Mitophagy in Aging Brains
The brain uses a great deal of energy, and thus, mitochondria are critical to brain health. Damaged or worn-out mitochondria are removed by mitophagy, and impaired mitophagy has been associated with Alzheimer’s and Parkinson’s diseases. The laboratory of Thomas McWilliams at the University of Helsinki showed that mitophagy levels increased differently in different areas of aging mouse brains. They increased in areas responsible for movement, but decreased in those for memory. These results provide insights that may be valuable for healthy brain aging.
Rappe A, Vihinen HA, Suomi F, Hassinen AJ, Ehsan H, Jokitalo ES, McWilliams TG (2024) Longitudinal autophagy profiling of the mammalian brain reveals sustained mitophagy throughout healthy aging. The EMBO Journal 43(23): 6199-6231.
Impaired Mitochondria Affect Skeletal Aging
Patients with impaired mitochondrial respiratory chain capacity often have show reduced levels of skeletal growth that are similar to premature degeneration of cartilage and aging. A research team led by Bent Brachvogel at the University of Cologne in Germany examined cartilage cells in mice with impaired cellular respiration. They found that those cells tended to lose the ability to regenerate and died to accelerate aging. In this way, they identified basic processes that might be therapeutic targets.
Bubb K, Etich J, Probst K, Parashar T, Schuetter M, Dethloff F, Reincke S, Nolte JL, Krüger M, Schlötzer-Schrehard U, Nüchel J (2025) Metabolic rewiring caused by mitochondrial dysfunction promotes mTORC1-dependent skeletal aging. Science Advances 11(16): eads1842.
Balancing Mitochondrial Transcription and Replication
The small mitochondrial genome serves as a template for replicating the DNA genome and for transcribing RNAs that encode proteins. The balance between these two functions is critical for mitochondrial health. However, the mechanism for this balancing is unknown. Recently, a team led by Takehiro Yasukawa at Kyushu University in Japan studied the role of mitochondrial transcription elongation factor (TEFM) in the process. They report that knockout of the TEFM gene resulted in reduced ability to make the transition from transcription to replication. This study provides additional insights into the biology of mitochondria.
Matsuda S, Nakayama M, Do Y, Ishiuchi T, Yagi M, Wanrooij S, Nakada K, Wei FY, Ichiyanagi K, Sasaki H, Kang D (2025) TEFM facilitates transition from RNA synthesis to DNA synthesis at H-strand replication origin of mtDNA. Communications Biology 8(1): 202.
Mitochondria at the Synapse
Mitochondria are important in synaptic transmission and plasticity, but how they do this is unknown. Sannon Farris and her team at Virginia Technical University looked at the mitochondrial calcium uniporter (MCU) that couples neuronal activity to ATP production. There is more MCU in hippocampal CA2 distal than proximal dendrites, and distal dendrites have more plasticity. However, the mechanism is unknown. Mice with a CA2-specific MCU knockout had fragmented mitochondria that might explain their functional deficits a synapses. These differences in MCU expression might be the mechanism used by different cell types to modulate mitochondrial function to different needs.
Pannoni KE, Fischer QS, Tarannum R, Cawley ML, Alsalman MM, Acosta N, Ezigbo C, Gil DV, Campbell LA, Farris S (2025) MCU expression in hippocampal CA2 neurons modulates dendritic mitochondrial morphology and synaptic plasticity. Scientific Reports 15(1): 4540.
Recycling Mitochondria
Cells with damaged mitochondrial DNA have been implicated in numerous diseases and aging. A team led by David Pla-Martín at Heinrich-Heine University Düsseldorf in Germany discovered a mechanism that deals with those mitochondria with damaged DNA. That mechanism depends on a protein complex called the retromer and lysosomes, which contain digestive enzymes. Together they recycle the mitochondrial components and eliminate the damaged mitochondria. The authors speculate that dysfunction in this recycling mechanism might be involved in disease pathogenesis.
Kakanj P, Bonse M, Kshirsagar A, Gökmen A, Gaedke F, Sen A, Mollá B, Vogelsang E, Schauss A, Wodarz A, Pla-Martín D (2025) Retromer promotes the lysosomal turnover of mtDNA. Science Advances 11(14): eadr6415.
How Mitochondria Produce Energy
Mitochondria have long been known to be the organelle that produces energy from sugar in the food we eat. However, the precise machinery that accomplishes this activity is unknown. Now, using cryo-electron microscopy, a team from the laboratory of Edmund Kunji at the MRC Mitochondrial Biology Unit, Cambridge determined the atomic structure of the mitochondrial pyruvate carrier that transports the key molecule pyruvate. This mechanism allows pyruvate to cross the normally impermeable inner mitochondrial membrane. Understanding this machine will allow scientists to develop drugs that interact with it and serve as therapies for a variety of diseases.
Sichrovsky M, Lacabanne D, Ruprecht JJ, Rana JJ, Stanik K, Dionysopoulou M, Sowton AP, King MS, Jones SA, Cooper L, Hardwick SW. (2025) Molecular basis of pyruvate transport and inhibition of the human mitochondrial pyruvate carrier. Science Advances 11(16): eadw1489.
Mitochondria have come to be more appreciated for their roles in health and disease. Mitochondrial dysfunction has been implicated in a direct role in more than 50 diseases. In others, mitochondrial dysfunction is an important contributing factor, including neurodegenerative diseases (e.g., Alzheimer’s disease, Parkinson’s disease), cancer, and metabolic diseases.
Mitochondria have increasingly become the focus of new biotech companies. One of those is Pretzel Therapeutics. Pretzel seeks to harness cellular energetics to treat a wide range of conditions now recognized as having a mitochondrial connection. Mitochondria are the powerhouses of the cell, and each cell contains hundreds of mitochondria. Interestingly, mitochondria have their own small genome that encodes enzymes that control mitochondrial activities. Several of the projects at Pretzel focus on controlling cellular energetics by modulating the cellular copy number of mitochondrial DNA (mtDNA).
The Pretzel researchers hope to restore cellular energetics to treat neurodegenerative and rare diseases associated with low mtDNA levels or to modify bioenergetics to treat obesity. In one program, they hope to restore energetics by activating the mitochondrial DNA polymerase. This program was disclosed in a recent Nature report (Valenzuela et al., 2025). In that publication, the researchers show that a small-molecule activator restored function to the most common mutants of the mtDNA polymerase, PolG. A lead therapeutic in this program was recently entered into Phase 1 clinical study. In another program, they hope to modulate the activity of the mitochondrial RNA polymerase. A third program employs gene editing to reduce mutated mtDNA and increase levels of healthy mtDNA.
Pretzel launched in 2022 with financing of $72.5 million. The founders include Gabriel Martinez, Claes Gustafsson, Maria Falkenberg, Michal Minczuk, Nils-Göran Larsson, and the late Paul Thurk. Their facilities are located in Waltham, Massachusetts, and Mölndal, Sweden.
For more information on Pretzel Therapeutics, visit their website (https://www.pretzeltx.com/).
Reference
Valenzuela S, Zhu X, Macao B, et al. (2025) Small molecules restore mutant mitochondrial DNA polymerase activity. Nature, doi.org/10.1038/s41586-025-08856-9
Questions for Pretzel Therapeutics
Pretzel researchers and others recently published an excellent paper in Nature that supports your therapeutic strategy. Congratulations on that publication.
Mitochondrial dysfunction has been implicated, directly or indirectly, in many diseases. What diseases do you see as the “low-hanging fruit” for treatments?
The lack of treatments for mitochondrial dysfunction and disease indicates to us that there really isn’t any “low-hanging fruit” in this area. That said, diseases with a clear and direct genetic cause, such as POLG disease, offer the clearest mechanistic rationale to drug discoverers.
Are you considering testing approved drugs for potential off-label uses for treating mitochondrial diseases?
We are focused on our own, novel targets and drug candidates. We do not work in re-purposing projects.
Neurodegenerative diseases are projected to increase rapidly with our aging population. Do you have any thoughts about how your therapies might fit into preventing or treating those diseases?
There has been substantial published research on the links between degenerative conditions of aging and declining mitochondrial function, far more than we can summarize here. We are of course interested in investigating how our novel mechanisms may influence these conditions. To this end, we have been awarded a grant from Parkinson’s-UK, a leading charity supporting research, to study our mechanisms in Parkinson’s disease.
Cytotoxic T cells are vital for eliminating cancer cells and thus for restraining tumour growth. However, many types of cancer cell blunt this immune attack by issuing signals that somehow cripple the attacking cell. Like almost everything in immunity, this undoubtedly has multiple dimensions. However, one crucial aspect seems to be centred on mitochondria. By rendering the mitochondria of the cytotoxic cell incapable of energizing the process(es) that kill the target cell, cancer cells survive immune attack. This concept has received support from studies showing that T cells that invade the tumour micro-environment exhibit signs of mitochondrial energetic dysfunction. However, the cellular and molecular mechanisms behind this phenomenon have, until recently, remained obscure.
This phenomenon could have serious consequences. Cancer cells frequently harbour mtDNA mutations that impair their own capacity for oxidative ATP generation (OXPHOS). Surprisingly, mitochondria from T cells use ‘tunnelling nanotubes,’ to transfer into cancer cells [2-4] and promote tumour survival. Even if only a small fraction of tumour cells are ‘empowered’ in this manner, it should be sufficient to protect them from cytotoxic drugs that target cells dependent on non-mitochondrial pathways for their energy supply, DNA repair and cell division. Thus, a pool of chemoresistant cells would survive to reconstitute the tumour in an even more aggressive form that is refractory to future treatments. The phenomenon of intercellular transfer of mitochondria remains somewhat controversial. However, it has been independently documented by multiple reputable laboratories and must now be considered physiological. Nevertheless, it could still be an enhanced property of tumour cells or other cells with metabolic impairment.
A recent paper from the lab of Yosuke Togashi in Japan shed light on one important part of this puzzle [1]. They report that mitochondrial transfer also occurs in the opposite direction, via tunnelling nanotubes and or/extracellular vesicles. OXPHOS-incompetent mitochondria from the tumour, bearing deleterious mutations in mtDNA, were detected in T cells that had invaded the tumour micro-environment. The origin of these mitochondria was confirmed by co-culture experiments with tumour cells expressing a mitochondrially targeted fluorescent reporter protein. Prolonged co-culture resulted in the almost complete replacement of endogenous T-cell mitochondria by those derived from the tumour cells. This replacement reflected the fact that tumour-derived mitochondria were resistant to mitophagy, whereas the original T-cell mitochondria were not.
T cells whose mitochondria had suffered this replacement showed OXPHOS impairment, increased dependence on non-mitochondrial ATP generation, downregulation of T-cell markers associated with cytotoxic functions, and signs of cellular senescence. Importantly, the acquisition of tumour- derived mitochondria impaired their activation and, hence, their ability to kill cancer cells, as shown by an in vivo model.
The work breaks new ground by showing that the interaction of immune cells with cancer cells can facilitate a bidirectional mitochondrial exchange that simultaneously favours cancer cell viability and immune evasion. However, this obviously raises many questions. The authors showed that the exchange can operate in at least three cancer-cell types (melanoma, breast and lung cancer), but does it work for all cancer types? Does the phenomenon depend on which surface markers are expressed by a given cancer cell, or by which antigen-receptors are expressed on the cytotoxic cell with which it interacts? The immune cell repertoire includes many different cell types, each with a role in anti-cancer immunity. Does mitochondrial exchange operate only in cytotoxic T cells, or is it seen in other immune cell-types, such as Treg and other CD4+ cells, dendritic cells, NK (natural killer) cells, phagocytes or B lymphocytes? If so, how is their function modified? Are those other immune cell functions secondarily deranged by impaired cytokine secretion from OXPHOS- deficient T cells? Does tumour mtDNA migrate to other cells in the body than immune cells, and if so, is their function also compromised?
The discovery of the mitochondrial fusion/fission cycle forced us to rethink the mitochondrial content of each cell as being akin to a single entity. The pioneering work of the Togashi lab and others now obliges us to revise another fundamental tenet of mitochondrial biology, recognizing that mitochondria and their DNA are not simply the chattels of single cells, but belong to populations of many cells and to tissues, organs and even the body as a whole.
References
- Ikeda H, et al. (2025) Immune evasion through mitochondrial transfer in the tumour microenvironment. Nature 638: 225–236. doi: 10.1038/s41586-024-08439-0.
- Tan AS, et al. (2015). Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA. Cell Metab. 21: 81-94. doi: 1016/j.cmet.2014.12.003.
- Saha T, et al. (2022) Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells. Nat Nanotechnol. 17: 98–106. doi: 10.1038/s41565-021-01000-4.
- Zhang H, et al (2023) Systematic investigation of mitochondrial transfer between cancer cells and T cells at single-cell resolution. Cancer Cell 41: 1788–1802.e10. doi: 10.1016/j.ccell.2023.09.003.
In a new paper in Nature, the laboratory of Martin Picard, PhD, mapped the location and activity of the many mitochondria in a human brain.1, 2 Dr. Picard is associate professor of behavioral medicine at Columbia University Irving Medical Center and directs the Mitochondrial Psychobiology Group. This project was conducted in collaboration with Michel Thiebaut de Shottten, PhD, at Director of research the CNRS in Bordeaux,
When most of us think about burning energy, we think of exercising our muscles. However, small as it is, the brain consumes an outsized proportion of our energy. It needs that energy to perform its myriad functions, and to provide all that energy, each brain cell contains thousands of mitochondria, the organelles that produce energy.
Dr. Picard wondered how the many mitochondria are located in the brain and how they relate to energy production and usage in different areas of the brain. How are the cellular components associated with brain anatomical structures? To explore these and other questions, they developed MitoBrainMap v1.0.3
They began by developing a method to physically divide or “voxelize” a frozen human brain into small cubes or voxels (3x3x3mm). Interestingly, magnetic resonance imaging (MRI) images small cubes of brain, virtually “voxelizing” the brain, but the voxelization of the brain into physical cubes had to await the development of new methods. This was spearheaded by Eugene Mosharov, PhD, first author on the paper.
Next the team had to define the different mitochondrial types across the brain. They found that the different regions of the gray and white matter had quite different mitochondrial densities and activities (measured as oxidative phosphorylation activity). Intriguingly, there was an intriguing correlation: regions with higher activity were those that developed later in evolution.
The final challenge was to create a computational model to assemble the information from a single slice to the whole brain. This was accomplished by Michel Thiebaut de Shotten, PhD, a neuroanatomist from the CNRS in Bordeaux. The team then expanded that model into a prediction model that allowed them to assign predictions to other regions of the brain, based on date from MRIs.
The map is only the beginning. The team can now use that map to further explore the relationship of mitochondria in different regions of the brain with psychosocial and cognitive factors; exploring the mind-mitochondria connection. This new tool will enable them to study how brain energy relates to anatomy, development, behavior, and neurodegeneration, and the nature of the mind and consciousness.
“This is the kind of project that is nearly impossible to do” said Picard, “we had to find an exceptionally high-quality brain, align engineering, computational, molecular biology, and neuroscience expertise, and find the right students courageous enough to undertake this massive effort! I am so grateful for this amazing team that came together to create MitoBrainMap v1.0.”
A Conversation with Martin Picard
How did the idea come about to create the mitochondria brain map?
One November evening in the lab, after discovering the diversity of mitochondria in the mouse brain (Nature Communications 2023) and imaging the brain of our patients with rare mitochondrial diseases. I dreamed of a way to systematically map mitochondria across the whole brain. Chatting with my engineer-at-heart neuroscientist colleague Eugene Mosharov, we came up with the technical approach to turn the brain into cubes to create the MitoBrainMap v1.0.
What were the steps needed to get the data?
1. Find a brain, 2. Develop a hardware/software approach to physically partition, or voxelize the brain, 3. Perform >18,000 laboratory assays, 4. Visualize the massive multi-modal dataset in the brain space, 5. Register that data to the standard human brain space used in neuroscience with Michel Thiebaut de Shotten.
Were there many debates and questions about how to get the right sample sizes and make sure they were representative?
We initially wanted to do the whole brain, but estimated that this would cost >5 million dollars and take multiple years. We abandoned that ambition, and focused on a single section of a single brain hemisphere.
What problems did you have to overcome?
The major challenge was one of scale. This was by far our largest mitochondrial phenotyping project. We developed a higher throughput lab platform and a robust data processing pipeline to overcome this challenge. The other challenge was the working across disciplines, combining molecular and single-cell RNA sequencing, mitochondrial biochemistry, neuroscience, and neuroanatomy.
How did the team work together?
Cohesive teamwork was critical. Students generated a ton of data. Eugene handled and integrated the unique dataset. Orian Shirihai’s team validated and enhanced our mitochondrial assays. And Michel Thiebaut de Shotten mapped this data onto the brain, developing an algorithm to extend to the whole brain.
How long did it take from inception to final paper?
November 2020 to March 2024 – 3.5 years! Plus a year to revise the paper.
What are the next steps on your research?
Cynthia Liu in our lab is now measuring mitochondrial content and OxPhos enzyme activities in 5,000 human samples. This will validate and extend this line of work.
Was there any correlation between mitochondria and regions of known brain functions (e.g., speech, vision, hearing)?
Great question. This is something to explore in future research.
The gap between molecular processes and macro-imaging often contains fascinating information. Your work bridged that gap. Might there be other organelle targets of your work?
Mitochondria are pretty special. They have their own genome, two membranes, and without them, mammalian cells cannot live at all. All cellular and molecular processes converge on their requirements for energy, and the human genome is actively regulated by signals from the Mitochondrial Information Processing System (MIPS) (Cell Metabolism 2022). That being said, one could certainly profile other organelles across the brain.
Can your work be correlated to mitochondrial brain work on neurodegenerative diseases, such as Parkinson’s or Alzheimer’s diseases?
Yes, this is a future direction some of our colleagues are interested in. But when we study diseased brains, it’s like trying to understand the causes of a car crash by looking at the totaled, permanently damaged vehicle. Without looking at the driver, we likely will never know why the vehicle/brain crashed in the first place. In the brain and living organisms, the driver is energy. And energy fluxes through our mitochondria.
You suggest that your method correlates with other imaging methods. Do you think these might have potential to eventually be used in diagnostic methods?
Yes, our hope is that we can quantify mitochondrial content and quality or health with standard neuroimaging methods. In the living human brain, non-invasively.
How did you come to be interested in mitochondria in the first place?
Mitochondria are the dynamic energetic portal between the physical processes that convert food and oxygen into subjective experiences, and consciousness. In graduate school, I became increasingly convinced that mitochondria were going to teach us something meaningful about mind-body processes. Now we study the mind-mitochondria connection, in and outside the brain.
References
1Mosharov EV, Rosenberg AM, Monzel AS, Osto CA, Stiles L, Rosoklija GB, Dwork AJ, Bindra S, Junker A, Zhang Y, Fujita M, Mariani MB, Bakalian M, Sulzer D, De Jager PL, Menon V, Shirihai OS, Mann JJ, Underwood M, Boldrini M, Thiebaut de Schotten M, Picard M (2025) A human brain map of mitochondrial respiratory capacity and diversity. Nature https://doi.org/10.1038/s41586-025-08740-6.
2Thiebaut de Schotten M, Picard M (2025) A map of mitochondrial biology reveals the energy
landscape of the human brain. Nature https://doi.org/10.1038/d41586-025-00872-z.
3Human MitoBrain Map (2025) Michel Thiebaut de Schotten. Retrieved from: http://humanmitobrainmap.bcblab.com/. April 1, 2025.
For more links and videos, visit http://humanmitobrainmap.bcblab.com/
Prediction of whole-brain mitochondrial density visualized along a red (high) yellow (low) gradient projected onto the lateral view of the brain. The map is overlaid on a tractographic representation of the brain, illustrating the connectional organization of white matter pathways.
Prediction of whole-brain mitochondrial density visualized along a standard heat gradient projected onto an axial slice. Warmer colors (e.g., red/yellow) indicate higher predicted mitochondrial density, whereas cooler colors (e.g., green/blue) denote lower density. The map is overlaid on a tractographic representation of the brain, illustrating the connectional organization of white matter pathways.
Since the days of van Leeuwenhoek and his microscope, new technologies have allowed researchers to look at life, quite literally, in new ways. As technologies have grown in power, they also increased in cost and complexity, often putting them beyond the means of individual researchers. Many research organizations solved this challenge by developing core facilities in which an expensive instrument is purchased for multiple researchers. Knowledgeable individuals are hired to operate it.
While many research organizations have core facilities, they typically focus on one instrument type (e.g., imaging, bioinformatics, HPLC). At UCLA, Orian Shirihai (professor of medicine and molecular and medical pharmacology) took this concept to the next level by combining advanced technologies of different modalities into a single integrative core. His Mitochondria and Metabolism Core (Core) brings together capabilities in bioenergetics, imaging, and biochemistry to facilitate the study of mitochondria metabolism. The Core may be unique in this comprehensive approach to research on mitochondria.
“Our mission is to empower scientists across academia, biotechnology, and pharmaceutical industries to design, execute, and interpret experiments related to mitochondria and metabolism. By doing so, we aim to accelerate scientific discovery and the development of novel therapies and diagnostics,” said Dr. Shirihai.
The Core comprises three sections. The bioenergetics section, led by Linsey Stiles, features several Seahorse instruments that measure oxygen consumption and extracellular acidification providing readouts of mitochondrial respiratory function, glycolysis, ATP production rates, and other metabolic measurements. The imaging section, led by Cristiane Beninca, has a broad selection of microscopes (brightfield, widefield, confocal, FLIM, and super-resolution microscopes as well as a transmission electron microscope) and techniques to study mitochondrial structure and function (e.g., membrane potential, reactive oxygen species, and NADH measurements, mitophagy flux, mitochondrial and cristae dynamics, organelle interactions) in live and fixed samples. A biochemistry section, led by Lucia Fernandez-del-Rio complements the other sections with enzyme assays, western blots, non-denaturing gels to examine mitochondria supercomplexes, and mtDNA-based assays.
The Core provides an exceptional resource for basic, clinical, and industry researchers who are interested in mitochondria research. It helps in all phases of mitochondrial research from study design to data interpretation. The Core operates on a fee-for-service recharge system, offering a range of options from full-service support to specialized training for autonomous use of advanced equipment, and everything in between.
In recent years, physicians and researchers have come to appreciate the intimate involvement of mitochondrial dysfunction in human disease. The Core provides a unique resource to accelerate research in this critical area. Its value is documented by its broad spectrum of users, including researchers at UCLA and many other organizations, such as CalTech, UC Santa Barbara, and USC.
David Shackelford, PhD (professor of medicine, UCLA), said, “The mitochondrial metabolism and the imaging cores at UCLA have been an incredible resource and been transformative with respect to our research.” Amy Wang, PhD (CEO, Enspire Bio Inc.), added, “The UCLA mitochondria cores are indispensable collaborators and have transformed the direction and focus of our research and were important for informing our scientific strategy.” Clearly, the Core is having an impact on research into metabolism and mitochondria.
For more information about the Core, please visit their website: https://medschool.ucla.edu/research/themed-areas/metabolism-research/metabolism-core.
The Core directors respond to our questions
The UCLA Core seems to be a unique resource for researchers. Are you aware of anyone else who has combined so many powerful technologies to the study of mitochondria?
Providing services as we do, no. We are the only place that can offer services and access to cutting-edge equipment to internal and external scientists in need of the techniques provided. There are certainly places with multiple researchers working in mitochondria research and sharing expertise and access to equipment, but the only way for other researchers to have access to their techniques is through collaborations. In the Core, we offer a fee-by-service system, so anybody can “hire” us to work for them.
Do you plan to add any new resources to your collection (e.g., mass spec, HPLC, new imaging methods)?
At UCLA, additional Core facilities (e.g., Lipidomics, Metabolomics, or Proteomics) are available to clients interested in a multi-omic approach, so we are not considering an expansion in that direction. In the Biochemistry Core, we are working to incorporate mitochondrial DNA (mtDNA)-based assays, such as mtDNA copy number, mutation frequency, or mtDNA nanopore sequencing, into our services. But our portfolio is always expanding as new techniques are constantly in development, and in certain cases, we even work with the clients to develop or improve the techniques they need. Particularly, in the Imaging Core, new dyes, imaging, and analysis techniques are always in development. And in the Bioenergetics Core, new ways to measure mitochondrial function or optimization of different samples are always happening. Once techniques are optimized, they can be added to our portfolio as a service to clients.
Can you describe the general idea of a couple of the studies that the Core has participated in where each of the three sections were involved?
One example is the research paper https://doi.org/10.15252/embj.2022111699. In this project, we started with a Sponsored Research Agreement with a Company that provided us with a compound to be tested. The story progressed into a scientific discovery that was published and even a new technique was developed 10.26508/lsa.202201628
The three sections work very closely together, and we also have researchers with Grants in need of the expertise of all three Cores, and we can work together with them almost as if we were part of their laboratory personnel too.
What would you estimate as the breakdown of your time spent in training, planning experiments and evaluating data, and actual experimenting?
Our top priority is to address the needs of our clients in the best way possible. So, we work together to find the best approach to do so. There are times when we have clients with more knowledge of the techniques needed, and they can be more independent on planning and even running the experiments, so training is just needed in the beginning. Other times, samples are shipped to us, and we are responsible for the whole process until the delivery of results.
How did each of your become interested in the study of mitochondria?
Cristiane Beninca: For me, it was during my PhD that my mentor discovered a new protein localizing at the mitochondria, and since that, everything became mitochondria related.
Lucia Fernandez del Rio: Back in my undergrad days, I joined the Cell Biology Department and started working in a lab that focused on oxidative stress. Since this phenomenon is deeply interconnected to mitochondria, that is where my mitochondria journey started.
Linsey Stiles: I did a rotation focused on the role of mitochondrial fusion and fission in erythropoiesis and knew immediately that I wanted to continue to study mitochondria for my PhD project.
In a paper published in Cell, a research group led by Agnel Sfeir at Memorial Sloan Kettering Cancer Center showed that gene editing can be used to engineer specific mtDNA mutations.
Mitochondria are unusual cell organelles in that they contain their own mitochondrial (mt)DNA, and like other DNA, mtDNA can develop mutations that result in diseases. Some mutations involve the deletion of parts of the coding sequences. Fortunately, cells typically have more than 1,000 mitochondria, and in many cases, a minimum number of healthy mitochondria are sufficient to avoid disease. Nevertheless, mitochondrial diseases can be highly debilitating.
Advances in genome engineering and editing (e.g., TALENS, CRISPR, zinc finger nucleases) have raised the possibility of reconstituting point mutations repairing defective mtDNA to curing the diseases. However, mtDNA deletions in human cells have been challenging. Recently, a research team at Memorial Sloan Kettering Cancer Center, led by Agnel Sfeir, succeeded in engineering mtDNA deletions with an innovative approach by expressing bacterial DNA end joining enzymes in human cells.
The Sfeir team developed a group of human cell lines with deletions to the mtDNA of varying extents (Fu et al., 2025). Cells with deletions in more than 75% of the mitochondria had affected mitochondrial function. With these cells, the team found two patterns of effects on nuclear genes. One was activated at the deletion threshold. The other was in response to increasing levels of mutations.
More importantly, this combination of tools facilitates modeling of disease-associated mtDNA deletions in different cell types. In addition, the panel of cell lines with the different levels of deletions will greatly benefit the research community and help in developing therapeutics.
A Discussion with Agnel Sfeir
What are the next steps in your research?
Our research focuses on elucidating the molecular pathways that govern the propagation versus elimination of mtDNA deletions. Understanding these mechanisms is crucial for identifying factors influencing mtDNA integrity and stability.
Additionally, we aim to investigate how cells, particularly neurons and muscle cells, respond to mtDNA deletions. These cell types are highly dependent on mitochondrial function and are disproportionately affected by mtDNA mutations, leading to severe pathologies. By studying their adaptive or maladaptive responses, we hope to uncover potential therapeutic targets that could mitigate the impact of mtDNA deletions in mitochondrial diseases.
You note that you can use this system to examine stem cells. It sounds like that is a great way to look at looks of cell types, especially muscle and brain. Maybe even iPS cells could be used.
Yes, that’s exactly what we are in the process of doing. IPS cells provide a powerful strategy for studying a wide range of cell types, including muscle and neuronal cells, which are particularly affected by mitochondrial dysfunction. By differentiating iPS cells into specific lineages, we can systematically investigate how mtDNA deletions impact various cell types and really model disease-relevant phenotypes. This approach also allows for high-throughput screening of potential therapeutic interventions in a controlled and physiologically relevant system.
The ability to switch the carbon source between galactose and glucose is a real advantage to your system.
Absolutely. The ability to switch between glucose and galactose as carbon sources is a key advantage of our system. Heteroplasmy refers to the proportion of mutated mtDNA within a cell, and when it surpasses 75%, cells typically exhibit severe respiratory defects and abnormal morphology. However, the RPE1 cells we used primarily rely on glycolysis, allowing them to proliferate normally in glucose-containing media—even with heteroplasmy levels exceeding 75%. The defect became evident only when glucose was replaced with galactose, forcing the cells to rely on oxidative phosphorylation. This metabolic shift was crucial in unmasking the mtDNA deletion-associated defects in galactose-containing media, whereas culturing cells in glucose allowed us to obtain cells with ~100% mtDNA deletion. In fact, to date, no cellular models of mtDNA deletions have been able to achieve near-homoplasmy.
You discuss the application of your method for the study of disease, but I assume it might also be used to explore mitochondrial regulation as well.
While our method is highly applicable to studying disease, it also provides a powerful tool for exploring mitochondrial regulation. By analyzing the dynamic behavior of intact and deleted mtDNA, we can gain valuable insights into key mitochondrial quality control mechanisms, such as mitophagy and selective replication. Specifically, our cellular system allows us to examine how cells manage mutant mtDNA through preferential degradation of dysfunctional mitochondria via mitophagy, or the selective replication of intact mitochondrial genomes. This, in turn, enables us to understand the mechanisms that maintain mitochondrial homeostasis under normal conditions and how they are altered in the presence of mtDNA deletions.
Moreover, our approach provides a window into the biochemical and metabolic adaptations that occur in response to mitochondrial stress. By assessing how cells adjust their energy metabolism, stress signaling pathways, and mitochondrial dynamics, we can uncover the requirements necessary for cellular survival and adaptation. This is particularly crucial for understanding the threshold at which mitochondrial dysfunction becomes pathological and identifying potential targets for therapeutic intervention.
Your work and that of others attempts to correct mutations in mtDNA. Are there any genetic diseases of mitochondria that involve genes in the host genome that are specific to mitochondria?
Nuclear-encoded mitochondrial genes play a crucial role in mitochondrial function, and their mutations can lead to severe disorders. Several mitochondrial diseases arise from mutations in nuclear-encoded genes that regulate mitochondrial DNA replication and transcription. For example, POLG mutations impair mtDNA replication, leading to disorders such as Alpers syndrome, and defects in TFAM reduce mtDNA copy number and transcription.
How did you get interested in mitochondria?
I am a chromosome biologist with a deep fascination for how cells repair DNA breaks to maintain genome integrity. During my training and early career, I focused on nuclear DNA repair pathways and how their deregulation contributes to cancer progression. However, after starting my lab, I felt the need for a new intellectual challenge—an opportunity to step outside my comfort zone and explore an entirely different aspect of biology (something I believe all scientists should do at least once in their careers).
mtDNA had always intrigued me, and I saw a unique opportunity to apply my expertise in nuclear DNA repair to studying this independent genome within a distinct organelle. To immerse myself in the field, I dedicated two months solely to reading the mtDNA literature, identifying key questions, outlining a research plan, and recruiting a student and a post-doc to pursue these challenges. Despite its unique regulatory mechanisms, mtDNA still adheres to fundamental principles of genome maintenance, making it an exciting system to study. Today, half of my lab is dedicated to studying mtDNA biology, and with every discovery we make our curiosity deepens. The same curiosity that led me to work on mtDNA in the first place continues to drive our research forward.
Reference
Fu Y, Land M, Cui R, Kavlashvili T, Kim M, Lieber T, Ryu KW, DeBitetto E, Masilionis I, Saha R, Takizawa M, Baker D, Tigano M, Lareau CA, Reznik E, Sharma R, Chaligne R, Thompson CB, Pe’er D, Sfeir A (2025) Engineering mtDNA deletions by reconstituting end-joining in human mitochondria. Cell
https://www.sciencedirect.com/science/article/pii/S0092867425001941?dgcid=author
Fu et al. (2025) used nucleases and an end-joining procedure to produce mutations in mitochondrial (mt) DNA. The mitochondria were then introduced into cells, and cell lines were produced with different numbers of mutated mitochondria. The resulting cell lines provide excellent models for testing the effects of the mutated mtDNA in diseases.
In a paper published in Cell, the laboratory of Isha Jain at Gladstone Institutes showed that a small molecule inhibitor can control excess oxygen levels resulting from dysfunctional mitochondria. These results point to a potential new therapeutic strategy for mitochondrial diseases.
Oxygen is well-known to be critical for human life, but too much oxygen can be toxic. Thus, the balance between supply and demand is important. For most of us, that is no problem. However, that careful balance is disrupted in those with some mitochondrial diseases so that excess oxygen is a common feature. Leigh syndrome is the most common pediatric mitochondrial disease.
Since tissue hyperoxia is problematic in these patients, one might wonder if reducing the concentration of oxygen in tissues would be beneficial. A recent study from the laboratory of at Gladstone Institutes and the University of California, San Francisco showed just that. The team, led by Isha Jain, took advantage of mice with a knockout of the Ndufs4 gene (Blume et al., 2025). That mutation results in the loss of an important complex in the electron transport chain that mimics the disease, and these mice are often used as a model for experimentation.
They exposed the mice to a small molecule called HypoxyStat that induces hypoxia by causing oxygen to bind more tightly to hemoglobin, leading to decreased release of oxygen into tissues. Chronic exposure of the mice to the molecule normalized tissue hyperoxia and resulted in significantly longer lifespans in the mice.
Thus, these findings suggest that drugs, such as HypoxyStat, that lower effective oxygen levels might be a promising therapeutic strategy for these diseases in general. Of course, manipulating oxygen levels is challenging, but these results provide valuable insights into mitochondrial diseases.
A video, Science in Seconds, produced by Gladstone tells the story of Jain and her team’s breakthrough, as does an article, Daily Drug Captures Health Benefits of High-Altitude, Low-Oxygen Living, published by Gladstone on the findings.
An interview with Dr. Isha Jain
Do the benefits result from improved mitochondrial function or the reduction of excess oxygen in tissues or both?
Currently, I would say we have the strongest evidence that the benefit occurs due to the alleviation of tissue hyperoxia (excess oxygen) in tissues. Future work is needed to fully understand the mechanism.
You noted that you wanted to determine if you could generalize your results beyond the Ndufs4 mice. What do you see as the next steps in this research?
The next steps include testing whether this compound (or further optimized compounds) work in additional mitochondrial disorders and additionally affected tissues beyond the brain.
Mitochondria have been around for a very long time, and oxygen levels have fluctuated in our atmosphere. Does that mean that mitochondria have adapted to the available oxygen?
It is indeed puzzling that oxygen is both a substrate and a toxin for mitochondrial ATP production. It appears that we have evolved to exist in a very delicate balance of too little or too much oxygen.
The findings are intriguing, but controlling oxygen levels might be a challenge in patients. For example, are those with mitochondrial diseases, such as Leigh syndrome, affected by changes in altitude (e.g., Denver)?
There is anecdotal evidence that this might be the case. However, mitochondrial diseases are rare enough and the altitudes we are talking about are fairly high, so it is difficult to amass enough epidemiological data to make this claim definitively.
You mention pulmonary hypertension as a complication of using drugs, such as these. Are there other potential complications and ways around the problems?
The side effects are the same as living at altitude: things like pulmonary hypertension and increased blood viscosity. A gradual acclimation to the drug (dose escalation) will likely be needed. Many more safety studies are needed before this compound or related compounds can be used in patients.
What initially interested you in the study of mitochondria?
I am broadly interested in developing creative new therapies for metabolic disorders. Of course, mitochondria are where much of metabolism takes place.
Reference
Blume SY, Garg A, Martí-Mateos Y, Midha AD, Chew BTL, Lin B, Yu C, Dick R, Lee PS, Situ E, Sarwaikar R, Green E, Ramanan Y, Grotenbreg G, Hoek M, Sinz C, Jain IH (2025) HypoxyStat, a small-molecule form of hypoxia therapy that increases oxygen-hemoglobin affinity. Cell https://doi.org/10.1016/j.cell.2025.01.029.
Ryu KW, Fung TS, Baker DC, Saoi M, Park J, Febres-Aldana CA, Aly RG, Cui R, Sharma A, Fu Y, Jones OL. Cai X, Pasolli HA, Cross JR, Rudin CM, Thompson CB (2024) Cellular ATP demand creates metabolically distinct subpopulations of mitochondria. Nature 6: 1–9.
You see one; you’ve seen them all. That has been pretty much the view of mitochondria for some time. But maybe that isn’t the case after all. The laboratory of Craig Thompson reports that, under stress conditions, mitochondria assume different roles. Dr. Thompson is the former president of Memorial Sloan Kettering Cancer Center and currently holds the Douglas A. Warner III Chair in the Cancer Biology and Genetics Program.
Dr. Thompson’s research team began their search with a careful rethinking of mitochondrial functions. While mitochondria have many key functions, they are best known for producing the energy that we all need from the food that we eat. However, the Thompson lab was interested in the role of mitochondria in wound healing. Repairing tissue requires energy and also proteins, lipids, nucleic acids and other biomolecules. Thus, part of our food goes for the raw materials to synthesize new biomolecules to repair and/or maintain our tissues. They discovered that the amino acid proline is critical for wound healing and that the mitochondria are important for its synthesis.
With that discovery, they became curious about how the mitochondria balance their efforts to make energy and proline. As often happens in science, a few simple experiments conducted by changing the cell culture conditions left them with even more questions. The cells seemed to be able to both make energy and raw materials at the same time. How could that be?
To tackle that question, they exposed cells to 2 different sets of stresses. In one set cells were made to need more ATP, and in the other to need more proline. Surprisingly, when cells were deprived of proline, some of the mitochondria changed their morphology. They developed a series of filaments within the inner membrane that increase their ability to make proline and ornithine. When cells needed more ATP their mitochondria also separated into two distinct subpopulations. In fact, the two types of mitochondria could be differentiated by simple microscopy. The bonus finding was that an enzyme called pyroline-5-carboxylate synthase or P5CS was the key to how glutamate was used to yield the two types.
The separation of activities is closely related to the ability of mitochondria to fuse together and separate by fission. When the cell’s need for oxidative synthesis increases, P5CS is segregated into a subset of mitochondria that lack cristae and ATP synthase, the enzyme that makes ATP.
Thus, the mitochondria can easily rearrange themselves to focus on one process or the other. Interfering with the fission-fusion cycle inhibits their ability to specialize. This paper shows that mitochondrial fission and fusion are intimately involved in maintaining the balance between the need for energy and synthesis of biomolecules.
Reference
Ryu KW, Fung TS, Baker DC, Saoi M, Park J, Febres-Aldana CA, Aly RG, Cui R, Sharma A, Fu Y, Jones OL. Cai X, Pasolli HA, Cross JR, Rudin CM, Thompson CB (2024) Cellular ATP demand creates metabolically distinct subpopulations of mitochondria. Nature 6: 1–9.
According to the CDC, about 38 million Americans suffer from diabetes, and it is the fourth leading cause of death in the United States. More concerning is that the incidence of diabetes—especially type 2 (T2D) or adult-onset diabetes—is increasing.
Diabetes is caused by the inability of pancreatic b-cells to make or other cells to use insulin. An autoimmune process has long been suspected to be involved. Skeletal muscle and b-cells in patients with T2D have multiple morphological and functional defects.1,2
Recently, a team of researchers at the University of Michigan explored this mitochondrial dysfunction and showed how it might be linked to diabetes.3 The team, led by Scott Soleimanpour, MD and Emily M. Walker, PhD, examined mitochondrial quality control that maintains appropriate mitochondrial function under stress conditions. More specifically, they looked at three pathways that lead to impairments in mitochondrial quality control: decreased mitoDNA levels, preventing elimination of damaged mitochondria, and inhibition of mitochondrial fusion. The initial experiments were done in mice, but the findings were repeated in human pancreatic islet cells.
Interestingly, the result was the same for all three pathways. In all cases, the stress response resulted in interference with the maturation of b-cells. Blocking the stress response allowed them to mature as normal. Thus, these insights suggest that blocking the stress response might be a beneficial therapeutic strategy.
A conversation with Dr. Walker.
T2D is linked to diet. Do your results offer any insight into that link?
In two of our models of impaired mitochondrial quality control, the animals were placed on a high-fat diet to model individuals eating a western-style diet. We saw a much stronger stress response and loss of b-cell maturity when the animals were on this diet. There are a lot of negative impacts for b-cells when they are required to produce more insulin to compensate for weight gain, and also, there are many studies showing that fatty acids themselves damage b-cells. In our study, the high-fat diet sped up the mitochondrial stress responses likely because of this increased damage due to the food they were eating.
Can you speculate on the nature of the signaling molecules that illicit the response?
Our study found that it was the integrated stress response that includes changes to signaling molecules, such as eIF2α, and a transcription factor called ATF4. Activation of this signaling pathway and these factors induced changes in the nucleus that changed what genes were expressed in both b-cells and liver cells. We saw increases in the stress response target genes and decreases in genes that help the cell to have mature function and identity.
Your results provide a window on the process of normal b-cell development. Do you have any thoughts on those mechanisms?
We know that b-cells need proper mitochondrial function to maintain cell maturity. We also know that, during differentiation of stem cells into b-cells, this doesn’t happen in a complete way. Other work in our lab is focused on how to improve mitochondrial function in stem cell-derived b-cells to help them mature better, and we’re just starting to figure out pathways that can help this happen.
The use of ISRIB to block the stress response is an intriguing result. Is that a viable possibility for a therapy?
Clinical trials for next generation drugs related to ISRIB are being performed to treat different diseases, including ALS. Additionally, another group has investigated the benefits of ISRIB on reducing the immune cells involved in type 1 diabetes. We are interested in following up on potential positive effects of ISRIB, or newer versions of the drug, on islets from T2D donors.
What do you see as the next steps in this research?
Our next steps include what I mentioned above in relation to inhibiting the integrated stress response (with ISRIB or other drugs) and seeing if they positively affect T2D donor islets. We would also like to determine if our findings in liver are applicable to human disease, such as metabolic dysfunction-associated steatotic liver disease.
What interested you in mitochondria in the first place?
We grow up learning about how mitochondria are the “powerhouse of the cell,” but what is really fascinating is that mitochondria do a whole lot of other things in the cell besides just making energy. In this work, we’ve shown that they can actually signal to the nucleus and change the way the DNA is packaged to make certain genes turn off and on! Because metabolism is so important to a cell, it’s really interesting to figure out how exactly the mitochondria direct changes in ways far beyond energy production.
References
1Flannick J, Mercader JM, Fuchsberger C, et al. (2019) Exome sequencing of 20,791 cases of type 2 diabetes and 24,440 controls. Nature 570: 71–76.
2Lowell BB, Shulman GI (2005) Mitochondrial dysfunction and type 2 diabetes. Science 307: 384–387.
3Walker EM, Pearson GL, Lawlor N, et al. (2025) Retrograde mitochondrial signaling governs the identity and maturity of metabolic tissues. Science. DOI: 10.1126/science.adf2034
Lee RG, Rudler DL, Raven SA, et al. (2024) Quantitative subcellular reconstruction reveals a lipid mediated inter-organelle biogenesis network. Nature Cell Biology 26: 57–71.
www.nature.com/articles/s41556-023-01297-4
To survive, cells rely on collections of organelles, such as mitochondria, endoplasmic reticulum, peroxisomes, Golgi apparatus, and more. Each has specific critical functions. However, they don’t work in isolation. They interact by communicating signals and exchanging materials in an orderly manner. Amazingly, little is known about how they interact.
The laboratory of Aleksandra Filipovska used an innovative strategy to explore the interrelationships of several key organelles with mitochondria. They first established cell lines that lacked genes specific for the production of peroxisomes, Golgi, and ER with mitochondria. Then they combined observations by scanning electron microscopy with multi-omics profiling to examine the RNAs, proteins, lipids, and glycogens that were affected by the perturbations. In this way, they could carefully examine how dysfunction in one organelle affected the mitochondria.
Because mitochondria influence so many cell activities, it wasn’t surprising that the researchers found effects in many systems. However, they found that lipid transfers were the most affected component. This very complete paper documents the many metabolic and morphological interactions within cells and provides insights into how they might be involved in diseases.
Questions for Dr. Filipovska
Congratulations on a tour-de-force paper! Your strategy here seems to be an interesting variation on the systems biology: eliminate one component and see what happens. Has anyone else tried this strategy?
Thank you! This specific strategy of genome-wide screening coupled with FIB-SEM imaging and quantification has not been tried previously, to the best of our knowledge. One of the reasons we decided to pursue this strategy was to get an unbiased account of inter-organelle interactions and changes as opposed to traditional targeted analyses.
Your calculations on the number of genes that might be involved in inter-organelle dysfunctions and diseases large enough to be concerning. How do you read that?
We were quite surprised to see so many gene changes, and in addition to the genes reported in Lee et al., we have also analyzed many more that are involved in inter-organelle communication and metabolite exchanges. These numbers are starting to make a lot of sense to us now, given how many of these genes are involved in metabolic and biogenesis pathways that are shared across different organelles. For example, the glycerophospholipid pathway spans the endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes. Systems biology methods are key to understanding changes across different organelles, providing a larger picture of metabolic processes and common dysfunctions that manifest in different diseases.
You gave a small hint about future experiments to look at tissues under different metabolic demands near the end of the Discussion. Could you expand on that a little?
We are particularly interested in understanding the tissue-specific defects in multi-systemic diseases, such as mitochondrial diseases where a common molecular defect can manifest in different pathologies depending on the affected tissue in a particular disease. This will enable us to design targeted therapeutics for specific organs. Our study also revealed that different molecular defects can cause mitochondrial dysfunction and that targeted treatments of mitochondrial dysfunction may help alleviate symptoms of other diseases that involve mitochondria.
How did you become interested in mitochondria in the first place?
As an undergraduate student, I became fascinated by the bacterial origin of mitochondria and how they retained a level of autonomy within eukaryotic cells, maintaining a very small genome that is essential for life. At the time, very little was known about the regulation of mitochondrial gene expression, despite the many diseases for which there were no cures or treatments that were caused by defects in the mitochondrial genome. I was inspired to understand how the small mitochondrial genome was regulated that could help identify much needed therapies for mitochondrial diseases that are devastating for the patients, often very young infants and children and their families. I started my career trying to modify the mitochondrial genome and find gene therapies, which of course at the time did not work, and the main reason for this was that there was so little known about the mitochondrial genome, transcriptome and proteome. My goal was to focus on understanding the regulation of the mitochondrial genome in my group to help us devise specific therapies for mitochondrial diseases.
Reference
Lee RG, Rudler DL, Raven SA, Peng L, Chopin A, Moh ES, McCubbin T, Siira SJ, Fagan SV, DeBono NJ, Stentenbach M, Brown J, Rackham FF, Li J, Simpson KJ, Marcellin E, Packer NH, Reid GE, Padman BS, Rackham O, Filipovska A (2024) Quantitative subcellular reconstruction reveals a lipid mediated inter-organelle biogenesis network. Nature Cell Biology 26: 57–71.