Ebselen as template for stabilization of A4V mutant dimer for motor neuron disease therapy.

Mutations to the gene encoding superoxide dismutase-1 (SOD1) were the first genetic elements discovered that cause motor neuron disease (MND). These mutations result in compromised SOD1 dimer stability, with one of the severest and most common mutations Ala4Val (A4V) displaying a propensity to monomerise and aggregate leading to neuronal death. We show that the clinically used ebselen and related analogues promote thermal stability of A4V SOD1 when binding to Cys111 only. We have developed a A4V SOD1 differential scanning fluorescence-based assay on a C6S mutation background that is effective in assessing suitability of compounds. Crystallographic data show that the selenium atom of these compounds binds covalently to A4V SOD1 at Cys111 at the dimer interface, resulting in stabilisation. This together with chemical amenability for hit expansion of ebselen and its on-target SOD1 pharmacological chaperone activity holds remarkable promise for structure-based therapeutics for MND using ebselen as a template.

A juvenile ALS-like phenotype dramatically improved after high-dose riboflavin treatment.

Riboflavin transporter deficiency (RTD) was recently characterized as a cause of genetic recessive childhood-onset motor neuron disease (MND) with hearing loss, formerly described as Brown-Vialetto-Van-Lear syndrome. We describe a 18-year-old woman with probable RTD mimicking juvenile Amyotrophic Lateral Sclerosis (ALS) who presented with an inaugural respiratory failure and moderate distal four limbs weakness. Only one heterozygous SLC52A3 mutation was detected, but presence of a sub-clinical auditory neuropathy and dramatic improvement under high dose riboflavin argued for a RTD. As RTD probably has a larger phenotypic spectrum than expected, a high dose riboflavin trial should be discussed in young-onset MND.

IqYmune® is an effective maintenance treatment for multifocal motor neuropathy: A randomised, double-blind, multi-center cross-over non-inferiority study vs Kiovig®-The LIME Study.

Intravenous immunoglobulin (IVIg) is the gold-standard for maintenance treatment of multifocal motor neuropathy (MMN). This phase III, randomised, double-blind, multi-centre, active-control, crossover study, aimed to evaluate the non-inferiority of IqYmune® relative to Kiovig®, primarily based on efficacy criteria. Twenty-two adult MMN patients, treated with any brand of IVIg (except Kiovig® or IqYmune®) at a stable maintenance dose within the range of 1 to 2 g/kg every 4 to 8 weeks, were randomised to receive either Kiovig® followed by IqYmune®, or IqYmune® followed by Kiovig®. Each product was administered for 24 weeks. The primary endpoint was the difference between IqYmune® and Kiovig® in mean assessments of modified Medical Research Council (MMRC) 10 sum score (strength of 5 upper-limb and 5 lower-limb muscle groups, on both sides, giving a score from 0 to 100) during the evaluation period (non-inferiority margin of Δ = 2). A linear mixed model analysis demonstrated the non-inferiority of IqYmune® relative to Kiovig®, independently of the covariates (value at baseline, treatment period, and treatment sequence). The estimated "IqYmune® - Kiovig®" difference was -0.01, with a 95% confidence interval (CI) -0.51 to 0.48. The number of adverse reactions (ARs) and the percentage of patients affected were similar for the two products: 39 ARs in 10 patients with IqYmune® vs 32 ARs in 11 patients with Kiovig®. No thromboembolic events nor haemolysis nor renal impairment were observed. In this first clinical trial comparing two IVIg brands for maintenance treatment of MMN, efficacy and tolerability of both brands were similar.

Fishing for causes and cures of motor neuron disorders.

Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development.

Enhancement of BDNF and activated-ERK immunoreactivity in spinal motor neurons after peripheral administration of BDNF.

Brain-derived neurotrophic factor (BDNF) shows neurotrophic effects on adult motor neurons when given systemically, But it is unknown whether systemically administered BDNF is transported to central cell bodies to affect them directly. Here we used immunohistochemistry to investigate the transport of peripherally injected BDNF to spinal motor neurons and the subsequent activation of a signaling pathway. We first injected BDNF into the flexor digitorum brevis (FDB) and analyzed the motor nucleus that projects to the FDB for BDNF immunoreactivity (BDNF-ir) and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 immunoreactivity (pERK1/2-ir). Both immunoreactivities were observed in the motor neuron cell bodies. Next, BDNF was injected subcutaneously (s.c.) into rats with a unilaterally axotomized sciatic nerve. pERK1/2-ir was detected in motor neurons of the lesioned side. BDNF-ir and pERK1/2-ir were also observed on the unlesioned side when a high dose of BDNF was injected. Therefore, we examined BDNF-ir and pERK1/2-ir after injecting BDNF s.c. into normal rats. Both immunoreactivities were observed in motor nuclei on both sides. Finally, we examined pERK1/2-ir after a lower dose of BDNF was injected, which prevents the decrease in choline acetyl transferase that occurs in the motor neuron upon axotomy. Spinal motor nuclei contained a few cell bodies with pERK1/2-ir. These findings represent the first direct evidence that subcutaneously injected BDNF is transported to motor neurons and that it activates a signaling pathway in the spinal cord and exhibits neurotrophic effects in vivo.

Increases in cortical glutamate concentrations in transgenic amyotrophic lateral sclerosis mice are attenuated by creatine supplementation.

Several lines of evidence implicate excitotoxic mechanisms in the pathogenesis of amyotrophic lateral sclerosis (ALS). Transgenic mice with a superoxide dismutase mutation (G93A) have been utilized as an animal model of familial ALS (FALS). We examined the cortical concentrations of glutamate using in vivo microdialysis and in vivo nuclear magnetic resonance (NMR) spectroscopy, and the effect of long-term creatine supplementation. NMDA-stimulated and Ltrans-pyrrolidine-2,4-dicarboxylate (LTPD)-induced increases in glutamate were significantly higher in G93A mice compared with littermate wild-type mice at 115 days of age. At this age, the tissue concentrations of glutamate were also significantly increased as measured with NMR spectroscopy. Creatine significantly increased longevity and motor performance of the G93A mice, and significantly attenuated the increases in glutamate measured with spectroscopy at 75 days of age, but had no effect at 115 days of age. These results are consistent with impaired glutamate transport in G93A transgenic mice. The beneficial effect of creatine may be partially mediated by improved function of the glutamate transporter, which has a high demand for energy and is susceptible to oxidative stress.

T-588, a novel neuroprotective agent, delays progression of neuromuscular dysfunction in wobbler mouse motoneuron disease.

R(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino) ethoxy]ethanol hydrochloride (T-588) enhances acetylcholine release from the frontal cortex and hippocampus in rats, and can ameliorate cognitive dysfunction in various amnesia models of rodents. T-588 protects rat cerebellar granule cells from glutamate neurotoxicity in culture. This agent also inhibits facilitation in the crayfish neuromuscular junction and mammalian cerebellum. Clinical trials of T-588 are underway in patients with Alzheimer's disease. We attempted to determine whether T-588 treatment ameliorates neuromuscular dysfunction in the wobbler mouse, an animal model of motoneuron disease (MND). After the initial diagnosis of MND at the age of 3-4 weeks, wobbler mice were orally administered T-588 (3, 10, 30 mg/kg) or vehicle daily for 4 weeks in a blinded fashion. We compared symptomatic, pathological and biochemical changes among the groups. In comparison with vehicle, T-588 administration potentiated grip strength, attenuated forelimb contracture and increased the weight of the biceps muscles. T-588-treated mice had retarded denervation muscle atrophy and elevated activities of choline acetyltransferase (ChAT) or lactate dehydrogenase in the biceps muscles. T-588 treatment also enhanced ChAT activities and promoted formation of cyclic adenosine monophosphate in the cervical cord. Pharmacokinetic study also showed that T-588 was transported efficiently into the cerebrum and spinal cord following oral administration. Thus, T-588 treatment delayed the progression of wobbler murine MND. Our findings suggest that this agent has therapeutic potential in human motor neuropathy or MND.

Treatment of pathological affect: variability of response for laughter and crying.

Pathological laughing and crying (PLC) is increasingly recognized to accompany diverse neurologic conditions, although it remains poorly understood. The authors describe 3 cases of amyotrophic lateral sclerosis (ALS) with an unusual change from a predominance of pathological crying to laughter following drug treatment. Possible explanations for this phenomenon are discussed.

Genetic transfer of the wobbler gene to a C57BL/6J x NZB hybrid stock: natural history of the motor neuron disease and response to CNTF and BDNF cotreatment.

Preclinical diagnosis of motor neuron disease (MND) in the wobbler mouse (wr/wr) has been impossible until recently. However, with the development of a new hybrid, the C57BL/6J x New Zealand Black (B6NZB) wr/wr mouse, the polymerase chain reaction (PCR) can be used to establish the preclinical diagnosis. We compared the clinical and histological features of MND and the effects of neurotrophic factor cotreatment between the hybrid B6NZB-wr/wr and the congenic C57BL/6J-wr/wr mice. Clinical assessments of body weight, grip strength, running speed, paw position, and walking pattern were made weekly from age 2 weeks through 8 weeks (n = 10, B6NZB-wr/wr; n = 15, C57BL/6J-wr/wr). Survival was analyzed (n = 7, each strain) as was C5 and C6 spinal cord motoneuron morphology and ventral root histometry (n = 7, each strain). For cotreatment, 8 B6NZB-wr/wr and 7 C57BL/6J-wr/wr mice received subcutaneous ciliary neurotrophic factor (1 mg/kg) and brain-derived neurotrophic factor (5 mg/kg) on alternate days, 6 days/week for 4 weeks. B6NZB-wr/wr mice could be distinguished from C57BL/6J-wr/wr mice at age 3 weeks by a more abnormal paw position (P < 0.01) and walking pattern (P < 0.05) and lower grip strength (P < 0.001) and running speed (P < 0.001). After 3 weeks, the changes continued to be greater in B6NZB-wr/wr mice. Although B6NZB-wr/wr mice were more severely affected early in the disease, their survival was comparable to C57BL/6J-wr/wr mice. Anterior horn cell vacuolar degeneration and myelinated fiber histometry were similar in both strains. The clinical response to CNTF/BDNF cotreatment was marked in both groups although it was weaker in B6NZB-wr/wr mice. Thus, the hybrid B6NZB-wr/wr mice have a more severe clinical phenotype and offer a unique opportunity to study the mechanisms of presymptomatic motor neuron degeneration and the effects of therapeutic agents for human MND.

Coadministration of interleukin-6 (IL-6) and soluble IL-6 receptor delays progression of wobbler mouse motor neuron disease.

Interleukin-6 (IL-6), a multipotential cytokine, initiates signal transduction pathways similar to those of ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF). These molecules share the signal transducing receptor component, gp130. IL-6 triggers homodimerization of gp130, whereas CNTF and LIF induce heterodimerization of gp130 and LIF receptor. Although CNTF or LIF treatment attenuates motor deficits in wobbler mouse motor neuron disease (MND), neuroprotective effects of IL-6 on this animal have not yet been clarified. Here we studied whether simultaneous treatment with IL-6 and soluble IL-6 receptor (sIL-6R) can ameliorate symptomatic and neuropathological changes in wobbler mouse MND. After clinical diagnosis at postnatal age 3-4 weeks, wobbler mice received subcutaneous injection with human recombinant IL-6 (1.0 mg/kg), human sIL-6R (0.5 mg/kg), IL-6 + sIL-6R or vehicle, daily for 4 weeks in a blind fashion. Compared to vehicle, coadministration with IL-6 and sIL-6R potentiated grip strength, attenuated muscle contractures in the forelimbs, reduced denervation muscle atrophy and prevented degeneration of spinal motor neurons. Single administration with IL-6 or sIL-6R did not retard the symptomatic and neuropathological progression, although IL-6-treated mice did not raise anti-IL-6 antibodies. Treatment with IL-6 + sIL-6R, but not with IL-6 or sIL-6R alone delayed progression of wobbler mouse MND. Our results indicate that the neuroprotective mechanism for IL-6/sIL-6R on wobbler mouse MND differs from that of CNTF or LIF alone. We hypothesize that IL-6/sIL-6R complex may function on motor neurons through activation and homodimerization of gp130.

Can nimodipine affect progression of motor neuron disease? A double-blind pilot study.

Recently, there has been some evidence suggesting that exposure to neuroexcitotoxic amino acids, culminating in excessive neuronal calcium influx, may cause nerve cell destruction in motor neuron disease (MND). We therefore hypothesized that central calcium channel blockade with nimodipine might slow-down progression of MND. Two patients with sporadic MND were treated in a double-blind placebo controlled cross-over study with oral nimodipine (120 mg/day) for 5 months. Disease progression was assessed by repeated computerized measurements of isometric force from 20 muscle groups, and calculation of global force score. Global force deterioration slopes during the 5-month periods of nimodipine or placebo therapy were highly linear and almost identical. Nimodipine therefore does not seem to be effective in changing the course of MND. The observed remarkable linearity of disease course also confirms that cross-over methodology and serial measurements of muscle force are constructive tools in the clinical evaluation of novel therapeutic strategies for this devastating disease.