Clinical and patient-reported outcomes and neurofilament response during tofersen treatment in SOD1-related ALS-A multicenter observational study over 18 months.

INTRODUCTION/AIMS: In amyotrophic lateral sclerosis (ALS) caused by SOD1 mutations (SOD1-ALS), tofersen received accelerated approval in the United States and is available via expanded access programs (EAP) outside the United States. This multicenter study investigates clinical and patient-reported outcomes (PRO) and serum neurofilament light chain (sNfL) during tofersen treatment in an EAP in Germany. METHODS: Sixteen SOD1-ALS patients receiving tofersen for at least 6 months were analyzed. The ALS progression rate (ALS-PR), as measured by the monthly change of the ALS functional rating scale-revised (ALSFRS-R), slow vital capacity (SVC), and sNfL were investigated. PRO included the Measure Yourself Medical Outcome Profile (MYMOP2), Treatment Satisfaction Questionnaire for Medication (TSQM-9), and Net Promoter Score (NPS). RESULTS: Mean tofersen treatment was 11 months (6-18 months). ALS-PR showed a mean change of -0.2 (range 0 to -1.1) and relative reduction by 25%. Seven patients demonstrated increased ALSFRS-R. SVC was stable (mean 88%, range -15% to +28%). sNfL decreased in all patients except one heterozygous D91A-SOD1 mutation carrier (mean change of sNfL -58%, range -91 to +27%, p < .01). MYMOP2 indicated improved symptom severity (n = 10) or yet perception of partial response (n = 6). TSQM-9 showed high global treatment satisfaction (mean 83, SD 16) although the convenience of drug administration was modest (mean 50, SD 27). NPS revealed a very high recommendation rate for tofersen (NPS +80). DISCUSSION: Data from this EAP supported the clinical and sNfL response to tofersen in SOD1-ALS. PRO suggested a favorable patient perception of tofersen treatment in clinical practice.

Loss-of-Function Variants in HOPS Complex Genes VPS16 and VPS41 Cause Early Onset Dystonia Associated with Lysosomal Abnormalities.

OBJECTIVES: The majority of people with suspected genetic dystonia remain undiagnosed after maximal investigation, implying that a number of causative genes have not yet been recognized. We aimed to investigate this paucity of diagnoses. METHODS: We undertook weighted burden analysis of whole-exome sequencing (WES) data from 138 individuals with unresolved generalized dystonia of suspected genetic etiology, followed by additional case-finding from international databases, first for the gene implicated by the burden analysis (VPS16), and then for other functionally related genes. Electron microscopy was performed on patient-derived cells. RESULTS: Analysis revealed a significant burden for VPS16 (Fisher's exact test p value, 6.9 × 109 ). VPS16 encodes a subunit of the homotypic fusion and vacuole protein sorting (HOPS) complex, which plays a key role in autophagosome-lysosome fusion. A total of 18 individuals harboring heterozygous loss-of-function VPS16 variants, and one with a microdeletion, were identified. These individuals experienced early onset progressive dystonia with predominant cervical, bulbar, orofacial, and upper limb involvement. Some patients had a more complex phenotype with additional neuropsychiatric and/or developmental comorbidities. We also identified biallelic loss-of-function variants in VPS41, another HOPS-complex encoding gene, in an individual with infantile-onset generalized dystonia. Electron microscopy of patient-derived lymphocytes and fibroblasts from both patients with VPS16 and VPS41 showed vacuolar abnormalities suggestive of impaired lysosomal function. INTERPRETATION: Our study strongly supports a role for HOPS complex dysfunction in the pathogenesis of dystonia, although variants in different subunits display different phenotypic and inheritance characteristics. ANN NEUROL 2020;88:867-877.

AAV.shRNA-mediated downregulation of ROCK2 attenuates degeneration of dopaminergic neurons in toxin-induced models of Parkinson's disease in vitro and in vivo.

Parkinson's disease (PD) is a neurodegenerative disorder with prominent neuronal cell death in the substantia nigra (SN) and other parts of the brain. Previous studies in models of traumatic and neurodegenerative CNS disease showed that pharmacological inhibition of Rho-associated kinase (ROCK), a molecule involved in inhibitory signaling in the CNS, by small-molecule inhibitors improves neuronal survival and increases regeneration. Most small-molecule inhibitors, however, offer only limited target specificity and also inhibit other kinases, including both ROCK isoforms. To establish the role of the predominantly brain-expressed ROCK2 isoform in models of regeneration and PD, we used adeno-associated viral vectors (AAV) to specifically knockdown ROCK2 in neurons. Rat primary midbrain neurons (PMN) were transduced with AAV expressing short-hairpin-RNA (shRNA) against ROCK2 and LIM-domain kinase 1 (LIMK1), one of the downstream targets of ROCK2. While knock-down of ROCK2 and LIMK1 both enhanced neurite regeneration in a traumatic scratch lesion model, only ROCK2-shRNA protected PMN against 1-methyl-4-phenylpyridinium (MPP+) toxicity. Moreover, AAV.ROCK2-shRNA increased levels of the pro-survival markers Bcl-2 and phospho-Erk1. In vivo, AAV.ROCK2-shRNA vectors were injected into the ipsilateral SN and a unilateral 6-OHDA striatal lesion was performed. After four weeks, behavioral, immunohistochemical and biochemical alterations were investigated. Downregulation of ROCK2 protected dopaminergic neurons in the SN from 6-OHDA-induced degeneration and resulted in significantly increased TH-positive neuron numbers. This effect, however, was confined to nigral neuronal somata as striatal terminal density, dopamine and metabolite levels were not significantly preserved. Interestingly, motor behavior was improved in the ROCK2-shRNA treated animals compared to control after four weeks. Our studies thus confirm ROCK2 as a promising therapeutic target in models of PD and demonstrate that neuron-specific inhibition of ROCK2 promotes survival of lesioned dopaminergic neurons.

Tofersen decreases neurofilament levels supporting the pathogenesis of the SOD1 p.D91A variant in amyotrophic lateral sclerosis patients.

BACKGROUND: Since the antisense oligonucleotide tofersen has recently become available for the treatment of amyotrophic lateral sclerosis (ALS) caused by mutations in SOD1, determining the causality of the over 230 SOD1 variants has become even more important. The most common SOD1 variant worldwide is p.D91A (c.272A > C), whose causality for ALS is contested when in a heterozygous state. The reason is the high allele frequency of SOD1D91A in Europe, exceeding 1% in Finno-Scandinavia. METHODS: We present the clinical disease course and serum neurofilament light chain (NfL) results of treating 11 patients either homo- or heterozygous for the SOD1D91A allele for up to 16 months with tofersen. RESULTS: Tofersen decreases serum neurofilament levels (sNFL), which are associated with the ALS progression rate, in the 6 ALS patients homozygous for SOD1D91A. We observe significantly lower sNfL levels in the 5 patients heterozygous for SOD1D91A. The results indicate that both mono- and bi-allelic SOD1D91A are causally relevant targets, with a possibly reduced effect size of SOD1D91Ahet. CONCLUSIONS: The finding is relevant for decision making regarding tofersen treatment, patient counseling and inclusion of SOD1D91A patients in drug trials. As far as we are aware, the approach is conceptually new since it provides evidence for the causality of an ALS variant based on a biomarker response to gene-specific treatment.

Amyotrophic lateral sclerosis (ALS) is a disease that can be inherited which affects nerve cells in the brain and spinal cord. Changes within a gene called SOD1 that result in a mutation named p.D91A can lead to the development of ALS. People have two copies of the SOD1 gene. It has been unclear whether the presence of only one copy of p.D91A can cause ALS. We treated ALS patients with the p.D91A variant of SOD1 with a drug called tofersen. We found that a marker of disease progression was reduced in patients with one or two copies of the p.D91A mutation. This suggests that the presence of just one p.D91A variant of SOD1 contributes to disease development. This information could be used to improve treatment decisions for people with ALS.

Safety, tolerability, and efficacy of fasudil in amyotrophic lateral sclerosis (ROCK-ALS): a phase 2, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Fasudil is a small molecule inhibitor of Rho-associated kinase (ROCK) and is approved for the treatment of subarachnoid haemorrhage. In preclinical studies, fasudil has been shown to attenuate neurodegeneration, modulate neuroinflammation, and foster axonal regeneration. We aimed to investigate the safety, tolerability, and efficacy of fasudil in patients with amyotrophic lateral sclerosis. METHODS: ROCK-ALS was a phase 2, randomised, double-blind, placebo-controlled trial conducted at 19 amyotrophic lateral sclerosis centres in Germany, France, and Switzerland. Individuals (aged 18-80 years) with at least probable amyotrophic lateral sclerosis (as per the revised El Escorial criteria), a disease duration of 6-24 months, and a slow vital capacity greater than 65% of predicted normal were eligible for inclusion. Patients were randomly assigned (1:1:1) to receive 30 mg (15 mg twice daily) or 60 mg (30 mg twice daily) fasudil or matched placebo intravenously for 20 days over a 4-week period. Follow-up assessments were performed at 45, 90, and 180 days after treatment initiation. The co-primary endpoints were safety until day 180 (defined as the proportion without drug-related serious adverse events) and tolerability during the treatment period (defined as the proportion who did not discontinue treatment due to suspected drug-related adverse events). The primary analyses were carried out in the intention-to-treat population, which included all participants who entered the treatment phase. This trial is registered at ClinicalTrials.gov (NCT03792490) and Eudra-CT (2017-003676-31) and is now completed. FINDINGS: Between Feb 20, 2019, and April 20, 2022, 120 participants were enrolled and randomised; two individuals assigned fasudil 30 mg withdrew consent before the baseline visit. Thus, the intention-to-treat population comprised 35 in the fasudil 30 mg group, 39 in the fasudil 60 mg group, and 44 in the placebo group. The estimated proportion without a drug-related serious adverse event was 1·00 (95% CI 0·91 to 1·00) with placebo, 1·00 (0·89 to 1·00) with fasudil 30 mg, and 1·00 (0·90 to 1·00) with fasudil 60 mg; the difference in proportions was 0·00 (95% CI -0·11 to 0·10; p>0·99) for fasudil 30 mg versus placebo and 0·00 (-0·10 to 0·10; p>0·99) for fasudil 60 mg versus placebo. Treatment tolerability (the estimated proportion who did not discontinue) was 0·93 (95% CI 0·81 to 0·99) with placebo, 1·00 (0·90 to 1·00) with fasudil 30 mg, and 0·90 (0·76 to 0·97) with fasudil 60 mg; the difference in proportions was 0·07 (95% CI -0·05 to 0·20; p=0·25) for fasudil 30 mg versus placebo, and -0·03 (-0·18 to 0·10; p=0·70) for fasudil 60 mg versus placebo. Eight deaths occurred: two in the placebo group, four in the fasudil 30 mg group, and two in the fasudil 60 mg group. The most common serious adverse events were respiratory failure (seven events), gastrostomy (five events), pneumonia (four events), and dysphagia (four events). No serious adverse events or deaths were attributed to study treatment. Adverse events, which were mainly related to disease progression, occurred in 139 participants in the placebo group, 108 in the fasudil 30 mg group, and 105 in the fasudil 60 mg group. INTERPRETATION: Fasudil was well tolerated and safe in people with amyotrophic lateral sclerosis. The effect of fasudil on efficacy outcomes should be explored in larger clinical trials with a longer treatment duration, oral administration, and potentially higher dose of the trial drug. FUNDING: Framework of the E-Rare Joint Transnational Call 2016 "Clinical research for new therapeutic uses of already existing molecules (repurposing) in rare diseases".

5qSMA: standardised retrospective natural history assessment in 268 patients with four copies of SMN2.

Newborn screening for 5qSMA offers the potential for early, ideally pre-symptomatic, therapeutic intervention. However, limited data exist on the outcomes of individuals with 4 copies of SMN2, and there is no consensus within the SMA treatment community regarding early treatment initiation in this subgroup. To provide evidence-based insights into disease progression, we performed a retrospective analysis of 268 patients with 4 copies of SMN2 from the SMArtCARE registry in Germany, Austria and Switzerland. Inclusion criteria required comprehensive baseline data and diagnosis outside of newborn screening. Only data prior to initiation of disease-modifying treatment were included. The median age at disease onset was 3.0 years, with a mean of 6.4 years. Significantly, 55% of patients experienced symptoms before the age of 36 months. 3% never learned to sit unaided, a further 13% never gained the ability to walk independently and 33% of ambulatory patients lost this ability during the course of the disease. 43% developed scoliosis, 6.3% required non-invasive ventilation and 1.1% required tube feeding. In conclusion, our study, in line with previous observations, highlights the substantial phenotypic heterogeneity in SMA. Importantly, this study provides novel insights: the median age of disease onset in patients with 4 SMN2 copies typically occurs before school age, and in half of the patients even before the age of three years. These findings support a proactive approach, particularly early treatment initiation, in this subset of SMA patients diagnosed pre-symptomatically. However, it is important to recognize that the register will not include asymptomatic individuals.

X-ray fluorescence analysis of iron and manganese distribution in primary dopaminergic neurons.

Transition metals have been suggested to play a pivotal role in the pathogenesis of Parkinson's disease. X-ray microscopy combined with a cryogenic setup is a powerful method for elemental imaging in low concentrations and high resolution in intact cells, eliminating the need for fixation and sectioning of the specimen. Here, we performed an elemental distribution analysis in cultured primary midbrain neurons with a step size in the order of 300 nm and ~ 0.1 ppm sensitivity under cryo conditions by using X-ray fluorescence microscopy. We report the elemental mappings on the subcellular level in primary mouse dopaminergic (DAergic) and non-DAergic neurons after treatment with transition metals. Application of Fe(2+) resulted in largely extracellular accumulation of iron without preference for the neuronal transmitter subtype. A quantification of different Fe oxidation states was performed using X-ray absorption near edge structure analysis. After treatment with Mn(2+) , a cytoplasmic/paranuclear localization of Mn was observed preferentially in DAergic neurons, while no prominent signal was detectable after Mn(3+) treatment. Immunocytochemical analysis correlated the preferential Mn uptake to increased expression of voltage-gated calcium channels in DAergic neurons. We discuss the implications of this differential elemental distribution for the selective vulnerability of DAergic neurons and Parkinson's disease pathogenesis.

Upregulation of reggie-1/flotillin-2 promotes axon regeneration in the rat optic nerve in vivo and neurite growth in vitro.

The ability of fish retinal ganglion cells (RGCs) to regenerate their axons was shown to require the re-expression and function of the two proteins reggie-1 and -2. RGCs in mammals fail to upregulate reggie expression and to regenerate axons after lesion suggesting the possibility that induced upregulation might promote regeneration. In the present study, RGCs in adult rats were induced to express reggie-1 by intravitreal injection of adeno-associated viral vectors (AAV2/1) expressing reggie-1 (AAV.R1-EGFP) 14d prior to optic nerve crush. Four weeks later, GAP-43-positive regenerating axons had crossed the lesion and grown into the nerve at significantly higher numbers and length (up to 5mm) than the control transduced with AAV.EGFP. Consistently, after transduction with AAV.R1-EGFP as opposed to AAV.EGFP, primary RGCs in vitro grew long axons on chondroitin sulfate proteoglycan (CSPG) and Nogo-A, both glial cell-derived inhibitors of neurite growth, suggesting that reggie-1 can provide neurons with the ability to override inhibitors of neurite growth. This reggie-1-mediated enhancement of growth was reproduced in mouse hippocampal and N2a neurons which generated axons 40-60% longer than their control counterparts. This correlates with the reggie-1-dependent activation of Src and PI3 kinase (PI3K), of the Rho family GTPase Rac1 and downstream effectors such as cofilin. This increased growth also depends on TC10, the GTPase involved in cargo delivery to the growth cone. Thus, the upregulation of reggie-1 in mammalian neurons provides nerve cells with neuron-intrinsic properties required for axon growth and successful regeneration in the adult mammalian CNS.

MicroRNA-124 protects against focal cerebral ischemia via mechanisms involving Usp14-dependent REST degradation.

MicroRNAs (miRNAs) are highly conserved non-coding RNAs modulating gene expression via mRNA binding. Recent work suggests an involvement of miRNAs in cardiovascular diseases including stroke. As such, the brain-abundant miR-124 and its transcriptional repressor RE1-silencing transcription factor (REST) do not only have elementary roles in the developing and the adult brain, but also alter expression upon cerebral ischemia. However, the therapeutic potential of miR-124 against stroke and the mechanisms involved remain elusive. Here, we analyzed the therapeutic potential of ectopic miR-124 against stroke and its underlying mechanisms with regard to the interaction between miR-124 and REST. Our results show that viral vector-mediated miR-124 delivery increased the resistance of cultured oxygen-glucose-deprived cortical neurons in vitro and reduced brain injury as well as functional impairment in mice submitted to middle cerebral artery occlusion. Likewise, miR-124 induced enhanced neurovascular remodeling leading to increased angioneurogenesis 8 weeks post-stroke. While REST abundance increased upon stroke, the increase was prevented by miR-124 despite a so far unknown negative feedback loop between miR-124 and REST. Rather, miR-124 decreased the expression of the deubiquitinating enzyme Usp14, which has two conserved miR-124-binding sites in the 3'UTR of its mRNA, and thereby mediated reduced REST levels. The down-regulation of REST by miR-124 was also mimicked by the Usp14 inhibitor IU-1, suggesting that miR-124 promotes neuronal survival under ischemic conditions via Usp14-dependent REST degradation. Ectopic miR-124 expression, therefore, appears as an attractive and novel tool in stroke treatment, mediating neuroprotection via a hitherto unknown mechanism that involves Usp14-dependent REST degradation.

Inhibition of rho kinase enhances survival of dopaminergic neurons and attenuates axonal loss in a mouse model of Parkinson's disease.

Axonal degeneration is one of the earliest features of Parkinson's disease pathology, which is followed by neuronal death in the substantia nigra and other parts of the brain. Inhibition of axonal degeneration combined with cellular neuroprotection therefore seem key to targeting an early stage in Parkinson's disease progression. Based on our previous studies in traumatic and neurodegenerative disease models, we have identified rho kinase as a molecular target that can be manipulated to disinhibit axonal regeneration and improve survival of lesioned central nervous system neurons. In this study, we examined the neuroprotective potential of pharmacological rho kinase inhibition mediated by fasudil in the in vitro 1-methyl-4-phenylpyridinium cell culture model and in the subchronic in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. Application of fasudil resulted in a significant attenuation of dopaminergic cell loss in both paradigms. Furthermore, dopaminergic terminals were preserved as demonstrated by analysis of neurite network in vitro, striatal fibre density and by neurochemical analysis of the levels of dopamine and its metabolites in the striatum. Behavioural tests demonstrated a clear improvement in motor performance after fasudil treatment. The Akt survival pathway was identified as an important molecular mediator for neuroprotective effects of rho kinase inhibition in our paradigm. We conclude that inhibition of rho kinase using the clinically approved small molecule inhibitor fasudil may be a promising new therapeutic strategy for Parkinson's disease.

Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calcium-sensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.

Spectrum and frequency of genetic variants in sporadic amyotrophic lateral sclerosis.

Therapy of motoneuron diseases entered a new phase with the use of intrathecal antisense oligonucleotide therapies treating patients with specific gene mutations predominantly in the context of familial amyotrophic lateral sclerosis. With the majority of cases being sporadic, we conducted a cohort study to describe the mutational landscape of sporadic amyotrophic lateral sclerosis. We analysed genetic variants in amyotrophic lateral sclerosis-associated genes to assess and potentially increase the number of patients eligible for gene-specific therapies. We screened 2340 sporadic amyotrophic lateral sclerosis patients from the German Network for motor neuron diseases for variants in 36 amyotrophic lateral sclerosis-associated genes using targeted next-generation sequencing and for the C9orf72 hexanucleotide repeat expansion. The genetic analysis could be completed on 2267 patients. Clinical data included age at onset, disease progression rate and survival. In this study, we found 79 likely pathogenic Class 4 variants and 10 pathogenic Class 5 variants (without the C9orf72 hexanucleotide repeat expansion) according to the American College of Medical Genetics and Genomics guidelines, of which 31 variants are novel. Thus, including C9orf72 hexanucleotide repeat expansion, Class 4, and Class 5 variants, 296 patients, corresponding to ∼13% of our cohort, could be genetically resolved. We detected 437 variants of unknown significance of which 103 are novel. Corroborating the theory of oligogenic causation in amyotrophic lateral sclerosis, we found a co-occurrence of pathogenic variants in 10 patients (0.4%) with 7 being C9orf72 hexanucleotide repeat expansion carriers. In a gene-wise survival analysis, we found a higher hazard ratio of 1.47 (95% confidence interval 1.02-2.1) for death from any cause for patients with the C9orf72 hexanucleotide repeat expansion and a lower hazard ratio of 0.33 (95% confidence interval 0.12-0.9) for patients with pathogenic SOD1 variants than for patients without a causal gene mutation. In summary, the high yield of 296 patients (∼13%) harbouring a pathogenic variant and oncoming gene-specific therapies for SOD1/FUS/C9orf72, which would apply to 227 patients (∼10%) in this cohort, corroborates that genetic testing should be made available to all sporadic amyotrophic lateral sclerosis patients after respective counselling.

Axonal degeneration as a therapeutic target in the CNS.

Degeneration of the axon is an important step in the pathomechanism of traumatic, inflammatory and degenerative neurological diseases. Increasing evidence suggests that axonal degeneration occurs early in the course of these diseases and therefore represents a promising target for future therapeutic strategies. We review the evidence for axonal destruction from pathological findings and animal models with particular emphasis on neurodegenerative and neurotraumatic disorders. We discuss the basic morphological and temporal modalities of axonal degeneration (acute, chronic and focal axonal degeneration and Wallerian degeneration). Based on the mechanistic concepts, we then delineate in detail the major molecular mechanisms that underlie the degenerative cascade, such as calcium influx, axonal transport, protein aggregation and autophagy. We finally concentrate on putative therapeutic targets based on the mechanistic prerequisites.

Glial fibrillary acidic protein in cerebrospinal fluid of patients with spinal muscular atrophy.

OBJECTIVE: Activated astroglia is involved in the pathophysiology of neurodegenerative diseases and has also been described in animal models of spinal muscular atrophy (SMA). Given the urgent need of biomarkers for treatment monitoring of new RNA-modifying and gene replacement therapies in SMA, we examined glial fibrillary acidic protein concentrations in cerebrospinal fluid (cGFAP) as a marker of astrogliosis in SMA. METHODS: 58 adult patients and 21 children with genetically confirmed 5q-associated SMA from four German motor neuron disease specialist care centers and 30 age- and sex-matched controls were prospectively included in this study. cGFAP was measured and correlated to motor performance and disease severity. Additionally, we compared cGFAP with neurofilament light chain concentrations in cerebrospinal fluid (cNfL). RESULTS: cGFAP concentrations did not differ from controls but showed higher levels in more severely affected patients after adjustment for patients' age. Normalized cNfL values were associated with disease severity. Within 14 months of nusinersen treatment, cGFAP concentrations did not change, while cNfL decreased significantly. INTERPRETATION: cGFAP is not an outstanding biomarker in SMA, but might support the hypothesis that glial activation is involved in SMA pathology. Unlike previously suggested, cNfL may be a promising biomarker also in adult patients with SMA, which should be subject to further investigations.

Safety and Effectiveness of Long-term Intravenous Administration of Edaravone for Treatment of Patients With Amyotrophic Lateral Sclerosis.

Importance: Intravenous edaravone is approved as a disease-modifying drug for patients with amyotrophic lateral sclerosis (ALS), but evidence for efficacy is limited to short-term beneficial effects shown in the MCI186-ALS19 study in a subpopulation in which efficacy was expected. Objective: To evaluate the long-term safety and effectiveness of intravenous edaravone therapy for patients with ALS in a real-world clinical setting. Design, Setting, and Participants: Multicenter, propensity score-matched cohort study conducted between June 2017 and March 2020 at 12 academic ALS referral centers associated with the German Motor Neuron Disease Network. Of 1440 patients screened, 738 were included in propensity score matching. Final analyses included 324 patients with ALS comprising 194 patients who started intravenous edaravone treatment (141 received ≥4 consecutive treatment cycles; 130 matched) and 130 propensity score-matched patients with ALS receiving standard therapy. All patients had probable or definite ALS according to the El Escorial criteria, with disease onset between December 2012 and April 2019. Subgroups were defined by applying the MCI186-ALS19 study inclusion criteria to evaluate whether patients would have been considered eligible (EFAS) or ineligible (non-EFAS). Exposures: Intravenous edaravone plus riluzole vs riluzole only. Main Outcomes and Measures: Patient characteristics and systematic safety assessment for patients who received at least 1 dose of intravenous edaravone. Effectiveness assessment of edaravone was conducted among patients who received at least 4 treatment cycles compared with propensity score-matched patients with ALS who received only standard therapy. Primary outcome was disease progression measured by decrease in the ALS Functional Rating Scale-Revised (ALSFRS-R) score. Secondary outcomes were survival probability, time to ventilation, and change in disease progression before vs during treatment. To account for the matched design, patients receiving edaravone and their corresponding matched controls were regarded as related samples in disease progression analyses; stratification for propensity score quintiles was used for survival probability and time to ventilation analyses. Results: A total of 194 patients started intravenous edaravone treatment; 125 (64%) were male, and the median age was 57.5 years (IQR, 50.7-63.8 years). Potential adverse effects were observed in 30 cases (16%), most notably infections at infusion sites and allergic reactions. Disease progression among 116 patients treated for a median of 13.9 months (IQR, 8.9-13.9 months) with edaravone did not differ from 116 patients treated for a median of 11.2 months (IQR, 6.4-20.0 months) with standard therapy (ALSFRS-R points/month, -0.91 [95% CI, -0.69 to -1.07] vs -0.85 [95% CI, -0.66 to -0.99]; P = .37). No significant differences were observed in the secondary end points of survival probability, time to ventilation, and change in disease progression. Similarly, outcomes between patients treated with edaravone and matched patients did not differ within the EFAS and non-EFAS subgroups. Conclusions and Relevance: This cohort study using propensity score matching found that, although long-term intravenous edaravone therapy for patients with ALS was feasible and mainly well tolerated, it was not associated with any disease-modifying benefit. Intravenous edaravone may not provide a clinically relevant additional benefit compared with standard therapy alone.

Challenges and opportunities for Multi-National Investigator-Initiated clinical trials for ALS: European and United States collaborations.

An inherent challenge to clinical trials that aim to test the efficacy of experimental therapeutics for patients with amyotrophic lateral sclerosis (ALS) is the relative rarity of the disease. A promising solution to this problem is a multi-center approach that ideally includes sites distributed across a broad geographic area. In support of such an approach, the European E-RARE program and the United States National Institutes of Health (NIH) partnered to support the investigator-initiated ROCK-ALS trial (Eudra-CT-Nr.: 2017-003676-31, NCT03792490) as a multi-national collaboration between centers in Europe and North America that is led by European investigators. During the set-up of this international trial, however, a number of unanticipated legal, administrative, and financial complexities emerged that required significant adaptation of the proposed trial scheme. Here, we report our experience navigating these obstacles and describe the potential solutions that we explored. Our experience may inform future efforts to implement multi-national investigator-initiated trials that involve both European and United States centers.

Serum creatine kinase and creatinine in adult spinal muscular atrophy under nusinersen treatment.

OBJECTIVE: To determine whether serum creatine kinase activity (CK) and serum creatinine concentration (Crn) are prognostic and predictive biomarkers for disease severity, disease progression, and nusinersen treatment effects in adult patients with 5q-associated spinal muscular atrophy (SMA). METHODS: Within this retrospective, multicenter observational study in 206 adult patients with SMA, we determined clinical subtypes (SMA types, ambulatory ability) and repeatedly measured CK and Crn and examined disease severity scores (Hammersmith Functional Motor Scale Expanded, Revised Upper Limb Module, and revised Amyotrophic Lateral Sclerosis Functional Rating Scale). Patients were followed under nusinersen treatment for 18 months. RESULTS: CK and Crn differed between clinical subtypes and correlated strongly with disease severity scores (e.g., for Hammersmith Functional Motor Scale Expanded: (CK) ρ = 0.786/ (Crn) ρ = 0.558). During the 18 months of nusinersen treatment, CK decreased (∆CK = -17.56%, p < 0.0001), whereas Crn slightly increased (∆Crn = +4.75%, p < 0.05). INTERPRETATION: Serum creatine kinase activity and serum creatinine concentration reflect disease severity of spinal muscular atrophy and are promising biomarkers to assess patients with spinal muscular atrophy during disease course and to predict treatment response. The decrease of creatine kinase activity, combined with the tendency of creatinine concentration to increase during nusinersen treatment, suggests reduced muscle mass wasting with improved muscle energy metabolism.

TGF-beta 1 enhances neurite outgrowth via regulation of proteasome function and EFABP.

Malfunction of the ubiquitin-proteasome system has been implicated as a causal factor in the pathogenesis of aggregation-related disorders, e.g. Parkinson's disease. We show here that Transforming growth factor-beta 1 (TGF-beta), a multifunctional cytokine and trophic factor for dopaminergic (DAergic) neurons modulates proteasome function in primary midbrain neurons. TGF-beta differentially inhibited proteasomal subactivities with a most pronounced time-dependent inhibition of the peptidyl-glutamyl peptide hydrolyzing-like and chymotrypsin-like subactivity. Regulation of proteasomal activity could be specifically quantified in the DAergic subpopulation. Protein blot analysis revealed an accumulation of ubiquitinated proteins after TGF-beta treatment. The identity of these enriched proteins was further analyzed by 2D-gel electrophoresis and mass spectrometry. We found epidermal fatty acid binding protein (EFABP) to be strongly increased and ubiquitinated after TGF-beta treatment and confirmed this finding by co-immunoprecipitation. While application of TGF-beta increased neurite regeneration in a scratch lesion model, downregulation of EFABP by siRNA significantly decreased this effect. We thus postulate that a differential regulation of proteasomal function, as demonstrated for TGF-beta, can result in an enrichment of proteins, such as EFABP, that mediate physiological functions, such as neurite regeneration.

Compassionate Use of the ROCK Inhibitor Fasudil in Three Patients With Amyotrophic Lateral Sclerosis.

The Rho kinase (ROCK) inhibitor Fasudil is a promising drug for a disease-modifying therapy of amyotrophic lateral sclerosis (ALS). In preclinical models, Fasudil was shown to increase motor neuron survival, inhibit axonal degeneration, enhance axonal regeneration and modulate microglial function in vitro and in vivo. It prolonged survival and improved motor function of SOD1-G93A-mice. Recently, a phase IIa clinical trial has been commenced to investigate the safety, tolerability, and efficacy of Fasudil in ALS patients at an early stage of disease (ROCK-ALS trial, NCT03792490, Eudra-CT-Nr.: 2017-003676-31). Although Fasudil has been approved in Japan for many years for the treatment of vasospasms following subarachnoid hemorrhage and is known to have a favorable side effect profile in these patients, there is no data on its use in human patients with ALS or any other neurodegenerative conditions. Here, we report the first three cases of compassionate use of Fasudil in patients with ALS. Between May 2017 and February 2019, one male (66 years old) and two female (62 and 68 years old) subjects with probable or definite ALS according to the El Escorial criteria (one of the females having a pathogenic SOD1 mutation) were administered Fasudil 30 mg intravenously twice daily over 45 min on 20 consecutive working days. Blood pressure, heart rate and routine laboratory tests were constantly controlled. All three subjects tolerated the Fasudil infusions well without any obvious side effects. Interestingly, the slow vital capacity showed a significant increase in one of the patients. Taken together, we report here the first compassionate use of the ROCK inhibitor Fasudil in three ALS patients, which was well-tolerated.