Neurofilaments and motor neuron disease

Amyotrophic lateral sclerosis (ALS) is an adult-onset and heterogeneous neurological disorder that affects primarily motor neurons in the brain and spinal cord. Although multiple genetic and environmental factors might be implicated in ALS, the striking similarities in the clinical and pathological features of sporadic ALS and familial ALS suggest that similar mechanisms of disease may occur. A common and perhaps universal pathological finding in ALS is the presence of abnormal accumulations of neurofilaments (often called spheroids or Lewy body-like deposits) in the cell body and proximal axon of surviving motor neurons. Such neurofilament deposits have been widely viewed as a consequence of neuronal dysfunction, perhaps reflecting axonal transport defects. This review discusses the emerging evidence, based primarily on transgenic mouse studies and on the discovery of deletion mutations in a neurofilament gene associated with ALS, that neurofilament proteins can play a causative role in motor neuron disease.     

Colivelin prolongs survival of an ALS model mouse

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease for which there is no sufficiently effective therapy. We have reported in our earlier study that intracerebroventricular (i.c.v.) injection of activity-dependent neurotrophic factor (ADNF) improves motor performance of G93A-SOD1 transgenic mice without significant prolongation in survival. Here, we found that i.c.v. injection of a synthetic hybrid peptide named Colivelin composed of ADNF and AGA-(C8R)HNG17, a potent derivative of Humanin that is a bioactive peptide with anti-Alzheimer's disease activity, dose-dependently improved motor performance and prolonged survival of ALS mice. Histological analysis, performed at the age of 120 days, demonstrated increased motoneuronal survival in spinal cords of Colivelin-treated mice as compared with saline- or ADNF-treated mice, indicating that Colivelin is a promising neurotrophic peptide for treatment of ALS.     

Motoneurons are altered in muscular dystrophy.

The activity of motoneurons supplying the brachial biceps muscle was examined in eight control subjects and 26 patients affected by Duchenne muscular dystrophy. The patients were subdivided into two groups: one whose motor units (MU) fired with normal rates (N group) and the other whose MU firing rates were higher as compared to controls (I group). Firing rates of motoneurons of patients from group I increased more rapidly with increasing force level. The relationship between the standard deviation of interspike intervals and their mean value, sigma(Tm), was shifted towards the shorter intervals and lower standard deviations in both groups of patients. The numerical values describing these changes correlated with the severity of disease. The MU recruitment was comparable for control subjects and for patients. Experimental results as well as computer simulations indicate that the break-point of the function sigma(Tm) is correlated with motoneuronal properties, and in particular with the afterhyperpolarization (AHP) duration. In muscular dystrophy this break-point corresponds to the shorter interspike intervals. Therefore, we propose that the motoneurons in muscular dystrophy are altered either in response to the muscle degeneration or as a result of the disease itself.     

Metals in motor neuron diseases

Degenerative processes within the nervous system are common features in disease entities such as dementia of Alzheimer type (DAT), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease with unknown etiology; widespread muscle wasting and respiratory failure lead to death within a few years. Denervation can be detected with electromyography and axonal deterioration monitored by motor unit number estimates. Several suggestions about the cause of ALS have emerged but no solid theory has yet precipitated. Lead or mercury exposure has been suggested. Exposure data alone cannot support this connection. Alterations in metal kinetics may underlie the deterioration of motor function observed in patients with ALS. In this review the role of metals in motor neuron disease is discussed. Both classic studies on exposure and recent understanding of metal binding proteins are considered. Aspects of peak exposure and excretion are merged toward an understanding of metal dynamics in ALS. An overview of chemical and electrophysiological investigations is given in the context of neurodegeneration.     

Pathways to motor neuron degeneration in transgenic mouse models

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disorder characterized by the selective loss of motor neurons. A pathological hallmark of both sporadic and familial ALS is the presence of abnormal accumulations of neurofilament and peripherin proteins in motor neurons. In the past decade, transgenic mouse approaches have been used to address the role of such cytoskeletal abnormalities in motor neuron disease and also to unravel the pathogenesis caused by mutations in the gene coding for superoxide dismutase 1 (SOD1) that account for ~20% of familial ALS cases. In mouse models, disparate effects could result from different types of intermediate filament (IF) aggregates. Perikaryal IF accumulations induced by the overexpression of any of the three wild-type neurofilament proteins were quite well tolerated by motor neurons. Indeed, perikaryal swellings provoked by NF-H overexpression can even confer protection against toxicity of mutant SOD1. Other types of IF aggregates seem neurotoxic, such as those found in transgenic mice overexpressing either peripherin or an assembly-disrupting NF-L mutant. Moreover, understanding the toxicity of SOD1 mutations has been surprisingly difficult. The analysis of transgenic mice expressing mutant SOD1 has yielded complex results, suggesting that multiple pathways may contribute to disease that include the involvement of non-neuronal cells.     

A model for steady isometric muscle activation

The present model of the motoneuronal (MN) pool – muscle complex (MNPMC) is deterministic and designed for steady isometric muscle activation. Time-dependent quantities are treated as time-averages. The character of the model is continuous in the sense that the motor unit (MU) population is described by a continuous density function. In contrast to most already published models, the wiring (synaptic weight) between the input fibers to the MNPMC and the MNs (about which no detailed data are known) is deduced, whereas the input–force relation is given. As suggested by experimental data, this relation is assumed to be linear during MU recruitment, but the model allows other, nonlinear relations. The input to the MN pool is defined as the number of action potentials per second in all input fibers, and the excitatory postsynaptic potential (EPSP) conductance in MNs evoked by the input is assumed to be proportional to the input. A single compartment model with a homogeneous membrane is used for a MN. The MNs start firing after passing a constant voltage threshold. The synaptic current–frequency relation is described by a linear function and the frequency–force transformation of a MU by an exponential function. The sum of the MU contraction forces is the muscle force, and the activation of the MUs obeys the size principle. The model parameters were determined a priori, i.e., the model was not used for their estimation. The analysis of the model reveals special features of the activation curve which we define as the relation between the input normalized by the threshold input of the MN pool and the force normalized by the maximal muscle force. This curve for any muscle turned out to be completely determined by the activation factor, the slope of the linear part of the activation curve (during MU recruitment). This factor determines quantitatively the relation between MU recruitment and rate modulation. This property of the model (the only known model with this property) allows a quantification of the recruitment gain (Kernell and Hultborn 1990). The interest of the activation factor is illustrated using two human muscles, namely the first dorsal interosseus muscle, a small muscle with a relatively small force at the end of recruitment, and the medial gastrocnemius muscle, a strong muscle with a relatively large force at the end of recruitment. It is concluded that the present model allows us to reproduce the main features of muscle activation in the steady state. Its analytical character facilitates a deeper understanding of these features. Received: 24 November 1997 / Accepted in revised form: 30 November 1998     

G93A SOD1 alters cell cycle in a cellular model of Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative multifactorial disease characterized, like other diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) or frontotemporal dementia (FTD), by the degeneration of specific neuronal cell populations. Motor neuron loss is distinctive of ALS. However, the causes of onset and progression of motor neuron death are still largely unknown. In about 2% of all cases, mutations in the gene encoding for the Cu/Zn superoxide dismutase (SOD1) are implicated in the disease. Several alterations in the expression or activation of cell cycle proteins have been described in the neurodegenerative diseases and related to cell death. In this work we show that mutant SOD1 can alter cell cycle in a cellular model of ALS. Our findings suggest that modifications in the cell cycle progression could be due to an increased interaction between mutant G93A SOD1 and Bcl-2 through the cyclins regulator p27. As previously described in post mitotic neurons, cell cycle alterations could fatally lead to cell death.     

Molecular biology of amyotrophic lateral sclerosis: insights from genetics

Amyotrophic lateral sclerosis (ALS) is a paralytic disorder caused by motor neuron degeneration. Mutations in more than 50 human genes cause diverse types of motor neuron pathology. Moreover, defects in five Mendelian genes lead to motor neuron disease, with two mutations reproducing the ALS phenotype. Analyses of these genetic effects have generated new insights into the diverse molecular pathways involved in ALS pathogenesis. Here, we present an overview of the mechanisms for motor neuron death and of the role of non-neuronal cells in ALS.     

4-hydroxynonenal and neurodegenerative diseases

The development of oxidative stress, in which production of highly reactive oxygen species (ROS) overwhelms antioxidant defenses, is a feature of many neurological diseases: ischemic, inflammatory, metabolic and degenerative. Oxidative stress is increasingly implicated in a number of neurodegenerative disorders characterized by abnormal filament accumulation or deposition of abnormal forms of specific proteins in affected neurons, like Alzheimer's disease (AD), Pick's disease, Lewy bodies related diseases, amyotrophic lateral sclerosis (ALS), and Huntington disease. Causes of neuronal death in neurodegenerative diseases are multifactorial. In some familiar cases of ALS mutation in the gene for Cu/Zn superoxide dismutase (SOD1) can be identified. In other neurodegenerative diseases ROS have some, usually not clear, role in early pathogenesis or implications on neuronal death in advanced stages of illness. The effects of oxidative stress on "post-mitotic cells", such as neurons may be cumulative, hence, it is often unclear whether oxidative damage is a cause or consequence of neurodegeneration. Peroxidation of cellular membrane lipids, or circulating lipoprotein molecules generates highly reactive aldehydes among which one of most important is 4-hydroxynonenal (HNE). The presence of HNE is increased in brain tissue and cerebrospinal fluid of AD patients, and in spinal cord of ALS patients. Immunohistochemical studies show presence of HNE in neurofibrilary tangles and in senile plaques in AD, in the cytoplasm of the residual motor neurons in sporadic ALS, in Lewy bodies in neocortical and brain stem neurons in Parkinson's disease (PD) and in diffuse Lewy bodies disease (DLBD). Thus, increased levels of HNE in neurodegenerative disorders and immunohistochemical distribution of HNE in brain tissue indicate pathophysiological role of oxidative stress in these diseases, and especially HNE in formation of abnormal filament deposites.     

VEGF: once regarded as a specific angiogenic factor, now implicated in neuroprotection

Both blood vessels and nerves are guided to their target. Vascular endothelial growth factor (VEGF)A is a key signal in the induction of vessel growth (a process termed angiogenesis). Though initial studies, now a decade ago, indicated that VEGF is an endothelial cell-specific factor, more recent findings revealed that VEGF also has direct effects on neural cells. Genetic studies showed that mice with reduced VEGF levels develop adult-onset motor neuron degeneration, reminiscent of the human neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Additional genetic studies confirmed that VEGF is a modifier of motor neuron degeneration in humans and in SOD1(G93A) mice--a model of ALS. Reduced VEGF levels may promote motor neuron degeneration by limiting neural tissue perfusion and VEGF-dependent neuroprotection. VEGF also affects neuron death after acute spinal cord or cerebral ischemia, and has also been implicated in other neurological disorders such as diabetic and ischemic neuropathy, nerve regeneration, Parkinson's disease, Alzheimer's disease and multiple sclerosis. These findings have raised growing interest in assessing the therapeutic potential of VEGF for neurodegenerative disorders.     

Genetics of amyotrophic lateral sclerosis: an update

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder involving both upper motor neurons (UMN) and lower motor neurons (LMN). Enormous research has been done in the past few decades in unveiling the genetics of ALS, successfully identifying at least fifteen candidate genes associated with familial and sporadic ALS. Numerous studies attempting to define the pathogenesis of ALS have identified several plausible determinants and molecular pathways leading to motor neuron degeneration, which include oxidative stress, glutamate excitotoxicity, apoptosis, abnormal neurofilament function, protein misfolding and subsequent aggregation, impairment of RNA processing, defects in axonal transport, changes in endosomal trafficking, increased inflammation, and mitochondrial dysfunction. This review is to update the recent discoveries in genetics of ALS, which may provide insight information to help us better understanding of the disease neuropathogenesis.     

Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration

To test the hypothesis that inhibition of axonal transport is sufficient to cause motor neuron degeneration such as that observed in amyotrophic lateral sclerosis (ALS), we engineered a targeted disruption of the dynein-dynactin complex in postnatal motor neurons of transgenic mice. Dynamitin overexpression was found to disassemble dynactin, a required activator of cytoplasmic dynein, resulting in an inhibition of retrograde axonal transport. Mice overexpressing dynamitin demonstrate a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, and denervation of muscle. Previous transgenic mouse models of ALS have shown abnormalities in microtubule-based axonal transport. In this report, we describe a mouse model that confirms the critical role of disrupted axonal transport in the pathogenesis of motor neuron degenerative disease.     

Neuromuscular effects of G93A-SOD1 expression in zebrafish

ABSTRACT: BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal disorder involving the degeneration and loss of motor neurons. The mechanisms of motor neuron loss in ALS are unknown and there are no effective treatments. Defects in the distal axon and at the neuromuscular junction are early events in the disease course, and zebrafish provide a promising in vivo system to examine cellular mechanisms and treatments for these events in ALS pathogenesis. RESULTS: We demonstrate that transient genetic manipulation of zebrafish to express G93A-SOD1, a mutation associated with familial ALS, results in early defects in motor neuron outgrowth and axonal branching. This is consistent with previous reports on motor neuron axonal defects associated with familial ALS genes following knockdown or mutant protein overexpression. We also demonstrate that upregulation of growth factor signaling is capable of rescuing these early defects, validating the potential of the model for therapeutic discovery. We generated stable transgenic zebrafish lines expressing G93A-SOD1 to further characterize the consequences of G93A-SOD1 expression on neuromuscular pathology and disease progression. Behavioral monitoring reveals evidence of motor dysfunction and decreased activity in transgenic ALS zebrafish. Examination of neuromuscular and neuronal pathology throughout the disease course reveals a loss of neuromuscular junctions and alterations in motor neuron innervations patterns with disease progression. Finally, motor neuron cell loss is evident later in the disease. CONCLUSIONS: This sequence of events reflects the stepwise mechanisms of degeneration in ALS, and provides a novel model for mechanistic discovery and therapeutic development for neuromuscular degeneration in ALS.     

The fatigability of two agonistic muscles in human isometric voluntary submaximal contraction: an EMG study. II. Motor unit firing rate and recruitment

The recruitment and firing rate of biceps brachii (BB) and brachioradialis (BR) motor units (MUs) were studied in the course of fatiguing isometric contractions at 20%-30% of maximal voluntary contraction (MVC). MU recruitment generally occurred throughout the maintained contraction and was similar for BB and BR muscles. Newly recruited MUs started to discharge in the form of bursts, the duration of which increased until a continuous rhythmical firing was achieved. Within each burst, the first interval between two consecutive discharges was usually the shortest. MU threshold was lowered just after the limit time of the maintained contraction. The MU's firing rate either increased or remained stable as a function of the elapsed time. It is concluded that (1) in fatiguing isometric contractions at 20%-30% MVC contractile failure is mainly compensated for by MU recruitment and a lowered MU threshold and (2) differences between in surface changes in the electromyogram of BB and BR muscles cannot easily be explained by related differences in MU firing rate and recruitment.     

Autophagy activation in glutamate-induced motor neuron loss

Recent literature demonstrated that exposure to excitatory amino acid in specific experimental conditions might produce a defect in the autophagy pathway. Such an effect was observed in motor neurons exposed chronically to glutamate agonists. On the other hand, it is well known that glutamate induces motor neuron death and this is supposed to play a key role in the physiopathology of motor neuron loss in amyotrophic lateral sclerosis (ALS). Similarly, a defective recruitment of autophagy was recently documented in ALS. In the present study we found that exposure of motor neurons to kainic acid produces intracellular changes associated with defective autophagy. In this experimental conditions, pharmacological activation of autophagy rescues the loss of motor neurons.     

Clinical neurophysiology in ALS

Amyotrophic lateral sclerosis (ALS) belongs to a group of disorders known as motor neuron diseases. Despite being one of the most devastating diseases known, there is little evidence for diagnosing and managing patients with ALS. Clinical neurophysiologic tests are essential, when no biological marker exists to aid early diagnosis, not only in relation to diagnosis, but also in the development of disease progression, and perhaps, in the future, in measuring patients' response to therapy. The electrophysiological features used in the diagnosis of ALS are based on Awaji-shima consensus recommendations for the application of electrophysiological tests, as applied to the revised El Escorial Criteria. Measurements of axonal excitability through nerve conduction study (ENG) is useful to evaluate axonal degeneration. Electromyography (EMG) recordings with needle examination are essential for confirming lower motor neuron involvement in the initial diagnosis of ALS. EMG abnormalities are frequent and these include fibrillation potentials or positive sharp wave potentials, or both, with fasciculation potentials in resting muscle, and an incomplete interference pattern, with abnormal motor unit potentials. Collateral or terminal nerve sprouting is common in ALS and is frequent large macro-motor unit potentials (MUPs). Motor unit number estimation (MUNE) may be useful in measuring loss of functioning motor units and is an attractive endpoint measure in clinical drug trials in ALS because it directly assesses loss of lower motor neurons and is sensitive to disease progression. Transcortical magnetic stimulation protocols, and cortical excitability may be useful to assess the involvement of upper motor neuron system. In this chapter the advantages, limitations and promise of these various methods are discussed, in order to indicate the direction for further neurophysiological studies in this disorder.     

Cytokine expression levels in ALS: A potential link between inflammation and BMAA-triggered protein misfolding

Recently, it has been shown that proinflammatory cytokines play a complex and important role in the pathogenesis of many neurological disorders, including amyotrophic lateral sclerosis (ALS). To help facilitate future discoveries and more effective treatment strategies, we highlight the role that both innate and adaptive immune systems play in ALS and summarize the main observations that relate to cytokine expression levels in this disease. Furthermore, we propose a mechanism by which a known neurotoxin, β-N-methylamino-l-alanine (BMAA), may trigger this cytokine expression profile through motor neuron protein misfolding and subsequent NLRP3 (nucleotide-binding domain (NOD)-like receptor protein 3) inflammasome activation.     

Edaravone,a Free Radical Scavenger,Delayed Symptomatic and Pathological Progression of Motor Neuron Disease in the Wobbler Mouse

Edaravone, a free radical scavenger is used widely in Japanese patients with acute cerebral infarction. This antioxidant could have therapeutic potentials for other neurological diseases. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the upper and the lower motor neuron, leading to death within 3–5 years after onset. A phase III clinical trial of edaravone suggested no significant effects in ALS patients. However, recent 2nd double-blind trial has demonstrated therapeutic benefits of edaravone in definite patients diagnosed by revised El Escorial diagnostic criteria of ALS. Two previous studies showed that edaravone attenuated motor symptoms or motor neuron degeneration in mutant superoxide dismutase 1-transgenic mice or rats, animal models of familial ALS. Herein we examined whether this radical scavenger can retard progression of motor dysfunction and neuropathological changes in wobbler mice, sporadic ALS-like model. After diagnosis of the disease onset at the postnatal age of 3–4 weeks, wobbler mice received edaravone (1 or 10 mg/kg, n = 10/group) or vehicle (n = 10), daily for 4 weeks by intraperitoneal administration. Motor symptoms and neuropathological changes were compared among three groups. Higher dose (10 mg/kg) of edaravone treatment significantly attenuated muscle weakness and contracture in the forelimbs, and suppressed denervation atrophy in the biceps muscle and degeneration in the cervical motor neurons compared to vehicle. Previous and the present studies indicated neuroprotective effects of edaravone in three rodent ALS-like models. This drug seems to be worth performing the clinical trial in ALS patients in the United States of American and Europe, in addition to Japan.     

Galectin-1 is a component of neurofilamentous lesions in sporadic and familial amyotrophic lateral sclerosis

In amyotrophic lateral sclerosis (ALS), abnormal accumulation of neurofilaments induces pathological changes such as axonal spheroids, cord-like neurite swellings, and perikaryal conglomerate inclusions in degenerating motor neurons of the spinal cord, and the accumulation seems to cause motor neuron degeneration in this disease. Such ALS lesions were intensely labeled with HepSS-1, a monoclonal antibody to heparan sulfate. Since the identification of HepSS-1-immunoreactive substance seems to be an important step for understanding the molecular pathology of ALS, we purified the substance from human spinal cord tissue to homogeneity. Amino acid sequence of the protein was consistent with that of galectin-1. Immunohistochemistry using antibodies against recombinant human galectin-1 showed that galectin-1 was accumulated in these lesions in ALS. Although HepSS-1 was believed to be specific for heparan sulfate, it reacted with recombinant human galectin-1 which has no heparan sulfate moiety. The results show that galectin-1 is a component of the neurofilamentous lesions in ALS. Since galectin-1 has axonal regeneration-enhancing activity, the abnormal accumulation of galectin-1 to the lesions seems to be related to the pathological process of ALS.     

Protein clearing pathways in ALS

In the present review a large amount of experimental and clinical studies on ALS are discussed in an effort to dissect common pathogenic mechanisms which may provide novel information and potential therapeutic strategies for motor neuron degeneration.Protein clearing systems play a critical role in motor neuron survival during excitotoxic stress, aging and neurodegenerative disorders. Among various mechanisms which clear proteins from the cell recent studies indicate autophagy as the most prominent pathway to promote survival of motor neurons.Autophagy regulates the clearance of damaged mitochondria, endoplasmic reticulum and misfolded proteins in eukaryotic cells. Upon recruitment of the autophagy pathway, an autophagosome is produced and directed towards lysosomal degradation.Here we provide evidence that in both genetic and sporadic amyotrophic lateral sclerosis (ALS, the most common motor neuron disorder) a defect in the autophagy machinery is common. In fact, swollen, disrupted mitochondria and intracellular protein aggregates accumulate within affected motor neurons. These structures localize within double membrane vacuoles, autophagosomes, which typically cluster in perinuclear position. In keeping with this, when using autophagy inhibitors or suppressing autophagy promoting genes, motor symptoms and motor neuron death are accelerated. Conversely stimulation of autophagy alleviates motor neuron degeneration.Therefore, autophagy represents an important target when developing novel treatments in ALS.