Mitochondria in Health and Disease

The average adult human may contain trillions of mitochondria, all of which may derive from a few maternally inherited mitochondria present in a primordial germ cell. As cells differentiate and become tissues, these mitochondria become structured into extensive networks, dynamically interchanging components with each other in a fluidity of motion that argues against a residual consideration of the mitochondrion as an individual entity. Mitochondrial networks also become the metabolic centers of the cell, coordinating the flow of metabolites and energy supplies in tight communication with both the endoplasmic reticulum and nucleus. Recent work has ascribed a conserved role for mitochondria in the regulation of stress resistance, lifespan, and metabolism across the organism. For the first time, such work is beginning to suggest a role for the mitochondria communicating across the organism outside, as opposed to just inside of, the cell. We examine the course that the mitochondrion has taken since its origin, what happens to the mitochondrion as the cell differentiates and ages, and the evidence for an evolved mitochondrial role in the homeostatic regulation of the distal function of tissues in a multi-cellular organism.

Mitochondrial dysfunction is a hallmark of aging, and underlies the development of many age-associated and metabolic disorders. Cells maintain mitochondrial health through a number of quality control systems that can detect dysfunctional mitochondria and repair or eliminate problematic organelles. We have now discovered a new mitochondrial quality control system conserved from yeast to humans, the Mitochondrial-Derived Compartment (MDC) pathway, which eliminates proteins from dysfunctional mitochondria by autophagy. Unlike many common mitochondrial autophagy pathways, this system selectively sorts and removes a subset of membrane proteins from the mitochondrial inner and outer membranes, while leaving the remainder of the organelle intact. Selective removal of preexisting proteins is achieved by membrane remodeling and sorting of proteins into a newly discovered mitochondrial-derived compartment, or MDC, followed by release through mitochondrial fission and elimination by autophagy. We have screened the mitochondrial proteome to catalog the substrates of this pathway, and found that the primary target of this system is a large class of inner membrane nutrient transporters called the SLC25A nutrient carrier protein family, which promote all nutrient exchange across the mitochondrial inner membrane. Based on this unique substrate selectivity, combined with our results that MDC formation is triggered by high levels of cytoplasmic amino acids, we propose that the MDC pathway functions to control levels of mitochondrial nutrient transporters to protect mitochondrial from metabolic stress. We are now working to understanding how cells form MDCs and selectively sort proteins into them, and how nutrient signals are relayed to the MDC activation machinery.

Mitochondria are double membrane-bounded organelles that perform a myriad of diverse and essential functions in cells. These functions are dependent on the collective intracellular behavior of the organelle. We have characterized key features of mitochondrial behaviors. Our work has addressed the physiological functions and mechanisms of mitochondrial division and fusion, which are important determinants of overall mitochondrial shape and distribution. We have characterized contact sites that intimately link mitochondria with the ER and determined their roles in mitochondrial positioning and dynamics. We have also addressed the fundamental question of how mitochondrial membranes are sub-compartmentalized to reveal how the complex internal architecture of the organelle is generated. Our current challenge is to determine how mitochondrial behaviors are integrated with one another and physiologically regulated within cells and organisms.

Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor activated through phosphorylation during inflammation and in cancer. STAT3 can also accumulate in mitochondria, increasing electron transport chain activity, ATP production and redox balance. STAT3 contributes to many human tumors, making it an attractive cancer therapeutic target. While a number or STAT3 inhibitors (STAT3i) are under development, little is known about their mechanisms of action (MOA) or factors determining differential sensitivity among cancer types. We are studying non-peptidic small molecule STAT3i and we recently showed that these compounds bind directly to STAT3 SH2-domain, and affect cell growth of multiple cancer cell lines in a STAT3- dependent and independent manner. Moreover, drug action required active mitochondria, since Rho0 cells were protected. Interestingly, low glucose and high oxygen augmented the cytostatic effects of these STAT3i and some metabolic perturbants modulated with STAT3i action, either increasing or decreasing their cytostatic effect. Growth inhibition involved rearrangement of the actin cytoskeleton and a release from anchorage attachment followed by cell death. STAT3i toxicity correlated with the overall ability of cancer cells to maintain a NADH/NAD+ ratio favoring NADH. We are currently investigating the effects of these STAT3i across a broad series of cancer subtypes and in combination with metabolic perturbants. These data will further define the MOA, optimal cancer types for therapeutic targeting, and potential combinatorial treatments.

Oxidative stress is believed to be critical in the development of neurodegenerative diseases, like Alzheimer’s disease (AD). Mitochondrial dysfunction, which can lead to the accumulation of reactive oxygen species (ROS), is one of the earliest features in the brain of AD patients. However, there is little mechanistic insight into the generation and contribution of mitochondrial ROS (mROS) in AD. ECSIT (Evolutionarily Conserved Signaling Intermediate in Toll pathways) regulates mitochondrial respiration and mROS generation, and interacts with redox and mitochondrial proteins involved in AD pathogenesis, such as PRDX2, ApoE and PSEN1/2. We hypothesized that ECSIT was a key factor in neurodegeneration through the regulation of mROS.

We observed that ECSIT was highly expressed in the Central Nervous System (CNS) in hippocampus and amygdala, both affected in AD, and localized in mitochondria in primary neurons. Using conditional ECSIT deletion in mature CNS neurons in mice, we investigated the function of ECSIT by characterizing mitochondrial homeostasis, oxidative stress and neurodegeneration. ECSIT deletion led to loss of mitochondrial Complex I, accumulation of damaged mitochondria and mROS. Moreover, mice developed abnormal cognitive behavior, synaptic dysfunction and neuropathology upon aging. Scavenging mROS by overexpressing catalase in mitochondria, rescued early cognitive defect and synaptic plasticity. Thus, ECSIT can suppress neurodegenerative oxidative stress in the CNS by regulating mitochondrial function and mROS. Our data suggests that dysregulation of ECSIT in the CNS could be a key contributor to early mitochondrial dysfunction in AD and cooperate with genetic susceptibilities to drive the disease.

Glaucoma is a leading cause of blindness worldwide and aging is a major risk factor. In glaucoma, retinal ganglion cells (RCGs), the neurons whose axons form the optic nerve, gradually become dysfunctional and die, by a mechanism that is poorly understood. Mitochondrial dysfunction is thought to play a key role. OPA1 codes for a mitochondrial dynamin-like GTPase essential for mitochondrial fusion, respiration, mtDNA stability, and RGC survival. OPA1 variants cause optic atrophy type 1, a blinding disease characterized by early-onset optic nerve degeneration. While OPA1 mutations cause rare early-onset forms of vision loss, it remains unclear whether OPA1 becomes sporadically inactivated in common lateonset glaucoma. Remarkably, mass spectrometry indicates that OPA1 is post-translationally modified by lysine acetylation in a cluster of OPA1 variants linked to vision loss. We show that Sirtuin 3 (SIRT3) activates OPA1 by Lys deacetylation. The deacetyl-mimetic OPA1 mutant exhibits increased GTPase activity and restores mitochondrial function in SIRT3-KO cells, while the acetyl-mimetic OPA1 mutant is inactive, similar to pathogenic OPA1 variants. SIRT3-KO causes retinal OPA1 Lys hyper-acetylation. SIRT3 reduction in aged DBA2/J (D2) mice with glaucoma is also linked with OPA1 inactivation by Lys hyper-acetylation. Our data provide evidence of SIRT3-mediated OPA1 activation by Lys deacetylation and erosion of this neuroprotective pathway in aging and glaucoma.

Targeting of damaged mitochondria for mitophagy involves ubiquitylation of mitochondrial outer membrane (MOM) proteins by the PARKIN ubiquitin ligase upon activation by PINK1. PARKIN recruitment to the MOM and UB chain assembly relies on phosphorylation of both PARKIN on S65 and UB chains on S65, which binds and further activates PARKIN UB chain synthesis in a feed-forward mechanism. Assembly of UB chains on the MOM then promotes recruitment of ubiquitin-binding autophagy adaptors such as OPTN in a process that is amplified by the associated TBK1 kinase. We are developing a quantitative framework for understanding the links between ubiquitin chain assembly, primary substrate ubiquitylation, and selective autophagy. We have developed a quantitative proteomics system for monitoring the kinetics and stoichiometry of primarily ubiquitylation on numerous MOM proteins simultaneously with measurement of UB chain synthesis and UB S65 phosphorylation. Mitochondrial substrates for PARKIN differ in total abundance by 2 orders of magnitude, which has major implications for underlying mechanisms of mitophagy, and a small number of substrates carry the majority of the ubiquitin molecules in response to depolarization. The majority of studies in this area have focused on the use of a HeLa cell model to study mitophagy. In order to understand this pathway in neurons, we have initiated an analysis in gene-edited ES cells that are subsequently converted to cortical or dopaminergic neurons. We find that some aspects of the pathway in neurons are distinct from that in the standard HeLa model, thereby providing a new paradigm for studying PARKIN-dependent mitophagy.

Mitochondria are ancient organelles that serve as cellular hubs for energy metabolism, signaling, and growth. Mitochondria contain their own genome (mtDNA) that encodes 13 proteins, while all remaining 1000+ proteins are encoded in the nuclear genome and imported. There is growing interest in mitochondrial biology since virtually all age associated diseases are associated with a decline in mitochondrial function. Our laboratory focuses on monogenic mitochondrial disorders – a large collection of inborn errors of metabolism, which can present in infancy or in adulthood, impacting virtually any organ system. Although next-generation sequencing has revolutionized the genetic diagnosis of these disorders, we still know precious little about pathogenesis, and we do not have a single proven therapy. Here I will share our latest efforts that combine human physiology, genetic screens, and functional genomic profiling to better understand mitochondrial pathogenesis. Our work is providing fundamentally new insights into mitochondrial homeostasis with potential implications for the therapy of these disorders.

Although mitochondria are known to be major sources of cellular ATP, their role in organ injury and repair remains inadequately understood. For example, Acute Lung Injury (ALI) is a severe inflammatory disease of the lung that causes high mortality and morbidity. But the significance of mitochondria in the pathogenesis of ALI remains unclear. Since ALI is characterized by severe damage to the alveolar epithelium at the gas-exchange interface, major loss of alveolar bioenergetics might underlie the damage and also inhibit epithelial repair. Our interest in this question arose since exogenous cell therapy with bone marrow-derived mesenchymal stromal cells (MSCs) protects against ALI. Optical imaging of live alveoli in a mouse model of ALI, indicated that the major protective mechanism was attributable to mitochondrial transfer from MSCs to the alveolar epithelium. The mitochondrial transfer took place within hours of MSC instillation through the airway and it was accompanied by increases in alveolar ATP levels and by recovery of surfactant secretion, a critical measure of homeostatic alveolar function. However, it is not clear how mitochondria of MSCs arriving in the inflammatory milieu remain sufficiently undamaged as to improve alveolar bioenergetics. I will discuss some of these issues in the background of studies conducted by my group involving confocal microscopy of the live lung. This approach has provided unique opportunity to directly visualize the live alveolar epithelium to enable real-time studies of mitochondrial biology during tissue inflammation.

For decades, the mitochondria have been primarily viewed as biosynthetic and bioenergetic organelles generating metabolites for the production of macromolecules and ATP, respectively. Our work has elucidated that mitochondria have a third distinct role whereby they release reactive oxygen species (ROS) and metabolites such as L-2-hydroxyglutarate to initiate physiological and pathological processes including hypoxic activation of HIF, stem cell differentiation, T cell activation and cancer cell proliferation. I will discuss our recent findings on how mitochondria as signaling organelles control regulatory T cell function.