Increased mitochondrial oxidative damage and oxidative DNA damage contributes to the neurodegenerative process in sporadic amyotrophic lateral sclerosis

To investigate the possibility that mitochondrial oxidative damage or oxidative DNA damage or both contribute to the neurodegenerative process of sporadic amyotrophic lateral sclerosis (sALS), this study used high-performance liquid chromatography with an electrochemical detector to measure the concentrations of the reduced and oxidized forms of coenzyme Q10 (CoQ10) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the cerebrospinal fluid (CSF) of 17 patients with sALS and 17 age-matched controls with no neurological diseases. The percentage of oxidized CoQ10 in the CSF of sALS patients was greater than that in the CSF of controls (p<0.002) and was negatively correlated with the duration of illness (rho=-0.61, p<0.01). The concentration of 8-OHdG in the CSF of sALS patients was greater than that in the CSF of controls (p<0.005) and was positively correlated with the duration of illness (rho=0.53, p<0.005). The percentage of oxidized CoQ10 was correlated with the concentrations of 8-OHdG in the CSF of sALS patients (rho=-0.53, p<0.05). These results suggest that both mitochondrial oxidative damage and oxidative DNA damage play important roles in the pathogenesis of sporadic amyotrophic lateral sclerosis.     

Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients Induces Mitochondrial and Lysosomal Dysfunction

In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS.     

Diverse Expression of Selected SMN Complex Proteins in Humans with Sporadic Amyotrophic Lateral Sclerosis and in a Transgenic Rat Model of Familial Form of the Disease

Background and Objective

There is circumstantial evidence linking sporadic amyotrophic lateral sclerosis (ALS) cases to a malfunction or deficit of a multimeric SMN complex that scrutinizes cellular RNAs; the core of this complex is survival motor neuron (SMN, or gemin 1) protein. We intended to verify this hypothesis by comparing the expression of both SMN and several other functionally associated gemins in the anterior horn motoneurons of patients who died of sporadic ALS (sALS), of transgenic rats with overexpression of the mutated human superoxide dismutase 1 gene (SOD1^G93A) that represent a model of familial ALS (fALS), and of the respective controls.

Methods

Using archival material of paraffin blocks with samples of human and rat spinal cords, immunohistochemical reactions with antibodies against SMN and gemins 2, 3, and 4 were performed and assessed by light microscopy.

Results

The expression of SMN and all other studied gemins was observed in motoneurons of sALS patients, fALS rats, and in all controls, although the intensity varied. The immunolabeling was most intense in sALS patients with relatively fast disease course, and decreased with increasing disease duration in both the human sALS and rat fALS material. Irrespective of the disease stage, sALS material showed no or very low gemin 2 immunoreactivity, while clear gemin 2 immnoreactivity was observed in all fALS rats and control material.

Conclusion

The deficient expression of gemin 2 in spinal cord motoneurons in human sALS may lead to a dysfunction and loss of neuroprotective action of the SMN complex.     

Biotransformation of nitric oxide in the cerebrospinal fluid of amyotrophic lateral sclerosis patients

Recent findings indicate that nitric oxide (NO*) over-production might be an important factor in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). We measured significantly higher concentrations of uric acid and thiol group-containing molecules (R-SH groups) in the cerebrospinal fluid (CSF) from SALS patients compared to controls. The above factors, together with a slightly increased free iron concentration found in the CSF, favour conditions necessary for the formation of the dinitrosyl iron complex, capable of NO* bio-transformation. Thus, we performed ex vivo saturation of CSF (from both SALS patients and controls) with NO*. A decrease in the level of R-SH was found. This was more pronounced in the CSF from SALS patients. In the CSF from SALS patients the production of nitrite and hydroxylamine was greater than that observed in the CSF from controls. Moreover, we also found increased Cu,Zn-SOD activity in the CSF from SALS patients (when compared to control subjects) but no activity corresponding to Mn-SOD in any CSF samples. As Cu,Zn-SOD can react with nitroxyl forming NO*, the conditions for a closed, but continuous, loop of NO* biotransformation are present in the CSF of ALS patients.     

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 miRNA signature in leukocytes from sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and seriously disabling adult-onset neurological disease. Accumulating evidence indicates that various miRNAs, expressed in a spatially and temporally controlled manner in the brain, play a key role in neuronal development. In addition, misregulation of microRNAs contributes to some mental disorders and neurodegeneration diseases. Here, we analyzed the expression profiles of 911 human miRNAs using microarray technology in leukocytes, the most readily available human tissue cells, obtained from 8 patients affected by sporadic amyotrophic lateral sclerosis (sALS) and 12 healthy controls. An independent group of 14 sALS patients and 14 controls was used for validation by TaqMan real-time polymerase chain reaction assay. We identified 8 miRNAs that were significantly up- or downregulated in sALS patients as compared to healthy controls. The significant variations in miRNAs profiles detected in leukocytes have been related to miRNAs predominantly expressed in the nervous system. One of these miRNAs, miR-338-3p, has previously been shown to be de-regulated in ALS brains. This study, for the first time, detected specific microRNAs disease-related changes at an earlier stage of sALS. We suggest that miRNAs profiles found in the peripheral blood leukocytes from sALS patients can be relevant to understand the pathogenesis of sALS and/or used as biomarkers of the disease.     

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.     

Increased ROS Level in Spinal Cord of Wobbler Mice due to Nmnat2 Downregulation

Amyotrophic lateral sclerosis is a devastating motor neuron disease and to this day not curable. While 5–10% of patients inherit the disease (familiar ALS), up to 95% of patients are diagnosed with the sporadic form (sALS). ALS is characterized by the degeneration of upper motor neurons in the cerebral cortex and of lower motor neurons in the brainstem and spinal cord. The wobbler mouse resembles almost all phenotypical hallmarks of human sALS patients and is therefore an excellent motor neuron disease model. The motor neuron disease of the wobbler mouse develops over a time course of around 40 days and can be divided into three phases: p0, presymptomatic; p20, early clinical; and p40, stable clinical phase. Recent findings suggest an essential implication of the NAD⁺-producing enzyme Nmnat2 in neurodegeneration as well as maintenance of healthy axons. Here, we were able to show a significant downregulation of both gene and protein expression of Nmnat2 in the spinal cord of the wobbler mice at the stable clinical phase. The product of the enzyme NAD⁺ is also significantly reduced, and the values of the reactive oxygen species are significantly increased in the spinal cord of the wobbler mouse at p40. Thus, the deregulated expression of Nmnat2 appears to have a great influence on the cellular stress in the spinal cord of wobbler mice.     

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.     

Increased Superoxide Dismutase Activity in Aged Human Cerebrospinal Fluid and Rat Brain Determined by Electron Spin Resonance Spectrometry Using the Spin Trap Method

Superoxide dismutase (SOD) activity in CSF of patients was determined by electron spin resonance spectrometry using the spin trap method. Variation in SOD activity was found among patients. SOD activity in CSF of subjects increased with age and this was identified as Cu,Zn-SOD activity by electrophoresis. In addition, animal experiments showed that SOD activities were higher in mitochondrial and cytosol fractions of aged rats than in those of adult rats. This finding on aged rat brain validates the increase of SOD activity in aged human CSF.     

Polymorphism of rs3737597 in DISC1 Gene on Chromosome 1q42.2 in sALS Patients: a Chinese Han Population Case-Control Study

Although lots of genes have been revealed to relate to sporadic amyotrophic lateral sclerosis (sALS), its genetic mechanisms still need to be further explored. We aimed to search the novel genetic factors of sALS and assess their contribution. We constructed an integrative dataset based on the 3227 subsignificant genes (P value?<?0.01) from two sALS-related genome-wide association studies (GWAS) (the US and Irish studies). A significant replication between both studies was confirmed by the gene set enrichment analysis in the integral level (P value?<?10^?4). Using the pathway overrepresentation analysis, we revealed the 34 shared Gene Ontology (GO) biological processes from the two independent studies (P value?<?0.01). Among these pathways, the nervous system developmental pathway (NSD function, GO:0007399) was further supported by the previously reported genes related to sALS (P value?=?3.28e?12). Importantly, four of 17 NSD-function-related target genes (disrupted-in-schizophrenia-1 (DISC1), CNTN4, NRXN3, and ERBB4) presented a considerable association with sALS in both studies. To further verify the association between the NSD function target genes and sALS, we preformed a two-stage case–control study based on 500 sALS patients and 500 controls of Chinese Han populations from mainland. A polymorphism of rs3737597 in DISC1 gene involved in the nervous system developmental pathway was closely associated with sALS. The nervous system developmental pathway is a potential pathogenesis of sALS, among them, the polymorphism of rs3737597 in DISC1 might play some roles.     

Angeli's Salt and Spinal Motor Neuron Injury

Nitroxyl anion or its conjugate acid (NO^-/HNO) and nitric oxide (NO) may both have pro-oxidative and cytotoxic properties. Superoxide dismutase (SOD) enzyme has been shown to convert reversibly HNO to NO. Mutations found in the SOD enzyme in some familial amyotrophic lateral sclerosis (ALS) patients affect redox properties of the SOD enzyme in a manner, which may affect the equilibrium between NO and HNO. Therefore, we studied the effects of HNO releasing compound, Angeli's salt (AS), on both motor and sensory functions after intrathecal administration in the lumbar spinal cord of a male rat. These functions were measured by rotarod, spontaneous activity, paw- and tail-flick tests. In addition, we compared the effect of AS to NO releasing papanonoate, old AS solution and sulphononoate in the motor performance test. The effect of intrathecal delivery of AS on the markers of the spinal cord injury and oxidative/nitrosative stress were further studied.

Results: Freshly prepared AS (5 or 10?μmol), but not papanonoate, caused a marked decrease in the rotarod performance 3–7 days after the intrathecal administration. The peak motor deficiency was noted 3 days after AS (5?μmol) delivery. Old, degraded, AS solution and nitrous oxide releasing sulphononoate did not decrease motor performance in the rotarod test. AS did not affect the sensory stimulus evoked responses as measured by the paw-flick and tail-flick tests. Immunohistological examination revealed that AS caused injury related changes in the expression of glial fibrillary acidic protein (GFAP), fibroblast growth factor (FGF-2) and laminins in the spinal cord. Moreover, AS increased nitrotyrosine immunoreactivity in the spinal motor neurons.

Therefore, we conclude that AS, but not NO releasing papanonoate, causes motor neuron injury but does not affect the function of sensory nerves in behavioural tests.     

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.     

Disease-related changes in the cerebrospinal fluid metabolome in amyotrophic lateral sclerosis detected by GC/TOFMS

Background/Aim

The changes in the cerebrospinal fluid (CSF) metabolome associated with the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) are poorly understood and earlier smaller studies have shown conflicting results. The metabolomic methodology is suitable for screening large cohorts of samples. Global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as CSF.

Methodology

Using gas chromatography coupled to mass spectrometry (GC/TOFMS) and multivariate statistical modeling, we simultaneously studied the metabolome signature of ∼120 small metabolites in the CSF of patients with ALS, stratified according to hereditary disposition and clinical subtypes of ALS in relation to controls.

Principal Findings

The study is the first to report data validated over two sub-sets of ALS vs. control patients for a large set of metabolites analyzed by GC/TOFMS. We find that patients with sporadic amyotrophic lateral sclerosis (SALS) have a heterogeneous metabolite signature in the cerebrospinal fluid, in some patients being almost identical to controls. However, familial amyotrophic lateral sclerosis (FALS) without superoxide dismutase-1 gene (SOD1) mutation is less heterogeneous than SALS. The metabolome of the cerebrospinal fluid of 17 ALS patients with a SOD1 gene mutation was found to form a separate homogeneous group. Analysis of metabolites revealed that glutamate and glutamine were reduced, in particular in patients with a familial predisposition. There are significant differences in the metabolite profile and composition among patients with FALS, SALS and patients carrying a mutation in the SOD1 gene suggesting that the neurodegenerative process in different subtypes of ALS may be partially dissimilar.

Conclusions/Significance

Patients with a genetic predisposition to amyotrophic lateral sclerosis have a more distinct and homogeneous signature than patients with a sporadic disease.     

Angeli's salt and spinal motor neuron injury

Nitroxyl anion or its conjugate acid (NO_-/HNO) and nitric oxide (NO) may both have pro-oxidative and cytotoxic properties. Superoxide dismutase (SOD) enzyme has been shown to convert reversibly HNO to NO. Mutations found in the SOD enzyme in some familial amyotrophic lateral sclerosis (ALS) patients affect redox properties of the SOD enzyme in a manner, which may affect the equilibrium between NO and HNO. Therefore, we studied the effects of HNO releasing compound, Angeli's salt (AS), on both motor and sensory functions after intrathecal administration in the lumbar spinal cord of a male rat. These functions were measured by rotarod, spontaneous activity, paw- and tail-flick tests. In addition, we compared the effect of AS to NO releasing papanonoate, old AS solution and sulphononoate in the motor performance test. The effect of intrathecal delivery of AS on the markers of the spinal cord injury and oxidative/nitrosative stress were further studied.

Results: Freshly prepared AS (5 or 10 μmol), but not papanonoate, caused a marked decrease in the rotarod performance 3-7 days after the intrathecal administration. The peak motor deficiency was noted 3 days after AS (5 μmol) delivery. Old, degraded, AS solution and nitrous oxide releasing sulphononoate did not decrease motor performance in the rotarod test. AS did not affect the sensory stimulus evoked responses as measured by the paw-flick and tail-flick tests. Immunohistological examination revealed that AS caused injury related changes in the expression of glial fibrillary acidic protein (GFAP), fibroblast growth factor (FGF-2) and laminins in the spinal cord. Moreover, AS increased nitrotyrosine immunoreactivity in the spinal motor neurons.

Therefore, we conclude that AS, but not NO releasing papanonoate, causes motor neuron injury but does not affect the function of sensory nerves in behavioural tests.     

Free Copper,Ferroxidase and SOD1 Activities,Lipid Peroxidation and NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> Content in the CSF. A Different Marker Profile in Four Neurodegenerative Diseases

The understanding of oxidative damage in different neurodegenerative diseases could enhance therapeutic strategies. Our objective was to quantify lipoperoxidation and other oxidative products as well as the activity of antioxidant enzymes and cofactors in cerebrospinal fluid (CSF) samples. We recorded data from all new patients with a diagnosis of either one of the four most frequent neurodegenerative diseases: Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD) and lateral amyotrophic sclerosis (ALS). The sum of nitrites and nitrates as end products of nitric oxide (NO) were increased in the four degenerative diseases and fluorescent lipoperoxidation products in three (excepting ALS). A decreased Cu/Zn-dependent superoxide dismutase (SOD) activity characterized the four diseases. A significantly decreased ferroxidase activity was found in PD, HD and AD, agreeing with findings of iron deposition in these entities, while free copper was found to be increased in CSF and appeared to be a good biomarker of PD.     

Analysis of smoking and LPO in ALS

Smoking has been suggested as one of the risk factor for amyotrophic lateral sclerosis (ALS) development. In order to investigate whether adverse effects of cigarette smoke in ALS have any association with increase in oxidative stress, disease severity, lipid hydroperoxides (LPO) and superoxide dismutase-1 (SOD1) levels were measured in biofluids of smoker and never smoker ALS patients and clinically correlated. Serum and CSF from sporadic ALS patients (n = 50) diagnosed with El Escorial criteria were collected in the study. Serum (n = 50) and CSF (n = 42) were also collected from normal healthy controls. The LPO levels were estimated using commercially available kits. Enzyme-linked immunosorbent assays (ELISAs) were used to quantitate SOD1. Their levels were further analyzed among smoker and never smoker subjects. Significantly elevated LPO in sera and CSF of ALS patients were observed (p < 0.05). There was considerably increased LPO in sera and CSF of smoker ALS subjects matched with disease severity as compared to never smoker ALS (p < 0.05). ALS group did not show any alteration in SOD1 when compared to controls (p > 0.05). In addition, no change has been observed in SOD1 levels in ALS subjects who smoke (p > 0.05). Increased LPO and unaltered SOD1 in ALS patients may suggest the neuro-pathological association of LPO with ALS disease independent of SOD1. With current findings, it may be proposed that LPO levels might constitute as probable biomarker for smoker ALS patients, however, it cannot be concluded without larger gender matched studies. Additional investigations are needed to determine whether LPO upregulation is primary or secondary to motor neuron degeneration in ALS.     

Development of novel small molecules for the treatment of ALS

Amyotrophic lateral sclerosis (ALS) is a rare and progressive neurodegenerative disease with unknown etiology. It is caused by the degeneration of motor neurons responsible for controlling voluntary muscles. It has been reported that mutations in the superoxide dismutase (SOD) 1 gene can lead to ALS. SOD1 abnormalities have been identified in both familial, as well as sporadic ALS cases. SOD2 is a highly inducible SOD that works in conjunction with SOD1. SOD2 can be induced through activation of NF-κBs. We previously reported that the novel small molecule, SRI-22818, increases NF-κB expression and activation and SOD2 levels in vitro and has activity in vivo in the SOD1-G93A reference model of ALS. We report herein the synthesis and biological evaluation of SRI-22818 analogs.