The goal of our research program is to uncover the relevant pathways and mechanisms through which mitochondria are established and maintained over the lifetime of neurons and oocytes, and how their stability contributes to cellular health and degeneration. We hypothesize that mitochondria, despite being often described as “highly dynamic”, evolved to create subcellular mitochondrial subtypes where parts of mitochondria are preserved to benefit the long-term homeostasis of the mitochondrial network in cells.

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The Life Cycle of a Powerhouse: From Mother’s Egg to Cellular Specialization of Mitochondria

Mitochondria are well known for their dynamic nature—just a quick peek under a microscope reveals a lively network of these organelles, some stationary, some on the move, while others merge, split, and rejoin in a dazzling dance. This fluidity is vital for maintaining a healthy network, allowing for rejuvenation, nutrient sharing, and the clearance of damaged components, all while delivering mitochondria to where they’re needed most.

But amidst this bustling activity, a fascinating question arises: how do mitochondria maintain their specialization over time? Each cell integrates a variety of intrinsic and external signals to shape a mitochondrial network tailored to its unique needs, yet how does it safeguard this specialization against years of degradation and remodeling?

Our lab’s main research areas center on:

  1. kinetics of mitochondrial biogenesis from the DNA to proteins to intact organelles
  2. mitochondrial proteome and genome turnover and replenishment along the development and aging continuum
  3. molecular and environmental determinants of mitochondrial specialization

Our approach

Our laboratory employs cutting-edge technologies encompassing mass spectrometry-based proteomics and metabolomics, biochemistry, cell biology, metabolic labelling and tracing, cross-linking MS and structural biology – in both in vitro and in vivo mouse models – to investigate lifelong fidelity of mitochondrial proteome and genome in healthy and diseased mammalian systems.

Why neurons and oocytes?

At first glance, neurons and oocytes might not seem to have much in common and are not often studied together. However, they do share a key similarities. First, both cells persist for months to years without dividing. Neurons are postmitotic, while oocytes are meiotically arrested. This means that, unlike rapidly dividing cells, their pool of mitochondria doesn’t get refreshed. As time passes, the risk of mitochondrial damage rises, leading to various forms of dysfunction. Secondly, our previous research efforts uncovered that a subset of mitochondrial proteins persist for an exceptionally long periods of time in both neuronal cells and oocytes, begging a question – why aren’t these long-lived cells, which rely heavily on fit mitochondria, do not renew their networks throughout their lifetime? Why are these cells holding on certain sets of proteins, while replacing others?

Previous Research

Continuous replenishment of mitochondria has long been thought to be essential for maintaining high-quality organelles throughout the lifespan of post-mitotic neurons. Impairments to the mechanisms governing various quality-control pathways are believed to lead to the progressive functional and structural degeneration of neurons. However, in recent years, largely due to advances in metabolic pulse-chase proteomic analyses, it has become clear that despite being highly dynamic organelles, a subset of the mitochondrial proteome can be exceptionally stable. Using in vivo dynamic metabolic stable isotope (15N) labeling of mice, I discovered that a discrete subset of the mitochondrial proteome in the mouse brain escapes classical first-order degradation kinetics and persists for at least 4 months. This is substantially longer than any previous report on the longevity of the mitochondrial proteome, given that the average half-life of mitochondrial proteins in the rodent brain has been estimated to be less than 3 weeks.

Previous research into long-lived proteins (LLPs), with lifetimes of months or more, has revealed that LLPs often serve as vital components of intracellular structures whose functions are commonly coupled to long-term stability. Accordingly, the mitochondrial LLPs, mt-LLPs, identified in my study include proteins that associate with the cristae sub-compartment, including OxPhos complexes, OPA1, MICOS, prohibitins, mt-DNA-associated proteins, chaperones, and cytochrome C. Mitochondrial cristae, similar to other architecturally stable and long-lived structures (e.g., the core of the nuclear pore), are recognized for their elaborate ultrastructure, the stability of which is coupled to mitochondrial function. Therefore, mt-LLPs could play a previously unrecognized role in the long-term stabilization of mitochondrial ultrastructure, which in turn might be imperative for mitochondrial fitness.

To gain a deeper understanding of how newly synthesized proteins integrate with existing pools, I developed a pioneering cross-linking mass spectrometry (XL-MS) method on intact immuno-isolated mitochondria from heart and brain extracts of dynamically 15N-pulse-labeled mice. Using this approach, I discovered that mt-LLPs are co-preserved with little to no subunit exchange and spatially restricted within cristae membranes in the brains of mice. Collectively, these initial findings have begun to shift the long-standing dogma that mitochondrial function is coupled to frequent protein turnover and organelle rejuvenation and point to a much more complex cross-talk between the “form and function” of mitochondria.

Bomba-Warczak, Ewa et al. Long-lived mitochondrial proteins and why they exist. Trends in Cell Biology, Volume 32, Issue 8, 646 – 654

The female reproductive system is the first to age in humans, characterized by a decrease in both gamete quantity and quality. Reproductive aging carries significant clinical implications as women increasingly delay childbearing, with advanced reproductive age being associated with infertility, spontaneous abortion, obstetrical complications, and an increased risk of birth defects. The mechanisms contributing to the general deterioration of reproductive function during aging are complex, with aberrant protein homeostasis, or proteostasis, identified as a major contributor. Therefore, given our growing knowledge of long-lived proteomes, which are thought to contribute to aberrant protein homeostasis in long-lived cells, we identified a critical need to identify ovarian and oocyte long-lived molecules and determine their functional role in aging.

We employed in vivo whole rodent metabolic stable isotope pulse-chase labeling combined with leading mass spectrometry (MS)-based approaches to visualize and identify long-lived macromolecules in ovarian and oocyte tissues along an aging continuum relevant to the reproductive system. First, we visualized the lifespan of ovarian macromolecules in mammals using a combination of stable isotope labeling and multi-isotope imaging mass spectrometry (MIMS). The data revealed that distinct macromolecular components within select ovarian cells and tissue regions persist throughout the healthy reproductive stage with limited renewal, and these long-lived molecules are present during the stage when ovaries manifest marked reproductive aging phenotypes.

Next, we conducted liquid chromatography MS (LC-MS/MS)-based proteomic analysis of ovarian tissues as well as purified oocytes isolated from metabolically labeled mice. We found that ovaries harbor a panel of exceptionally long-lived proteins, including cytoskeletal components, mitochondrial proteins, and oocyte-derived proteins. By isolating fully grown oocytes from the ovary, we established the long-lived proteome of a purified germ cell population across the reproductive lifespan. Our findings revealed that mitochondrial proteins and myosins were the predominant long-lived proteins in isolated oocytes across the reproductive lifespan.

Overall, the exceptional persistence of these long-lived molecules may play a critical role in both the lifelong maintenance and age-dependent deterioration of reproductive tissues. In an example of a life history trade-off, the turnover of mitochondrial LLPs later in the reproductive lifespan may serve as a biological timer of aging. Because mitochondria are maternally inherited, the mitochondrial LLPs formed during fetal development in the mother are likely transferred to the embryo and impact subsequent generations.

Bomba-Warczak E., Velez K., Zhou L., Guillermier C., Edassery S., Steinhauser M., Savas J., Duncan F. Exceptional longevity of mammalian ovarian and oocyte macromolecules throughout the reproductive lifespan. Elife. (accepted 10/16/2023; manuscript available upon request)