Abstract
BackgroundAll cells accumulate insoluble protein aggregates throughout their lifespan. While many studies have characterized the canonical disease-associated protein aggregates, such as those associated with amyloid plaques, additional, undefined proteins aggregate in the brain and may be directly associated with disease and lifespan.MethodsA proteomics approach was used to identify a large subset of insoluble proteins in the mild cognitively impaired (MCI) and Alzheimer’s disease (AD) human brain. Cortical samples from control, MCI, and AD patients were separated into detergent-soluble and detergent-insoluble fractions, and high-resolution LC/MS/MS technology was used to determine which proteins became more insoluble in the disease state. Bioinformatics analyses were used to determine if the alteration of protein aggregation between AD and control patients was associated with any specific biological process. Western blots were used to validate the proteomics data and to assess the levels of secondary protein modifications in MCI and AD.ResultsThere was a stage-dependent increase in detergent-insoluble proteins, with more extreme changes occurring in the AD cohort. Glycolysis was the most significantly overrepresented gene ontology biological process associated with the alteration of protein aggregation between AD and control patients. It was further shown that many low molecular weight proteins that were enriched in the AD brain were also highly aggregated, migrating on SDS-PAGE far above their predicted molecular masses. Glucose-6-phosphate isomerase, ubiquitin carboxyl-terminal hydrolase isoenzyme L1 (UCHL1/PARK5), and the DNA damage repair enzyme KU70 were among the top insoluble proteins identified by proteomics and validated by Western blot to be increased in the insoluble fractions of both MCI and AD brain samples.ConclusionsDiverse proteins became more detergent-insoluble in the brains of both MCI and AD patients compared to age-matched controls, suggesting that multiple proteins aggregate in these diseases, likely posing a direct toxic insult to neurons. Furthermore, detergent-insoluble proteins included those with important biological activities for critical cellular processes such as energetics, proteolysis, and DNA damage repair. Thus, reduced protein solubility likely promotes aggregation and limits functionality, reducing the efficiency of multiple aspects of cell physiology. Pharmaceutical interventions that increase autophagy may provide a useful therapeutic treatment to combat protein aggregation.
Highlights
All cells accumulate insoluble protein aggregates throughout their lifespan
Diverse proteins became more detergent-insoluble in the brains of both mild cognitively impaired (MCI) and Alzheimer’s disease (AD) patients compared to age-matched controls, suggesting that multiple proteins aggregate in these diseases, likely posing a direct toxic insult to neurons
Because of the demonstrable relevance of this subset of cellular proteins to lifespan and disease [1,2,3, 5, 6], we asked if an analogous set of detergent-insoluble aggregates could be identified in human brain tissue and if they changed in kind or amount with MCI and AD
Summary
While many studies have characterized the canonical disease-associated protein aggregates, such as those associated with amyloid plaques, additional, undefined proteins aggregate in the brain and may be directly associated with disease and lifespan As cells age, they accumulate insoluble protein aggregates. There is evidence that additional undefined, detergent-insoluble, aggregated proteins accumulate in the brain during the aging process and may be directly associated with disease and lifespan. Our laboratory identified a new class of detergent-insoluble proteins in flies whose levels dramatically increase in the brain with age [2] When these proteins were removed by increasing autophagy in the brain, the flies lived longer, while increasing aggregated proteins by decreasing autophagy in the brain decreased lifespan [2]. Detergentinsoluble aggregates accumulate with age in Alzheimer’s disease (AD) transgenic mice and are reduced by neuroprotective AD drug candidates that extend lifespan in worms and flies [4,5,6]
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