Long-lived proteins as pillars of mitochondrial architecture in rodent brains

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ABSTRACT Mitochondria are multifaceted organelles that play vital roles in a myriad of cellular functions, including energy production, metabolism, calcium homeostasis, and cell death. It is generally accepted that a decline in mitochondria quality is a key contributor to mitochondrial dysfunction, aging, and represents a key point of convergence for several neurological disorders. Yet, precisely how dysfunctional mitochondria contribute to these conditions remains elusive. Proper mitochondrial function and fitness depends on a healthy proteome. Therefore, the mitochondrial proteome is monitored by an elaborate and integrated protein quality control network. Recent studies in mice have found that, on average, half-lives of mitochondrial proteins in the brain vary from minutes to days. Notably, our own recent discovery-based proteomic analysis revealed that a small subset of mitochondrial proteome persists for months in brain, heart, and eyeball in mice and rats. Given the vital role of mitochondria in cell health and survival, and the highly dynamic nature of mitochondria our discovery that mitochondrial proteins can persist for months in healthy tissues is unexpected and of potential importance. The overarching goal of this project is to characterize mitochondrial long-lived proteins (mt-LLPs) in the context of mitochondrial homeostasis. Our current understanding of mt-LLPs are based on composite measures from tissue homogenates and we lack an understanding of which specific cell types harbor these exceptional proteins. Several lines of evidence point to an inevitable dichotomy of proteins with exceptionally long lifespans. On one hand, due to their persistence, mt-LLPs serve as pillars of mitochondrial architecture, providing structural stability ensure a compact energy generating chemical reactor. At the same time, their long-term persistence puts LLPs at an inherently increased risk for age-related deterioration. Thus, our overall objectives are to use whole- animal stable isotope pulse labelling combined with biochemical and proteomic analyses to identify the brain cell types harboring mt-LLPs (Aim 1) and in parallel investigate mtDNA lifetime (Aim 2). Finally, we aim to determine if cristae structural integrity is required for mt-LLP persistence (Aim 3.1) and correlate the presence of mt-LLPs with mitochondrial membrane potential (Aim 3.2). Understanding the cells and structures harboring mt-LLPs, and the effect on mitochondrial fitness, could open new avenues of research and provide molecular targets for modulating mitochondrial network dynamics in the process of age-related neurodegeneration.