Mitochondria and Medicine: The 2017 Dr. Paul Janssen Award Symposium

2017 Dr. Paul Janssen Award for Biomedical Research Lecture — The Mitochondrian: Our Origins, Our Diseases

Highlights

• Mitochondria power the metabolism of eukaryotic cells, and mitochondrial dysfunction has been implicated as a cause or contributor to numerous diseases.
• The cell's nuclear DNA encodes most mitochondrial proteins, but mitochondria also have independent DNA (mtDNA) that encodes subunits of the electron transport chain and RNAs.
• Mitochondrial DNA (mtDNA) is maternally inherited; mtDNA mutations can cause genetic diseases.
• The high mutation rate of mtDNA and its maternal inheritance pattern makes it ideal for tracing ancient human migrations.

Power for Life

In the 20th century, medical research produced a steady stream of major breakthroughs. Antimicrobial compounds and modern vaccines vanquished many of humanity's deadliest diseases, progress in epidemiology and public health revealed effective methods to prevent many more, and innovations in medicinal chemistry yielded a pharmacopoeia of new treatments. From 1900 to 2000, the average life expectancy increased by about 30 years in developed countries.

Recently, though, progress has slowed to a crawl. As people live longer and adopt new, often sedentary lifestyles, they develop chronic health problems that researchers have found hard to explain, let alone treat. "It's astonishing how much effort we've put into trying to understand the common complex diseases, and regrettably how little progress we've made to actually helping people's lives, curing diseases," said 2017 Janssen Award recipient Douglas Wallace.

Wallace listed some of the toughest challenges in medicine today: metabolic disorders, cancer, aging, heart failure, Alzheimer's disease, attention-deficit disorder, and autism. Each remains cloaked in mystery even as the diagnosed populations grow. The crux of the problem, Wallace argued, is that medical researchers have clung to outdated paradigms based on anatomy and Mendelian genetics, which insufficiently explain these chronic conditions that often involve multiple organ systems and a complex interplay of genes and the environment. According to Wallace, energy is the missing piece of the puzzle: "We have to think about life as not only anatomy but energy ... maybe [one] could have a systemic energy defect that would then preferentially affect those organs that have high energy demands."

In eukaryotes, energy in the form of ATP is produced by mitochondria, the descendants of ancient bacteria that became symbiotic residents inside larger cells. The human cell nuclear DNA codes for over 20,000 genes, 2,000 protein genes of which make up the mitochondrial structure. However, a separate DNA carried inside mitochondria (mtDNA), encodes for 13 critical proteins for the enzyme complexes that form the electron transport chain and ATP synthase, which are critical to the organelles' energy production role. These protein complexes effectively burn hydrogen in food with inhaled oxygen to generate an electrical potential, which then powers our metabolism and life. "The mitochondrion is pivotal to almost every physiological process that occurs in your body," said Wallace. When the mitochondrial energy flow stops, we die.

Given its importance, one might expect the DNA to be highly stable, but the opposite is true. This allows mitochondria to be genetically diverse and adaptable, but may lead to the accumulation of deleterious mutations. Dr. Wallace described findings that mtDNA, which is maternally inherited, is subject to strong selection to weed out such deleterious mutations in the female germline. Oogenesis produces a population of oocytes with diverse mtDNAs, but only metabolically functional cells are ovulated with the potential to be fertilized. Each oocyte has a unique set of mitochondrial gene variants, but all of the resulting proteins have been selected to work properly together in that cell. During fertilization, the egg selectively destroys sperm mitochondria to prevent them from contributing incompatible components. Interestingly, Dr. Wallace also showed that divergent mtDNA haplogroups are incompatible by creating mice with mtDNAs from two different strains. These mice had reduced physical activity and severe cognitive dysfunction, potentially explaining the advantage of uniparental inheritance. Over generations, mtDNA from one strain was lost while the other was selectively enriched.

Mitochondria carry their own DNA, but rely on nuclear encoded genes for most of their structures.

Mitochondria exhibit heteroplasmy—each cell has thousands of copies of mtDNA, and they can differ in their sequence within cells. The same mutation may cause disease in one individual and not another, depending on the proportion of mtDNA in each individual that carries the mutation. In addition, different organs have differing susceptibility to mitochondrial defects—for example, the brain, heart, and muscles, require more energy and therefore slight mitochondrial defects will result in a stronger phenotype in these tissues than others. To complicate things even further, mitochondria accumulate mutations as cells replicate and age so different organs and tissues often have genetically distinct populations of mitochondria, which in turn can cause confusing phenotypes.

Wallace described patients he and his colleagues saw in the 1980s with an odd assortment of metabolic and neurological problems. Though they varied in their presentations, the researchers found that the patients all shared a common mutation in their mtDNA. Individuals with 20%–30% of their mitochondria carrying the mutation developed diabetes and autism; those with 70% developed cardiomyopathy; and those with 100% died in infancy from severe psychomotor regression and respiratory failure.

A mouse model harboring a mtDNA mutation that causes Leigh's syndrome revealed that the mutation caused a drastic increase in oxidative stress in the  energy-hungry nerve cells. "In this case, the disease is primarily about oxygen radical toxicity, not about energy deficiency," said Wallace.

Wallace has also used mtDNA to identify past human migration patterns and uncover adaptations that helped humans colonize cold climates, high altitudes, and resist infections. Some of those adaptations may help explain different groups' predispositions to chronic diseases.

Highlights

• Defects in mitochondrial protein targeting underlie many diseases.
• The structure of mitochondria changes in response to the cell's environment.
• Mitochondrial DNA may serve as a stress sensor, triggering a cell's protective responses when it becomes damaged or lost.
• Recent discoveries about mitochondrial metabolism are already producing promising new disease treatments.

Mitochondrial Protein Transport and Disease: Another Block in the Wall

While mtDNA encodes the core energy-generating machinery, the rest of the organelle's components come from nuclear genes. These proteins must be imported through or into the mitochondrial membrane. To study this crucial process, Carla Koehler and her colleagues are developing small molecules that can modify specific components of the mitochondrial import system.

Having descended from bacteria, mitochondria have two membrane layers—the inner and outer mitochondrial membrane, with an intermembrane space (IMS) in between these membranes. Several protein complexes are involved in transporting proteins through these membranes. "All of these [transport] proteins are essential for viability, so this tells you how important protein translocation is," said Koehler. Humans carrying mutations in these genes develop a range of serious diseases.

The TOM transport complex on the outer mitochondrial membrane has a very small opening, forcing proteins to move through it unfolded before folding into their proper shapes inside the mitochondrion. Once proteins reach the intermembrane space, the Erv1 enzyme of the MIA pathway catalyzes oxidization of new disulfide bonds in cysteine-containing proteins, facilitating protein folding and formation of oligomeric structures. Using an assay that detects the oxidation of a sulfur-containing compound and thereby measures Erv1 activity, Koehler and her team screened a library of thousands of chemicals and identified a handful that appeared to inhibit Erv1. Subsequent experiments in yeast led them to focus on a compound they named MitoBlock-6, which appeared to inhibit Erv1 very specifically.

Mitochondria must import proteins through two separate membranes.

Dr. Koehler’s group then looked for the role of this pathway in mammals. MitoBlock-6 had no effect on differentiated cells, it caused apoptosis (a form of programmed cell death) in embryonic stem cells. "So there's something about the pluripotent state that will require [Erv1]," said Koehler. Exploiting these findings could provide a boost to the field of stem cell therapy, where stem cells are differentiated into a cell type of choice and transplanted into patients. A major concern with this therapy is that stem cells that fail to differentiate could develop into teratomas, tumors involving multiple tissue types, in patients. Drugs that target Erv1 might be able to eliminate stem cells that have not differentiated before being transplanted, preventing teratomas.

Mistargeted proteins are a common problem in many other diseases, such as Parkinson's disease. The protein PINK1 typically functions in mitochondrial quality control: it accumulates on the outer mitochondrial membrane of dysfunctional mitochondria and recruits Parkin proteins, which then target mitochondria for degradation through mitophagy. Some Parkinson’s Disease patients have mutations in genes encoding PINK1 or Parkin which prevent PINK1 recruitment of Parkin, thereby inhibiting mitophagy and leading to cell death of dopamine-producing neurons—which is believed to contribute to or cause the disease. Interestingly, in another of Koehler's discoveries, a compound called MitoBlock-10 inhibits Parkin recruitment, while a low dose of MitoBlock-12 stimulates it, without damaging the mitochondria. The ability to separate Parkin recruitment from mitochondrial degradation now allows her lab to study the underlying mechanisms of the disease in more detail.

Mitochondrial Stresses Signalling in Disease, Aging, and Immunity: Well-warned Genes

For simplicity, textbooks and researchers' slides often illustrate mitochondria as distinct oblong capsules, but their structure within cells is much more fluid and complex. "Mitochondria are not usually the shape of little submarines in the cytoplasm, they're actually a network that's constantly fusing and dividing, you can kind of think of it like a lava lamp," said Gerald Shadel.

These dynamic structures connect to some of the most important signaling pathways in the cell. Using chemical signals that indicate the cell's energy level, mitochondria can prompt major changes in nuclear gene expression and regulate—or dysregulate—the cell's metabolism. In response to a variety of stressors, mitochondria can also activate innate immune responses and inflammation.

Shadel and his colleagues study how mitochondria package and regulate their own genes. Mitochondrial transcription factor A (TFAM) is a very abundant protein that fully coats and packages circular mtDNA into nucleoids within the organelle. Mice lacking both copies of the gene for TFAM die embryonically. Mice lacking only one copy (Tfam+/− ) have half the normal amount of mtDNA in their cells, but are viable. "So why do we have so much mtDNA if you don't actually need it to be alive?" asked Shadel.

While the mutant animals are superficially healthy, more detailed analysis reveals that they carry hidden problems, including increased oxidative mtDNA damage and mis-packaging of mtDNA. Crossing heterozygous TFAM-deficient (Tfam+/− ) mice with a mouse strain called APC^Min/+, an animal model for intestinal cancer, yields offspring with increased tumor number and growth in the small intestine, which appears to be due to increased mitochondrial reactive oxygen species production. The mitochondria in the cells of these mice have a more fused, elongated structure than those in APC^Min/+ parental mice with both wild-type copies of TFAM. These experiments highlight one way that mitochondrial dysfunction can contribute to cancer.

In further experiments, gene expression profiling revealed that interferon-stimulated genes, normally activated as part of the innate immune response, are among the top targets induced in cells from Tfam+/−  mice. mtDNA shares some molecular traits with viral DNA, but because it's compartmentalized inside the organelles it doesn't normally trigger the cell's innate immune response. Through a series of rigorous cell fractionation studies, Shadel's team found that the mtDNA stress caused by TFAM depletion in these cells promotes accumulation of aberrant mtDNA, which is released into the cytoplasm at double or triple the normal amount, activating the antiviral response.

Further work, however, showed that the TFAM depletion response only induces a subset of the antiviral response, representing the genes that normally help the cell resist DNA damage. Many tumors stimulate the same pathway, which enables them to resist chemotherapy. Indeed, the researchers found that TFAM-mutant cells resist DNA damaging agents.

Mitochondria can activate a cell's innate immune response.

"We think what we're actually doing by creating mtDNA stress is activating this subset of interferon-stimulated genes that's signaling to the nucleus to be prepared to protect your genome," said Shadel.

Returning to his original question,  When mtDNA becomes damaged or goes missing, mitochondria signal that loss to the nucleus to induce genes that help protect against DNA damage. The same process might occur in conditions associated with mitochondrial instability, such as lupus, autoimmunity, and aging.

"We need to understand those pathways and the tissue specificity of mitochondrial function ... if we're going to ever have any chance of using mitochondria as a therapeutic target for diseases," said Shadel.

Understanding Genetics and Treatment Strategies in Mitochondrial Disease: Born This Way

Emphasizing that point, Michio Hirano presented his group's work on rare mitochondrial diseases in humans, in which discoveries about the inner workings of mitochondria are already yielding promising new clinical strategies. Hirano is part of the North American Mitochondrial Disease Consortium (NAMDC), one of 22 rare disease consortia funded by the National Institutes of Health. The NAMDC now has a registry of over a thousand patients with mitochondrial diseases.

A wide range of mutations can cause mitochondrial dysfunction. About two-thirds of the patients in the NAMDC registry have mutations in mitochondrial genes, but "we have mutations in more than 50 different nuclear genes, and many of these are represented by only one patient, so there are many, very rare nuclear [mutations] that cause mitochondrial diseases," said Hirano. Complicating matters further, around one in 200 people carries mtDNA mutations that can cause disease, but only a small subset of those individuals become symptomatic.

One major determinant of disease severity is mitochondrial heteroplasmy—a disease-causing mutation can be asymptomatic if present only in a low proportion of the total mtDNA in cells. "This could be the Achilles' heel of mitochondrial mutations," said Hirano, explaining that gene therapies that repair even a small proportion of a patient's mitochondria could be life-saving. Mothers who are asymptomatic can be carriers of mitochondrial disease if the disease-causing mutation is present in a low percentage of their mtDNA; their children would develop the disease if they inherit the mutation at a higher percentage.

Hirano and his colleagues aim to treat mitochondrial disease, and have focused on thymidine kinase 2 (TK2) deficiency. TK2 localizes to the mitochondria and functions in the production and maintenance of mtDNA: it is required to generate deoxypyrimidine monophosphates, dTMP and dCMP (nucleotides). Patients with mutations in the gene that encodes TK2 have mtDNA depletion leading to impaired mitochondrial function and disease, which manifests as progressive muscle weakness (myopathy) typically beginning in early childhood. The mean age of death of TK2 patients is 2.6 years. Using a mouse model of the condition lacking TK2 (TK2−/−), Hirano's team found that simply supplementing mutant animals with oral dTMP and dCMP prolongs their lifespan two- to three-fold. Another group found that giving more stable nucleoside precursors to dTMP and dCMP also increases lifespan, while requiring fewer doses.

Mitochondrial defects can affect every major organ system.

Taking this finding straight to the clinic, Hirano got an emergency approval from the US FDA to start deoxypyrimidine monophosphate therapy on a patient with TK2-deficiency and severe mtDNA depletion. "He's been on that therapy now for almost five years ... he's not cured but he's significantly better," said Hirano. A total of 20 patients worldwide are now receiving either deoxypyrimidine monophosphates or nucleosides as therapy for TK2-deficiency, and all are still alive with no obvious side effects. Other mitochondrial diseases, such as ribonucleotide reductase deficiency, might be amenable to similar therapy.

A more dramatic intervention would be to prevent inheritance of mitochondrial mutations through mitochondrial replacement therapy: by transferring the chromosomal DNA either 1) from an unfertilized egg that has mutant mtDNA to an unfertilized donor egg that has had its nucleus removed (maternal spindle transfer), or 2) from a fertilized egg with mutant mitochondrial DNA to a healthy donor egg that has had its pronucleus removed. There is one report in the literature of a successful live birth from such a procedure in Mexico, and in December 2016 the UK approved use of the procedure. It remains illegal in the US, but a report from the Institute of Medicine at the US National Academies of Sciences, Engineering, and Medicine, a nonprofit that advises the government on science policy, concluded it would be ethical and safe as long as it was restricted to male embryos who would not pass the donor mitochondria to their own offspring.

Highlights

• Over-nutrition disrupts normal mitochondrial turnover in the liver.
• Drugs that impact mitochondrial quality control might help treat metabolic liver disease.
• Gene therapy that corrects defects in only a subset of cells could be life-changing for patients.

Liver Free or Diet

"Cardiometabolic disease is all around us, I don't think there's anyone in this room who hasn't been impacted by it in some way," said Lisa Norquay. She and her colleagues are particularly interested in non-alcoholic steatohepatitis (NASH), an increasingly prevalent metabolic disorder with no approved therapy.

When the body detects excess nutrients in the bloodstream, it stores the extra calories as fat. That response was highly adaptive for most of human evolutionary history, when food availability varied drastically from day to day. However, chronic over-nutrition, far more common in modern humans, pushes hepatocytes in the liver towards fat accumulation. Over time, excess fat in the liver can lead to the condition of NASH as hepatocytes balloon and the tissue becomes inflamed and fibrotic. Under normal conditions, a cycle of mitochondrial fusion and fission allows the exchange of proteins between mitochondria and regulates mitochondrial quality within a cell. Using fluorescent probes to track the behavior of mitochondria in cultured cells, Norquay and her colleagues found that a prolonged excess of nutrients disrupts this "quality control" cycle. "There's actually an impairment of the [mitochondrial] dynamics under excess nutrients," said Norquay.

The obesity pandemic is increasing the prevalence of liver disease.

In addition to decreasing the mitochondrial fission-fusion cycle, over-nourishment renders cells’ lysosomes more alkaline, reducing mitophagy, or mitochondrial breakdown. Normally, the process of mitophagy entails the digestion of mitochondria in acidic lysosomes. In overfed cells, aged or damaged mitochondria are not digested in lysosomes, but instead accumulate within the cell. As a result of these changes, the cells develop a fragmented, dysfunctional mitochondrial pool with lower energy-producing capacity. This reduced energy-capacity is also observed in animal models of over-nourishment, leading Dr. Norquay to look for ways to correct these defects to develop therapies that may help patients.

To that end, the investigators obtained light-activated nanoparticles that selectively lower the pH of lysosomes. Activating these nanoparticles in overfed cells enabled them to resume the process of mitophagy. Acidifying lysosomes also boosted energy production in the cells. "It's restoring the protein degradation, that cleanup process that you need, that mitophagy, and you can see that with this tool," said Norquay.

Light-activated nanoparticles aren't a practical drug for treating liver disease, but the experiments did reveal that the underlying process of NASH pathogenesis can be reversed, and highlighted mitophagy as a critical component of it. Norquay's team next turned to identifying portions of the mitochondrial life cycle that they could target therapeutically.

Other researchers have found that overexpressing the cardiolipin-modifying enzyme ALCAT1 can cause mitochondrial fragmentation in muscle and liver cells. Cardiolipin is an important component of the inner mitochondrial membrane, and ALCAT1 modifies cardiolipin into forms that are more sensitive to oxidative damage. Based on their earlier work, Norquay and her colleagues found ALCAT1 intriguing.

The investigators found that ALCAT1 expression increases in the liver when mice are fed a diet containing high fat and high sucrose. They also observed that knocking down ALCAT1 expression reduces mitochondrial fragmentation in hepatocytes, but doesn't seem to damage the cells. It is still too early to understand the potential of ALCAT1 as a drug test, but Dr. Norquay and her team continue to evaluate the pathway of mitochondrial quality control in the search to identify novel therapeutic targets for metabolic disease.

Emerging Gene Therapies for Inherited Disorders: A Little Goes a Long Way

Shifting the discussion back to genetic conditions, Beverly Davidson discussed her work on Huntington's and other genetic diseases. About 45,000 individuals in the US are at risk of developing Huntington's disease, which causes a progressive loss of neural tissue, accompanying disability, and death. Current therapies are purely palliative. The disease is caused by an expansion of a repeat sequence in the huntingtin gene, Mutant alleles are autosomal dominant, so inheriting one mutant copy, with the other wild type, causes Huntington’s disease.

Several groups have identified promising gene therapy strategies in mouse models of Huntington's disease, generally by deleting or editing the mutant copy of huntingtin using various gene editing technologies. Davidson's team uses the bacterially-derived CRISPR/Cas9 system to remove specifically the defective copy of the gene, targeting single-nucleotide polymorphisms found only in the mutated versions of huntingtin.

Huntington's disease causes a progressive loss of brain tissue.

After getting promising results in various cultured cell lines, the team created a viral gene therapy vector expressing the required Cas9 enzyme and guide RNA sequences, and injected it into the brains of mice carrying a mutant version of human huntingtin. The treatment decreased mutant Huntingtin protein expression by half. "We know [from earlier work] if we can achieve reduction in about 50% of the cells of the basal ganglia, we have a profound impact on the behavior of these animals and actual recovery of some of the neuropathogenic phenotypes," said Davidson. She and her colleagues are now developing both viral and non-viral vectors to deliver a similar therapy to patients.

The meeting concluded with a short panel discussion in which the speakers took questions from the audience. Attendees talked about the possibility of noncoding RNA inhibition in mitochondria, the prospects for using gene editing to cure mitochondrial diseases in embryos, and the effects of environmental factors and the microbiome on mitochondrial biology. Though many aspects of mitochondria remain mysterious, the group seemed to agree that these crucial organelles are finally receiving the attention they deserve.