Submission
| Title: | Age-dependent changes in the molecular signatures of mouse brain elucidated by multi-protease proteomic and phosphoproteomic analyses |
| Presenter: | Rodrigo Mohallem |
| Institution: | Purdue University |
| Authors: | Rodrigo Mohallem, Allison Schaser, Uma K Aryal |
Abstract
| Background/Significance/Rationale: | With the growing elderly population, aging and age-related diseases are at the forefront of the medical challenges faced in the 21st century. The prevalence of age-related diseases, in particular neurodegenerative illnesses, is currently at an all-time high. It is estimated that one fourth of Americans over the age of 55 suffer from a mental disorder, and worldwide one person is diagnosed with dementia worldwide in every 3 seconds. Despite the increasing numbers of patients suffering from dementia, there are currently no treatments or therapeutic strategies available. In fact, the process of neurodegeneration remains poorly understood, and the mechanisms underlying the pathobiology of aging brains are complex yet not fully explored. In this study, we focus on unveiling the changes in the proteome, kinome and phosphoproteome of aging mice brains to elucidate the molecular signatures of age-related neurodegenerative processes. |
| Methods: | The brain from Adult (3–4-month-old), Middle-aged (10-month-old) and Old (19-2-month-old) wild-type mice were harvested, homogenized and separetely digested with Trypsin, Chymotrypsin, AspN and GluC (Sigma-Aldrich, USA). Peptides from each enzyme digestion were desalted using C18 spin columns (Thermo Fisher Scientific, USA) before the enrichment of phosphopeptides using the PolyMaC spin tips. (Phospho)peptides were then separated with a reverse phase column and analyzed with the Orbitrap Fusion Lumos mass spectrometer. Data was analyzed with the MaxQuant and Perseus software. |
| Results/Findings: | Our multi-protease digestion strategy greatly improved the sequence coverage of identified proteins, and, by extent, drastically increased the number of quantified phosphosites. Using this approach, we were able to identify 18492 phosphorylated peptides, from which 10474 were class I phosphosites (phosphosites with probability >0.75). By employing a wider range of enzymatic digestions, including Chymotrypsin, AspN, and GluC, we were able to map 40% of all identified class I phosphosites that would have been entirely missed in studies relying solely on Trypsin. This underscores the significant advantages of this approach for comprehensively surveying phosphorylation events relevant to aging research. Our global proteomics results suggested a marked increase in proteins involved in neuroinflammation, synaptic functions and protein folding were among the top enriched terms for clusters in which protein levels were significantly elevated in old mice. All of these pathways underly neurodegeneration and declined synaptic functions. Furthermore, “Post-Translational protein phosphorylation” were among the top upregulated pathways in old mice, prompting the exploration of the age-associated changes in the mouse kinome and phosphoproteome. U |
| Conclusions/Discussion: | We found an upregulation of the PI3K-AKT-mTOR-p53 signaling pathway, further corroborated with hyperphosphorylation of several proteins, including key proteins in the onset of Alzheimer’s and Parkinson’s diseases, such as Mapt and Dpysl2. Our results further suggest an interplay between Cdk5 and Gsk3b signaling pathways in the brain of old mice, a crosstalk which has been previously suggested to underline AD pathogenesis. |
| Translational/Human Health Impact: | Our data provides a new perspective of age-related changes in the brain kinome and phosphoproteome |
