Motor neuron degeneration in amyotrophic lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death

The mechanisms of human mutant superoxide dismutase-1 (mSOD1) toxicity to motor neurons (MNs) are unresolved. We show that MNs in G93A-mSOD1 transgenic mice undergo slow degeneration lacking similarity to apoptosis structurally and biochemically. It is characterized by somal and mitochondrial swelling and formation of DNA single-strand breaks prior to double-strand breaks occurring in nuclear and mitochondrial DNA. p53 and p73 are activated in degenerating MNs, but without nuclear import. The MN death is independent of activation of caspases-1, -3, and -8 or apoptosis-inducing factor within MNs, with a blockade of apoptosis possibly mediated by Aven up-regulation. MN swelling is associated with compromised Na,K-ATPase activity and aggregation. mSOD1 mouse MNs accumulate mitochondria from the axon terminals and generate higher levels of superoxide, nitric oxide, and peroxynitrite than MNs in control mice. Nitrated and aggregated cytochrome c oxidase subunit-I and alpha-synuclein as well as nitrated SOD2 accumulate in mSOD1 mouse spinal cord. Mitochondria in mSOD1 mouse MNs accumulate NADPH diaphorase and inducible nitric oxide synthase (iNOS)-like immunoreactivity, and iNOS gene deletion extends significantly the life span of G93A-mSOD1 mice. Prior to MN loss, spinal interneurons degenerate. These results identify novel mechanisms for mitochondriopathy and MN degeneration in amyotrophic lateral sclerosis (ALS) mice involving blockade of apoptosis, accumulation of MN mitochondria with enhanced toxic potential from distal terminals, NOS localization in MN mitochondria and peroxynitrite damage, and early degeneration of alpha-synuclein(+) interneurons. The data support roles for oxidative stress, protein nitration and aggregation, and excitotoxicity as participants in the process of MN degeneration caused by mSOD1.     

A novel mutation of SOD-1 (Gly 108 Val) in familial amyotrophic lateral sclerosis

A novel mutation of the SOD-1 gene which encodes the enzyme copper-zinc superoxide dismutase was identified in a family manifesting amyotrophic lateral sclerosis (ALS) in three generations. The mutation is a heterozygote point mutation in exon 4, codon 108 (GGA to GTA), predicting the substitution of valine for glycine. The mutation creates a new restriction site for the endonuclease AccI. The mutation was demonstrated in two affected members of the family, who show features of autosomal dominant inheritance of ALS, but variable age at onset ranging from 48 to 72 years. Over 30 different mutations of SOD-1 have now been identified in families with ALS. The definition of the different mutations causing human disease may allow further investigation of their pathogenicity in transgenic animal models, and also offers insight into the variable phenotypic disease expression both within and between genotypes.     

Superoxide dismutase (SOD-1) activity in erythrocytes of patients with multiple sclerosis]

The purpose of the study was determination of the activity of superoxide dismutase (SOD-1) in the erythrocytes of multiple sclerosis patients taking into consideration certain aspects of the disease, such as its duration, clinical form and course, age and sex of the patients. The study included 34 patients (17 males and 17 females) aged 19 to 56 years. The control group comprised 64 healthy subjects (25 females and 38 males) aged 19-59 years. A significant reduction was found of SOD-1 activity in the erythrocytes of the patients which may suggest a lower enzymatic defense mechanisms against oxidative stress. No difference in the activity of SOD-1 was found between clinical forms and different courses of the disease, although the activity changed in relation to age, sex and disease duration.     

Serotonergic mechanisms in amyotrophic lateral sclerosis

Serotonin (5-HT) has been intimately linked with global regulation of motor behavior, local control of motoneuron excitability, functional recovery of spinal motoneurons as well as neuronal maturation and aging. Selective degeneration of motoneurons is the pathological hallmark of amyotrophic lateral sclerosis (ALS). Motoneurons that are preferentially affected in ALS are also densely innervated by 5-HT neurons (e.g., trigeminal, facial, ambiguus, and hypoglossal brainstem nuclei as well as ventral horn and motor cortex). Conversely, motoneuron groups that appear more resistant to the process of neurodegeneration in ALS (e.g., oculomotor, trochlear, and abducens nuclei) as well as the cerebellum receive only sparse 5-HT input. The glutamate excitotoxicity theory maintains that in ALS degeneration of motoneurons is caused by excessive glutamate neurotransmission, which is neurotoxic. Because of its facilitatory effects on glutaminergic motoneuron excitation, 5-HT may be pivotal to the pathogenesis and therapy of ALS. 5-HT levels as well as the concentrations 5-hydroxyindole acetic acid (5-HIAA), the major metabolite of 5-HT, are reduced in postmortem spinal cord tissue of ALS patients indicating decreased 5-HT release. Furthermore, cerebrospinal fluid levels of tryptophan, a precursor of 5-HT, are decreased in patients with ALS and plasma concentrations of tryptophan are also decreased with the lowest levels found in the most severely affected patients. In ALS progressive degeneration of 5-HT neurons would result in a compensatory increase in glutamate excitation of motoneurons. Additionally, because 5-HT, acting through presynaptic 5-HT1B receptors, inhibits glutamatergic synaptic transmission, lowered 5-HT activity would lead to increased synaptic glutamate release. Furthermore, 5-HT is a precursor of melatonin, which inhibits glutamate release and glutamate-induced neurotoxicity. Thus, progressive degeneration of 5-HT neurons affecting motoneuron activity constitutes the prime mover of the disease and its progression and treatment of ALS needs to be focused primarily on boosting 5-HT functions (e.g., pharmacologically via its precursors, reuptake inhibitors, selective 5-HT1A receptor agonists/5-HT2 receptor antagonists, and electrically through transcranial administration of AC pulsed picotesla electromagnetic fields) to prevent excessive glutamate activity in the motoneurons. In fact, 5HT1A and 5HT2 receptor agonists have been shown to prevent glutamate-induced neurotoxicity in primary cortical cell cultures and the 5-HT precursor 5-hydroxytryptophan (5-HTP) improved locomotor function and survival of transgenic SOD1 G93A mice, an animal model of ALS.     

Inflammatory mechanisms in amyotrophic lateral sclerosis

Neuroinflammation is a pathological hallmark in human amyotrophic lateral sclerosis (ALS) patients and in the transgenic models of the disease. The importance of glial cell activation and pro-inflammatory cytokines in ALS has been confirmed by numerous studies. For instance, tumor necrosis factor-α (TNF-α), a major pro-inflammatory cytokine, activates microglia and cause neurotoxicity in motor neurons. More recently, the relationship of nuclear factor-κB (NF-κB) and motor neuron degeneration has garnered attention since optineurin (OPTN) mutations were reported in familial ALS. OPTN negatively regulates TNF-α-induced NF-κB activation, but OPTN mutations can lead to dysinhibition of NF-κB-induced neurotoxicity. Notably, OPTN-positive inclusions are observed not only in familial ALS with OPTN mutation but also in sporadic ALS and in familial ALS with SOD1 and fused in sarcoma mutations, suggesting that OPTN- and NF-κB-related pathways are relevant to the general pathomechanisms of ALS. In this review, we discuss inflammatory aspects of ALS comprising the roles of cytokines, glial cells, and T cells.     

Nonoxidative protein glycation is implicated in familial amyotrophic lateral sclerosis with superoxide dismutase-1 mutation

To assess a role for oxidative stress in the pathogenesis of amyotrophic lateral sclerosis (ALS), we analyzed the immunohistochemical localization of 8-hydroxy-2′-deoxyguanosine (OHdG) as a nucleic acid oxidation product, acrolein-protein adduct and 4-hydroxy-2-nonenal (HNE)-protein adduct as lipid peroxidation products, N ^ɛ-carboxymethyl-lysine (CML) as a lipid peroxidation or protein glycoxidation product, pentosidine as a protein glycoxidation product, and imidazolone and pyrraline as nonoxidative protein glycation products in the spinal cord of three familial ALS patients with superoxide dismutase-1 (SOD1) A4V mutation, six sporadic ALS patients, and six age-matched control individuals. The spinal cord sections of the control cases did not show any distinct immunoreactivities for these examined products. In the familial ALS cases, intense immunoreactivities for pyrraline and CML were confined to the characteristic Lewy body-like hyaline inclusions, and imidazolone immunoreactivity was located in the cytoplasm of the residual motor neurons. No significant immunoreactivities for other examined products were detected in the familial ALS spinal cords. In the sporadic ALS cases, intense immunoreactivities for pentosidine, CML and HNE-protein adduct were seen in the cytoplasm of the degenerated motor neurons, and OHdG immunoreactivity was located in the cell nuclei of the residual neurons and glial cells. The present results indicate that oxidative reactions are involved in the disease processes of sporadic ALS, while there is no evidence for increased oxidative damage except for CML deposition in the familial ALS spinal cords. Furthermore, it is likely that the accumulation of pyrraline and imidazolone supports a nonoxidative mechanism in SOD1-related motor neuron degeneration. Received: 18 August 1999 / Revised, accepted: 17 November 1999     

Cerebral degeneration predicts survival in amyotrophic lateral sclerosis

Objective

To determine the relationship of cerebral degeneration with survival in amyotrophic lateral sclerosis (ALS).

Methods

Patients with probable or definite ALS underwent magnetic resonance spectroscopic imaging (MRSI) of the brain between July 1996 and May 2002, and were followed prospectively until March 2004. Creatine (Cr), choline (Cho) and the neuronal marker N‐acetylaspartate (NAA) were quantified as ratios in the motor cortex.

Results

In 63 patients compared with 18 healthy people, NAA/Cho was reduced by 13% (p<0.001), NAA/Cr was reduced by 5% (p = 0.01) and Cho/Cr was increased by 8% (p = 0.01). NAA/Cho was used for survival analysis, given its larger effect size and superior test accuracy (a sensitivity of 67% and a specificity of 83%). Median survival after MRSI was 24 months. Multivariate analysis showed reduced survival for lower NAA/Cho (hazard ratio (HR) 0.24, 95% confidence interval (CI) 0.08 to 0.72, p = 0.01), older age (HR 1.03, 95% CI 1.00 to 1.06, p = 0.04) and shorter symptom duration (HR 0.96, 95% CI 0.93 to 0.99, p = 0.01). Patients with NAA/Cho <2.11 had a reduced survival of 19.4 v 31.9 months (HR 2.05, 95% CI 1.12 to 4.03, p = 0.02).

Conclusions

Cerebral degeneration is predictive of reduced survival in ALS.The extent to which cerebral involvement contributes to shortened life expectancy in amyotrophic lateral sclerosis (ALS) has been difficult to elucidate. The clinical evaluation of upper motor neurone (UMN) integrity as an index of motor cortex degeneration falls short owing to the possible masking of UMN signs by the loss of lower motor neurones (LMNs). Indeed, autopsy series have shown UMN degeneration in cases of LMN‐predominant motor neurone disease that would not have otherwise met the clinical criteria for ALS.¹ The identification of prognostic indicators would aid in selecting patients for clinical trials who are predicted to behave in a homogeneous manner with respect to disease progression and, potentially, pathogenesis. Prognostic indicators would also have clinical value in counselling patients.Proton magnetic resonance spectroscopy (MRS) studies in ALS have consistently shown evidence of neuronal loss or dysfunction in the motor cortex by the finding of decreased N‐acetylaspartate (NAA), a neuronal marker.^2,3We aimed to determine whether cerebral neurochemical abnormalities quantified by MRS correlated with survival.     

Heterogeneous distribution of amyotrophic lateral sclerosis patients with SOD-1 gene mutations: preliminary data on an Italian survey

We report the absence of superoxide dismutase (SOD-1) gene mutations in 30 patients with amyotrophic lateral sclerosis (ALS) including individuals with a confirmed family history of ALS (familial ALS/FALS), ALS with an unclear family history (UFALS) and sporadic ALS (SALS). Single strand conformation polymorphism (SSCP) and sequence analysis of the 5 SOD-1 gene exons were undertaken to improve the accuracy of the mutation detection. Our preliminary data appear to diverge from the results of studies by other groups using different populations. We discuss the possible reasons for this disparity and the apparent heterogeneous distribution of ALS with SOD-1 gene mutations among different ethnic groups.     

Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis

The most common cause of amyotrophic lateral sclerosis (ALS) is mutations in superoxide dismutase-1 (SOD1). Since there is evidence for the involvement of non-neuronal cells in ALS, we searched for signs of SOD1 abnormalities focusing on glia. Spinal cords from nine ALS patients carrying SOD1 mutations, 51 patients with sporadic or familial ALS who lacked such mutations, and 46 controls were examined by immunohistochemistry. A set of anti-peptide antibodies with specificity for misfolded SOD1 species was used. Misfolded SOD1 in the form of granular aggregates was regularly detected in the nuclei of ventral horn astrocytes, microglia, and oligodendrocytes in ALS patients carrying or lacking SOD1 mutations. There was negligible staining in neurodegenerative and non-neurological controls. Misfolded SOD1 appeared occasionally also in nuclei of motoneurons of ALS patients. The results suggest that misfolded SOD1 present in glial and motoneuron nuclei may generally be involved in ALS pathogenesis.     

Familial amyotrophic lateral sclerosis: Features of multisystem degeneration

Summary Two sibling cases of familial amyotrophic lateral sclerosis (ALS) revealed degerneration usually associated with other systemic degenerative disorders. The changes in the 41-year-old sister were compatible with those reported in other familial ALS cases affecting the upper and lower motor neurons, posterior columns, and spinocerebellar tracts. The 45-year-old sister revealed more wide-spread degenerative changes involving not only motor neuron systems, but also proprioceptive, general somatic afferent and spinocerebellar afferent systems. Intracytoplasmic hyaline inclusions were observed in the oculomotor nuclei. Clinical manifestations of urinary disturbance and oculomotor imparirment seldom seen in sporadic ALS were interpreted to be due to the unusual distribution of the morbid process. These pathologic findings suggest that familial ALS may be a multisystemic degenerative disorder, frequently involving the spinocerebellar tracts, but occasionally involving other systems as well.Supported in part by a Research Grant for the Intractable Disease from the Ministry of Health and Welfare of Japan     

Paraoxonase gene mutations in amyotrophic lateral sclerosis

Three clustered, homologous paraoxonase genes (PON1, PON2, and PON3) have roles in preventing lipid oxidation and detoxifying organophosphates. Recent reports describe a genetic association between the PON genes and sporadic amyotrophic lateral sclerosis (ALS). We now report that in genomic DNA from individuals with familial and sporadic ALS, we have identified at least 7 PON gene mutations that are predicted to alter PON function. ANN NEUROL 2010     

Familial amyotrophic lateral sclerosis (FALS) with a novel SOD1 gene mutation: a clinicopathological study]

We describe a patient with familial amyotrophic lateral sclerosis (FALS) in whom we identified a substitution of G for CGTTTA at codon 144 in the Cu/Zn superoxide dismutase 1 (SOD1) gene, causing amino acid changes from leucine to phenylalanine, valine and a stop codon (L144FVX). This mutation is novel, and so we report the clinical and neuropathological features of this case compared with those of other FALS cases with SOD1 mutations. A 39-year-old woman developed muscle weakness and atrophy in the hands, which rapidly progressed and expanded to other muscles, resulting in respiratory insufficiency and death at only 10 months after the onset Her grandmother, father and uncle had also been diagnosed as having ALS. The most noticeable neuropathological findings in the present case were marked loss of large motor neurons in the anterior horns associated with the frequent appearance of cord-like swollen, partially SOD 1- and ubiquitin-immunopositive axons. These findings together with the absence of Bunina bodies are compatible with the neuropathology of FALS with SOD1 gene mutation, although Lewy body-like inclusions characteristic for this condition were not observed.     

Animal models of amyotrophic lateral sclerosis]

Dominant mutation in the gene of superoxide dismutase 1 (SOD1) leads amyotrophic lateral sclerosis (ALS), an adult-onset progressive fatal motor neuron disease. Recent research progress in ALS has been made by the use of transgenic mouse model of familial ALS, which expresses mutant form of SOD1 and recapitulates the phenotype and pathology of motor neuron disease. There is accumulating evidence indicating non-cell-autonomous motor neuron death in ALS mouse model. In this symposium, I review the recent advance of ALS research focusing on the development of animal models, the role of glial cells in ALS, and therapeutic intervention of rodent models and discuss their prospect.