Lecture Summaries

1 Introduction of instructor and students and course overview Let's get to know each other and discuss what is expected for this course. We will discuss some general strategies about how to read a research article. We will also discuss how to conduct literature searches on PubMed. Lastly, the instructor will introduce the basics of mitochondrial function, mitochondrial DNA, and reactive oxygen species.
2 The vulnerability of mtDNA and consequences of oxidative damage Mitochondria are the cell's primary source of reactive oxygen species (ROS). These ROS are potent damaging molecules that can react with DNA to chemically alter the bases and create DNA damage that can cause problems for the cell. One of the most well-studied and abundant DNA lesions formed by ROS is 8-oxoguanine (8-oxoG). The detection and quantitation of the amount of 8-oxoG has become an important way to asses oxidative damage. Because mitochondrial DNA (mtDNA) is located near source of ROS, the amount of oxidative damage that it incurs is extensive and can be indicated by the amount of 8-oxoG. Lesions such as 8-oxoG can cause mutations, because the mitochondrial DNA polymerase Pol γ will incorrectly insert an "A" across 8-oxoG instead of a "C." Therefore, oxidative damage can have mutagenic consequences for the mitochondrial DNA.
3 Repair of 8-oxoG from the mitochondrial genome: The importance of OGG1 and base excision repair pathways In the last class we learned about how ROS leads to the formation of 8-oxoguanine in mtDNA and how that has mutagenic consequences. How do cells cope with this damage to protect genomic integrity? In this class, we will learn about how cells use a repair pathway called Base Excision Repair to excise damaged bases and repair DNA. An important enzyme that deals with 8-oxoguanine specifically is called OGG1 (Oxoguanine glycosylase). OGG1 is classified as a bifunctional "DNA glycosylase," which means it can both break the glycosidic bond of the 8-oxoG lesion and cause a strand break in the DNA so that the lesion can be removed. These two papers use a combination of genetics and biochemistry to determine the importance of OGG1 in repairing mitochondrial DNA that has suffered oxidative damage.
4 The mitochondrial theory of aging and the importance of OGG1 Oxidative damage accumulates with age. One debated theory about the molecular mechanism of aging is called "The mitochondrial theory of aging." This theory postulates that oxidative damage accumulates in mtDNA over time and can lead to mitochondrial dysfunction through mutation of mtDNA, which subsequently causes more ROS production and therefore more oxidative damage ("the vicious cycle"). However, there is some disagreement about how deleterious 8-oxoG is to the mtDNA of cells, thus raising questions about how important OGG1 and other types of oxidative-lesion repair systems actually are. We will look at two papers that investigate what happens to cells devoid of OGG1. One investigation uses a mouse model, while the other uses human fibroblast cells. Please note the disparity in the conclusions of these two papers.
5 Mouse models for mtDNA mutation and aging In week 2 you read about the mutagenicity of 8-oxoG when it is a template of the mtDNA replicative polymerase Pol γ during DNA synthesis in vitro. Like many DNA polymerases, Pol γ can make an occasional mistake by incorporating the wrong nucleotide across undamaged template or by incorporating an adenine residue across 8-oxoG instead of a guanine. Like most replicative polymerases, Pol γ has a 3'-5' exonuclease function, sometimes referred to as the "proofreading" function, because if Pol γ makes a mistake during DNA synthesis, the exonuclease can remove the incorrect nucleotide and incorporate a new one. In this class we will discuss two investigations that have deliberately perturbed the proofreading function of Pol γ to create mice that incur a large mutational load to their mtDNA. This allows the authors to explore the consequences of enhanced mtDNA mutagenesis to aging. We will also be introduced to how mitochondria are involved in apoptosis. Apoptosis, also known as "programmed cell death," is a way for organisms to eliminate unhealthy cells in a controlled manner. While beneficial in some cases, apoptosis can be a problem when it affects terminally differentiated cells such as neurons. Collectively, these two papers investigate how accumulation of mtDNA mutations impacts the aging process in mice.
6 Oxidative damage and base excision repair in Alzheimer's disease Alzheimer's disease has been linked to mitochondrial dysfunction. We know that ROS-induced base damage can be properly countered by DNA repair mechanisms. An interesting realm of research is how the process of ROS-induced mtDNA damage and the function of base excision repair are involved in neurodegenerative diseases such as Alzheimer's. In this class, we will discuss two articles that examine the extent of oxidative damage in nuclear and mtDNA and the amount of base excision repair in the brains of Alzheimer's patients.
7 mtDNA deletions in neurons and the importance of oxidative phosphorylation A few weeks ago, we discussed mtDNA point mutations (base substitutions) and the theories on how they may or may not contribute to aging. Deletions of mtDNA segments are another type of mutation. This week we will discuss a landmark paper that uses an innovative approach to provide conclusive evidence that deletions in mtDNA accumulate in a region of the brain as part of the aging process. The technique used allowed the investigators to recognize that one region of the brain suffered mtDNA deletions while another did not. These findings have some important revelations for aging and Parkinson's disease. We will also discuss an article that establishes the importance of oxidative phosphorylation for protecting cells against ROS accumulation. This article establishes that the act of respiration, known to be a critical process for ATP production, reduces ROS and therefore protects cells against ROS-induced DNA damage.
8 Quality control of mtDNA: The bottleneck and mitochondrial fusion Eukaryotic cells have hundreds to thousands of mitochondria, each housing several copies of mtDNA. We have discussed numerous articles that demonstrate that mutations in mtDNA accumulate and that base excision repair is one system designed to preserve the integrity of DNA by excising damaged bases. We will discuss two other quality control mechanisms employed by cells to preserve mtDNA integrity. The first one, mitochondrial "fusion," occurs when two mitochondria combine and exchange mtDNA segments, which can eliminate mtDNA molecules that are badly mutated. We will also discuss how mutant mtDNA molecules are eliminated from the female germline, thus preventing their spread to the progeny. In this process, the number of mtDNA molecules passed into the germline is dramatically reduced and the deleterious mtDNA molecules eliminated.
9 From yeast to humans: Conservation of Pol γ One allelic variant of Pol γ, Y955C, has been found in patients with diseases such as progressive opthalmoplegia (PEO) and Parkinson's disease. The first article is an investigation of the structural and biochemical abnormalities caused by Y955C. This article demonstrates how a genetic mutation in a nuclear-encoded mitochondrial DNA metabolism gene can have consequences for mtDNA. Secondly, we will discuss an article that uses the yeast Saccharomyces cerevisiae to engineer and then study mutant forms of Pol γ analogous to those found in numerous human diseases. Using S. cerevisiae to make predictions of the in vivo consequences of different mutant forms of Pol γ found in human diseases is possible because of the similarities, or "conservation," of Pol γ in both species. Therefore, yeast can be used to model how allelic variants of Pol γ affect mtDNA copy number and mtDNA mutagenesis.
10 Determination of nuclear encoded genes that contribute to mitochondrial disorders Mitochondrial DNA depletion syndromes (MDDS) are characterized by diminution of mtDNA copy number over time, which can lead to organ failure of the affected tissue. Mitochondrial energy-generation disorders, on the other hand, include several diseases are often caused by a defect in Complex I of the respiratory chain. Both types of disorders can be caused by mutations in one of several nuclear-encoded mitochondrial genes. We will discuss two investigations that identify and characterize the defective gene in patients with an MDDS and mitochondrial energy-generation disorder. Dr. Vamsi Mootha, whose lab we will visit next week, contributed to both of these investigations.
11 Field trip to the Laboratory of Vamsi Mootha at Massachusetts General Hospital

Dr. Vamsi Mootha is an Associate Professor of Systems Biology and of Medicine at Harvard Medical School. His lab is based at both the Center for Human Genetic Research at Massachusetts General Hospital and at the Broad Institute. He investigates numerous aspects of mitochondrial function and how they affect human diseases. The Mootha lab combines biochemistry, genomics, and computational techniques to investigate mitochondria. We will meet with Dr. Mootha and several of his lab members. We will learn about some of the projects that are being carried out in the lab and learn about the methods they use to conduct their research.

12 mtDNA in cancer and chemotherapeutic resistance

mtDNA mutations and ROS accumulation have been found in numerous cancers. Mutations in mtDNA of cancer cells can promote tumoroigenesis and also contribute to resistance to chemotherapies. We will discuss two articles that explore these two phenomena. The first article is a clinical study that investigates how leukemia patients' mtDNA mutates after chemotherapy. You will notice that the number of mutations and the number of affected genes is extremely variable among patients. We will discuss the selective advantage that these mutations have for cancer cells. Mutant mtDNA molecules can also confer resistance to anticancer drugs, which is explored in the second paper. The second paper employs the "cybrid" technique and a mouse model to show that transferring mtDNA from a chemoresistant cancer cell to a non-cancer cell renders it resistant to apoptosis that would be induced by several chemotherapies.

*Note about the first paper- don't get bogged down with the list of mutations and what genes are affected. Notice its complexity, but do not attempt to memorize anything on the list.

13 Levels of ROS in the mitochondria of cancer cells Here are two examples of mtDNA mutations affecting cancer development. In one case, the offending mtDNA mutation contributes to resistance to apoptosis and correlates with an increase in ROS production. In the second article, the mtDNA mutation contributes to metastasis but does not lead to alterations in ROS production.
14 Oral presentations Students will make oral presentations about a research article provided by the instructor. There will also be a discussion of the overall course.