Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans
| Autor: | Raimund Jung, Nicole Groh, Della C. David, Ivan Gallotta, Chaolie Huang, Marie C. Lechler |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Gene knockdown General Immunology and Microbiology General Chemical Engineering General Neuroscience Transgene Biology Protein aggregation biology.organism_classification General Biochemistry Genetics and Molecular Biology Genetically modified organism Cell biology 03 medical and health sciences 030104 developmental biology Proteostasis Protein folding Gene Caenorhabditis elegans |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional. |
| Databáze: | OpenAIRE |
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