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Biochimica et Biophysica Acta 1842 (2014) 1198–1207

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Biochimica et Biophysica Acta

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Review Mitochondrial changes and neurodegenerative diseases☆

Milena Pinto a,c,CarlosT.Moraesa,b,c,⁎

a Department of , University of Miami Miller School of Medicine, Miami, FL 33136, USA b Neuroscience Graduate Program, University of Miami Miller School of Medicine, Miami, FL 33136, USA c Department of Cell Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA

article info abstract

Article history: Mitochondria are essential within the cell where most of the energy production occurs by the oxida- Received 31 July 2013 tive phosphorylation system (OXPHOS). Critical components of the OXPHOS are encoded by the mitochondrial Received in revised form 6 November 2013 DNA (mtDNA) and therefore, involving this genome can be deleterious to the cell. Post-mitotic tissues, Accepted 8 November 2013 such as muscle and brain, are most sensitive to mtDNA changes, due to their high energy requirements and non- Available online 16 November 2013 proliferative status. It has been proposed that mtDNA biological features and location make it vulnerable to mu- tations, which accumulate over time. However, although the role of mtDNA damage has been conclusively con- Keywords: nected to neuronal impairment in mitochondrial diseases, its role in age-related neurodegenerative diseases mtDNA remains speculative. Here we review the pathophysiology of mtDNA mutations leading to and discuss the insights obtained by studying mouse models of mtDNA dysfunction. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases. © 2013 Published by Elsevier B.V.

1. Introduction All the proteins encoded by the mtDNA are components of the four OXPHOS multi-subunit complexes. Complex II is the only exception, 1.1. The mtDNA as all its components are encoded by the nDNA. The proteins involved in mtDNA transcription, translation and replication as well as the other 1.1.1. Structure OXPHOS components are all encoded by the nDNA, so that mutations in Mitochondrial DNA (mtDNA) was discovered in 1963 [1] and the these nuclear genes can also affect the stability of the mtDNA (Table 1). human mtDNA fully sequenced in 1981 [2]. The human mitochondrial The replication of mtDNA is independent from the cell cycle and, to genome is a circular, double-stranded, supercoiled molecule composed date, only few are known to participate in the process, among of 16569 bp and encoding for 37 genes. them, POLG (mitochondrial DNA polymerase γ, that has also a 3′–5′ Because of their nucleotide compositions, the DNA strands were exonuclease/proofreading activity), and Twinkle (a DNA helicase) are named “H-strand” and “L-strand”, as the heavy strand is rich in , particularly important, as mutations in these genes have been found and light strand is rich in cytosines. The H-strand encodes for 28 genes, in different mitochondrial diseases [4]. whereas the L-strands encodes for the remaining 9 (Fig. 1). mtDNA does not contain introns and the majority of the genome is composed by coding regions, with only a small non-coding portion 1.1.2. Mutations (1.1 kb), called the displacement loop (D-loop). The D-loop is essential for the mtDNA replication and transcription since it contains the origin The mtDNA is particularly prone to damage with a rate 10 of H-strand replication (OH) and the promoter regions of the two strands times higher than that of nDNA [5]. The reasons for this high susceptibility (HSP and LSP). The two polycistronic RNAs transcribed from the two are several. Unlike nDNA, mtDNA is organized in nucleoids but it is not strands are processed to obtain 22 tRNAs molecules, 2 mitochondrial protected by histones and its proximity to the mitochondrial respiratory rRNA and translated into 13 proteins [3]. chain (in particular Complex I and III) makes it also close to a source of radicaloxygenspeciesthatarewell-knowngenotoxicagents.Moreover, it replicates more often than the nDNA and the efficiency of repair mech- anisms appear to be less efficient than the one for nDNA [6]. POLG has an 3′–5′ exonuclease/proofreading activity and TFAM (mitochondrial transcription factor A) seems to also act as a chaperone protecting it ☆ This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial from oxidative damage [6]. Dysfunction, and Neurodegenerative Diseases. The first pathogenic mtDNA mutations were identified by Holt ⁎ Corresponding author at: Dept. of Neurology, University of Miami, 1420 NW 9th ave, Miami, FL 33133, USA. at al. and Wallace et al. in 1988 [7,8] and were considered to be E-mail address: [email protected] (C.T. Moraes). very rare in the total populations. In the last decades an increasing

0925-4439/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bbadis.2013.11.012 M. Pinto, C.T. Moraes / Biochimica et Biophysica Acta 1842 (2014) 1198–1207 1199

Fig. 1. Schematic representation of human mitochondrial DNA. Color codes represent: Grey, the two mitochondrial rRNAs; Yellow, the D-loop containing the H-strand origin of replication (OH); Light blue, subunits of the Complex I of the mitochondrial ; Orange, subunits of (Complex IV); Violet, subunits of the ATP synthase (Complex V); Green, the cytochrome b gene, which is part of the Complex III. The black arrows represent the mitochondrial tRNA genes. The red squares indicate the location of the most common mutations discussed in this review. NSHL: non-syndromic hearing loss; MELAS: Mitochondrial Encephalomyopathy, , and -like episodes; MIDD: Maternally Inherited ; LHON: Leber Hereditary Optic Neuropathy; MERRF: Myoclonic and Ragged Red Muscle Fibers; NARP: Neurogenic muscle weakness, , and Retinitis Pigmentosa. number of studies showed that the incidence of pathogenic mutations is 1.1.5. Pathogenic mtDNA changes much higher than previously expected (N1/200 live births), although in most cases they are present in low, non-pathogenic levels [9]. MtDNA defects can be maternally inherited or sporadic. Point muta- tions are, in general, maternally inherited and heteroplasmic, with an 1.1.3. Homoplasmy and heteroplasmy estimated incidence of 1:5000 [16]. They can affect mtDNA-encoded proteins, tRNA or rRNA, and eventually ATP production. Multiple copies of mtDNA (approximately 100 to 10,000 copies) MtDNA rearrangements, like large-scale deletions, remove large exist in most cells, and these levels can vary depending on energy portions of the mtDNA, with consequent ablation of various genes, demands [10]. If all the mtDNA molecules present in a cell are iden- depending on the site and size of the deletion. They are consistently tical (all wild-type or all carrying a mutation), this condition is heteroplasmic and sporadic and even though the exact mechanism of known as homoplasmic. When mtDNA with different sequences formation is still controversial, it is believed that they can derive from (pathogenic or not) are present in a single cell, the condition is errors in replication or inefficiency of the mtDNA repair system [17,18]. known as heteroplasmy. The latter is common for pathogenic mu- Their levels may increase during life as deleted mtDNA molecules were tations, as only a portion of the cellular mtDNA content is affected reported to have a replicative advantage [19,20]. They manifest a (Table 2). pathogenic effect at a lower threshold (approximately 60%) compared Heteroplasmy is a major factor that determines the clinical severity to most point mutations [13]. of mitochondrial diseases as mitochondrial function only begins to be affected when there is a relative high number of mutated mtDNA 2. Neuronal susceptibility: post-mitotic tissues sensitivity to compared to wt, usually N70–80% [11,12]. This phenomenon is known mitochondrial dysfunctions as “threshold effect” [13] and it can vary depending on the mutation, the cell type, the tissue or even depending on affected individual. Mitochondrial diseases caused by mtDNA mutations are in general Heteroplasmy can be dynamic, changing during the lifetime in both multi-symptomatic diseases with dysfunctions affecting different sys- mitotic and postmitotic tissues, due to the cell-cycle-independent tems and tissues (loss of muscle coordination, muscle weakness, visual mtDNA replication [14,15]. problems, hearing problems, learning disabilities, disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, diabetes, autonomic dysfunction and ). 1.1.4. Maternal inheritance The most affected tissues are the post-mitotic ones, such as myocytes and neurons. The reasons for this particular susceptibility lie in the The lack of paternal inheritance of mtDNA in vetebrates is believed non-proliferative nature of these cells, making it difficult to eliminate to be due to both a dilution effect, since sperm contains 1000 times cells with high levels of damaged or mutated mtDNA. less mitochondria compared to the unfertilized egg, and to the so- The cell-cycle-independent replication of mtDNA and the co- called mtDNA bottleneck. During the phases of primordial germ cells, existence of wt and mutated molecules of mtDNA are the cause of the mtDNA content falls to very low levels, preventing the transmission of different load of heteroplasmy in different cell types and tissues. In mi- pathogenic mtDNA mutations. totic tissues not all the cells will contain the same amount of damaged Maternal inheritance is a very important factor to take in consid- mtDNA so that the daughter cells with high levels of mtDNA mutation eration during the diagnosis of a since the could be in energetic disadvantage and replaced during the lifespan of transmission occurs only through the mother. an individual [14]. On the other hand, in post-mitotic cells, like 1200 M. Pinto, C.T. Moraes / Biochimica et Biophysica Acta 1842 (2014) 1198–1207

Table 1 List of mitochondrial tRNAs gene mutations associated with phenotypes affecting predominantly the CNS (Source: MITOMAP).

Mutation Gene Syndromes Symptoms

Deafness/neurosensory hearing loss A7445G tRNASer(UCN) Deafness/Neurosensory Hearing Loss Neurosensory Hearing Loss T7511C tRNASer(UCN) Deafness/Neurosensory Hearing Loss Neurosensory Hearing Loss T7510C tRNA Ser (UCN) Deafness; Neurosensory Hearing Loss Neurosensory Hearing Loss T7511C tRNA Ser(UCN) Deafness; Neurosensory Hearing Loss Neurosensory Hearing Loss 7472insC tRNA Ser(UCN) Deafness; cerebellar dysfunction Neurosensory Hearing Loss and Cerebellar Dysfunction T14709C tRNA Glu Deafness; Ataxia and MR Neurosensory Hearing Loss, Mental Retardation, Cerebellar Dysfunction Encephalomyopathy G583A tRNA Phe Encephalomyopathy, MELAS MELAS/ & exercise intolerance G1606A tRNA Val Encephalomyopathy, ataxia, , and deafness Ataxia, Myoclonus and Deafness A3243G tRNALeu(UUR) Encephalomyopathy, MELAS MELAS/ C3256T tRNALeu(UUR) Encephalomyopathy, MELAS MELAS T3271C tRNALeu(UUR) Encephalomyopathy, MELAS MELAS T3291C tRNALeu(UUR) Encephalomyopathy, MELAS MELAS/Myopathy/Deafness, Cognitive Impairment G4332A tRNA Gln Encephalomyopathy, MELAS MELAS/encephalopamyopathy A5537insT tRNA Trp Encephalomyopathy, Leigh syndrome Maternally Inherited Leigh Syndrome C7472insC tRNA Ser (UCN) Encephalomyopathy Progressive Encephalomyopathy/Ataxia, Myoclonus and Deafness/-like A8344G tRNA Lys Encephalomyopathy, MERRF MERRF T8356C tRNA Lys Encephalomyopathy, MERRF MERRF G8363A tRNA Lys Encephalomyopathy, MERRF MERRF/Maternally Inherited Cardiomyopathy/ deafness/Autism/Leigh Syndrome/Ataxia/Lipomas T10010C tRNA Gly Encephalomyopathy Progressive Encephalomyopathy G12147A tRNA His Encephalomyopathy, MERRF MERRF-MELAS/ C1624T tRNA Val Encephalomyopathy, Leigh Syndrome Leigh Syndrome G1644T tRNA Val Encephalomyopathy, Leigh Syndrome Adult Leigh Syndrome A5537insT tRNA Trp Encephalomyopathy Leigh Syndrome Maternally Inherited Leigh Syndrome G611A tRNA Phe Encephalomyopathy MERRF MERRF G8361A tRNA Lys Encephalomyopathy MERRF MERRF G8363A tRNA Lys Encephalomyopathy MERRF MERRF/Maternally Inherited Cardiomyopathy, deafness/Autism G3255A tRNALeu (UUR) Encephalomyopathy MERRF MERRF/KSS overlap A7543G tRNA Asp Encephalomyopathy Myoclonus and Psychomotor Regression and Psychomotor Regression G1606A tRNA Val Encephalomyopathy Ataxia, Myoclonus and Deafness Ataxia, Myoclonus and Deafness G3244A tRNALeu (UUR) Encephalomyopathy MELAS MELAS A3252G tRNALeu (UUR) Encephalomyopathy MELAS MELAS T3258C tRNALeu (UUR) Encephalomyopathy MELAS MELAS/Myopathy T3291C tRNALeu (UUR) Encephalomyopathy MELAS MELAS G1642A tRNA Val Encephalomyopathy MELAS MELAS G583A tRNA Phe Encephalomyopathy MELAS MELAS C3093G 16S rRNA Encephalomyopathy MELAS MELAS T3271delT tRNALeu(UUR) Encephalomyopathy Progressive Encephalomyopathy C3287A tRNALeu (UUR) Encephalomyopathy Encephalomyopathy T4290C tRNA Ile Encephalomyopathy Progressive Encephalomyopathy C4320T tRNA Ile Encephalomyopathy Encephalocardiomyopathy G5540A tRNA Trp Encephalomyopathy Encephalomyopathy T5693C tRNA Asn Encephalomyopathy Encephalomyopathy T5814C tRNA Cys Encephalomyopathy Encephalopathy A5816G tRNA Cys Encephalomyopathy Progressive T7512C tRNA Ser (UCN) Encephalomyopathy Progressive Encephalomyopathy, MERRF/MELAS G8328A tRNA Lys Encephalomyopathy Encephalopathy A8332G tRNA Lys Encephalomyopathy Dystonia and stroke-like episodes T10010C tRNA Gly Encephalomyopathy Progressive Encephalomyopathy G15915A tRNA Thr Encephalomyopathy Encephalomyopathy C2835T rRNA 16S Encephalomyopathy Rett Syndrome A10044G tRNA Gly Encephalomyopathy Gastrointestinal Reflux/Sudden Infant Death Syndrome G8313A tRNA Lys Mitochondrial Myopathy Mitochondrial Neurogastrointestinal Neurogastrointestinal encephalomyopathy Encephalomyopathy Encephalopathy A10438G tRNA Arg Encephalopathy Progressive Encephalopathy C14680A tRNA Glu Encephalopathy Mitochondrial Encephalopathy A14696G tRNA Glu Encephalopathy Progressive Encephalopathy G14724A tRNA Glu Encephalopathy Mitochondrial Leukoencephalopathy G14740A tRNA Glu Encephalopathy Encephalopathy + Retinopathy G15967A tRNA Pro Encephalopathy MERRF-like disease C15975T tRNA Pro Encephalopathy Ataxia+retinopathy+deafness G4284A tRNA Ile Multisystem Disease Varied familial presentation G12207A tRNA Ser (AGY) Mitochondrial Myopathy/Encephalopathy Myopathy/Encephalopathy A4269G tRNA Ile Fatal Infantile Cardiomyopathy Plus (MELAS) Fatal Infantile Cardiomyopathy Plus A4317G tRNA Ile Fatal Infantile Cardiomyopathy Plus (MELAS) Fatal Infantile Cardiomyopathy Plus Other T1659C tRNA Val Movement Disorder G3196A rRNA 16S Alzheimer & Parkinson Disease Alzheimer & Parkinson Disease T4336C tRNA Gln Alzheimer & Parkinson Disease; Deafness & Alzheimer & Parkinson Disease/Hearing loss and migraine G5549A tRNA Trp Dementia and Dementia and Chorea T5728C tRNA Asn Multi-organ Failure Multi-organ failure M. Pinto, C.T. Moraes / Biochimica et Biophysica Acta 1842 (2014) 1198–1207 1201

Table 2 List of protein-encoding mtDNA mutations associated with phenotypes affecting predominantly the CNS (Source: MITOMAP).

Mutation Gene Syndromes Symptoms

Dystonia/Leigh syndrome A3796G MTND1 Adult-onset dystonia Dystonia T8993C MTATP6 Leigh Syndrome/Neuropathy Ataxia and Retinitis Pigmentosa (LS/NARP) Leigh syndrome/Neuropathy Ataxia and Retinitis Pigmentosa T8993G MTATP6 NARP Neuropathy Ataxia and Retinitis Pigmentosa T9176G MTATP6 LS Leigh syndrome T9176C MTATP6 LS/familial bilateral striatal Leigh syndrome T9185C MTATP6 LS/Ataxia/NARP-like disease Leigh syndrome/Neuropathy Ataxia and Retinitis Pigmentosa T9191C MTATP6 LS Leigh Syndrome C9537insC MTCO3 LS-like Leigh Syndrome T10158C MTND3 LS Leigh syndrome T10191C MTND3 LS/LS-like Disease/Epilepsy, , Optic atrophy, and Cognitive decline Leigh syndrome G10197A MTND3 LS/Dystonia/Stroke Leigh syndrome C11777A MTND4 LS Leigh syndrome T12706C MTND5 LS Leigh syndrome G14459A MTND6 Leber hereditary optic neuropathy and dystonia/LS Dystonia/Leigh syndrome T14487C MTND6 LS/dystonia/ataxia Dystonia/Leigh syndrome Encephalomyopathy T3308C MTND1 MELAS Encephalomyopathy, MELAS G3376A MTND1 MELAS/LHON Encephalomyopathy, MELAS G3697A MTND1 MELAS/LS Encephalomyopathy, MELAS G3946A MTND1 MELAS Encephalomyopathy, MELAS T3949C MTND1 MELAS Encephalomyopathy, MELAS A11084G MTND4 MELAS Encephalomyopathy, MELAS A12770G MTND5 MELAS Encephalomyopathy, MELAS A13045C MTND5 MELAS/LHON/LS overlap syndrome Encephalomyopathy, MELAS, optic neuropathy G13513A MTND5 MELAS/LS Encephalomyopathy, MELAS A13514G MTND5 MELAS Encephalomyopathy, MELAS A13084T MTND5 MELAS/LS Encephalomyopathy, MELAS G14453A MTND6 MELAS Encephalomyopathy, MELAS 14787del4 MTCYB MELAS/Parkinson's-like Encephalomyopathy, MELAS, C6489A MTCO1 Therapy-resistant epilepsy Encephalomyopathy, Epilepsy G6930A MTCO1 Multisystem disorder Encephalomyopathy, Multisystem Disorder 6015del5 MTCOI Myopathy and cortical Encephalomyopathy, Multisystem Disorder T7587C MTCO2 Encephalomyopathy Encephalomyopathy G7896A MTCO2 Multisystem disorder Encephalomyopathy, Multisystem Disorder 8042del2 MTCO2 Lactic acidosis Encephalomyopathy, Lactic Acidosis G9952A MTCO3 Encephalomyopathy Encephalomyopathy T9957C MTCO3 MELAS/progressive encephalopathy/non‐arteritic ischaemic optic neuropathy Encephalomyopathy, MELAS 9205del2 MTATP6 Lactic acidosis/ Encephalomyopathy, Lactic Acidosis A15579G MTCYB Multisystem disorder Encephalomyopathy, Multisystem Disorder T14849C MTCYB Septo-optic dysplasia Encephalomyopathy, Septo-Optic Dysplasia Deafness/sensorineural hearing loss G11778A MTND4 LHON Optic neuropathy, Sensory Neural Hearing Loss G3460A MTND1 LHON Optic neuropathy, Sensory Neural Hearing Loss T14484C MTND6 LHON Optic neuropathy, Sensory Neural Hearing Loss G7444A MTCO1 Sensory neural hearing loss/LHON ,Sensory Neural Hearing Loss, optic neuropathy A8108G MTCO2 Sensory neural hearing loss Sensory Neural Hearing Loss C14340T MTND6 Sensory neural hearing loss Sensory Neural Hearing Loss Alzheimer and Parkinson-like presentations A3397G MTND1 ADPD Alzheimer & Parkinson disease G5460A MTND2 AD Alzheimer’sdisease myocytes and neurons, the mutated mtDNA cannot be eliminated by dysfunction. Those who survive infancy are expected to develop KSS mitotic segregation. The replication of mtDNA in post-mitotic tissues [22–24]. CPEO is characterized by ptosis and ophthalmoplegia with causes a clonal expansion and consequent accumulation of somatic some patients showing also oropharyngeal and proximal muscle pathogenic mtDNA mutations (Table 3). weakness. Patients with CPEO can have brain, inner ear, and retinal Myocytes and neurons are also cells with a high-energy demand. disease in later stages of the disease, depending on the age of onset This characteristic makes them more vulnerable to the ATP depletion andonthelevelofheteroplasmy[25]. CPEO is commonly seen as a that can derive from the pathogenic mutations in the mtDNA that affect milder form of the disease, and clinical presentations can involve proteins involved in the respiratory chain [21]. other muscles or symptoms and are sometimes referred to as CPEO Plus [26]. 3. Neurodegeneration in mitochondrial diseases Although it is a multisytem disorder, CNS involvement is evident in KSS. The syndrome is defined by onset prior to 20 years of age, progres- 3.1. Mitochondrial caused by MtDNA deletions sive external ophthalmoplegia (PEO), and a pigmentary retinopathy (usually rod-cone dystrophy). Moreover, patients may also show cardiac Patients with mtDNA large deletions commonly show one of conduction block (usually the cause of death in young adulthood), three classic phenotypes: , chronic progressive elevated cerebrospinal fluid protein level, or cerebellar ataxia [26]. external ophthalmoplegia (CPEO) and Kearns–Sayre syndrome Other neurologic problems may include proximal myopathy, exercise (KSS). Patients with Pearson Syndrome show a multi-symptomatic intolerance, ptosis, oropharyngeal andesophagealdysfunction,sensori- disease from birth with a 50% surviving rate after 4 years of age. neural hearing loss, dementia, and choroid plexus dysfunction resulting The main symptoms are sideroblastic anemia and exocrine pancreas in cerebral folate deficiency [27]. 1202 M. Pinto, C.T. Moraes / Biochimica et Biophysica Acta 1842 (2014) 1198–1207

Table 3 List of nuclear DNA-encoded genes involved in mtDNA integrity associated with phenotypes affecting predominantly the CNS.

Name Function Inheritance Clinical phenotype

POLG1 Polymerase gamma, mtDNA replication AD-AR Alpers syndrome, AD-PEO and AR-PEO, male infertility, SANDO* syndrome, SCAE* POLG2 Catalytic subunit of DNA polymerase gamma, mtDNA replication AD AD-PEO ANT1 Adenine nucleotide translocator isoform 1, dNTPs pool maintenance AD AD-PEO, multiple mtDNA deletions MPV17 Unknown, involved in the metabolism of reactive oxygen species and AR Hepatocerebral MDDS in the regulation of mtDNA copy number Twinkle (C10orf2) Twinkle helicase, mtDNA replication AD AD-PEO, SANDO syndrome TYMP Thymidine phosphorylase, dNTPs pool maintenance AR MNGIE, mtDNA depletion DGUOK Deoxyguanosine kinase Mitochondrial, dNTPs pool maintenance AR Hepatocerebral mtDNA depletion syndrome RRM2B Ribonucleotide reductase, dNTPs pool maintenance AR Encephalomyopathic Renal tubulopathy MNGIE, AD-PEO SUCLA2 Succinate-CoA ligase, ADP-forming, beta subunit AR Encephalomyopathy with methylmalonic aciduria SUCLG1 Succinate-CoA ligase, alpha subunit AR Encephalomyopathy with methylmalonic aciduria MGME1 (C20orf72) mitochondrial genome maintenance exonuclease 1 AR Mitochondrial DNA depletion syndrome 11 DNA2 helicase/nuclease, mtDNA replication, long-patch base-excision repair AD PEO pathway TK2 Thymidine kinase mitochondrial, dNTPs pool maintenance AR Myopathic mtDNA depletion

3.2. Mitochondrial encephalopathies caused by mtDNA point mutations lactic acidosis and stroke-like symptoms, patients can also show deafness, diabetes, , gut immobility and seizures [39]. Almost 600 pathogenic point mutations have been identified in the Multiple strokes affect the patients, mainly in the cerebral cortex or last 25 years, involving most of the mtDNA molecule (according to in the subcortical white matter, causing multifocal necrosis with lesions MITOMAP, 299 involving tRNA–rRNA and control that do not respect vascular territories and are often accompanied by regions and 274 involving OXPHOS proteins). profound neuronal cell loss, neuronal eosinophilia, astrogliosis and The most common mitochondrial encephalopathies caused by point spongiform degeneration [40]. There is also a loss of Purkinje cells causing mutations can be classified in clinical categories: LHON (Leber Heredi- cerebellar degeneration and a particularly prominent calcification in tary Optic Neuropathy), Leigh Syndrome, MELAS (Mitochondrial myop- the . Depending on the portions of the brain affected by the athy, encephalomyopathy, lactic acidosis, stroke-like symptoms), MIDD ischemic lesions, symptoms may vary from focal and (maternally inherited diabetes and deafness), MERRF (Myoclonic Epi- epilepsia partialis continua to just occipital migraines and a visual aura. lepsy with Ragged Red Fibers), non-syndromic hearing loss and NARP A3243G is also the most frequent mutation associated with MIDD (Neuropathy, ataxia, retinitis pigmentosa). (maternally inherited diabetes and deafness) [41] in fact, patients diag- Approximately 95% of LHON cases show a mutation in one of three nosed with this disease may also have typical symptoms of MELAS mtDNA genes: G11778A, G3460A or T14484C, respectively encoding for including myopathy, cardiomyopathy, neuropsychiatric symptoms, ND4, ND1 and ND6, all subunits of Complex I of the electron transport and renal disease and patients belonging to the same family can have chain (Fig. 1). The main characteristic of the disease is a painless, MELAS or MIDD. The level of heteroplasmy can explain in part the bilateral, subacute or acute visual failure, prevalently in young male different clinical outcome [42]. adults, caused by the atrophy of the optic nerve [28].Neurodegeneration The neurodegeneration that typically causes the sensorineural deaf- is limited to the retinal ganglion cell (RGC) layer with cell body and axonal ness involves the auditory nerve or the brain, with defect in oxidative degeneration, demyelination and atrophy observed from the optic nerves phosphorylation in the beta cells that sense and diminished to the lateral geniculate bodies [29]. One possible explanation for the in- ATP production by strial marginal cells of the inner ear [43]. volvement of the optic nerve alone is the disruption of glutamate trans- In 90% of the cases of MERRF, the mutation responsible is an A8344G port out of the inner retina with consequent excitotoxic damage [30]. transition, in the tRNALys, [44] (Fig. 1). The myoclonic epilepsy is the main There is a correlation between genotype and the severity of the disease: symptom associated with this disease together with the presence of patients with the G11778A mutation seem to have the most severe clumps of diseased mitochondria accumulation in the sub-sarcolemmal phenotype, with the least favorable visual outcome, while the region of the muscle fiber called “Ragged Red Fibers.” Other symptoms T14484C mutation is associated with the mildest pathogenicity [31]. like ataxia, neuropathy, and cardiac abnormalities can be present. Curi- Leigh Syndrome can be caused by different mutations, both in ously, many patients with the A8344G mutation also show multiple lipo- mtDNA and in nDNA genes. Mutations can occur in genes encoding for mas in the back region [45]. The main neuropathological signs involve the tRNAs as well as in genes encoding for complexes I, IV, or V of the olivocerebellar pathway, with severe neuron loss from the inferior olivary OXPHOS [32–37] and are commonly present in heteroplasmy. The nucleus, Purkinje cells, and . Neurodegeneration is heterogenic symptoms include motor and intellectual developmental present also in the gracile and cuneate nuclei, moreover there are delay, bilateral disease, , elevated signs of demyelination of the [46]. In the surviving neurons or CSF lactate levels, , spasticity, chorea and other movement there are evidences of respiratory chain deficiency and presence of en- disorders, cerebellar ataxia, , and respiratory larged mitochondria containing inclusions [47]. No clear correlation has failure secondary to brainstem dysfunction [35].Theconditionmay been identified between genotype or heteroplasmy and clinical pheno- also affect the liver, heart (including hypertrophic cardiomyopathy), type, nor it is clear why typical MERRF is associated with mutations in kidneys, and pancreas [38]. tRNALys [48]. About 80% of MELAS cases are caused by a very common A3243G Non-syndromic hearing loss, that means severe and profound deaf- mutation in the mitochondrial tRNALeu(UUR) gene, whereas 10% of ness not associated to other neurologic or systemic diseases, can be cases carry the T3271C mutation in the same tRNALeu(UUR) (Fig. 1), caused by mutations in mitochondrial tRNAs like A3243G, A7443/4/5G, although other mtDNA point mutations have been also associated and in rRNAs like 961delT, and A1555G [49] (Fig. 1). with this phenotype (MITOMAP). As the vast majority of the mito- NARP is associated mainly with the mutation T8993G (or T8993C) chondrial diseases, this is a multisystemic disorder with symptoms in the mtDNA encoding for the MTATP6 gene, even though a family varying depending on the heteroplasmy status and on the age of with a G8989C mutation has also been described [50] (Fig. 1). This onset. Other than mitochondrial myopathy, encephalomyopathy, disease is characterized by sensory or sensorimotor axonal neuropathy, M. Pinto, C.T. Moraes / Biochimica et Biophysica Acta 1842 (2014) 1198–1207 1203 neurogenic muscle weakness, ataxia, cerebral or cerebellar atrophy, particular of Complexes I and IV of the electron transport chain, has and retinitis pigmentosa. Other non-typical neurological symptoms, in- been proposed in the last decades and extensively reviewed [72–75]. Dif- cluding seizures, learning problems, hearing loss, progressive external ferent hypotheses have been proposed to explain the high suscepti- ophthalmoplegia, and anxiety can be present. bility of SN to mitochondrial stress and to mtDNA damage, and the anatomical characteristics of these neurons, that have particularly 3.3. Mitochondrial diseases caused by mutations in nDNA genes affecting long unmyelinated , seem to be the most accredited [76,77]. mtDNA stability More specifically, different groups have also analyzed mtDNA changes in patients' post-mortem brain: high levels of mutations and deletions MtDNA changes can be a consequence of mutations in nDNA- are found within the neurons of the SN both in patients with PD and encoded genes involved in the maintenance of mtDNA integrity and during normal aging [78,79]. mtDNA copy number. The most common mutations affect POLG, the If mtDNA changes are sufficient to confer PD symptoms is still gene encoding for the catalytic subunit of the mitochondrial DNA controversial, studies on animal models [80–82] and the fact that polymerase gamma, and PEO1 which encodes for the DNA helicase patients with POLG mutations also show Parkinsonism similar to cases Twinkle [51,52], both genes involved in mtDNA replication. Mutations of juvenile PD [57,83] suggest that they play a role in the pathogenesis. in these genes provoke an accumulation of mtDNA point mutations Moreover, it has also been suggested that different mtDNA haplotypes and deletions eventually leading to a different clinical manifestation. can confer higher or lower risk factors to develop PD, because different Over 200 mutations in POLG associated with mitochondrial diseases polymorphisms can show subtle differences of the respiratory chain have been identified, causing a plethora of heterogeneous disorders activity and ROS production. In particular, the super-haplogroup JT involving different tissues, time of onset and severity. At least five seems to confer a reduced risk of PD while the super-haplogroup HV major phenotypes can be distinguished: Alpers–Huttenlocher syndrome, seems to be associated with an increased risk [84]. childhood myocerebrohepatopathy spectrum, myoclonic epilepsy AD is the most common late-onset progressive neurodegenerative myopathy sensory ataxia, the ataxia neuropathy spectrum, and progres- disease, clinically characterized by memory loss, impairment of cogni- sive external ophthalmoplegia (PEO) with or without sensory ataxic tive functions and changes in behavior and personality. The neurode- neuropathy and dysarthria [53–56]. Patients with mutations in POLG generation that occurs in these patients affect mostly the cortex and also show defects in complex I and complex IV in dopaminergic neurons the hippocampus. Here there is an accumulation of senile plaques, of the substantia nigra and neuronal cell loss [57,58]. composed mainly by beta-amyloid peptide, and intraneuronal tau Recessive mutations in Twinkle protein cause severe, early-onset deposition as neurofibrillary tangles. The most studied pathogenetic disorders due also to defects in mtDNA maintenance, such as infantile- model for this disease is the “beta-amyloid cascade”, where an unbal- onset and a hepatocerebral mtDNA depletion ance in the cleavage sequence of APP (amyloid precursor protein) disorder with severe epilepsy, migraine, and psychiatric symptoms [55]. leads to an accumulation of toxic Aβ fragment, cytotoxic plaques, and Also TYMP, that encodes for thymidine phosphorylase and is essential consequent neurodegeneration. in the nucleotide salvage pathway for mtDNA replication, is typically Mitochondrial dysfunctions, in particular cytochrome c oxidase mutated (multiple deletions and single base changes) in mitochondrial defects, have also been implicated in the development and progression neurogastrointestinal encephalomyopathy [59,60]. of AD [85,86].Aβ fragments negatively affect mitochondrial function, Mutations in OPA1, a protein involved in the mitochondrial fusion suggesting that mitochondrial dysfunction is a consequence of the Aβ process, cause autosomal dominant optic atrophy with loss of retinal toxicity, moreover studies in AD mouse models show also that Aβ ganglion cells and progressive optic nerve degeneration with cases of oligomerization impairs mitochondrial function [87–90]. Moreover, more severe neuromuscular complications [61,62]. RRM2B encodes a the γ-secretase activity and so the APP cleavage, occurs predominantly ribonucleotide reductase and is mutated in cases of autosomal dominant in the MAM (mitochondria-associated ER membranes), a compartment OPA [63,64]. Mutations in both genes cause mtDNA instability and of the endoplasmic reticulum physically and biochemically connected multiple deletions. to mitochondria. MAM function and ER–mitochondrial connectivity Changes in mtDNA copy number are also associated with clinical are increased in AD, so that AD is also proposed to be an ER–mitochon- syndromes. The MDS (mtDNA depletion syndromes) are autosomal drial communication and MAM dysfunction disease [91,92].Thishy- recessive disorders caused by mtDNA depletion in clinically affected pothesis, other than the β-amyloid cascade one, would explain also tissues [65]. Most of them derive from a mutation in nDNA genes in- other biochemical changes occurring in the disease like mitochondrial volved in mtDNA replication machinery (POLG, Twinkle) or in different dysfunction, elevated levels of cholesterol, altered metabolism of fatty mitochondrial functions like nucleotide metabolism (TYMP, TK2, DGUOK acids and phospholipids, and aberrant calcium homeostasis [93]. and MPV17) or fission–fusion machinery (Mfn2) [66–70]. Phenotypically The presence of increased mutations in mtDNA of AD patients is still they are very heterogeneous with myopathic, encephalomyopathic, a controversial subject: different laboratories with various groups of hepatocerebral or neurogastrointestinal manifestations, with a common patients and dissimilar techniques have obtained debatable results neurological involvement [69,71]. [94–98]. It has also been suggested that inherited haplogroups may influence AD risk but to date, no clear result has been found [99]. 4. mtDNA changes in age-related neurodegenerative diseases ALS is a motor neuron disease, characterized by rapidly progressive weakness, muscle atrophy and fasciculation, muscle spasticity, dysar- MtDNA changes have been hypothesized to have a role also in age- thria, , dyspnea caused by the degeneration of the upper related neurodegenerative diseases like Parkinson's (PD), Alzheimer's and lower motor neurons. Increased mtDNA deletions have been (AD), amyotrophic lateral sclerosis (ALS) and (MS). found in muscle and brain of ALS patients, although the levels are PD is the second most common progressive neurodegenerative disorder, still relatively low and of unknown consequence [100–102]. Moreover mainly characterized by motor (resting , postural instability, rigid- in some cases of patients with mtDNA mutations, an ALS phenotype ity and bradykinesia) and non-motor symptoms (fatigue, depression, has been diagnosed [103–106]. Also in this case the association of anxiety, sleep disturbances, autonomic disturbances, decreased motiva- haplogroup with increased risk factor is still controversial [107]. tion, apathy and a decline in cognition, dementia). The cause of the MS is a chronic inflammatory autoimmune disease caused by loss of motor symptoms is a depletion of dopamine in the striatum, derived and . The etiology of this disease is largely unknown and from a specific degeneration of dopaminergic neurons located in the hypothesis on genetic, environmental and infective agents have been midbrain and forming the substantia nigra (SN). The etiology of this disor- analyzed without a clear response. A mitochondrial role has been der is still unknown, but a role of mitochondrial dysfunction, and in proposed since changes in the expression of TFAM, PGC1α and nuclear 1204 M. Pinto, C.T. Moraes / Biochimica et Biophysica Acta 1842 (2014) 1198–1207 respiratory factor 1 (NRF1) have been found in cortical neurons of MS As previously described, Twinkle has a helicase activity essential for patients [108,109]. Moreover in patients' post-mortem brains, there is mtDNA replication and maintenance. The Twinkle A360T mutation and a reduction of Complex I and Complex III activity [110]. Different aa 353–365 duplication are typical of PEO so that Twinkle mice have mtDNA mutations have been reported in patients with MS [111] even been generated knocking in the mutated gene [125] resulting in an though the direct role of mtDNA single mutation or deletion in the accumulation of multiple large mtDNA deletions mostly in brain and pathogenesis of this disease is also still controversial [112]. muscle. These transgenic mice show progressive mitochondrial myopathy and mitochondrial impairment in cerebellar Purkinje cells and hippocampal CA2 pyramidal neurons. 5. Mouse models of mtDNA alterations in neurodegenerative Other nDNA-encoded genes have been manipulated to indirectly conditions affect mtDNA stability. Among them, TYMP/UP KO mice, who lack thymidine phosphorylase activity, showed partial mtDNA depletion in As described in the previous paragraphs, mtDNA changes have been brain, late-onset vacuoles in cerebral and cerebellar white matter with- implicated in a vast group of diseases and possibly normal aging. In out demyelination or axonal loss [126]. MPV17 KO mice showed mtDNA order to study the mechanisms behind these pathologies, the use of depletion in different tissues including brain [127] TK2 KO [128],mu- mouse models is compelling. Unfortunately, engineering and integrating tant TK2 knock in mice [129] and RRM2B KO mice [64] also showed specific mtDNA mutations in mice, in particular pathogenic mutations, is mtDNA depletion. A novel mouse model is also expressing a mutated technically challenging, most of all because of the “bottleneck effect” version of UNG1 in neurons (that removes , in addition to uracil [113]. The specific mutation in mice will be diluted in the progeny so from mtDNA), showing signs of neurodegeneration and impaired be- that to date, mouse models with pathological mtDNA mutations are havior [130]. rare [114] and, if not lethal, kept in homoplasmy [115]. As detailed below, the modeling of mtDNA changes has been 5.2. Direct damage to mtDNA approached in two alternative ways: by engineering nuclear genes or by direct manipulation of mtDNA [116]. On one side, mouse models with The “mito-mice” [131] have been generated by introducing exoge- mtDNA mutations have been created by genetic modification of nDNA nous mitochondria carrying mutated mtDNA into mouse zygotes. Three encoded genes involved in mtDNA maintenance like POLG, Tfam and mito-micehavebeengeneratedsofar,aΔmtDNA (carrying a 4696 bp de- Twinkle, on the other hand specific deletions have been created in the letion), mtDNA-COI (carrying T6589C mutation in mtDNA gene encoding mtDNA by the introduction of defective mitochondria into mouse zygotes cytochrome c oxidase subunit I) and mtDNA-ND6 (with A13997G muta- or by the expression of restriction mitochondria-targeted endonucleases. tion in mtDNA gene encoding NADH dehydrogenase 6) [132–134]. ΔmtDNA mice show various percentage of heteroplasmy in different tis- 5.1. Manipulation of nDNA encoded genes that affect mtDNA sues and clinical defects resembling the ones of mitochondrial diseases like low body weight, lactic acidosis, systemic ischemia, hearing loss, One of the first nDNA-encoded genes to be targeted in order to renal failures, and male infertility. Mice with more than 60% ΔmtDNA induce an indirect damage to mtDNA was TFAM. It is a transcriptional show mitochondrial respiration defects in brain and a downregulation activator that binds mtDNA promoters and activates transcription. It is of Calmodulin-dependent Protein Kinase II subunit alpha (CamKIIa) lead- also necessary for mtDNA replication since it provides the RNA primers ing to impairment of spatial remote memory. MtDNA-COI and mtDNA- for initiation and it has an important histone-like role in mtDNA main- ND6 are homoplasmic and show typical signs of COXI or Complex I defi- tenance since it binds to mtDNA coating it [117]. Tfam KO mice ciencies [135]. The disadvantage of the use of these mice is the colony completely lack mtDNA and die during embryogenesis. Tissue-specific maintenance: ΔmtDNA is inherited by progeny for only about three preg- disruption of Tfam shows different clinical evidences, in particular nancies, since the amount of ΔmtDNA in eggs decreases with the aging of neurodegeneration is present when Tfam is knocked out in forebrain the females. Moreover it is difficult to obtain a large population of mito- neurons (MILON mice) and in dopaminergic cells (MitoPark mouse). miceΔ with the same ΔmtDNA load. In MILON mice, Tfam was deleted post-natally, showing no sign of Recently, Lin et al. created a novel animal model of LHON by disease until 6–8 months of age, when deteriorating conditions and introducing the homoplasmic mtDNA ND6P25L mutation into the death appeared in 2–3 weeks caused by significant mtDNA depletion mouse [136]. Mice with this mutation exhibited reduction in retinal [118]. In Mito-Park mice, Tfam was deleted in dopaminergic neurons, function, decline in optic nerve fibers, neuronal accumulation of causing a Parkinson-like phenotype with dopamine depletion, neurode- abnormal mitochondria, axonal swelling, and demyelination. generation of dopaminergic cells of substantia nigra and motor deficits The mito-PstI mouse expresses a mitochondria-targeted restriction [119]. endonuclease, mito-PstI, in an inducible and tissue-specificway,de- POLG is also essential for mtDNA replication, so that POLG KO mice pending on the promoter that controls the expression of the transgene are embryonically lethal [120]. In order to induce mtDNA damage in [137].Mito-PstI cleaves the mtDNA in two different sizes, leading to different tissues, Polg gene has been manipulated to impair its proof- the accumulation of multiple deletions and eventual mtDNA depletion. reading activity. Two different groups created the “mutator mouse” by When expressed in neurons (under the control of CamKIIα promoter), knocking-in PolgA gene carrying two different mutations: the D257A it causes different phenotype, depending on the level and timing of mutation [121] or the AC → CT substitution in positions 1054 and expression. On a high expression from birth, the mice show an acute 1055 [122]. These mutations provoked an accumulation of mtDNA neurodegeneration with an abnormal ‘limb-clasping behavior’ at 2 point mutations in different tissues [123], causing two very similar clin- months of age. With a high expression starting at P21, mice progressive- ical phenotypes. The mutator mice showed signs typical of premature ly become less active and died before 100-day-old with a severe reduc- aging, with reduced life span, kyphosis, alopecia, weight loss, reduced tion of COX activity in the forebrain. When mito-PstIisexpressedat fat content, osteoporosis, thymic involution, testicular atrophy, loss of lower levels, mice show no differences in lifespan up to 16 months of intestinal crypts, progressive decrease in circulating red blood cells, age with no obvious phenotypic abnormalities until 6–8 months, hearing loss and sarcopenia. Surprisingly these mice did not show strik- when they displayed abnormal and progressively worse motor behav- ing signs of neurodegeneration [124]. Nonetheless, detailed studies of ior. This phenotype is caused by a progressive neurodegeneration brain mtDNA by NextGen sequencing show that brain of mutator mice particularly severe in the striatum and cortex due to a decreased COXI accumulate mtDNA species with abnormal D-loop structures. These activity [138,139]. 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