of evidence suggest that toxicity to motor neurons requires damage to non-neuronal cells. In line with this observation, primary astrocytes isolated from mutant hSOD1 over-expressing rodents induce motor neuron death in co-culture. Mitochondrial alterations have been documented in both neuronal and glial cells from ALS patients as well as in ALS-animal models. In addition, mitochondrial dysfunction and increased oxidative stress have been linked to the toxicity of mutant hSOD1 in astrocytes and neurons. In mutant SOD1-linked ALS, mitochondrial alterations may be partially due to the increased association of mutant SOD1 with the outer membrane and intermembrane space of the mitochondria, where it can affect several critical aspects of mitochondrial function. We have previously shown that decreasing glutathione levels, which is crucial for peroxide detoxification in the mitochondria, significantly accelerates motor neuron death in hSOD1^G93A mice. Here we employed a catalase targeted to the mitochondria to investigate the effect of increased mitochondrial peroxide detoxification capacity in models of mutant hSOD1-mediated motor neuron death. The over-expression of mitochondria-targeted catalase improved mitochondrial antioxidant defenses and mitochondrial function in hSOD1^G93A astrocyte cultures. It also reverted the toxicity of hSOD1^G93A-expressing astrocytes towards co-cultured motor neurons, however ALS-animals did not develop the disease later or survive longer. Hence, while increased oxidative stress and mitochondrial dysfunction have been extensively documented in ALS, these results suggest that preventing peroxide-mediated mitochondrial damage alone is not sufficient to delay the disease.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

A Novel Locus for Familial Amyotrophic Lateral Sclerosis, on Chromosome 18q

Amyotrophic lateral sclerosis (ALS) is an adult-onset degenerative disorder characterized by the death of motor neurons in the cortex, brain stem, and spinal cord. Despite intensive research the basic pathophysiology of ALS remains unclear. Although most cases are sporadic, approximately 10% of ALS cases are familial (FALS). Mutations in the Cu/Zn superoxide dismutase (SOD1) gene cause approximately 20% of FALS. The gene(s) responsible for the remaining 80% of FALS remain to be found. Using a large European kindred without SOD1 mutation and with classic autosomal dominant adult-onset ALS, we have identified a novel locus by performing a genome scan and linkage analysis. The maximum LOD score is 4.5 at recombination fraction 0.0, for polymorphism D18S39. Haplotype analysis has identified a 7.5-cM, 8-Mb region of chromosome 18q21, flanked by markers D18S846 and D18S1109, as a novel FALS locus.