Fasciculation electromechanical latency is prolonged in amyotrophic lateral sclerosis.

OBJECTIVE: In amyotrophic lateral sclerosis (ALS), motor neurons become hyperexcitable and spontaneously discharge electrical impulses causing fasciculations. These can be detected by two noninvasive methods: high-density surface electromyography (HDSEMG) and muscle ultrasonography (MUS). We combined these methods simultaneously to explore the electromechanical properties of fasciculations, seeking a novel biomarker of disease. METHODS: Twelve ALS patients and thirteen healthy participants each provided up to 24 minutes of recordings from the right biceps brachii (BB) and gastrocnemius medialis (GM). Two automated algorithms (Surface Potential Quantification Engine and a Gaussian mixture model) were applied to HDSEMG and MUS data to identify correlated electromechanical fasciculation events. RESULTS: We identified 4,197 correlated electromechanical fasciculation events. HDSEMG reliably detected electromechanical events up to 30 mm below the skin surface with an inverse correlation between amplitude and depth in ALS muscles. Compared to Healthy-GM muscles (mean = 79.8 ms), electromechanical latency was prolonged in ALS-GM (mean = 108.8 ms; p = 0.0458) and ALS-BB (mean = 112.0 ms; p = 0.0128) muscles. Electromechanical latency did not correlate with disease duration, symptom burden, sum muscle power score or fasciculation frequency. CONCLUSIONS: Prolonged fasciculation electromechanical latency indicates impairment of the excitation-contraction coupling mechanism, warranting further exploration as a potential novel biomarker of disease in ALS. SIGNIFICANCE: This study points to an electromechanical defect within the muscles of ALS patients.

Demystifying the spontaneous phenomena of motor hyperexcitability.

Possessing a discrete functional repertoire, the anterior horn cell can be in one of two electrophysiological states: on or off. Usually under tight regulatory control by the central nervous system, a hierarchical network of these specialist neurons ensures muscular strength is coordinated, gradated and adaptable. However, spontaneous activation of these cells and their axons can result in abnormal muscular twitching. The muscular twitch is the common building block of several distinct clinical patterns, namely fasciculation, myokymia and neuromyotonia. When attempting to distinguish these entities electromyographically, their unique temporal and morphological profiles must be appreciated. Detection and quantification of burst duration, firing frequency, multiplet patterns and amplitude are informative. A common feature is their persistence during sleep. In this review, we explain the accepted terminology used to describe the spontaneous phenomena of motor hyperexcitability, highlighting potential pitfalls amidst a bemusing and complex collection of overlapping terms. We outline the relevance of these findings within the context of disease, principally amyotrophic lateral sclerosis, Isaacs syndrome and Morvan syndrome. In addition, we highlight the use of high-density surface electromyography, suggesting that more widespread use of this non-invasive technique is likely to provide an enhanced understanding of these motor hyperexcitability syndromes.

Preprocessing surface EMG data removes voluntary muscle activity and enhances SPiQE fasciculation analysis.

OBJECTIVES: Fasciculations are a clinical hallmark of amyotrophic lateral sclerosis (ALS). The Surface Potential Quantification Engine (SPiQE) is a novel analytical tool to identify fasciculation potentials from high-density surface electromyography (HDSEMG). This method was accurate on relaxed recordings amidst fluctuating noise levels. To avoid time-consuming manual exclusion of voluntary muscle activity, we developed a method capable of rapidly excluding voluntary potentials and integrating with the established SPiQE pipeline. METHODS: Six ALS patients, one patient with benign fasciculation syndrome and one patient with multifocal motor neuropathy underwent monthly thirty-minute HDSEMG from biceps and gastrocnemius. In MATLAB, we developed and compared the performance of four Active Voluntary IDentification (AVID) strategies, producing a decision aid for optimal selection. RESULTS: Assessment of 601 one-minute recordings permitted the development of sensitive, specific and screening strategies to exclude voluntary potentials. Exclusion times (0.2-13.1 minutes), processing times (10.7-49.5 seconds) and fasciculation frequencies (27.4-71.1 per minute) for 165 thirty-minute recordings were compared. The overall median fasciculation frequency was 40.5 per minute (10.6-79.4 IQR). CONCLUSION: We hereby introduce AVID as a flexible, targeted approach to exclude voluntary muscle activity from HDSEMG recordings. SIGNIFICANCE: Longitudinal quantification of fasciculations in ALS could provide unique insight into motor neuron health.

Establishing the UK DNA Bank for motor neuron disease (MND).

In 2003 the Motor Neurone Disease (MND) Association, together with The Wellcome Trust, funded the creation of a national DNA Bank specific for MND. It was anticipated that the DNA Bank would constitute an important resource to researchers worldwide and significantly increase activity in MND genetic research. The DNA Bank houses over 3000 high quality DNA samples, all of which were donated by people living with MND, family members and non-related controls, accompanied by clinical phenotype data about the patients. Today the primary focus of the UK MND DNA Bank still remains to identify causative and disease modifying factors for this devastating disease.

Proteomic analyses reveal that loss of TDP-43 affects RNA processing and intracellular transport.

Transactive response DNA-binding protein 43 (TDP-43) is a predominantly nuclear, ubiquitously expressed RNA and DNA-binding protein. It recognizes and binds to UG repeats and is involved in pre-mRNA splicing, mRNA stability and microRNA metabolism. TDP-43 is essential in early embryonic development but accumulates in cytoplasmic aggregates in amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal lobar degeneration (FTLD). It is not known yet whether cytoplasmic aggregates of TDP-43 are toxic or protective but they are often associated with a loss of TDP-43 from the nucleus and neurodegeneration may be caused by a loss of normal TDP-43 function or a gain of toxic function. Here we present a proteomic study to analyze the effect of loss of TDP-43 on the proteome. MS data are available via ProteomeXchange with identifier PXD001668. Our results indicate that TDP-43 is an important regulator of RNA metabolism and intracellular transport. We show that Ran-binding protein 1 (RanBP1), DNA methyltransferase 3 alpha (Dnmt3a) and chromogranin B (CgB) are downregulated upon TDP-43 knockdown. Subsequently, transportin 1 level is increased as a result of RanBP1 depletion. Improper regulation of these proteins and the subsequent disruption of cellular processes may play a role in the pathogenesis of the TDP-43 proteinopathies ALS and FTLD.

Transportin 1 colocalization with Fused in Sarcoma (FUS) inclusions is not characteristic for amyotrophic lateral sclerosis-FUS confirming disrupted nuclear import of mutant FUS and distinguishing it from frontotemporal lobar degeneration with FUS inclusions.

AIMS: Transportin 1 (TNPO 1) is an abundant component of the Fused in Sarcoma (FUS)-immunopositive inclusions seen in a subgroup of frontotemporal lobar degeneration (FTLD-FUS). TNPO 1 has been shown to bind to the C-terminal nuclear localizing signal (NLS) of FUS and mediate its nuclear import. Amyotrophic lateral sclerosis (ALS)-linked C-terminal mutants disrupt TNPO 1 binding to the NLS and impair nuclear import in cell culture. If this held true for human ALS then we predicted that FUS inclusions in patients with C-terminal FUS mutations would not colocalize with TNPO 1. METHODS: Expression of TNPO 1 and colocalization with FUS was studied in the frontal cortex of FTLD-FUS (n = 3) and brain and spinal cord of ALS-FUS (n = 3), ALS-C9orf72 (n = 3), sporadic ALS (n = 7) and controls (n = 7). Expression levels and detergent solubility of TNPO 1 was measured by Western blot. RESULTS: Aggregates of TNPO 1 were abundant and colocalized with FUS inclusions in the cortex of all FTLD-FUS cases. In contrast, no TNPO 1-positive aggregates or FUS colocalization was evident in two-thirds, ALS-FUS cases and was rare in one ALS-FUS case. Nor were they present in C9orf72 or sporadic ALS. No increase in the levels of TNPO 1 was seen in Western blots of spinal cord tissues from all ALS cases compared with controls. CONCLUSIONS: These findings confirm that C-terminal FUS mutations prevent TNPO 1 binding to the NLS, inhibiting nuclear import and promoting cytoplasmic aggregation. The presence of TNPO 1 in wild-type FUS aggregates in FTLD-FUS distinguishes the two pathologies and implicates different disease mechanisms.

Mutational analysis reveals the FUS homolog TAF15 as a candidate gene for familial amyotrophic lateral sclerosis.

FUS, EWS, and TAF15 belong to the TET family of structurally similar DNA/RNA-binding proteins. Mutations in the FUS gene have recently been discovered as a cause of familial amyotrophic lateral sclerosis (FALS). Given the structural and functional similarities between the three genes, we screened TAF15 and EWS in 263 and 94 index FALS cases, respectively. No coding variants were found in EWS, while we identified six novel changes in TAF15. Of these, two 24 bp deletions and a R388H missense variant were also found in healthy controls. A D386N substitution was shown not to segregate with the disease in the affected pedigree. A single A31T and two R395Q changes were identified in FALS cases but not in over 1,100 controls. Interestingly, one of the R395Q FALS cases also harbors a TARDBP mutation (G384R). Altogether, these results suggest that additional studies are needed to determine whether mutations in the TAF15 gene represent a cause of FALS.

An estimate of amyotrophic lateral sclerosis heritability using twin data.

BACKGROUND: Causative gene mutations have been identified in about 2% of those with amyotrophic lateral sclerosis (ALS), often, but not always, when there is a strong family history. There is an assumption that there is a genetic component to all ALS, but genome-wide association studies have yet to produce a robustly replicated result. A definitive estimate of ALS heritability is therefore required to determine whether ongoing efforts to find susceptibility genes are worth while. METHODS: The authors performed two twin studies, one population- and one clinic-based. The authors used structural equation modelling to perform a meta-analysis of data from these studies and an existing twin study to estimate ALS heritability, and identified 171 twin pairs in which at least one twin had ALS. RESULTS AND DISCUSSION: Five monozygotic twin pairs were concordant-affected, and 44 discordant-affected. No dizygotic twin pairs were concordant-affected, and 122 discordant-affected. The heritability of sporadic ALS was estimated as 0.61 (0.38 to 0.78) with the unshared environmental component 0.39 (0.22 to 0.62). ALS has a high heritability, and efforts to find causative genes should continue.

Tau levels do not influence human ALS or motor neuron degeneration in the SOD1G93A mouse.

BACKGROUND: The microtubule-associated protein tau is thought to play a pivotal role in neurodegeneration. Mutations in the tau coding gene MAPT are a cause of frontotemporal dementia, and the H1/H1 genotype of MAPT, giving rise to higher tau expression levels, is associated with progressive supranuclear palsy, corticobasal degeneration, and Parkinson disease (PD). Furthermore, tau hyperphosphorylation and aggregation is a hallmark of Alzheimer disease (AD), and reducing endogenous tau has been reported to ameliorate cognitive impairment in a mouse model for AD. Tau hyperphosphorylation and aggregation have also been described in amyotrophic lateral sclerosis (ALS), both in human patients and in the mutant SOD1 mouse model for this disease. However, the precise role of tau in motor neuron degeneration remains uncertain. METHODS: The possible association between ALS and the MAPT H1/H2 polymorphism was studied in 3,540 patients with ALS and 8,753 controls. Furthermore, the role of tau in the SOD1(G93A) mouse model for ALS was studied by deleting Mapt in this model. RESULTS: The MAPT genotype of the H1/H2 polymorphism did not influence ALS susceptibility (odds ratio = 1.08 [95% confidence interval 0.99-1.18], p = 0.08) and did not affect the clinical phenotype. Lowering tau levels in the SOD1(G93A) mouse failed to delay disease onset (p = 0.302) or to increase survival (p = 0.557). CONCLUSION: These findings suggest that the H1/H2 polymorphism in MAPT is not associated with human amyotrophic lateral sclerosis, and that lowering tau levels in the mutant SOD1 mouse does not affect the motor neuron degeneration in these animals.

Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

Four novel SPG3A/atlastin mutations identified in autosomal dominant hereditary spastic paraplegia kindreds with intra-familial variability in age of onset and complex phenotype.

Mutation of the atlastin gene (SPG3A) is responsible for approximately 10% of autosomal dominant hereditary spastic paraplegia (AD-HSP) cases. The goal of this study was to identify novel disease causing atlastin mutations. Atlastin nucleotide variations were detected by direct sequencing of all 14 exons in 70 autosomal dominant (AD), 16 single sibship and 14 sporadic spastic paraplegia patients. Six mis-sense mutations (four of which were novel) were identified in six unrelated AD-HSP kindreds in exons 4, 7 and 8 of the atlastin gene. One kindred with a novel mutation showed variability in clinical phenotype and age of onset. Mutations are predicted to decrease GTPase activity, cause morphological abnormalities of the endoplasmic reticulum and prevent maturation of the Golgi complex resulting in impaired vesicle trafficking. Our study significantly adds to the spectrum of mutations and clinical phenotype of SPG3A. We advocate that all spastin mutation negative AD-HSP kindreds should be screened for pathogenic atlastin mutations regardless of age of onset or phenotypic complexity.

A large-scale international meta-analysis of paraoxonase gene polymorphisms in sporadic ALS.

BACKGROUND: Six candidate gene studies report a genetic association of DNA variants within the paraoxonase locus with sporadic amyotrophic lateral sclerosis (ALS). However, several other large studies, including five genome-wide association studies, have not duplicated this finding. METHODS: We conducted a meta-analysis of 10 published studies and one unpublished study of the paraoxonase locus, encompassing 4,037 ALS cases and 4,609 controls, including genome-wide association data from 2,018 ALS cases and 2,425 controls. RESULTS: The combined fixed effects odds ratio (OR) for rs662 (PON1 Q192R) was 1.09 (95% confidence interval [CI], 1.02-1.16, p = 0.01); the genotypic OR for RR homozygotes at Q192R was 1.25 (95% CI, 1.07-1.45, p = 0.0004); the combined OR for rs854560 (PON1 L55M) was 0.97 (95% CI, 0.86-1.10, p = 0.62); the OR for rs10487132 (PON2) was 1.08 (95% CI, 0.92-1.27, p = 0.35). Although the rs662 polymorphism reached a nominal level of significance, no polymorphism was significant after multiple testing correction. In the subanalysis of samples with genome-wide data from which population outliers were removed, rs662 had an OR of 1.06 (95% CI, 0.97-1.16, p = 0.22). CONCLUSIONS: In contrast to previous positive smaller studies, our genetic meta-analysis showed no significant association of amyotrophic lateral sclerosis (ALS) with the PON locus. This is the largest meta-analysis of a candidate gene in ALS to date and the first ALS meta-analysis to include data from whole genome association studies. The findings reinforce the need for much larger and more collaborative investigations of the genetic determinants of ALS.

SOD1 and cognitive dysfunction in familial amyotrophic lateral sclerosis.

BACKGROUND: Sporadic Amyotrophic Lateral Sclerosis (sALS) is associated with frontotemporal dementia (ALS-FTD) or milder deficits of cognitive (predominantly executive) dysfunction (ALSCi) in some patients. Some forms of familial ALS (FALS) have a family history of FTD, ALS-FTD, or both, but there have been few reports of ALS-FTD in FALS patients with mutations of the gene superoxide dismutase-1 (SOD1 FALS). The aim of this study was to test the hypothesis that ALSCi may be found in non-SOD1 FALS, but that SOD1 FALS patients would show little or no evidence of cognitive change. METHODS: A neuropsychological test battery was administered to 41 SALS patients, 35 control participants, 7 FALS patients with a SOD1 mutation (SOD1 FALS) and 10 FALS patients without a SOD1 mutation (non-SOD1 FALS). RESULTS: Relative to control participants, non-SOD1 FALS patients had impaired performance on written verbal fluency and confrontation naming, and reported higher levels of executive behavioural problems. These deficits were absent in SOD1 FALS patients. SALS patients performed poorer than controls only on the Graded Naming Test. All ALS groups had higher levels of behavioural apathy and emotional lability than were found in control participants. Cognitive domains of memory, receptive language, and visuospatial perception were spared. Groups were matched for age, gender, premorbid full-scale IQ, anxiety and depression. DISCUSSION: Individuals with SOD1 gene mutations are less likely to have significant cognitive changes compared to non-SOD1 FALS patients. Cognitive abnormalities in ALS are heterogeneous and may reflect underlying genetic variations rather than a simple spectrum of extra-motor involvement.

Natural history and clinical features of the flail arm and flail leg ALS variants.

OBJECTIVE: We sought to define the significance of brachial amyotrophic diplegia (flail arm syndrome [FA]) and the pseudopolyneuritic variant (flail leg syndrome [FL]) of amyotrophic lateral sclerosis (ALS; motor neuron disease). METHODS: We analyzed survival in clinic cohorts in London, UK (1,188 cases), and Melbourne, Australia (432 cases). Survival from disease onset was analyzed using the Kaplan- Meier method and Cox proportional hazards model. RESULTS: In the London cohort, the FA syndrome represented 11% and the FL syndrome 6% of the sample. Median survival was 35 months for limb onset and 27 months for bulbar onset ALS, whereas this was 61 months for FA syndrome (p < 0.001) and 69 months for FL syndrome (p < 0.001). Five-year survival in this cohort was 8.8% for bulbar onset, 20% for limb onset, 52% for FA syndrome, and 64% for FL syndrome. The ratio of men to women was 4:1 in the FA group compared to 2:1 in other limb onset cases. Excluding lower motor neuron FA and FL cases, progressive muscular atrophy comprised 4% of the sample and had a prognosis similar to typical limb onset ALS. In the Melbourne cohort, median survival for limb onset ALS was 31 months, bulbar onset 27 months, FA syndrome 66 months (p < 0.001), and FL syndrome 71 months (p = 0.001). CONCLUSIONS: The flail arm (FA) and flail leg (FL) syndromes had significantly better survival than typical amyotrophic lateral sclerosis (ALS) or progressive muscular atrophy cases that were not classified as FA or FL. Our findings underline the clinical and prognostic importance of the FA and FL variants of ALS.

New VAPB deletion variant and exclusion of VAPB mutations in familial ALS.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disorder involving upper and lower motor neurons. The vesicle-associated membrane protein B (VAPB) gene has been genetically linked to ALS in several large Brazilian families in which the disorder is caused by a proline to serine mutation at codon 56 (P56S). No additional mutations have been identified. METHODS: To establish the prevalence of VAPB mutations, we screened 80 familial ALS samples by DNA sequencing. RESULTS: Our study failed to identify any novel VAPB gene mutations but identified a single Brazilian family harboring the P56S mutation. In a second familial ALS case, we identified a three-base pair deletion within exon 5 of the VAPB gene that deleted the serine residue at position 160 (Delta S160). This variant is detected in a normal population at low frequency (0.45%). Analyses of homology alignment and secondary structure predict that this deletion significantly alters the structure of VAPB, although a GFP-Delta S160 VAPB fusion protein demonstrates a wild-type subcellular localization. This contrasts the aberrant localization observed in a GFP-P56S VAPB fusion protein. The allele frequency of Delta S160 in patients with sporadic ALS does not differ significantly from that in the normal population. CONCLUSIONS: Mutations in the VAPB gene are rare and the Delta S160 variant does not contribute to the development of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis in South-East England: a population-based study. The South-East England register for amyotrophic lateral sclerosis (SEALS Registry).

BACKGROUND/AIMS: We aimed to estimate the incidence and prevalence of amyotrophic lateral sclerosis (ALS) in the South East of England. The reported incidence of ALS varies between 0.44 and 3.2 per 100,000 person years. This can partly be explained by differences in design and diagnostic criteria used. There is little population data concerning England, particularly the South East. METHODS: A population study of South-East England (total population: 2,890,482) was carried out and multiple sources including our tertiary centre and district general hospitals were used for complete case ascertainment. RESULTS: Between 1 January 2002 and 30 June 2006 we identified 138 people (76 males; 62 females) with a new diagnosis of ALS, giving a crude incidence of 1.06 per 100,000 person years. The projected age- and gender-adjusted annual incidence rate for England and Wales was 1.10 (95% CI 0.80-1.40). 142 people were alive on 30 June 2006, giving a point prevalence of 4.91 per 100,000 population. CONCLUSION: Our incidence and prevalence rates are similar to those reported in comparable studies from other countries. This argues against the role of a specific exogenous factor in the aetiology of ALS in South-East England.

Proteome-based plasma biomarkers for Alzheimer's disease.

Alzheimer's disease is a common and devastating disease for which there is no readily available biomarker to aid diagnosis or to monitor disease progression. Biomarkers have been sought in CSF but no previous study has used two-dimensional gel electrophoresis coupled with mass spectrometry to seek biomarkers in peripheral tissue. We performed a case-control study of plasma using this proteomics approach to identify proteins that differ in the disease state relative to aged controls. For discovery-phase proteomics analysis, 50 people with Alzheimer's dementia were recruited through secondary services and 50 normal elderly controls through primary care. For validation purposes a total of 511 subjects with Alzheimer's disease and other neurodegenerative diseases and normal elderly controls were examined. Image analysis of the protein distribution of the gels alone identifies disease cases with 56% sensitivity and 80% specificity. Mass spectrometric analysis of the changes observed in two-dimensional electrophoresis identified a number of proteins previously implicated in the disease pathology, including complement factor H (CFH) precursor and alpha-2-macroglobulin (alpha-2M). Using semi-quantitative immunoblotting, the elevation of CFH and alpha-2M was shown to be specific for Alzheimer's disease and to correlate with disease severity although alternative assays would be necessary to improve sensitivity and specificity. These findings suggest that blood may be a rich source for biomarkers of Alzheimer's disease and that CFH, together with other proteins such as alpha-2M may be a specific markers of this illness.

Racial and ethnic disparities in survival in lung transplant candidates with idiopathic pulmonary fibrosis.

Minority patients have worse outcomes than nonminority patients in a variety of pulmonary diseases. We aimed to compare the survival of Black and Hispanic patients to that of others with idiopathic pulmonary fibrosis (IPF). We performed a retrospective cohort study of patients with IPF who were evaluated for lung transplantation at our center. Kaplan-Meier survival curves and Cox proportional hazards models were used to compare survival between groups. Black and Hispanic patients had spirometry, lung volumes and diffusion capacity that were similar to others, but had worse exercise capacity. Minority patients had a significantly increased risk of death compared to others independent of transplantation status (hazard ratio = 3.3, 95% CI 1.2-8.9, p = 0.02). Differences in exercise capacity, pulmonary hemodynamics and socioeconomic factors appeared to account for some of the differences in survival. Black and Hispanic patients with IPF had an increased risk of death following referral for lung transplantation. This finding may be due to differences in disease progression and/or differences in access to medical care among minority patients. Future studies should confirm our findings in a larger cohort. The elimination of racial and ethnic disparities in outcome should be a priority for clinicians and researchers in this field.