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International audience; Background: Rasagiline, a monoamine oxidase B inhibitor with neuroprotective potential in Parkinson's disease, has shown a disease-modifying effect in the SOD1-Gly93Ala low-expressing mouse model of amyotrophic lateral sclerosis, both alone and in combination with riluzole. We sought to test whether or not rasagiline 1 mg/day can prolong survival in patients with amyotrophic lateral sclerosis also receiving riluzole.Methods: Patients with possible, probable, or definite amyotrophic lateral sclerosis were enrolled to our randomised, placebo-controlled, parallel-group, double-blind, phase 2 trial from 15 German network for motor neuron diseases (MND-NET) centres (university hospitals or clinics). Eligible patients were aged at least 18 years, had onset of progressive weakness within the 36 months before the study, had disease duration of more than 6 months and less than 3 years, and had a best-sitting slow vital capacity of at least 50%. After a 4-week screening period, eligible patients were randomly assigned (1:1) to receive either rasagiline (1 mg/day) or placebo in addition to riluzole (100 mg/day), after stratification for site of onset (bulbar or spinal) and study centre. Patients and all personnel assessing outcome parameters were masked to treatment allocation. Patients were followed up 2, 6, 12, and 18 months after randomisation. The primary endpoint was survival time, defined as the time to death or time to study cutoff date (ie, the last patient's last visit plus 14 days). Analyses of primary outcome and safety measures were done in all patients who received at least one dose of trial treatment (intention-to-treat population). The trial is registered with ClinicalTrials.gov, number NCT01879241.Findings: Between July 2, 2013, and Nov 11, 2014, 273 patients were screened for eligibility, and 252 patients were randomly assigned to receive rasagiline (n=127) or placebo (n=125). 126 patients taking rasagiline and 125 taking placebo were included in the intention-to-treat analysis. For the primary outcome, the survival probability at the end of the study was 0·43 (95% CI 0·25-0·59) in the rasagiline group (n=126) and 0·53 (0·43-0·62) in the placebo group (n=125). The estimated effect size (hazard ratio) was 0·91 (one-sided 97·5% CI -infinity to 1·34; p=0·31). Rasagiline was well tolerated, and most adverse events were due to amyotrophic lateral sclerosis disease progression rather than treatment; the most frequent of these were dysphagia (32 [25%] taking rasagiline vs 24 [19%] taking placebo) and respiratory failure (25 [20%] vs 31 [25%]). Frequency of adverse events were comparable between both groups.Interpretation: Rasagiline was safe in patients with amyotrophic lateral sclerosis. There was no difference between groups in the primary outcome of survival, although post-hoc analysis suggested that rasagiline might modify disease progression in patients with an initial slope of Amyotrophic Lateral Sclerosis Functional Rating Scale Revised greater than 0·5 points per month at baseline. This should be confirmed in another clinical trial.Funding: Teva Pharmaceutical Industries.

Objectives Botulinum neurotoxin serotypes A and B (BoNT/A & B) are highly effective medicines to treat hyperactive cholinergic neurons. Due to neutralizing antibody formation, some patients may become non-responders. In these cases, the serotypes BoNT/C-G might become treatment alternatives. BoNT/D is genetically least related to BoNT/A & B and thereby circumventing neutralisation in A/B non-responders. We produced BoNT/D and compared its pharmacology with BoNT/A ex vivo in mice tissue and in vivo in human volunteers. Methods BoNT/D was expressed recombinantly in E. coli, isolated by chromatography and its ex vivo potency was determined at mouse phrenic nerve hemidiaphragm preparations. Different doses of BoNT/D or incobotulinumtoxinA were injected into the extensor digitorum brevis (EDB) muscles (n = 30) of human volunteers. Their compound muscle action potentials were measured 11 times by electroneurography within 220 days. Results Despite a 3.7-fold lower ex vivo potency in mice, a 110-fold higher dosage of BoNT/D achieved the same clinical effect as incobotulinumtoxinA while showing a 50% shortened duration of action. Conclusions BoNT/D blocks dose-dependently acetylcholine release in human motoneurons upon intramuscular administration, but its potency and duration of action is inferior to approved BoNT/A based drugs. Significance BoNT/D constitutes a potential treatment alternative for BoNT/A & B non-responders.

Cellular therapy represents a novel option for the treatment of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Its major aim is the generation of a protective environment for degenerating motor neurons. Mesenchymal stromal cells secrete different growth factors and have antiapoptotic and immunomodulatory properties. They can easily and safely be isolated from human bone marrow and are therefore considered promising therapeutic candidates. In the present study, we compared intraventricular application of human mesenchymal stromal cells (hMSCs) versus single and repeated intraspinal injections in the mutant SOD1G93A transgenic ALS mouse model. We observed significant reduction of lifespan of animals treated by intraventricular hMSC injection compared with the vehicle treated control group, accompanied by changes in weight, general condition, and behavioural assessments. A potential explanation for these rather surprising deleterious effects lies in increased microgliosis detected in the hMSC treated animals. Repeated intraspinal injection at two time points resulted in a slight but not significant increase in survival and significant improvement of motor performance although no hMSC-induced changes of motor neuron numbers, astrogliosis, and microgliosis were detected. Quantitative real time polymerase chain reaction showed reduced expression of endothelial growth factor in animals having received hMSCs twice compared with the vehicle treated control group. hMSCs were detectable at the injection site at Day 20 after injection into the spinal cord but no longer at Day 70. Intraspinal injection of hMSCs may therefore be a more promising option for the treatment of ALS than intraventricular injection and repeated injections might be necessary to obtain substantial therapeutic benefit.

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 G93A -dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.

Motor unit number estimation (MUNE) techniques such as motor unit number index (MUNIX) have been used to quantify lower motor neuron loss and disease progression in amyotrophic lateral sclerosis (ALS). We investigated the consistency of reproducibility of MUNIX in 30 ALS-patients during the course of the disorder.MUNIX was recorded in abductor pollicis brevis and tibialis anterior muscles bilaterally in ALS-patients by two measurements at the first and at one follow-up visit and once in healthy controls. Intra-rater reproducibility was evaluated by three statistical methods: interclass correlation coefficient (ICC), correlation coefficient analysis (CCA), and coefficient of variation (CV).We found significant correlation between the first and second measurement of MUNIX in all tested muscles and at the follow-up visit (r⩾0.891, p0.01) and good statistically significant reproducibility of MUNIX in all four measured muscles at the follow-up visit (ICC⩾0.946, p0.01). The CV of MUNIX at the follow-up visit ranged from 13.90% to 32.95%.This study shows good consistency of reproducibility of MUNIX in the course of ALS.This study suggests that MUNIX can be used to track the progression of the disorder both in clinical routine and in treatment trials.

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disorder that leads to progressive paralysis of skeletal muscles and death by respiratory failure. There is increasing evidence that ALS is at least in part an axonopathy and that mechanisms regulating axonal degeneration and regeneration might be pathogenetically relevant. Semaphorin 3A (Sema3A) is an axon guidance protein; it acts as an axon repellent and prevents axonal regeneration. Increased Sema3A expression has been described in a mouse model of ALS in which it may contribute to motor neuron degeneration. This study aimed to investigate Sema3A mRNA and protein expression in human CNS tissues. We assessed Sema3A expression using quantitative real-time PCR, in situ hybridization, and immunohistochemistry in motor cortex and spinal cord tissue of 8 ALS patients and 6 controls. We found a consistent increase of Sema3A expression in the motor cortex of ALS patients by all 3 methods. In situ hybridization further confirmed that Sema3A expression was present in motor neurons. These findings indicate that upregulation of Sema3A may contribute to axonal degeneration and failure of regeneration in ALS patients. The inhibition of Sema3A therefore might be a promising future therapeutic option for patients with this disease.

Cellular therapy represents a novel option for the treatment of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Its major aim is the generation of a protective environment for degenerating motor neurons. Mesenchymal stromal cells secrete different growth factors and have antiapoptotic and immunomodulatory properties. They can easily and safely be isolated from human bone marrow and are therefore considered promising therapeutic candidates. In the present study, we compared intraventricular application of human mesenchymal stromal cells (hMSCs) versus single and repeated intraspinal injections in the mutant SOD1

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons and skeletal muscle atrophy. The majority of ALS cases are acquired spontaneously, with inherited disease accounting for only 10 % of all cases. Recent studies provide compelling evidence that aggregates of misfolded proteins underlie both types of ALS. Small molecules such as artificial chaperones can prevent or even reverse the aggregation of proteins associated with various human diseases. However, their very high active concentration (micromolar range) severely limits their utility as drugs. We synthesized several ester and amide derivatives of chemical chaperones. The lead compound 14, 3-((5-((4,6-dimethylpyridin-2-yl)methoxy)-5-oxopentanoyl)oxy)-N,N-dimethylpropan-1-amine oxide shows, in the micromolar concentration range, both neuronal and astrocyte protective effects in vitro; at daily doses of 10 mg kg(-1) 14 improved the neurological functions and delayed body weight loss in ALS mice. Members of this new chemical chaperone derivative class are strong candidates for the development of new drugs for ALS patients.

Neural stem or progenitor cells are considered to be a novel therapeutic strategy for amyotrophic lateral sclerosis (ALS), based on their potential to generate a protective environment rather than to replace degenerating motor neurons. Following local injection to the spinal cord, neural progenitor cells may generate glial cells and release neurotrophic factors. In the present study, human spinal cord-derived neural progenitor cells (hscNPCs) were injected into the lumbar spinal cord of G93A-SOD1 ALS transgenic mice. We evaluated the potential effect of hscNPC treatment by survival analysis and behavioural/phenotypic assessments. Immunohistological and real-time PCR experiments were performed at a defined time point to study the underlying mechanisms. Symptom progression in hscNPC-injected mice was significantly delayed at the late stage of disease. On average, survival was only prolonged for 5 days. Animals treated with hscNPCs performed significantly better in motor function tests between weeks 18 and 19. Increased production of GDNF and IGF-1 mRNA was detectable in spinal cord tissue of hscNPC-treated mice. In summary, treatment with hscNPCs led to increased endogenous production of several growth factors and increased the preservation of innervated motor neurons but had only a small effect on overall survival. Copyright © 2015 John Wiley & Sons, Ltd.

Neural stem or progenitor cells are considered to be a novel therapeutic strategy for amyotrophic lateral sclerosis (ALS), based on their potential to generate a protective environment rather than to replace degenerating motor neurons. Following local injection to the spinal cord, neural progenitor cells may generate glial cells and release neurotrophic factors. In the present study, human spinal cord-derived neural progenitor cells (hscNPCs) were injected into the lumbar spinal cord of G93A-SOD1 ALS transgenic mice. We evaluated the potential effect of hscNPC treatment by survival analysis and behavioural/phenotypic assessments. Immunohistological and real-time PCR experiments were performed at a defined time point to study the underlying mechanisms. Symptom progression in hscNPC-injected mice was significantly delayed at the late stage of disease. On average, survival was only prolonged for 5 days. Animals treated with hscNPCs performed significantly better in motor function tests between weeks 18 and 19. Increased production of GDNF and IGF-1 mRNA was detectable in spinal cord tissue of hscNPC-treated mice. In summary, treatment with hscNPCs led to increased endogenous production of several growth factors and increased the preservation of innervated motor neurons but had only a small effect on overall survival. Copyright © 2015 John WileySons, Ltd.

Background/Aims: Sirtuins (SIRT1–7; class III histone deactylases) modulate fundamental mechanisms in age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We assessed the expression levels of sirtuins in human postmortem ALS and control brain and spinal cord. Methods and Results: By quantitative real-time PCR, a significant reduction of SIRT1 and SIRT2 was detected in homogenates of the primary motor cortex (white and gray matter), while there were no differences in spinal cord homogenates. When specifically analyzing mRNA and protein expression in the gray matter (cortical layers I–VI of the precentral gyrus, ventral/dorsal horn of the spinal cord) by in situ hybridization histochemistry and immunohistochemistry, we found increased levels of SIRT1, SIRT2 and SIRT5 in ALS which were significant for SIRT1 and SIRT5 mRNA in the spinal cord. Conclusion: Our results indicate a general reduction of SIRT1 and SIRT2 in ALS primary motor cortex, while in situ hybridization histochemistry and immunohistochemistry showed neuron-specific upregulation of SIRT1, SIRT2 and SIRT5, particularly in the spinal cord. Opposed effects have been described for SIRT1 and SIRT2: while SIRT1 activation is mainly associated with neuroprotection, SIRT2 upregulation is toxic to neuronal cells. Novel therapeutic approaches in ALS could therefore target SIRT1 activation or SIRT2 inhibition.