Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1

Analysis of transgenic mice expressing familial amyotrophic lateral sclerosis (ALS)-linked mutations in the enzyme superoxide dismutase (SOD1) have shown that motor neuron death arises from a mutant-mediated toxic property or properties. In testing the disease mechanism, both elimination and elevation of wild-type SOD1 were found to have no effect on mutant-mediated disease, which demonstrates that the use of SOD mimetics is unlikely to be an effective therapy and raises the question of whether toxicity arises from superoxide-mediated oxidative stress. Aggregates containing SOD1 were common to disease caused by different mutants, implying that coaggregation of an unidentified essential component or components or aberrant catalysis by misfolded mutants underlies a portion of mutant-mediated toxicity.     

Protective effect of neurofilament heavy gene overexpression in motor neuron disease induced by mutant superoxide dismutase

To investigate the role of neurofilaments in motor neuron disease caused by superoxide dismutase (SOD1) mutations, transgenic mice expressing a amyotrophic lateral sclerosis-linked SOD1 mutant (SOD1(G37R)) were mated with transgenic mice expressing human neurofilament heavy (NF-H) subunits. Unexpectedly, expression of human NF-H transgenes increased by up to 65%, the mean lifespan of SOD1(G37R) mice. Microscopic examination corroborated the protective effect of NF-H protein against SOD1 toxicity. Although massive neurodegeneration occurred in 1-yr-old mice expressing SOD1(G37R) alone, spinal root axons and motor neurons were remarkably spared in doubly SOD1(G37R);NF-H-transgenic littermates.     

Oxidative stress and motor neurone disease

The effects of oxidative stress within post mitotic cells such as neurones may be cumulative, and injury by free radical species is a major potential cause of the age-related deterioration in neuronal function seen in several neurodegenerative diseases. There is strong evidence that oxidative stress plays an important role in the pathogenesis of motor neurone disease (MND). Point mutations in the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) are found in some pedigrees with the familial form of MND. How mutations in this ubiquitous enzyme cause the relatively selective cell death of specific groups of motor neurones is not clear, although a number of hypotheses have been forwarded. These include (1) the formation of hydroxyl radicals, (2) the catalysis of reactions of the nitrogen centred oxidant species peroxynitrite, (3) toxicity of copper or zinc and (4) protein aggregation. Some experimental support for these different hypotheses has been produced by manipulating cells in culture to express the mutant SOD1 proteins and by generating transgenic mice which over-express mutant SOD1. Observations in these model systems are, in some cases at least, supported by observations made on pathological material from patients with similar SOD1 mutations. Furthermore, there are reports of evidence of free radical mediated damage to neurones in the sporadic form of MND. Several lines of evidence suggest that alterations in the glutamatergic neurotransmitter system may also play a key role in the injury to motor neurones in sporadic MND. There are several important subcellular targets, which may be preferentially impaired within motor neurones, including neurofilament proteins and mitochondria. Future research will need to identify the aspects of the molecular and physiological phenotype of human motor neurones that makes them susceptible to degeneration in MND, and to identify those genetic and environmental factors which combine to cause this disease in individuals and in familial pedigrees.     

The reorganization of motor units in motor neuron disease

We studied 78 patients with motor neuron disease (MND) using concentric needle electromyography. Analysis on weak and maximal effort was performed using our own, fully automated, computer method, EMG-LAB. In addition to the conventional parameters of single motor unit action potentials (MUAPs) and interference pattern, new criteria were applied: the range of the acting motor units and the functional recruitment order. A total of 375 muscles of MND patients and 120 control muscles were investigated. The electromyographic data were analyzed separately in five groups of muscles, classified A, B, C, D, and E according to their clinical condition. Those results allowed us to discern six neurophysiological stages (N(0,1,2,3,4,5)) from the early to the most advanced phase. It has been confirmed that reinnervation in MND is adequate to compensate for the loss of over 50% of motor neurons but it is only a transitory phase in the morbid course. At stages N(O-5), the electrophysiological data reflect structural and functional integrity of the functioning motor units. Evaluation of not only single MUAPs but also of the full range of acting motor units and their recruitment order allowed a deeper look into the underlying pathophysiological mechanisms.     

Antibody panels in idiopathic polyneuropathy and motor neuron disease

We prospectively evaluated patients with idiopathic polyneuropathy (PN) and motor neuron disease (MND) with commercial antibody (Ab) panels. Patients with sensorimotor PN received a "sensorimotor neuropathy profile" [3-sulfated glucuronyl paragloboside (SGPG)/myelin-associated glycoprotein (MAG), GM1, asialo-GM1, GD1b, Hu, sulfatide]. Motor neuropathy or MND patients underwent a "motor neuropathy profile" (SGPG/MAG, GM1, asialo-GM1). Seven of 78 patients (9.0%) with sensorimotor PN and 3 of 44 patients (6.8%) with MND had abnormal panels. None of 60 patients with axonal sensory or sensorimotor PN had antisulfatide Ab. Seven of 13 patients (54%) with multifocal motor neuropathy had abnormal panels, with 6 seropositive to GM1. We found abnormal Ab panels in fewer than 10% of patients with idiopathic sensorimotor PN and MND. Moreover, abnormal Ab tests often did not relate to the clinical context. Our data do not support the use of commercial Ab panels in the evaluation of patients with idiopathic PN or MND.     

Progressive neuronopathy in transgenic mice expressing the human neurofilament heavy gene: a mouse model of amyotrophic lateral sclerosis

Equatorial regions of chicken intrafusal fibres were examined with a panel of monoclonal antibodies against intracellular proteins and components of extracellular matrix to identify structural associations at points of contact between sensory terminals and intrafusal fibres, and at points which lacked them. One aspect of this study was to establish whether the known morphological differences between myosensory and neuromuscular junctions also extended to the molecular level. As viewed in cross-sections, myosensory junctions at the equator are restricted to approximately one-half of the intrafusal fibre circumference, a region referred to as the sensory sector. The diametrically opposite region which lacks sensory terminals is referred to as the non-sensory sector. The basal lamina over the sensory sector was positive for chondroitin sulphate, while that part which covered the non-sensory sector was negative. Staining for collagen type IV was very faint at the sensory sector and stronger at the non-sensory sector, but immunoreactivity for heparan sulphate proteoglycan and laminin was moderate to strong in all parts of the basal lamina. Within intrafusal fibres, filamin and alpha-actinin were largely limited to the sensory sector. The major feature of the non-sensory sector was a sharply delineated, narrow intrafibre crescent of vinculin, and colocalized with it, a crescent of talin. The plasmalemma of intrafusal fibres at the non-sensory sector reacted positively for the beta 1 subunit of the integrin family of receptors. Immunolocalization of these receptors was not observed to any significant extent in the sensory sector. Towards the end of the equator and the initial portion of the juxtaequator, chondroitin sulphate, vinculin and the other proteins came gradually to be distributed equally all the way round the intrafusal fibres. This changeover in distribution of connective tissue proteins and structural intracellular proteins parallels the decreasing number of contacts made by sensory terminals.     

Differential features of motor neuron disease mortality in Spain

BACKGROUND: The objective of this study was to describe the temporal and spatial patterns of motor neuron disease (MND) in Spain. METHODS: We studied data where MND was stated as the principal cause of death in official statistics from Spain. Time trends were analysed for age-, sex-specific and age-adjusted rates for the period 1951-1990. Age-adjusted mortality and relative risk, obtained by Poisson regression adjusting for age, were calculated for each province from deaths during the period 1975-1988. Maps were constructed using log transformed rates. Statistical significance of spatial aggregation was assessed using the Ohno et al. test. RESULTS: The 1951-1990 mortality rate, age- and sex-adjusted to the European population, for the population aged > or = 40 years was 1.49 per 100,000; 1.90 and 1.21 for males and females respectively. In general, mortality increased with age. Age-adjusted rates rose until 1960, dropped by 70% during the 1960s and declined slightly over the 1951-1990 period as a whole. From 1970 onwards MND mortality rose evenly, particularly in the 60-69 age group. A North-South gradient was suggested for both sexes with statistically significant clustering in the Northern coastal regions and--for males alone--in the Midwest provinces. CONCLUSIONS: Mortality from MND in Spain displayed a magnitude and recently rising temporal trend similar to that described in several other countries. Specific traits were: a decrease during the 1960s, which has been described for Japan only, as well as spatial heterogeneity and a predominant recent increase among the 60-69 age group. The determinants of these unusual MND mortality patterns are unknown.     

Kinesin and cytoplasmic dynein in spinal spheroids with motor neuron disease

OBJECTIVE: Gene therapy is a promising strategy to modify ischemia-reperfusion injury and rejection after transplantation. We evaluated variables that may affect ex vivo gene transfer to rat lung isografts. METHODS: Left lungs were harvested and perfused via the pulmonary vein with chloramphenicol acetyltransferase complementary deoxyribonucleic acid complexed with cationic liposomes. Several variables were examined: (1) Influence of temperature: In group I (n = 4), grafts were stored for 4 hours at 23 degrees C and transplanted. Chloramphenicol acetyltransferase activity was assessed on postoperative day 2. In groups II and III (n = 4), grafts were stored at 10 degrees and 4 degrees C, respectively. Arterial oxygen tension and inflammatory infiltrate were also determined. (2) Influence of storage time: Grafts were preserved at 10 degrees C for 1, 2, 3, 4 (n = 4), and 10 hours (n = 5). chloramphenicol acetyltransferase activity was assessed on postoperative day 2. (3) Rapidity and duration of transgene expression: Grafts were preserved at 10 degrees C for 1 hour and then transplanted. Chloramphenicol acetyltransferase activity was assessed 2, 4, 6, 12, and 24 hours and 2, 7, 14, 21, and 28 days after implantation. RESULTS: Chloramphenicol acetyltransferase expression was apparently less in lungs transfected at 4 degrees C than in those transfected at 10 degrees and 23 degrees C. Storage for 1 hour at 10 degrees C was sufficient to yield significant expression. Increasing the exposure time to 10 hours did not increase toxicity. There were no differences in arterial oxygen tension between transfected and nontransfected lungs. Chloramphenicol acetyltransferase expression was detected for at least 28 days. CONCLUSION: Ex vivo liposome-mediated transfection of lung isografts can be achieved after a short time of cold storage, with minimal toxicity.     

Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1

Amyotrophic lateral sclerosis (ALS) involves motor neuron degeneration, skeletal muscle atrophy, paralysis, and death. Mutations in Cu,Zn superoxide dismutase (SOD1) are one cause of the disease. Mice transgenic for mutated SOD1 develop symptoms and pathology similar to those in human ALS. To understand the disease mechanism, we developed a simple behavioral assay for disease progression in mice. Using this assay, we defined four stages of the disease in mice expressing G93A mutant SOD1. By studying mice with defined disease stages, we tied several pathological features into a coherent sequence of events leading to motor neuron death. We show that onset of the disease involves a sharp decline of muscle strength and a transient explosive increase in vacuoles derived from degenerating mitochondria, but little motor neuron death. Most motor neurons do not die until the terminal stage, approximately 9 weeks after disease onset. These results indicate that mutant SOD1 toxicity is mediated by damage to mitochondria in motor neurons, and this damage triggers the functional decline of motor neurons and the clinical onset of ALS. The absence of massive motor neuron death at the early stages of the disease indicates that the majority of motor neurons could be rescued after clinical diagnosis.     

Organization and development of facial motor neurons in the kreisler mutant mouse

The adult facial nerve contains the axons from two populations of efferent neurons. First, the branchiomotor efferent neurons that innervate the muscles of the second arch. These neurons project out of the hindbrain in the motor root and form the facial motor nuclei. Second, the preganglionic efferent neurons that innervate the submandibular and pterygopalatine ganglia. These neurons project from the hindbrain via the intermediate nerve and form the superior salivatory nucleus. The motor neurons of the facial nerve are known to originate within rhombomeres 4 and 5. In the kreisler mouse mutant there is a specific disruption of the hindbrain rhombomeres 5 and 6 appear to be absent. To investigate changes in the organization of the facial motor neurons in this mutant, we have used lipophilic dyes to trace the facial motor components both retrogradely and anterogradely. As expected, facial motor neurons are missing from rhombomere 5 in this mutant. In addition, the loss of these neurons correlates with the specific loss of the superior salivatory nucleus. In contrast, the branchiomeric neurons, that originate in rhombomere 4, appear to develop normally. This includes the caudal migration of their cell bodies forming the genu of the facial nerve. Our studies confirm that rhombomeres are critical to hindbrain development and that they are the fundamental unit at which motor neurons are specified.     

Oxidative stress and mitochondrial DNA mutations in human aging

The effects of chemical sympathectomy on the mucosal compartments of the immune system were examined in adult rats. Ablation of the sympathetic nervous system using 6-hydroxydopamine in recipient animals reduced the migration into Peyer's patches and mesenteric lymph nodes (MLN) of adoptively transferred cells from MLN of normal donors. The mucosal immune response to ovalbumin (OVA), assessed by enumeration of anti-OVA antibody containing cells (AOCC) in the lamina propria after intestinal immunisation, was reduced in animals sympathectomized prior to immunization. In order to identify whether this reduction in AOCC response in intestinally immunized sympathectomized animals was due to a defect in migration of AOCC precursors to the intestinal lamina propria, the effect of chemical sympathectomy on the appearance of AOCC in the gut of immunized animals after adoptive transfer of AOCC precursors was investigated. The IgA-specific AOCC response was significantly reduced in sympathectomized recipients compared to the control group. Taken together these results demonstrate that the peripheral sympathetic nervous system influences the migration and accumulation in vivo of both naive and memory/effector lymphocytes in mucosal lymphoid tissues.     

Arrest of motor neuron disease in wobbler mice cotreated with CNTF and BDNF

Ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) each promote the survival and differentiation of developing motor neurons, but do so through distinct cellular signaling pathways. Administration of either factor alone has been shown to slow, but not to arrest, progression of motor neuron dysfunction in wobbler mice, an animal model of motor neuron disease. Because CNTF and BDNF are known to synergize in vitro and in ovo, the efficacy of CNTF and BDNF cotreatment was tested in the same animal mode. Subcutaneous injection of the two factors on alternate days was found to arrest disease progression in wobbler mice for 1 month, as measured by several behavioral, physiological, and histological criteria.     

A heart segmental defect in the anterior-posterior axis of a transgenic mutant mouse

A recessive lethal insertional mutation on chromosome 13 has been identified in a transgenic mouse line that displays a segmental form of cardiac defect along the anterior-posterior axis in all homozygous mice identified. The most anterior segment (future conus and right ventricle) of the single heart tube fails to develop normally and the endocardial cushions in both the conus and the atrioventricular regions are missing. Analysis of the beta-galactosidase reporter portion of the transgene during embryonic development shows a segmental expression of activity primarily in the defective outlet of the primitive heart. In addition to expression in the heart tube, hemizygous embryos show transgene expression in the chondrogenic regions of first and second branchial arches, the appendicular skeleton, and the dermal papillae of the vibrissae. The restricted pattern of beta-galactosidase expression in the heart can be disrupted with retinoic acid exposure and extended posteriorly along the anterior-posterior axis in hemizygous mice. Although cushion mesenchyme fail to form in the homozygous mutant, the myocardial and endothelial cells explanted from the mutant atrioventricular, but not the conus, are capable of forming mesenchyme in vitro. Mice trisomic for chromosome 13 have also been shown to display segmental anomalies associated with the anterior primitive outlet segments of the heart. Our data show that this insertional mutation identifies a new gene locus, hdf (heart defect), on mouse chromosome 13 that may be required for mechanisms that initially establish and/or maintain continued development of the anterior limb of the developing heart. The hdf mouse mutation also provides a new model system to evaluate the molecular requirements of normal endocardial cushion formation and the segmental interactions that form the adult heart.     

Root stimulation studies in the evaluation of patients with motor neuron disease

Nerve root stimulation may be employed in patients with motor neuron disease (MND) to rule out motor neuropathy with conduction block. The diagnostic utility of these studies is unknown, in part because the range of amplitude changes across nerve root segments in patients with active neuronal degeneration has not been well studied. We reviewed root stimulation studies in 32 patients (59 nerves) with MND and found segmental amplitude reduction from 0 to 45%, a range similar to values reported for normal subjects; there was no suggestion of conduction block based on our usual criteria.     

Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes-survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.     

Geographical epidemiology of residence of patients with motor neuron disease in Lancashire and south Cumbria

OBJECTIVES: To seek objective evidence for geographical clustering of places of residence of patients with motor neuron disease (MND). METHODS: A complete residential history from birth to onset of disease was obtained from a cohort of 130 patients with MND from Lancashire and south Cumbria presenting to the Department of Neurology in Preston between 1 January 1989 and 31 December 1993. These data were compared with population based reference data from the 1991 UK Census. RESULTS: Some areal units showed a greater, others a lesser, number of MND patient residences than expected. The results suggest that the background population incidence of MND is relatively low and that the overall incidence figures previously quoted have been skewed upwards by areas in which the incidence of MND is relatively increased. These findings were further tested by Poisson modelling. The Poisson model provided a poor fit for the data at postcode district and sector levels confirming that patients with MND were significantly more likely to have lived in some areas than others after allowing for variation in population of the different areal units and for variation in duration of residence. CONCLUSIONS: These findings reinforce the results of previous work, much of which has been qualitative rather than quantitative. The results presented here suggest a low background incidence of MND in the context of generally quoted overall incidence figures. This low background incidence is, however, skewed upwards by some areal units with a relatively high incidence, thus achieving overall incidence rates comparable with generally quoted figures. We conclude that there is prima facie evidence of spatial patterns in the distribution of places of residence of patients with MND. Further examination of occupational and environmental factors in the lives of the patients with MND is required to obtain a better understanding of the importance of these findings.     

CNS tissue Cu/Zn superoxide dismutase (SOD1) mutations in motor neurone disease (MND)

DNA extracted from CNS tissue of 79 cases of motor neurone disease (MND) was screened by single strand conformation analysis (SSCA) and heteroduplex analysis (HA) for mutations in the Cu/Zn superoxide dismutase (SOD1) gene. The aims were to determine whether somatic mutations of SOD1 may underlie some cases of MND and to characterize the genetic abnormalities by sequencing, for subsequent correlation with the molecular pathological phenotype. In 3 cases a point mutation was found in exon 4: E100G in one familial case, and I113T in two cases (one familial, one sporadic). Two cases had previously undescribed mutations in the 3' untranslated region (3'UTR) of SOD1 and one case had a single base substitution in the intronic sequence upstream from exon 2. None of these patients had a positive family history. Non-CNS tissue was available for 3 out of the 6 cases in whom changes were found. In all 3 the same changes were consistently found in both CNS and non-CNS tissue, excluding the presence of somatic mutations in SOD1. We investigated many MND blood samples and normal controls for the presence of the 3'UTR deletions. We found the 4 bp deletion in 1/90 sporadic MND patients and 1/209 non-MND controls. If the 3'UTR deletions are pathogenic, they would have to operate via a loss of the function mechanism, and further work is necessary to define their significance.     

Angiogenesis and apoptosis are cellular parameters of neoplastic progression in transgenic mouse models of tumorigenesis

The epidemiology and histopathology of human cancers and studies of animal models of tumorigenesis have led to a widely-accepted notion that multiple genetic and epigenetic changes have to accrue for the successful development of a malignant phenotype. Tumor growth and expansion requires an ability not only to proliferate, but also to down-modulate cell death (apoptosis) and activate angiogenesis to produce a tumor neovasculature. This review will describe the interplay between apoptosis and proliferation, as well as the characteristics of the angiogenic phenotype in two transgenic mouse models of multi-step tumorigenesis, namely, pancreatic islet cell carcinomas and squamous cell carcinomas of the skin.     

Association between plasma vitamin E concentration and the risk of equine motor neuron disease

Venous ulceration is a common problem in western countries and results in large costs to healthcare systems. A number of hypotheses of the mechanisms of development of venous ulceration have been advanced, but this question has not been fully resolved. In recent years research effort has focused on the microcirculation of the skin and many methods of investigation have been employed to study this. Some of the principal findings described in published work are reviewed in this article. It seems unlikely from the available evidence that venous ulceration is attributable solely to failure of diffusion of oxygen and other small nutritional molecules to the tissues of the skin. The microvascular changes in the skin are characterised by activated endothelium and perivascular inflammatory cells. It is much more likely that leucocytes attach themselves to the cutaneous microcirculation, become activated and produce endothelial injury. Repeated over many months or years, this chronic inflammatory process leads to be tissues changes of lipodermatosclerosis. Although there is evidence of leucocyte involvement in the pathogenesis of venous ulceration, the exact mechanisms remain to be resolved. Improved treatment for patients may be devised once a better understanding of the basic causes of this condition has been reached.