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Mutations in the gene encoding the RNA-binding protein fused in sarcoma (FUS) account for 4 – 5% of familial cases of amyotrophic lateral sclerosis (ALS). We describe the identification and in vitro cellular characterization of a genetic mutation in a family in which the index case, and subsequently her two children, each developed rapidly progressive ALS at a young age and died within a year of onset. Exome capture and sequencing revealed a mutation in the FUS gene consisting of a 2-bp deletion, c.1509_1510delAG, resulting in a predicted truncated protein, p.G504Wfs * 12, lacking the nuclear localization signal. Expression of this mutation in HEK293 and NSC-34 cells demonstrated severe cytoplasmic mislocalization of mutant FUS, and colocalization with stress granules when compared to wild-type, R521C and P525L mutant FUS. This study provides further evidence of a broad correlation between clinical severity of FUS-related ALS and mislocalization of the protein to the cytoplasm.

Spinal muscular atrophy (SMA) is characterized by the selective loss of spinal motor neurons owing to reduced levels of survival motor neuron (Smn) protein. In addition to its well-established role in assembling constituents of the spliceosome, diverse cellular functions have been proposed for Smn, but the reason why low levels of this widely expressed protein result in selective motor neuron pathology is still debated. In longitudinal studies of exon-level changes in SMA mouse model tissues, designed to determine the contribution of splicing dysfunction to the disease, we have previously shown that a generalized defect in splicing is unlikely to play a causative role in SMA. Nevertheless, we identified a small subset of genes that were alternatively spliced in the spinal cord compared with control mice before symptom onset, indicating a possible mechanistic role in disease. Here, we have performed functional studies of one of these genes, chondrolectin (Chodl), known to be highly expressed in motor neurons and important for correct motor axon outgrowth in zebrafish. Using in vitro and in vivo models of SMA, we demonstrate altered expression of Chodl in SMA mouse spinal motor neurons, show that Chodl has distinct effects on cell survival and neurite outgrowth and that increasing the expression of chodl can rescue motor neuron outgrowth defects in Smn-depleted zebrafish. Our findings thus link the dysregulation of Chodl to the pathophysiology of motor neuron degeneration in SMA.

SummaryExome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis.Video Abstract

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease with clinical, pathological and genetic overlap with frontotemporal dementia (FTD). No longer viewed as one disease with a single unified cause, ALS is now considered to be a clinicopathological syndrome resulting from a complex convergence of genetic susceptibility, age-related loss of cellular homeostasis, and possible environmental influences. The rapid increase in recent years of the number of genes in which mutations have been associated with ALS has led to in vitro and in vivo models that have generated a wealth of data indicating disruption of specific biochemical pathways and sub-cellular compartments. Data implicating pathways including protein misfolding, mRNA splicing, oxidative stress, proteosome and mitochondrial dysfunction in the pathogenesis of ALS reinforce a disease model based on selective age-dependent vulnerability of a specific population of cells. To the clinical neurologist, however, ALS presents as a disease of focal onset and contiguous spread. Characteristic regional patterns of involvement and progression suggest that the disease does not proceed randomly but via a restricted number of anatomical pathways. These clinical observations combined with electrophysiological and brain-imaging studies underpin the concept of ALS at the macroscopic level as a 'system degeneration'. This dichotomy between cellular and systems neurobiology raises the fundamental questions of what initiates the disease process in a specific anatomical site and how the disease is propagated. Is the essence of ALS a cell-to-cell transmission of pathology with, for example, a 'prion-like' mechanism, or does the cellular pathology follow degeneration of specific synaptic networks? Elucidating the interaction between cellular degeneration and system level degeneration will aid modeling of the disease in the earliest phases, improve the development of sensitive markers of disease progression and response to therapy, and expand our understanding of the biological basis of clinical and pathological heterogeneity. © 2013 Anatomical Society.

Background: Identifying factors influencing phenotypic heterogeneity in Parkinson's Disease is crucial for understanding variability in disease severity and progression. Age and gender are two most basic epidemiological characteristics, yet their effect on expression of PD symptoms is not fully defined. We aimed to delineate effects of age and gender on the phenotype in an incident cohort of PD patients and healthy controls from the Oxford Parkinson Disease Centre (OPDC). Methods: Clinical features, including demographic and medical characteristics and non-motor and motor symptoms, were analyzed in a group of PD patients within 3 years of diagnosis and a group of healthy controls from the OPDC cohort. Disease features were stratified according to age and compared between genders, controlling for effects of common covariates. Results: 490 PD patients and 176 healthy controls were analyzed. Stratification by age showed increased disease severity with age on motor scales. Some non-motor features showed similar trend, including cognition and autonomic features. Comparison across genders highlighted a pattern of increased severity and greater symptom symmetricality in the face, neck and arms in men with women having more postural problems. Amongst the non-motor symptoms, men had more cognitive impairment, greater rate of REM behavior disorder (RBD), more orthostatic hypotension and sexual dysfunction. Conclusions: Age in PD is a strong factor contributing to disease severity even after controlling for the effect of disease duration. Gender-related motor phenotype can be defined by a vertical split into more symmetrical upper-body disease in men and disease dominated by postural symptoms in women. © 2013 Elsevier Ltd.

An 18-year-old-man was referred to the Neurology Department complaining of headache and double vision. The headache was exquisitely sensitive to postural changes, being completely abolished by lying at. Neurological examination revealed an isolated right sixth nerve palsy. Some 5 weeks previously he had been seen in the Emergency Department of his local hospital after being assaulted with a screwdriver, leading to a penetrating injury of the back at the level of the ninth thoracic vertebra. A plain X-ray was performed and the attending doctor removed the screwdriver. Three days after this injury he represented to the same hospital complaining of sudden onset of headache and vomiting. A diagnosis of migraine was made and he was discharged the next day. The headache persisted and 10 days later he represented because of double vision, mu‚ed hearing, mild neck sti€ness and photophobia, at which time the neurological referral was made. Computed tomography (CT) of the brain on this occasion was normal except for a dilated straight sinus (Fig. 1). A lumbar puncture was not performed. There was no signi®cant past medical history. Brain MRI was performed 4 weeks after injury. The T2-weighted axial images showed dilatation of the dural venous sinuses (Fig. 2a) and the proton density-weighted axial images revealed small subdural and subtentorial collections which were of a higher signal than CSF (Fig. 2b). A sagittal T1-weighted image showed the cerebellar tonsils at the foramen magnum, and post gadolinium-DTPA enhanced coronal T1-weighted images showed homogeneous dural enhancement (Fig. 2c). A diagnosis of probable intracranial hypotension was made and, due to the penetrating nature of the trauma, MRI of the spine was performed. A marker was placed at the site of the skin wound. Sagittal T2-weighted fat-saturated images revealed bright signal posterior to the thecal sac and no evidence of cord injury (Fig. 3a). Axial T2-weighted fat-saturated images showed the spinal cord surrounded by a reduced volume of CSF and an extradural uid collection, the same signal as CSF (Fig. 3b). From this imaging it was concluded that there was a traumatic dural CSF leak causing symptomatic intracranial hypotension. Since the patient had not improved with conservative management, an autologous epidural blood patch was performed, at the site of penetrating injury, and his symptoms resolved completely.

The spinal muscular atrophies are a group of mostly inherited disorders selectively affecting the lower motor neuron. There is a wide degree of clinical and genetic heterogeneity that must be taken into account when giving prognostic information. Autosomal recessive childhood proximal SMA is the commonest form and is due to mutations in a gene encoding a novel protein, SMN, that appears to play a critical role in RNA metabolism but has also been shown to interact with actin-binding proteins and mediators of programmed cell death. The identification of the genetic basis of SMA has resulted in advances for prenatal diagnosis and in new insights into motor neuron biology. The chromosomal location of two of the rarer dominant forms of SMA has been found. Identification of the molecular pathophysiology of lower motor neuron syndromes can be expected to aid in the development of therapy for these disabling disorders.

Spinal muscular atrophy (SMA) is caused by a drastic reduction in the ubiquitously expressed SMN protein, which is critical for the correct assembly of the snRNP complexes required for RNA splicing. However, it is unclear why loss of SMN and altered snRNP assembly only seem to affect motor neurons. Reporting in this issue, Zhang et al. (2008) challenge prior assumptions about the housekeeping function of SMN and demonstrate that loss of SMN leads to highly tissue-specific effects on splicing.

Mutations in the gene encoding the Survival Motor Neuron (SMN) protein are responsible for autosomal recessive proximal spinal muscular atrophy (SMA). SMN orthologues have been identified in the nematode worm Caenorhabditis elegans and the yeast Schizosaccharomyces pombe but, to date, no human paralogues have been described. Here we describe identification and characterization of an SMN-related protein (SMNrp) gene that encodes a novel protein of 239 amino acids, which has recently been identified as a constituent of the spliceosome complex and designated SPF30. Significant similarity to the SMN protein is apparent only within a central region of SMNrp that represents a tudor domain. The SMNrp/SPF30 gene has been mapped to chromosome 10q23. It is differentially expressed, with abundant levels in skeletal muscle. An exclusively nuclear localization for SMNrp in cultured cells and muscle sections was revealed using GFP fusion constructs and thereafter confirmed with a polyclonal antibody raised against SMNrp. Overexpression of SMNrp as a fusion protein in HeLa cells in culture induced dose-dependent apoptosis with positive TUNEL staining. In addition to a possible role for this protein as a pro-apoptotic factor, SMN and its related protein share significant similarities in sequence and cellular function.

We screened 100 patients with inherited and sporadic lower motor neuron degeneration and identified three novel missense mutations in the glycyl-tRNA synthetase (GARS) gene. One mutation was in the anticodon binding domain and associated with onset in early childhood and predominant involvement of the lower limbs, thus extending the phenotype associated with GARS mutations.

Mitogen-activated protein kinase phosphatases (MKPs) play a central role in a variety of signaling pathways. We recently described a novel murine MKP, M3/6, which is uniquely specific for c-Jun N-terminal kinase/stress-activated protein kinase and p38 kinase. Here we report the localization of the human orthologue of this gene, HB5, to within 150 kb of H19 on human chromosome 11p15.5. The gene consists of six exons. Two of the introns in HB5 are not found in other genes of this family, suggesting an evolutionary split between MKPs displaying specificity toward different MAP kinases. An intronless pseudogene is present on chromosome 10q11.2. Although 11p15.5 is an imprinted region, HB5 is almost entirely unmethylated on both alleles in lymphocytes. Chromosome 11p15 has been implicated in the development of a number of tumor types, including lung, a tissue known to express this gene. Loss of heterozygosity was found in one of eight informative lung tumors studied.

Autosomal recessive proximal spinal muscular atrophy (SMA) is a disease of motor neuron death and a common cause of morbidity in childhood. It has been mapped to 5q13 and shown to be associated with deletions in a gene which has been called the survival motor neuron (SMN) gene. SMN exists in two copies in 5q13 and deletions in exon 7 and 8 of the telomeric copy (SMN tel ) occur in over 90% of patients regardless of disease severity. In contrast, deletion of exon 7 and 8 of the centromeric copy of SMN is present in 3–5% of the normal population. In a minority of patients, exon 7 but not exon 8 of SMN tel appears deleted. The purpose of this study was to analyse this latter type of deletion in more detail. In all patients where there was absence of PCR amplification of exon 7 but not exon 8 of SMN tel this was found to be due to replacement with the homologous copy of SMN cen by a possible gene conversion event. This type of mutation occurred in all grades of severity of SMA.

Primary lateral sclerosis (PLS) has been regarded as a rare, extreme form of amyotrophic lateral sclerosis (ALS). Like ALS, it is a clinical diagnosis without established biomarkers. We sought to explore loss of cerebral myelin in relation to clinical features, including cognitive impairment, in cases of both ALS and PLS. A novel MRI sequence (mcDESPOT) sensitive to water pools within myelin and intra- and extra-cellular spaces was applied to 23 ALS patients, seven PLS patients and 12 healthy controls, with interval follow-up in 15 ALS and four PLS patients. Results demonstrated that PLS patients were distinguished by widespread cerebral myelin water fraction reductions, independent of disease duration and clinical upper motor neuron burden. ALS patients showed a significant increase in intra- and extra-cellular water, indirectly linked to neuroinflammatory activity. Limited measures of cognitive impairment in the ALS group were associated with myelin changes within the anterior corpus callosum and frontal lobe projections. Longitudinal changes were only significant in the PLS group. In conclusion, in this exploratory study, myelin imaging has potential to distinguish PLS from ALS, and may have value as a marker of extramotor involvement. PLS may be a more active cerebral pathological process than its rate of clinical deterioration suggests.

A 65 year old female was treated with Onyx embolisation of a superior cerebellar aneurysm for subarachnoid haemorrhage, resulting in mild residual left leg spasticity, but independent mobility. Eight years later, her left ankle started to turn inwards, and gradually the foot became inverted, ultimately becoming fixed due to tendon shortening from spasticity. There was no fluctuation in the position of the foot and it could not be easily corrected with passive stretching, as might be expected in dystonia. Examination revealed weakness of left ankle dorsiflexion, with normal sensation, reflexes and a flexor plantar response. MRI/MRA of the brain showed a treated aneurysm with no evidence of recurrence. Her weakness continued to progress and over the next 6 months she went on to develop brisk left knee and ankle jerks and a left extensor plantar response. Though there was no clinical evidence of wasting or fasciculation, an EMG of the left lower limb showed active denervation. A diagnosis of ALS was suspected, but with changes only confined to one limb there was insufficient evidence for an unequivocal diagnosis. In an attempt to alleviate severe pain associated with fixed flexion deformity, she was treated with 1,000 units of botulinum toxin A (Dysport) into the left plantar flexor muscles. Four days later, she rapidly deteriorated, developing generalised limb weakness with loss of ambulation, dysphagia and dysarthria. Her clinical course over the next year was entirely consistent with ALS, involving progressive weakness of all four limbs and respiratory failure, and ultimately requiring hospice care. In ALS a pre-clinical phase of motor neuron degeneration is likely to occur before motor symptoms develop. Botulinum toxin prevents acetylcholine-containing vesicles from binding to the pre-synaptic membrane. In its use in the treatment of sialorrhoea in ALS, there are no published reports of generalised exacerbation of limb muscle weakness. Anecdotal side effects include bilateral temporomandibular joint subluxation [1] and facial weakness [2]. In one case a woman with ALS who received botulinum toxin for sialorrhoea became anarthric and aphagic [3]. Botulinum toxin injection has also unmasked Lambert– Eaton myasthenic syndrome [4] and caused clinical deterioration in myasthenis gravis [5] and mitochondrial cytopathies [6]. The fact that symptoms were remote to the treatment site suggests a systemic effect of the toxin, a well-recognised phenomenon that has been demonstrated electrophysiologically in patients without clinical weakness [7]. Denervation and incomplete re-innervation in ALS may predispose to decompensation of the neuromuscular junction even with small amounts of toxin. It is perhaps surprising that more frequent side effects have not been reported in ALS, and the clinical course in this case may relate to the fact that this patient was treated for spasticity with a much larger dose than that generally used for sialorrhoea [8]. Clinicians should be aware of this potentially serious complication of the use of high-dose botulinum toxin in ALS. L. Kent P. Davies R. Kennett S. Wimalaratna R. Kerr M. R. Turner K. Talbot (&) Department of Neurology, John Radcliffe Hospital, Oxford, UK e-mail: Kevin.talbot@ndcn.ox.ac.uk

BACKGROUND: Lithium has neuroprotective effects in cell and animal models of amyotrophic lateral sclerosis (ALS), and a small pilot study in patients with ALS showed a significant effect of lithium on survival. We aimed to assess whether lithium improves survival in patients with ALS. METHODS: The lithium carbonate in amyotrophic lateral sclerosis (LiCALS) trial is a randomised, double-blind, placebo-controlled trial of oral lithium taken daily for 18 months in patients with ALS. Patients aged at least 18 years who had ALS according to the revised El Escorial criteria, had disease duration between 6 and 36 months, and were taking riluzole were recruited from ten centres in the UK. Patients were randomly assigned (1:1) to receive either lithium or matched placebo tablets. Randomisation was via an online system done at the level of the individual by block randomisation with randomly varying block sizes, stratified by study centre and site of disease onset (limb or bulbar). All patients and assessing study personnel were masked to treatment assignment. The primary endpoint was the rate of survival at 18 months and was analysed by intention to treat. This study is registered with Eudract, number 2008-006891-31. FINDINGS: Between May 26, 2009, and Nov 10, 2011, 243 patients were screened, 214 of whom were randomly assigned to receive lithium (107 patients) or placebo (107 patients). Two patients discontinued treatment and one died before the target therapeutic lithium concentration could be achieved. 63 (59%) of 107 patients in the placebo group and 54 (50%) of 107 patients in the lithium group were alive at 18 months. The survival functions did not differ significantly between groups (Mantel-Cox log-rank χ(2) on 1 df=1·64; p=0·20). After adjusting for study centre and site of onset using logistic regression, the relative odds of survival at 18 months (lithium vs placebo) was 0·71 (95% CI 0·40-1·24). 56 patients in the placebo group and 61 in the lithium group had at least one serious adverse event. INTERPRETATION: We found no evidence of benefit of lithium on survival in patients with ALS, but nor were there safety concerns, which had been identified in previous studies with less conventional designs. This finding emphasises the importance of pursuing adequately powered trials with clear endpoints when testing new treatments. FUNDING: The Motor Neurone Disease Association of Great Britain and Northern Ireland.

In 2011 Les Halpin was diagnosed with motor neuron disease (MND), also known as amyotrophic lateral sclerosis (ALS). Since his diagnosis, Les has been committed to accelerating the development, approval and licensing of novel therapies for people with life threatening illnesses. Two years ago he also founded Halpin Neurosciences, a privately owned biotechnology company, to focus on accelerating the development of therapies for MND. In 2012 Les formed a campaigning organisation, ‘Access to Medicine’ http://www.accesstomedicine.co.uk/, a platform to open up a wider debate about the lack of drug development for people with life threatening illnesses. A very good friend of mine died of motor neuron disease (MND) 20 years ago, as did a non-familial uncle 15 years ago. They received no therapies which could slow the disease progress, and from my perspective not a lot has changed since that time. This disease has affected me in a profound way, leaving me unable to walk and to use my limbs for everyday activities, affecting my speech, swallowing and breathing and robbing me of independence. The single licensed drug for MND, riluzole, is having no measurable impact on the progression of my disease nor on my quality of life. The current drug approval process is cumbersome—it can take years, and requires extremely large and risky investment, to take a drug from the development stage through to Phase III and beyond. As an example, no new drug has been approved for MND since riluzole, almost 20 years ago. The drug regulatory regime, therefore, must change in order to help people with life threatening and rare illnesses. For such individuals, the ‘risk-return ratio’ is different compared with patients with more benign conditions and drug regulations should be adapted to allow such people the opportunity to try out new combinations of drugs. Currently drug companies do not …

The 8th annual UK spinal muscular atrophy (SMA) Dr. Eduardo Tizzano (Hospital de Santa Creu i Sant research conference, jointly organised and sponsored by the SMA Trust and the Jennifer Trust for SMA, was held onOctober 3–4, 2011 inOxford, UK.Hosted by Prof. Kevin Talbot at the Oxford Belfry Hotel, the meeting was attended by over 40 researchers and clinicians from across Europe and a total of 17 platform presentations were given. The first day of talks provided a new perspective on the basic functions of the survival motor neuron (SMN) protein, challenged the general assumption that SMA is simply a disorder of the lower motor neuron, and provided interesting ideas on the cause of motor neuron specificity seen in the disease; the second day concentrated on practical aspects of clinical trials in SMA and explored some emerging therapies that are at the cusp of translation to human trials. Presentations on the first day were given by Prof. Michael Sendtner (University of Wurzburg, Germany) on a neuronal specific function of SMN, Dr. Thomas Wishart (University of Edinburgh, UK) on SMN protein expression at the synapse, Dr. Zameel Cader (University of Oxford, UK) on a non-cell autonomous mechanism of neuronal toxicity in distal SMA type V, Dr. Judith Sleeman (University of St. Andrews, UK) on small nuclear ribonucleoprotein dynamics and splicing, Prof. Thomas Gillingwater (University of Edinburgh, UK) on the differential susceptibility of tissues to SMN depletion, Dr. Simon Parson (University of Edinburgh, UK) on the microvasculature in a severe mouse model of SMA, Miss. Maria Gabriela Boza-Moran (University of London, UK) on using stem cells to elucidate pathological mechanisms of SMA, Dr. Heidi Fuller (Keele University, UK) on the proteomic effects of salbutamol treatment on SMA fibroblasts, and

Spinal muscular atrophy (SMA) is the most common lethal recessive disease in childhood, and there is currently no effective treatment to halt disease progression. The translation of scientific advances into effective therapies is hampered by major roadblocks in clinical trials, including the complex regulatory environment in Europe, variations in standards of care, patient ascertainment and enrolment, a narrow therapeutic window and a lack of biomarkers of efficacy. In this context, SMA-Europe organized its first international workshop in July 2012 in Rome, gathering 34 scientists, clinicians and representatives of patient organizations to establish recommendations for improving clinical trials for SMAa.

Two decades after the discovery that 20% of familial amyotrophic lateral sclerosis (ALS) cases were linked to mutations in the superoxide dismutase-1 (SOD1) gene, a substantial proportion of the remainder of cases of familial ALS have now been traced to an expansion of the intronic hexanucleotide repeat sequence in C9orf72. This breakthrough provides an opportunity to re-evaluate longstanding concepts regarding the cause and natural history of ALS, coming soon after the pathological unification of ALS with frontotemporal dementia through a shared pathological signature of cytoplasmic inclusions of the ubiquitinated protein TDP-43. However, with profound clinical, prognostic, neuropathological, and now genetic heterogeneity, the concept of ALS as one disease appears increasingly untenable. This background calls for the development of a more sophisticated taxonomy, and an appreciation of ALS as the breakdown of a wider network rather than a discrete vulnerable population of specialised motor neurons. Identification of C9orf72 repeat expansions in patients without a family history of ALS challenges the traditional division between familial and sporadic disease. By contrast, the 90% of apparently sporadic cases and incomplete penetrance of several genes linked to familial cases suggest that at least some forms of ALS arise from the interplay of multiple genes, poorly understood developmental, environmental, and age-related factors, as well as stochastic events.

Two candidate genes (NAIP and SMN) have recently been reported for childhood onset spinal muscular atrophy (SMA). Although affected subjects show deletions of these genes, these deletions can lead to either a very mild or a severe phenotype. We have analysed a large number of clinically well defined patients, carriers, and normal controls to assess the frequency and extent of deletions encompassing both of these genes. A genotype analysis indicates that more extensive deletions are seen in the severe form of SMA than in the milder forms. In addition, 1 center dot 9% of phenotypically normal carriers are deleted for the NAIP gene; no carriers were deleted for the SMN gene. Our data suggest that deletions in both of these genes, using the currently available assays, are associated with both a severe and very mild phenotype.

Background Guidelines caution against percutaneous endoscopic gastrostomy (PEG) in MND patients with forced vital capacity (FVC) below 50% predicted. However, radiological approaches (RIG) may be equally challenging in patients with advanced disease, and less reliable. Objectives To review Oxford MND Centre PEG patient characteristics, including the introduction of a risk stratification tool to identify those vulnerable to sedation-related complications, in whom a procedural adaptation was undertaken. Methods MND patients undergoing PEG insertion over the last four years were analysed. Cases stratified as higher risk underwent insertion in a semi-supine position, by a consultant gastroenterologist and experienced team using reduced sedation, and with intra-operative nasal non-invasive ventilation available. Results PEG was undertaken in 107 patients. All were successful in terms of immediate placement and recovery. Four deaths occurred within one month of insertion. Across the entire cohort one third had pre-procedure FVC Conclusions PEG can be safely performed in MND patients with advanced respiratory involvement. Screening and procedural adaptation for higher risk cases is associated with lower sedative doses.

Background Amyotrophic lateral sclerosis (ALS) is a progressive and usually fatal neurodegenerative disease. Survival from diagnosis varies considerably. Several prognostic factors are known, including site of onset (bulbar or limb), age at symptom onset, delay from onset to diagnosis and the use of riluzole and non-invasive ventilation (NIV). Clinicians and patients would benefit from a practical way of using these factors to provide an individualised prognosis. Methods 575 consecutive patients with incident ALS from a population-based registry in South-East England register for ALS (SEALS) were studied. Their survival was modelled as a two-step process: the time from diagnosis to respiratory muscle involvement, followed by the time from respiratory involvement to death. The effects of predictor variables were assessed separately for each time interval. Findings Younger age at symptom onset, longer delay from onset to diagnosis and riluzole use were associated with slower progression to respiratory involvement, and NIV use was associated with lower mortality after respiratory involvement, each with a clinically significant effect size. Riluzole may have a greater effect in younger patients and those with longer delay to diagnosis. A patient's survival time has a roughly 50% chance of falling between half and twice the predicted median. Interpretation A simple and clinically applicable graphical method of predicting an individual patient's survival from diagnosis is presented. The model should be validated in an independent cohort, and extended to include other important prognostic factors.

A hexanucleotide repeat expansion mutation in the C9orf72 locus is the commonest genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Currently, the biological function of C9orf72 remains unknown. However, bioinformatic analysis has revealed that the C9orf72 gene product has a differentially expressed in normal and neoplasia (DENN)-like domain and might regulate membrane trafficking as a Rab-GTPase GDP/GTP exchange factor. Here, using comprehensive GST-Rab pulldown analysis, we show that C9orf72 physically interacts with the Rab29 GTPase (also known as Rab7L1), suggesting that C9orf72 is a Rab29 effector. Further, we show that loss of the C9orf72/Rab29 interaction leads to dramatically reduced trafficking of extracellular vesicles (EVs), impaired intracellular trans-Golgi trafficking, and a consequent deficiency of autophagy in fibroblasts and iPSC-derived neurons from patients with C9ALS/FTD. Both overexpression of C9orf72 and antisense oligonucleotides targeting repeat-containing C9orf72 transcripts to upregulate normal variant 1 transcript levels are able independently to rescue the defective EV, trans-Golgi vesicle trafficking and basal autophagy phenotype. Our findings identify C9orf72 haploinsufficiency as a major contributing factor in ALS/FTD neurodegenerative disorders and directly link ALS/FTD to EV biogenesis. Furthermore, we demonstrate that this novel ALS/FTD pathogenic mechanism can be rescued by an antisense-based intervention.

Autosomal recessive spinal muscular atrophy is a motor neuron disease which affects about 1 in 10,000 births. Recent evidence shows that the candidate region contains multiple copies of genes and pseudogenes and is characterised by genome instability. We have analysed the frequency of deletions in a recently characterised candidate survival motor neuron (SMN) gene. Our data confirm previous analyses and show that this gene is disrupted by deletion in SMA patients. The same deletion frequency is observed in the milder variants of the disease as in patients with the severe form. In addition, we observed one case of a new mutation in a family previously thought not to be segregating for a chromosome 5 linked form of SMA. This assay is a very good diagnostic for SMA although no direct correlation between phenotype and genotype is apparent and carrier status cannot be determined. The implications for the identification of the gene or genes causing the disease are discussed.

Background Novel biomarker discovery and validation are essential to the development and appraisal of emerging therapies for the devastating cerebral neurodegenerative disorder motor neuron disease (MND), the commonest phenotype of which is amyotrophic lateral sclerosis (ALS). MND pathology (and strong genetic risk of MND prior to symptoms) was hypothesised to be manifested by changes in movement-related cortical neuronal β-oscillations. Methods We used magnetoencephalography (MEG) to investigate both symptomatic patients and asymptomatic mutation carriers at risk of developing ALS. MEG data were acquired during a visually cued lateralised motor preparation task that included infrequent movement inhibition trials. Data from 11 patients with ALS, ten with the upper motor neuron-only phenotype primary lateral sclerosis (PLS), and 12 asymptomatic mutation carriers were age-matched against healthy controls. Source-space MEG analysis used a combination of functionally defined voxels of interest and whole brain data to measure β-power before, during, and after movement execution or inhibition. Findings Patients with PLS were slower to respond to targets relative to controls (663 ms [SD 189] vs 440 [124], p=0·008), but the task was otherwise comparably performed across groups. Source reconstructed MEG data revealed excessive β-desynchronisation relative to controls in the motor cortices of both patients with ALS (p=0·012) and in asymptomatic mutation carriers (p=0·043). In only those patients with PLS, β-desynchronisation was inadequately lateralised to the contralateral motor cortex during response preparation. Transition to β-rebound was slowed in patients with ALS relative to controls (p=0·028). During successfully performed inhibition trials asymptomatic mutation carrier (p=0·046) groups demonstrated reduced inhibitory β-power relative to controls immediately before their expected response time to GO trials. Interpretation MEG is sensitive to cortical dysfunction in MND through changes in neural β-oscillations related to motor pathology. These abnormalities differ by phenotype, yet may be detectable before the development of overt symptoms in those at genetic risk. Funding Wellcome Trust, Medical Research Council.

As non-neurophysiologists we enjoyed this informative review of electromyography (EMG), and benefit enormously from the service provided by our local colleagues. We suggest, however, that EMG is neither ‘essential’ nor ‘diagnostic’ for motor neurone disease (MND). Rather, for at least the 85% of cases with classical amyotrophic lateral sclerosis, MND is a clinical diagnosis. The average delay in diagnosis of MND from the onset of symptoms has stubbornly remained around 1 year over the last two decades.1 Over-reliance on investigations, including neurophysiology, excluding implausible mimic conditions may be a contributory factor. The first …

Written and designed to provide comprehensive, easily accessible advice for all healthcare professionals involved in the care of patients with this challenging condition, this resource addresses the entire care pathway from presentation to diagnosis to symptom management and end of life issues.

Background Motor neurone disease (MND) is a devastating illness which leads to muscle weakness and death, usually within 2-3 years of symptom onset. Respiratory insufficiency is a common cause of morbidity, particularly in later stages of MND and respiratory complications are the leading cause of mortality in MND patients. Non Invasive Ventilation (NIV) is the current standard therapy to manage respiratory insufficiency. Some MND patients however do not tolerate NIV due to a number of issues including mask interface problems and claustrophobia. In those that do tolerate NIV, eventually respiratory muscle weakness will progress to a point at which intermittent/overnight NIV is ineffective. The NeuRx RA/4 Diaphragm Pacing System was originally developed for patients with respiratory insufficiency and diaphragm paralysis secondary to stable high spinal cord injuries. The DiPALS study will assess the effect of diaphragm pacing (DP) when used to treat patients with MND and respiratory insufficiency. Method/Design 108 patients will be recruited to the study at 5 sites in the UK. Patients will be randomised to either receive NIV (current standard care) or receive DP in addition to NIV. Study participants will be required to complete outcome measures at 5 follow up time points (2, 3, 6, 9 and 12 months) plus an additional surgery and 1 week post operative visit for those in the DP group. 12 patients (and their carers) from the DP group will also be asked to complete 2 qualitative interviews. Discussion The primary objective of this trial will be to evaluate the effect of Diaphragm Pacing (DP) on survival over the study duration in patients with MND with respiratory muscle weakness. The project is funded by the National Institute for Health Research, Health Technology Assessment (HTA) Programme (project number 09/55/33) and the Motor Neurone Disease Association and the Henry Smith Charity. Trial Registration: Current controlled trials ISRCTN53817913. The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the HTA programme, NIHR, NHS or the Department of Health.

Charcot-Marie-Tooth (CMT) disease comprises a heterogeneous group of peripheral neuropathies characterized by muscle weakness and wasting, and impaired sensation in the extremities. Four genes encoding an aminoacyl-tRNA synthetase (ARS) have been implicated in CMT disease. ARSs are ubiquitously expressed, essential enzymes that ligate amino acids to cognate tRNA molecules. Recently, a p.Arg329His variant in the alanyl-tRNA synthetase (AARS) gene was found to segregate with dominant axonal CMT type 2N (CMT2N) in two French families; however, the functional consequence of this mutation has not been determined. To investigate the role of AARS in CMT, we performed a mutation screen of the AARS gene in patients with peripheral neuropathy. Our results showed that p.Arg329His AARS also segregated with CMT disease in a large Australian family. Aminoacylation and yeast viability assays showed that p.Arg329His AARS severely reduces enzyme activity. Genotyping analysis indicated that this mutation arose on three distinct haplotypes, and the results of bisulfite sequencing suggested that methylation-mediated deamination of a CpG dinucleotide gives rise to the recurrent p.Arg329His AARS mutation. Together, our data suggest that impaired tRNA charging plays a role in the molecular pathology of CMT2N, and that patients with CMT should be directly tested for the p.Arg329His AARS mutation.

Historically, there has been a surprising level of uncertainty about whether amyotrophic lateral sclerosis (ALS), in its typical form, has a genetic basis. No less a figure than Jean-Martin Charcot (1825–93), at least initially, held the view that the disease he first named was never familial (Charcot and Joffroy, 1869). Some years earlier, in fact, familial cases of progressive muscular atrophy, a disorder that is now generally considered to be part of the same clinicopathological spectrum as ALS, were clearly described (Aran, 1850). Twenty years after Charcot, William Osler (1849–1919) reported typical ALS with autosomal dominant inheritance in a family from Vermont, which in the modern era, has been shown to have a mutation in SOD1 (Osler, 1880; Rouleau and Meijer, 2007). The view that the genetic contribution to ALS as a whole is relatively insignificant prevailed in the first half of the 20th century, but the development of modern molecular biology was a major impetus to the collection of familial kindreds with ALS in the 1980s and 1990s, leading ultimately to the identification in 1993 of mutations in SOD1 using classical linkage analysis (Rosen et al ., 1993). In recent years, the genetic landscape of ALS has been transformed. TDP43 has emerged as a signature protein in ALS and frontotemporal dementia (FTD) pathology (Neumann et al ., 2006) but also as a gene in which mutations have been found in 3–4% of familial ALS patients (Sreedharan et al ., 2008). In the last 2 years a further five genes have been associated with classical ALS ( FUS , OPTN , ANG , VCP and most recently UBQL2 : Greenway et al ., 2006; Vance et al ., …

Amyotrophic lateral sclerosis (ALS) appears to be a sporadic disorder in 95% of cases. Although few personal characteristics associated with developing ALS are known, identification of those at risk is essential to any vision of early intervention. There is persistent anecdotal observation that those with ALS are premorbidly physically 'fitter', although such observations are susceptible to bias. Hospital admission for coronary heart disease (CHD) might serve as an objective marker of reduced cardiovascular fitness.A record linkage study of two large databases of hospital admissions, the Oxford Record Linkage Study (ORLS) and an English national record linkage dataset of Hospital Episode Statistics was undertaken. The ratio of the rate of ALS in people without a record of CHD to that in those with a record of CHD was calculated, factoring out premature death in both cohorts. Similar analysis for Parkinson's disease (PD) and multiple sclerosis (MS) was undertaken.In the English population, the rate ratio for ALS in the non-CHD cohort, compared with the CHD cohort, was 1.14 (95% CI 1.05 to 1.22); for PD it was 0.95 (95% CI 0.93 to 0.98); and for MS 0.95 (95% CI 0.88 to 1.04). The ORLS data yielded similar findings.Those without a record of CHD were at modestly higher risk of ALS, but not for PD or MS. This lends support to the assertion that ALS arises within a population who may have relatively higher levels of cardiovascular fitness.

Background The objective of this research was to develop a disease-specific measure for fatigue in patients with motor neurone disease (MND) by generating data that would fit the Rasch measurement model. Fatigue was defined as reversible motor weakness and whole-body tiredness that was predominantly brought on by muscular exertion and was partially relieved by rest. Methods Qualitative interviews were undertaken to confirm the suitability of a previously identified set of 52 neurological fatigue items as relevant to patients with MND. Patients were recruited from five U.K. MND clinics. Questionnaires were administered during clinic or by post. A sub-sample of patients completed the questionnaire again after 2-4 weeks to assess test-retest validity. Exploratory factor analyses and Rasch analysis were conducted on the item set. Results Qualitative interviews with ten MND patients confirmed the suitability of 52 previously identified neurological fatigue items as relevant to patients with MND. 298 patients consented to completing the initial questionnaire including this item set, with an additional 78 patients completing the questionnaire a second time after 4-6 weeks. Exploratory Factor Analysis identified five potential subscales that could be conceptualised as representing: 'Energy', 'Reversible muscular weakness' (shortened to 'Weakness'), 'Concentration', 'Effects of heat' and 'Rest'. Of the original five factors, two factors 'Energy' and 'Weakness' met the expectations of the Rasch model. A higher order fatigue summary scale, consisting of items from the 'Energy' and 'Weakness' subscales, was found to fit the Rasch model and have acceptable unidimensionality. The two scales and the higher order summary scale were shown to fulfil model expectations, including assumptions of unidimensionality, local independency and an absence of differential item functioning. Conclusions The Neurological Fatigue Index for MND (NFI-MND) is a simple, easy-to-administer fatigue scale. It consists of an 8-item fatigue summary scale in addition to separate scales for measuring fatigue experienced as reversible muscular weakness and fatigue expressed as feelings of low energy and whole body tiredness. The underlying two factor structure supports the patient concept of fatigue derived from qualitative interviews in this population. All three scales were shown to be reliable and capable of interval level measurement.

HspB8, a small heat-shock protein implicated in autophagy, is mutated in patients with distal hereditary motor neuropathy type II (dHMNII). Autophagy is essential for maintaining protein homeostasis in the central nervous system, but its role has not been investigated in peripheral motor neurons. We used a novel, multispectral-imaging flow cytometry assay to measure autophagy in cells. This assay revealed that over-expression of wild-type HspB8 in motor neuron-like NSC34 cells led to an increased co-localisation of autophagosomes with the lysosomes. By contrast, over-expression of mutant HspB8 resulted in autophagosomes that co-localised with protein aggregates but failed to co-localise with the lysosomes. A similar impairment of autophagy could also be demonstrated in peripheral blood mononuclear cells from two dHMNII patients with the HspB8(K141E) mutation. We conclude that defects in HspB8-mediated autophagy are likely to contribute to dHMNII pathology and their detection in peripheral blood mononuclear cells could be a useful, accessible biomarker for the disease.

Background The Hospital Anxiety and Depression Scale (HADS) is commonly used to assess symptoms of anxiety and depression in motor neurone disease (MND). The measure has never been specifically validated for use within this population, despite questions raised about the scale's validity. This study seeks to analyse the construct validity of the HADS in MND by fitting its data to the Rasch model. Methods The scale was administered to 298 patients with MND. Scale assessment included model fit, differential item functioning (DIF), unidimensionality, local dependency and category threshold analysis. Results Rasch analyses were carried out on the HADS total score as well as depression and anxiety subscales (HADS-T, D and A respectively). After removing one item from both of the seven item scales, it was possible to produce modified HADS-A and HADS-D scales which fit the Rasch model. An 11-item higher-order HADS-T total scale was found to fit the Rasch model following the removal of one further item. Conclusion Our results suggest that a modified HADS-A and HADS-D are unidimensional, free of DIF and have good fit to the Rasch model in this population. As such they are suitable for use in MND clinics or research. The use of the modified HADS-T as a higher-order measure of psychological distress was supported by our data. Revised cut-off points are given for the modified HADS-A and HADS-D subscales.

Although traditionally regarded as spared, a range of oculomotor dysfunction has been recorded in patients with amyotrophic lateral sclerosis (ALS). Most frequent is ophthalmoparesis, particularly in patients with prolonged survival; however, pursuit, nystagmus, and saccadic impairments have also been reported. The apparent resistance to pathologic involvement of oculomotor (and sphincter) control pathways in most patients with ALS has prompted comparative study to establish the key pathways that underlie motor neuronal vulnerability, with the hope of generating novel therapeutic strategies. Developments in the assessment of oculomotor function, including portable eye-tracking devices, have revealed more subtle impairments in ALS in relation to phenotype, which can now be better understood through parallel elucidation of the normal cerebral oculomotor control network. Given the clinicopathologic overlap between ALS and some types of frontotemporal dementia, the study of oculomotor function has particular value in probing the variable but consistent cognitive impairment seen in ALS and that reflects frontotemporal extramotor cerebral abnormalities. By transcending the requirement to write or speak, loss of which precludes standard neuropsychological testing in some patients with advanced ALS, cognitive tests performed using only oculomotor functions offer additional potential, allowing the study of patients much later in their disease course. The study of oculomotor dysfunction holds significant promise as an additional source of much needed prognostic, monitoring, and mechanistic biomarkers for ALS.

The depolarising neuromuscular blocking agent suxamethonium chloride, frequently used during endotracheal intubation, is contraindicated in patients with chronic denervation in whom it can cause a life-threatening hyperkalaemic reaction, thought to be mediated through upregulation of nicotinic alpha7 acetylcholine receptors. An underlying neuromuscular disorder should be considered in all patients with acute respiratory insufficiency, and an alternative neuromuscular blocking drug must be used if there is any possibility of widespread denervation.

Charcot-Marie-Tooth disease type 2D (CMT2D) is a dominantly inherited peripheral neuropathy caused by missense mutations in the glycyl-tRNA synthetase gene (GARS). In addition to GARS, mutations in three other tRNA synthetase genes cause similar neuropathies, although the underlying mechanisms are not fully understood. To address this, we generated transgenic mice that ubiquitously over-express wild-type GARS and crossed them to two dominant mouse models of CMT2D to distinguish loss-of-function and gain-of-function mechanisms. Over-expression of wild-type GARS does not improve the neuropathy phenotype in heterozygous Gars mutant mice, as determined by histological, functional, and behavioral tests. Transgenic GARS is able to rescue a pathological point mutation as a homozygote or in complementation tests with a Gars null allele, demonstrating the functionality of the transgene and revealing a recessive loss-of-function component of the point mutation. Missense mutations as transgene-rescued homozygotes or compound heterozygotes have a more severe neuropathy than heterozygotes, indicating that increased dosage of the disease-causing alleles results in a more severe neurological phenotype, even in the presence of a wild-type transgene. We conclude that, although missense mutations of Gars may cause some loss of function, the dominant neuropathy phenotype observed in mice is caused by a dose-dependent gain of function that is not mitigated by over-expression of functional wild-type protein., Author Summary Mutations in the glycyl-tRNA synthetase gene (GARS) cause Charcot-Marie-Tooth disease type 2D, a disease characterized by neuronal axon loss in the arms and legs, resulting in weakness and sensory problems. The GARS protein is essential for protein synthesis in every cell, and it has been difficult to determine whether the mutations result in disease because they impair this function or whether GARS somehow becomes toxic when it is mutated. We generated mice that overexpress normal GARS and mated these to two different mouse models of the disease to determine whether a restoration of normal function could prevent the disease. These crosses demonstrated that the mutant forms of GARS are toxic, and this toxic effect increases as the amount of mutant protein increases. Furthermore, this toxicity cannot be reduced or prevented by providing additional normal GARS. Therefore, these results suggest that, for most patients, therapies need to specifically target the mutant form of GARS or the toxic function.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are hypothalamic peptides sharing considerable sequence homology which are postulated to be hypophysiotrophic releasing factors. When infused into man, PACAP has no effect on anterior pituitary hormone levels, while VIP causes a significant increase in circulating prolactin concentrations. However, PACAP has recently been shown to augment the release of LH and FSH in response to LHRH in rat anterior pituitary cell culture. In order to ascertain if either peptide has a similar effect in man, PACAP and VIP were infused at 3·6 pmol/kg per min into six healthy male volunteers, and an LHRH test was performed 30 min after the infusion was commenced. Infusion of PACAP did not alter the gonadotrophin response to LHRH significantly. However, VIP augmented the release of LH significantly, both during the infusion and for 30 min thereafter, although there was no effect on FSH release. Thus VIP, but not PACAP, potentiates the release of LH after LHRH injection in man. Journal of Endocrinology (1993) 137, 529–532

Charcot-Marie-Tooth (CMT) disease comprises a genetically and clinically heterogeneous group of peripheral nerve disorders characterized by impaired distal motor and sensory function. Mutations in three genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease primarily associated with an axonal pathology. ARSs are ubiquitously expressed, essential enzymes responsible for charging tRNA molecules with their cognate amino acids. To further explore the role of ARSs in CMT disease, we performed a large-scale mutation screen of the 37 human ARS genes in a cohort of 355 patients with a phenotype consistent with CMT. Here we describe three variants (p.Leu133His, p.Tyr173SerfsX7, and p.Ile302Met) in the lysyl-tRNA synthetase (KARS) gene in two patients from this cohort. Functional analyses revealed that two of these mutations (p.Leu133His and p.Tyr173SerfsX7) severely affect enzyme activity. Interestingly, both functional variants were found in a single patient with CMT disease and additional neurological and non-neurological sequelae. Based on these data, KARS becomes the fourth ARS gene associated with CMT disease, indicating that this family of enzymes is specifically critical for axon function.

Our objective was to establish the pattern of spread in lower limb-onset ALS (contra- versus ipsi-lateral) and its contribution to prognosis within a multivariate model. Pattern of spread was established in 109 sporadic ALS patients with lower limb-onset, prospectively recorded in Oxford and Sheffield tertiary clinics from 2001 to 2008. Survival analysis was by univariate Kaplan-Meier log-rank and multivariate Cox proportional hazards. Variables studied were time to next limb progression, site of next progression, age at symptom onset, gender, diagnostic latency and use of riluzole. Initial progression was either to the contralateral leg (76%) or ipsilateral arm (24%). Factors independently affecting survival were time to next limb progression, age at symptom onset, and diagnostic latency. Time to progression as a prognostic factor was independent of initial direction of spread. In a regression analysis of the deceased, overall survival from symptom onset approximated to two years plus the time interval for initial spread. In conclusion, rate of progression in lower limb-onset ALS is not influenced by whether initial spread is to the contralateral limb or ipsilateral arm. The time interval to this initial spread is a powerful factor in predicting overall survival, and could be used to facilitate decision-making and effective care planning.

Background: Juvenile amyotrophic lateral sclerosis (ALS) with basophilic inclusions is a form of ALS characterized by protein deposits in motor neurons that are morphologically and tinctorially distinct from those of classic sporadic ALS. The nosologic position of this type of ALS in the molecular pathologic and genetic classification of ALS is unknown. Methods: We identified neuropathologically 4 patients with juvenile ALS with basophilic inclusions and tested the hypothesis that specific RNA binding protein pathology may define this type of ALS. Immunohistochemical findings prompted us to sequence the fused in sarcoma ( FUS ) gene. Results: Motor symptoms began between ages 17 and 22. Disease progression was rapid without dementia. No family history was identified. Basophilic inclusions were strongly positive for FUS protein but negative for TAR DNA binding protein 43 (TDP-43). Granular and compact FUS deposits were identified in glia and neuronal cytoplasm and nuclei. Ultrastructure of aggregates was in keeping with origin from fragmented rough endoplasmic reticulum. Sequencing of all 15 exons of the FUS gene in 3 patients revealed a novel deletion mutation (c.1554_1557delACAG) in 1 individual and the c.1574C>T (P525L) mutation in 2 others. Conclusion: Juvenile ALS with basophilic inclusions is a FUS proteinopathy and should be classified as ALS-FUS. The FUS c.1574C>T (P525L) and c.1554_1557delACAG mutations are associated with this distinct phenotype. The molecular genetic relationship with frontotemporal lobar degeneration with FUS pathology remains to be clarified.

Motor neurons are large, highly polarised cells with very long axons and a requirement for precise spatial and temporal gene expression. Neurodegenerative disorders characterised by selective motor neuron vulnerability include various forms of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). A rapid expansion in knowledge on the pathophysiology of motor neuron degeneration has occurred in recent years, largely through the identification of genes leading to familial forms of ALS and SMA. The major emerging theme is that motor neuron degeneration can result from mutation in genes that encode factors important for ribonucleoprotein biogenesis and RNA processing, including splicing regulation, transcript stabilisation, translational repression and localisation of mRNA. Complete understanding of how these pathways interact and elucidation of specialised mechanisms for mRNA targeting and processing in motor neurons are likely to produce new targets for therapy in ALS and related disorders. © Cambridge University Press 2010.