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

In spite of prodigious efforts to identify nuclear DNA (nDNA) genetic variants associated with common diseases, the genetic and physiological bases of these disorders remains unclear. One reason for this dilemma may be that most studies have overlooked the mitochondrial genetic system. The mitochondrion contains its own mitochondrial DNA (mtDNA) and independent biogenesis system. The mtDNA codes for the 13 most important genes for mitochondrial oxidative phosphorylation (OXPHOS), the primary cellular energy generative system, plus the tRNAs and rRNAs for mtDNA gene expression. Different organs have different mitochondrial energy dependency, in decreasing order of the brain, heart, muscle, renal, and endocrine systems. Hence, subtle changes in mitochondrial function preferentially affect the brain and then other organ systems. Mitochondrial structure and function also require one to two thousand nDNA genes making for complex genetic interactions. The mitochondrion regulates a host of cellular functions besides energy production including redox control and reactive oxygen species (ROS) production, Ca⁺⁺ regulation, and apoptosis. The mitochondrion also controls cellular and nuclear function by generating the substrates for the cellular signal transduction pathways and the epigenome. This is required because no cellular function can proceed without energy.

The mtDNA in maternally inherited and has a very high mutation rate, generating great inter-individual sequence diversity. This is achieved without high genetic load by the severe restriction of the number of mtDNAs introduced into each female primordial germ cell followed by selection for the most energetically robust follicles at ovulation which harbor the best mtDNAs.

Analysis of mtDNA variation of aboriginal populations has revealed that they harbor groups of related mtDNA haplotypes, designated haplogroups. Haplogroups are founded by functional mutations that modified energy metabolism to adapt to local environmental demands. Hence, the mtDNA is our adaptive engine. The mtDNA mutational tree originated in Africa ~200,000 years before present (YBP). After radiating in Africa for ~150,000 years to generate multiple African-specific haplogroups, encompassed by macrohaplogroup L; only two mtDNAs successfully left Africa about 65,000 YBP to found macrohaplogroups M and N and colonize all of Eurasia and the Americas. While beneficial in one environment, foundation haplogroup variants may be maladaptive in another environment. Hence, haplogroups have been found to predispose to a wide range of metabolic and degenerative diseases such as autism, Alzheimer and Parkinson Disease, cardiovascular and inflammatory disease.

The maternal mtDNA is present in thousands of copies per cell and its high mutation rate inevitably yields deleterious mutations.  When a new mutation arises within the female germline or a cell it creates a mixture of mutant and normal mtDNAs, known as heteroplasmy. As the proportion of mutant mtDNA heteroplasmy increases by replicative segregation, bioenergetic function declines until it falls below the minimum energy for that organ causing disease. Hundreds of pathogenic, maternally-inherited, homoplasmic or heteroplasmic, mtDNA mutations have been identified. The first maternal mutation identified causes Leber Hereditary Optic Neuropathy (LHON), whose penitence is modulated by the background mtDNA haplogroup.  Other mtDNA mutations have been associated with complex array of metabolic and degenerative diseases and various cancers.

mtDNA mutations also accumulate in cells and tissues during development and with age. These somatic mutations progressively erode energy production providing the aging clock and explaining the delayed onset and progressive course of many metabolic and degenerative diseases and cancer.

Proof that mitochondrial defects are sufficient to cause the common diseases comes from the generation of mice which harbor mutations in nDNA and mtDNA coded mitochondrial genes.  Creation of mice harboring the human pathogenic mtDNA ND6 P25L mutation resulted in neurodegenerative diseases. Mice harboring the mtDNA COI V421A mutation manifest cardiomyopathy, myopathy, and metabolic disease. Simply mixing two normal but different mouse mtDNAs resulted in reduced activity, hyper-excitability, and a severe learning defect.

Mutations in nDNA-coded mitochondrial genes also cause disease. Mutations in the human brain-heart-muscle isoform of the adenine nucleotide translocator (ANT1) result in autosomal recessive cardiomyopathy and myopathy, the severity of cardiomyopathy being determined by the background mtDNA haplogroup.  Ant1-deficient mice also develop myopathy and cardiomyopathy, and the severity of their cardiomyopathy is markedly modulated by different mtDNA backgrounds.

Mitochondrial genetic variation also effects the mouse response to mild environmental stressors. Furthermore, mitochondrial variation can differentially affect the cortical developmental migration of GABAergic inhibitory interneurons and glutamatergic excitatory pyramidal neurons. This can result in excitation-inhibition imbalance and autism associated phenotypes.

These observations suggest that the mitochondria are the environmental sensors. They respond to environmental change and signal the nucleus to modify bioenergetic gene expression to maintain homeostasis. However, sever environmental changes or mitochondrial gene mutations can exceed the capacity of mitochondrial-nuclear interaction to maintain homeostasis resulting in disease and ultimately death.

Mitochondrial dysfunction is a contributing factor in degenerative diseases. Modulation of the mitochondrial protein import pathways can have regulatory effects on mitochondrial function. Studying these pathways by conventional methods such as RNAi in mammalian cells can be difficult because it takes several days to knock-down proteins coupled with an overall loss of mitochondrial function. Therefore, we have developed several approaches to develop small molecule modulators for mitochondrial protein translocation. To date, we have conducted screens to identify modulators for the TOM-TIM23, TIM22, and MIA protein import pathways. Our efforts now focus on identifying the specific target of the small molecules in mitochondria using structural and genetic approaches and then using the modulators in model systems to understand how defects in mitochondrial protein translocation impact the rest of the cell.  In some cases, we find that specific stress pathways, including mitophagy and apoptosis pathways, are activated. In addition, we also show that these probes are a valuable platform for therapeutic strategies, because the small molecules can modulate mitochondrial stress pathways. Therefore, our small molecule screening strategy has been useful in generating a toolbox of small molecule modulators for mitochondrial translocation that can be used in a variety of experimental systems and for regulating mitochondrial stress pathways.

Mitochondria are multi-faceted organelles in eukaryotic cells that function at the nexus of energy metabolism, oxidative stress, and apoptosis. Consequently, circumstances (genetics, environmental factors, aging) that result in mitochondrial dysfunction disrupt a multitude of cellular processes that can cause human disease pathology. Changes in redox balance due to altered mitochondria reactive oxygen species production, decline or rewiring of cellular energy metabolism, and cell death are some of the major downstream cellular consequences leading to mitochondrial-based pathology, ranging from heart, skeletal muscle and nerve dysfunction to metabolic diseases, blindness, and deafness. I will discuss our latest interrogations of mitochondria-to-nucleus stress signaling pathways and some novel connection to disease, aging and immunity.

Mitochondria are unique mammalian organelles by virtue of having their own DNA, mitochondrial DNA (mtDNA), a 16,569 base-pair circular molecule that encodes only 37 genes that are essential for mitochondrial oxidative phosphorylation. Each mitochondrion contains 2–10 copies of mtDNA and in turn, each cell contains numerous mitochondria; therefore, there are hundreds to thousands of copies of mtDNA in each cell. Alterations of mtDNA may be present in some of the mtDNA molecules (heteroplasmy) or in all of the molecules (homoplasmy). As a consequence of heteroplasmy, the proportion of a deleterious mtDNA mutation can vary widely among individuals and the clinical manifestations may be diverse. Another unique feature of mtDNA is maternal inheritance. Recent developments in oocyte nuclear genome transfer as well as pronuclear transfer between zygotes offer new options to prevent transmission of mtDNA-disease.

Because maintenance of mtDNA is entirely dependent upon genes encoded by the nuclear DNA (nDNA), it is not surprising that primary nuclear gene defects can cause secondary mtDNA instability. Among these disorders is deficiency of thymidine kinase 2 (TK2), a mitochondrial protein required for synthesis of pyrimidine deoxynucleoside triphosphate building blocks for mtDNA replication. Autosomal recessive mutations of TK2 cause predominantly myopathy. In our Tk2 H126N knockin mouse model, substrate enhancement therapy with dT+dC nucleosides extends the lifespan of the mutant mice by 2–3 fold. Based on our Tk2 knockin mouse studies, 18 TK2-deficient patients worldwide have been treated with nucleosides on a compassionate use basis; all have shown clinical stabilization or improvements. This therapy may be applicable to other mtDNA maintenance disorders.

Mitochondria have evolved to efficiently produce energy from available nutrients, but this proficiency is impeded when substrate availability persistently exceeds energetic demand. In cardiometabolic diseases, such as diabetes, diabetic nephropathy, and NASH, disease progression is coupled to reduced mitochondrial oxidative capacity. The Cardiovascular and Metabolism Therapeutic Area at Janssen is actively pursuing novel drug targets that aim to restore aberrant mitochondrial function in our patients. We have recently partnered with the mitochondrial and metabolism core at the David Geffen School of Medicine in UCLA. Utilizing a diverse range of cellular assays we seek to understand the impact of our targets on fundamental regulatory processes such as mitochondrial dynamics, mitoautophagy, bioenergetic capacity, and fuel metabolism, and ultimately, determine their potential to prevent, intercept, and treat cardiometabolic disease.