Gene therapy for primary myopathies: literature review and prospects.

Gene therapy has emerged as a promising frontier in the pursuit of effective treatments for primary myopathies. This scientific review explores the application of viral vectors and more specifically of recombinant adeno-associated virus (rAAV) vectors as a potent gene delivery tool in the context of primary myopathies, highlighting its transformative potential. Focusing on primary myopathies, including Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophies (LGMDs), X-linked myotubular myopathy (XLMTM), and Pompe disease, we review the ongoing pre-clinical and clinical trials that underscore the therapeutic promise of rAAV-based gene therapies. Recent developments in gene therapy have unveiled innovative gene transfer approaches, particularly with rAAV vectors. These vectors offer a well-tolerated and efficient means of delivering corrective genetic material to diseased muscles, thereby addressing the root causes of primary myopathies. Encouraging data from pre-clinical studies and early clinical trials have demonstrated the potential to ameliorate muscle function, reduce pathological manifestations, and enhance the quality of life for patients afflicted with these devastating diseases. However, the transition from bench to bedside is not without challenges. This review emphasizes the critical need for a comprehensive risk management strategy to better handle potential side effects and immune responses associated with gene therapy. As the field of gene therapy for primary myopathies is advancing, it is imperative to refine and optimize safety measures, ensuring that the transformative potential of these therapies is realized while the risks are minimized. © 2023 Published by Elsevier Masson SAS on behalf of French Society of Pediatrics.

The value of serum creatinine as biomarker of disease progression in spinal and bulbar muscular atrophy (SBMA).

Serum creatinine has been indicated as a potential marker of motor function in SBMA and results form previous longitudinal studies pointed to its decline over time. This is a longitudinal retrospective study investigating creatinine changes over a 36-month-period in 73 patients with SBMA. Severity and progression of the disease was assessed according to serum creatine kinase (CK) values, manual muscle testing (MMT), SBMA functional rating scale (SBMAFRS) score, 6-min-walk test (6MWT) value, and spirometry (forced vital capacity, fVC%) obtained at the baseline and at each of the annual follow-up visits. Baseline serum creatinine concentrations positively correlated with 6MWT, the MMT megascore score of both the upper (ULM) and lower (LLM) limbs and SBMAFRS. No correlation was found with CK or fVC% values. Similar correlation results were achieved at all the subsequent time points. Longitudinal assessments conducted by the generalized estimating equations (GEE) method returned significant changes for SBMAFRS (- 1.41 points per year, p < 0.001), ULM and LLM (- 0.69, p = 0.01; and - 1.07, p < 0.001, respectively), 6MWT (- 47 m, p < 0.001) but not for creatinine (- 0.82, p > 0.05). We also observed that creatinine levels at baseline did not correlate with changes in the other measures from baseline at each annual visit. Our data do not support a role for serum creatinine as sensitive biomarker of disease progression, and possibily prognosis, in SBMA.

Phenotype Presentation and Molecular Diagnostic Yield in Non-5q Spinal Muscular Atrophy.

Background and Objectives: Spinal muscular atrophy (SMA) is mainly caused by homozygous SMN1 gene deletions on 5q13. Non-5q SMA patients' series are lacking, and the diagnostic yield of next-generation sequencing (NGS) is largely unknown. The aim of this study was to describe the clinical and genetic landscape of non-5q SMA and evaluate the performance of neuropathy gene panels in these disorders. Methods: Description of patients with non-5q SMA followed in the different neuromuscular reference centers in France as well as in London, United Kingdom. Patients without a genetic diagnosis had undergone at least a neuropathy or large neuromuscular gene panel. Results: Seventy-one patients from 65 different families were included, mostly sporadic cases (60.6%). At presentation, 21 patients (29.6%) showed exclusive proximal weakness (P-SMA), 35 (49.3%) showed associated distal weakness (PD-SMA), and 15 (21.1%) a scapuloperoneal phenotype (SP-SMA). Thirty-two patients (45.1%) had a genetic diagnosis: BICD2 (n = 9), DYNC1H1 (n = 7), TRPV4 (n = 4), VCP, HSBP1, AR (n = 2), VRK1, DNAJB2, MORC2, ASAH1, HEXB, and unexpectedly, COL6A3 (n = 1). The genetic diagnostic yield was lowest in P-SMA (6/21, 28.6%) compared with PD-SMA (16/35, 45.7%) and SP-SMA (10/15, 66.7%). An earlier disease onset and a family history of the disease or consanguinity were independent predictors of a positive genetic diagnosis. Neuropathy gene panels were performed in 59 patients with a 32.2% diagnostic yield (19/59). In 13 additional patients, a genetic diagnosis was achieved through individual gene sequencing or an alternative neuromuscular NGS. Discussion: Non-5q SMA is genetically heterogeneous, and neuropathy gene panels achieve a molecular diagnosis in one-third of the patients. The diagnostic yield can be increased by sequencing of other neuromuscular and neurometabolic genes. Nevertheless, there is an unmet need to cluster these patients to aid in the identification of new genes.

Anoctamin-5 related muscle disease: clinical and genetic findings in a large European cohort.

Anoctamin-5 related muscle disease is caused by biallelic pathogenic variants in the anoctamin-5 gene (ANO5) and shows variable clinical phenotypes: limb-girdle muscular dystrophy type 12 (LGMD-R12), distal muscular dystrophy type 3 (MMD3), pseudometabolic myopathy or asymptomatic hyperCKaemia. In this retrospective, observational, multicentre study we gathered a large European cohort of patients with ANO5-related muscle disease to study the clinical and genetic spectrum and genotype-phenotype correlations. We included 234 patients from 212 different families, contributed by 15 centres from 11 European countries. The largest subgroup was LGMD-R12 (52.6%), followed by pseudometabolic myopathy (20.5%), asymptomatic hyperCKaemia (13.7%) and MMD3 (13.2%). In all subgroups, there was a male predominance, except for pseudometabolic myopathy. Median age at symptom onset of all patients was 33 years (range 23-45 years). The most frequent symptoms at onset were myalgia (35.3%) and exercise intolerance (34.1%), while at last clinical evaluation most frequent symptoms and signs were proximal lower limb weakness (56.9%) and atrophy (38.1%), myalgia (45.1%) and atrophy of the medial gastrocnemius muscle (38.4%). Most patients remained ambulatory (79.4%). At last evaluation, 45.9% of patients with LGMD-R12 additionally had distal weakness in the lower limbs and 48.4% of patients with MMD3 also showed proximal lower limb weakness. Age at symptom onset did not differ significantly between males and females. However, males had a higher risk of using walking aids earlier (P = 0.035). No significant association was identified between sportive versus non-sportive lifestyle before symptom onset and age at symptom onset nor any of the motor outcomes. Cardiac and respiratory involvement that would require treatment occurred very rarely. Ninety-nine different pathogenic variants were identified in ANO5 of which 25 were novel. The most frequent variants were c.191dupA (p.Asn64Lysfs*15) (57.7%) and c.2272C>T (p.Arg758Cys) (11.1%). Patients with two loss-of function variants used walking aids at a significantly earlier age (P = 0.037). Patients homozygous for the c.2272C>T variant showed a later use of walking aids compared to patients with other variants (P = 0.043). We conclude that there was no correlation of the clinical phenotype with the specific genetic variants, and that LGMD-R12 and MMD3 predominantly affect males who have a significantly worse motor outcome. Our study provides useful information for clinical follow up of the patients and for the design of clinical trials with novel therapeutic agents.

HINT1 neuropathy: Expanding the genotype and phenotype spectrum.

Inherited peripheral neuropathy (IPN) is a heterogeneous group of disorders due to pathogenic variation in more than 100 genes. In 2012, the first cases of IPN associated with HINT1 pathogenic variations were described in 33 families sharing the same phenotype characterized by an axonal neuropathy with neuromyotonia and autosomal recessive inheritance (NMAN: OMIM #137200). Histidine Triad Nucleotide Binding Protein 1 regulates transcription, cell-cycle control, and is possibly involved in neuropsychiatric pathophysiology. Herein, we report seven French patients with NMAN identified by Next Generation Sequencing. We conducted a literature review and compared phenotypic and genotypic features with our cohort. We identified a new HINT1 pathogenic variation involved in NMAN: c.310G>C p.(Gly104Arg). This cohort is comparable with literature data regarding age of onset (7,4yo), neuronal involvement (sensorimotor 3/7 and motor pure 4/7), and skeletal abnormalities (scoliosis 3/7, feet anomalies 6/7). We expand the phenotypic spectrum of HINT1-related neuropathy by describing neurodevelopmental or psychiatric features in six out of seven individuals such as generalized anxiety disorder (GAD), obsessive-compulsive disorder (OCD), mood disorder and attention deficit hyperactivity disorder (ADHD). However, only 3/128 previously described patients had neuropsychiatric symptomatology or neurodevelopmental disorder. These features could be part of HINT1-related disease, and we should further study the clinical phenotype of the patients.

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles.

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.

Biomarkers for C9orf7-ALS in Symptomatic and Pre-symptomatic Patients: State-of-the-art in the New Era of Clinical Trials.

The development of new possible treatments for C9orf72-related ALS and the possibility of early identification of subjects genetically at risk of developing the disease is creating a critical need for biomarkers to track neurodegeneration that could be used as outcome measures in clinical trials. Current candidate biomarkers in C9orf72-ALS include neuropsychology tests, imaging, electrophysiology as well as different circulating biomarkers. Neuropsychology tests show early executive and verbal function involvement both in symptomatic and asymptomatic mutation carriers. At brain MRI, C9orf72-ALS patients present diffuse white and grey matter degeneration, which are already identified up to 20 years before symptom onset and that seem to be slowly progressive over time, while regions of altered connectivity at fMRI and of hypometabolism at [18F]FDG PET have been described as well. At the same time, spinal cord MRI has also shown progressive decrease of FA in the cortico-spinal tract over time. On the side of wet biomarkers, neurofilament proteins are increased both in the CSF and serum just before symptom onset and tend to slowly increase over time, while poly(GP) protein can be detected in the CSF and probably used as target engagement marker in clinical trials.

Deep phenotyping of an international series of patients with late-onset dysferlinopathy.

BACKGROUND: To describe the clinical, pathological, and molecular characteristics of late-onset (LO) dysferlinopathy patients. METHODS: Retrospective series of patients with LO dysferlinopathy, defined by an age at onset of symptoms ≥30 years, from neuromuscular centers in France and the International Clinical Outcome Study for dysferlinopathy (COS). Patients with early-onset (EO) dysferlinopathy (<30 years) were randomly selected from the COS study as a control group, and the North Star Assessment for Dysferlinopathy (NSAD) and Activity Limitation (ACTIVLIM) scores were used to assess functionality. Muscle biopsies obtained from 11 LO and 11 EO patients were revisited. RESULTS: Forty-eight patients with LO dysferlinopathy were included (28 females). Median age at onset of symptoms was 37 (range 30-57) years and most patients showed a limb-girdle (n = 26) or distal (n = 10) phenotype. However, compared with EO dysferlinopathy patients (n = 48), LO patients more frequently showed atypical phenotypes (7 vs. 1; p = 0.014), including camptocormia, lower creatine kinase levels (2855 vs. 4394 U/L; p = 0.01), and higher NSAD (p = 0.008) and ACTIVLIM scores (p = 0.016). Loss of ambulation in LO patients tended to occur later (23 ± 4.4 years after disease onset vs. 16.3 ± 6.8 years; p = 0.064). Muscle biopsy of LO patients more frequently showed an atypical pattern (unspecific myopathic changes) as well as significantly less necrosis regeneration and inflammation. Although LO patients more frequently showed missense variants (39.8% vs. 23.9%; p = 0.021), no differences in dysferlin protein expression were found on Western blot. CONCLUSIONS: Late-onset dysferlinopathy patients show a higher frequency of atypical presentations, are less severely affected, and show milder dystrophic changes in muscle biopsy.

Development of new outcome measures for adult SMA type III and IV: a multimodal longitudinal study.

OBJECTIVE: The aim of this study was the comprehensive characterisation of longitudinal clinical, electrophysiological and neuroimaging measures in type III and IV adult spinal muscular atrophy (SMA) with a view to propose objective monitoring markers for future clinical trials. METHODS: Fourteen type III or IV SMA patients underwent standardised assessments including muscle strength testing, functional evaluation (SMAFRS and MFM), MUNIX (abductor pollicis brevis, APB; abductor digiti minimi, ADM; deltoid; tibialis anterior, TA; trapezius) and quantitative cervical spinal cord MRI to appraise segmental grey and white matter atrophy. Patients underwent a follow-up assessment with the same protocol 24 months later. Longitudinal comparisons were conducted using the Wilcoxon-test for matched data. Responsiveness was estimated using standardized response means (SRM) and a composite score was generated based on the three most significant variables. RESULTS: Significant functional decline was observed based on SMAFRS (p = 0.019), pinch and knee flexion strength (p = 0.030 and 0.027), MUNIX and MUSIX value in the ADM (p = 0.0006 and 0.043) and in TA muscle (p = 0.025). No significant differences were observed based on cervical MRI measures. A significant reduction was detected in the composite score (p = 0.0005, SRM = -1.52), which was the most responsive variable and required a smaller number of patients than single variables in the estimation of sample size for clinical trials. CONCLUSIONS: Quantitative strength testing, SMAFRS and MUNIX readily capture disease progression in adult SMA patients. Composite multimodal scores increase predictive value and may reduce sample size requirements in clinical trials.

Beyond the Traditional Clinical Trials for Amyotrophic Lateral Sclerosis and The Future Impact of Gene Therapy.

Amyotrophic lateral sclerosis (ALS) is a devastating and incurable motor neuron (MN) disorder affecting both upper and lower MNs. Despite impressive advances in the understanding of the disease's pathological mechanism, classical pharmacological clinical trials failed to provide an efficient cure for ALS over the past twenty years. Two different gene therapy approaches were recently approved for the monogenic disease Spinal muscular atrophy, characterized by degeneration of lower MNs. This milestone suggests that gene therapy-based therapeutic solutions could be effective for the treatment of ALS. This review summarizes the possible reasons for the failure of traditional clinical trials for ALS. It provides then a focus on the advent of gene therapy approaches for hereditary forms of ALS. Specifically, it describes clinical use of antisense oligonucleotides in three familial forms of ALS, caused by mutations in SOD1, C9orf72 and FUS genes, respectively.. Clinical and pre-clinical studies based on AAV-mediated gene therapy approaches for both familial and sporadic ALS cases are presented as well. Overall, this overview highlights the potential of gene therapy as a transforming technology that will have a huge impact on treatment perspective for ALS patients and on the design of future clinical trials.

Neural Correlates of Motor Imagery of Gait in Amyotrophic Lateral Sclerosis.

BACKGROUND: Gait impairment is poorly characterized in amyotrophic lateral sclerosis (ALS), despite increasing evidence of extrapyramidal and cerebellar dysfunction. Gait impairment adds to the considerable motor disability of ALS patients and requires targeted multidisciplinary interventions. PURPOSE: To assess gait imagery-specific networks and functional adaptation in ALS. STUDY TYPE: Prospective. POPULATION: Seventeen ALS patients with lower motor neuron predominant (LMNp) disability, 14 patients with upper motor neurons predominant (UMNp) disease, and 14 healthy controls were included. FIELD STRENGTH/SEQUENCES: 3T / gradient echo echo planar (GE-EPI). ASSESSMENT: Subjects performed a dual motor imagery task: normal and precision gait. The Movement Imagery Questionnaire - Revised Second Version (MIQ-rs) was used to appraise movement imagery in each participant. Study group-specific activation patterns were evaluated during motor imagery of gait. Additional generalized psychophysiological interaction analyses were carried out using the supplementary motor area, caudate, cerebellum, and superior parietal lobule as seed regions. STATISTICAL TESTS: Repeated-measures analysis of variance (ANOVA) was used to compare time imagery and MIQ-rs scores between groups. Size effects were also reported as partial eta squared (η2). One-way ANOVA was performed to explore differences in terms of connexions during motor imagery tasks. RESULTS: A significant increase in imagery time in UMNp patients compared to controls (P < 0.05) and LMNp (P < 0.05) during imagined gait was demonstrated. UMNp patients exhibited altered supplementary motor area, precentral gyrus, superior parietal lobule, and dorsolateral prefrontal cortex activation and increased orbitofrontal (pFDR(False Discovery Rate) < 0.05), posterior parietal (pFDR < 0.05) caudate (pFDR < 0.05), and cerebellar (pFDR < 0.05) signal during imagined locomotion. Increased effective connectivity of the striato-cerebellar and parieto-cerebellar circuits was also demonstrated. Additional activation was detected in the insula and cingulate cortex. DATA CONCLUSION: Enhanced striato- and parieto-cerebellar networks in UMNp ALS patients are likely to represent a compensatory response to impaired postural control. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 5.

The Identification of Novel Biomarkers Is Required to Improve Adult SMA Patient Stratification, Diagnosis and Treatment.

Spinal muscular atrophy (SMA) is currently classified into five different subtypes, from the most severe (type 0) to the mildest (type 4) depending on age at onset, best motor function achieved, and copy number of the SMN2 gene. The two recent approved treatments for SMA patients revolutionized their life quality and perspectives. However, upon treatment with Nusinersen, the most widely administered therapy up to date, a high degree of variability in therapeutic response was observed in adult SMA patients. These data, together with the lack of natural history information and the wide spectrum of disease phenotypes, suggest that further efforts are needed to develop precision medicine approaches for all SMA patients. Here, we compile the current methods for functional evaluation of adult SMA patients treated with Nusinersen. We also present an overview of the known molecular changes underpinning disease heterogeneity. We finally highlight the need for novel techniques, i.e., -omics approaches, to capture phenotypic differences and to understand the biological signature in order to revise the disease classification and device personalized treatments.

Cerebellar degeneration in adult spinal muscular atrophy patients.

BACKGROUND: Spinal muscular atrophy (SMA) is a genetic motor neuron disease related to deletions in the SMN1 gene. There is mounting evidence that the disease is not restricted to motor neurons. In this neuroimaging study, we aimed to investigate the presence of in-vivo cerebellar damage in adult SMA patients not treated with disease-modifying treatment. METHODS: Twenty-five molecularly confirmed patients with SMA type III or IV and 25 healthy controls underwent MRI with cerebellar focused structural analysis by the CERES automated pipeline. Volumetry (total and gray matter-GM) as well as cortical thickness of the cerebellar lobules were compared in both groups. Full clinical and demographic data were then assessed for correlations with cerebellar imaging findings. RESULTS: Volumes of cerebellar lobules VIIIB (right), IX and X were significantly smaller in patients with SMA. Lobule IX also had GM atrophy in comparison to controls. We found no significant correlation between clinical findings and cerebellar damage. CONCLUSIONS: Neuroimaging detects cerebellar structural changes in adult SMA patients, suggesting that neurodegeneration is not confined to the lower motor neurons in the disease.

The French national protocol for Kennedy's disease (SBMA): consensus diagnostic and management recommendations.

BACKGROUND: Kennedy's disease (KD), also known as spinal and bulbar muscular atrophy (SBMA), is a rare, adult-onset, X-linked recessive neuromuscular disease caused by CAG expansions in exon 1 of the androgen receptor gene (AR). The objective of the French national diagnostic and management protocol is to provide evidence-based best practice recommendations and outline an optimised care pathway for patients with KD, based on a systematic literature review and consensus multidisciplinary observations. RESULTS: The initial evaluation, confirmation of the diagnosis, and management should ideally take place in a tertiary referral centre for motor neuron diseases, and involve an experienced multidisciplinary team of neurologists, endocrinologists, cardiologists and allied healthcare professionals. The diagnosis should be suspected in an adult male presenting with slowly progressive lower motor neuron symptoms, typically affecting the lower limbs at onset. Bulbar involvement (dysarthria and dysphagia) is often a later manifestation of the disease. Gynecomastia is not a constant feature, but is suggestive of a suspected diagnosis, which is further supported by electromyography showing diffuse motor neuron involvement often with asymptomatic sensory changes. A suspected diagnosis is confirmed by genetic testing. The multidisciplinary assessment should ascertain extra-neurological involvement such as cardiac repolarisation abnormalities (Brugada syndrome), signs of androgen resistance, genitourinary abnormalities, endocrine and metabolic changes (glucose intolerance, hyperlipidemia). In the absence of effective disease modifying therapies, the mainstay of management is symptomatic support using rehabilitation strategies (physiotherapy and speech therapy). Nutritional evaluation by an expert dietician is essential, and enteral nutrition (gastrostomy) may be required. Respiratory management centres on the detection and treatment of bronchial obstructions, as well as screening for aspiration pneumonia (chest physiotherapy, drainage, positioning, breath stacking, mechanical insufflation-exsufflation, cough assist machnie, antibiotics). Non-invasive mechanical ventilation is seldom needed. Symptomatic pharmaceutical therapy includes pain management, endocrine and metabolic interventions. There is no evidence for androgen substitution therapy. CONCLUSION: The French national Kennedy's disease protocol provides management recommendations for patients with KD. In a low-incidence condition, sharing and integrating regional expertise, multidisciplinary experience and defining consensus best-practice recommendations is particularly important. Well-coordinated collaborative efforts will ultimately pave the way to the development of evidence-based international guidelines.

Absence of hyperexcitability of spinal motoneurons in patients with amyotrophic lateral sclerosis.

KEY POINTS: •Amyotrophic lateral sclerosis (ALS) motoneurons become hypoexcitable with disease progression in experimental models, raising questions about the neural hyperexcitability supported by clinical observations. •A variant of the ∆F method, based on motor unit discharge frequency modulations during recruitment and derecruitment, has been developed to investigate the motoneuron capacity to self-sustained discharge in patients. •The modulation of motor unit firing rate during ramp contraction and vibration-induced recruitment are modified in ALS, suggesting lower motoneuron capacity to self-sustained discharge, which is a sign of hypoexcitability. •∆F-D decreases with functional impairment and its reduction is more pronounced in fast progressors. •In patients with ALS, motoneurons exhibit hypoexcitability, which increases with disease progression. ABSTRACT: Experimental models have primarily revealed spinal motoneuron hypoexcitability in amyotrophic lateral sclerosis (ALS), which is contentious considering the role of glutamate-induced excitotoxicity in neurodegeneration and clinical features rather supporting hyperexcitability. This phenomenon was evaluated in human patients by investigating changes in motor unit firing during contraction and relaxation. Twenty-two ALS patients with subtle motor deficits and 28 controls performed tonic contractions of extensor carpi radialis, triceps brachialis, tibialis anterior and quadriceps, aiming to isolate a low-threshold unit (U1) on the electromyogram (EMG). Subsequently, they performed a stronger contraction or tendon vibration was delivered, to recruit higher threshold unit (U2) for 10 s before they relaxed progressively. EMG and motor unit potential analyses suggest altered neuromuscular function in all muscles, including those with normal strength (Medical Research Council score at 5). During the preconditioning tonic phase, U1 discharge frequency did not differ significantly between groups. During recruitment, the increase in U1 frequency (∆F-R) was comparable between groups both during contraction and tendon vibration. During derecruitment, the decrease in U1 frequency (∆F-D) was reduced in ALS regardless of the recruitment mode, particularly for ∆F-R <8 Hz in the upper limbs, consistent with the muscle weakness profile of the group. ∆F-D was associated with functional disability and its reduction was more pronounced in patients with more rapid disease progression rate. This in vivo study has demonstrated reduced motoneuron capacity for self-sustained discharge, and further supports that motoneurons are normo- to hypoexcitable in ALS patients, similar to observations in experimental models.

Presymptomatic spinal cord pathology in c9orf72 mutation carriers: A longitudinal neuroimaging study.

OBJECTIVE: C9orf72 hexanucleotide repeats expansions account for almost half of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases. Recent imaging studies in asymptomatic C9orf72 carriers have demonstrated cerebral white (WM) and gray matter (GM) degeneration before the age of 40 years. The objective of this study was to characterize cervical spinal cord (SC) changes in asymptomatic C9orf72 hexanucleotide carriers. METHODS: Seventy-two asymptomatic individuals were enrolled in a prospective study of first-degree relatives of ALS and FTD patients carrying the c9orf72 hexanucleotide expansion. Forty of them carried the pathogenic mutation (C9+ ). Each subject underwent quantitative cervical cord imaging. Structural GM and WM metrics and diffusivity parameters were evaluated at baseline and 18 months later. Data were analyzed in C9+ and C9- subgroups, and C9+ subjects were further stratified by age. RESULTS: At baseline, significant WM atrophy was detected at each cervical vertebral level in C9+ subjects older than 40 years without associated changes in GM and diffusion tensor imaging parameters. At 18-month follow-up, WM atrophy was accompanied by significant corticospinal tract (CST) fractional anisotropy (FA) reductions. Intriguingly, asymptomatic C9+ subjects older than 40 years with family history of ALS (as opposed to FTD) also exhibited significant CST FA reduction at baseline. INTERPRETATION: Cervical SC imaging detects WM atrophy exclusively in C9+ subjects older than 40 years, and progressive CST FA reductions can be identified on 18-month follow-up. Cervical SC magnetic resonance imaging readily captures presymptomatic pathological changes and disease propagation in c9orf72-associated conditions. ANN NEUROL 2019;86:158-167.

Comparative Analysis of C9orf72 and Sporadic Disease in a Large Multicenter ALS Population: The Effect of Male Sex on Survival of C9orf72 Positive Patients.

We investigated whether the C9orf72 repeat expansion is associated with specific clinical features, comorbidities, and prognosis in patients with amyotrophic lateral sclerosis (ALS). A cohort of 1417 ALS patients, diagnosed between January 1, 2009 and December 31, 2013 by 13 Italian ALS Referral Centers, was screened for the C9orf72 repeat expansion, and the analyses were performed comparing patients carrying this expansion (ALS-C9Pos) to those negative for this and other explored ALS-related mutations (ALS without genetic mutations, ALSwoGM). Compared to the ALSwoGM group, ALS-C9Pos patients (n = 84) were younger at disease onset, at the first clinical observation and at diagnosis (p < 0.001). After correcting for these differences, we found that ALS-C9Pos patients had higher odds of bulbar onset, diagnosis of frontotemporal dementia (FTD) and family history of ALS, FTD, and Alzheimer's disease and had lower odds of spinal onset, non-invasive ventilation, hypertension and psychiatric diseases than ALSwoGM patients. Among these variables, those related to shorter survival time were: bulbar onset, presence of FTD, hypertension, psychiatric disease, and family history of ALS (p < 0.05). Cox proportional hazards regression multivariate analysis suggested that carrying the C9orf72 repeat expansion was an independent factor negatively impacting on survival time in men (HR 1.58, 95% CI 1.07-2.33, p = 0.021), but not in women (p > 0.05) as well as in the whole sample (p > 0.05). When compared to ALSwoGM, ALS-C9Pos showed an earlier disease onset, no significant diagnostic delay and a higher odds of bulbar onset, FTD and family history of ALS and dementia. Moreover, male sex drove the negative effect of expanded variant on survival, confirming the hypothesis that sex is likely to be a crucial factor in the biology of C9orf72-related disease.

Results from Phase I Clinical Trial with Intraspinal Injection of Neural Stem Cells in Amyotrophic Lateral Sclerosis: A Long-Term Outcome.

The main objective of this phase I trial was to assess the feasibility and safety of microtransplanting human neural stem cell (hNSC) lines into the spinal cord of patients with amyotrophic lateral sclerosis (ALS). Eighteen patients with a definite diagnosis of ALS received microinjections of hNSCs into the gray matter tracts of the lumbar or cervical spinal cord. Patients were monitored before and after transplantation by clinical, psychological, neuroradiological, and neurophysiological assessment. For up to 60 months after surgery, none of the patients manifested severe adverse effects or increased disease progression because of the treatment. Eleven patients died, and two underwent tracheotomy as a result of the natural history of the disease. We detected a transitory decrease in progression of ALS Functional Rating Scale Revised, starting within the first month after surgery and up to 4 months after transplantation. Our results show that transplantation of hNSC is a safe procedure that causes no major deleterious effects over the short or long term. This study is the first example of medical transplantation of a highly standardized cell drug product, which can be reproducibly and stably expanded ex vivo, comprising hNSC that are not immortalized, and are derived from the forebrain of the same two donors throughout this entire study as well as across future trials. Our experimental design provides benefits in terms of enhancing both intra- and interstudy reproducibility and homogeneity. Given the potential therapeutic effects of the hNSCs, our observations support undertaking future phase II clinical studies in which increased cell dosages are studied in larger cohorts of patients. Stem Cells Translational Medicine 2019;8:887&897.