Biochemical Pharmacology Discussion Group
The New York Academy of Sciences
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Posted May 03, 2018
Biochemical Pharmacology Discussion Group
The New York Academy of Sciences
Found within almost every cell and in almost every organism, mitochondria are crucially responsible for the production of energy from food and oxygen. Within the intricate inner structure of each mitochondrion, electrons from the byproducts of food are transferred through a series of protein complexes ultimately yielding molecules of ATP, the main energy currency in a cell. Yet, the functions of mitochondria extend far beyond the production of energy to important roles in cellular signaling pathways and in the synthesis of metabolites. On November 2, 2017 the Biochemical Pharmacology Discussion Group at the New York Academy of Sciences presented Mitochondria in Health and Disease, a daylong conference exploring the many facets of mitochondrial function in physiology and medicine, across diverse disciplines including: neurology, aging, oncology, autophagy, membrane morphogenesis, structural biology, and bioenergetics.
The most prominent theory of aging involves the accumulation of reactive oxygen species in the cell, leading to mutations in mitochondrial DNA and a shortened lifespan. However, some mitochondrial mutations, Andy Dillin of University of California, Berkeley observed, can extend lifespan. To determine this, Dillin and his team first conducted a genome-wide screen of the worm Caenorhabditis elegans, in which they inactivated every single gene in turn and asked whether this manipulation increased the lifespan of the worm. They identified 100 genes from this experiment, all of which were strikingly involved in coding for electron transport chain proteins within mitochondria. They further specified inactivation of the genes in different tissues (i.e. skin, muscle, neural) and found that mutation of the genes particularly in neurons extended the lifespan of the worms. Damage to these genes, he observed, led to the activation of the mitochondrial unfolded protein response (UPR), which signals mitochondrial damage to the nucleus leading to the production of proteins to correct the misfolded proteins. The UPR serves as the first line of defense against mitochondrial stress before mitophagy, the removal of the damaged mitochondria, or autophagy, complete cell death. Blocking the UPR pathway, they found, blocked longevity. They reasoned that the worms were living longer because they were mounting defense responses.
Dillin and his colleagues then sought to understand how an activated UPR in neurons could confer protection to other cells in the organism. They found that under stress, neurons release a ligand, Wnt, which signals damage at long distances thus activating the UPR in far reaching cells.
Beyond ATP production, mitochondria have a variety of other functions; they are critically involved in a number of signaling pathways and in the synthesis of cellular metabolites. With aging, the structure and function of mitochondria changes. These changes, Adam Hughes of University of Utah School of Medicine said, are linked to changes in the lysosomes (or vacuoles in yeast), organelles responsible for the degradation of molecules. Many mechanisms exist to maintain mitochondrial health and thus preserve these functions over an organism’s lifetime. Hughes and his team discovered of one such mechanism, the formation of a structure which they named the Mitochondrial-Derived Compartment (MDC).
The MDC, they observed, is a 200–500nm membranous-bound structure that is contiguous with the mitochondria but can eventually detach and travel to the vacuole for degradation. Hughes and his group screened 500 proteins to determine which ones get incorporated into an MDC. They found a class of transporters that is responsible for all metabolite exchange across the inner membrane. Amino acids can move into the mitochondria and induce stress. Upon removal of amino acids from the cell media, the development of MDCs was blocked while adding in amino acids restored the formation of MDCs. Hughes and his group are now asking what the sensing mechanism for amino acid levels is and how the membrane is remodeled to create an MDC.
In normal cell metabolism, cells take in nutrients and excrete waste. Marcia Haigis of Harvard Medical School described how these metabolic processes are reprogrammed in tumor cells, which utilize larger amounts of nutrients than healthy cells. As a consequence, tumor cells produce an excess of metabolic waste. Haigis and her group were interested in investigating what tumor cells do to process a particular waste product, ammonia. There were two possibilities: the ammonia could either be excreted or it could be assimilated back into the cell. To distinguish between these possibilities, they first examined the levels of ammonia assimilation enzymes within mitochondria in normal and tumor cells. They found that two of these enzymes, Gs and Gdh, were elevated in breast tumor cells, indicating that assimilation was the more likely option. Then, to determine where the ammonia was re-incorporated, the team tracked the fate of heavy nitrogen molecules within cells and calculated the weights they should observe in metabolites if that nitrogen had been incorporated. With this method, they discovered that the ammonia was efficiently recycled back into molecules of glutamate. Tumor cells so efficiently utilized excess ammonia to create glutamate, Haigis said, that proliferation of the tumor is accelerated. Her team is now exploring the mechanism by which ammonia can lead to this proliferation.
Mitochondria are dynamic; they are in constant flux, dividing, changing location, and morphing into different shapes. In her presentation, Jodi Nunnari of University of California, Davis discussed the active mechanisms that determine mitochondrial arrangement both during division and in its location within the cell. Mitochondria replicate independently of the cell cycle depending on the energy demands of a cell. The machinery that divides mitochondria is localized to the outer membrane. In examining this region, Nunnari and her collaborators discovered that the endoplasmic reticulum is a major determinant of where a future mitochondrial division will occur. A complex, the ER-Mitochondria Encounter Structure (ERMES), forms between the endoplasmic reticulum (ER) and mitochondria through transmembrane proteins and a linker molecule in the middle. The formation of ERMES leads to the recruitment of other proteins to this site and results in a fission event. When they disrupted the ER, the group observed that mitochondrial DNA replication was reduced. Now, Nunnari is trying to understand how the events of division are orchestrated around ERMES contact sites.
Harvard Medical School
Damaged mitochondria are marked and degraded in a process called mitophagy. J Wade Harper of Harvard Medical School described how the ubiquitin system is involved in this turnover of mitochondria. When mitochondria are damaged, PINK1 kinase accumulates in the mitochondrial outer membrane and leads to the recruitment of parkin, a ubiquitin ligase. Parkin then mediates the formation of a ubiquitin coat on the damaged mitochondria which recruits autophagasomes around the mitochondria that degrade the organelle. Harper and his team were interested in understanding the mechanisms that drive this ubiquitylation process: the kinetics, the sites on proteins that are modified, and the abundance of the recruited proteins. They found that in HeLa cells and in neurons, depolarizing the cells results in the accumulation of PINK1 and phospho-ubiquitin accumulation in the cell. A variety of proteins receive ubiquitin from parkin with over 2 log difference in the abundance of these parkin targeting substrates in damaged mitochondria. To recruit the autophagasome, a series of adapter proteins recognize and bind to the ubiquitin coat. Harper and his colleagues examined one such adaptor protein, optineurin (OPTN), and found that it localized to the outer membrane of mitochondria, forming a bridge to the autophagosome membrane. Using proteomics, they also identified a host of the other adaptor proteins that colocalize with OPTN within the autophagosome recruiting complex.
There are over 200 monogenic or single gene mitochondrial diseases that can lead to a wide variety of dysfunction in humans, from blindess to diabetes. Yet, with the exception of a few cases, “we have no proven therapies,” said Vamsi Mootha of Harvard Medical School. Mootha and his group set out to identify novel therapeutic strategies by using a genome-wide CRISPR strategy. More specifically, they used CRISPR to remove individual genes in the genome that would allow cells to proliferate even when a mitochondrial poison is added to their environment. They identified only one gene from the screen that had this effect, the von Hippel Lindau (VHL) gene. VHL encodes a ubiquitin ligase which plays a role in the body's oxygen sensing pathway to activate a program that is beneficial when oxygen is low.
Mootha and his team then applied this idea whether hypoxia itself may be beneficial as a new treatment for mitochondrial disease. They focused on Leigh syndrome, the most common pediatric instance of mitochondrial disease, which results in bilateral neurodegeneration of the brainstem and basal ganglia. Mouse models created to mimic the symptoms of Leigh syndrome were placed in either a hypoxic environment (11% oxygen) or normal oxygen conditions (21% oxygen). The team were shocked (and delighted) to observe that the hypoxic mice displayed longer lifespan as well as more normal levels of temperature, body weight, and activity. “The in vivo effects of hypoxia are far better than we could have ever dreamt,” Mootha said. Now, he and his team are trying to elucidate the full in vivo mechanism by which hypoxia is alleviating disease in this mouse model, and given the heterogeneity of mitochondrial disease, to determine whether hypoxia extends to any other mouse models. While encouraging, it’s still too early to contemplate studies in human patients, he said.
Jahar Bhattacharya of Columbia University presented work from his lab examining the role of mitochondria in acute inflammatory lung disease. This disease is characterized by the rapid filling of the lungs with fluid and is associated with high mortality in humans. Bhattacharya explained that epithelial cells in the lung depend on mitochondria to produce a surfactant, which is crucial for normal lung function. Secretion of the surfactant reduces the surface tension in the air-filled alveoli, preventing the lungs from collapsing. Bhattacharya and colleagues found that exposing the alveoli tissue to bone derived mesenchymal cells harboring normal mitochondria can rescue mitochondrial function. Mouse models of acute lung injury were protected from a lethal dose of bacterial lipopolysaccharide (LPS) when treated with this mitochondrial transfer.
The group then monitored the function of mitochondria in normal lungs, to understand what mitochondria are doing during LPS-induced acute lung injury. They observed an increase in both cytosolic and mitochondrial calcium in the alveolar epithelium when the lungs were inflated. Lung injury, or the injection of LPS, blocked this mitochondrial calcium influx. Thus they concluded that there was a correlation in the ability of mitochondria to pick up calcium and recovery from inflammation. At higher magnification, they observed that LPS causes mitochondrial fragmentation within the cell rather than being localized to surfactant releasing vesicles. This led the group to think about Drp1, a protein that plays a role in mitochondrial distribution. When they deleted Drp1 in LPS cells, calcium influx in mitochondria was restored.
Navdeep S. Chandel of Northwestern University discussed the role of mitochondria as signaling platforms within the cell, receiving input from the environment and orchestrating the appropriate pathways in response. One example of mitochondrial signaling involves the release of hydrogen peroxide (H2O2) in the cytoplasm to activate gene expression during cellular differentiation. Most stem cells are in a low metabolic state. When induced to develop in a certain direction, however, mitochondria proliferate and release H2O2 which, along with other signaling molecules, helps to differentiate the cell. To test this model in vivo, the group first looked at keratinocyte differentiation. They deleted the gene Tfam, which is necessary for mitochondrial replication and maintenance, and found that knockout mice were hairless. In addition, differentiation markers in the skin were less differentiated than in wild type mice. When they added H2O2, keratinocyte differentiation was rescued indicating that H2O2 is an important differentiation stimulus.
In another system, Chandel and his colleagues knocked out RISP protein, a subunit of mitochondrial complex III, specifically in hematopoetic stem cells. They observed that a deficiency in complex III caused anemia and an inability to create progenitor cells. An RNA sequencing analysis on these cells showed that genes were dysregulated.
The disease sarcopenia is characterized by the loss of skeletal muscle with aging. David J. Glass of Novartis discussed the role that mitochondrial signaling has in this process. In a rat model of sarcopenia, Glass and his team observed that the mass of the gastrocnemius muscle in the animals decreased with age. In addition, the force that could be evoked by the muscle decreased. When they examined the genetic changes that co-occurred with the onset of sarcopenia, they noticed that all of the genes were mitochondrial. This finding focused their attention to mitochondrial loss and what could lead to muscle mass loss. Over the last 20 years, the group has uncovered a complex pathway underlying signaling that underlies muscle mass degeneration including a decrease in IGF-1; a decrease in AKT signaling; and a correlated decrease in ATP citric lyase, all signaling proteins that are involved with the growth and survival of muscle cells. Increasing protein synthesis, mitochondrial activity and mitochondrial repair on the other hand, can increase skeletal muscle mass.