BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments.Methodology/Principal FindingsWe used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC), a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%), between two levels of disease severity (90%), and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing.Conclusions/SignificanceOur results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms.
The prion protein (PrP) is a glycosylphosphatidylinositolanchored membrane glycoprotein that plays a vital role in prion diseases, a class of fatal neurodegenerative disorders of humans and animals. Approximately 20% of human prion diseases display autosomal dominant inheritance and are linked to mutations in the PrP gene on chromosome 20. PrP mutations are thought to favor the conformational conversion of PrP into a misfolded isoform that causes disease by an unknown mechanism. The PrP mutation D178N/Met-129 is linked to fatal familial insomnia, which causes severe sleep abnormalities and autonomic dysfunction. We showed by immunoelectron microscopy that this mutant PrP accumulates abnormally in the endoplasmic reticulum and Golgi of transfected neuroblastoma N2a cells. To investigate the impact of intracellular PrP accumulation on cellular homeostasis, we did a two-dimensional gel-based differential proteomics analysis. We used wide range immobilized pH gradient strips, pH 4 -7 and 6 -11, to analyze a large number of proteins. We found changes in proteins involved in energy metabolism, redox regulation, and vesicular transport. Rab GDP dissociation inhibitor ␣ (GDI) was one of the proteins that changed most. GDI regulates vesicular protein trafficking by acting on the activity of several Rab proteins. We found a specific reduction in the level of functional Rab11 in mutant PrP-expressing cells associated with impaired post-Golgi trafficking. Our data are consistent with a model by which mutant PrP induces overexpression of GDI, activating a cytotoxic feedback loop that leads to protein accumulation in the secretory pathway. Molecular & Cellular Proteomics 9:611-622, 2010.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive upper and lower motor neuron degeneration. One of the peculiar clinical characteristics of ALS is the wide distribution in age of onset, which is probably caused by different combinations of intrinsic and exogenous factors. We investigated whether these modifying factors are converging into common pathogenic pathways leading either to an early or a late disease onset. This would imply the identification of phenotypic biomarkers, that can distinguish the two populations of ALS patients, and of relevant pathways to consider in a therapeutic intervention. Toward this aim a differential proteomic analysis was performed in peripheral blood mononuclear cells (PBMC) from a group of 16 ALS patients with an age of onset ≤55 years and a group of 16 ALS patients with an age of onset ≥75 years, and matched healthy controls. We identified 43 differentially expressed proteins in the two groups of patients. Gene ontology analysis revealed that there was a significant enrichment in annotations associated with protein folding and response to stress. We next validated a selected number of proteins belonging to this functional group in 85 patients and 83 age- and sex-matched healthy controls using immunoassays. The results of the validation study confirmed that there was a decreased level of peptidyl-prolyl cis-trans isomerase A (also known as cyclophilin A), heat shock protein HSP 90-alpha, 78 kDa glucose-regulated protein (also known as BiP) and protein deglycase DJ-1 in PBMC of ALS patients with an early onset. Similar results were obtained in PBMC and spinal cord from two SOD1G93A mouse models with an early and late disease onset. This study suggests that a different ability to upregulate proteins involved in proteostasis, such as foldase and chaperone proteins, may be at the basis of a different susceptibility to ALS, putting forward the development of therapeutic approaches aiming at boosting the protein quality control system.
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