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1.  An Evaluation of a SVA Retrotransposon in the FUS Promoter as a Transcriptional Regulator and Its Association to ALS 
PLoS ONE  2014;9(3):e90833.
Genetic mutations of FUS have been linked to many diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration. A primate specific and polymorphic retrotransposon of the SINE-VNTR-Alu (SVA) family is present upstream of the FUS gene. Here we have demonstrated that this retrotransposon can act as a classical transcriptional regulatory domain in the context of a reporter gene construct both in vitro in the human SK-N-AS neuroblastoma cell line and in vivo in a chick embryo model. We have also demonstrated that the SVA is composed of multiple distinct regulatory domains, one of which is a variable number tandem repeat (VNTR). The ability of the SVA and its component parts to direct reporter gene expression supported a hypothesis that this region could direct differential FUS expression in vivo. The SVA may therefore contribute to the modulation of FUS expression exhibited in and associated with neurological disorders including ALS where FUS regulation may be an important parameter in progression of the disease. As VNTRs are often clinical associates for disease progression we determined the extent of polymorphism within the SVA. In total 2 variants of the SVA were identified based within a central VNTR. Preliminary analysis addressed the association of these SVA variants within a small sporadic ALS cohort but did not reach statistical significance, although we did not include other parameters such as SNPs within the SVA or an environmental factor in this analysis. The latter may be particularly important as the transcriptional and epigenetic properties of the SVA are likely to be directed by the environment of the cell.
doi:10.1371/journal.pone.0090833
PMCID: PMC3946630  PMID: 24608899
2.  The widening spectrum of C9ORF72-related disease; genotype/phenotype correlations and potential modifiers of clinical phenotype 
Acta Neuropathologica  2014;127:333-345.
The GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common cause of familial amyotrophic lateral sclerosis (ALS), frontotemporal lobar dementia (FTLD) and ALS–FTLD, as well as contributing to sporadic forms of these diseases. Screening of large cohorts of ALS and FTLD cohorts has identified that C9ORF72-ALS is represented throughout the clinical spectrum of ALS phenotypes, though in comparison with other genetic subtypes, C9ORF72 carriers have a higher incidence of bulbar onset disease. In contrast, C9ORF72-FTLD is predominantly associated with behavioural variant FTD, which often presents with psychosis, most commonly in the form of hallucinations and delusions. However, C9ORF72 expansions are not restricted to these clinical phenotypes. There is a higher than expected incidence of parkinsonism in ALS patients with C9ORF72 expansions, and the G4C2 repeat has also been reported in other motor phenotypes, such as primary lateral sclerosis, progressive muscular atrophy, corticobasal syndrome and Huntington-like disorders. In addition, the expansion has been identified in non-motor phenotypes including Alzheimer’s disease and Lewy body dementia. It is not currently understood what is the basis of the clinical variation seen with the G4C2 repeat expansion. One potential explanation is repeat length. Sizing of the expansion by Southern blotting has established that there is somatic heterogeneity, with different expansion lengths in different tissues, even within the brain. To date, no correlation with expansion size and clinical phenotype has been established in ALS, whilst in FTLD only repeat size in the cerebellum was found to correlate with disease duration. Somatic heterogeneity suggests there is a degree of instability within the repeat and evidence of anticipation has been reported with reducing age of onset in subsequent generations. This variability/instability in expansion length, along with its interactions with environmental and genetic modifiers, such as TMEM106B, may be the basis of the differing clinical phenotypes arising from the mutation.
doi:10.1007/s00401-014-1251-9
PMCID: PMC3925297  PMID: 24493408
Amyotrophic lateral sclerosis; Frontotemporal lobar dementia; C9ORF72; G4C2 expansion; Phenotypic variation; Genetic modifiers
3.  Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis 
PLoS ONE  2014;9(1):e87508.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes death within a mean of 2–3 years from symptom onset. There is no diagnostic test and the delay from symptom onset to diagnosis averages 12 months. The identification of prognostic and diagnostic biomarkers in ALS would facilitate earlier diagnosis and faster monitoring of treatments. Gene expression profiling (GEP) can help to identify these markers as well as therapeutic targets in neurological diseases. One source of genetic material for GEP in ALS is peripheral blood, which is routinely accessed from patients. However, a high proportion of globin mRNA in blood can mask important genetic information. A number of methods allow safe collection, storage and transport of blood as well as RNA stabilisation, including the PAXGENE and TEMPUS systems for the collection of whole blood and LEUKOLOCK which enriches for the leukocyte population. Here we compared these three systems and assess their suitability for GEP in ALS. We collected blood from 8 sporadic ALS patients and 7 controls. PAXGENE and TEMPUS RNA extracted samples additionally underwent globin depletion using GlobinClear. RNA was amplified and hybridised onto Affymetrix U133 Plus 2.0 arrays. Lists of genes differentially regulated in ALS patients and controls were created for each method using the R package PUMA, and RT-PCR validation was carried out on selected genes. TEMPUS/GlobinClear, and LEUKOLOCK produced high quality RNA with sufficient yield, and consistent array expression profiles. PAXGENE/GlobinClear yield and quality were lower. Globin depletion for PAXGENE and TEMPUS uncovered the presence of over 60% more transcripts than when samples were not depleted. TEMPUS/GlobinClear and LEUKOLOCK gene lists respectively contained 3619 and 3047 genes differentially expressed between patients and controls. Real-time PCR validation revealed similar reliability between these two methods and gene ontology analyses revealed similar pathways differentially regulated in disease compared to controls.
doi:10.1371/journal.pone.0087508
PMCID: PMC3903649  PMID: 24475299
4.  A new zebrafish model produced by TILLING of SOD1-related amyotrophic lateral sclerosis replicates key features of the disease and represents a tool for in vivo therapeutic screening 
Disease Models & Mechanisms  2013;7(1):73-81.
Mutations in the superoxide dismutase gene (SOD1) are one cause of familial amyotrophic lateral sclerosis [ALS; also known as motor neuron disease (MND)] in humans. ALS is a relentlessly progressive neurodegenerative disease and, to date, there are no neuroprotective therapies with significant impact on the disease course. Current transgenic murine models of the disease, which overexpress mutant SOD1, have so far been ineffective in the identification of new therapies beneficial in the human disease. Because the human and the zebrafish (Danio rerio) SOD1 protein share 76% identity, TILLING (‘targeting induced local lesions in genomes’) was carried out in collaboration with the Sanger Institute in order to identify mutations in the zebrafish sod1 gene. A T70I mutant zebrafish line was characterised using oxidative stress assays, neuromuscular junction (NMJ) analysis and motor function studies. The T70I sod1 zebrafish model offers the advantage over current murine models of expressing the mutant Sod1 protein at a physiological level, as occurs in humans with ALS. The T70I sod1 zebrafish demonstrates key features of ALS: an early NMJ phenotype, susceptibility to oxidative stress and an adult-onset motor neuron disease phenotype. We have demonstrated that the susceptibility of T70I sod1 embryos to oxidative stress can be used in a drug screening assay, to identify compounds that merit further investigation as potential therapies for ALS.
doi:10.1242/dmm.012013
PMCID: PMC3882050  PMID: 24092880
MND; ALS; SOD1; Zebrafish
5.  Assessing social isolation in motor neurone disease: A Rasch analysis of the MND Social Withdrawal Scale☆ 
Journal of the Neurological Sciences  2013;334(1-2):112-118.
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.
doi:10.1016/j.jns.2013.08.002
PMCID: PMC3837185  PMID: 24011605
Social withdrawal; Rasch analysis; Psychological distress; ALS; Amyotrophic lateral sclerosis; MND; Motor neurone disease
6.  Wild-type but not mutant SOD1 transgenic astrocytes promote the efficient generation of motor neuron progenitors from mouse embryonic stem cells 
BMC Neuroscience  2013;14:126.
Background
The efficient derivation of mature (Hb9+) motor neurons from embryonic stem cells is a sought-after goal in the understanding, and potential treatment, of motor neuron diseases. Conditions that promote the robust generation of motor neuron progenitors from embryonic stem cells and that promote the survival of differentiated motor neurons ex vivo are likely, therefore, to be critical in future biological/therapeutic/screening approaches. Previous studies have shown that astrocytes have a protective effect on differentiated motor neurons (in vivo and ex vivo), but it remains unclear whether astrocytes also play a beneficial role in the support of motor neuron progenitors. Here we explore the effect of murine astrocyte-conditioned medium on monolayer cultures of mouse embryonic stem cell-derived motor neuron progenitors.
Results
Our data show that wild-type astrocyte-conditioned medium significantly increases the number of Olig2+/Hb9- progenitors, which subsequently differentiate into Hb9+/Islet1+ post-mitotic motor neurons. Intriguingly, while astrocyte-conditioned medium derived from mice transgenic for wild-type human SOD1 mimics the effect of wild-type astrocytes, conditioned medium derived from astrocytes carrying an amyotrophic lateral sclerosis-related SOD1-G93A mutation shows no such beneficial effect. The effect of astrocyte-conditioned medium, moreover, is specific to motor neurons: we find that interneurons generated from mouse embryonic stem cells are unaffected by conditioned media from any type of astrocyte.
Conclusions
Our study indicates that conditioned medium derived from wild type astrocytes enhances the efficient generation of motor neurons from mouse embryonic stem cells by enhancing motor neuron progenitors. In contrast, conditioned medium from SOD1-G93A astrocytes does not show a similar enhancing effect.
doi:10.1186/1471-2202-14-126
PMCID: PMC3853012  PMID: 24134124
ES cells; G93A astrocytes; Motor neuron; Progenitor; Differentiation; ALS
7.  Residual association at C9orf72 suggests an alternative amyotrophic lateral sclerosis-causing hexanucleotide repeat 
Neurobiology of Aging  2013;34(9):2234.e1-2234.e7.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons. Single-nucleotide polymorphism rs3849942 is associated with ALS, tagging a hexanucleotide repeat mutation in the C9orf72 gene. It is possible that there is more than 1 disease-causing genetic variation at this locus, in which case association might remain after removal of cases carrying the mutation. DNA from patients with ALS was therefore tested for the mutation. Genome-wide association testing was performed first using all samples, and then restricting the analysis to samples not carrying the mutation. rs3849942 and rs903603 were strongly associated with ALS when all samples were included (rs3849942, p = [3 × 2] × 10−6, rank 7/442,057; rs903603, p = [7 × 6] × 10−8, rank 2/442,057). Removal of the mutation-carrying cases resulted in loss of association for rs3849942 (p = [2 × 6] × 10−3, rank 1225/442,068), but had little effect on rs903603 (p = [1 × 9] × 10−5, rank 8/442,068). Those with a risk allele of rs903603 had an excess of apparent homozygosity for wild type repeat alleles, consistent with polymerase chain reaction failure of 1 allele because of massive repeat expansion. These results indicate residual association at the C9orf72 locus suggesting a second disease-causing repeat mutation.
doi:10.1016/j.neurobiolaging.2013.03.003
PMCID: PMC3753508  PMID: 23587638
Amyotrophic lateral sclerosis; Genetics; C9orf72; Hexanucleotide repeat mutation; GWAS; Residual association
8.  S[+] Apomorphine is a CNS penetrating activator of the Nrf2-ARE pathway with activity in mouse and patient fibroblast models of amyotrophic lateral sclerosis☆ 
Free Radical Biology & Medicine  2013;61(100):438-452.
Compelling evidence indicates that oxidative stress contributes to motor neuron injury in amyotrophic lateral sclerosis (ALS), but antioxidant therapies have not yet achieved therapeutic benefit in the clinic. The nuclear erythroid 2-related-factor 2 (Nrf2) transcription factor is a key regulator of an important neuroprotective response by driving the expression of multiple cytoprotective genes via its interaction with the antioxidant response element (ARE). Dysregulation of the Nrf2-ARE system has been identified in ALS models and human disease. Taking the Nrf2-ARE pathway as an attractive therapeutic target for neuroprotection in ALS, we aimed to identify CNS penetrating, small molecule activators of Nrf2-mediated transcription in a library of 2000 drugs and natural products. Compounds were screened extensively for Nrf2 activation, and antioxidant and neuroprotective properties in vitro. S[+]-Apomorphine, a receptor-inactive enantiomer of the clinically approved dopamine-receptor agonist (R[–]-apomorphine), was identified as a nontoxic Nrf2 activating molecule. In vivo S[+]-apomorphine demonstrated CNS penetrance, Nrf2 induction, and significant attenuation of motor dysfunction in the SOD1G93A transgenic mouse model of ALS. S[+]-apomorphine also reduced pathological oxidative stress and improved survival following an oxidative insult in fibroblasts from ALS patients. This molecule emerges as a promising candidate for evaluation as a potential neuroprotective agent in ALS patients in the clinic.
doi:10.1016/j.freeradbiomed.2013.04.018
PMCID: PMC3684770  PMID: 23608463
ALS, amyotrophic lateral sclerosis; ARE, antioxidant response element; carboxy-H2DCFDA, 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate; CNS, central nervous system; DCF, dichlorofluorescein; MEFs, mouse embryonic fibroblasts; Nrf2, nuclear erythroid 2-related-factor 2; PBPK, physiologically based pharmacokinetic; Q-RTPCR, quantitative RT-PCR; Amyotrophic lateral sclerosis; motor neurone disease; Nrf2; preclinical pharmacology; neurodegeneration
9.  Tardbpl splicing rescues motor neuron and axonal development in a mutant tardbp zebrafish 
Human Molecular Genetics  2013;22(12):2376-2386.
Mutations in the transactive response DNA binding protein-43 (TARDBP/TDP-43) gene, which regulates transcription and splicing, causes a familial form of amyotrophic lateral sclerosis (ALS). Here, we characterize and report the first tardbp mutation in zebrafish, which introduces a premature stop codon (Y220X), eliminating expression of the Tardbp protein. Another TARDBP ortholog, tardbpl, in zebrafish is shown to encode a Tardbp-like protein which is truncated compared with Tardbp itself and lacks part of the C-terminal glycine-rich domain (GRD). Here, we show that tardbp mutation leads to the generation of a novel tardbpl splice form (tardbpl-FL) capable of making a full-length Tardbp protein (Tardbpl-FL), which compensates for the loss of Tardbp. This finding provides a novel in vivo model to study TDP-43-mediated splicing regulation. Additionally, we show that elimination of both zebrafish TARDBP orthologs results in a severe motor phenotype with shortened motor axons, locomotion defects and death at around 10 days post fertilization. The Tardbp/Tardbpl knockout model generated in this study provides an excellent in vivo system to study the role of the functional loss of Tardbp and its involvement in ALS pathogenesis.
doi:10.1093/hmg/ddt082
PMCID: PMC3658164  PMID: 23427147
10.  Neuronal dark matter: the emerging role of microRNAs in neurodegeneration 
MicroRNAs (miRNAs) are small, abundant RNA molecules that constitute part of the cell's non-coding RNA “dark matter.” In recent years, the discovery of miRNAs has revolutionised the traditional view of gene expression and our understanding of miRNA biogenesis and function has expanded. Altered expression of miRNAs is increasingly recognized as a feature of many disease states, including neurodegeneration. Here, we review the emerging role for miRNA dysfunction in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and Huntington's disease pathogenesis. We emphasize the complex nature of gene regulatory networks and the need for systematic studies, with larger sample cohorts than have so far been reported, to reveal the most important miRNA regulators in disease. Finally, miRNA diversity and their potential to target multiple pathways, offers novel clinical applications for miRNAs as biomarkers and therapeutic agents in neurodegenerative diseases.
doi:10.3389/fncel.2013.00178
PMCID: PMC3794211  PMID: 24133413
microRNA; neurodegeneration; Alzheimer's disease; Parkinson's disease; amyotrophic lateral sclerosis; Huntington's disease
11.  Investigating cell death mechanisms in amyotrophic lateral sclerosis using transcriptomics 
Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by degeneration and loss of upper and lower motor neurons from the motor cortex, brainstem and spinal cord although evidence is suggesting that there is further involvement of other cell types in the surrounding tissue. Transcriptomic analysis by gene expression profiling using microarray technology has enabled the determination of patterns of cell death in the degenerating tissues. This work has examined gene expression at the level of the tissue and individual cell types in both sporadic and familial forms of the disease. In addition, further studies have examined the differential vulnerability of neuronal cells in different regions of the central nervous system. Model systems have also provided further information to help unravel the mechanisms that lead to death of the motor neurons in disease and also provided novel insights. In this review we shall describe the methods that have been used in these investigations and describe how they have contributed to our knowledge of the cell death mechanisms in ALS.
doi:10.3389/fncel.2013.00259
PMCID: PMC3865770  PMID: 24381542
transcriptomics; cell death; amyotrophic lateral sclerosis; microarray
12.  Unravelling the enigma of selective vulnerability in neurodegeneration: motor neurons resistant to degeneration in ALS show distinct gene expression characteristics and decreased susceptibility to excitotoxicity 
Acta Neuropathologica  2012;125(1):95-109.
A consistent clinical feature of amyotrophic lateral sclerosis (ALS) is the sparing of eye movements and the function of external sphincters, with corresponding preservation of motor neurons in the brainstem oculomotor nuclei, and of Onuf’s nucleus in the sacral spinal cord. Studying the differences in properties of neurons that are vulnerable and resistant to the disease process in ALS may provide insights into the mechanisms of neuronal degeneration, and identify targets for therapeutic manipulation. We used microarray analysis to determine the differences in gene expression between oculomotor and spinal motor neurons, isolated by laser capture microdissection from the midbrain and spinal cord of neurologically normal human controls. We compared these to transcriptional profiles of oculomotor nuclei and spinal cord from rat and mouse, obtained from the GEO omnibus database. We show that oculomotor neurons have a distinct transcriptional profile, with significant differential expression of 1,757 named genes (q < 0.001). Differentially expressed genes are enriched for the functional categories of synaptic transmission, ubiquitin-dependent proteolysis, mitochondrial function, transcriptional regulation, immune system functions, and the extracellular matrix. Marked differences are seen, across the three species, in genes with a function in synaptic transmission, including several glutamate and GABA receptor subunits. Using patch clamp recording in acute spinal and brainstem slices, we show that resistant oculomotor neurons show a reduced AMPA-mediated inward calcium current, and a higher GABA-mediated chloride current, than vulnerable spinal motor neurons. The findings suggest that reduced susceptibility to excitotoxicity, mediated in part through enhanced GABAergic transmission, is an important determinant of the relative resistance of oculomotor neurons to degeneration in ALS.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-012-1058-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-012-1058-5
PMCID: PMC3535376  PMID: 23143228
13.  Dysregulation of astrocyte–motoneuron cross-talk in mutant superoxide dismutase 1-related amyotrophic lateral sclerosis 
Brain  2011;134(9):2627-2641.
Amyotrophic lateral sclerosis is a neurodegenerative disease in which death of motoneurons leads to progressive failure of the neuromuscular system resulting in death frequently within 2–3 years of symptom onset. Focal onset and propagation of the disease symptoms to contiguous motoneuron groups is a striking feature of the human disease progression. Recent work, using mutant superoxide dismutase 1 murine models and in vitro culture systems has indicated that astrocytes are likely to contribute to the propagation of motoneuron injury and disease progression. However, the basis of this astrocyte toxicity and/or failure of motoneuron support has remained uncertain. Using a combination of in vivo and in vitro model systems of superoxide dismutase 1-related amyotrophic lateral sclerosis, linked back to human biosamples, we set out to elucidate how astrocyte properties change in the presence of mutant superoxide dismutase 1 to contribute to motoneuron injury. Gene expression profiling of spinal cord astrocytes from presymptomatic transgenic mice expressing mutant superoxide dismutase 1 revealed two striking changes. First, there was evidence of metabolic dysregulation and, in particular, impairment of the astrocyte lactate efflux transporter, with resultant decrease of spinal cord lactate levels. Second, there was evidence of increased nerve growth factor production and dysregulation of the ratio of pro-nerve growth factor to mature nerve growth factor, favouring p75 receptor expression and activation by neighbouring motoneurons. Functional in vitro studies showed that astrocytes expressing mutant superoxide dismutase 1 are toxic to normal motoneurons. We provide evidence that reduced metabolic support from lactate release and activation of pro-nerve growth factor-p75 receptor signalling are key components of this toxicity. Preservation of motoneuron viability could be achieved by increasing lactate provision to motoneurons, depletion of increased pro-nerve growth factor levels or p75 receptor blockade. These findings are likely to be relevant to human amyotrophic lateral sclerosis, where we have demonstrated increased levels of pro-nerve growth factor in cerebrospinal fluid and increased expression of the p75 receptor by spinal motoneurons. Taken together, these data confirm that altered properties of astrocytes are likely to play a crucial role in the propagation of motoneuron injury in superoxide dismutase 1-related amyotrophic lateral sclerosis and indicate that manipulation of the energy supply to motoneurons as well as inhibition of p75 receptor signalling may represent valuable neuroprotective strategies.
doi:10.1093/brain/awr193
PMCID: PMC3170534  PMID: 21908873
amyotrophic lateral sclerosis; astrocytes; microarray; lactate; nerve growth factor
14.  Development of a patient reported outcome measure for fatigue in motor neurone disease: the Neurological Fatigue Index (NFI-MND) 
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.
doi:10.1186/1477-7525-9-101
PMCID: PMC3282643  PMID: 22107756
15.  Pattern of spread and prognosis in lower limb-onset ALS 
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.
doi:10.3109/17482960903420140
PMCID: PMC3182546  PMID: 20001488
Epidemiology; prognostic; survival
16.  Rasch analysis of the hospital anxiety and depression scale (hads) for use in motor neurone 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.
doi:10.1186/1477-7525-9-82
PMCID: PMC3192662  PMID: 21955749
17.  Protocol for a double-blind randomised placebo-controlled trial of lithium carbonate in patients with amyotrophic Lateral Sclerosis (LiCALS) [Eudract number: 2008-006891-31] 
BMC Neurology  2011;11:111.
Background
Amyotrophic lateral sclerosis is a rapidly progressive neurodegenerative disorder characterised by loss of motor neurons leading to severe weakness and death from respiratory failure within 3-5 years. Riluzole prolongs survival in ALS. A published report has suggested a dramatic effect of lithium carbonate on survival. 44 patients were studied, with 16 randomly selected to take LiCO3 and riluzole and 28 allocated to take riluzole alone. In the group treated with lithium, no patients had died (i.e., 100% survival) at the end of the study (15 months from entry), compared to 71% surviving in the riluzole-only group. Although the trial can be criticised on several grounds, there is a substantial rationale from other laboratory studies that lithium is worth investigating therapeutically in amyotrophic lateral sclerosis.
Methods/Design
LiCALS is a multi-centre double-blind randomised parallel group controlled trial of the efficacy, safety, and tolerability of lithium carbonate (LiCO3) at doses to achieve stable 'therapeutic' plasma levels (0.4-0.8 mmol/L), plus standard treatment, versus matched placebo plus standard treatment, in patients with amyotrophic lateral sclerosis. The study will be based in the UK, in partnership with the MND Association and DeNDRoN (the Dementias and Neurodegnerative Diseases Clinical Research Network). 220 patients will be recruited. All patients will be on the standard treatment for ALS of riluzole 100 mg daily. The primary outcome measure will be death from any cause at 18 months defined from the date of randomisation. Secondary outcome measures will be changes in three functional rating scales, the ALS Functional Rating Scale-Revised, The EuroQOL (EQ-5D), and the Hospital Anxiety and Depression Scale.
Eligible patients will have El Escorial Possible, Laboratory-supported Probable, Probable or Definite amyotrophic lateral sclerosis with disease duration between 6 months and 36 months (inclusive), vital capacity ≥ 60% of predicted within 1 month prior to randomisation and age at least18 years.
Discussion
Patient recruitment began in June 2009 and the last patient is expected to complete the trial protocol in November 2011.
Trial registration
Current controlled trials ISRCTN83178718
doi:10.1186/1471-2377-11-111
PMCID: PMC3189869  PMID: 21936930
18.  Diagnosis and management of motor neurone disease 
BMJ : British Medical Journal  2008;336(7645):658-662.
doi:10.1136/bmj.39493.511759.BE
PMCID: PMC2270983  PMID: 18356234
19.  Phosphatase and tensin homologue/protein kinase B pathway linked to motor neuron survival in human superoxide dismutase 1-related amyotrophic lateral sclerosis 
Brain  2011;134(2):506-517.
Gene expression profiling has been used previously with spinal cord homogenates and laser capture microdissected motor neurons to determine the mechanisms involved in neurodegeneration in amyotrophic lateral sclerosis. However, while cellular and animal model work has focused on superoxide dismutase 1-related amyotrophic lateral sclerosis, the transcriptional profile of human mutant superoxide dismutase 1 motor neurons has remained undiscovered. The aim of this study was to apply gene expression profiling to laser captured motor neurons from human superoxide dismutase 1-related amyotrophic lateral sclerosis and neurologically normal control cases, in order to determine those pathways dysregulated in human superoxide dismutase 1-related neurodegeneration and to establish potential pathways suitable for therapeutic intervention. Identified targets were then validated in cultured cell models using lentiviral vectors to manipulate the expression of key genes. Microarray analysis identified 1170 differentially expressed genes in spinal cord motor neurons from superoxide dismutase 1-related amyotrophic lateral sclerosis, compared with controls. These genes encoded for proteins in multiple functional categories, including those involved in cell survival and cell death. Further analysis determined that multiple genes involved in the phosphatidylinositol-3 kinase signalling cascade were differentially expressed in motor neurons that survived the disease process. Functional experiments in cultured cells and primary motor neurons demonstrate that manipulating this pathway by reducing the expression of a single upstream target, the negative phosphatidylinositol-3 kinase regulator phosphatase and tensin homology, promotes a marked pro-survival effect. Therefore, these data indicate that proteins in the phosphatidylinositol-3 kinase pathway could represent a target for therapeutic manipulation in motor neuron degeneration.
doi:10.1093/brain/awq345
PMCID: PMC3030763  PMID: 21228060
amyotrophic lateral sclerosis; SOD1; PTEN; PI3K; AKT
20.  Mutant SOD1 G93A microglia have an inflammatory phenotype and elevated production of MCP-1 
Neuroreport  2009;20(16):1450-1455.
The inflammatory response in amyotrophic lateral sclerosis (ALS) is well documented but the underlying cellular mechanisms have not been fully elucidated. We report that microglia isolated from the mutant human SOD1 G93A transgenic mouse model of ALS, have an increased response to the inflammatory stimulus, lipopolysaccharide. Cell surface area and F4/80 surface marker, both indicators of cell activation, are increased relative to transgenic wild-type human SOD1 microglia (SOD1 WT). Monocyte chemoattractant protein-1 (MCP-1), known to be increased in ALS, is produced at 3 fold higher levels by SOD1 G93A than by SOD1 WT microglia, under activating conditions. This novel finding implicates ALS microglia as a source of the increased MCP-1 levels detected in ALS patients and the ALS mouse model.
doi:10.1097/WNR.0b013e328331e8fa
PMCID: PMC2889291  PMID: 19752764
microglia; amyotrophic lateral sclerosis; monocyte chemoattractant protein 1
21.  An in vitro screening cascade to identify neuroprotective antioxidants in ALS 
Free Radical Biology & Medicine  2009;46(8):1127-1138.
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.
doi:10.1016/j.freeradbiomed.2009.01.019
PMCID: PMC2742740  PMID: 19439221
5-LOX, 5-lipoxygenase; AAPH, 2,2′-azobis(2-methylpropionamidine) dihydrochloride; ALS, amyotrophic lateral sclerosis; ARE, antioxidant response element; BBB, blood–brain barrier; CAPE, caffeic acid phenethyl ester; CNS, central nervous system; DCF, dichlorofluorescein; DMSO, dimethyl sulfoxide; Esc, esculetin; EthD1, ethidium homodimer-1; EGFP, enhanced green fluorescent protein; LTB4, leukotriene B4; MN, motor neuron; MTT, methylthiazolyldiphenyl tetrazolium bromide; NDGA, nordihydroguaiaretic acid; Nrf2, nuclear factor erythroid 2-related factor 2; OTCA, 2-oxo-l-thiazolidine-4-carboxylic acid; PBS, phosphate-buffered saline; PI, prediction interval; PSA, polar surface area; Res, resveratrol; R-PE, R-phycoerythrin; SOD1, superoxide dismutase 1; TK, thymidine kinase promoter; TRAP, total radical-trapping antioxidant parameter.; Antioxidant; Amyotrophic lateral sclerosis; Mouse; NSC34; Superoxide dismutase; Free radicals
22.  Screening of the transcriptional regulatory regions of vascular endothelial growth factor receptor 2 (VEGFR2) in amyotrophic lateral sclerosis 
BMC Medical Genetics  2007;8:23.
Background
Vascular endothelial growth factor (VEGF) has neurotrophic activity which is mediated by its main agonist receptor, VEGFR2. Dysregulation of VEGF causes motor neurone degeneration in a mouse model of amyotrophic lateral sclerosis (ALS), and expression of VEGFR2 is reduced in motor neurones and spinal cord of patients with ALS.
Methods
We have screened the promoter region and 4 exonic regions of functional significance of the VEGFR2 gene in a UK population of patients with ALS, for mutations and polymorphisms that may affect expression or function of this VEGF receptor.
Results
No mutations were identified in the VEGFR2 gene. We found no association between polymorphisms in the regulatory regions of the VEGFR2 gene and ALS.
Conclusion
Mechanisms other than genetic variation may downregulate expression or function of the VEGFR2 receptor in patients with ALS.
doi:10.1186/1471-2350-8-23
PMCID: PMC1868706  PMID: 17456229
23.  C9ORF72 expansions, parkinsonism, and Parkinson disease 
Neurology  2013;81(9):808-811.
Objective:
To determine the histopathologic bases for the observed incidence of parkinsonism in families with C9ORF72 expansions, which typically cause amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia.
Methods:
DNA was extracted from 377 brains with the histopathologic diagnosis of idiopathic Parkinson disease or related disorders and analyzed for C9ORF72 expansions. α-Synuclein and p62 immunohistochemistry of the substantia nigra (SN) was undertaken in brains of 17 ALS cases with (C9ORF72+) and 51 without (C9ORF72−) the C9ORF72 expansion.
Results:
Only 1 of 338 cases with pathologically confirmed idiopathic Parkinson disease had a C9ORF72 expansion. Similarly, only 1 of 17 C9ORF72+ brains displayed features suggestive of α-synucleinopathy. In contrast, p62-positive, TDP-43–negative neuronal cytoplasmic inclusions within the SN were considerably more frequent in C9ORF72+ brain tissue than in the C9ORF72− brains (p = 0.005). Furthermore, there was a more marked loss of dopaminergic neurons in the SN of C9ORF72+ ALS brains than C9ORF72− ALS brains (p = 0.029).
Conclusions:
SN involvement is common in C9ORF72+ ALS but can be clearly distinguished from Parkinson disease–related mechanisms by the presence of p62-positive inclusions and the absence of α-synuclein–positive Lewy bodies or Lewy neurites.
doi:10.1212/WNL.0b013e3182a2cc38
PMCID: PMC3908460  PMID: 23884045
24.  Motor neurone disease 
BMJ : British Medical Journal  1999;318(7191):1118-1121.
PMCID: PMC1115517  PMID: 10213726
25.  Early interneuron dysfunction in ALS: Insights from a mutant sod1 zebrafish model 
Annals of neurology  2012;73(2):246-258.
Objectives
To determine, when, how and which neurons initiate the onset of pathophysiology in ALS using a transgenic mutant sod1 zebrafish model and identify neuroprotective drugs.
Methods
Proteinopathies like ALS involve mutant proteins that misfold and activate the heatshock stress response (HSR). The HSR is indicative of neuronal stress and we use a fluorescent hsp70-DsRed reporter in our transgenic zebrafish to track neuronal stress and to measure functional changes in neurons and muscle over the course of the disease.
Results
We show that mutant sod1 fish first exhibit the HSR in glycinergic interneurons at 24 hours post fertilization (hpf). By 96 hpf, we observe a significant reduction in spontaneous glycinergic currents induced in spinal motor neurons. The loss of inhibition is followed by increased stress in the motor neurons of symptomatic adults and concurrent morphological changes at the neuromuscular junction (NMJ) indicative of denervation. Riluzole, the only approved ALS drug and apomorphine, an NRF2 activator, reduce the observed early neuronal stress response.
Interpretation
The earliest event in the pathophysiology of ALS in the mutant sod1 zebrafish model involves neuronal stress in inhibitory interneurons, resulting from mutant Sod1 expression. This is followed by a reduction in inhibitory input to motor neurons. The loss of inhibitory input may contribute to the later development of neuronal stress in motor neurons and concurrent inability to maintain the NMJ. Riluzole, the approved drug for use in ALS, modulates neuronal stress in interneurons, indicating a novel mechanism of riluzole action.
doi:10.1002/ana.23780
PMCID: PMC3608830  PMID: 23281025

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