Mitochondrial DNA ,structure,function,and related diseases.

The presentation describes mitochondrial DNA,structure,mutations , and relation to diseases

1. Mitochondrial DNA Ekbal Mohamed Abohashem (MD) Professor of Clinical Pathology Mansoura University -Egypt بسم الله الرحمن الرحيم

2. Mitochondria are small, mobile, and plastic organelles located in the cytoplasm of most eukaryotic cells. These organelles are responsible for important cellular processes, such as regulation of apoptosis, calcium homeostasis, and reactive oxygen species (ROS) production. However, the main function of mitochondria is energy production through oxidative phosphorylation (OXPHOS), which takes place in the mitochondrial respiratory chain (MRC). Introduction: Mitochondria, the Powerhouses of the Cell

3. Mitochondria contain their own DNA, the mitochondrial DNA ( mtDNA ), which is circular and double stranded. The mitochondrial genome consists of 16,569 nucleotide pairs that encode 13 proteins, two ribosomal RNA components, and 22 transfer RNAs (tRNAs) .

4. Regarding mitochondrial structure, these organelles are composed by two membranes, the inner and the outer mitochondrial membranes (IMMs and OMMs, respectively) that delimit two main compartments: the internal matrix and the intermembrane space. The IMM contains many folds named cristae that protrude into the matrix and enlarge the IMM surface. This membrane can be subdivided into two compartments, the inner boundary membrane (IBM) and the cristae membrane (CM), that are connected via cristae junctions.

5. Mitochondrial structure

6. Although IMM is considered a continuous membrane, lateral diffusion of membrane proteins is restricted and IBM and CM exhibit an asymmetric protein distribution. This heterogeneity is important for efficient OXPHOS, mitochondrial biogenesis, and remodeling. Defects in mitochondrial function have been linked not only to genetic mitochondrial diseases but also to cardiovascular diseases ,and neurodegenerative disorders such as Huntington’s and Parkinson’s diseases .

7. History of mitochondrial DNA : Mitochondrial DNA was discovered in the 1960s by Margit M. K. Nass and Sylvan Nass by electron microscopy as DNase-sensitive threads inside mitochondria, and by Ellen Haslbrunner , Hans Tuppy and Gottfried Schatz by biochemical assays on highly purified mitochondrial fractions

8. Mitochondrial DNA ( mtDNA or mDNA ) is the DNA located in mitochondria , It represents only a small portion of the DNA in an eukaryotic cell; most of the DNA is found in the cell nucleus . Human mitochondrial DNA was the first significant part of the human genome to be sequenced. This sequencing revealed that the human mtDNA includes 16,569 base pairs and encodes 13 proteins . Since animal mtDNA evolves faster than nuclear genetic markers, it represents a mainstay of phylogenetics and evolutionary biology . It also permits an examination of the relatedness of populations, and so has become important in anthropology and biogeography . Mitochondrial DNA:

9. The two strands of the human mitochondrial DNA are distinguished as the heavy strand and the light strand. The heavy strand is rich in guanine and encodes 12 subunits of the oxidative phosphorylation system, two ribosomal RNAs (12S and 16S), and 14 transfer RNAs ( tRNAs ). The light strand encodes one subunit, and 8 tRNAs . So, altogether mtDNA encodes for two rRNAs , 22 tRNAs , and 13 protein subunits , all of which are involved in the oxidative phosphorylation process . Genes on the human mtDNA and their transcription

10. The promoters for the initiation of the transcription of the heavy and light strands are located in the main non-coding region of the mtDNA called the displacement loop, the D-loop . There is evidence that the transcription of the mitochondrial rRNAs is regulated by the heavy-strand promoter 1 (HSP1), and the transcription of the polycistronic transcripts coding for the protein subunits are regulated by HSP2 . Regulation of transcription

11. Map of human mtDNA

12. Map of human mtDNA The genome encodes for 13 mRNA (green), 22 tRNA (violet), and 2 rRNA (pale blue) molecules. There is also a major noncoding region (NCR), which is shown enlarged at the top. The major NCR contains the heavy strand promoter (HSP), the light strand promoter (LSP), three conserved sequence boxes (CSB1-3, orange), the H-strand origin of replication (O H ), and the termination-associated sequence (TAS, yellow). The triple-stranded displacement-loop (D-loop) structure is formed by premature termination of nascent H-strand DNA synthesis at TAS. The short H-strand replication product formed in this manner is termed 7S DNA. A minor NCR, located approximately 11,000 bp downstream of O H , contains the L-strand origin of re

13. Mitochondrial inheritance In most multicellular organisms , mtDNA is inherited from the mother (maternally inherited). Mechanisms for this include * simple dilution (an egg contains on average 200,000 mtDNA molecules, whereas a healthy human sperm has been reported to contain on average 5 molecules), * degradation of sperm mtDNA in the male genital tract and in the fertilized egg; and, * at least in a few organisms, failure of sperm mtDNA to enter the egg. Whatever the mechanism, this single parent ( uniparental inheritance ) pattern of mtDNA inheritance is found in most animals, most plants and also in fungi.

14. Heteroplasmy Heteroplasmy is the presence of more than one type of organellar genome ( mitochondrial DNA ) within a cell or individual. It is an important factor in considering the severity of mitochondrial diseases . Because most eukaryotic cells contain many hundreds of mitochondria with hundreds of copies of mitochondrial DNA, it is common for mutations to affect only some mitochondria, leaving most unaffected.

15. Female inheritance In sexual reproduction , mitochondria are normally inherited exclusively from the mother; the mitochondria in mammalian sperm are usually destroyed by the egg cell after fertilization. Also, mitochondria are only in the sperm tail, which is used for propelling the sperm cells and sometimes the tail is lost during fertilization. In 1999 it was reported that paternal sperm mitochondria (containing mtDNA ) are marked with ubiquitin to select them for later destruction inside the embryo . Some in vitro fertilization techniques, particularly injecting a sperm into an oocyte , may interfere with this

16. Similar to the nuclear genome, the mitochondrial genome is built of double-stranded DNA, and it encodes genes . However, the mitochondrial genome differs from the nuclear genome in several ways : The mitochondrial genome is circular, whereas the nuclear genome is linear . The mitochondrial genome is built of 16,569 DNA base pairs, whereas the nuclear genome is made of 3.3 billion DNA base pairs. Mitochondrial vs. Nuclear DNA

17. The mitochondrial genome contains 37 genes that encode 13 proteins, 22 tRNAs, and 2 rRNAs. The 13 mitochondrial gene-encoded proteins all instruct cells to produce protein subunits of the enzyme complexes of the oxidative phosphorylation system, which enables mitochondria to act as the powerhouses of our cells.

18. The small mitochondrial genome is not able to independently produce all of the proteins needed for functionality; thus, mitochondria rely heavily on imported nuclear gene products. One mitochondrion contains dozens of copies of its mitochondrial genome. In addition, each cell contains numerous mitochondria. Therefore, a given cell can contain several thousand copies of its mitochondrial genome, bu t only one copy of its nuclear genome.

19. The mitochondrial genome is not enveloped, and is it not packaged into chromatin . The mitochondrial genome contains few, if any, noncoding DNA sequences. (Three percent of the mitochondrial genome is noncoding DNA, whereas 93% of the nuclear genome is noncoding DNA). Some mitochondrial coding sequences (triplet codons) do not follow the universal codon usage rules when they are translated into proteins.

20. Some mitochondrial nucleotide bases exhibit functional overlap between two genes; in other words, the same nucleotide can sometimes function as both the last base of one gene and the first base of the next gene. The mitochondrial mode of inheritance is strictly maternal, whereas nuclear genomes are inherited equally from both parents. Therefore, mitochondria-associated disease mutations are also always inherited maternally.

21. Mitochondrial genes on both DNA strands are transcribed in a polycistronic manner: Large mitochondrial mRNAs contain the instructions to build many different proteins, which are encoded one after the next along the mRNA. In contrast, nuclear genes are usually transcribed one at a time from their own mRNA

22. Each cell contains numerous mitochondria, and each mitochondrion contains dozens of copies of the mitochondrial genome. Moreover, the mitochondrial genome has a higher mutation rate (about 100-fold higher) than the nuclear genome. This leads to a heterogeneous population of mitochondrial DNA within the same cell, and even within the same mitochondrion; as a result, mitochondria are considered heteroplasmic . Mitochondrial DNA Mutations

23. When a cell divides, its mitochondria are partitioned between the two daughter cells. However, the process of mitochondrial segregation occurs in a random manner and is much less organized than the highly accurate process of nuclear chromosome segregation during mitosis. As a result, daughter cells receive similar, but not identical, copies of their mitochondrial DNA

24. Due to the multicopy nature of mtDNA , these mutations can be homoplasmic or heteroplasmic . Thus, MELAS and MERRF syndromes are heteroplasmic , which means that mutant and wild-type mtDNA copies coexist within the same cell . Mitochondrial disease may become clinically apparent once the number of affected mitochondria reaches a certain level; this phenomenon is called " threshold expression "

25. Mitochondrial diseases are a heterogeneous group of maternally inherited rare genetic disorders caused by a partial or total dysfunction of mitochondria. These illnesses can be caused by mutations in nDNA or mtDNA . These mutations affect not only genes encoding for mitochondrial respiratory chain( MRC) components but also those that are involved in protein translation and assembly, mtDNA stability, as well as mutations in those nDNA -encoded proteins involved in the maintenance of mitochondrial nucleotide pools, nucleotide transport, mtDNA replication, RNA transcription, and mitochondrial dynamics . Mitochondrial Diseases :

26. More than 400 pathogenic mutations in mitochondrial transfer RNA have been characterized . Some frequent mitochondrial disorders caused by a point mutation in mtDNA are MELAS and MERRF syndromes. In most of the cases, MELAS syndrome is caused by a transition from adenine to guanine in the position 3243 in the mt-tRNA Leu (UUR) (MT-TL1) gene .This affects mt-tRNA structure stabilization, methylation, aminoacylation , and triplet recognition . In the case of MERRF syndrome, the m.8344A > G mutation in the mt-tRNA Lys (MT-TK) gene is the most frequently associated with the disease .It affects both AAA and AAG codon translation, causing a defect of whole mitochondrial protein synthesis.

27. How common is mitochondrial disease? An estimated 1 in 5,000 people has a genetic mitochondrial disease. It’s common for mitochondrial diseases to receive a misdiagnosis due to the number and type of symptoms and organ systems involved, so this number may be underestimated. What are the types of mitochondrial disease? There are many types of mitochondrial diseases. Some of the most common include: Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome . Leber hereditary optic neuropathy (LHON). Leigh syndrome . Kearns-Sayre syndrome (KSS). Myoclonic epilepsy and ragged-red fiber disease (MERRF).

28. Mitochondrial diseases are clinically heterogeneous; they may occur at any age, and patients manifest a wide variety of symptoms . However, all of them share morphological and biochemical features. As a consequence of the MRC deficiency, cells manifest a reduced enzymatic function of MRC components, a reduction in oxygen consumption and ATP synthesis, and ROS overproduction.

29. Patients suffer from lactic acidosis and elevated serum pyruvate levels at rest and, specially, after moderate exercise. Additionally, patients’ muscle biopsies usually show ragged red fibers that reflect the proliferation of oxidative phosphorylation ( OXPHOS-defective mitochondria .)

30. Both MELAS and MERRF syndromes are associated with neurological symptoms. MELAS syndrome affects several organs, and some of its manifestations include stroke-like episodes, dementia, epilepsy, lactic acidemia , myopathy, recurrent headaches, hearing impairment, diabetes, and short stature . In the case of MERRF syndrome, the first symptom is usually myoclonus that is followed by generalized epilepsy, ataxia, weakness, and dementia. Other findings are hearing loss, short stature, optic atrophy, and cardiomyopathy .

31. Role of mitochondrial DNA in diseases

32. ). Mitochondrial dysfunctions contribute to diabetes, obesity, and metabolic syndromes ). Mitochondria play diverse roles in cancer, such as providing energy and biosynthetic products for rapid proliferation, supporting metabolic adaptation to the tumor microenvironment, and regulating oncogenic signaling and apoptosis ). Mitochondria support cellular immune functions. However, dysfunctions in mitochondria or trauma could release immunogenic mtDNA and mtRNA in the cytosol or circulation to cause severe or chronic inflammation

33. ). Mitochondrial genetics, metabolism, and inflammation significantly impact age-associated pathologies ) and neurodegenerative diseases such as Alzheimer's and Parkinson's diseases

34. The development of useful therapies for mitochondrial diseases is challenging due to : *the difficulty of correcting the lack or dysfunction of essential mitochondrial proteins, * the phenotypical heterogeneity of the diseases, and *multisystem alteration. Furthermore, the brain, one of the most affected organs, is difficult to reach by potential therapies because it is protected by the blood–brain barrier. For those reasons, there are no effective treatments available for mitochondrial diseases and management of these diseases is mainly symptomatic. Therapeutic Management of Mitochondrial Diseases:

35. Pharmacological treatment options are generally focused on targeting cellular pathways, such as mitochondrial biogenesis or autophagy, or preventing oxidative damage. For these reasons, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1) signaling have been the main targets of these strategies. Particularly in mtDNA mutations, several supplements as antioxidants and cofactors are being used.

36. Given the diversity of mutations and the different therapeutic options, a personalized therapeutic approach is required in mitochondrial diseases. For this reason, the development of cellular models derived from patients can be useful for both the evaluation of new drugs and the repositioning of existing ones. Gene therapy is a promising alternative for treating mitochondrial diseases. Since pathogenic mtDNA mutations are usually heteroplasmic , reducing mutational load can be used as a therapeutic approach. There are several tools that could target mtDNA , but only two of them have been demonstrated to be successful: zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Both tools are delivered into mitochondria using a mitochondria localization signal, and they selectively target mtDNA sequences to create double-strand breaks.

37. TALENs are artificial restriction enzymes and can cut DNA strands at any desired sequence,which makes them ideal for genetic engineering and targeted genome editing . They have been widely used for genetic manipulation in different organisms. This tool has been demonstrated to reduce mutant mtDNA load and improve the pathophysiology in a cellular model of MERRF syndrome as well as to eliminate the m.3243A > G mutation in MELAS inexperimental models and porcine oocytes.

38. Zinc-finger nucleases have been demonstrated to reduce mutant mtDNA and consequently restore mitochondrial respiratory function in cytoplasmic hybrid (cybrid) cell models . In addition, TALENs have been able to reduce mutant mtDNA load in a mouse model harboring a mutation in a mt-tRNA, reverting disease-related phenotypes .

39. The only option available is transferring embryos below the threshold of clinical expression in order to avoid or at least reduce the risk of transmission of mtDNA mutations. The selection of these embryos is based on preimplantation genetic diagnosis (PGD) . In addition, there is a new strategy, the mitochondrial donation, that consists of the substitution of mutant maternal mitochondria using enucleated donor oocytes .However, this technique has raised ethical issues and remains controversial). Prevention of mitochondrial diseases,

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