Characterization of Detergent-Insoluble Proteins in ALS Indicates a Causal Link between Nitrative Stress and Aggregation in Pathogenesis

Abstract

BackgroundAmyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited.Methodology/Principal FindingsWe carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages. We identified several proteins enriched in the detergent-insoluble fraction already at a preclinical stage, including intermediate filaments, chaperones and mitochondrial proteins. Aconitase, HSC70 and cyclophilin A were also significantly enriched in the insoluble fraction of spinal cords of ALS patients. Moreover, we found that the majority of proteins in mice and HSP90 in patients were tyrosine-nitrated. We therefore investigated the role of nitrative stress in aggregate formation in fALS-like murine motor neuron-neuroblastoma (NSC-34) cell lines. By inhibiting nitric oxide synthesis the amount of insoluble proteins, particularly aconitase, HSC70, cyclophilin A and SOD1 can be substantially reduced.Conclusion/SignificanceAnalysis of the insoluble fractions from cellular/mouse models and human tissues revealed novel aggregation-prone proteins and suggests that nitrative stress contribute to protein aggregate formation in ALS.

Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase

“…Insoluble aggregates of GAPDH are found in many diseases (11)(12)(13)(14)(15)(16)(17)(18)(19). No prior study has ascribed a structural/functional role for Met-46 in GAPDH biology.…”

“…In vivo studies show that GAPDH converts from its native soluble state into a non-native high molecular weight insoluble state during disease (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). For instance, insoluble aggregates of GAPDH have been observed in the affected tissues of patients with Alzheimer disease (13) and alcoholic liver cirrhosis (12).…”

“…Interestingly, GAPDH is also a susceptibility locus for late onset Alzheimer disease (21). Furthermore, robust aggregation of GAPDH has been detected in rodent models of motor neuron disease (17) and methamphetamine abuse (11). However, whether GAPDH aggregation is a causative factor of disease remains to be determined.…”

Redox Proteomics in Selected Neurodegenerative Disorders: From Its Infancy to Future Applications

“…One of the sites of Eno1 nitration at Tyr (43) is also a target of phosphorylation, suggesting the nitration of the protein may affect the function of the proteins as discussed earlier. In addition, a study by Basso et al (32) showed that nitration of the proteins play an important role in the aggregation of proteins, suggesting that oxidation/nitration has a key role in aggregation. All these data together may indicate that multiple oxidative modifications extensively and simultaneously affect certain proteins, inducing misfolding and finally aggregation.…”

Nitric oxide in neurodegeneration: potential benefits of non-steroidal anti-inflammatories

“…As such, reducing NO helps prevent neuronal death in these circumstances, again linking elevated NO to neurodegeneration. In models of amyotrophic lateral sclerosis, nitrosative stress increases the likelihood of protein aggregation for key proteins [115] , neuronal loss in the 6-OHDA-lesioned rat model of PD [84] .…”