Role of mitochondria in mutant SOD1 linked amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an adult onset characterized by loss of both upper and lower motor neurons. In ~ 10% of cases, patients developed ALS with an apparent genetic linkage (familial ALS or fALS). Approximately 20% of fALS displays mutations in the SOD1 gene encoding superoxide dismutase 1. There are many proposed cellular and molecular mechanisms among which, mitochondrial dysfunctions occur early, prior to symptoms occurrence. In this review, we modeled the effect of mutant SOD1 protein via the formation of a toxic complex with Bcl2 on mitochondrial bioenergetics. Furthermore, we discuss that the shutdown of ATP permeation through mitochondrial outer membrane could lead to both respiration inhibition and temporary mitochondrial hyperpolarization. Moreover, we reviewed mitochondrial calcium signaling, oxidative stress, fission and fusion, autophagy and apoptosis in mutant SOD1-linked ALS. Functional defects in mitochondria appear early before symptoms are manifested in ALS. Therefore, mitochondrial dysfunction is a promising therapeutic target in ALS. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

Macroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1^G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1^G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1^G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS.     

Insoluble mutant SOD1 is partly oligoubiquitinated in amyotrophic lateral sclerosis mice

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS) through an unknown gain-of-function mechanism. Mutant SOD1 aggregation may be the toxic property. In fact, proteinaceous inclusions rich in mutant SOD1 have been found in tissues from the familial form of ALS patients and in mutant SOD1 animals, before disease onset. However, very little is known of the constituents and mechanism of formation of aggregates in ALS. We and others have shown that there is a progressive accumulation of detergent-insoluble mutant SOD1 in the spinal cord of G93A SOD1 mice. To investigate the mechanism of SOD1 aggregation, we characterized by proteome technologies SOD1 isoforms in a Triton X-100-insoluble fraction of spinal cord from G93A SOD1 mice at different stages of the disease. This showed that at symptomatic stages of the disease, part of the insoluble SOD1 is unambiguously mono- and oligoubiquitinated, in spinal cord and not in hippocampus, and that ubiquitin branches at Lys(48), the major signal for proteasome degradation. At presymptomatic stages of the disease, only insoluble unmodified SOD1 is recovered. Partial ubiquitination of SOD1-rich inclusions was also confirmed by immunohistochemical and electron microscopy analysis of lumbar spinal cord sections from symptomatic G93A SOD1 mice. On the basis of these results, we propose that ubiquitination occurs only after SOD1 aggregation and that oligoubiquitination may underline alternative mechanisms in disease pathogenesis.     

Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis

Familial amyotrophic lateral sclerosis (FALS)-linked mutations in copper-zinc superoxide dismutase (SOD1) cause motor neuron death through one or more acquired toxic properties. We analyzed the molecular mechanism underlying motor neuron degeneration in the transgenic mouse model expressing the SOD1 gene with G93A mutation. Using cDNA microarray, the differentially expressed genes were identified in the spinal cords of G93A mice, 30 being elevated and seven decreased. cDNA microarray analysis to monitor gene expression during neurodegeneration revealed an up-regulation of genes related to an inflammatory process, such as the tumor necrosis factor-alpha (TNF-alpha) gene, resulting from glial cell activation, together with the change in apoptosis-related gene expression, such as caspase-1. The increased expression of the inflammation- and apoptosis-related genes occurred at 11 weeks of age in the presymptomatic stage prior to motor neuron death. These results suggest a mechanism of neurodegeneration that includes an inflammatory response as an important component. Thus, ALS has paralleled other neurodegenerative disorders, such as Alzheimer's and prion diseases, in which the inflammatory process is believed to participate directly in neuronal death.     

Muscle cells and motoneurons differentially remove mutant SOD1 causing familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuronal disease which occurs in sporadic or familial forms, clinically indistinguishable. About 15% of familial ALS cases are linked to mutations of the superoxide dismutase 1 (SOD1) gene that may induce misfolding in the coded protein, exerting neurotoxicity to motoneurons. However, other cell types might be target of SOD1 toxicity, because muscle-restricted expression of mutant SOD1 correlates with muscle atrophy and motoneurons death. We analysed the molecular behaviour of mutant SOD1 in motoneuronal NSC34 and muscle C2C12 cells. We found that misfolded mutant SOD1 clearance is much more efficient in muscle C2C12 than in motoneuronal NSC34 cells. Mutant SOD1 forms aggregates and impairs the proteasome only in motoneuronal NSC34 cells. Interestingly, NSC34 cells expressing mutant SOD1 are more sensitive to a superoxide-induced oxidative stress. Moreover, in muscle C2C12 cells mutant SOD1 remains soluble even when proteasome is inhibited with MG132. The higher mutant SOD1 clearance in muscle cells correlates with a more efficient proteasome activity, combined with a robust autophagy activation. Therefore, muscle cells seem to better manage misfolded SOD1 species, not because of an intrinsic property of the mutant protein, but in function of the cell environment, indicating also that the SOD1 toxicity at muscle level may not directly depend on its aggregation rate.     

Structural consequences of the familial amyotrophic lateral sclerosis SOD1 mutant His46Arg

The His46Arg (H46R) mutant of human copper-zinc superoxide dismutase (SOD1) is associated with an unusual, slowly progressing form of familial amyotrophic lateral sclerosis (FALS). Here we describe in detail the crystal structures of pathogenic H46R SOD1 in the Zn-loaded (Zn-H46R) and metal-free (apo-H46R) forms. The Zn-H46R structure demonstrates a novel zinc coordination that involves only three of the usual four liganding residues, His 63, His 80, and Asp 83 together with a water molecule. In addition, the Asp 124 "secondary bridge" between the copper- and zinc-binding sites is disrupted, and the "electrostatic loop" and "zinc loop" elements are largely disordered. The apo-H46R structure exhibits partial disorder in the electrostatic and zinc loop elements in three of the four dimers in the asymmetric unit, while the fourth has ordered loops due to crystal packing interactions. In both structures, nonnative SOD1-SOD1 interactions lead to the formation of higher-order filamentous arrays. The disordered loop elements may increase the likelihood of protein aggregation in vivo, either with other H46R molecules or with other critical cellular components. Importantly, the binding of zinc is not sufficient to prevent the formation of nonnative interactions between pathogenic H46R molecules. The increased tendency to aggregate, even in the presence of Zn, arising from the loss of the secondary bridge is consistent with the observation of an increased abundance of hyaline inclusions in spinal motor neurons and supporting cells in H46R SOD1 transgenic rats.     

Therapeutic gene silencing delivered by a chemically modified small interfering RNA against mutant SOD1 slows amyotrophic lateral sclerosis progression

Inherited neurodegenerative diseases, such as Huntington disease and subset of Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis, are caused by the mutant genes that have gained undefined properties that harm cells in the nervous system, causing neurodegeneration and clinical phenotypes. Lowering the mutant gene expression is predicted to slow the disease progression and produce clinical benefit. Administration of small interfering RNA (siRNA) can silence specific genes. However, long term delivery of siRNA to silence the mutant genes, a requirement for treatment of these chronic central nervous system (CNS) diseases, remains a critical unsolved issue. Here we designed and tested a chemically stabilized siRNA against human Cu,Zn-superoxide dismutase (SOD1) in a mouse model for amyotrophic lateral sclerosis. We show that the modified siRNA has enhanced stability and retains siRNA activity. Administration of this siRNA at the disease onset by long term infusion into the CNS resulted in widespread distribution of this siRNA, knocked down the mutant SOD1 expression, slowed the disease progression, and extended the survival. These results bring RNA interference therapy one step closer to its clinical application for treatment of chronic, devastating, and fatal CNS disorders.     

Rapamycin treatment augments motor neuron degeneration in SOD1G93A mouse model of amyotrophic lateral sclerosis

Aberrant protein misfolding may contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS) but the detailed mechanisms are largely unknown. Our previous study has shown that autophagy is altered in the mouse model of ALS. In the present study, we systematically investigated the correlation of the autophagic alteration with the motor neurons (MNs) degeneration in the ALS mice. We have demonstrated that the autophagic protein marker LC3-II is markedly and specifically increased in the spinal cord MNs of the ALS mice. Electron microscopy and immunochemistry studies have shown that autophagic vacuoles are significantly accumulated in the dystrophic axons of spinal cord MNs of the ALS mice. All these changes in the ALS mice appear at the age of 90 d when the ALS mice display modest clinical symptoms; and they become prominent at the age of 120 d. The clinical symptoms are correlated with the progression of MNs degeneration. Moreover, we have found that p62/SQSTM1 is accumulated progressively in the spinal cord, indicating that the possibility of impaired autophagic flux in the SOD1^G93A mice. Furthermore, to our surprise, we have found that treatment with autophagy enhancer rapamycin accelerates the MNs degeneration, shortens the life span of the ALS mice, and has no obvious effects on the accumulation of SOD1 aggregates. In addition, we have demonstrated that rapamycin treatment in the ALS mice causes more severe mitochondrial impairment, higher Bax levels and greater caspase-3 activation. These findings suggest that selective degeneration of MNs is associated with the impairment of the autophagy pathway and that rapamycin treatment may exacerbate the pathological processing through apoptosis and other mechanisms in the ALS mice.     

Over-expression of Hsp27 does not influence disease in the mutant SOD1(G93A) mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a chronic, adult-onset neurodegenerative disorder characterized by the selective loss of upper and lower motor neurons, resulting in severe atrophy of muscles and death. Although the exact pathogenic mechanism of mutant superoxide dismutase 1 (SOD1) causing familial ALS is still elusive, toxic protein aggregation leading to insufficiency of chaperones is one of the main hypotheses. In this study, we investigated the effect of over-expressing one of these chaperones, heat shock protein 27 (Hsp27), in ALS. Mice over-expressing the human, mutant SOD1^G93A were crossed with mice that ubiquitously over-expressed human Hsp27. Even though the single transgenic hHsp27 mice showed protection against spinal cord ischemia, the double transgenic SOD1^G93A/hHsp27 mice did not live longer, and did not show a significant delay in the onset of disease compared to their SOD1^G93A littermates. There was no protective effect of hHsp27 over-expression on the motor neurons and on the mutant SOD1 aggregates in the double transgenic SOD1^G93A/hHsp27 mice. In conclusion, despite the protective action against acute motor neuron injury, Hsp27 alone is not sufficient to protect against the chronic motor neuron injury due to the presence of mutant SOD1.     

Lack of effect of methylene blue in the SOD1 G93A mouse model of amyotrophic lateral sclerosis

Background

Methylene blue (MB) is a drug with a long history and good safety profile, and with recently-described features desirable in a treatment for ALS.

Methodology/Principal Findings

We tested oral MB in inbred high-copy number SOD1 G93A mice, at 25 mg/kg/day beginning at 45 days of age. We measured disease onset, progression, and survival. There was no difference in disease onset between MB-treated mice and controls, although subgroup analysis showed a modest but statistically significant delay in disease onset in MB-treated female mice only (control 122±10.2 versus MB 129±10.0 days). MB-treated mice of both sexes spent more time in less severe stages of disease, and less time in later, more severe stages of disease. There was a non-significant trend to longer survival in MB-treated animals (control males reached endpoint at 161±14.1 days, versus 166±10.0 days for MB-treated animals, and control females reached endpoint at 171±6.2 days versus 173±13.4 days for MB-treated animals).

Conclusions/Significance

In spite of a strong theoretical rationale, MB had no significant effects on onset or survival in the inbred SOD1 G93A mouse model of ALS.     

Respiratory impairment in a mouse model of amyotrophic lateral sclerosis.

Amyothrophic lateral sclerosis (ALS) is a progressive, lethal neuromuscular disease that is associated with the degeneration of cortical and spinal motoneurons, leading to atrophy of limb, axial, and respiratory muscles. Patients with ALS invariably develop respiratory muscle weakness and most die from pulmonary complications. Overexpression of superoxide dismutase 1 (SOD1) gene mutations in mice recapitulates several of the clinical and pathological characteristics of ALS and is therefore a valuable tool to study this disease. The present study is intended to evaluate an age-dependent progression of respiratory complications in SOD1(G93A) mutant mice. In each animal, baseline measurements of breathing pattern [i.e., breathing frequency and tidal volume (VT)], minute ventilation (VE), and metabolism (i.e., oxygen consumption and carbon dioxide production) were repeatedly sampled at variable time points between 10 and 20 wk of age with the use of whole-body plethysmographic chambers. To further characterize the neurodegeneration of breathing, VE was also measured during 5-min challenges of hypercapnia (5% CO(2)) and hypoxia (10% O(2)). At baseline, breathing characteristics and metabolism remained relatively unchanged from 10 to 14 wk of age. From 14 to 18 wk of age, there were significant (P < 0.05) increases in baseline VT, VE, and the ventilatory equivalent (VE/oxygen consumption). After 18 wk of age, there was a rapid decline in VE due to significant (P < 0.05) reductions in both breathing frequency and VT. Whereas little change in hypoxic VE responses occurred between 10 and 18 wk, hypercapnic VE responses were significantly (P < 0.05) elevated at 18 wk due to an augmented VT response. Like baseline breathing characteristics, hypercapnic VE responses also declined rapidly after 18 wk of age. The phenotypic profile of SOD1(G93A) mutant mice was apparently unique because similar changes in respiration and metabolism were not observed in SOD1 controls. The present results outline the magnitude and time course of respiratory complications in SOD1(G93A) mutant mice as the progression of disease occurs in this mouse model of ALS.     

SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis

A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention.     

Bim links ER stress and apoptosis in cells expressing mutant SOD1 associated with amyotrophic lateral sclerosis

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.     

The effect of leukaemia inhibitory factor on SOD1 G93A murine amyotrophic lateral sclerosis

Before potential therapeutic strategies for the treatment of amyotrophic lateral sclerosis (ALS) can be advanced to human clinical trials, there is a need to assess them in an animal model that best resembles the disease process. SOD1 G93A mice have close resemblance to familial ALS (fALS) and have been used in this study to evaluate the therapeutic potential of leukaemia inhibitory factor (LIF). LIF action was investigated by assessing three delivery methods: (1) daily subcutaneous injection; (2) through LIF rods placed adjacent to hind limb skeletal muscle and (3) continuous intrathecal infusion. The effect on disease progression was assessed by semi-quantitative and quantitative functional measurements, and histologically on the survival of motor neurons and number of reactive astrocytes. The results show that LIF had no beneficial effects when administered using the three methods of drug delivery. These results suggest that further evaluation of LIF in this transgenic model is required to fully characterize its' therapeutic potential.     

Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis.     

S‐nitrosylated protein disulfide isomerase contributes to mutant SOD1 aggregates in amyotrophic lateral sclerosis

A major hallmark of mutant superoxide dismutase (SOD1)‐linked familial amyotrophic lateral sclerosis is SOD1‐immunopositive inclusions found within motor neurons. The mechanism by which SOD1 becomes aggregated, however, remains unclear. In this study, we aimed to investigate the role of nitrosative stress and S‐nitrosylation of protein disulfide isomerase (PDI) in the formation of SOD1 aggregates. Our data show that with disease progression inducible nitric oxide synthase (iNOS) was up‐regulated, which generated high levels of nitric oxide (NO) and subsequently induced S‐nitrosylation of PDI in the spinal cord of mutant SOD1 transgenic mice. This was further confirmed by in vitro observation that treating SH‐SY5Y cells with NO donor S‐nitrosocysteine triggered a dose‐dependent formation of S‐nitrosylated PDI. When mutant SOD1 was over‐expressed in SH‐SY5Y cells, the iNOS expression was up‐regulated, and NO generation was consequently increased. Furthermore, both S‐nitrosylation of PDI and the formation of mutant SOD1 aggregates were detected in the cells expressing mutant SOD1^G93A. Blocking NO generation with the NOS inhibitor N‐nitro‐l ‐arginine attenuated the S‐nitrosylation of PDI and inhibited the formation of mutant SOD1 aggregates. We conclude that NO‐mediated S‐nitrosylation of PDI is a contributing factor to the accumulation of mutant SOD1 aggregates in amyotrophic lateral sclerosis.     

Rapamycin treatment augments motor neuron degeneration in SOD1(G93A) mouse model of amyotrophic lateral sclerosis

Aberrant protein misfolding may contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS) but the detailed mechanisms are largely unknown. Our previous study has shown that autophagy is altered in the mouse model of ALS. In the present study, we systematically investigated the correlation of the autophagic alteration with the motor neurons (MNs) degeneration in the ALS mice. We have demonstrated that the autophagic protein marker LC3-II is markedly and specifically increased in the spinal cord MNs of the ALS mice. Electron microscopy and immunochemistry studies have shown that autophagic vacuoles are significantly accumulated in the dystrophic axons of spinal cord MNs of the ALS mice. All these changes in the ALS mice appear at the age of 90 d when the ALS mice display modest clinical symptoms; and they become prominent at the age of 120 d. The clinical symptoms are correlated with the progression of MNs degeneration. Moreover, we have found that p62/SQSTM1 is accumulated progressively in the spinal cord, indicating that the possibility of impaired autophagic flux in the SOD1(G93A) mice. Furthermore, to our surprise, we have found that treatment with autophagy enhancer rapamycin accelerates the MNs degeneration, shortens the life span of the ALS mice, and has no obvious effects on the accumulation of SOD1 aggregates. In addition, we have demonstrated that rapamycin treatment in the ALS mice causes more severe mitochondrial impairment, higher Bax levels and greater caspase-3 activation. These findings suggest that selective degeneration of MNs is associated with the impairment of the autophagy pathway and that rapamycin treatment may exacerbate the pathological processing through apoptosis and other mechanisms in the ALS mice.     

Informatics-assisted protein profiling in a transgenic mouse model of amyotrophic lateral sclerosis

One of the causes of amyotrophic lateral sclerosis (ALS) is due to mutations in Cu,Zn-superoxide dismutase (SOD1). The mutant protein exhibits a toxic gain of function that adversely affects the function of neurons in the spinal cord, brain stem, and motor cortex. A proteomic analysis of protein expression in a widely used mouse model of ALS was undertaken to identify differences in protein expression in the spinal cords of mice expressing a mutant protein with the G93A mutation found in human ALS. Protein profiling was done on soluble and particulate fractions of spinal cord extracts using high throughput two-dimensional liquid chromatography coupled to tandem mass spectrometry. An integrated proteomics-informatics platform was used to identify relevant differences in protein expression based upon the abundance of peptides identified by database searching of mass spectrometry data. Changes in the expression of proteins associated with mitochondria were particularly prevalent in spinal cord proteins from both mutant G93A-SOD1 and wild-type SOD1 transgenic mice. G93A-SOD1 mouse spinal cord also exhibited differences in proteins associated with metabolism, protein kinase regulation, antioxidant activity, and lysosomes. Using gene ontology analysis, we found an overlap of changes in mRNA expression in presymptomatic mice (from microarray analysis) in three different gene categories. These included selected protein kinase signaling systems, ATP-driven ion transport, and neurotransmission. Therefore, alterations in selected cellular processes are detectable before symptomatic onset in ALS mouse models. However, in late stage disease, mRNA expression analysis did not reveal significant changes in mitochondrial gene expression but did reveal concordant changes in lipid metabolism, lysosomes, and the regulation of neurotransmission. Thus, concordance of proteomic and mRNA expression data within multiple categories validates the use of gene ontology analysis to compare different types of "omic" data.     

Comparative study of behavioural tests in the SOD1G93A mouse model of amyotrophic lateral sclerosis

In preclinical trials, a sensitive functional test is required to detect changes in the motor behaviour of the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS). We evaluated changes in body weight and motor impairment in behavioural tests, such as the rotarod, the hanging-wire test and the treadmill, of transgenic and wild type mice. We found differences in detection of the onset of symptoms and progression of the disease between the different tests assessed. Moreover, the data showed significant gender differences in the motor behaviour of this mouse model. The rotarod and the hanging-wire test were more sensitive to detect early motor impairment. Moreover, the results suggested that the rotarod and hanging-wire became the most accurate tests rather than treadmill to characterise the ALS disease phenotype.     

Spinal cord pathology is ameliorated by P2X7 antagonism in a SOD1-mutant mouse model of amyotrophic lateral sclerosis

In recent years there has been an increasing awareness of the role of P2X7, a receptor for extracellular ATP, in modulating physiopathological mechanisms in the central nervous system. In particular, P2X7 has been shown to be implicated in neuropsychiatry, chronic pain, neurodegeneration and neuroinflammation. Remarkably, P2X7 has also been shown to be a ‘gene modifier’ in amyotrophic lateral sclerosis (ALS): the receptor is upregulated in spinal cord microglia in human and rat at advanced stages of the disease; in vitro, activation of P2X7 exacerbates pro-inflammatory responses in microglia that have an ALS phenotype, as well as toxicity towards neuronal cells. Despite this detrimental in vitro role of P2X7, in SOD1-G93A mice lacking P2X7, the clinical onset of ALS was significantly accelerated and disease progression worsened, thus indicating that the receptor might have some beneficial effects, at least at certain stages of disease. In order to clarify this dual action of P2X7 in ALS pathogenesis, in the present work we used the antagonist Brilliant Blue G (BBG), a blood-brain barrier permeable and safe drug that has already been proven to reduce neuroinflammation in traumatic brain injury, cerebral ischemia-reperfusion, neuropathic pain and experimental autoimmune encephalitis. We tested BBG in the SOD1-G93A ALS mouse model at asymptomatic, pre-symptomatic and late pre-symptomatic phases of disease. BBG at late pre-onset significantly enhanced motor neuron survival and reduced microgliosis in lumbar spinal cord, modulating inflammatory markers such as NF-κB, NADPH oxidase 2, interleukin-1β, interleukin-10 and brain-derived neurotrophic factor. This was accompanied by delayed onset and improved general conditions and motor performance, in both male and female mice, although survival appeared unaffected. Our results prove the twofold role of P2X7 in the course of ALS and establish that P2X7 modulation might represent a promising therapeutic strategy by interfering with the neuroinflammatory component of the disease.KEY WORDS: ALS, Brilliant Blue G, Microglia, Motor neuron, P2X7