Section 1 Mitochondrial Diseases

#### **Chapter 1**

## Introductory Chapter: Mitochondrial Diseases - Advances and Perspectives - My Point of View

*Angel Catala*

#### **1. Introduction**

There is only one metabolic pathway that is under the dual control of the mitochondrial genome (mtDNA) and the nuclear genome (nDNA). Disorders in the mitochondrial respiratory chain are called by convention "mitochondrial diseases." Mitochondrial disorders symbolize a major challenge in medicine. Much of the mitochondrial proteins are encoded by nuclear DNA (nDNA), while only a few are encoded by mitochondrial DNA (mtDNA). Mutations in mtDNA or mitochondrialrelated nDNA genes can cause a mitochondrial disorder. The disorder can affect multiple organs in different locations and severity; but there are some ways that involve only one organ. Modifications of the mitochondrial oxidative phosphorylation system can generate mutations in both mitochondrial DNA and nuclear DNA that lead to mitochondrial diseases. Mitochondrial diseases comprise a diverse group of genetic disorders, which appear at any age and have a wide spectrum of clinical symptoms. This leads to highly changeable cases, making it difficult to diagnose mitochondrial diseases. The latest advances in genetic testing and original reproductive options hold great promise for improving the clinical recognition and treatment of mitochondrial diseases. In this chapter we discuss developments in the recognition and diagnosis of mitochondrial diseases. In the last five decades, the effect of mitochondrial diseases on biological systems began to be widely investigated. This chapter explains the most important aspects in our opinion of mitochondrial diseases.

#### **2. Brief history of mitochondria**

The generation of adenosine triphosphate by oxidative phosphorylation occurs in the mitochondria; about 90% of the cell's energy need is satisfied during the hydrolysis of ATP produced in this way. In addition, mitochondria are also involved in other processes including, but not limited to, the formation of iron and sulfur groups, the citric acid cycle, the regulation of apoptosis1, and calcium homeostasis in conjunction with the endoplasmic reticulum.

Mitochondria do not have nearly the amount of DNA necessary to encode all the specific proteins of mitochondria; however, millions of years of evolution could explain a progressive loss of autonomy. The endosymbiotic hypothesis could be called a theory, but no experimental reason can be offered to test it. Only indirect confirmation can be accessed in support of the proposal, which is the most likely justification for the mitochondria starting point. The verification necessary to change the model from hypothesis to theory is probably forever lacking in ancient times.

#### **3. Mitochondrial diseases**

Studies of genetic pathologies that affect mitochondrial metabolism as a consequence of modifications in genes encoded by mitochondrial DNA or genes encoded by nuclear DNA for dynamic proteins inside the mitochondria began in 1988. Since that year, a new notional "mitochondrial genetics" has become visible; based on three attributes of mtDNA: (1) polyplasmy; (2) maternal inheritance; and (3) mitotic segregation. Diagnosis of mtDNA-connected diseases was completed through genetic analysis and experimental advances that incorporated histochemical staining of muscle or brain sections, single-fiber polymerase chain reaction (PCR) of mtDNA, and the design of a "hybrid" Immortal (cytoplasmic hybrid) derivative from patient fibroblast cell lines.

#### **4. My participation in studies with mitochondria**

In the last three decades, our laboratory has investigated the lipid peroxidation of biological membranes of various tissues and different species, as well as liposomes prepared with phospholipids with a high content of polyunsaturated fatty acids. We analyzed the effect of various antioxidants such as alpha tocopherol, vitamin A, melatonin and its structural analogues and conjugated linoleic acid, among others [1, 2]. The integrity of the mitochondrial membranes and the function of numerous protein complexes in the ETC [3] are determined by cardiolipin, which is a unique class of specific mitochondrial phospholipids that exist almost exclusively in the inner membrane of mitochondria (IMM). In most mammalian tissues, tetralinoleoylcardiolipin (L4CL) is the main form of cardiolipins, this molecule contains four chains of structural linoleic acid. The incorporation of four LA side chains into L4CL and their presence in mitochondria allow L4CL to be easily oxidized by reactive oxygen species and then to generate an electrophilic reaction **Figure 1** [4].

#### **5. Conclusions**

Aging and age-related diseases have been connected with mitochondrial uncoupling and elevated ROS formation. Dysfunctional mitochondria incline to modified lipid metabolism and augmented lipid peroxidation products. Mitochondrial antioxidants that can re-establish function and prevent pathological lipid peroxidation are showing guarantee in diminishing biological aging and therefore they may offer advantage for slowing the development to age-related diseases such as neurodegeneration. In parallel, novel drug groups are providing a unusual strategy to delay aging during elimination of senescent cells. By mean of these drugs as instruments offer a chance to amplify our understanding of whether the alterations in reactive oxygen species, lipid metabolism and mitochondrial lipids detected during aging and diseases are due to the increase of senescent cells.

*Introductory Chapter: Mitochondrial Diseases - Advances and Perspectives - My Point of View DOI: http://dx.doi.org/10.5772/intechopen.98510*

#### **Figure 1.**

*Chemical mechanisms for 4-HNE formation from lipid peroxidation. (A) General scheme for the formation of 4-HNE from decomposition of lipid hydroperoxides that can be generated from free radical oxidation of ω-6 PUFA or enzymatic oxidation by lipoxygenases. (B) Lipid electrophiles generated from oxidation of mitochondrial cardiolipin: Oxidation of L4CL by the peroxidase activity of cyt c and CL complex in the presence of H2O2 results in the formation of hydroperoxyoctadecadienoic acid (HpODE), 9-HpODE-CL and 13-HpODE-CL. During this process, through intra-molecular peroxyl radical addition and decomposition of an unstable intermediate, several reactive aldehydes are produced including epoxyalcohol-aldehyde-CL (EAA-CL), 4-HNE, and 4-oxo-2-nonenal (4-ONE). Reproduced from Redox Biol. 2015 Apr; 4: 193–199. Rights Managed by Elsevier.*

#### **6. General remarks, and perspectives**

It has been fascinating to follow the field of mitochondrial diseases research during almost five decades. From my experience, it is impossible to predict which aspects in this area of research will dominate in the future.

### **Acknowledgements**

The author is grateful to his mentors: Rodolfo Brenner and Phillip Strittmatter, all his Ph.D. students, and post docs.

### **Author details**

Angel Catala1,2

1 Facultad de Ciencias Exactas, Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA-CCT La Plata-CONICET), Universidad Nacional de La Plata, Argentina

2 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina

\*Address all correspondence to: catala@inifta.unlp.edu.ar

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Introductory Chapter: Mitochondrial Diseases - Advances and Perspectives - My Point of View DOI: http://dx.doi.org/10.5772/intechopen.98510*

#### **References**

[1] Catala A. (2013) Five Decades with Polyunsaturated Fatty Acids: Chemical Synthesis, Enzymatic Formation, Lipid Peroxidation and Its Biological Effects J Lipids. Available online: https://www. hindawi.com/journals/jl/2013/710290/ [PMC free article]

[2] Catala A. (2009). Lipid peroxidation of membrane phospholipids generates hydroxyl-alkenals and oxidized phospholipids active in physiological and/or pathological conditions. Chem. Phys. Lipids 157, 1-11

[3] Gonzalvez F., Gottlieb E. (200/) Cardiolipin: setting the beat of apoptosis. Apoptosis: An International Journal on Programmed Cell Death. 12:877-885.

[4] Yin H., Zhu M. (2012) Free radical oxidation of cardiolipin: chemical mechanisms, detection and implication in apoptosis, mitochondrial dysfunction and human diseases. Free Radical Research. 46:959-974

### **Chapter 2** Mitochondria and Eye

*Lata Singh and Mithalesh Kumar Singh*

#### **Abstract**

Mitochondria are essential subcellular organelles and important key regulators of metabolism. Mammalian mitochondria contain their own DNA (mtDNA). Human mtDNA is remarkably small (16,569 bp) compared to nuclear DNA. Mitochondria promote aerobic respiration, an important part of energy metabolism in eukaryotes, as the site of oxidative phosphorylation (OXPHOS). OXPHOS occurs in the inner membrane of the mitochondrion and involves 5 protein complexes that sequentially undergo reduction-oxygen reactions ultimately producing adenosine triphosphate (ATP). Tissues with high metabolic demand such as lungs, central nervous system, peripheral nerves, heart, adrenal glands, renal tubules and the retina are affected preferentially by this critical role in energy production by mitochondrial disorders. Eye-affected mitochondrial disorders are always primary, but the role of mitochondrial dysfunction is now best understood in acquired chronic progressive ocular diseases. Recent advances in mitochondrial research have improved our understanding of ocular disorders. In this chapter, we will discuss the mitochondria in relation to eye diseases, ocular tumors, pathogenesis, and treatment modalities that will help to improve the outcomes of these conditions.

**Keywords:** mitochondria, LHON, biomarkers, mutations, tumors

#### **1. Introduction**

#### **1.1 Mitochondria**

Mitochondria are essential sub cellular mammalian organelles found in eukaryotes. It is surrounded by two lipid bilayers which is commonly associated with oxidative phosphorylation, a process that meets the majority of cellular energy demands. It is involved in many other cellular functions such as fatty acids oxidation, apoptosis, heme biosynthesis, metabolism of amino acids and lipids, and signal transduction [1]. They are central organelles controlling the life and death of the cell. Mitochondria contain their own DNA, which is maternally inherited. Mitochondrial density varies from one tissue to another [2]. Mitochondrial diseases are heterogeneous group of disorders, often characterized by morphological changes in the mitochondria, a defective respiratory chain and variable symptoms, ranging from severe metabolic disorders with onset in early infancy or childhood to late onset adult myopathies [3]. Mutations in mitochondrial DNA (mtDNA) are the most frequent cause of mitochondrial diseases in adults. However, the mtDNA encodes only a subset of proteins of the different complexes of the respiratory chain [4]. Nuclear genes encode all the other mitochondrial proteins and most of the mitochondrial disorders are caused by mutations in the nuclear genes [5].

Mitochondria are ~0.5 to ~3 μm long tubular organelles that undergo continuous remodeling of their network by fusion and fission events [6]. Mitochondria forms an extensive network preserved in many cells by an intricate balance between fission and fusion, mitochondrial biogenesis and mitophagy [7, 8]. Mitochondria was identified as the main source of cell energy, and indeed mitochondria is a major site of ATP and macromolecule development. Equivalent-reducing electrons are fuelled by the ETC to produce an electrochemical gradient required for both the production of ATP and the active transport of selective metabolites, such as pyruvate and ATP, through the IMM [9]. Mitochondria, however, plays a variety of roles beyond energy production, including generation of reactive oxygen species (ROS), redox molecules and metabolites, control of cell signaling and cell death, and biosynthetic metabolism.

While mitochondria is best known for harvesting and storage of energy released by oxidation of organic substrates under aerobic conditions by respiration, their many anabolic functions are often ignored [7]. Biosynthetic functions of mitochondria are essential for tumorigenesis and tumor progression [10]. Tumor cells easily survive under hypoxic conditions by recycling NADH to NAD+ through lactate dehydrogenase (LDH) and plasma membrane electron transport (PMET) to enable continued production of glycolytic ATP [11].

#### **2. Mitochondrial genetics**

The human mitochondrial genome consists of 16,569 pairs of nucleotides of double-stranded, closed-circular molecules. It was first sequenced in 1981 and updated in 1999 [12, 13]. mtDNA contains no introns and only encodes 13 polypeptides, 22 transfer RNAs (tRNAs), and the mitochondrial protein synthesis genes 12S and 16S rRNA [14]. The 13 polypeptides of the respiratory complexes (RC) encode subunits (7 of 45 for RC-I, 1 of 11 for RC-III, 3 of 13 for RC-IV, and 2 of 16 for RC-V). Along with the remaining 85% of the other RC subunits, the four subunits that make up RC-II are nuclear-encoded [14]. About 22,000 proteins are encoded by nuclear DNA, about 1,500 of which contribute to the mitochondrial proteome. These nuclear encoded proteins include TCA cycle enzymes, amino acids, nucleic acid and lipid biosynthesis, mtDNA and RNA polymerases, transcription factors, and ribosomal proteins, in addition to all DNA pathway repair components. In the cytoplasm, these proteins are expressed and folded through the TOM/TIM complex upon entry through the mitochondrial outer membrane. From there, they find the outer mitochondrial membrane (OMM), the IMM, the intermembrane space (IMS) or the mitochondrial matrix at their specific positions [15]. There is no structural association of mtDNA with histones, as is nuclear DNA. Rather, it is closely associated with a variety of proteins, about 100 nm in diameter, in discrete nucleoids.

Germline mutations resulting in reduced or lost expression of succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase have been identified in inherited paragangliomas, gastrointestinal stromal tumors, pheochromocytomas, myomas, SDH, papillary renal cell cancer (FH) and gliomas [16]. mtDNA mutations have been involved in neuromuscular and neurodegenerative mitochondrial disease [17–19] and complex diseases such as diabetes [20], cardiovascular disease [21, 22], gastrointestinal disorders [23], skin disorders [24], aging [25, 26] and cancer. Different human populations have different human mtDNA haplotypes, each with a specific mtDNA polymorphism fingerprint, transmitted through the maternal germline. These haplotypes are associated with the geographic

origin of the population. Some human haplotypes are at greater risk of developing a certain form of cancer or neurodegenerative disorder during their lifetime than others [27–29]. The 22 mitochondrial tRNA genes have more than 50 percent of the mtDNA mutations involved in carcinogenesis [29].

The single nucleotide polymorphism, 3243A > G, which alters leucine mt-tRNA and thus affects the translation of 13 respiratory subunits, leading to fewer mitochondrial subunits and impaired OXPHOS, is the most common mtDNA mutation [30, 31]. Individuals can develop maternally inherited diabetes and deafness with 10–30 percent defective copies of tRNALeu. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) are likely to occur in people with 50–90% defective copies [20, 30–35]. The mutation of tRNALeu results in variable types of mitochondrial RC deficiency in various patients. By far, complex I (RC-I) deficiency is the most common finding in MELAS, although some patients have combined RC-I, RC-III and RC-IV deficiencies [30, 36]. Other mutations in mt-tRNA that play a role in human disease include: tRNAlys, which is associated with myoclonal epilepsy, tRNASer with deafness, and tRNAIle with cardiomyopathy [21].

#### **3. Drivers of mtDNA mutations**

mtDNA mutations are caused by ROS-mediated oxidative damage [28, 37]. ROS generation in the respiratory chain is an inherent part of OXPHOS. ROS plays an important role in many signaling processes and their levels are regulated by the antioxidant enzyme systems in the mitochondrial matrix and the IMS. However, in situations where OXPHOS is compromised due to misshapen respiratory complexes resulting in increased leakage of electrons to oxygen, ROS levels can overwhelm the antioxidant protection system and damage to nearby mtDNA [38, 39]. DeBalsi and colleagues suggest that errors produced by mtDNA replication and repair machines may also cause mtDNA mutations [40].

Human cells contain 17 different human DNA polymerases, but in mtDNA replication and repair, only polymerase gamma (Pol-γ) functions. A catalytic subunit and an accessory subunit consist of a nuclear-encoded Pol-γ holo-enzyme [40]. Pol-γ replicates high fidelity mtDNA with one misinsertion in every 500,000 new base pairs due to nucleotide selectivity and proofreading capacity [41]. More than 300 Pol-γ mutations have been associated with human illness, some of which occur in adulthood and are associated with aging, including different types of progressive external ophthalmoplegia (PEO) and Parkinson's disease (PD) [40]. The role of Pol-γ in restricting mtDNA mutations has been demonstrated by homozygous, but not heterozygous, mutator mice with re-reading-deficient Pol-g developing multiple agerelated disorders and shortening their lifespan. As their antioxidant capacities were the same and the degree of oxidative damage was comparable to wild-type mice, they acquired mtDNA mutations that were not caused by oxidative damage.

Somatic point mutations, great deletions and several linear deleted mtDNA fragments were acquired by the mutator mice. The mtDNA-specific Twinkle helicase, which unwinds mtDNA for Pol-γ synthesis, is another n-mitoprotein involved in mtDNA replication [42]. Overexpression of Twinkle in transgenic mice resulted in increased copy number of mtDNA and OXPHOS and some twinkle mutations are associated with mitochondrial myopathy [40]. Oxidative damage and defective replication are both likely to add to the overall mutational load of the mtDNA cell, and the contribution of each mutational driver is likely to change over time. Inevitable respiratory electron leakage from complexes I and III results in the formation of superoxide, O2 − that can react with lipids, proteins and DNA [43–46]. Superoxide can be quickly converted to H2O2 either naturally or through a manganese superoxide dismutase (MnSOD) dysmutation reaction, a resident of the mitochondrial matrix. In the presence of redox active metal ions, H2O2 can generate a highly reactive hydroxyl radical through the Fenton reaction (OH-) [47]. Multiple mtDNA damage sites, including single and double-strand breaks, abasic sites and base changes, are responsible for the OH-radical. Another oxidative burden is caused by damage to mitochondrial protein centers caused by O2 − to Fe-S and involves subunits of complexes I, II and III as well as aconitase [48–50]. A significant target for ROS is provided by Labile Fe-S enzymes such as mitochondrial aconitase.

Mitochondria located in cells exposed to visible light generate ROS through interactions with mitochondrial photosensitizers, such as cytochrome c oxidase, of particular relevance to the eye, to produce ROS and mtDNA damage [50, 51]. Transferring energy from photoactivated chromophores to oxygen contributes to the formation of singlet oxygen, <sup>1</sup> O2, which occurs in an excited state. <sup>1</sup> O2 can produce ROS, such as O2 − by interacting with diatomic oxygen and directly reacting with dual-bond electrons without the formation of free radical intermediates [52]. It is also important to remember that, from non-mitochondrial sources, various tissues within the eye may also produce substantial amounts of ROS. For instance, lipofuscin (an age-related pigment that accumulates with age in RPE cells) is a potent photoinducible ROS generator, and NADPH oxidase is considered to be a major source of superoxide in microvascular endothelial cells. Studies indicate that ROS may also contribute to exogenous mitochondrial oxidative damage, exacerbating mitochondrial dysfunction [51, 53, 54].

#### **4. Ophthalmologic mitochondrial dysfunction**

Mitochondrial disease can manifest in any organ at any age. In general terms, tissues and organs (retina, optic nerve, brain, heart, testis, muscle, etc.) that are heavily dependent upon oxidative phosphorylation bear the brunt of the pathology. It is also puzzling that many mitochondrial disorders affect multiple organ systems, whereas others have a highly stereotyped and organ specific phenotype. These subtle interactions between nuclear and mitochondrial genes in health and disease will have broader relevance for our understanding of many inherited and sporadic disorders.

Mitochondrial disorder can be categorized according to several different criteria in the manifestations of ophthalmology diseases. They may be defined as isolated or nonisolated, occurring in combination with other manifestations of the organ. The dominant trait of the phenotype or a nondominant attribute can be ophthalmologic manifestations. Mitochondrial disorders with ophthalmic manifestations may be caused either by mutations in mtDNA or nuclear DNA. Ophthalmologic symptoms may be unique to syndromic mitochondrial disorder (e.g. Leber hereditary optic neuropathy) or nonspecific to syndromic mitochondrial disorder (eg, cataract). The cornea, iris, lens, ciliary body, retina, choroid, uvea, or optic nerve may be the primary manifestations of ophthalmologic mitochondrial disorder. There is growing evidence supporting an association between mitochondrial dysfunction and a number of ophthalmic diseases causing defects in OXPHOS and increased production of ROS triggering the activation of cell death pathway.

### **5. Corneal dystrophy**

Some evidence has been given in recent years that the cornea may be involved in mitochondrial disorders. However, systematic studies have not been performed on this matter. Astigmatism, corneal dystrophy, corneal clouding, or corneal endothelial dysfunction are corneal disorders associated with mitochondrial dysfunction [55, 56]. Loss of SLC4A11 gene activity which is localized to the inner mitochondrial membrane of corneal endothelium, induces oxidative stress and cell death, resulting in Congenital Hereditary Endothelial Dystrophy (CHED) with corneal edema and vision loss [57]. Fuchs endothelial corneal dystrophy (FECD) is characterized by progressive and non-regenerative corneal endothelial loss. Variations in mtDNA affect the susceptibility of FECD. Mitochondrial variant A10398G and Haplogroup I were significantly associated with FECD [58]. There are few studies showing the role of mtDNA in the pathogenesis of FECD. Mitophagy activation leads to decrease in Mfn2 gene level and loss of mitochondrial mass in FECD [59]. In a study of 20 patients, keratoconus was related to increased oxidative stress due to mitochondrial respiratory chain complex-I sequence variation [60]. Progressive external ophthalmoplegia secondarily led to persistent conjunctivitis and keratitis in a patient with Kearns-Sayre Syndrome [61]. Corneal clouding has been documented occasionally in Kearns-Sayre syndrome due to structural changes in the endothelium or Descemet membrane [62]. Numerous distended mitochondria were present in the corneal epithelium in a child with Leigh syndrome due to the m.8993 T > G mutation [63]. There are also non-specific corneal alterations in a patient with Neurogastrointestinal mitochondrial encephalomyopathy [64]. Pathogenesis of type 2 granular corneal dystrophy (GCD2) is associated with alteration of mitochondrial features and functions that causes mutated GCD2 keratocytes, particularly in older cells [65].

#### **6. Mitochondrial encephalomyopathy, lactic acidosis, and episodic stroke-like syndrome (MELAS)**

Early onset of the disease and higher level of mtDNA heteroplasmy are associated with a worse prognosis in mitochondrial encephalomyopathy, lactic acidosis, and episodic stroke-like syndrome (MELAS). Iris involvement in mitochondrial disorders has been rarely mentioned in MELAS [66]. The m.3243A > G variant is the most common heteroplasmic mtDNA mutation in MELAS and underlies a spectrum of diseases. Patchy iris stroma atrophy has been identified in a patient carrying the m.3243A > G mutation in the tRNA (Lys) gene [66]. MNRR1 (CHCHD2) is a bi-organellar regulator of mitochondrial function, found to be depleted in MELAS and significantly associated with m.3243A > G mutation (heteroplasmic) in the mtDNA at a level of ∼50 to 90% [67]. Ability of the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), improves the mitochondrial biogenesis/respiratory functions in MELAS cybrid cells containing >90% of the m.3243A > G mutation that found to be novel therapies to treat this disease [68]. Induced pluripotent stem cells (iPSCs) are appropriate for studying mitochondrial diseases caused by mtDNA mutations in MELAS. Increase of autophagy inpatient-specific iPSCs generated from fibroblasts are associated with mtDNA mutations and OXPHOS defects in patients with MELAS [69]. Studies demonstrated that defective MRM2 gene causes a MELAS-like phenotype which suggests the genetic screening of the MRM2 gene in patients with a m.3243 A > G negative MELAS-like presentation [70]. Mutations caused by mitochondrial complex I deficiencies by alleviating ketone bodies are also associated with MELAS that leads to recurrent cerebral insults resembling strokes [71].

#### **7. Cataract**

Cataracts are the most common lenticular defects of mitochondrial disorders. In mitochondrial disorders, cataract is typically of the posterior subcapsular type [66]. Autophagic dysfunction and abnormal oxidative stress are associated with cataract. Cataract may be a phenotypic characteristic of MELAS syndrome, but a patient with nonsyndromic mitochondrial disorder due to mtDNA deletion has also been documented as an initial manifestation [66, 72, 73]. Oxidative stress plays an important role in cataractogenesis [74, 75]. Mitochondria are found in the epithelium and superficial fiber cells of the lens and it is extremely sensitive to ROS. Interestingly, mitochondria have been confirmed as the main source of ROS generation in these cell types [76]. A number of in vitro studies have shown that human lens cells are particularly sensitive to oxidative insults, where antioxidant activity was inversely proportional to the severity of cataracts [77]. Proteins, lipids and DNA oxidation have been found in cataract lenses [78–80]. Under high glucose conditions, fluctuations in autophagy and oxidative stress are found in mouse lens epithelial cells (LECs) that might attenuate high glucose-induced oxidative injury to LECs [81]. Cataract proteins lose sulfhydryl groups, contain oxidized residues, produce aggregates of high molecular weight and become insoluble [75]. In addition, cataract has been shown to be a symptom of a newly identified mitochondrial disorder called autosomal recessive myopathy, caused by growth factor mutations, increased liver regeneration gene, which affects protein levels of mitochondrial intermembrane space region [82].

#### **8. Leigh syndrome**

In mitochondrial disorders, involvement of ciliary body has rarely been reported. Leigh's syndrome is the most common pediatric syndrome, characterized by symmetrical brain lesions, hypotonia, motor and respiratory deficits, and premature death are associated with pathways involved in mitochondrial diseases [83]. A case report showed ocular histopathological finding such as thinning of nerve fibers and ganglion cell layers in the nasal aspect of the macula, mild atrophy of the temporal aspect of the optic nerve head, and numerous distended mitochondria, non-pigmented cilla are associated with the m.8993 T > G mutation in the ATPase6 gene of mtDNA in patient with Leigh's syndrome [63]. In addition, ciliary epithelium was also found to be impaired by a long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency [84]. Dysfunction of mitochondrial complex I are also associated with many brain pathologies including Leigh's syndrome. Mitochondrial complex I activity facilitates organismal survival by its regeneration potential of NAD+, while optimal motor regulation involves mitochondrial complex I bioenergetic function in Leigh's syndrome [85].

#### **9. Retinitis pigmentosa**

Retinitis pigmentosa is a central characteristic of Kearns-Sayre syndrome and neuropathic ataxia retinitis pigmentosa syndrome [72]. Typical for Kearns-Sayre

#### *Mitochondria and Eye DOI: http://dx.doi.org/10.5772/intechopen.96368*

syndrome is 'salt and pepper' retinitis, with areas of increased and decreased pigmentation, especially in the equatorial fundus [62]. Pigment retinopathy is only an uncommon characteristic of progressive external ophtalmoplegia and can be milder than in Kearns-Sayre syndrome [72, 86]. Only certain patients with MELAS or MERRF syndrome have mild posterior pole pigment retinopathy [72]. Mild pigmentary defects were also observed in 2 of 20 patients with Leber hereditary optic neuropathy due to mutation m.11778G > A [72]. Small pigment retinal defects have been identified in a 4-year-old female with a COX deficiency [87]. In addition, because of the mutation m.8993 T > GG retinitis pigmentosa has been identified in patients with Leigh syndrome [88].

In a sample of 44 Korean Leigh syndrome patients, pigmentary retinopathy was also observed in 22% of Korean patients [89]. In a study of 14 patients with pontocerebellar hypoplasia, 4 patients presented with retinopathy without disclosing information [90]. Occasionally, retinal dystrophy can manifest with photophobia. In a report of 46 mitochondrial disease patients, 4 had photophobia. Two patients had Leigh syndrome, 1 of which had rod-cone dystrophy on electroretinography, 1 had Kearns-Sayre syndrome with regular electroretinography, and 1 had MERRF syndrome with isoelectric electroretinography [91].

#### **10. Diabetic retinopathy**

It has been shown that mitochondrial dysfunction plays a significant role in diabetic retinopathy [92, 93]. Hyperglycemia causes retinal mitochondrial damages that plays a central role in the development of diabetic retinopathy. Retinal mitochondria undergo elevated oxidative stress in diabetes, and complex III is one of the key causes of increased O2 − [94]. Superoxide levels are elevated in in the retina of diabetic rats and in retinal vascular endothelial cells incubated in high-glucose media [95] and the content of hydrogen peroxide is also increased in the retina of diabetic rats [96]. In diabetes, membrane lipid peroxidation and oxidative DNA damage, the effects of ROS-induced injury, are elevated in the retina [97]. Chronic overproduction of ROS in the retina results in aberrant mitochondrial functions in diabetes [92]. Overproduction of superoxide by the mitochondrial electron transport chain caused by hyperglycemia is considered to cause major hyperglycemic damage pathways by inhibiting the action of GAPDH. However, it is not yet fully understood the mechanism by which hyperglycemia induces an increase in mitochondrial ROS, with some suggesting a direct effect and others an indirect function via high-glucose-induced cytokines [98–101].

Elevated levels of O2 − activate caspase 3 in retinal capillaries contributes to cell death [92]. Upregulation of superoxide dismutase (SOD2) inhibited increased mitochondrial O2-induced diabetes, restored mitochondrial function, and prevented both in vitro and in vivo vascular pathology [94, 102–104]. However, the timing of such therapies is important because animal studies have shown that oxidative stress not only leads to the development of diabetic retinopathy, but also to the resistance of retinopathy to reversal [105]. The resistance to reversal of diabetic retinopathy may be due to the accumulation of weakened mitochondrial molecules and ROS-induced damage that is not readily removed even after the restoration of high glycemic control. However, the accumulation of advanced glycation end products is also involved in metabolic memory [106]. The mtDNA variation has also been associated with resistance to type 1 diabetes. A single nucleotide modification (C5173A) is associated

with resistance to type 1 diabetes in the Japanese population, resulting in a leucineto-methionine amino acid substitution in the mitochondrially encoded NADH dehydrogenase subunit 2 gene [107]. Similarly, in comparison with the diabetes-prone nonobese diabetic mouse strain, orthologous polymorphism (C4738A), resulting in L-to-M substitution, offers resistance against the development of spontaneous diabetes [108]. Gusdon et al., have shown that the replacement of methionine results in a lower level of development of ROS from complex III [109].

The product of mtDNA mutations is also known to result in many syndromic central nervous system diseases. The most common retinal pathology is pigmentary retinopathy, while optic neuropathy is an uncommon finding in these disorders. Neurogenic atrophy and retinitis pigmentosa syndrome results from point mutations in the mtDNA ATPase-6 gene, usually T8993G variation. Patients usually present with retinitis pigmentosa with or without optic neuropathy and may develop dystonia [110]. Several mtDNA point mutations may result from MELAS, although the A3243G mutation in the tRNALeu gene is the most common. Patients with MELAS undergo stroke-like episodes leading to recurrent retrochiasmal vision loss, but sometimes even to pigmentary retinopathy without optic atrophy [111]. Its contribution to the pathogenesis of maternally inherited diabetes and deafness is also evidenced by the spectrum of disease resulting from the A3243G point mutation [112–114]. This is a multisystemic disease characterized by sensorineural deafness, retinal defects and diabetes, generally occurring in the third to fourth decades of life [115]. The second phenotype is a pattern dystrophy, with diffuse granularity and pigment clumping, marked by relative sparing of the fovea, and retinal pigment epithelium within the vascular retinal arcades. However, with a strong prognosis, visual acuity is retained, despite the degree of atrophy [116, 117].

#### **11. Macular degeneration**

Age-related macular degeneration is a neurodegenerative late-onset disorder that shares certain characteristics of Alzheimer's disease. In most cases, the build-up of protein plaques, known as drusen, in the central macular area of the retina involves age-related macular degeneration. Both age-related macular degeneration and Alzheimer's disease pathogenesis can be driven by stress stimuli, including oxidative stress, aging, genetic factors and inflammation, including the deposition of protein plaques in the retina or brain [98]. Similarities in these two disorders are also found in the risk factor gene polymorphisms, APOE, associated with age-related macular degeneration [99, 100] and Alzheimer's disease [101, 102]. The APOE gene controls the homeostasis of triglycerides and cholesterol [103], and the loss of function of APOE has been correlated with the deposit of senile plaques, consisting mainly of amyloid beta peptide [104], which is produced in drusen [105, 106] and is also associated with an additional risk factor for age-related macular degeneration, i.e. complement protein [107, 108]. Evidence shows that the APOE genotype can dictate the risk of stress stimuli, including oxidative stress, aging, genetic factors and inflammation, including the deposition of protein plaques in the retina or brain, can drive both age-related macular degeneration and Alzheimer's disease pathogenesis. Alzheimer's disease and other chronic disorders, primarily because of its effect on regulation of oxidative stress [109]. Age-related macular degeneration is split into two main forms, i.e. the "wet" form induced by leakage into the subretinal space from choroidal neovascularization and the more common "dry" form associated with the accumulation

#### *Mitochondria and Eye DOI: http://dx.doi.org/10.5772/intechopen.96368*

of drusen in the macula [75]. In patients with age-related macular degeneration, there is an increased incidence of large-scale mtDNA rearrangements and deletions in blood [76] and retinas [77, 78]. In the non-coding mtDNA control area (d-loop) in retinas with age-related macular degeneration, which has been found in Alzheimer's disease and other conditions of oxidative stress, there are also increased rates of single nucleotide polymorphisms [79]. An increased rate of mtDNA deletions and single nucleotide polymorphisms are likely to decrease the amount and density of mitochondria [80].

Other than pigmentary retinopathy or macular degeneration, retinal anomalies include retinal dystrophy, retinal hypertrophy, and pigmentary maculopathy. Patients with Kearns-Sayre syndrome, Leigh syndrome, MELAS syndrome, MERRF syndrome, and Leber hereditary optic neuropathy will find retinal dystrophies that are most easily measured by electroretinography [91]. Retinal hypertrophy has been identified in patients with autosomal recessive spastic ataxia with leukoencephalopathy and autosomal recessive spastic ataxia with Charlevoix-Saguenay (ARSAL/ ARSACS) [118]. Six affected males in a family with Mohr-Tranebjaerg syndrome had blindness resulting from unexplained retinal degeneration [119]. Treatment options for retinopathy are usually limited.

#### **12. Choroidal dystrophy**

Choroid and uvea are occasionally affected by mitochondrial disorders. Choroid atrophy is the most common manifestation of mitochondrial disorders [66]. Choroidal atrophy was especially identified in the sense of MELAS syndrome [66]. Choroid pigment epithelium atrophy also occurs in maternally inherited deafness and diabetes [120]. Central choroidal dystrophy was identified in 1 patient with Mohr-Tranebjaerg syndrome as confirmed by electroretinography [119]. In addition, chorioretinal dystrophy was reported in a single patient with a significant deletion of mtDNA [121].

#### **13. Uveitis**

A significant causative factor causing blindness from retinal photoreceptor degeneration is intraocular inflammation, also referred to as uveitis. Activated macrophages, which generate various cytotoxic agents, including inducible nitric oxide generated by inducible nitric oxide synthase, O2 − and other ROS, are responsible for oxidative retinal damage in uveitis [122]. Oxidative stress plays an important role in the early stages of experimental autoimmune uveitis (EAU) in the photoreceptor mitochondria. mtDNA damage has been shown to occur early in the EAU; interestingly, nDNA damage occurred later in the EAU [123]. In addition, peroxynitrite-mediated nitration modifies mitochondrial proteins in the inner segments of the photoreceptor, which, in turn, contributes to increased mitochondrial ROS generation [124]. MnSOD has been shown to be upregulated during EAU to promote an increased state of mitochondrial oxidative stress, possibly to combat ROS [125]. In the early phase of the EAU, before leukocyte infiltration, recent data seem to indicate a causative function of oxidative mtDNA harm. Such mitochondrial oxidative damage can be the initial event that contributes to retinal degeneration in uveitis [123].

#### **14. Optic atrophy**

Optic atrophy is the principal mitochondrial dysfunction manifestation of the optic nerve. Optic atrophy is a prevalent manifestation of mitochondrial disorder but is often overlooked or misinterpreted. This is due to the difficulties of optic atrophy diagnosis. Funduscopy can more reliably determine optic atrophy if the distal portion of the optic nerve is impaired, or if the more proximal portions of the nerve are affected by orbital magnetic resonance imaging (MRI). A decreased amplitude of visually evoked potential is a sign of optic nerve atrophy [126]. Optic atrophy has been specifically identified in Leber hereditary optic neuropathy and autosomal dominant optic atrophy among syndromic mitochondrial disorders, conditions in which optic atrophy is the dominant phenotypic function [127]. MELAS syndrome, Kearns-Sayre syndrome, Pearson syndrome, pontocerebellar hypoplasia, Mohr-Tranebjaerg syndrome, Alpers-Huttenlocher disease or Wolfram syndrome have been documented more rarely, with optic atrophy [62, 90, 91, 127]. In patients with MERRF syndrome, partial or complete optic atrophy has also been identified [72, 91, 128]. Optical atrophy is a common phenotypic characteristic of inherited motor and sensory neuropathy type VI (HMSN-IV) due to MFN1 mutations [127]. In addition, C12orf65 (COXPD7) mutations manifest phenotypicly with optical atrophy and Leigh-like phenotype [129]. Optical atrophy associated with neuropathy ataxia retinitis pigmentosa syndrome due to m.8993 T > G mutation in the ATPase6 gene was only seen in a single family [110]. In a study of 44 Korean patients with Leigh Syndrome, 22.5 per cent of optical atrophy was identified [89]. Optical disk alterations have been observed only in a single patient with mitochondrial neurogastrointestinal encephalomyopathy [64]. Optical atrophy can also be a characteristic of childhood-onset spinocerebellar ataxia [130] or mitochondrial depletion syndrome. 39 Non-syndromic mitochondrial optic atrophy disorders is attributed to ACI1 mutation [131], due to ND5 mutation with cataract and retinopathy [132].

#### **15. Glaucoma**

Increased intraocular pressure (Glaucoma) is an unusual phenotypic characteristic of mitochondrial disorders. There are two primary types of glaucoma that can be distinguished, open-angle glaucoma and closed-angle glaucoma. In addition, normotensive and hypertensive glaucoma are distinguished. Open-angle glaucoma is seldom observed in patients with Leber inherited optic neuropathy or autosomal dominant optic atrophy. Funduscopic findings can indicate a mixture of abnormalities common for glaucoma retinopathy and an inherited Leber optic neuropathy fundus [133]. In a single patient with mitochondrial neurogastrointestinal encephalomyopathy, glaucomatous changes in the optic disc were observed by visual field assessment and optical coherence tomography [64]. In a study of 14 patients with pontocerebellar hypoplasia, one presented with glaucoma [90]. Normal pressure glaucoma is associated with polymorphism in the OPA1 gene [134].

Glaucoma has also been identified in a family with Wolfram Syndrome. There are signs that ND5 mutations are associated with the development of open-angle glaucoma. Glaucoma in mitochondrial disorders may be eligible for treatment with drugs or surgery [135, 136]. There is evidence in glaucoma that mitochondrial dysfunction can reduce the bioenergetic status of retinal ganglion cells, leading to increased susceptibility to oxidative stress and apoptotic cell death [93, 137]. Light exposure may also be an oxidative risk factor, reducing mitochondrial function and increasing the

development of ROS in ganglion cells [138]. A defective mitochondria has been highly implicated in neuronal apoptosis in the experimental models of glaucoma [139, 140]. The mtDNA abnormalities further support the importance of mitochondrial dysfunction-associated stress as a risk factor for glaucoma patients [141].

#### **16. Nystagmus**

The central nervous system or vestibular involvement in mitochondrial disorders may cause nystagmus or roving eye movements and are the most common ophthalmological manifestations as a symptom in patients with pediatric mitochondrial disorder [142]. A Gaze-evoked nystagmus identified in a single patient with "Leber hereditary optic neuropathy plus" who not only possessed the "m.11778G > A" mutation in the hereditary Leber hereditary optic neuropathy gene but also the "m.3394 T > C" mutation [143]. Since patients with MELAS may display irregular eye movements on an eye movement cueing task, ultrasound records of eye movement may show abnormally slow saccadic reactions, prolonged saccades, impaired suppression of reflex eye movements, prolonged reaction during antisaccades, square-wave jerks, or impaired chase [144]. Patients have epilepsy due to MELAS may have epileptic nystagmus, disrupted smooth pursuit, or transient eye divergence, none of which are outward signs [145]. In addition, nystagmus was documented in a patient carrying a point mutation in the DGUOK gene who also had retinal blindness. Nystagmus, which is a common symptom of the disease along with retinitis pigmentosa, was also reported in a patient with nonsyndromic mitochondrial disorder due to the m.15995G > A mutation in the tRNA (Pro) gene manifesting as ataxia, deafness, and leukoencephalopathy [146]. Nystagmus was part of the phenotype in a study of 7 Czech patients with autosomal dominant optic atrophy [147]. Nystagmus is also a common characteristic of ARSAL/ARSACS [148]. Nystagmus was observed in 14 percent in a study of 44 Korean patients with Leigh syndrome [88].

#### **17. Strabismus**

Strabismus was the most common ophthalmologic abnormality in a study of 44 Korean patients with Leigh syndrome and was present in 41% of patients [89]. Of the strabismus patients, 13 had exotropia and 5 had esotropia [89]. In some patients with X-linked sideroblast anemia with ataxia, strabismus has also been identified [149]. In 25 percent of juvenile mitochondrial disorders, divergent strabismus has been identified as the presenting manifestation [150]. In a study of 14 patients with pontocerebellar hypoplasia, of whom 13 had a CASK mutation, 2 had strabismus. 9 Strabismus was also identified without knowing the underlying mutation in other patients with pontocerebellar hypoplasia [151, 152]. The initial presentation at birth was cataract and strabismus in a child with a significant mtDNA deletion. Later on, he experienced Leigh-like pathologies and episodes of stroke [153]. In certain instances, surgery can have a beneficial effect on strabism.

#### **18. Progressive external ophthalmoplegia**

In mitochondrial disorders, affectation of the extraocular muscles results in progressive external ophthalmoplegia. The recurrent ophthalmologic manifestation of mitochondrial disorders is progressive external ophthalmoplegia. It may be complete, resulting in, or partial, walled-in bulbs. Both directions of bulb movements or only some of them can be affected. One eye or both eyes can be affected by it. Single or multiple mtDNA deletions are most often associated with progressive external ophthalmoplegia. Progressive external ophthalmoplegia, Kearns-Sayre syndrome or Pearson syndrome can cause single mtDNA deletions [154]. Multiple deletions of mtDNA may be due to mutations in nuclear genes such as PEO1, POLG1, SLC25A4, RRM2B, POLG2, or OPA1, along with progressive external ophthalmoplegia [154]. In addition, progressive external ophthalmoplegia, especially in the transfer of RNA (eg, tRNA(Lys)) genes, may be due to mtDNA point mutations [154]. Transfer RNA mutations with progressive external ophthalmoplegia are mostly sporadically similar to mtDNA deletions and can only be observed in muscle deletions [155]. The sole manifestation of the m.3243A > G mutation, which often manifests as MELAS syndrome, may be progressive external ophthalmoplegia [156]. In a patient with mitochondrial neurogastrointestinal encephalomyopathy, progressive external ophthalmoplegia was a phenotypic feature [64], Wolfram syndrome [157], Leigh syndrome, autosomal dominant optic atrophy, and mitochondrial recessive ataxia syndrome. In MERRF syndrome, progressive external ophthalmoplegia has also been described [158].

Infantile-onset spinocerebellar ataxia is a Finnish disorder, with some of the 24 cases identified to date developing ophthalmoplegia [130]. Ophthalmoparesis is a hallmark of sensory ataxic neuropathy with ophthalmoparesis syndrome and dysarthria [159]. Sensory ataxic neuropathy with dysarthria and ophthalmoparesis is due to mutations in either the POLG1 or PEO1 gene resulting in multiple mtDNA deletions [159]. Furthermore, ophthalmoparesis can be observed in patients with mitochondrial depletion syndrome [160] or nonsyndromal mitochondrial disorders [161]. In patients with Leber inherited optic neuropathy and progressive external ophthalmoplegia, ultrastructural variations in muscle biopsy from the extraocular muscles clearly differ [162].

#### **19. Eyelid**

Ptosis is one of the most common forms of mitochondrial dysfunction. It can occur unilaterally at onset, but during the course of the disease, it usually becomes bilateral. Ptosis can be the sole manifestation, particularly at the onset of the disease, of a mitochondrial disorder or associated with other manifestations. Particularly at the onset of the disease, ptosis can show dynamic alterations, leading to misinterpretation as myasthenia gravis [163]. Ptosis may be discrete, especially at initiation, so that it is missed on clinical review. Progressive external ophthalmoplegia or other ocular symptoms of mitochondrial disease can be associated with ptosis. Ptosis of syndromic as well as nonsyndromic mitochondrial disorders may be a phenotypic manifestation. In particular, ptosis was identified in progressive external ophthalmoplegia, MELAS, MERRF, Kearns-Sayre syndrome, sensory ataxic neuropathy with dysarthria and ophthalmoparesis [164], Pearson syndrome, mitochondrial neurogastrointestinal encephalomyopathy, and autosomal dominant optic atrophy, among the syndromic mitochondrial disorders [91]. Ptosis was present in 16 percent in a group of 44 Korean patients with Leigh syndrome [89]. Ptosis was also present in isolated cases of maternally inherited deafness and diabetes [156], mitochondrial neurogastrointestinal encephalomyopathy [64], or mitochondrial depletion syndrome [160]. Poor lid closure was found in a Persian Jew with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia due to a PUS1 mutation [165].

#### **20. Leber hereditary optic neuropathy**

Leber hereditary optic neuropathy is a maternally inherited blindness condition caused by gene mutations encoding the respiratory-chain complex I subunits. Nearly 90 percent of all cases of Leber inherited optic neuropathy contain mutations in 3 genes [128]. The m.3460A > G mutation in the ND1 gene, the m.11778G > A mutation in the ND4 gene and the m.14484 T > C mutation in the ND6 gene are the 3 most common Leber hereditary optic neuropathy mutations (primary Leber hereditary optic neuropathy mutations) [128]. Leber inherited optic neuropathy is clinically characterized as bilateral, painless, subacute vision impairment that occurs during young adult life [134].

Compared with women, Leber hereditary optic neuropathy is 4 to 5 times more common in males. Individuals affected are usually completely asymptomatic until they experience visual blurring in 1 eye affecting the central visual field [134]. On average, 2 to 3 months later, similar signs develop in the other eye. In most cases, visual acuity is greatly diminished or even worse when counting fingers, and visual field examination reveals an expanded central or ceco-central thick scotoma [134]. After the acute process, the optical disks become atrophic. Funduscopic findings characteristic of Leber inherited optic neuropathy include microangiopathy, hyperemic disks, retinal telangiectasis (ectatic capillaries), peripapillary microangiopathy, and tortuosity of vessels (twisted vessels). (twisted vessels). The orbital MRI can display atrophy of the nerve with a compensated widening of the space below the optic sheath. Mutations in mitochondrial ND3, ND4, or ND6 genes can cause hereditary Leber optic neuropathy with dystonia [166].

#### **21. Autosomal dominant optic atrophy**

Autosomal dominant optic atrophy is a blindness condition which does not display a gender disparity, unlike Leber inherited optic neuropathy [127]. It is caused by mutations in the nuclearly encoded OPA1 gene [127]. Autosomal dominant optic atrophy can also be due to OPA3 mutations that are associated with cataract [167]. Progressive, painless, bilateral symmetrical vision loss clinically characterizes autosomal dominant optic atrophy [154]. Central, ceco-central, or para-central scotomas, consistent with early involvement of the papillo-macular bundle, are the most common visual field anomalies in autosomal dominant optic atrophy [154]. OPA1 mutations can manifest not only with optic atrophy in some families, but also with progressive external ophthalmoplegia, ptosis, and hypoacusis [168]. Since glaucoma neuropathy, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy often have similar changes in the topographic optic disc, they cannot be discriminated against alone by disc evaluation [169]. There is currently no appropriate treatment available.

#### **22. Retinoblastoma**

Retinoblastoma (Rb) is the most common intraocular cancer in children that arise from retinal precursor cells. Electron microscopy revealed numerous morphological and pathological changes in mitochondria of retinoblastoma patients. Cristolysis and degenerated mitochondria were the most frequently observed features in Rb [170]. A study suggested that T16519C, C16223T, A263G and A73G mtDNA D-Loop mutations plays a significant role in the etiology of retinoblastoma. This was the first study to examine the mtDNA D-loop mutation in retinoblastoma and its correlation with various parameters and patient outcome [171]. Their findings imply a strong inhibition of mitochondrial oxidative phosphorylation complexes in these patients. Loss of mitochondrial complex I was found in majority of the cases whereas expression of mitochondrial complex III, IV and V were found in more than 50% of the cases. Expression of mitochondrial complex I was associated with good prognosis and better overall survival [172]. Another consequence of alteration in OXPHOS complexes is an increased production of reactive oxygen species (ROS). NADPH oxidases (NOX4) are a major intracellular source of ROS and it was found to be overexpressed in retinoblastoma [173]. Increased expression of ROS and decreased expression of OXPHOS complexes modulates the apoptotic pathway involved in mitochondria by altering BCl-2 family proteins. Singh et al. showed a differential expression of apoptotic regulatory proteins (Bax, BCl-2, PUMA and p53) where they found increased expression of BCl-2 and PUMA along with loss of Bax and p53, which might contribute to carcinogenesis in Rb [174].

### **23. Conclusion**

Researchers found that these findings are important because they indicate that mtDNA damage can be caused by both spontaneous ROS and by inherited mtDNA mutations. Continued study in this clinically important area would certainly provide a better understanding of how deficiencies/mutations of the mitochondrial genome contribute to the pathogenesis of ocular diseases. The biggest problems with the future of mitochondria are the advancement of therapeutic strategies to target mitochondria and modify its DNA using nucleotide precursors to retain mitochondrial integrity. These therapeutic strategies can potentially be used to block or slow down the effects of mitochondrial disease in future.

### **Author details**

Lata Singh1 \* and Mithalesh Kumar Singh<sup>2</sup>

1 Department of Pediatric, All India Institute of Medical Sciences, New Delhi, India

2 Department of Dermatology, University of Wisconsin, Madison, USA

\*Address all correspondence to: lata.aiims@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Mitochondria and Eye DOI: http://dx.doi.org/10.5772/intechopen.96368*

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#### **Chapter 3**

## Mitochondrial Cytopathies of the Renal System

*Lovelesh K. Nigam, Aruna V. Vanikar, Rashmi Dalsukhbhai Patel, Kamal V. Kanodia, Kamlesh Suthar and Umang Thakkar*

#### **Abstract**

Mitochondria are major intracellular organelles with a variety of critical roles like adenosine triphosphate production, metabolic modulation, generation of reactive oxygen species, maintenance of intracellular calcium homeostasis, and the regulation of apoptosis. Mitochondria often undergo transformation in both physiological and pathological conditions. New concepts point that mitochondrial shape and structure are intimately linked with their function in the kidneys and diseases related to mitochondrial dysfunction have been identified. Diseases associated with mitochondrial dysfunction are termed as "mitochondrial cytopathies". Evidence support that there is a role of mitochondrial dysfunction in the pathogenesis of two common pathways of end-stage kidney disease, namely, chronic kidney disease (CKD) and acute kidney injury (AKI). Mitochondrial cytopathies in kidneys mainly manifest as focal segmental glomerular sclerosis, tubular defects, and as cystic kidney diseases. The defects implicated are mutations in mtDNA and nDNA. The proximal tubular cells are relatively vulnerable to oxidative stress and are therefore apt to suffer from respiratory chain defects and manifest as either loss of electrolyte or low-molecular-weight proteins. Patients with mitochondrial tubulopathy are usually accompanied by myoclonic epilepsy and ragged red muscle fibers (MERRF), and Pearson's, Kearns-Sayre, and Leigh syndromes. The majority of genetic mutations detected in these diseases are fragment deletions of mtDNA. Studies have shown significantly increased ROS production, upregulation of COX I and IV expressions, and inactivation of complex IV in peripheral blood mononuclear cells of patients with stage IV–V CKD, thereby demonstrating the close association between mitochondrial dysfunction and progression to CKD. Furthermore, the mechanisms that translate cellular cues and demands into mitochondrial remodeling and cellular damage, including the role of microRNAs and lncRNAs, are examined with the final goal of identifying mitochondrial targets to improve treatment of patients with chronic kidney diseases.

**Keywords:** mitochondrial cytopathies, renal, glomerular, mitophagy, fission, fusion

#### **1. Introduction**

Mitochondria, also called as the "power house" of the cell, are double membraned cell organelles involved with converting the energy derived from oxidative phosphorylation into a "fuel" in the form of adenosine triphosphate (ATP) [1, 2]. These also are involved in calcium storage, regulation of metabolism and apoptosis, and cell signaling. The energy demand of an organ is directly proportional to the number of mitochondria present in the organ, so heart is the organ with maximum number of mitochondria followed by kidneys [3, 4]. In the kidneys, renal tubular cells are richest in mitochondria, so as to facilitate the energy-consuming task of reabsorption of the majority of the glomerular filtrate. The renal function depends on interplay between multiple cell types, including endothelial cells, podocytes, mesangial cells, and tubulointerstitial cells, and is energetically demanding and relying on mitochondrial function [5].

Renal dysfunction is a multifactorial entity and manifests as a sequel to an acute or chronic insult to the organ. Recently it has been proposed that renal inflammation and tissue damage during acute kidney injury (AKI) and chronic kidney disease (CKD) have been linked to mitochondrial structural and functional alterations [4, 6].

#### **2. Definition**

Diseases related to mitochondrial alterations are known as 'mitochondrial cytopathies' (MC) and encompass a group of disorders characterized by mutations either in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) genes that encode for mitochondrial proteins [6]. Mitochondrial cytopathies affecting the kidneys are broadly classified as [6, 7]:


Mitochondrial cytopathies can also present as:

1.Tubular defects affecting the:

	- a.Glomerular diseases
	- b.Tubulointerstitial nephritis
	- c.Renal cystic diseases
	- d.Neoplasia

#### **3. Mechanism of mitochondrial cytopathies**

Various studies indicate that mitochondrial dysfunction can arises due to disturbances in the regulation of the mitochondrial electron transport chain, proton gradient, and membrane potential [4, 7]. These disturbances lead to reduction in concentration of adenosine triphosphate (ATP) and increase in production of mitochondrial-derived reactive oxygen species (mROS). These reactive oxygen species promote kidney injury and inflammation [4, 7, 8].

Structural changes of mitochondrial swelling and fragmentation occur earlier than rise in serum creatinine which is largely used as a marker for kidney injury. These changes also indicate that impaired mitochondrial metabolism is directly linked to the deterioration of kidney function [4–9].

Inherited forms of mitochondrial cytopathies are associated with fair number of mutations with mitochondrial DNA (mtDNA) as many nuclear genes are responsible for proper maintenance of mtDNA. Mutations in these genes cause quantitative (mtDNA depletion) and qualitative defects (mtDNA deletions) in mtDNA leading to renal impairment [10].

The equilibrium between mitochondrial fusion and fission maintains the healthy mitochondrial structure and functions [4, 11]. Disruption of this balance leads to mitochondrial fragmentation, loss of mitochondrial DNA (mtDNA) integrity, and cell death [12, 13].

Mitochondrial cytopathies encompasses a group of disorders characterized by mitochondrial or nuclear DNA mutations in genes encoding for mitochondrial proteins [7]. Mitochondrial dysfunction, characterized by a loss of efficiency in the electron transport chain and reductions in the synthesis of high-energy molecules, such as adenosine-5′-triphosphate (ATP), is characteristic of aging, and essentially, of all chronic diseases [1–4]. Mitochondrial dysfunction arises from an inadequate number of mitochondria, an inability to provide necessary substrates to mitochondria, or a dysfunction in their electron transport and ATP-synthesis machinery [10, 11].

The number and functional status of mitochondria in a cell can be changed by [10, 12, 14].


#### **3.1 Fission and fusion**

The mitochondrial homeostasis is maintained because of the balance between fission and fusion. Fission leads to production of short rods or spheres whereas fusion leads to production of long and filamentous mitochondria. The balance between the two processes is disrupted under stress that leads to mitochondrial fragmentation. The both two processes are mediated by following factors: [15–19]


#### **3.2 The process of fission**

Fission is regulated by two main mediators: Drp1 and Fis1. The Drp1 is a GTPase of dynamin superfamily and is mainly present in the cytoplasm and later localizes to the outer membrane of the mitochondria. It has been seen that this shuffling of Drp1 is regulated by phosphorylation, ubiquilation and sumoylation.

Fis1 is a small membrane protein anchored at the outer mitochondrial membrane and overexpression of Fis1 promoted mitochondrial fission causes fragmentation of the mitochondria.

#### **3.3 The process of fusion**

Mitochondrial fusion is mediated by mitofusin 1 (Mfn1), mitofusin 2 (Mfn2) and optic atrophy factor 1 (OPA1). All three proteins are GTPases belonging to dynamin superfamily like Drp1. Mfn1 and Mfn2 are also localized to outer mitochondrial membrane whereas OPA1 is present on the inner mitochondrial membrane.

#### **3.4 Mitophagy**

Mitophagy, an autophagy process by which dysfunctional or superfluous mitochondria are selectively eliminated. Defective mitophagy has been implicated in various human diseases, such as aging, neurodegenerative disease, cardiovascular disease, cancers and many other renal diseases. Altered mitophagy related mechanisms are implicated in the pathogenesis of acute kidney injury, diabetic kidney disease, and lupus nephritis. The process includes initiation, priming of mitochondria for recognition by autophagy machinery, formation of the autophagosome, followed by lysosomal sequestration and hydrolytic degradation [17–19].

Mitophagy as described by Palikaras, can be described as three types: basal, programmed and stress-induced. Basal mitophagy is a steady-state, continuous, process responsible for elimination and recycling of aged and damaged mitochondria. This type of mitophagy exhibits tissue-specific distribution, with low levels in the thymus and high levels in the heart and kidneys [20, 21]. Stress induced mitophagy facilitates mitochondrial quality control to mediate metabolic adjustments to external challenges [20, 21].

Mitophagy is largely explained by molecular pathways and is mediated by either PINK1/Parkin-pathway or via the receptors. Mitophagy receptors are localized in the outer and inner mitochondrial membranes, and can directly induce mitophagy. Proteins that promote mitophagy are FUN14 domain-containing protein 1, BNIP3 and BCL2 interacting protein 3 like, and FKBP prolyl isomerase 8 [22–24].

Recently it was described that BNIP3/NIX, atypical members of the pro-apoptotic BCL2 family, contain an atypical BH3 domain which under hypoxic stress, get upregulated by hypoxia-inducible factor 1 (HIF-1). This in turn causes initiation of LC3 dependent mitophagy and overproduction of mtROS overproduction [4, 6, 9, 20, 23].

Overall, it is believed that impairment of mitophagy is responsible for mitochondrial dysfunction and progressive accumulation of defective organelles, leading to cell death and tissue damage. Blockade of mitophagy leads to the accumulation of damaged, ROS-generating mitochondria which activate the NLRP3 inflammasome [25].

Thus, mitochondrial cytopathies result due to disturbances in the process related to mitophagy or due to imbalance between the processes of fusion and fission.

#### **4. Mitochondrial dysfunction and kidney injury**

In this section we will discuss about the diseases that affect the kidney due to mitochondrial dysfunction. As described before renal mitochondrial cytopathies can manifests either as glomerular or tubular diseases, or as renal cysts or neoplasia.

#### **4.1 Glomerular involvement in renal mitochondrial cytopathies**

#### *4.1.1 Diabetic nephropathy*

Diabetic nephropathy results from microvascular complications, leading to chronic kidney disease that develops in approximately 30% of patients with type 1 diabetes mellitus (DM1) and approximately 40% of patients with type 2 diabetes mellitus (DM2) [26–28]. Various mitochondrial defects seen include impaired respiratory chain functions, structural and networking abnormalities, disrupted cellular signaling and increased reactive oxygen species generation [4, 29].

Coughlan et al. demonstrated that a deficiency in apoptosis inducing factor (AIF) results in changes in mitochondrial function, networking, and production of reactive oxygen species that precipitate renal disease. Along with the diabetic milieu, switch from mitochondrial fusion to fission, impaired OXPHOS, and a depleted mitochondrial ATP pool, all accelerate towards a more advanced renal injury [30].

Studies have implicated impaired mitophagy as the cause of mitochondrial dysfunction in diabetic kidneys and also showed that with progression of disease, concomitant accumulation of fragmented and swollen mitochondria occurs [22, 30, 31].

Experimental studies have shown that there is decrease in PINK1 and Parkin in the tubules of diabetic mice [32]. In study on streptozotocin-induced diabetic rat models, the authors demonstrated that in early stages of diabetes there is increase in expression of PINK1 in the renal cortex. This provided an evidence that mitophagy could be activated to clear dysfunctional mitochondria from the kidney during early diabetes and as the disease progresses there is accumulation of fragmented mitochondria and induction of cell death [4]. Thioredoxin-interacting protein (TXNIP) - dependent activation of the mammalian target of rapamycin (mTOR) signaling pathway contributes to dysfunctional mitophagy in the diabetic kidney [4, 30, 33, 34].

Studies have evaluated presence of cell-free mtDNA in urine in patients of diabetic nephropathy and reported an inverse relationship in levels of urinary mtDNA and intra-renal mtDNA leading to increase in interstitial fibrosis and reduction in estimated glomerular filtration rate (eGFR) [35].

It has also been demonstrated that damaged mitochondria generate excess mitochondrial superoxide, and glycation of mitochondrial proteins also contributes to mROS generation. Advanced glycation end products as well as the receptors for these, play a vital role in generation of ROS that contribute in progression of diabetic nephropathy [33, 36].

#### *4.1.2 IgA nephropathy (IgAN)*

IgAN is one of the most common glomerulonephritis and a leading cause of CKD that can progress to ESRD. Kidney biopsy from a patient with IgAN may show varied morphological affection ranging from mesangial proliferation to focal segmental mesangial sclerosis, crescents with dominant mesangial IgA deposition [4, 37]. The disease is characterized by presence of circulating and glomerular immune complexes comprised of galactose-deficient IgA1, an IgG autoantibody directed against the hinge region O-glycans, and C3 [38]. Nishida et al. demonstrated an increased number of abnormal mitochondria in the proximal tubular epithelial cells and an elevated urinary mtDNA levels in patients with IgAN. An association between five common single-nucleotide polymorphisms and ESRD, suggests that mitochondrial defects have an essential role in the progression to CKD in patients with IgAN [4, 39]. Interestingly, higher expression and interaction between the mitochondrial protein induced in high glucose-1 (IHG-1) and cold shock protein Y-box binding protein-1 are associated with renal inflammation, tubulointerstitial inflammation, and glomerulosclerosis in IgAN. Defects in the mitochondrial genome and functions play a critical role in worsening glomerular inflammation and disease progression [40].

#### *4.1.3 Polycystic kidney disease*

Autosomal dominant PKD (ADPKD), is characterized by presence of multiple cysts in the renal parenchyma and is associated with mutations in the genes PKD1 and PKD2, which encode polycystin 1 (PC1) and PC2, respectively [41]. The PC1-PC2 complex modulates mitochondrial Ca2+ uptake and directly regulate oxidative phosphorylation and indirectly affect mitochondrial function by maintaining the mtDNA copy number and mitochondrial morphology [42]. Mutations in PKD1 and PKD2 lead to mitochondrial dysfunction and metabolic imbalance. Proinflammatory cytokine TNFα promote cyst formation, increased MCP-1 in cyst-lining cells and excretion of urinary MCP-1, and renal profibrotic macrophages in experimental ARPKD which might be associated with defects in mitophagy are also reported in patients with ADPKD. Loss of PC2 enhanced mitochondrial Ca2+ uptake, mitochondrial bioenergetics, and mitochondrial-ER tethering associated with increased Mfn2, and knockdown of Mfn2 rescued ER-dependent mitochondrial Ca2+ signaling are associated with reduced cyst proliferation [4]. Mitochondria of cyst-lining cells in the kidney of a mouse model of ADPKD display morphological abnormalities and decreased mtDNA. There is reduced peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), which regulates mitochondrial biogenesis. Functional mitochondrial abnormalities and increased mROS production indicate that mitochondrial dysfunction plays a functional role in cystogenesis [4, 43].

#### *4.1.4 Lupus nephritis*

Renal involvement in systemic lupus erythematosus (SLE) occurs in 40–50% of adult patients, and results in end stage renal disease (ESRD) about 10% of patients despite modifications in therapeutic strategies [44]. LN is the most common severe manifestation of systemic lupus erythematosus. The pathogenesis of LN is multifactorial, and includes aberrant T-cell and B-cell signaling, autoantibody production, and deregulated cytokine secretion. Various genetic as well as environmental factors also contribute [45, 46]. The T-cells of SLE show increased mitochondrial mass

#### *Mitochondrial Cytopathies of the Renal System DOI: http://dx.doi.org/10.5772/intechopen.96850*

(megamitochondria), mitochondrial hyper-polarization, and ATP depletion which lead to aberrant activation and enhanced necrosis of T-cells. This leads to release of extracellular mitochondria and their components and are recognized as damageassociated molecular patterns (DAMPs) that initiate innate and adaptive immune responses to elicit an inflammatory response that triggers organ damage [47]. Nitric oxide (NO)-dependent mitochondrial biogenesis could account for megamitochondria leading to sustained T-cell activation. On the other hand, increased T-cell mitochondria in SLE have also been attributed to insufficient mitophagy. Sequestration and successful clearance of damaged mitochondria by mitophagy suppresses mtROS accumulation, prevents inflammation and generation of autoantigens by intracellular oxidation suggesting that mitophagy is a potential therapeutic target for SLE and LN [46–48].

Gkirtzimanaki et al. observed that IFNα damages mitochondrial metabolism and mediates lysosomal dysfunction, impeding mitochondrial clearance and leading to cytosolic accumulation of mtDNA in monocytes [49]. Caspase-1 gets activated in the podocytes of both lupus nephritis patients and lupus-prone mice and inhibit mitophagy and amplify mitochondrial damage, mediated by cleavage of the key mitophagy regulator Parkin in lipopolysaccharide (LPS)-primed bone-marrow-derived macrophages [50].

Drp1, fusion mediator of mitochondria, is reduced in T cells from SLE patients and lupus-prone mice, concomitant with the accumulation of mitochondria.

Mitochondrial hyperpolarization and reactive oxygen intermediates production have been detected in peripheral blood T-lymphocytes from SLE patients, together with diminished levels of intracellular ATP, indicating dysfunction in mitochondria of T-cells in patients with lupus nephritis. CD4þT cells from SLE exhibit an increased mitochondrial mass and size due to increased mitochondrial biogenesis and defective mitophagy [51].

#### *4.1.5 Membranous nephropathy & focal and segmental glomerulosclerosis*

Membranous nephropathy (MN) is a most common cause of adult nephrotic syndrome. Various podocytic autoantigens have been implicated in the pathogenesis of the disease. Phospholipase A2 receptor (PLA2R) is the major autoantigen on podocytes in primary MN, whereas thrombospondin type-I domain-containing 7A (THSD7A) is the minor antigen, the antibodies to which are predominantly of the IgG4 subclass [52].

Cultured podocytes when exposed to sera from patients with MN revealed mitochondrial fragmentation, loss of membrane potential, and mROS production [53]. Patients with MN also show increased glomerular mitochondrial fission proteins, DRP1, phosphorylated-DRP1 (Ser-616), and FIS1. The observation of these studies show that podocytic injury in MN is secondary to mitochondrial dysfunction [53].

Focal segmental glomerular sclerosis (FSGS), also a common cause of nephrotic syndrome in pediatric as well as adults, is one of the major renal complication of mitochondrial cytopathies.

The mitochondrial DNA (mtDNA) encodes for 13 structural genes of OXPHOS enzymes, two ribosomal RNAs, and 22 transfer RNAs. Glomerular involvement of an A-to-G transition at mtDNA position 3243 in the gene for tRNALeu(UUR) has been implicated in FSGS. Recent studied with a mouse model carrying mutant mtDNA with a 4696-bp deletion, developed focal and segmental glomerulosclerosis and died within 6 months due to renal failure [54, 55].

Puromycin aminonucleoside nephrosis (PAN) model to study FSGS reveals reduction of respiratory chain enzymatic activities, oxygen consumption, and the swelling of renal tubular mitochondria [56]. Reduction of the intraglomerular mtDNAencoded protein, COX I, suggests that there is either an induction of mtDNA damage or a reduction in mtDNA copy number during the progression of PAN. Several studies have described mitochondrial dysfunction and/or mtDNA changes in glomerular diseases like accumulation of oxidative damage of mtDNA in the kidney of streptozotocin-induced diabetes rats, and downregulation of respiratory chain complex in patients with the congenital nephrotic syndrome of the Finnish type [57].

MCs comprise one of the causes of primary FSGS, among which mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke like episodes (MELAS) syndrome account for a large proportion. MELAS syndrome is mainly caused by point mutations in the *MTTL1* gene, encoding mitochondrial tRNALEU. Renal biopsies from patients with coexistence of MELAS and FSGS often manifest with numerous dysmorphic mitochondria in podocytes and effacement of foot processes [58].

#### *4.1.6 Tubular defects and role of mitochondria*

Renal tubules comprise one of the major victims of MCs, of which the most frequently reported is proximal tubular defects. Proximal tubular cells are relatively vulnerable to oxidative stress and are therefore apt to suffer from respiratory chain defects. Renal tubule defects mainly manifest as loss of electrolytes and low-molecular-weight proteins, which are frequently characterized as Fanconi syndrome and Bartter-like syndrome. Patients with mitochondrial tubulopathy are usually accompanied by myoclonic epilepsy and ragged red muscle fibers (MERRF), and Pearson's, Kearns-Sayre, and Leigh syndromes. The majority of genetic mutations detected in these diseases are fragment deletions of mtDNA [6, 7, 59–61].

#### *4.1.7 Acute kidney injury (AKI)*

AKI is defined as an abrupt (within hours) decrease in kidney function, which encompasses both injury (structural damage) and impairment (loss of function). AKI is common (8–16% of hospital admissions) and many aspects of its natural history remain uncertain [62]. Classification of AKI includes pre-renal AKI, acute post-renal obstructive nephropathy and intrinsic acute kidney diseases. Of these, only 'intrinsic' AKI represents true kidney disease, and the most common etiologies are toxins, ischemia, sepsis and obstructive injury [63]. Disruption of mitochondrial integrity in renal tubular cells is considered as the common findings in all forms of AKI [64]. In AKI, mitochondrial damage contributes critically to sublethal and lethal injury of kidney tubules, and the consequent loss of renal function. In various models of AKI, mitochondrial dynamics are disrupted, resulting in mitochondrial fragmentation, membrane permeabilization, mitochondrial dysfunction, energetic failure, and ROS production [9]. There is decreased antioxidant defenses, injured mitochondrial respiration, intrarenal inflammatory response and oxidative stress along with downregulation of protein expression during mitochondrial metabolism and decreased oxygen are seen [65]. Elevated mitochondrial DNA levels in the urine has been considered as a novel non-invasive biomarker for detecting mitochondrial dysfunction. Eirin et al. revealed that increased urinary mtDNA (UmtDNA) in hypertensive patients correlated with other biomarkers of renal dysfunction and glomerular hyperfiltration [66, 67]. Derangements of mitochondrial integrity may be associated with the

#### *Mitochondrial Cytopathies of the Renal System DOI: http://dx.doi.org/10.5772/intechopen.96850*

detectable release of UmtDNA in sepsis-induced AKI has never been determined. Sepsis activates several pathological mechanisms linked to mitochondria, including hypoperfusion, oxidative stress, and the inflammatory response. Ultrastructural changes observed in the kidney tubular cells include mitochondrial impairment, swelling and cellular death. Disruption of mitochondrial integrity in the renal tubular epithelial cells leads to release of mitochondrial DAMPs into the urine which can be used as a surrogate biomarker of renal mitochondrial damage [68].

Expression of genes involved in oxidative phosphorylation are reduced as demonstrated by Parikh et al [69]. There is proportional decrease in expression of PGC-1a expression with reduction of renal function. Activation of PGC-1a promotes recovery from AKI caused by sepsis. cGAS–STING pathway activation is involved in autoimmune and inflammatory reactions, that activate by self-genomic DNA damage. Cyclic GMP–AMP synthase (cGAS) is a pattern recognition receptor that recognizes doublestranded DNA in the cytoplasm and then binds to the trans-membrane protein, a stimulator of interferon genes (STING) localized on the endoplasmic reticulum (ER). A relationship between mitochondrial damage and induction of cGAS–STING pathway in inflamed proximal tubular cells has been postulated in Cisplatin induced AKI. In ischemic and cisplatin nephrotoxic AKI, the fusion-fission mitochondrial dynamics in proximal tubules reveal that mitochondrial fission initiated by Drp1 occurs immediately after the injury [69–72].

#### *4.1.7.1 Mitophagy and acute kidney injury*

Recent literature suggests that mitophagy is involved in the pathophysiological processes of AKI. PINK1/Parkin-mediated mitophagy has a protective role for mitochondrial quality control in the context of tubular cell survival and function. Tang et al. demonstrated both PINK1 and Parkin are upregulated in renal tubular epithelial cells during ischemic AKI *in vitro* and *in vivo*, PINK1 and/or Parkin deficiency results in increased mitochondrial damage, ROS production, and inflammation causing increased tubular damage and aggravated AKI [73]. Boston University mouse proximal tubular cell line (BUPMT cells) show upregulation of BNIP3 following oxygen–glucose deprivation-reperfusion, and in kidney tissues of mouse models. BNIP3-deficient mice renal tubular epithelial cells show accumulation of damaged mitochondria, increased ROS production, enhanced cell apoptosis, and inflammation. These findings strongly support the involvement of multiple mitophagy regulatory pathways in the pathogenesis of AKI [74].

Wang et al. stated that Bax inhibitor-1 (BI1) promotes mitochondrial retention of PHB2 and improves mitophagy, preserving mitochondrial homeostasis in a murine AKI model. He also demonstrated that renal functional loss, tissue damage, and apoptosis are aggravated in cisplatin-treated *Pink1*−/− and *Parkin*−/− mice relative to cisplatin-treated wild-type mice, suggesting that activation of PINK1/Parkinmediated mitophagy plays a protective role against cisplatin nephrotoxicity [3]. A recent study by Zhu et al. demonstrated that trehalose administration attenuates mitochondrial dysfunction through activating transcriptional factor EB (TFEB) mediated autophagy and mitophagy in cisplatin-induced AKI *in vitro* and *in vivo*. The study sheds lights on the roles of TFEB on mitophagy and provides a novel promising therapeutic target for AKI [75].

Notably, preservation of mitochondrial dynamics, prevention of mitochondrial membrane permeabilization, and/or promotion of mitochondrial biogenesis can protect kidney tubular cells and tissues in AKI.

#### *4.1.8 Tubulointerstitial fibrosis*

Tubulointerstitial fibrosis follows following aberrant kidney repair following AKI, eventually progressing to CKD. Suppression of the proinflammatory cytokines interleukin (IL)-18 and IL-1β and nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation, inhibits progression to CKD following prolonged ischemia. Studies indicate that mitochondrial dysfunction plays a role in inflammation leading to tubulointerstitial fibrosis and development of end-stage renal disease. Analysis of genome-wide transcriptome-based analyses revealed that human fibrotic kidneys have lower expression of various mitochondrial enzymes and regulators of fatty acid oxidation along with higher intracellular lipid deposition. Fibrosis is mediated by monocyte chemoattractant protein 1 (MCP-1), a chemokine that promotes the infiltration of monocytes, inflammation, and fibrosis, the levels of which are increased with decreased renal expression of mitophagy regulators (PINK1, MFN2, and Parkin) in experimental and human kidney fibrosis. Mitophagy impairment led to an accumulation of abnormal mitochondria, augmented macrophage induced fibrotic response, superoxide production, and reduced ATP synthesis. Deficiency of mitophagy by Pink1 or Park2 gene deletion markedly increased mROS production and mitochondrial damage, which worsened renal fibrosis. These effects were rescued by a mitochondria-targeted antioxidant. Defective mitochondrial metabolism and reduced expression of mitophagy regulators have been shown to enhance the renal inflammatory and fibrotic responses and mediate the progression of CKD [4, 76–78].

### **Author details**

Lovelesh K. Nigam\*, Aruna V. Vanikar, Rashmi Dalsukhbhai Patel, Kamal V. Kanodia, Kamlesh Suthar and Umang Thakkar Department of Pathology, Laboratory Medicine, Transfusion Services and Immunohematology, G.R. Doshi and K.M. Mehta Institute of Kidney Diseases and Research Centre and Dr. H.L. Trivedi Institute of Transplantation Sciences, Ahmedabad, India

\*Address all correspondence to: drloveleshnigam@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Mitochondrial Cytopathies of the Renal System DOI: http://dx.doi.org/10.5772/intechopen.96850*

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#### **Chapter 4**

## Maneuvering Mitochondria for Better Understanding of Therapeutic Potential of mtDNA Mutation

*Sanket Tembe*

#### **Abstract**

Heterogeneity of mitochondrial diseases in terms of genetic etiology and clinical management makes their diagnosis challenging. Mitochondrial genome, basic mitochondrial genetics, common mutations, and their correlation with human diseases is well-established now and advances in sequencing is accelerating the molecular diagnostics of mitochondrial diseases. Major research focus now is on development of mtDNA intervention techniques like mtDNA gene editing, transfer of exogenous genes (sometimes even entire mtDNA) that would compensate for mtDNA mutations responsible for mitochondrial dysfunction. Although these genetic manipulation techniques have good potential for treatment of mtDNA diseases, research on such mitochondrial manipulation fosters ethical issues. The present chapter starts with an introduction to the factors that influence the clinical features of mitochondrial diseases. Advancement in treatments for mitochondrial diseases are then discussed followed by a note on methods for preventing transmission of these diseases.

**Keywords:** mitochondrial diseases, mtDNA intervention techniques, mitochondrial donation, genomics advancements, reproductive techniques

#### **1. Introduction**

Mitochondria are synonymized with energy thanks to their ability to produce most of the Adenosine Triphosphate (ATP) through the process of Oxidative Phosphorylation. In addition to ATP production, several metabolic processes like tricarboxylic acid cycle (TCA), fatty acid oxidation, ketogenesis, urea cycle (partly), heme and phospholipid synthesis take place in mitochondria [1, 2]. Role of mitochondria in cell death (apoptosis) is also well-established [3]. Recent research suggests new role of mitochondria in calcium homeostasis, iron and copper metabolism and inflammation and immunity [4]. Though oxidative phosphorylation puts aerobes at higher level in terms of efficiency of energy production, one unpleasant consequence of this important process is production of reactive oxygen species (ROS) also known as mitochondrial ROS (mtROS). The culprit for formation of these reactive species

is proton leak at the inner mitochondrial membrane. Formation of such species pose great threat to mitochondrial DNA (mtDNA) and may lead to mitochondrial dysfunction [5]. Once thought to be uncommon, mtDNA diseases are now known to be quite prevalent and their definition is no more restricted to defects in oxidative phosphorylation alone but also include defects in molecular processes like mitochondrial fission, fusion and translation [6–8].

The list of common mitochondrial diseases and syndromes is quite lengthy that include mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS syndrome), Leber's hereditary optic neuropathy (LHON), myoclinic epilepsy with ragged-red fibers (MERRF), Leigh syndrome and Pearson syndrome, Kearne-Sayre syndrome (KSS), chronic progressive external ophthalmoloplegia (CPEO) and neuropathy, ataxia and retinitis pigmentosa (NARP) [9–17]. Mutations in the mitochondrially-encoded genes are the most common cause of these diseases. Several mutations have been reported such as m.3243A > G, m.3271 T > C, m.1642G > A, m.9957 T > C, m.3272 T > C, m.1642G > A, m.1277A > G, m.13045A > C, m.13513G > A and m.13514A > G (all reported in MELAS [18–26]), m.8344A > G, m.8356G > A, m.3291 T > C, m.4279A > G (reported in MERRF [27–29]), G3460A, T14484C in LHON [30]. Recent review describes a comprehensive approach to study mitochondrial disorders caused by mutations through an example of m.3243 A > G [31].

Reviews on basic mitochondrial genetics, mutations and their correlation with human diseases are available [32–34]. Starting with unique features of mitochondria that decide the clinical presentation of mitochondrial diseases, this review focusses on advancement in mitochondrial DNA manipulation. Methods for preventing transmission of these diseases are discussed at the end.

#### **2. Factors that govern clinical features of mitochondrial diseases**

#### **2.1 Heteroplasmy**

Presence of several thousand copies of mitochondrial genome (mtGenome) per cell creates two conditions; homoplasmy and heteroplasmy. When all copies of mtGenome are identical, the scenario is described as homoplasmy. Heteroplasmy is a situation in which more than one mtDNA variants exist between the cells of an individual or within a same cell. Often this is due to de novo mutations either in germ line or in somatic cells. As a result, mitochondrial dysfunction can be seen only in specific cells, tissues, or organs. The rate at which the regions in the mtGenome evolves is much higher than that of nuclear genes. This reduces the possibility of all mtDNA molecules to be identical in an individual's cells. Considering the large copy number of mtGenome present, detection of mtDNA mutation is difficult until it is spread among enough mtDNA molecules in a given cell. Only when mutated mtDNA exceeds threshold levels, clinical consequences of such mutations are seen [35]. Absence of fixed functional threshold level makes the analysis of mtDNA results even more complicated. Variations in threshold frequencies have been reported for different types of tissues and mtDNA mutations.

#### **2.2 Mitochondrial DNA bottleneck**

Mitochondrial genome, unlike its nuclear counterpart, shows uniparental transmission. Considering a single-parent origin, theoretically, mitochondrial DNA of a *Maneuvering Mitochondria for Better Understanding of Therapeutic Potential of mtDNA… DOI: http://dx.doi.org/10.5772/intechopen.96915*

mother and her progeny should not show any variations. But, in reality, extensive variations have been reported in humans [36, 37]. Accumulation and enrichment of mutant mitochondria thus suggests presence of mitochondrial bottleneck; a concept that describes why mtDNA of an embryo may differ significantly from that of its mother [38].

#### **3. Manipulation of mitochondrial DNA**

Diagnosis and monitoring clinical progression of mtDNA diseases is difficult due to multi-copy nature of mtGenome. Fortunately, many harmful mtDNA mutations are heteroplasmic and this paves the way for curing these disorders. If mutated copies of mtDNAmolecules can be removed selectively from the pool of wild type molecules, heteroplasmy can be reduced and cellular biochemical defects can be cured. However, manipulating heteroplasmy has been challenging due to several barriers. Some of these barriers and attempts to overcome them are discussed in this section.

#### **3.1 First barrier: difficulty in mitochondrial transfection**

Mitochondria have two lipid bilayers that includes outer and inner membranes. While outer membrane allows easy transport of small molecules like ATP, proteins less than 10 KDaand ions, the inner mebrane brings selectivity barrier. Hydrophilic molecules cannot cross this barrier due to presence of cardiolipin; a hallmark mitochondrial lipid with four alkyl tails. It is this impermeability of inner membrane to the hydrophilic molecules that makes the passage of DNA through mitochondrial membranes difficult.

#### **3.2 Strategies to overcome mitochondrial membrane barrier**

One of the effective ways to treat mitochondrial diseases is to introduce wild type genes into the mitochondria. The approaches for introducing genes can be broadly classified into three categories namely physical, chemical, and biological methods. Physical methods are relatively simple and straightforward. Methods like microinjection, particle bombardment, electroporation and sonication have been used for delivering exogenous genes into the mitochondria [39, 40]. Separate carrier molecules are not required in these methods which eliminates the toxicity problems of such molecules. However, drawbacks of these methods include random distribution of DNA in mitochondrial matrix and the risk of damage of target cell during cell membrane penetration [40].

Many chemical-based methods have been reported for mitochondrial gene delivery. Considering hydrophobicity and presence of negative charges on mitochondrial membrane, cationic and amphiphilic carrier molecules have been used to enclose the negatively charged DNA [41]. Plasmid DNA was introduced to mitochondria using rhodamine-pDNA-nanoparticle complex [42] where the dye facilitated movement of nanoparticles across the plasma membrane and mitochondrial membrane. Mitochondria-specific liposomes were used for successful release of plasmid DNA in mitochondrial matrix [43], however, certain limitations like cytotoxicity and low transfection efficiency were noted. Improved version of liposome-based nanocarrier came in the form of MITO-Porter [44, 45]. Current research focuses on improving

the mitochondrial targeting and reducing the toxicity to target cells. New ligands are being explored and linked to chemically synthesized carrier molecules that target the mitochondrial receptors.

Understanding of mitochondrial targeting signal peptide (MTS)-mediated translocation has provided a new biological approach for specific mitochondrial gene delivery. Carrier molecules having DNA-binding ability were conjugated to MTS. DNA oligomer peptide nucleic acid (PNA) that has polyamide bond rather than usual sugar-phosphate backbone, was conjugated to MTS and this MTS-mediated PNA could successfully enter the mitochondrial matrix through the translocase of outer membrane (TOM) and that of inner membrane (TIM) [46, 47]. Though this approach has some shortcomings like low mitochondrial targeting (as PNA tends to be localized in nucleus) and the restricted size of genes-to be-transferred, this is a clear indication that MTS can be successfully applied in mitogene delivery in near future. Use of viral vectors, especially adeno-associated virus (AAV), have been tested for mitochondrial gene delivery [48]. The wild type human mitochondrial genes were added to MTS-AAV complex to compensate mutated and defective NADH ubiquinone oxidoreductase subunit 4 (ND4) gene which is the culprit for LHON [49]. In addition to these physical, chemical, and biological methods, there are several combinatorial approaches that have been tested. A recent review [50] gives details of these methods and also discusses the need for new approaches.

#### **3.3 Barrier 2: eliminating mutant mtDNA molecules**

Elimination of mutant mtDNA molecules can reduce the threshold of mutant molecule load. Total elimination of mutant mtDNA is not required because a small reduction in mutant mtDNA load just below the threshold can improve the clinical scenario of a diseased person.

#### **3.4 Strategies to selectively target mutant molecules**

Construction and characterization of mitoApaLI; one of the several mitochondriatargeted restriction endonucleases developed so far, and its significant role in shifting heteroplasmy towards one of the two mtDNA haplotypes is explained in detail in a recent book chapter [51]. The prerequisite (also a limiting factor) of using mitoREs is that the target mutation should result in a unique restriction site to avoid breaking of wild type mtDNA. Different methods of mitochondrial transfection and strategies to deal with heteroplasmy are summerized in **Figure 1.**

Two recent gene editing systems namely mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) and mitochondria-targeted zinc finger nucleases (mtZFN) can selectively target single nucleotide mutations and can degrade them. Minczuk and Gammage laboratories have extensively used mtZFN to shift heteroplasmy [52, 53]. The mitoTALENs have been used to target specific mutations from animal and human-derived cells [54]. Although these gene therapy approaches are quite promising, we need to be careful because of the risk involved in this approach. mtDNA copy number may go down significantly and there may be undesirable off-target effects while attempting elimination of mutated copies. Crisper-Cas 9 cannot be used for this purpose because it needs single-guide RNA for gene editing and RNA import in mitochondria is restricted [55].

*Maneuvering Mitochondria for Better Understanding of Therapeutic Potential of mtDNA… DOI: http://dx.doi.org/10.5772/intechopen.96915*

#### **Figure 1.**

*The figure is a schematic representation of different methods of mitochondrial transfection and strategies to deal with heteroplasmy. Abbreviations used in the figure are: Outer mitochondrial membrane (OMM), intermembrane space (IMS), inner mitochondrial membrane (IMM), mitochondrial target signal peptide (MTS), translocase of outer membrane (TOM), translocase of inner membrane (TIM), adeno-associated virus (AAV) and mitochondrial DNA (mtDNA).*

#### **4. Decrease in NAD+ levels**

Nicotinamide adenine dinucleotide oxidized (NAD+ ) is a coenzyme required for action of many enzymes like polyADP ribose polymerase (PARP) and sirtuin deacetylases. Substantial decrease in NAD+ concentration and the ratio of NAD+ /NADH was reported in the cells having defective mitochondria [56]. Defective respiratory chain cannot reoxidize NADH to oxygen. This results in reduction of pyruvate to lactate by lactate dehydrogenase generating NAD+ . Transport of excess lactate outside the cell leads to lactate acidemia, which is a common feature of mitochondrial diseases. Increasing the cellular levels of NAD+ either through supplementation or through bringing changes in enzymes involved in its synthesis have been reported [57].

A recent approach tested in mice was to reoxidize extracellular lactate to pyruvate and bring it back to the cell for its re-reduction by lactate dehydrogenase thus increasing NAD+ /NADH ratio [58].

#### **5. Prevention of transmission of mitochondrial diseases**

#### **5.1 Options to prevent transmission**

Mitochondrial DNA is maternally inherited and genetic bottleneck makes it even more peculiar. Therefore, options different from those with nuclear genetic defects must be considered. It is important to know which mutation a woman carries and its level; especially in those cases who harbor heteroplasmic mtDNA mutations. Genetic diagnosis and expert counseling is invaluable for such cases. Post-counseling options include voluntary childlessness and adoption. Prenatal testing and preimplantation genetic diagnosis (PGD) are recently available alternatives. PGD includes in vitro fertilization (IVF) and embryo development to blastocyst stage. Because of inherent issues with IVF, PGD has limited chance to succeed.

#### **5.2 Mitochondrial replacement therapy (MRT) or mitochondrial donation**

MRT is probably the only way available to those couples who are suffering from mitochondrial disease and wish to have a healthy child. In such cases, nucleus is taken from a mother carrying defective mitochondria and transferred to an enucleated oocyte or egg of a woman with healthy mitochondria. Embryo formed after this procedure (also called as three parent embryo) will have nuclear DNA from both parents but mitochondrial DNA from another mother. Ideally such embryo should be free from defective mitochondria. Using this technique in human oocytes, good quality embryos could be formed as reported by several workers [59, 60]. Though potentially this is a great advancement, mitochondrial donation may raise ethical issues [61]. Also some workers observed that the nucleus which was transferred to enucleated oocyte/egg showed presence of contaminating defective mitochondria. Enrichment of such contaminating mitochondria may cause mitochondrial disease in individuals generated through MRT. This issue becomes more sensitive when female embryos are generated after MRT because they will be passing on their defective mitochondria to the next generation. MRT females may show same mitochondrial disease and infertility as their mothers. In future, better understanding of maternal inheritance of mitochondria will improve the efficacy of this therapeutic method and make it a sustainable approach for betterment of individuals across the generations. Another issue that may hamper the progress of mitochondrial donation is availability of oocyte donors because this involves hormonal treatment.

#### **6. Conclusions**

Advances in DNA sequencing are accelerating the diagnosis of mitochondrial diseases and helping in assessment of heteroplasmy levels. Although molecular diagnosis is crucial, it can only identify the problem but cannot solve it. Input from reproductive biologists are equally important for comprehensive analysis and personal care of diseased individuals. Development of new treatments through further advancements in gene therapy holds great promise for the sufferers of mitochondrial disease.

*Maneuvering Mitochondria for Better Understanding of Therapeutic Potential of mtDNA… DOI: http://dx.doi.org/10.5772/intechopen.96915*

#### **Acknowledgements**

I am grateful to the Principal, Fergusson College (Autonomous) and Head, Department of Biotechnology, Fergusson College for their support. I thank my colleague Monica Joshi for her help in proofreading the manuscript. I am thankful to my friend Mithila Shukla from Purdue University for sending me research articles. I also thank my undergraduate student Hrishikesh Hardikar for his help in preparing illustration and in formatting-related work.

### **Author details**

Sanket Tembe Department of Biotechnology, Fergusson College (Autonomous), Pune, India

\*Address all correspondence to: sankettembe@fergusson.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 2
