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1.  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
2.  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
3.  Mutations in CHMP2B in Lower Motor Neuron Predominant Amyotrophic Lateral Sclerosis (ALS) 
PLoS ONE  2010;5(3):e9872.
Background
Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3–10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis.
Methodology/Principal Findings
Sequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p.Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B.
Conclusions/Significance
We conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS.
We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype.
doi:10.1371/journal.pone.0009872
PMCID: PMC2844426  PMID: 20352044

Results 1-3 (3)