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Despite the intense research focus on the molecular and cellular mechanisms of motor neuron degeneration using models based on specific genetic mutations, there are good reasons to consider amyotrophic lateral sclerosis (ALS) as a ‘system degeneration’, which arises through multiple biological triggers. ALS experts generally agree that loss of function has a clinically focal onset and a non-random pattern of spread, which favours the ‘vulnerable network’ hypothesis, in which the degenerative process propagates through compartmentalised anatomical and functional networks.1 Furthermore, evidence from neuropathological studies suggests that the burden of TDP-43 proteinopathy reflects an orderly progression of spread through anatomically contiguous pathways.2 All of which raises the critical question of the nature of the anatomical substrate of …

Summary Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in axonal transport have been identified in several ALS models, but in vivo evidence remains limited, thus their pathogenetic importance remains to be fully resolved. We therefore analyzed the in vivo dynamics of retrogradely transported, neurotrophin-containing signaling endosomes in nerve axons of two ALS mouse models with mutations in the RNA processing genes TARDBP and FUS. TDP-43M337V mice, which show neuromuscular pathology without motor neuron loss, display axonal transport perturbations manifesting between 1.5 and 3 months and preceding symptom onset. Contrastingly, despite 20% motor neuron loss, transport remained largely unaffected in FusΔ14/+ mice. Deficiencies in retrograde axonal transport of signaling endosomes are therefore not shared by all ALS-linked genes, indicating that there are mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes., Graphical Abstract, Highlights • Mutant TDP-43 mice display in vivo defects in signaling endosome axonal transport • Despite motor neuron loss, mutant FUS mice display no such deficiency • Axonal transport disruption is thus not common to all ALS mouse models, Sleigh et al. address the importance of disturbances in axonal transport in two mouse models for ALS. They find that deficiencies in the in vivo axonal delivery of target tissue-derived survival factors are not a common feature of all ALS mouse models, suggesting mechanistic distinctions in different ALS-linked genes.

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in the basic neuronal process of axonal transport have been identified in several ALS models. However, in vivo evidence of early/pre-symptomatic deficiencies in neuronal cargo trafficking remains limited, thus the pathogenic importance of axonal transport to the ALS disease spectrum remains to be fully resolved. We therefore analysed the in vivo dynamics of retrogradely transported, neurotrophin-containing signalling endosomes in motor neuron axons of two new mouse models of ALS that have mutations in different RNA processing genes (Tardbp and Fus). TDP-43M337V mice, which show neuromuscular pathology but no overt motor neuron loss, displayed in vivo perturbations in axonal transport that manifested between 1.5 and 3 months and preceded motor symptom onset. In contrast, signalling endosome transport remained largely unaffected in mutant FusΔ14/+ mice, despite 20% motor neuron loss. These findings indicate that deficiencies in retrograde neurotrophin signalling and axonal transport are not common to all ALS-linked genes, and that there are inherent and mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes.

Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of ‘sporadic’ cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS.

Spinocerebellar ataxia type 14 (SCA14) is a subtype of the autosomal dominant cerebellar ataxias that is characterized by slowly progressive cerebellar dysfunction and neurodegeneration. SCA14 is caused by mutations in the PRKCG gene, encoding protein kinase C gamma (PKCγ). Despite the identification of 40 distinct disease-causing mutations in PRKCG, the pathological mechanisms underlying SCA14 remain poorly understood. Here we report the molecular neuropathology of SCA14 in post-mortem cerebellum and in human patient-derived induced pluripotent stem cells (iPSCs) carrying two distinct SCA14 mutations in the C1 domain of PKCγ, H36R and H101Q. We show that endogenous expression of these mutations results in the cytoplasmic mislocalization and aggregation of PKCγ in both patient iPSCs and cerebellum. PKCγ aggregates were not efficiently targeted for degradation. Moreover, mutant PKCγ was found to be hyper-activated, resulting in increased substrate phosphorylation. Together, our findings demonstrate that a combination of both, loss-of-function and gain-of-function mechanisms are likely to underlie the pathogenesis of SCA14, caused by mutations in the C1 domain of PKCγ. Importantly, SCA14 patient iPSCs were found to accurately recapitulate pathological features observed in post-mortem SCA14 cerebellum, underscoring their potential as relevant disease models and their promise as future drug discovery tools.

BackgroundThe thalamus is a major neural hub, with selective connections to virtually all cortical regions of the brain. The multisystem neurodegenerative syndrome amyotrophic lateral sclerosis (ALS) has pathogenic overlap with frontotemporal dementia, and objective in vivo markers of extra-motor pathological spread are lacking. To better consider the role of the thalamus in neurodegeneration, the present study assessed the integrity of the thalamus and its connectivity to major cortical regions of the brain in a longitudinal manner.MethodsDiffusion-based MRI tractography was used to parcellate the thalamus into distinct regions based on structural thalamo-cortical connectivity in 20 patients with ALS, half of whom were scanned at two time points, and 31 matched controls scanned on a single occasion.ResultsAt baseline, widespread diffusivity alterations in motor- and extramotor-associated thalamic parcellations were detectable. Longitudinal decline selectively affected thalamic regions associated with frontal and temporal lobe connectivity. Diffusivity measures were significantly correlated with clinical measures of disease burden. Progression of functional disability, as indicated by change on the ALS functional rating scale, was associated with longitudinal change in mean diffusivity of the right frontal lobe thalamic parcellation (r=0.59, p=0.05).ConclusionsRegional thalamic connectivity changes mirror the progressive frontotemporal cortical involvement associated with the motor functional decline in ALS. Longitudinal MRI thalamic parcellation has potential as a non-invasive surrogate marker of cortical dysfunction in ALS.

As clinical evidence supports a negative impact of dysfunctional energy metabolism on the disease progression in amyotrophic lateral sclerosis, it is vital to understand how the energy metabolic pathways are altered and whether they can be restored to slow disease progression. Possible approaches include increasing or rerouting catabolism of alternative fuel sources to supplement the glycolytic and mitochondrial pathways such as glycogen, ketone bodies and nucleosides. To analyse the basis of the catabolic defect in amyotrophic lateral sclerosis we used a novel phenotypic metabolic array. We profiled fibroblasts and induced neuronal progenitor-derived human induced astrocytes from C9orf72 amyotrophic lateral sclerosis patients compared to normal controls, measuring the rates of production of reduced nicotinamide adenine dinucleotides from 91 potential energy substrates. This approach shows for the first time that C9orf72 human induced astrocytes and fibroblasts have an adenosine to inosine deamination defect caused by reduction of adenosine deaminase, which is also observed in induced astrocytes from sporadic patients. Patient-derived induced astrocyte lines were more susceptible to adenosine-induced toxicity, which could be mimicked by inhibiting adenosine deaminase in control lines. Furthermore, adenosine deaminase inhibition in control induced astrocytes led to increased motor neuron toxicity in co-cultures, similar to the levels observed with patient derived induced astrocytes. Bypassing metabolically the adenosine deaminase defect by inosine supplementation was beneficial bioenergetically in vitro, increasing glycolytic energy output and leading to an increase in motor neuron survival in co-cultures with induced astrocytes. Inosine supplementation, in combination with modulation of the level of adenosine deaminase may represent a beneficial therapeutic approach to evaluate in patients with amyotrophic lateral sclerosis.

Cerebrospinal fluid (CSF) extracellular vesicles (EVs) show promise as a source of neurological disease biomarkers, though their precise origin is poorly understood. Current extraction techniques produce disappointing yield and purity. This study describes the application of ultrafiltration liquid chromatography (UFLC) to CSF-EVs, compared with ultracentrifugation (UC), and explores CSF-EV origin. EVs were extracted from human CSF by UC and UFLC and characterized using nanoparticle tracking analysis, electron microscopy and immunoblotting. EV and CSF proteomes were analysed by LC-MS/MS. UFLC-isolated particles have size, morphology and marker expression characteristic of EVs. UFLC provides greater EV yield (UFLC 7.90x108± SD 1.31x108 EVs/mL CSF, UC 1.06x108±0.57x108 p

Background The identification of blood-based biomarkers specific to the diagnosis of amyotrophic lateral sclerosis (ALS) is an active field of academic and clinical research. While inheritance studies have advanced the field, a majority of patients do not have a known genetic link to the disease, making direct sequence-based genetic testing for ALS difficult. The ability to detect biofluid-based epigenetic changes in ALS would expand the relevance of using genomic information for disease diagnosis. Methods Assessing differences in chromosomal conformations (i.e. how they are positioned in 3-dimensions) represents one approach for assessing epigenetic changes. In this study, we used an industrial platform, EpiSwitch™, to compare the genomic architecture of healthy and diseased patient samples (blood and tissue) to discover a chromosomal conformation signature (CCS) with diagnostic potential in ALS. A three-step biomarker selection process yielded a distinct CCS for ALS, comprised of conformation changes in eight genomic loci and detectable in blood. Findings We applied the ALS CCS to determine a diagnosis for 74 unblinded patient samples and subsequently conducted a blinded diagnostic study of 16 samples. Sensitivity and specificity for ALS detection in the 74 unblinded patient samples were 83∙33% (CI 51∙59 to 97∙91%) and 76∙92% (46∙19 to 94∙96%), respectively. In the blinded cohort, sensitivity reached 87∙50% (CI 47∙35 to 99∙68%) and specificity was 75∙0% (34∙91 to 96∙81%). Interpretations The sensitivity and specificity values achieved using the ALS CCS identified and validated in this study provide an indication that the detection of chromosome conformation signatures is a promising approach to disease diagnosis and can potentially augment current strategies for diagnosing ALS. Fund This research was funded by Oxford BioDynamics and Innovate UK. Work in the Oxford MND Care and Research Centre is supported by grants from the Motor Neurone Disease Association and the Medical Research Council. Additional support was provided by the Northeast ALS Consortium (NEALS).

Summary\ud Background\ud Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive, fatal motor neuron disease with a variable natural history. There are no accurate models that predict the disease course and outcomes, which complicates risk assessment and counselling for individual patients, stratification of patients for trials, and timing of interventions. We therefore aimed to develop and validate a model for predicting a composite survival endpoint for individual patients with ALS.\ud \ud Methods\ud We obtained data for patients from 14 specialised ALS centres (each one designated as a cohort) in Belgium, France, the Netherlands, Germany, Ireland, Italy, Portugal, Switzerland, and the UK. All patients were diagnosed in the centres after excluding other diagnoses and classified according to revised El Escorial criteria. We assessed 16 patient characteristics as potential predictors of a composite survival outcome (time between onset of symptoms and non-invasive ventilation for more than 23 h per day, tracheostomy, or death) and applied backward elimination with bootstrapping in the largest population-based dataset for predictor selection. Data were gathered on the day of diagnosis or as soon as possible thereafter. Predictors that were selected in more than 70% of the bootstrap resamples were used to develop a multivariable Royston-Parmar model for predicting the composite survival outcome in individual patients. We assessed the generalisability of the model by estimating heterogeneity of predictive accuracy across external populations (ie, populations not used to develop the model) using internal–external cross-validation, and quantified the discrimination using the concordance (c) statistic (area under the receiver operator characteristic curve) and calibration using a calibration slope.\ud \ud Findings\ud Data were collected between Jan 1, 1992, and Sept 22, 2016 (the largest data-set included data from 1936 patients). The median follow-up time was 97·5 months (IQR 52·9–168·5). Eight candidate predictors entered the prediction model: bulbar versus non-bulbar onset (univariable hazard ratio [HR] 1·71, 95% CI 1·63–1·79), age at onset (1·03, 1·03–1·03), definite versus probable or possible ALS (1·47, 1·39–1·55), diagnostic delay (0·52, 0·51–0·53), forced vital capacity (HR 0·99, 0·99–0·99), progression rate (6·33, 5·92–6·76), frontotemporal dementia (1·34, 1·20–1·50), and presence of a C9orf72 repeat expansion (1·45, 1·31–1·61), all p

ObjectiveWe sought to assess cortical function in amyotrophic lateral sclerosis (ALS) using noninvasive neural signal recording.MethodsResting-state magnetoencephalography was used to measure power fluctuations in neuronal oscillations from distributed cortical parcels in 24 patients with ALS and 24 healthy controls. A further 9 patients with primary lateral sclerosis and a group of 15 asymptomatic carriers of genetic mutations associated with ALS were also studied.ResultsIncreased functional connectivity, particularly from the posterior cingulate cortex, was demonstrated in both patient groups compared to healthy controls. Directionally similar patterns were also evident in the asymptomatic genetic mutation carrier group.ConclusionIncreased cortical functional connectivity elevation is a quantitative marker that reflects ALS pathology across its clinical spectrum, and may develop during the presymptomatic period. The amelioration of pathologic magnetoencephalography signals might be a marker sensitive enough to provide proof-of-principle in the development of future neuroprotective therapeutics.

© 2018 Elsevier Inc., To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.

Spinal muscular atrophy (SMA) is a devastating motor neuron disease that predominantly affects children and represents the most common cause of hereditary infant mortality. The condition results from deleterious variants in SMN1, which lead to depletion of the survival motor neuron protein (SMN). Now, 20 years after the discovery of this genetic defect, a major milestone in SMA and motor neuron disease research has been reached with the approval of the first disease-modifying therapy for SMA by US and European authorities - the antisense oligonucleotide nusinersen. At the same time, promising data from early-stage clinical trials of SMN1 gene therapy have indicated that additional therapeutic options are likely to emerge for patients with SMA in the near future. However, the approval of nusinersen has generated a number of immediate and substantial medical, ethical and financial implications that have the potential to resonate beyond the specific treatment of SMA. Here, we provide an overview of the rapidly evolving therapeutic landscape for SMA, highlighting current achievements and future opportunities. We also discuss how these developments are providing important lessons for the emerging second generation of combinatorial ('SMN-plus') therapies that are likely to be required to generate robust treatments that are effective across a patient's lifespan.

Motor neuron disease (MND) is characterised by progressive neurological deterioration and coexistence of upper and lower motor neuron signs. Over the past decade, evidence has emerged of unique pathophysiological processes, including glutamate-mediated excitotoxicity, which has resulted in the development of novel diagnostic investigations and uncovered potential therapeutic targets. Advances in genetics, including the recently discovered C9orf72 gene, have radically changed the pathological mindset, from MND being classified as a neuromuscular disease to one that MND forms a continuum with other primary neurodegenerative disorders, including frontotemporal dementia. The present review will highlight the improvements that have occurred in clinical care, in conjunction with recent scientific developments.

The transactive response DNA binding protein (TDP-43) is a major component of the characteristic neuronal cytoplasmic inclusions seen in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, are found in sporadic and familial ALS cases. To study the molecular mechanisms of cellular toxicity due to TDP-43 mutations we generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying one of two ALS-associated mutations, A382T or M337V, which were used to generate site-specific bacterial artificial chromosome (BAC) human stable cell lines and BAC transgenic mice. In cell lines and primary motor neurons in culture, TDP-M337V mislocalized to the cytoplasm more frequently than wild-type TDP (wt-TDP) and TDP-A382T, an effect potentiated by oxidative stress. Expression of mutant TDP-M337V correlated with increased apoptosis detected by cleaved caspase-3 staining. Cells expressing mislocalized TDP-M337V spontaneously developed cytoplasmic aggregates, while for TDP-A382T aggregates were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin. Lowering Ca(2+) concentration in the ER of wt-TDP cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca(2+) dysregulation as a potential mediator of pathology through alterations in Bcl-2 protein levels. Ca(2+) signaling from the ER was impaired in immortalized cells and primary neurons carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca(2+) stores content and delayed Ca(2+) release compared with cells carrying wt-TDP. The deficits in Ca(2+) release in human cells correlated with the upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored the amplitude of Ca(2+) oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 and Bcl-2 mediated ER Ca(2+) signaling dysregulation.

This scientific commentary refers to ‘Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia’, by Vatsavayai et al. (doi:10.1093/brain/aww250) . The C9orf72 hexanucleotide repeat expansion mutation is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for up to 12% of all patients in an average ALS clinic population. At post-mortem, patients with the C9orf72 hexanucleotide repeat expansion have the typical pathology seen in sporadic ALS and FTD, in which transactive response DNA-binding protein of 43 kDa (TDP-43; encoded by TARDBP ) is translocated from its normal nuclear location to form cytoplasmic aggregates. However, they also show additional pathological features in the form of RNA foci, containing the transcribed hexanucleotide repeats, and aggregates, which stain with antibodies against dipeptide repeat proteins, the product of non-ATG translation. The order in which these distinct molecular abnormalities develop, and whether each of these pathological features is a necessary or sufficient condition for the development of clinical disease, are currently key questions in ALS/FTD research. In this issue of Brain , Vatsavayai et al. describe two strikingly different pathological cases of C9orf72 -positive FTD with excellent ante-mortem characterization that provide novel and provocative contributions to this debate (Vatsavayai et al. , 2016). Transcription of the C9orf72 hexanucleotide repeat expansion can be detected as sense and antisense RNA foci in the nucleus and cytoplasm. Despite being located in the non-coding region of the C9orf72 gene, the repeat sequence is translated through a non-canonical process called ‘repeat associated non-ATG’ (RAN) translation, into five different species of dipeptide …

The neurodegenerative disease, amyotrophic lateral sclerosis (ALS), is a heterogeneous clinical syndrome involving multiple molecular pathways. The development of biomarkers for use in therapeutic trials is a priority. We sought to use a high-throughput proteomic method to identify novel biomarkers in individual cerebrospinal fluid (CSF) samples.Liquid chromatography/tandem mass spectrometry with label-free quantification was used to identify CSF proteins using samples from a well-characterized longitudinal cohort comprising patients with ALS (n = 43), the upper motor neuron variant, primary lateral sclerosis (PLS; n = 6), and cross-sectional healthy (n = 20) and disease controls (Parkinsons' disease, n = 20; ALS mimic disorders, n = 12).Three macrophage-derived chitinases showed increased abundance in ALS: chitotriosidase (CHIT1), chitinase-3-like protein 1 (CHI3L1), and chitinase-3-like protein 2 (CHI3L2). Elevated CHI3L1 was common to ALS and PLS, whereas CHIT1 and CHI3L2 levels differed. Chitinase levels correlated with disease progression rate (CHIT1, r = 0.56, p0.001; CHI3L1, r = 0.31; p = 0.028; CHI3L2, r = 0.29, p = 0.044). CHIT1, CHI3L1, and CHI3L2 levels correlated with phosphorylated neurofilament heavy chain (pNFH; r = 0.62, p0.001; r = 0.49, p0.001; r = 0.41, p0.001). CHI3L1 levels, but not CHIT1 or CHI3L2, increased over time in those with low initial levels (gradient = 0.005 log abundance units/month, p = 0.001). High CHIT1 was associated with shortened survival (hazard ratio [HR] 2.84; p = 0.009). Inclusion of pNFH in survival models left only an association of pNFH and survival (HR 1.26; p = 0.019).Neuroinflammatory mechanisms have been consistently implicated through various experimental paradigms. These results support a key role for macrophage activity in ALS pathogenesis, offering novel target engagement and pharmacodynamic biomarkers for neuroinflammation-focused ALS therapy. Ann Neurol 2018;83:258-268.

A 41-year-old woman was diagnosed with Susac’s syndrome in 2012, having presented with confusion, headache, hearing loss and retinal arteriolar occlusions. Her MR scan of the brain showed several T2 hyperintensities in the corpus callosum and she was treated with cyclophosphamide. She was admitted 3 years later with a 4-week history of cognitive deterioration and visual loss, but with no new MR brain scan changes. Her Montreal Cognitive Assessment (MoCA) had fallen from a baseline of 22/30 to 16/30, while her Snellen central visual acuity had fallen from 6/6 bilaterally to 6/24 in the right eye and 6/36 in the left eye. Retinal photography and fundus fluorescein angiography showed bilateral, superotemporal branch retinal artery occlusions with macular ischaemia (figures 1 and 2). Figure 1 Colour retinal image of the right eye (Optomap) showing pallor and nerve fibre layer swelling due to retinal ischaemia within the distribution of the superotemporal …

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.

The increasing complexity of the genetic landscape in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) presents a significant resource and physician training challenge. At least 10% of those diagnosed with ALS or FTD are known to carry an autosomal dominant genetic mutation. There is no consensus on what constitutes a positive family history, and ascertainment is unreliable for many reasons. However, symptomatic individuals often wish to understand as much as possible about the cause of their disease, and to share this knowledge with their family. While the right of an individual not to know is a key aspect of patient autonomy, and despite the absence of definitive therapy, many newly diagnosed individuals are likely to elect for genetic testing if offered. It is incumbent on the practitioner to ensure that they are adequately informed, counselled and supported in this decision.

Patients with Charcot-Marie-Tooth disease with predominant axonal loss (CMT2) show extensive genetic heterogeneity. Benoy et al. demonstrate a link between CMT2 and histone deacetylase 6 (HDAC6), which controls the acetylation of α-tubulin, and propose that pharmacological inhibition of HDAC6 has therapeutic potential in CMT2 genetic variants., Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

© 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way., Neurologists of the ENCALS centers throughout Europe have discussed the potential of edaravone as a new therapy for amyotrophic lateral sclerosis (ALS, Motor Neuron Disease, MND) at the ENCALS meeting, 18–20 May 2017, in Ljubljana, Slovenia. In May 2017, the US Food and Drug Administration (FDA) granted a license for the drug known as edaravone (licensed in Japan in 2015 as Radicut) for the treatment of ALS in the United States (to be marketed as Radicava). We are not aware of any official request from Mitsubishi Tanabe Pharma, the manufacturer of edaravone, to the European Medicines Agency (EMA) to register the drug for use in ALS in Europe. However, edaravone can be imported to Europe from Japan or the United States.

Polyglutamine expansions in the ataxin-2 gene (ATXN2) cause autosomal dominant spinocerebellar ataxia type 2 (SCA2), but have recently also been associated with amyotrophic lateral sclerosis (ALS). We present clinical and pathological features of a family in which a pathological ATXN2 expansion led to frontotemporal lobar degeneration with ALS (FTLD-ALS) in the index case, but typical SCA2 in a son, and compare the neuropathology with a case of typical SCA2. The index case shares the molecular signature of SCA2 with prominent polyglutamine and p62-positive intranuclear neuronal inclusions mainly in the pontine nuclei, while harbouring more pronounced neocortical and spinal TDP-43 pathology. We conclude that ATXN2 mutations can cause not only ALS, but also a neuropathological overlap syndrome of SCA2 and FTLD presenting clinically as pure FTLD-ALS without ataxia. The cause of the phenotypic heterogeneity remains unexplained, but the presence of a CAA-interrupted CAG repeat in the FTLD case in this family suggests that one potential mechanism may be variation in repeat tract composition between members of the same family.

Item does not contain fulltext Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein-protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G-associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.

Summary Motor neurone disease (MND), the commonest clinical presentation of which is amyotrophic lateral sclerosis (ALS), is regarded as the most devastating of adult-onset neurodegenerative disorders. The last decade has seen major improvements in patient care, but also rapid scientific advances, so that rational therapies based on key pathogenic mechanisms now seem plausible. ALS is strikingly heterogeneous in both its presentation, with an average one-year delay from first symptoms to diagnosis, and subsequent rate of clinical progression. Although half of patients succumb within 3–4 years of symptom onset, typically through respiratory failure, a significant minority survives into a second decade. Although an apparently sporadic disorder for most patients, without clear environmental triggers, recent genetic studies have identified disease-causing mutations in genes in several seemingly disparate functional pathways, so that motor neuron degeneration may need to be understood as a common final pathway with a number of upstream causes. This apparent aetiological and clinical heterogeneity suggests that therapeutic studies should include detailed biomarker profiling, and consider genetic as well as clinical stratification. The most common mutation, accounting for 10% of all Western hemisphere ALS, is a hexanucleotide repeat expansion in C9orf72. This and several other genes implicate altered RNA processing and protein degradation pathways in the core of ALS pathogenesis. A major gap remains in understanding how such fundamental processes appear to function without obvious deficit in the decades prior to symptom emergence, and the study of pre-symptomatic gene carriers is an important new initiative.

Increased serum neurofilament is associated with CNS axonal loss from a range of causes. We assessed its potential as a minimally-invasive indicator of active CNS pathology in those with neurological symptoms.GP referrals to the Neurology Rapid Access Clinic (RAC) at the John Radcliffe Hospital, Oxford were approached to take part. Informed consent was taken under a local Research Ethics Committee-approved protocol. Serum was taken prior to clinical assessment and tested for phosphorylated neurofilament heavy chain (pNFH) using a commercial ELISA (Euroimmun, Germany). Participants were followed up for final diagnosis.The 62 participants had a range of symptoms, with final diagnoses including migraine, functional, neurodegenerative and inflammatory conditions. Receiver Operating Curve analysis defined cut-off levels based on serum pNfH levels in healthy controls and patients with motor neurone disease measured as part of an independent local biomarker study. pNFH levels in the ‘pathological’ range were associated with serious underlying pathology in several RAC referrals. Ongoing analysis of the cohort will be presented.Alongside clinical reasoning, serum pNFH may have significant value for the improved triage of those presenting to primary care with neurological symptoms. Further study in larger populations, including healthy individuals, is now warranted.

Therapeutic options for the fatal neurodegenerative disorder ALS are urgently needed. Trials rely on blunt outcome measures such as survival because of a lack of objective markers of disease activity and progression. The C9orf72 hexanucleotide repeat expansion (HRE) is associated with 10% of all cases of ALS, bringing the near-future prospect of oligonucleotide therapeutic trials.The development of biomarkers will reduce trial duration and cost by providing more sensitive measures of disease-slowing and/or evidence of target engagement. ALS is consistently associated with cortical hyperexcitability (CE), based on transcranial magnetic paired stimulation (TMS) to induce short-interval cortical inhibition (SICI). This project builds on the potential shown by non-invasive neuroimaging to provide biomarkers in ALS. We develop functional neuroimaging biomarkers that reflect the phenomenon of CE, including markers of pre-symptomatic pathology.Affected ALS patients carrying the C9orf72 HRE and a group of asymptomatic carriers are studied using a combined functional MRI (FMRI and MRS) and neurophysiological (MEG) readout. This study offers novel non-invasive biomarkers based on a consistent neurophysiological mechanism in ALS to advance therapeutic development

BACKGROUND: Concomitant REM sleep behaviour disorder (RBD) is commonly observed in patients with Parkinson's disease (PD). Although the brainstem structures responsible for the symptoms of RBD correspond to the premotor stages of PD, the association of RBD with motor and non-motor features in early PD remains unclear. METHODS: The study evaluated 475 patients with PD within 3.5 years of diagnosis for the presence of probable RBD (pRBD) using the REM Sleep Behaviour Disorder Screening Questionnaire (RBDSQ). A neurologist and a trained research nurse carried out evaluation of each participant blinded to the results of the RBDSQ. Standardised rating scales for motor and non-motor features of PD, as well as health-related quality of life measures, were assessed. Multiple linear and logistic regression analyses were used to determine the relationship between pRBD and a variety of outcomes, controlling for confounding factors. RESULTS: The overall frequency of pRBD was 47.2% (95% CI 42.7% to 51.9%). None of the patients had a previous diagnosis of RBD. Patients with PD and concomitant pRBD did not differ on motor phenotype and scored comparably on the objective motor scales, but reported problems with motor aspects of daily living more frequently. Adjusted for age, sex, disease duration and smoking history, pRBD was associated with greater sleepiness (p=0.001), depression (p=0.001) and cognitive impairment (p=0.006). CONCLUSIONS: pRBD is common and under-recognised in early PD. It is associated with increased severity and frequency of non-motor features, poorer subjective motor performance and a greater impact on health-related quality of life.

Previous imaging studies that investigated morphometric group differences of subcortical regions outside the substantia nigra between non-demented Parkinson's patients and controls either did not find any significant differences, or reported contradictory results. Here, we performed a comprehensive morphometric analysis of 20 cognitively normal, early-stage PD patients and 19 matched control subjects. In addition to relatively standard analyses of whole-brain grey matter volume and overall regional volumes, we examined subtle localized surface shape differences in striatal and limbic grey matter structures and tested their utility as a diagnostic marker. Voxel-based morphometry and volumetric comparisons did not reveal significant group differences. Shape analysis, on the other hand, demonstrated significant between-group shape differences for the right pallidum. Careful diffusion tractography analysis showed that the affected parts of the pallidum are connected subcortically with the subthalamic nucleus, the pedunculopontine nucleus, and the thalamus and cortically with the frontal lobe. Additionally, microstructural measurements along these pathways, but not along other pallidal connections, were significantly different between the two groups. Vertex-wise linear discriminant analysis, however, revealed limited accuracy of pallidal shape for the discrimination between patients and controls. We conclude that localized disease-related changes in the right pallidum in early Parkinson's disease, undetectable using standard voxel-based morphometry or volumetry, are evident using sensitive shape analysis. However, the subtle nature of these changes makes it unlikely that shape analysis alone will be useful for early diagnosis.

Our objective was to evaluate the direct and indirect relationships between psychosocial variables, fatigue and quality of life for patients with motor neuron disease (MND). A cross-sectional sample of 147 MND patients was recruited from five neurological care centres in England. Variables included anxiety, coping, depression, fatigue, functional status, social withdrawal and quality of life. Direct and indirect relationships between study variables were assessed using structural equation modelling (SEM), using linear values derived from Rasch analyses of study questionnaires. Following some modification, Rasch analysis confirmed the suitability of all measures for use in this population. The final SEM model consisting of anxiety, coping, depression, fatigue, social withdrawal and quality of life showed excellent fit to the data. The model accounted for 59% of the variance in quality of life and 50% of the variance in depression. In conclusion, our data support a model that explains a large degree of the variance in quality of life for MND patients. Coping was most strongly related to quality of life, with the largest proportion of its influence mediated by anxiety and depression. Significant direct effects upon quality of life were exhibited by depression, fatigue and social withdrawal.

The progression of motor neurone disease (MND) is currently irreversible, and the grave implications of diagnosis naturally fuels concern among neurologists over missing a potential mimic disorder. There is no diagnostic test for MND but in reality there are few plausible mimics in routine clinical practice. In the presence of a progressive pure motor disorder, signs such as florid fasciculations, bilateral tongue wasting, the 'split hand', head drop, emotionality, and cognitive or behavioural impairment carry high positive predictive value. MND is clinically heterogeneous, however, with some important chameleon-like presentations and considerable variation in clinical course. Lack of confidence about the scope of such variation, or an approach to diagnosis emphasising investigations over clinical common sense, has the potential to exacerbate diagnostic delay in MND and impede timely planning of the care which is essential to maximising quality of life.

As neurons die cholesterol is released in the central nervous system (CNS), hence this sterol and its metabolites may represent a biomarker of neurodegeneration, including in amyotrophic lateral sclerosis (ALS) in which altered cholesterol levels have been linked to prognosis. More than 40 different sterols were quantified in serum and cerebrospinal fluid (CSF) from ALS patients and healthy controls. In CSF the concentration of cholesterol was found to be elevated in ALS samples. When CSF metabolite levels were normalised to cholesterol, the cholesterol metabolite 3beta,7alpha-dihydroxycholest-5-en-26-oic acid, along with its precursor 3beta-hydroxycholest-5-en-26-oic acid and product 7alpha-hydroxy-3-oxocholest-4-en-26-oic acid were reduced in concentration, whereas metabolites known to be imported from the circulation into the CNS were not found to differ in concentration between groups. Analysis of serum revealed that (25R)26-hydroxycholesterol, the immediate precursor of 3beta-hydroxycholest-5-en-26-oic acid was reduced in concentration in ALS patients compared to controls. We conclude that the acidic branch of bile acid biosynthesis, known to operative in-part in brain, is defective in ALS leading to a failure of the CNS to remove excess cholesterol which may be toxic to neuronal cells, compounded by a reduction in neuroprotective 3beta,7alpha-dihydroxycholest-5-en-26-oic acid.

We describe a family with an autosomal dominant familial dyskinesia resembling myoclonus-dystonia associated with a novel missense mutation in ADCY5, found through whole-exome sequencing. A tiered analytical approach was used to analyse whole-exome sequencing data from an affected grandmother-granddaughter pair. Whole-exome sequencing identified 18,000 shared variants, of which 46 were non-synonymous changes not present in a local cohort of control exomes (n = 422). Further filtering based on predicted splicing effect, minor allele frequency in the 1000 Genomes Project and on phylogenetic conservation yielded 13 candidate variants, of which the heterozygous missense mutation c.3086T>G, p. M1029R in ADCY5 most closely matched the observed phenotype. This report illustrates the utility of whole-exome sequencing in cases of undiagnosed movement disorders with clear autosomal dominant inheritance. Moreover, ADCY5 mutations should be considered in cases with apparent myoclonus-dystonia, particularly where SCGE mutations have been excluded. ADCY5-related dyskinesia may manifest variable expressivity within a single family, and affected individuals may be initially diagnosed with differing neurological phenotypes. Electronic supplementary material The online version of this article (doi:10.1007/s10048-017-0510-z) contains supplementary material, which is available to authorized users.

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, affecting 1% of the population over 65 years characterized clinically by both motor and non-motor symptoms accompanied by the preferential loss of dopamine neurons in the substantia nigra pars compacta. Here, we sequenced the exomes of 244 Parkinson’s patients selected from the Oxford Parkinson’s Disease Centre Discovery Cohort and, after quality control, 228 exomes were available for analyses. The PD patient exomes were compared to 884 control exomes selected from the UK10K datasets. No single non-synonymous (NS) single nucleotide variant (SNV) nor any gene carrying a higher burden of NS SNVs was significantly associated with PD status after multiple-testing correction. However, significant enrichments of genes whose proteins have roles in the extracellular matrix were amongst the top 300 genes with the most significantly associated NS SNVs, while regions associated with PD by a recent Genome Wide Association (GWA) study were enriched in genes containing PD-associated NS SNVs. By examining genes within GWA regions possessing rare PD-associated SNVs, we identified RAD51B. The protein-product of RAD51B interacts with that of its paralogue RAD51, which is associated with congenital mirror movements phenotypes, a phenotype also comorbid with PD.

Charcot-Marie-Tooth disease type 2D (CMT2D) is a peripheral nerve disorder caused by dominant, toxic, gain-of-function mutations in the widely expressed, housekeeping gene,GARS. The mechanisms underlying selective nerve pathology in CMT2D remain unresolved, as does the cause of the mild-to-moderate sensory involvement that distinguishes CMT2D from the allelic disorder distal spinal muscular atrophy type V. To elucidate the mechanism responsible for the underlying afferent nerve pathology, we examined the sensory nervous system in CMT2D mice. We show that the equilibrium between functional subtypes of sensory neuron in dorsal root ganglia is distorted byGarsmutations, leading to sensory defects in peripheral tissues and correlating with overall disease severity. CMT2D mice display changes in sensory behaviour concordant with the afferent imbalance, which is present at birth and non-progressive, indicating that sensory neuron identity is prenatally perturbed and that a critical developmental insult is key to the afferent pathology. This suggests that both neurodevelopmental and neurodegenerative mechanisms contribute to CMT2D pathogenesis, and thus has profound implications for the timing of future therapeutic treatments.Significance StatementCharcot-Marie-Tooth disease (CMT) is a collection of genetically diverse inherited nerve disorders with the unifying feature of peripheral neuron degeneration. The mechanisms triggering this motor and sensory nerve dysfunction remain unresolved, as does the reason for the lack of sensory pathology observed in distal hereditary motor neuropathies, which can be associated with CMT genes. To unravel the mechanisms leading to afferent deterioration, we have studied the sensory nervous system of CMT Type 2D mice. Our work indicates that the specific cellular identity of sensory nerves is perturbed in mutant mice pre-natally. CMT therefore manifests through the complex interplay between malfunctioning developmental, maturation, and survival programs, which has important ramifications for therapeutic timing.

Objective Social withdrawal is described as the condition in which an individual experiences a desire to make social contact, but is unable to satisfy that desire. It is an important issue for patients with motor neurone disease who are likely to experience severe physical impairment. This study aims to reassess the psychometric and scaling properties of the MND Social Withdrawal Scale (MND-SWS) domains and examine the feasibility of a summary scale, by applying scale data to the Rasch model. Methods The MND Social Withdrawal Scale was administered to 298 patients with a diagnosis of MND, alongside the Hospital Anxiety and Depression Scale. The factor structure of the MND Social Withdrawal Scale was assessed using confirmatory factor analysis. Model fit, category threshold analysis, differential item functioning (DIF), dimensionality and local dependency were evaluated. Results Factor analysis confirmed the suitability of the four-factor solution suggested by the original authors. Mokken scale analysis suggested the removal of item five. Rasch analysis removed a further three items; from the Community (one item) and Emotional (two items) withdrawal subscales. Following item reduction, each scale exhibited excellent fit to the Rasch model. A 14-item Summary scale was shown to fit the Rasch model after subtesting the items into three subtests corresponding to the Community, Family and Emotional subscales, indicating that items from these three subscales could be summed together to create a total measure for social withdrawal. Conclusion Removal of four items from the Social Withdrawal Scale led to a four factor solution with a 14-item hierarchical Summary scale that were all unidimensional, free for DIF and well fitted to the Rasch model. The scale is reliable and allows clinicians and researchers to measure social withdrawal in MND along a unidimensional construct. © 2013 The Authors.

Background: Vasculitis of the peripheral nervous system (PNS) is rare. There are no controlled treatment trials, and clinical practice is guided by experience from case series and indirectly by analogy with systemic vasculitis. Methods: We identified patients ( n = 212) with possible vasculitic peripheral neuropathy (VPN) from the neuropathology and neurophysiology records of two centres over 28 years. Case-notes were available for 181, from which, 106 cases of clinicopathological VPN were identified. Adequate treatment data were available in 100; follow-up data, in 93. Results: Of 106 cases, 95 had systemic vasculitis and 11 had vasculitis confined to the PNS. Pharmacological treatment (94/100 cases) was corticosteroid-based, and included cyclophosphamide in 54; 17 received additional agents. Initial stabilization was achieved in all but six. One-year survival was 90.3%. Of the nine who died in the first years (mean age 73 years), seven had received cyclophosphamide, and all but two had severe, multisystem vasculitis. The neurological relapse rate was 10%. Only one relapse occurred after cyclophosphamide treatment. Outcome was reported as good in 72% (78% in those who relapsed). Discussion: Death and relapse were infrequent in treated patients. Relapse occurred almost exclusively in patients treated with prednisolone alone. Aggressive early treatment with cyclophosphamide may prevent relapse. The current management approach to VPN appears largely effective, especially if cyclophosphamide is used.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease characterised by progressive loss of spinal motor neurons. Mutations in the genes underlying spontaneous bovine and feline models of SMA have recently been described. The clinical and pathological features of these disorders are similar to human forms of SMA making both genes excellent candidates in patients with motor neuron loss of no known aetiology. Here we report that a screen for mutations in coding regions and splice sites of the LIX1 and FVT1 genes in a cohort of 96 non-5q SMA patients and 119 familial and sporadic Amyotrophic Lateral Sclerosis patients identified no obvious pathogenic changes. This study indicates that mutations in these genes do not contribute significantly to the cause of motor neuron diseases in the human population.

α-Synuclein (α-Syn), a protein synthesized by neurons, as the major protein component of Lewy body inclusions, undoubtedly has a prominent role in the pathogenesis of Parkinson's Disease (PD). In an attempt to enable pre-symptomatic and definitive diagnosis, numerous attempts have been made to align assayed total α-Syn levels in serum (where there is a native presence) to PD disease status. Results have been conflicting. The status of circulating and potentially neuroprotective α-Syn autoantibodies in PD subjects is also unclear. In previous work we demonstrated that electrochemically assayed autoantibody levels were higher in PD patients compared to controls and, significantly, noted that this differentiation was most marked early in disease. Herein we report a robust (coefficient of variation 3.0%) single step and label free analysis of 90 subjects, including 60 PD patients, with a mean disease duration of 1.4 years and 29 control subjects. In this cross sectional cohort we observe a statistically significant (p < 0.05; Mann-Whitney U test) difference in autoantibody levels in PD patients versus controls, although there was no resolved scaling with symptomatic disease stage (p > 0.05; Kruskal-Wallis test).

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes responsible for ligating amino acids to cognate tRNA molecules. Mutations in four genes encoding an ARS have been implicated in inherited peripheral neuropathy with an axonal pathology, suggesting that all ARS genes are relevant candidates for disease in patients with related phenotypes. Here, we present results from a mutation screen of the histidyl-tRNA synthetase (HARS) gene in a large cohort of patients with peripheral neuropathy. These efforts revealed a rare missense variant (c.410G>A/p.Arg137Gln) that resides at a highly conserved amino acid, represents a loss-of-function allele when evaluated in yeast complementation assays, and is toxic to neurons when expressed in a worm model. In addition to the patient with peripheral neuropathy, p.Arg137Gln HARS was detected in three individuals by genome-wide exome sequencing. These findings suggest that HARS is the fifth ARS locus associated with axonal peripheral neuropathy. Implications for identifying ARS alleles in human populations and assessing them for a role in neurodegenerative phenotypes are discussed. © 2012 Wiley Periodicals, Inc.

Objective: While the hallmark of amyotrophic lateral sclerosis (ALS) is corticospinal tract in combination with lower motor neuron degeneration, the clinical involvement of both compartments is characteristically variable and the site of onset debated. We sought to establish whether there is a consistent signature of cerebral white matter abnormalities in heterogeneous ALS cases. Methods: In this observational study, diffusion tensor imaging was applied in a whole-brain analysis of 24 heterogeneous patients with ALS and well-matched healthy controls. Tract-based spatial statistics were used, with optimized voxel-based morphometry of T1 images to determine any associated gray matter involvement. Results: A consistent reduction in fractional anisotropy was demonstrated in the corpus callosum of the ALS group, extending rostrally and bilaterally to the region of the primary motor cortices, independent of the degree of clinical upper motor neuron involvement. Matched regional radial diffusivity increase supported the concept of anterograde degeneration of callosal fibers observed pathologically. Gray matter reductions were observed bilaterally in primary motor and supplementary motor regions, and also in the anterior cingulate and temporal lobe regions. A post hoc group comparison model incorporating significant values for fractional anisotropy, radial diffusivity, and gray matter was 92% sensitive, 88% specific, with an accuracy of 90%. Conclusion: Callosal involvement is a consistent feature of ALS, independent of clinical upper motor neuron involvement, and may reflect independent bilateral cortical involvement or interhemispheric spread of pathology. The predominantly rostral corticospinal tract involvement further supports the concept of independent cortical degeneration even in those patients with ALS with predominantly lower motor neuron involvement clinically. Neurology ® 2010;75:1645–1652

Dominant mutations in GARS, encoding the essential enzyme glycyl-tRNA synthetase (GlyRS), result in a form of Charcot-Marie-Tooth disease, type 2D (CMT2D), predominantly characterized by lower motor nerve degeneration. GlyRS charges the amino acid glycine with its cognate tRNA and is therefore essential for protein translation. However, the underlying mechanisms linking toxic gain-of-function GARS mutations to lower motor neuron degeneration remain unidentified. The neuromuscular junction (NMJ) appears to be an early target for pathology in a number of peripheral nerve diseases and becomes denervated at later stages in two mouse models of CMT2D. We therefore performed a detailed longitudinal examination of NMJs in the distal lumbrical muscles and the proximal transversus abdominis (TVA) muscles of wild-type and Gars mutant mice. We determined that mutant lumbrical NMJs display a persistent defect in maturation that precedes a progressive, age-dependent degeneration. Conversely, the TVA remains relatively unaffected, with only a subtle, short-lived impairment in pre- and post-synaptic development and no reduction in lower motor neuron connectivity to muscle. Together, these observations suggest that mutant Gars is associated with compromised development of the NMJ prior to synaptic degeneration and highlight the neuromuscular synapse as an important site of early, selective pathology in CMT2D mice.

Mutant superoxide dismutase 1 (SOD1) action within non-neuronal cells is implicated in damage to spinal motor neurons in a genetic form of amyotrophic lateral sclerosis (ALS). Central nervous system glial cells such as astrocytes and microglia drive progression in transgenic mutant SOD1 mice, however, the role of myelinating glia remains unclear. Specifically, peripheral myelinating glial cells are likely candidates for mediating degeneration of distal synapses and axons of motor neurons in ALS. Here, we examine the potential contribution of peripheral axon ensheathing Schwann cells to ALS by constructing transgenic mice expressing dismutase active mutant SOD1(G93A) driven by the myelin protein zero (P0) promoter. In this model, mutant SOD1 accumulation in Schwann cells was comparable to levels in mice ubiquitously expressing a SOD1(G93A) transgene that become paralysed. Growth, locomotion and survival of these P0-SOD1(G93A) mice were indistinguishable from normal animals. There was no evidence for spinal motor neuron loss, distal axonal degeneration and p75 neurotrophin receptor (p75(NTR)) upregulation in the periphery of P0-SOD1(G93A) mice, unlike transgenic SOD1(G93A) mice with presymptomatic p75(NTR) induction and death-signalling. Furthermore, Schwann cells were resistant to mutant SOD1 aggregation in vivo and in transfected primary cultures. Increasing mutant SOD1 synthesis in Schwann cells by cross-breeding transgenic P0-SOD1(G93A) and SOD1(G93A) mice did not affect disease onset or survival. We conclude that dismutase-competent mutant SOD1 accumulation within Schwann cells is not pathological to spinal motor neurons or deleterious to disease course in transgenic ALS model mice, in contrast to astrocytes and microglia.

Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the ‘diagnostic odyssey’ for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1–3, 6, 7 and Friedrich’s ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3–35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.