Oxidative Damage and Motor Neurone Disease Difficulties in the Measurement of Protein Carbonyls in Human Brain Tissue

It has been suggested in the literature that elevated oxidative protein damage, measured as protein carbonyls, is present in the nervous system of patients with sporadic motor neurone disease (MND). However, the actual reported levels of brain protein carbonyls vary over a wide range. We show here that this is probably due to the use of different protocols for the carbonyl assay; results differ depending on when the dinitrophenylhydrazine reagent is added and at what stage in the procedure protein is assayed for the calculation of carbonyls on a unit protein basis. Using a range of different procedures, we were unable to confirm reports of elevated protein carbonyls in motor cortex from brains of patients with MND. We also measured thiobarbituric acid-reactive material in the brain samples using an HPLC-based TBA test in the presence of butylated hydroxytoluene. In general, there was no significant elevation of TBARS in MND motor cortex. However, four patients showed values higher than any of the control patients (both 'normal' control and 'disease control'). There was no correlation of TBARS with protein carbonyl values. We suggest that oxidative damage in motor cortex in sporadic MND, if it occurs, may be confined to a small group of patients and may affect different molecular targets in each patient.     

Non-spiking local interneurones mediate abdominal extension related descending control of uropod motor neurones in the crayfish terminal abdominal ganglion

The role of non-spiking local interneurones in the synaptic interactions between abdominal extension-evoking descending interneurones and uropod motor neurones in the terminal abdominal ganglion of the crayfish Procambarus clarkii (Girard) was investigated electrophysiologically. Continuous electrical stimulation of the lateral region of the 3rd-4th abdominal connective that included abdominal extension evoking interneurones excited the opener motor neurones and inhibited the closer, reductor motor neurone. Spikes from a single descending interneurone evoked consistent and short latency (0.8–0.9 ms) excitatory postsynaptic potentials (e.p.s.ps) in the opener motor neurones, and evoked rather long-latency (1.5–2.7 ms) inhibitory postsynaptic potentials (i.p.s.ps) in the reductor motor neurone. Many non-spiking interneurones also received depolarizing p.s.ps (0.8–2.5 ms in latency) that were usually faster than i.p.s.ps of the reductor motor neurone if both neurones were recorded sequentially in the same preparation. Non-spiking interneurones received convergent inputs from several descending interneurones and made inverting connection with the reductor motor neurone. Elimination of descending inputs to a particular non-spiking interneurone could reduce the inhibitory response of the reductor motor neurone. These observations strongly suggested that descending inhibitory inputs to the closer, reductor motor neurone were mediated by non-spiking interneurones. Furthermore, some non-spiking interneurones made output connections with the opener motor neurones. The disynaptic pathway through non-spiking interneurones is significant to control and modulate the opening pattern of the uropod during abdominal extension. Accepted: 27 December 1996     

F responses and central motor conduction in multiple sclerosis

Two electrophysiological investigations were used to study 18 patients with multiple sclerosis — F wave characteristics including amplitude, persistence and frequency, which can provide a measure of motoneurone excitability, and magnetic stimulation of the cortex, which enables measurement of conduction along central motor pathways. There was an increase in the mean amplitude and persistence of the F response in patients with abnormal central motor conduction (CMC), although no correlation between the degree of abnormality of CMC and increase in F response amplitude was found. Increase in mean amplitude and persistence of the F response were also found in patients with normal CMC but clinical evidence of a UMN disorder (spasticity and/or weakness); there was no correlation, however, between any single F response characteristic and any particular clinical sign. CMC appears to be the preferred test for detecting subclinical motor lesions in MS: of the patient sides with normal clinical examination, 36% showed abnormal CMC, whereas 23% showed abnormal F responses.     

Protocol for diaphragm pacing in patients with respiratory muscle weakness due to motor neurone disease (DiPALS): a randomised controlled trial

ABSTRACT: BACKGROUND: Motor neurone disease (MND) is a devastating illness which leads to muscle weakness and death, usually within 2-3 years of symptom onset. Respiratory insufficiency is a common cause of morbidity, particularly in later stages of MND and respiratory complications are the leading cause of mortality in MND patients. Non Invasive Ventilation (NIV) is the current standard therapy to manage respiratory insufficiency. Some MND patients however do not tolerate NIV due to a number of issues including mask interface problems and claustrophobia. In those that do tolerate NIV, eventually respiratory muscle weakness will progress to a point at which intermittent/overnight NIV is ineffective. The NeuRx RA/4 Diaphragm Pacing System was originally developed for patients with respiratory insufficiency and diaphragm paralysis secondary to stable high spinal cord injuries. The DiPALS study will assess the effect of diaphragm pacing (DP) when used to treat patients with MND and respiratory insufficiency. Method/Design 108 patients will be recruited to the study at 5 sites in the UK. Patients will be randomised to either receive NIV (current standard care) or receive DP in addition to NIV. Study participants will be required to complete outcome measures at 5 follow up time points (2, 3, 6, 9 and 12 months) plus an additional surgery and 1 week post operative visit for those in the DP group. 12 patients (and their carers) from the DP group will also be asked to complete 2 qualitative interviews. DISCUSSION: The primary objective of this trial will be to evaluate the effect of Diaphragm Pacing (DP) on survival over the study duration in patients with MND with respiratory muscle weakness. The project is funded by the National Institute for Health Research, Health Technology Assessment (HTA) Programme (project number 09/55/33) and the Motor Neurone Disease Association and the Henry Smith Charity. Trial Registration: Current controlled trials ISRCTN53817913. The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the HTA programme, NIHR, NHS or the Department of Health.     

Metabolomic analysis and signatures in motor neuron disease

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

Trace elements in cerebrospinal fluid in motor neurone disease.

Concentrations of sodium, chlorine, potassium, chromium, iron, cobalt, zinc, rubidium, silver, caesium, and selenium in cerebrospinal fluid from 14 control subjects and 20 patients with motor neurone disease were measured by in vitro neutron activation analysis. No statistically significant correlation was found between the concentrations of any two elements other than sodium and chlorine in either the patient or control group (r = 0.9905; p less than 0.001). The mean cobalt concentration was significantly lower in the patients (p = 0.0015). No other statistically significant difference was shown. The relevance of this finding was examined in relation to current concepts of the pathogenesis of motor neurone disease and the role of cobalt in cellular metabolism.     

Development and characterisation of a glutamate-sensitive motor neurone cell line

Modification of the growth conditions of NSC-34 mouse neuroblastoma x motor neurone cells by serum depletion promotes the expression of functional glutamate receptors as the cells mature into a form that bears the phenotypic characterisation of motor neurones. Immunocytochemical studies demonstrated the presence of the glutamate receptor proteins NMDAR1, NMDAR2A/B, GluR1, GluR2, GluR2/3, GluR4, GluR6/7, and KA2. Toxicity assays using cell counting techniques demonstrated a mild but significant cell death (approximately 30%, p < 0.01) following a 24-h exposure to 1 mM glutamate that could be prevented by the presence of the glutamate receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (10 microM) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (1 microM). As an indication of glutamate receptor functional activity a novel approach was used to detect the production of free radicals following stimulation with glutamate receptor agonists. The release of superoxide free radicals was detected using a micro-electrochemical sensor following addition of glutamate receptor agonists to the cell bathing solution. Alterations in intracellular calcium concentrations were examined using fura-2 imaging. Exposure of the differentiated NSC-34 cells to glutamate leads to an increase in intracellular calcium concentrations that is prevented by the presence of glutamate receptor antagonists. The motor neurone origin of these cells makes them particularly useful for investigating the potential role of glutamatergic toxicity in motor neurone degeneration.     

Animal models for motor neuron disease.

Motor neuron disease is a general term applied to a broad class of neurodegenerative diseases that are characterized by fatally progressive muscular weakness, atrophy, and paralysis attributable to loss of motor neurons. At present, there is no cure for most motor neuron diseases, including amyotrophic lateral sclerosis (ALS), the most common human motor neuron disease--the cause of which remains largely unknown. Animal models of motor neuron disease (MND) have significantly contributed to the remarkable recent progress in understanding the cause, genetic factors, and pathologic mechanisms proposed for this class of human neurodegenerative disorders. Largely driven by ALS research, animal models of MND have proven their usefulness in elucidating potential causes and specific pathogenic mechanisms, and have helped to advance promising new treatments from "benchside to bedside." This review summarizes important features of selected established animal models of MND: genetically engineered mice and inherited or spontaneously occurring MND in the murine, canine, and equine species.     

Vitamin E deficiency and risk of equine motor neuron disease

Background  

Equine motor neuron disease (EMND) is a spontaneous neurologic disorder of adult horses which results from the degeneration of motor neurons in the spinal cord and brain stem. Clinical manifestations, pathological findings, and epidemiologic attributes resemble those of human motor neuron disease (MND). As in MND the etiology of the disease is not known. We evaluated the predisposition role of vitamin E deficiency on the risk of EMND.     

Communication aids in patients with motor neurone disease.

The study aimed at determining the main factors responsible for the speech and communication problems in motor neurone disease to try to assess the suitability of commercially available aids. Sixteen patients suffering from motor neurone disease with communication problems had their speech assessed by using the Frenchay dysarthria assessment. It was found essential to offer a choice of aids to the patient since suitability of a particular aid could not be evaluated without the patient practising with it.     

A novel locus for distal motor neuron degeneration maps to chromosome 7q34-q36

The motor neuron diseases (MND) are a group of related neurodegenerative diseases that cause the relative selective progressive death of motor neurons. These diseases range from slowly progressive forms including hereditary motor neuropathy (HMN), to the rapidly progressive disorder amyotrophic lateral sclerosis (ALS). There is clinical and genetic overlap among these MNDs, implicating shared pathogenic mechanisms. We recruited a large family with a MND that was previously described as juvenile ALS and distal HMN. We identified a novel MND/HMN locus on chromosome 7q34-q36 following a genome-wide scan for linkage in this family. The disease causing mutation maps to a 26.2 cM (12.3 Mb) interval flanked by D7S2513 and D7S637 on chromosome 7q34-q36. Recombinant haplotype analysis including unaffected individuals suggests that the refined candidate interval spans 14.3 cM (6.3 Mb) flanked by D7S2511 and D7S798. One gene in the candidate interval, CDK5, was selected for immediate mutation analysis based upon its known association with an ALS-like phenotype in mice however, no mutations were identified. Identification of genes causing familial MND will lead to a greater understanding of the biological basis of both familial and sporadic motor neuron degeneration including ALS. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of the glutathione S-transferase P1 gene Ile105Val polymorphism in the patients with sporadic motor neuron disease from Russia

Ile105Val polymorphism in exon 5 of glutathione S-transferase (GSTP1) gene was examined in a group of patients with motor neuron disease (MND) and control sample. No statistically significant differences in the allele and genotype frequency distributions between the samples examined were demonstrated. We conclude that Ile105Val polymorphism is not associated with the risk of the disease development in the patients from Russia with sporadic form of MND.     

Polymorphism of glutathione S-transferase genes M1 and T1 in patients with motor neuron disease from the city of Moscow

Comparison of the frequency distributions of alleles, genotypes, and genotype combinations of genes GSTM1 and GSTT1 did not show statistically significant differences between patients with motor neuron disease (MND) and a random sample from the Moscow population. Apparently, these genes are not involved in MND pathogenesis in these patients.     

Sequences specific for enterovirus detected in spinal cord from patients with motor neurone disease.

OBJECTIVE--To investigate the association of enteroviruses with motor neurone disease, also known as amyotrophic lateral sclerosis. DESIGN--Analysis by enterovirus polymerase chain reaction of wax embedded material from spinal cords taken at necropsy from subjects with motor neurone disease and from age and sex matched controls. SETTING--Specimens were collected in the west of Scotland and in London between 1982 and 1992. RESULTS--Sequences specific for a non-poliovirus type enterovirus were detected in spinal cord tissue from subjects with motor neurone disease. Amplification of a 414 base RNA target sequence in the conserved enterovirus 5'' untranslated region from wax embedded tissue sections was successful in tissue from eight of 11 cases of sporadic motor neurone disease, one of two cases of familial motor neurone disease, and the one case of poliomyelitis, but not in the six matched controls or one case of antecedent poliomyelitis. In addition, sequences were detected in spinal cords from one monkey infected with wild type poliovirus and one monkey infected with polio vaccine. Comparison of sequences from cases of motor neurone disease with sequences of corresponding regions of the 5'' untranslated regions of known picornaviruses showed them to be tightly grouped within the enterovirus genus closely related to coxsackievirus type B but not to polioviruses. Sequences derived from different parts of the spinal cord of the same subjects were identical, but sequences differed between individual subjects. CONCLUSIONS--Conserved enteroviral sequences closely related to coxsackie B virus sequences were detectable in spinal cords from subjects with sporadic motor neurone disease and from one subject with possible familial motor neurone disease.     

In-vivo proton MR-spectroscopy of the human brain: assessment of N-acetylaspartate (NAA) reduction as a marker for neurodegeneration

Summary.  Proton magnetic resonance spectroscopy (¹H-MRS) is a non-invasive method to investigate changes in brain metabolite composition in different cerebral diseases. We performed proton spectroscopy in patients with dementia of the Alzheimer's type (AD) and in patients with motor neuron disease (MND) with the aim to detect the specific metabolic pattern for these neurodegenerative disorders. In the MND group we found a significant reduction of NAA/tCr metabolite ratios in the motor cortex, which correlates with the disease severity and the clinical lateralization of neurological symptoms and further decreases in the time course of the disease. In AD patients a reduction of NAA/tCr was observed in the medial temporal lobe. Since NAA is exclusively expressed in neurons as shown by immunohistochemical studies, reduced NAA levels suggest neuronal loss or dysfunction in the observed regions. The observed regional metabolic alterations reflect the neuronal basis of the characteristic neurological symptoms in AD (dementia) and MND (muscular palsy) and mirrors the disease progress over time. Received June 29, 2001 Accepted August 6, 2001 Published online August 9, 2002     

Experimental approach to the gene therapy of motor neuron disease with the use of genes hypoxia-inducible factors

Motor neuron disease (MND), or amyotrophic lateral sclerosis, is a fatal neurodegenerative disorder characterized by a progressive loss of motor neurons in the spinal cord and the brain. Several angiogenic and neurogenic growth factors, such as the vascular endothelial growth factor (VEGF), angiogenin (ANG), insulin-like growth factor (IGF) and others, have been shown to promote survival of the spinal motor neurons during ischemia. We constructed recombinant vectors using human adenovirus 5 (Ad5) carrying the VEGF, ANG or IGF genes under the control of the cytomegalovirus promoter. As a model for MND, we employed a transgenic mice strain, B6SJL-Tg(SOD1*G93A)dl1 Gur/J that develops a progressive degeneration of the spinal motor neurons caused by the expression of a mutated Cu/Zn superoxide dismutase gene SOD1 ^G93A. Delivery of the therapeutic genes to the spinal motor neurons was done using the effect of the retrograde axonal transport after multiple injections of the Ad5-VEGF, Ad5-ANG and Ad5-IGF vectors and their combinations into the limbs and back muscles of the SOD1 ^G93A mice. Viral transgene expression in the spinal cord motor neurons was confirmed by immunocytochemistry and RT-RCR. We assessed the neurological status, motor activity and lifespan of experimental and control animal groups. We discovered that SOD1 ^G93A mice injected with the Ad5-VEGF + Ad5-ANG combination showed a 2–3 week delay in manifestation of the disease, higher motor activity at the advanced stages of the disease, and at least a 10% increase in the lifespan compared to the control and other experimental groups. These results support the safety and therapeutic efficacy of the tested recombinant treatment. We propose that the developed experimental MND treatment based on viral delivery of VEGF + AGF can be used as a basis for gene therapy drug development and testing in the preclinical and clinical trials of the MND.     

THE EFFECT OF SPIKE ACTIVITY VERSUS SYNAPTIC ACTIVATION ON THE METABOLISM OF RIBONUCLEIC ACID IN A MOLLUSCAN GIANT NEURONE

—Previous experiments on a giant neurone (R2) from Aplysia californica have shown that a prolonged electrical stimulation of ganglionic nerves, strong enough to elicit post-synaptic spikes in the giant neurone, caused a marked increase in the uptake of labelled nucleosides into the neuronal RNA. The results described in the present paper very strongly indicate that these effects of synaptic activation were not due to the discharge of spikes in the giant neurone itself. Spikes which were directly elicited in the giant neurone by current pulses injected into the cell through an intracellular microelectrode had no significant effect on RNA labelling. Weak stimulation of ganglionic nerves, eliciting post-synaptic potentials but few spikes in the giant neurone, produced a small but significant increase of RNA labelling.     

Diagnostic and prognostic value of CSF neurofilaments in a cohort of patients with motor neuron disease: A cross-sectional study

Motor neuron disease (MND) is a rare group of disorders characterized by degeneration of motor neurons (MNs). The most common form of MND, amyotrophic lateral sclerosis (ALS), is an incurable disease with a variable rate of progression. The search of robust biomarkers able to discriminate among different ALS forms is paramount to properly stratify patients, and to identify those who could most likely benefit from experimental therapies. Phosphorylated-neurofilament heavy chain (p-NfH) and neurofilament light chain (NfL) are neuron-specific components of the cytoskeleton and may represent reliable markers of neuronal injury in neurological disorders. In this study, we described our cohort of ALS patients in order to investigate whether and how cerebrospinal fluid (CSF) p-NfH and NfL levels may reflect progression rate, MN involvement and the extent of neurodegeneration. CSF p-NfH and NfL were significantly increased in ALS compared with healthy and disease controls, including patients with other forms of MND, and were higher in patients with more aggressive disease course, reflecting progression rate. We also evaluated neurofilament diagnostic accuracy in our centre, identifying with high sensitivity and 100% specificity cut-off values of 0.652 ng/mL for CSF p-NfH (P < .0001) and of 1261 pg/mL for NfL (P < .0001) in discriminating ALS from healthy controls. CSF neurofilaments were significantly correlated with ALS progression rate. Overall, CSF neurofilaments appear to reflect the burden of neurodegeneration in MND and represent reliable diagnostic and prognostic biomarkers in ALS.     

Analysis of the possible involvement of the glutamate transporter gene EAAT2 and the glutamate receptor genes GRIA1 and GRIA2 in the pathogenesis of motor neuron disease in the Russian population

Polymorphisms of the genes of the glutamatergic system EAAT2, GRIA1, and GRIA2 have been analyzed in patients with sporadic motor neuron disease (MND) from Russia. The disease is not associated with polymorphic alleles of the genes studied, which indicates that EAAT2, GRIA1, and GRIA2 play an insignificant role in the pathogenesis of sporadic MND.     

Neuropathy target esterase gene mutations cause motor neuron disease

The possibility that organophosphorus (OP) compounds contribute to motor neuron disease (MND) is supported by association of paraoxonase 1 polymorphisms with amyotrophic lateral sclerosis (ALS) and the occurrence of MND in OP compound-induced delayed neuropathy (OPIDN), in which neuropathy target esterase (NTE) is inhibited by organophosphorylation. We evaluated a consanguineous kindred and a genetically unrelated nonconsanguineous kindred in which affected subjects exhibited progressive spastic paraplegia and distal muscle wasting. Affected subjects resembled those with OPIDN and those with Troyer Syndrome due to SPG20/spartin gene mutation (excluded by genetic linkage and SPG20/spartin sequence analysis). Genome-wide analysis suggested linkage to a 22 cM homozygous locus (D19S565 to D19S884, maximum multipoint LOD score 3.28) on chromosome 19p13 to which NTE had been mapped (GenBank AJ004832). NTE was a candidate because of its role in OPIDN and the similarity of our patients to those with OPIDN. Affected subjects in the consanguineous kindred were homozygous for disease-specific NTE mutation c.3034A-->G that disrupted an interspecies conserved residue (M1012V) in NTE's catalytic domain. Affected subjects in the nonconsanguineous family were compound heterozygotes: one allele had c.2669G-->A mutation, which disrupts an interspecies conserved residue in NTE's catalytic domain (R890H), and the other allele had an insertion (c.2946_2947insCAGC) causing frameshift and protein truncation (p.S982fs1019). Disease-specific, nonconserved NTE mutations in unrelated MND patients indicates NTE's importance in maintaining axonal integrity, raises the possibility that NTE pathway disturbances contribute to other MNDs including ALS, and supports the role of NTE abnormalities in axonopathy produced by neuropathic OP compounds.