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1.  The Extracellular Domain of Neurotrophin Receptor p75 as a Candidate Biomarker for Amyotrophic Lateral Sclerosis 
PLoS ONE  2014;9(1):e87398.
Objective biomarkers for amyotrophic lateral sclerosis would facilitate the discovery of new treatments. The common neurotrophin receptor p75 is up regulated and the extracellular domain cleaved from injured neurons and peripheral glia in amyotrophic lateral sclerosis. We have tested the hypothesis that urinary levels of extracellular neurotrophin receptor p75 serve as a biomarker for both human motor amyotrophic lateral sclerosis and the SOD1G93A mouse model of the disease. The extracellular domain of neurotrophin receptor p75 was identified in the urine of amyotrophic lateral sclerosis patients by an immuno-precipitation/western blot procedure and confirmed by mass spectrometry. An ELISA was established to measure urinary extracellular neurotrophin receptor p75. The mean value for urinary extracellular neurotrophin receptor p75 from 28 amyotrophic lateral sclerosis patients measured by ELISA was 7.9±0.5 ng/mg creatinine and this was significantly higher (p<0.001) than 12 controls (2.6±0.2 ng/mg creatinine) and 19 patients with other neurological disease (Parkinson's disease and Multiple Sclerosis; 4.1±0.2 ng/mg creatinine). Pilot data of disease progression rates in 14 MND patients indicates that p75NTRECD levels were significantly higher (p = 0.0041) in 7 rapidly progressing patients as compared to 7 with slowly progressing disease. Extracellular neurotrophin receptor p75 was also readily detected in SOD1G93A mice by immuno-precipitation/western blot before the onset of clinical symptoms. These findings indicate a significant relation between urinary extracellular neurotrophin receptor p75 levels and disease progression and suggests that it may be a useful marker of disease activity and progression in amyotrophic lateral sclerosis.
doi:10.1371/journal.pone.0087398
PMCID: PMC3903651  PMID: 24475283
2.  Practice Parameter update: The care of the patient with amyotrophic lateral sclerosis: Multidisciplinary care, symptom management, and cognitive/behavioral impairment (an evidence-based review) 
Neurology  2009;73(15):1227-1233.
Objective:
To systematically review evidence bearing on the management of patients with amyotrophic lateral sclerosis (ALS).
Methods:
The authors analyzed studies from 1998 to 2007 to update the 1999 practice parameter. Topics covered in this section include breaking the news, multidisciplinary clinics, symptom management, cognitive and behavioral impairment, communication, and palliative care for patients with ALS.
Results:
The authors identified 2 Class I studies, 8 Class II studies, and 30 Class III studies in ALS, but many important areas have been little studied. More high-quality, controlled studies of symptomatic therapies and palliative care are needed to guide management and assess outcomes in patients with ALS.
Recommendations:
Multidisciplinary clinic referral should be considered for managing patients with ALS to optimize health care delivery and prolong survival (Level B) and may be considered to enhance quality of life (Level C). For the treatment of refractory sialorrhea, botulinum toxin B should be considered (Level B) and low-dose radiation therapy to the salivary glands may be considered (Level C). For treatment of pseudobulbar affect, dextromethorphan and quinidine should be considered if approved by the US Food and Drug Administration (Level B). For patients who develop fatigue while taking riluzole, withholding the drug may be considered (Level C). Because many patients with ALS demonstrate cognitive impairment, which in some cases meets criteria for dementia, screening for cognitive and behavioral impairment should be considered in patients with ALS (Level B). Other management strategies all lack strong evidence.
GLOSSARY
= amyotrophic lateral sclerosis;
= amyotrophic lateral sclerosis with a dementia meeting the Neary criteria for frontotemporal dementia;
= amyotrophic lateral sclerosis with behavioral impairment;
= amyotrophic lateral sclerosis with cognitive impairment;
= botulinum toxin type A;
= botulinum toxin type B;
= dextromethorphan;
= Food and Drug Administration;
= frontotemporal dementia;
= noninvasive ventilation;
= percutaneous endoscopic gastrostomy;
= quinidine.
doi:10.1212/WNL.0b013e3181bc01a4
PMCID: PMC2764728  PMID: 19822873
3.  A patient with amyotrophic lateral sclerosis and atypical clinical and electrodiagnostic features: a case report 
Introduction
Amyotrophic lateral sclerosis is a rapidly progressive, fatal neurodegenerative disorder for which there is no effective treatment. The diagnosis is dependent on the clinical presentation and consistent electrodiagnostic studies. Typically, there is a combination of upper and lower motor neuron signs as well as electrodiagnostic studies indicative of diffuse motor axonal injury. The presentation of amyotrophic lateral sclerosis, however, may be variable. At the same time, the diagnosis is essential for patient prognosis and management. It is therefore important to appreciate the range of possible presentations of amyotrophic lateral sclerosis.
Case presentation
We present the case of a 57-year-old Caucasian man with pathological findings on postmortem examination consistent with amyotrophic lateral sclerosis but atypical clinical and electrodiagnostic features. He died after a rapid course of progressive weakness. The patient did not respond to immunosuppressive therapy.
Conclusion
Amyotrophic lateral sclerosis should be considered in patients with a rapidly progressive, unexplained neuropathic process. This should be true even if there are atypical clinical and electrodiagnostic findings. Absence of response to therapy and the development of upper motor neuron signs should reinforce the possibility that amyotrophic lateral sclerosis may be present. Since amyotrophic lateral sclerosis is a fatal illness, however, the possibility of this disease in patients with atypical clinical features should not diminish the need for a thorough diagnostic evaluation and treatment trials.
doi:10.1186/1752-1947-5-538
PMCID: PMC3240922  PMID: 22047468
4.  Clinical characteristics of patients with familial amyotrophic lateral sclerosis carrying the pathogenic GGGGCC hexanucleotide repeat expansion of C9ORF72 
Brain  2012;135(3):784-793.
A large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72, a gene located on chromosome 9p21, has been recently reported to be responsible for ∼40% of familial amyotrophic lateral sclerosis cases of European ancestry. The aim of the current article was to describe the phenotype of amyotrophic lateral sclerosis cases carrying the expansion by providing a detailed clinical description of affected cases from representative multi-generational kindreds, and by analysing the age of onset, gender ratio and survival in a large cohort of patients with familial amyotrophic lateral sclerosis. We collected DNA and analysed phenotype data for 141 index Italian familial amyotrophic lateral sclerosis cases (21 of Sardinian ancestry) and 41 German index familial amyotrophic lateral sclerosis cases. Pathogenic repeat expansions were detected in 45 (37.5%) patients from mainland Italy, 12 (57.1%) patients of Sardinian ancestry and nine (22.0%) of the 41 German index familial amyotrophic lateral sclerosis cases. The disease was maternally transmitted in 27 (49.1%) pedigrees and paternally transmitted in 28 (50.9%) pedigrees (P = non-significant). On average, children developed disease 7.0 years earlier than their parents [children: 55.8 years (standard deviation 7.9), parents: 62.8 (standard deviation 10.9); P = 0.003]. Parental phenotype influenced the type of clinical symptoms manifested by the child: of the 13 cases where the affected parent had an amyotrophic lateral sclerosis–frontotemporal dementia or frontotemporal dementia, the affected child also developed amyotrophic lateral sclerosis–frontotemporal dementia in nine cases. When compared with patients carrying mutations of other amyotrophic lateral sclerosis-related genes, those with C9ORF72 expansion had commonly a bulbar onset (42.2% compared with 25.0% among non-C9ORF72 expansion cases, P = 0.03) and cognitive impairment (46.7% compared with 9.1% among non-C9ORF72 expansion cases, P = 0.0001). Median survival from symptom onset among cases carrying C9ORF72 repeat expansion was 3.2 years lower than that of patients carrying TARDBP mutations (5.0 years; 95% confidence interval: 3.6–7.2) and longer than those with FUS mutations (1.9 years; 95% confidence interval: 1.7–2.1). We conclude that C9ORF72 hexanucleotide repeat expansions were the most frequent mutation in our large cohort of patients with familial amyotrophic lateral sclerosis of Italian, Sardinian and German ancestry. Together with mutation of SOD1, TARDBP and FUS, mutations of C9ORF72 account for ∼60% of familial amyotrophic lateral sclerosis in Italy. Patients with C9ORF72 hexanucleotide repeat expansions present some phenotypic differences compared with patients with mutations of other genes or with unknown mutations, namely a high incidence of bulbar-onset disease and comorbidity with frontotemporal dementia. Their pedigrees typically display a high frequency of cases with pure frontotemporal dementia, widening the concept of familial amyotrophic lateral sclerosis.
doi:10.1093/brain/awr366
PMCID: PMC3286333  PMID: 22366794
amyotrophic lateral sclerosis; familial ALS, C9ORF72 gene; phenotype–genotype correlation
5.  Dietary BMAA Exposure in an Amyotrophic Lateral Sclerosis Cluster from Southern France 
PLoS ONE  2013;8(12):e83406.
Background
Dietary exposure to the cyanotoxin BMAA is suspected to be the cause of amyotrophic lateral sclerosis in the Western Pacific Islands. In Europe and North America, this toxin has been identified in the marine environment of amyotrophic lateral sclerosis clusters but, to date, only few dietary exposures have been described.
Objectives
We aimed at identifying cluster(s) of amyotrophic lateral sclerosis in the Hérault district, a coastal district from Southern France, and to search, in the identified area(s), for the existence of a potential dietary source of BMAA.
Methods
A spatio-temporal cluster analysis was performed in the district, considering all incident amyotrophic lateral sclerosis cases identified from 1994 to 2009 by our expert center. We investigated the cluster area with serial collections of oysters and mussels that were subsequently analyzed blind for BMAA concentrations.
Results
We found one significant amyotrophic lateral sclerosis cluster (p = 0.0024), surrounding the Thau lagoon, the most important area of shellfish production and consumption along the French Mediterranean coast. BMAA was identified in mussels (1.8 µg/g to 6.0 µg/g) and oysters (0.6 µg/g to 1.6 µg/g). The highest concentrations of BMAA were measured during summer when the highest picocyanobacteria abundances were recorded.
Conclusions
While it is not possible to ascertain a direct link between shellfish consumption and the existence of this ALS cluster, these results add new data to the potential association of BMAA with sporadic amyotrophic lateral sclerosis, one of the most severe neurodegenerative disorder.
doi:10.1371/journal.pone.0083406
PMCID: PMC3862759  PMID: 24349504
6.  The risk to relatives of patients with sporadic amyotrophic lateral sclerosis 
Brain  2011;134(12):3451-3454.
Amyotrophic lateral sclerosis is a neurodegenerative disease of motor neurons with a median survival of 2 years. Most patients have no family history of amyotrophic lateral sclerosis, but current understanding of such diseases suggests there should be an increased risk to relatives. Furthermore, it is a common question to be asked by patients and relatives in clinic. We therefore set out to determine the risk of amyotrophic lateral sclerosis to first degree relatives of patients with sporadic amyotrophic lateral sclerosis attending a specialist clinic. Case records of patients with sporadic amyotrophic lateral sclerosis seen at a tertiary referral centre over a 16-year period were reviewed, and pedigree structures extracted. All individuals who had originally presented with sporadic amyotrophic lateral sclerosis, but who subsequently had an affected first degree relative, were identified. Calculations were age-adjusted using clinic population demographics. Probands (n = 1502), full siblings (n = 1622) and full offspring (n = 1545) were identified. Eight of the siblings and 18 offspring had developed amyotrophic lateral sclerosis. The unadjusted risk of amyotrophic lateral sclerosis over the observation period was 0.5% for siblings and 1.0% for offspring. Age information was available for 476 siblings and 824 offspring. For this subset, the crude incidence of amyotrophic lateral sclerosis was 0.11% per year (0.05–0.21%) in siblings and 0.11% per year (0.06–0.19%) in offspring, and the clinic age-adjusted incidence rate was 0.12% per year (0.04–0.21%) in siblings. By age 85, siblings were found to have an 8-fold increased risk of amyotrophic lateral sclerosis, in comparison to the background population. In practice, this means the risk of remaining unaffected by age 85 dropped from 99.7% to 97.6%. Relatives of people with sporadic amyotrophic lateral sclerosis have a small but definite increased risk of being affected.
doi:10.1093/brain/awr248
PMCID: PMC3235555  PMID: 21933809
amyotrophic lateral sclerosis; sporadic case; family history; risk to relatives
7.  Clinico-pathological features in amyotrophic lateral sclerosis with expansions in C9ORF72 
Brain  2012;135(3):751-764.
Intronic expansion of the GGGGCC hexanucleotide repeat within the C9ORF72 gene causes frontotemporal dementia and amyotrophic lateral sclerosis/motor neuron disease in both familial and sporadic cases. Initial reports indicate that this variant within the frontotemporal dementia/amyotrophic lateral sclerosis spectrum is associated with transactive response DNA binding protein (TDP-43) proteinopathy. The amyotrophic lateral sclerosis/motor neuron disease phenotype is not yet well characterized. We report the clinical and pathological phenotypes associated with pathogenic C9ORF72 mutations in a cohort of 563 cases from Northern England, including 63 with a family history of amyotrophic lateral sclerosis. One hundred and fifty-eight cases from the cohort (21 familial, 137 sporadic) were post-mortem brain and spinal cord donors. We screened DNA for the C9ORF72 mutation, reviewed clinical case histories and undertook pathological evaluation of brain and spinal cord. Control DNA samples (n = 361) from the same population were also screened. The C9ORF72 intronic expansion was present in 62 cases [11% of the cohort; 27/63 (43%) familial, 35/500 (7%) cases with sporadic amyotrophic lateral sclerosis/motor neuron disease]. Disease duration was significantly shorter in cases with C9ORF72-related amyotrophic lateral sclerosis (30.5 months) compared with non-C9ORF72 amyotrophic lateral sclerosis/motor neuron disease (36.3 months, P < 0.05). C9ORF72 cases included both limb and bulbar onset disease and all cases showed combined upper and lower motor neuron degeneration (amyotrophic lateral sclerosis). Thus, clinically, C9ORF72 cases show the features of a relatively rapidly progressive, but otherwise typical, variant of amyotrophic lateral sclerosis associated with both familial and sporadic presentations. Dementia was present in the patient or a close family member in 22/62 cases with C9ORF72 mutation (35%) based on diagnoses established from retrospective clinical case note review that may underestimate significant cognitive changes in late disease. All the C9ORF72 mutation cases showed classical amyotrophic lateral sclerosis pathology with TDP-43 inclusions in spinal motor neurons. Neuronal cytoplasmic inclusions and glial inclusions positive for p62 immunostaining in non-motor regions were strongly over-represented in the C9ORF72 cases. Extra-motor pathology in the frontal cortex (P < 0.0005) and the hippocampal CA4 subfield neurons (P < 0.0005) discriminated C9ORF72 cases strongly from the rest of the cohort. Inclusions in CA4 neurons were not present in non-C9ORF72 cases, indicating that this pathology predicts mutation status.
doi:10.1093/brain/awr365
PMCID: PMC3286332  PMID: 22366792
amyotrophic lateral sclerosis; C9ORF72; dementia; neurodegeneration
8.  Corticomotoneuronal function and hyperexcitability in acquired neuromyotonia 
Brain  2010;133(9):2727-2733.
Acquired neuromyotonia encompasses a group of inflammatory disorders characterized by symptoms reflecting peripheral nerve hyperexcitability, which may be clinically confused in the early stages with amyotrophic lateral sclerosis. Despite a clear peripheral nerve focus, it remains unclear whether the ectopic activity in acquired neuromyotonia receives a central contribution. To clarify whether cortical hyperexcitability contributes to development of clinical features of acquired neuromyotonia, the present study investigated whether threshold tracking transcranial magnetic stimulation could detect cortical hyperexcitability in acquired neuromyotonia, and whether this technique could differentiate acquired neuromyotonia from amyotrophic lateral sclerosis. Cortical excitability studies were undertaken in 18 patients with acquired neuromyotonia and 104 patients with amyotrophic lateral sclerosis, with results compared to 62 normal controls. Short-interval intracortical inhibition in patients with acquired neuromyotonia was significantly different when compared to patients with amyotrophic lateral sclerosis (averaged short interval intracortical inhibition acquired neuromyotonia 11.3 ± 1.9%; amyotrophic lateral sclerosis 2.6 ± 0.9%, P < 0.001). In addition, the motor evoked potential amplitudes (acquired neuromyotonia 21.0 ± 3.1%; amyotrophic lateral sclerosis 38.1 ± 2.2%, P < 0.0001), intracortical facilitation (acquired neuromyotonia −0.9 ± 1.3%; amyotrophic lateral sclerosis −2.3 ± 0.6%, P < 0.0001), resting motor thresholds (acquired neuromyotonia 62.2 ± 1.6%; amyotrophic lateral sclerosis 57.2 ± 0.9%, P < 0.05) and cortical silent period durations (acquired neuromyotonia 212.8 ± 6.9 ms; amyotrophic lateral sclerosis 181.1 ± 4.3 ms, P < 0.0001) were significantly different between patients with acquired neuromyotonia and amyotrophic lateral sclerosis. Threshold tracking transcranial magnetic stimulation established corticomotoneuronal integrity in acquired neuromyotonia, arguing against a contribution of central processes to the development of nerve hyperexcitability in acquired neuromyotonia.
doi:10.1093/brain/awq188
PMCID: PMC2929332  PMID: 20736187
acquired neuromyotonia; amyotrophic lateral sclerosis; cortical excitability
9.  Gray matter perfusion correlates with disease severity in ALS 
Neurology  2010;74(10):821-827.
Objective:
The goal of this study is to determine if regional brain perfusion, as measured by arterial spin labeling (ASL) MRI, is correlated with clinical measures of amyotrophic lateral sclerosis (ALS) disease severity. The presence of such a relationship would indicate a possible role for ASL perfusion as a marker of disease severity and upper motor neuron involvement in ALS.
Methods:
Disease severity was assessed in 16 subjects with ALS (age 54 ± 11) using the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS) and the pulmonary function measure, forced vital capacity (FVC). Upper motor neuron involvement was assessed by testing rapid tapping of the fingers and feet. Magnetic resonance perfusion images were coregistered with structural T1-weighted MRI, corrected for partial volume effects using the structural images and normalized to a study-specific atlas. Correlations between perfusion and ALS disease severity were analyzed, using statistical parametric mapping, and including age as a factor. Analyses were adjusted for multiple clusters.
Result:
ALS severity, as measured by the ALSFRS and FVC, was correlated with gray matter perfusion. This correlation was predominantly observed in the hemisphere contralateral to the more affected limbs. ALSFRS scores correlated with perfusion in the contralateral frontal and parietal lobe (p < 0.001) and ipsilateral frontal lobe (p < 0.02). FVC scores correlated with gray matter perfusion in contralateral frontal lobe (p < 0.001). Upper motor neuron involvement, as measured by rapid finger tapping, correlated bilaterally with perfusion in the middle cingulate gyrus (p < 0.001).
Conclusion:
Amyotrophic lateral sclerosis (ALS) severity is correlated with brain perfusion as measured by arterial spin labeling (ASL) perfusion. This correlation appears to be independent of brain atrophy. ASL perfusion may be a useful tool for monitoring disease progression and assessing treatment effects in ALS.
GLOSSARY
= amyotrophic lateral sclerosis;
= Amyotrophic Lateral Sclerosis Functional Rating Scale;
= arterial spin labeling;
= Brodmann areas;
= fluorodeoxyglucose PET;
= forced vital capacity;
= Tc-hexamethylpropyleneamine oxime SPECT;
= N-acetylaspartate;
= voxel-based morphometry.
doi:10.1212/WNL.0b013e3181d3e2dd
PMCID: PMC2839193  PMID: 20147656
10.  Leukocyte-derived microparticles and scanning electron microscopic structures in two fractions of fresh cerebrospinal fluid in amyotrophic lateral sclerosis: a case report 
Introduction
Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder characterized by degeneration of motoneuron cells in anterior spinal horns. There is a need for early and accurate diagnosis with this condition. In this case report we used two complementary methods: scanning electron microscopy and fluorescence-activated cell sorting. This is the first report to our knowledge of microparticles in the cerebrospinal fluid of a patient with amyotrophic lateral sclerosis.
Case presentation
An 80-year-old Swedish man of Caucasian ethnicity presented to our facility with symptoms of amyotrophic lateral sclerosis starting a year before his first hospital examination, such as muscle weakness and twitching in his right hand progressing to arms, body and leg muscles. Electromyography showed classical neurophysiological findings of amyotrophic lateral sclerosis. Routine blood sample results were normal. A lumbar puncture was performed as a routine investigation and his cerebrospinal fluid was normal with regard to cell count and protein levels, and there were no signs of inflammation. However, scanning electron microscopy and fluorescence-activated cell sorting showed pronounced abnormalities compared to healthy controls. Flow cytometry analysis of two fractions of cerebrospinal fluid from our patient with amyotrophic lateral sclerosis was used to measure the specific binding of antibodies to CD42a, CD144 and CD45, and of phosphatidylserine to lactadherin. Our patient displayed over 100 times more phosphatidylserine-positive microparticles and over 400 times more cell-derived microparticles of leukocyte origin in his cerebrospinal fluid compared to healthy control subjects. The first cerebrospinal fluid fraction contained about 50% more microparticles than the second fraction. The scanning electron microscopy filters used with cerebrospinal fluid from our patient were filled with compact aggregates of spherical particles of lipid appearance, sticking together in a viscous batter. The quantitative increase in scanning electron microscopy findings corresponded to the flow cytometry result of an increase in leukocyte-derived microparticles.
Conclusions
Microparticles represent subcellular arrangements that can influence the pathogenesis of amyotrophic lateral sclerosis and may serve as biomarkers for underlying cellular disturbances. The increased number of leukocyte-derived microparticles with normal cell counts in cerebrospinal fluid may contribute to the amyotrophic lateral sclerosis inflammatory process by formation of immune complexes of prion-like propagation, possibly due to misfolded proteins. The two complementary methods used in this report may be additional tools for revealing the etiology of amyotrophic lateral sclerosis, for early diagnostic purposes and for evaluation of clinical trials, long-term follow-up studies and elucidating the pathophysiology in amyotrophic lateral sclerosis.
doi:10.1186/1752-1947-6-274
PMCID: PMC3492039  PMID: 22943439
Amyotrophic lateral sclerosis; Antibodies; Cerebrospinal fluid; Complementary methods; Electromyography; Flow cytometry; Microparticles; Phosphatidylserine; Scanning electron microscopy
11.  Clinical and pathological features of familial frontotemporal dementia caused by C9ORF72 mutation on chromosome 9p 
Brain  2012;135(3):709-722.
Frontotemporal dementia and amyotrophic lateral sclerosis are closely related clinical syndromes with overlapping molecular pathogenesis. Several families have been reported with members affected by frontotemporal dementia, amyotrophic lateral sclerosis or both, which show genetic linkage to a region on chromosome 9p21. Recently, two studies identified the FTD/ALS gene defect on chromosome 9p as an expanded GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 gene (C9ORF72). In the present study, we provide detailed analysis of the clinical features and neuropathology for 16 unrelated families with frontotemporal dementia caused by the C9ORF72 mutation. All had an autosomal dominant pattern of inheritance. Eight families had a combination of frontotemporal dementia and amyotrophic lateral sclerosis while the other eight had a pure frontotemporal dementia phenotype. Clinical information was available for 30 affected members of the 16 families. There was wide variation in age of onset (mean = 54.3, range = 34–74 years) and disease duration (mean = 5.3, range = 1–16 years). Early diagnoses included behavioural variant frontotemporal dementia (n = 15), progressive non-fluent aphasia (n = 5), amyotrophic lateral sclerosis (n = 9) and progressive non-fluent aphasia–amyotrophic lateral sclerosis (n = 1). Heterogeneity in clinical presentation was also common within families. However, there was a tendency for the phenotypes to converge with disease progression; seven subjects had final clinical diagnoses of both frontotemporal dementia and amyotrophic lateral sclerosis and all of those with an initial progressive non-fluent aphasia diagnosis subsequently developed significant behavioural abnormalities. Twenty-one affected family members came to autopsy and all were found to have transactive response DNA binding protein with Mr 43 kD (TDP-43) pathology in a wide neuroanatomical distribution. All had involvement of the extramotor neocortex and hippocampus (frontotemporal lobar degeneration-TDP) and all but one case (clinically pure frontotemporal dementia) had involvement of lower motor neurons, characteristic of amyotrophic lateral sclerosis. In addition, a consistent and relatively specific pathological finding was the presence of neuronal inclusions in the cerebellar cortex that were ubiquitin/p62-positive but TDP-43-negative. Our findings indicate that the C9ORF72 mutation is a major cause of familial frontotemporal dementia with TDP-43 pathology, that likely accounts for the majority of families with combined frontotemporal dementia/amyotrophic lateral sclerosis presentation, and further support the concept that frontotemporal dementia and amyotrophic lateral sclerosis represent a clinicopathological spectrum of disease with overlapping molecular pathogenesis.
doi:10.1093/brain/awr354
PMCID: PMC3286328  PMID: 22344582
frontotemporal dementia; frontotemporal lobar degeneration; amyotrophic lateral sclerosis; C9ORF72, TDP-43
12.  Is SOD1 loss of function involved in amyotrophic lateral sclerosis? 
Brain  2013;136(8):2342-2358.
Mutations in the gene superoxide dismutase 1 (SOD1) are causative for familial forms of the neurodegenerative disease amyotrophic lateral sclerosis. When the first SOD1 mutations were identified they were postulated to give rise to amyotrophic lateral sclerosis through a loss of function mechanism, but experimental data soon showed that the disease arises from a—still unknown—toxic gain of function, and the possibility that loss of function plays a role in amyotrophic lateral sclerosis pathogenesis was abandoned. Although loss of function is not causative for amyotrophic lateral sclerosis, here we re-examine two decades of evidence regarding whether loss of function may play a modifying role in SOD1–amyotrophic lateral sclerosis. From analysing published data from patients with SOD1–amyotrophic lateral sclerosis, we find a marked loss of SOD1 enzyme activity arising from almost all mutations. We continue to examine functional data from all Sod1 knockout mice and we find obvious detrimental effects within the nervous system with, interestingly, some specificity for the motor system. Here, we bring together historical and recent experimental findings to conclude that there is a possibility that SOD1 loss of function may play a modifying role in amyotrophic lateral sclerosis. This likelihood has implications for some current therapies aimed at knocking down the level of mutant protein in patients with SOD1–amyotrophic lateral sclerosis. Finally, the wide-ranging phenotypes that result from loss of function indicate that SOD1 gene sequences should be screened in diseases other than amyotrophic lateral sclerosis.
doi:10.1093/brain/awt097
PMCID: PMC3722346  PMID: 23687121
amyotrophic lateral sclerosis; motor neuron disease; superoxide dismutase 1; loss of function
13.  Needle EMG of the tongue: motor unit action potential versus peak ratio analysis in limb and bulbar onset amyotrophic lateral sclerosis 
OBJECTIVES—to find out if conventional and automatic needle EMG of the tongue can be helpful in the diagnosis and differentiation of limb and bulbar onset amyotrophic lateral sclerosis.
METHODS—Motor unit action potential (MUAP) analysis and peak ratio interference pattern analysis were performed in the right genioglossus muscle of 30 healthy subjects aged 30-81 years, 10 patients aged 49-73 years with limb onset amyotrophic lateral sclerosis, and eight patients aged 52-75 years with bulbar onset amyotrophic lateral sclerosis. Electrical activity was sampled via standard concentric needle electrodes with a commercially available EMG recorder.
RESULTS—Normal mean (2SD) MUAP duration was 6.6 (1.5) ms. Normal mean (2SD) MUAP amplitude was 224 (97.4) µV. Normal mean (2SD) peak ratio (PR), turns/second (T/s), amplitude/turn (A/T), and time intervals (TI1, TI2, TI3) were 1.68 (0.56), 732 (303.9), 446 (180.3) µV, 2.62 (0.34), 2.31 (0.14), and 1.01 (0.50) respectively. Mean MUAP duration and amplitude were significantly increased in limb onset (P=0.0001 and P=0.013) and bulbar onset amyotrophic lateral sclerosis (P=0.0001 and P=0.017). Peak ratio indices stayed unchanged in limb onset amyotrophic lateral sclerosis but were significantly decreased (PR, T/s, A/T, TI1, and TI2) or increased (TI3) in bulbar onset disease. The sensitivity of the MUAP analysis was 70% in limb and 75% in bulbar onset amyotrophic lateral sclerosis. The sensitivity of the peak ratio interference pattern analysis was 20% in limb and 100% in bulbar onset amyotrophic lateral sclerosis. Subclinical involvement of the tongue was found in 20% of the patients with limb onset amyotrophic lateral sclerosis and could be more accurately assessed with MUAP analysis than with automatic EMG.
CONCLUSIONS—both conventional and automatic needle EMG of the tongue are valuable electrophysiological devices to assess the clinical and subclinical involvement of the tongue in patients with limb and bulbar onset amyotrophic lateral sclerosis.


PMCID: PMC2169683  PMID: 9285455
14.  Patient-reported Outcomes as a Source of Evidence in Off-Label Prescribing: Analysis of Data From PatientsLikeMe 
Background
Evaluating a new use for an existing drug can be expensive and time consuming. Providers and patients must all too often rely upon their own individual-level experience to inform clinical practice, which generates only anecdotal and unstructured data. While academic-led clinical trials are occasionally conducted to test off-label uses of drugs with expired patents, this is relatively rare. In this work, we explored how a patient-centered online research platform could supplement traditional trials to create a richer understanding of medical products postmarket by efficiently aggregating structured patient-reported data. PatientsLikeMe is a tool for patients, researchers, and caregivers (currently 82,000 members across 11 condition-based communities) that helps users make treatment decisions, manage symptoms, and improve outcomes. Members enter demographic information, longitudinal treatment, symptoms, outcome data, and treatment evaluations. These are reflected back as longitudinal health profiles and aggregated reports. Over the last 3 years, patients have entered treatment histories and evaluations on thousands of medical products. These data may aid in evaluating the effectiveness and safety of some treatments more efficiently and over a longer period of time course than is feasible through traditional trials.
Objective
The objective of our study was to examine the illustrative cases of amitriptyline and modafinil – drugs commonly used off-label.
Methods
We analyzed patient-reported treatment histories and drug evaluations for each drug, examining prevalence, treatment purpose, and evaluations of effectiveness, side effects, and burden.
Results
There were 1948 treatment histories for modafinil and 1394 treatment reports for amitriptyline reported across five PatientsLikeMe communities (multiple sclerosis, Parkinson's disease, mood conditions, fibromyalgia/chronic fatigue syndrome, and amyotrophic lateral sclerosis). In these reports, the majority of members reported taking the drug for off-label uses. Only 34 of the 1755 (1%) reporting purpose used modafinil for an approved purpose (narcolepsy or sleep apnea). Only 104 out of 1197 members (9%) reported taking amitriptyline for its approved indication, depression. Members taking amitriptyline for off-label purposes rated the drug as more effective than those who were taking it for its approved indication. While dry mouth is a commonly reported side effect of amitriptyline for most patients, 88 of 220 (40%) of people with amyotrophic lateral sclerosis on the drug reported taking advantage of this side effect to treat their symptom of excess saliva.
Conclusions
Patient-reported outcomes, like those entered within PatientsLikeMe, offer a unique real-time approach to understand utilization and performance of treatments across many conditions. These patient-reported data can provide a new source of evidence about secondary uses and potentially identify targets for treatments to be studied systematically in traditional efficacy trials.
doi:10.2196/jmir.1643
PMCID: PMC3221356  PMID: 21252034
Off-label; Internet; research; patient platform; methods; online community
15.  High signal intensity on T1 weighted MRI of the anterolateral column of the spinal cord in amyotrophic lateral sclerosis. 
OBJECTIVE: To investigate MRI abnormalities in patients with amyotrophic lateral sclerosis. METHODS: Fourteen patients with amyotrophic lateral sclerosis underwent MRI of the head and spinal cord using T1 and T2 weighted images. Forty age matched controls (29 with other neurological diseases, 11 with non-neurological diseases) underwent MRI of the cervical spinal cord using T1 and T2 weighted images. RESULTS: In all the control patients, the signal intensity of the posterior column was equal or slightly hypointense compared with the anterolateral column of the cervical spinal cord on T1 weighted images. However, eight of 14 patients with amyotrophic lateral sclerosis showed pronounced high signal intensity in the anterolateral column of the spinal cord on T1 weighted MRI, which also disclosed high signal intensity of the intracranial corticospinal tract in two of the 14 patients. T2 weighted MRI demonstrated high signal intensity of the lateral corticospinal tract of the spinal cord in two, high signal intensity of the intracranial corticospinal tract in five, and low signal intensity of the motor cortex in six of the 14 patients. Two of the 14 patients showed no abnormal findings on MRI. CONCLUSIONS: High signal intensity of the anterolateral column of the spinal cord of patients with amyotrophic lateral sclerosis is a new imaging abnormality and may be useful for the diagnosis of this disease.
Images
PMCID: PMC486702  PMID: 9010407
16.  White matter alterations differ in primary lateral sclerosis and amyotrophic lateral sclerosis 
Brain  2011;134(9):2642-2655.
Primary lateral sclerosis is a sporadic disorder characterized by slowly progressive corticospinal dysfunction. Primary lateral sclerosis differs from amyotrophic lateral sclerosis by its lack of lower motor neuron signs and long survival. Few pathological studies have been carried out on patients with primary lateral sclerosis, and the relationship between primary lateral sclerosis and amyotrophic lateral sclerosis remains uncertain. To detect in vivo structural differences between the two disorders, diffusion tensor imaging of white matter tracts was carried out in 19 patients with primary lateral sclerosis, 18 patients with amyotrophic lateral sclerosis and 19 age-matched controls. Fibre tracking was used to reconstruct the intracranial portion of the corticospinal tract and three regions of the corpus callosum: the genu, splenium and callosal fibres connecting the motor cortices. Both patient groups had reduced fractional anisotropy, a measure associated with axonal organization, and increased mean diffusivity of the reconstructed corticospinal and callosal motor fibres compared with controls, without changes in the genu or splenium. Voxelwise comparison of the whole brain white matter using tract-based spatial statistics confirmed the differences between patients and controls in the diffusion properties of the corticospinal tracts and motor fibres of the callosum. This analysis further revealed differences in the regional distribution of white matter alterations between the patient groups. In patients with amyotrophic lateral sclerosis, the greatest reduction in fractional anisotropy occurred in the distal portions of the intracranial corticospinal tract, consistent with a distal axonal degeneration. In patients with primary lateral sclerosis, the greatest loss of fractional anisotropy and mean diffusivity occurred in the subcortical white matter underlying the motor cortex, with reduced volume, suggesting tissue loss. Clinical measures of upper motor neuron dysfunction correlated with reductions in fractional anisotropy in the corticospinal tract in patients with amyotrophic lateral sclerosis and increased mean diffusivity and volume loss of the corticospinal tract in patients with primary lateral sclerosis. Changes in the diffusion properties of the motor fibres of the corpus callosum were strongly correlated with changes in corticospinal fibres in patients, but not in controls. These findings indicate that degeneration is not selective for corticospinal neurons, but affects callosal neurons within the motor cortex in motor neuron disorders.
doi:10.1093/brain/awr178
PMCID: PMC3170531  PMID: 21798965
diffusion tensor imaging; diffusion tensor tractography; motor neuron disorders; primary lateral sclerosis; corpus callosum
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.  A proposed staging system for amyotrophic lateral sclerosis 
Brain  2012;135(3):847-852.
Amyotrophic lateral sclerosis is a neurodegenerative disorder characterized by progressive loss of upper and lower motor neurons, with a median survival of 2–3 years. Although various phenotypic and research diagnostic classification systems exist and several prognostic models have been generated, there is no staging system. Staging criteria for amyotrophic lateral sclerosis would help to provide a universal and objective measure of disease progression with benefits for patient care, resource allocation, research classifications and clinical trial design. We therefore sought to define easily identified clinical milestones that could be shown to occur at specific points in the disease course, reflect disease progression and impact prognosis and treatment. A tertiary referral centre clinical database was analysed, consisting of 1471 patients with amyotrophic lateral sclerosis seen between 1993 and 2007. Milestones were defined as symptom onset (functional involvement by weakness, wasting, spasticity, dysarthria or dysphagia of one central nervous system region defined as bulbar, upper limb, lower limb or diaphragmatic), diagnosis, functional involvement of a second region, functional involvement of a third region, needing gastrostomy and non-invasive ventilation. Milestone timings were standardized as proportions of time elapsed through the disease course using information from patients who had died by dividing time to a milestone by disease duration. Milestones occurred at predictable proportions of the disease course. Diagnosis occurred at 35% through the disease course, involvement of a second region at 38%, a third region at 61%, need for gastrostomy at 77% and need for non-invasive ventilation at 80%. We therefore propose a simple staging system for amyotrophic lateral sclerosis. Stage 1: symptom onset (involvement of first region); Stage 2A: diagnosis; Stage 2B: involvement of second region; Stage 3: involvement of third region; Stage 4A: need for gastrostomy; and Stage 4B: need for non-invasive ventilation. Validation of this staging system will require further studies in other populations, in population registers and in other clinic databases. The standardized times to milestones may well vary between different studies and populations, although the stages themselves and their meanings are likely to remain unchanged.
doi:10.1093/brain/awr351
PMCID: PMC3286327  PMID: 22271664
amyotrophic lateral sclerosis; staging; motor neuron disease; natural history; El Escorial criteria
19.  Endogenous regulatory T lymphocytes ameliorate amyotrophic lateral sclerosis in mice and correlate with disease progression in patients with amyotrophic lateral sclerosis 
Brain  2011;134(5):1293-1314.
Amyotrophic lateral sclerosis is a relentless and devastating adult-onset neurodegenerative disease with no known cure. In mice with amyotrophic lateral sclerosis, CD4+ T lymphocytes and wild-type microglia potentiate protective inflammatory responses and play a principal role in disease pathoprogression. Using this model, we demonstrate that endogenous T lymphocytes, and more specifically regulatory T lymphocytes, are increased at early slowly progressing stages, augmenting interleukin-4 expression and protective M2 microglia, and are decreased when the disease rapidly accelerates, possibly through the loss of FoxP3 expression in the regulatory T lymphocytes. Without ex vivo activation, the passive transfer of wild-type CD4+ T lymphocytes into amyotrophic lateral sclerosis mice lacking functional T lymphocytes lengthened disease duration and prolonged survival. The passive transfer of endogenous regulatory T lymphocytes from early disease stage mutant Cu2+/Zn2+ superoxide dismutase mice into these amyotrophic lateral sclerosis mice, again without ex vivo activation, were substantially more immunotherapeutic sustaining interleukin-4 levels and M2 microglia, and resulting in lengthened disease duration and prolonged survival; the stable disease phase was extended by 88% using mutant Cu2+/Zn2+ superoxide dismutase regulatory T lymphocytes. A potential mechanism for this enhanced life expectancy may be mediated by the augmented secretion of interleukin-4 from mutant Cu2+/Zn2+ superoxide dismutase regulatory T lymphocytes that directly suppressed the toxic properties of microglia; flow cytometric analyses determined that CD4+/CD25+/FoxP3+ T lymphocytes co-expressed interleukin-4 in the same cell. These observations were extended into the amyotrophic lateral sclerosis patient population where patients with more rapidly progressing disease had decreased numbers of regulatory T lymphocytes; the numbers of regulatory T lymphocytes were inversely correlated with disease progression rates. These data suggest a cellular mechanism whereby endogenous regulatory T lymphocytes are immunocompetent and actively contribute to neuroprotection through their interactions with microglia. Furthermore, these data suggest that immunotherapeutic interventions must begin early in the pathogenic process since immune dysfunction occurs at later stages. Thus, the cumulative mouse and human amyotrophic lateral sclerosis data suggest that increasing the levels of regulatory T lymphocytes in patients with amyotrophic lateral sclerosis at early stages in the disease process may be of therapeutic value, and slow the rate of disease progression and stabilize patients for longer periods of time.
doi:10.1093/brain/awr074
PMCID: PMC3097891  PMID: 21596768
regulatory T lymphocytes; microglia; inflammation; IL-4; FoxP3
20.  Lithium in patients with amyotrophic lateral sclerosis (LiCALS): a phase 3 multicentre, randomised, double-blind, placebo-controlled trial 
Lancet Neurology  2013;12(4):339-345.
Summary
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 χ2 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.
doi:10.1016/S1474-4422(13)70037-1
PMCID: PMC3610091  PMID: 23453347
21.  ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation 
PLoS Genetics  2013;9(10):e1003895.
The gene encoding a DNA/RNA binding protein FUS/TLS is frequently mutated in amyotrophic lateral sclerosis (ALS). Mutations commonly affect its carboxy-terminal nuclear localization signal, resulting in varying deficiencies of FUS nuclear localization and abnormal cytoplasmic accumulation. Increasing evidence suggests deficiencies in FUS nuclear function may contribute to neuron degeneration. Here we report a novel FUS autoregulatory mechanism and its deficiency in ALS-associated mutants. Using FUS CLIP-seq, we identified significant FUS binding to a highly conserved region of exon 7 and the flanking introns of its own pre-mRNAs. We demonstrated that FUS is a repressor of exon 7 splicing and that the exon 7-skipped splice variant is subject to nonsense-mediated decay (NMD). Overexpression of FUS led to the repression of exon 7 splicing and a reduction of endogenous FUS protein. Conversely, the repression of exon 7 was reduced by knockdown of FUS protein, and moreover, it was rescued by expression of EGFP-FUS. This dynamic regulation of alternative splicing describes a novel mechanism of FUS autoregulation. Given that ALS-associated FUS mutants are deficient in nuclear localization, we examined whether cells expressing these mutants would be deficient in repressing exon 7 splicing. We showed that FUS harbouring R521G, R522G or ΔExon15 mutation (minor, moderate or severe cytoplasmic localization, respectively) directly correlated with respectively increasing deficiencies in both exon 7 repression and autoregulation of its own protein levels. These data suggest that compromised FUS autoregulation can directly exacerbate the pathogenic accumulation of cytoplasmic FUS protein in ALS. We showed that exon 7 skipping can be induced by antisense oligonucleotides targeting its flanking splice sites, indicating the potential to alleviate abnormal cytoplasmic FUS accumulation in ALS. Taken together, FUS autoregulation by alternative splicing provides insight into a molecular mechanism by which FUS-regulated pre-mRNA processing can impact a significant number of targets important to neurodegeneration.
Author Summary
FUS/TLS is a frequently mutated gene in amyotrophic lateral sclerosis (ALS). ALS, also known as Lou Gehrig's disease, is characterized by a progressive degeneration of motor neurons. The abnormal cytoplasmic accumulation of mutant FUS protein is a characteristic pathology of ALS; however, recent evidence increasingly suggests deficiencies in FUS nuclear function may also contribute to neurodegeneration in ALS. Here we report a novel autoregulatory mechanism of FUS by alternative splicing and nonsense mediated decay (NMD). We show FUS binds to exon 7 and flanking introns of its own pre-mRNAs. This results in exon skipping, inducing a reading frame shift and subsequent degradation of the splice variants. As such, this mechanism provides a feedback loop that controls the homeostasis of FUS protein levels. This balance is disrupted in ALS-associated FUS mutants, which are deficient in nuclear localization and FUS-dependent alternative splicing. As a result, the abnormal accumulation of mutant FUS protein in ALS neurons goes unchecked and uncontrolled. Our study provides novel insight into the molecular mechanism by which FUS regulates gene expression and new understanding of the role of FUS in disease at the molecular level. This may lead to new potential therapeutic targets for the treatment of ALS.
doi:10.1371/journal.pgen.1003895
PMCID: PMC3814325  PMID: 24204307
22.  A Yeast Model of FUS/TLS-Dependent Cytotoxicity 
PLoS Biology  2011;9(4):e1001052.
FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression.
Author Summary
Of all the thousand natural shocks that flesh is heir to, one of the most devastating is amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's Disease. This disorder, which comes in both inherited and random forms, is characterized by degeneration of spinal motor neurons, leading to paralysis and death. The cause of the sporadic form is unknown, but new insight has come from studying the genetic variations that lead to the rarer familial forms. One such gene, accounting for 5%–10% of inherited ALS, is FUS/TLS, which encodes a protein that normally lives in the nucleus of the cell and is involved in the life-cycle of messenger RNA (mRNA). ALS-associated mutations in FUS/TLS cause the protein to mislocalize outside the nucleus into stress granules. Understanding the basis for the toxicity of mislocalized FUS/TLS could lead to new approaches to the treatment of ALS. We have made a yeast model for FUS/TLS cellular toxicity that recapitulates the mislocalization, granular accumulation, and cell death. We have exploited the yeast model to obtain information about what part of the protein is required for proper localization and what part is essential for toxicity. We have also identified several human genes that, when over-expressed in yeast, are able to rescue the cell from the toxicity of mislocalized FUS/TLS. These genes all have functions in mRNA quality control, implicating changes in this pathway in the pathology of ALS.
doi:10.1371/journal.pbio.1001052
PMCID: PMC3082520  PMID: 21541368
23.  New considerations in the design of clinical trials for amyotrophic lateral sclerosis 
Clinical investigation  2011;1(10):1375-1389.
Amyotrophic lateral sclerosis is a devastating neurodegenerative disease caused by loss of motor neurons. Its pathophysiology remains unknown, but progress has been made in understanding its genetic and biochemical basis. Clinical trialists are working to translate basic science successes into human trials with more efficiency, in the hope of finding successful treatments. In the future, new preclinical models, including patient-derived stem cells may augment transgenic animal models as preclinical tools. Biomarker discovery projects aim to identify markers of disease onset and progression for use in clinical trials. New trial designs are reducing study time, improving efficiency and helping to keep pace with the increasing rate of basic and translational discoveries. Ongoing trials with novel designs are paving the way for amyotrophic lateral sclerosis clinical research.
doi:10.4155/cli.11.127
PMCID: PMC3335743  PMID: 22545191
antisense oligonucleotide; continual reassessment model; futility design study; induced pluripotent stem cells; selection design study
24.  Blockade of Gap Junction Hemichannel Suppresses Disease Progression in Mouse Models of Amyotrophic Lateral Sclerosis and Alzheimer's Disease 
PLoS ONE  2011;6(6):e21108.
Background
Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.
Methods and Findings
In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer's disease mouse model.
Conclusions
Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.
doi:10.1371/journal.pone.0021108
PMCID: PMC3119678  PMID: 21712989
25.  Non-human primate model of amyotrophic lateral sclerosis with cytoplasmic mislocalization of TDP-43 
Brain  2012;135(3):833-846.
Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by progressive motoneuron loss. Redistribution of transactive response deoxyribonucleic acid-binding protein 43 from the nucleus to the cytoplasm and the presence of cystatin C-positive Bunina bodies are considered pathological hallmarks of amyotrophic lateral sclerosis, but their significance has not been fully elucidated. Since all reported rodent transgenic models using wild-type transactive response deoxyribonucleic acid-binding protein 43 failed to recapitulate these features, we expected a species difference and aimed to make a non-human primate model of amyotrophic lateral sclerosis. We overexpressed wild-type human transactive response deoxyribonucleic acid-binding protein 43 in spinal cords of cynomolgus monkeys and rats by injecting adeno-associated virus vector into the cervical cord, and examined the phenotype using behavioural, electrophysiological, neuropathological and biochemical analyses. These monkeys developed progressive motor weakness and muscle atrophy with fasciculation in distal hand muscles first. They also showed regional cytoplasmic transactive response deoxyribonucleic acid-binding protein 43 mislocalization with loss of nuclear transactive response deoxyribonucleic acid-binding protein 43 staining in the lateral nuclear group of spinal cord innervating distal hand muscles and cystatin C-positive cytoplasmic aggregates, reminiscent of the spinal cord pathology of patients with amyotrophic lateral sclerosis. Transactive response deoxyribonucleic acid-binding protein 43 mislocalization was an early or presymptomatic event and was later associated with neuron loss. These findings suggest that the transactive response deoxyribonucleic acid-binding protein 43 mislocalization leads to α-motoneuron degeneration. Furthermore, truncation of transactive response deoxyribonucleic acid-binding protein 43 was not a prerequisite for motoneuronal degeneration, and phosphorylation of transactive response deoxyribonucleic acid-binding protein 43 occurred after degeneration had begun. In contrast, similarly prepared rat models expressed transactive response deoxyribonucleic acid-binding protein 43 only in the nucleus of motoneurons. There is thus a species difference in transactive response deoxyribonucleic acid-binding protein 43 pathology, and our monkey model recapitulates amyotrophic lateral sclerosis pathology to a greater extent than rodent models, providing a valuable tool for studying the pathogenesis of sporadic amyotrophic lateral sclerosis.
doi:10.1093/brain/awr348
PMCID: PMC3286326  PMID: 22252998
TDP-43; Bunina bodies; cystatin C; cynomolgus monkeys; amyotrophic lateral sclerosis

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