Mitochondrial genome changes and neurodegenerative diseases.

BBADIS-63838; No. of pages: 10; 4C: 2 Biochimica et Biophysica Acta xxx (2013) xxx–xxx

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Review

Mitochondrial genome changes and neurodegenerative diseases☆ Milena Pinto a,c, Carlos T. Moraes a,b,c,⁎ a b c

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

a r t i c l e

i n f o

Article history: Received 31 July 2013 Received in revised form 6 November 2013 Accepted 8 November 2013 Available online xxxx Keywords: Mitochondrion mtDNA Encephalopathy

a b s t r a c t Mitochondria are essential organelles within the cell where most of the energy production occurs by the oxidative phosphorylation system (OXPHOS). Critical components of the OXPHOS are encoded by the mitochondrial DNA (mtDNA) and therefore, mutations involving this genome can be deleterious to the cell. Post-mitotic tissues, such as muscle and brain, are most sensitive to mtDNA changes, due to their high energy requirements and nonproliferative status. It has been proposed that mtDNA biological features and location make it vulnerable to mutations, which accumulate over time. However, although the role of mtDNA damage has been conclusively connected to neuronal impairment in mitochondrial diseases, its role in age-related neurodegenerative diseases remains speculative. Here we review the pathophysiology of mtDNA mutations leading to neurodegeneration 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 1.1. The mtDNA 1.1.1. Structure Mitochondrial DNA (mtDNA) was discovered in 1963 [1] and the human mtDNA fully sequenced in 1981 [2]. The human mitochondrial genome is a circular, double-stranded, supercoiled molecule composed of 16569 bp and encoding for 37 genes. Because of their nucleotide compositions, the DNA strands were named “H-strand” and “L-strand”, as the heavy strand is rich in guanines, and light strand is rich in cytosines. The H-strand encodes for 28 genes, 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 kb), called the displacement loop (D-loop). The D-loop is essential for the mtDNA replication and transcription since it contains the origin of H-strand replication (OH) and the promoter regions of the two strands (HSP and LSP). The two polycistronic RNAs transcribed from the two strands are processed to obtain 22 tRNAs molecules, 2 mitochondrial rRNA and translated into 13 proteins [3]. All the proteins encoded by the mtDNA are components of the four OXPHOS multi-subunit complexes. Complex II is the only exception, as all its components are encoded by the nDNA. The proteins involved in ☆ This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction and Neurodegenerative Diseases. ⁎ Corresponding author at: Dept. of Neurology, University of Miami, 1420 NW 9th ave, Miami, FL 33133, USA. E-mail address: [email protected] (C.T. Moraes).

mtDNA transcription, translation and replication as well as the other OXPHOS components are all encoded by the nDNA, so that mutations in these nuclear genes can also affect the stability of the mtDNA (Table 1). The replication of mtDNA is independent from the cell cycle and, to date, only few enzymes are known to participate in the process, among them, POLG (mitochondrial DNA polymerase γ, that has also a 3′–5′ exonuclease/proofreading activity), and Twinkle (a DNA helicase) are particularly important, as mutations in these genes have been found in different mitochondrial diseases [4].

1.1.2. Mutations The mtDNA is particularly prone to damage with a mutation rate 10 times higher than that of nDNA [5]. The reasons for this high susceptibility are several. Unlike nDNA, mtDNA is organized in nucleoids but it is not protected by histones and its proximity to the mitochondrial respiratory chain (in particular Complex I and III) makes it also close to a source of radical oxygen species that are well-known genotoxic agents. Moreover, it replicates more often than the nDNA and the efficiency of repair mechanisms 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 from oxidative damage [6]. The first pathogenic mtDNA mutations were identified by Holt at al. and Wallace et al. in 1988 [7,8] and were considered to be very rare in the total populations. In the last decades an increasing number of studies showed that the incidence of pathogenic mutations is much higher than previously expected (N1/200 live births), although in most cases they are present in low, non-pathogenic levels [9].

0925-4439/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bbadis.2013.11.012

Please cite this article as: M. Pinto, C.T. Moraes, Mitochondrial genome changes and neurodegenerative diseases, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbadis.2013.11.012

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M. Pinto, C.T. Moraes / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

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 electron transport chain; Orange, subunits of cytochrome c oxidase (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, Lactic Acidosis, and Stroke-like episodes; MIDD: Maternally Inherited Diabetes and Deafness; LHON: Leber Hereditary Optic Neuropathy; MERRF: Myoclonic Epilepsy and Ragged Red Muscle Fibers; NARP: Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa.

1.1.3. Homoplasmy and heteroplasmy Multiple copies of mtDNA (approximately 100 to 10,000 copies) exist in most cells, and these levels can vary depending on energy demands [10]. If all the mtDNA molecules present in a cell are identical (all wild-type or all carrying a mutation), this condition is known as homoplasmic. When mtDNA with different sequences (pathogenic or not) are present in a single cell, the condition is known as heteroplasmy. The latter is common for pathogenic mutations, as only a portion of the cellular mtDNA content is affected (Table 2). Heteroplasmy is a major factor that determines the clinical severity of mitochondrial diseases as mitochondrial function only begins to be affected when there is a relative high number of mutated mtDNA compared to wt, usually N70–80% [11,12]. This phenomenon is known as “threshold effect” [13] and it can vary depending on the mutation, the cell type, the tissue or even depending on affected individual. Heteroplasmy can be dynamic, changing during the lifetime in both mitotic and postmitotic tissues, due to the cell-cycle-independent mtDNA replication [14,15]. 1.1.4. Maternal inheritance The lack of paternal inheritance of mtDNA in vetebrates is believed to be due to both a dilution effect, since sperm contains 1000 times less mitochondria compared to the unfertilized egg, and to the socalled mtDNA bottleneck. During the phases of primordial germ cells, mtDNA content falls to very low levels, preventing the transmission of pathogenic mtDNA mutations. Maternal inheritance is a very important factor to take in consideration during the diagnosis of a mitochondrial disease since the transmission occurs only through the mother. 1.1.5. Pathogenic mtDNA changes MtDNA defects can be maternally inherited or sporadic. Point mutations are, in general, maternally inherited and heteroplasmic, with an

estimated incidence of 1:5000 [16]. They can affect mtDNA-encoded proteins, tRNA or rRNA, and eventually ATP production. MtDNA rearrangements, like large-scale deletions, remove large portions of the mtDNA, with consequent ablation of various genes, depending on the site and size of the deletion. They are consistently heteroplasmic and sporadic and even though the exact mechanism of formation is still controversial, it is believed that they can derive from errors in replication or inefficiency of the mtDNA repair system [17,18]. Their levels may increase during life as deleted mtDNA molecules were reported to have a replicative advantage [19,20]. They manifest a pathogenic effect at a lower threshold (approximately 60%) compared to most point mutations [13]. 2. Neuronal susceptibility: post-mitotic tissues sensitivity to mitochondrial dysfunctions Mitochondrial diseases caused by mtDNA mutations are in general multi-symptomatic diseases with dysfunctions affecting different systems and tissues (loss of muscle coordination, muscle weakness, visual problems, hearing problems, learning disabilities, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, diabetes, autonomic dysfunction and dementia). The most affected tissues are the post-mitotic ones, such as myocytes and neurons. The reasons for this particular susceptibility lie in the non-proliferative nature of these cells, making it difficult to eliminate cells with high levels of damaged or mutated mtDNA. The cell-cycle-independent replication of mtDNA and the coexistence of wt and mutated molecules of mtDNA are the cause of the different load of heteroplasmy in different cell types and tissues. In mitotic tissues not all the cells will contain the same amount of damaged mtDNA so that the daughter cells with high levels of mtDNA mutation could be in energetic disadvantage and replaced during the lifespan of an individual [14]. On the other hand, in post-mitotic cells, like myocytes and neurons, the mutated mtDNA cannot be eliminated by mitotic segregation. The replication of mtDNA in post-mitotic tissues causes a clonal expansion and consequent accumulation of somatic pathogenic mtDNA mutations (Table 3).


M. Pinto, C.T. Moraes / Biochimica et Biophysica Acta xxx (2013) xxx–xxx Table 1 List of mitochondrial tRNAs gene mutations associated with phenotypes affecting predominantly the CNS (Source: MITOMAP). Mutation

Gene

A7445G T7511C T7510C T7511C 7472insC T14709C

Deafness/neurosensory hearing loss tRNASer(UCN) Deafness/Neurosensory Hearing Loss tRNASer(UCN) Deafness/Neurosensory Hearing Loss tRNA Ser (UCN) Deafness; Neurosensory Hearing Loss tRNA Ser(UCN) Deafness; Neurosensory Hearing Loss tRNA Ser(UCN) Deafness; cerebellar dysfunction tRNA Glu Deafness; Ataxia and MR

G583A G1606A A3243G C3256T T3271C T3291C G4332A A5537insT C7472insC

tRNA Phe tRNA Val tRNALeu(UUR) tRNALeu(UUR) tRNALeu(UUR) tRNALeu(UUR) tRNA Gln tRNA Trp tRNA Ser (UCN)

Encephalomyopathy Encephalomyopathy, MELAS Encephalomyopathy, ataxia, myoclonus, and deafness Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, Leigh syndrome Encephalomyopathy

A8344G T8356C G8363A

tRNA Lys tRNA Lys tRNA Lys

Encephalomyopathy, MERRF Encephalomyopathy, MERRF Encephalomyopathy, MERRF

T10010C G12147A C1624T G1644T A5537insT G611A G8361A G8363A G3255A A7543G G1606A G3244A A3252G T3258C T3291C G1642A G583A C3093G T3271delT C3287A T4290C C4320T G5540A T5693C T5814C A5816G T7512C G8328A A8332G T10010C G15915A C2835T A10044G G8313A

tRNA Gly tRNA His tRNA Val tRNA Val tRNA Trp tRNA Phe tRNA Lys tRNA Lys tRNALeu (UUR) tRNA Asp tRNA Val tRNALeu (UUR) tRNALeu (UUR) tRNALeu (UUR) tRNALeu (UUR) tRNA Val tRNA Phe 16S rRNA tRNALeu(UUR) tRNALeu (UUR) tRNA Ile tRNA Ile tRNA Trp tRNA Asn tRNA Cys tRNA Cys tRNA Ser (UCN) tRNA Lys tRNA Lys tRNA Gly tRNA Thr rRNA 16S tRNA Gly tRNA Lys

A10438G C14680A A14696G G14724A G14740A G15967A C15975T G4284A G12207A A4269G A4317G T1659C G3196A T4336C G5549A T5728C

Syndromes

Encephalomyopathy Encephalomyopathy, MERRF Encephalomyopathy, Leigh Syndrome Encephalomyopathy, Leigh Syndrome Encephalomyopathy Leigh Syndrome Encephalomyopathy MERRF Encephalomyopathy MERRF Encephalomyopathy MERRF Encephalomyopathy MERRF Encephalomyopathy Myoclonus and Psychomotor Regression Encephalomyopathy Ataxia, Myoclonus and Deafness Encephalomyopathy MELAS Encephalomyopathy MELAS Encephalomyopathy MELAS Encephalomyopathy MELAS Encephalomyopathy MELAS Encephalomyopathy MELAS Encephalomyopathy MELAS Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Encephalomyopathy Rett Syndrome Encephalomyopathy Mitochondrial Myopathy Mitochondrial Neurogastrointestinal Encephalomyopathy Encephalopathy tRNA Arg Encephalopathy tRNA Glu Encephalopathy tRNA Glu Encephalopathy tRNA Glu Encephalopathy tRNA Glu Encephalopathy tRNA Pro Encephalopathy tRNA Pro Encephalopathy tRNA Ile Multisystem Disease tRNA Ser (AGY) Mitochondrial Myopathy/Encephalopathy tRNA Ile Fatal Infantile Cardiomyopathy Plus (MELAS) tRNA Ile Fatal Infantile Cardiomyopathy Plus (MELAS) Other tRNA Val Movement Disorder rRNA 16S Alzheimer & Parkinson Disease tRNA Gln Alzheimer & Parkinson Disease; Deafness & Migraine tRNA Trp Dementia and Chorea tRNA Asn Multi-organ Failure

Symptoms Neurosensory Hearing Loss Neurosensory Hearing Loss Neurosensory Hearing Loss Neurosensory Hearing Loss Neurosensory Hearing Loss and Cerebellar Dysfunction Neurosensory Hearing Loss, Mental Retardation, Cerebellar Dysfunction MELAS/mitochondrial myopathy & exercise intolerance Ataxia, Myoclonus and Deafness MELAS/Leigh Syndrome MELAS MELAS MELAS/Myopathy/Deafness, Cognitive Impairment MELAS/encephalopamyopathy Maternally Inherited Leigh Syndrome Progressive Encephalomyopathy/Ataxia, Myoclonus and Deafness/Motor neuron disease-like MERRF MERRF MERRF/Maternally Inherited Cardiomyopathy/ deafness/Autism/Leigh Syndrome/Ataxia/Lipomas Progressive Encephalomyopathy MERRF-MELAS/cerebral edema Leigh Syndrome Adult Leigh Syndrome Maternally Inherited Leigh Syndrome MERRF MERRF MERRF/Maternally Inherited Cardiomyopathy, deafness/Autism MERRF/KSS overlap Myoclonic Epilepsy and Psychomotor Regression Ataxia, Myoclonus and Deafness MELAS MELAS MELAS/Myopathy MELAS MELAS MELAS MELAS Progressive Encephalomyopathy Encephalomyopathy Progressive Encephalomyopathy Encephalocardiomyopathy Encephalomyopathy Encephalomyopathy Encephalopathy Progressive Dystonia Progressive Encephalomyopathy, MERRF/MELAS Encephalopathy Dystonia and stroke-like episodes Progressive Encephalomyopathy Encephalomyopathy Rett Syndrome Gastrointestinal Reflux/Sudden Infant Death Syndrome Neurogastrointestinal encephalomyopathy

Progressive Encephalopathy Mitochondrial Encephalopathy Progressive Encephalopathy Mitochondrial Leukoencephalopathy Encephalopathy + Retinopathy MERRF-like disease Ataxia+retinopathy+deafness Varied familial presentation Myopathy/Encephalopathy Fatal Infantile Cardiomyopathy Plus Fatal Infantile Cardiomyopathy Plus Movement Disorder Alzheimer & Parkinson Disease Alzheimer & Parkinson Disease/Hearing loss and migraine Dementia and Chorea Multi-organ failure


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

Gene

A3796G T8993C T8993G T9176G T9176C T9185C T9191C C9537insC T10158C T10191C G10197A C11777A T12706C G14459A T14487C

MTND1 MTATP6 MTATP6 MTATP6 MTATP6 MTATP6 MTATP6 MTCO3 MTND3 MTND3 MTND3 MTND4 MTND5 MTND6 MTND6

T3308C G3376A G3697A G3946A T3949C A11084G A12770G A13045C G13513A A13514G A13084T G14453A 14787del4 C6489A G6930A 6015del5 T7587C G7896A 8042del2 G9952A T9957C 9205del2 A15579G T14849C

MTND1 MTND1 MTND1 MTND1 MTND1 MTND4 MTND5 MTND5 MTND5 MTND5 MTND5 MTND6 MTCYB MTCO1 MTCO1 MTCOI MTCO2 MTCO2 MTCO2 MTCO3 MTCO3 MTATP6 MTCYB MTCYB

G11778A G3460A T14484C G7444A A8108G C14340T

MTND4 MTND1 MTND6 MTCO1 MTCO2 MTND6

A3397G G5460A

MTND1 MTND2

Syndromes

Symptoms

Dystonia/Leigh syndrome Adult-onset dystonia Leigh Syndrome/Neuropathy Ataxia and Retinitis Pigmentosa (LS/NARP) NARP LS LS/familial bilateral striatal necrosis LS/Ataxia/NARP-like disease LS LS-like LS LS/LS-like Disease/Epilepsy, Strokes, Optic atrophy, and Cognitive decline LS/Dystonia/Stroke LS LS Leber hereditary optic neuropathy and dystonia/LS LS/dystonia/ataxia Encephalomyopathy MELAS MELAS/LHON MELAS/LS MELAS MELAS MELAS MELAS MELAS/LHON/LS overlap syndrome MELAS/LS MELAS MELAS/LS MELAS MELAS/Parkinson's-like Therapy-resistant epilepsy Multisystem disorder Myopathy and cortical lesions Encephalomyopathy Multisystem disorder Lactic acidosis Encephalomyopathy MELAS/progressive encephalopathy/non‐arteritic ischaemic optic neuropathy Lactic acidosis/seizures Multisystem disorder Septo-optic dysplasia Deafness/sensorineural hearing loss LHON LHON LHON Sensory neural hearing loss/LHON Sensory neural hearing loss Sensory neural hearing loss Alzheimer and Parkinson-like presentations ADPD AD

Myocytes and neurons are also cells with a high-energy demand. This characteristic makes them more vulnerable to the ATP depletion that can derive from the pathogenic mutations in the mtDNA that affect proteins involved in the respiratory chain [21]. 3. Neurodegeneration in mitochondrial diseases 3.1. Mitochondrial encephalopathies caused by MtDNA deletions Patients with mtDNA large deletions commonly show one of three classic phenotypes: Pearson Syndrome, chronic progressive external ophthalmoplegia (CPEO) and Kearns–Sayre syndrome (KSS). Patients with Pearson Syndrome show a multi-symptomatic disease from birth with a 50% surviving rate after 4 years of age. The main symptoms are sideroblastic anemia and exocrine pancreas dysfunction. Those who survive infancy are expected to develop KSS [22–24]. CPEO is characterized by ptosis and ophthalmoplegia with some patients showing also oropharyngeal and proximal muscle weakness. Patients with CPEO can have brain, inner ear, and retinal disease in later stages of the

Dystonia Leigh syndrome/Neuropathy Ataxia and Retinitis Pigmentosa Neuropathy Ataxia and Retinitis Pigmentosa Leigh syndrome Leigh syndrome Leigh syndrome/Neuropathy Ataxia and Retinitis Pigmentosa Leigh Syndrome Leigh Syndrome Leigh syndrome Leigh syndrome Leigh syndrome Leigh syndrome Leigh syndrome Dystonia/Leigh syndrome Dystonia/Leigh syndrome Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS, optic neuropathy Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS Encephalomyopathy, MELAS, Parkinsonism Encephalomyopathy, Epilepsy Encephalomyopathy, Multisystem Disorder Encephalomyopathy, Multisystem Disorder Encephalomyopathy Encephalomyopathy, Multisystem Disorder Encephalomyopathy, Lactic Acidosis Encephalomyopathy Encephalomyopathy, MELAS Encephalomyopathy, Lactic Acidosis Encephalomyopathy, Multisystem Disorder Encephalomyopathy, Septo-Optic Dysplasia Optic neuropathy, Sensory Neural Hearing Loss Optic neuropathy, Sensory Neural Hearing Loss Optic neuropathy, Sensory Neural Hearing Loss ,Sensory Neural Hearing Loss, optic neuropathy Sensory Neural Hearing Loss Sensory Neural Hearing Loss Alzheimer & Parkinson disease Alzheimer’s disease

disease, depending on the age of onset and on the level of heteroplasmy [25]. CPEO is commonly seen as a milder form of the disease, and clinical presentations can involve other muscles or symptoms and are sometimes referred to as CPEO Plus [26]. Although it is a multisytem disorder, CNS involvement is evident in KSS. The syndrome is defined by onset prior to 20 years of age, progressive external ophthalmoplegia (PEO), and a pigmentary retinopathy (usually rod-cone dystrophy). Moreover, patients may also show cardiac conduction block (usually the cause of death in young adulthood), elevated cerebrospinal fluid protein level, or cerebellar ataxia [26]. Other neurologic problems may include proximal myopathy, exercise intolerance, ptosis, oropharyngeal and esophageal dysfunction, sensorineural hearing loss, dementia, and choroid plexus dysfunction resulting in cerebral folate deficiency [27]. 3.2. Mitochondrial encephalopathies caused by mtDNA point mutations Almost 600 pathogenic point mutations have been identified in the last 25 years, involving most of the mtDNA molecule (according to



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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

POLG2 ANT1 MPV17

Catalytic subunit of DNA polymerase gamma, mtDNA replication Adenine nucleotide translocator isoform 1, dNTPs pool maintenance Unknown, involved in the metabolism of reactive oxygen species and in the regulation of mtDNA copy number Twinkle helicase, mtDNA replication Thymidine phosphorylase, dNTPs pool maintenance Deoxyguanosine kinase Mitochondrial, dNTPs pool maintenance Ribonucleotide reductase, dNTPs pool maintenance Succinate-CoA ligase, ADP-forming, beta subunit Succinate-CoA ligase, alpha subunit mitochondrial genome maintenance exonuclease 1 helicase/nuclease, mtDNA replication, long-patch base-excision repair pathway Thymidine kinase mitochondrial, dNTPs pool maintenance

AD AD AR

Alpers syndrome, AD-PEO and AR-PEO, male infertility, SANDO* syndrome, SCAE* AD-PEO AD-PEO, multiple mtDNA deletions Hepatocerebral MDDS

AD AR AR AR AR AR AR AD

AD-PEO, SANDO syndrome MNGIE, mtDNA depletion Hepatocerebral mtDNA depletion syndrome Encephalomyopathic Renal tubulopathy MNGIE, AD-PEO Encephalomyopathy with methylmalonic aciduria Encephalomyopathy with methylmalonic aciduria Mitochondrial DNA depletion syndrome 11 PEO

AR

Myopathic mtDNA depletion

Twinkle (C10orf2) TYMP DGUOK RRM2B SUCLA2 SUCLG1 MGME1 (C20orf72) DNA2 TK2

MITOMAP, 299 point mutation involving tRNA–rRNA and control regions and 274 involving OXPHOS proteins). The most common mitochondrial encephalopathies caused by point mutations can be classified in clinical categories: LHON (Leber Hereditary Optic Neuropathy), Leigh Syndrome, MELAS (Mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms), MIDD (maternally inherited diabetes and deafness), MERRF (Myoclonic Epilepsy with Ragged Red Fibers), non-syndromic hearing loss and NARP (Neuropathy, ataxia, retinitis pigmentosa). Approximately 95% of LHON cases show a mutation in one of three mtDNA genes: G11778A, G3460A or T14484C, respectively encoding for ND4, ND1 and ND6, all subunits of Complex I of the electron transport chain (Fig. 1). The main characteristic of the disease is a painless, bilateral, subacute or acute visual failure, prevalently in young male adults, caused by the atrophy of the optic nerve [28]. Neurodegeneration is limited to the retinal ganglion cell (RGC) layer with cell body and axonal degeneration, demyelination and atrophy observed from the optic nerves to the lateral geniculate bodies [29]. One possible explanation for the involvement of the optic nerve alone is the disruption of glutamate transport out of the inner retina with consequent excitotoxic damage [30]. There is a correlation between genotype and the severity of the disease: patients with the G11778A mutation seem to have the most severe phenotype, with the least favorable visual outcome, while the T14484C mutation is associated with the mildest pathogenicity [31]. Leigh Syndrome can be caused by different mutations, both in mtDNA and in nDNA genes. Mutations can occur in genes encoding for tRNAs as well as in genes encoding for complexes I, IV, or V of the OXPHOS [32–37] and are commonly present in heteroplasmy. The heterogenic symptoms include motor and intellectual developmental delay, bilateral brainstem disease, basal ganglia disease, elevated blood or CSF lactate levels, hypotonia, spasticity, chorea and other movement disorders, cerebellar ataxia, peripheral neuropathy, and respiratory failure secondary to brainstem dysfunction [35]. The condition may also affect the liver, heart (including hypertrophic cardiomyopathy), kidneys, and pancreas [38]. About 80% of MELAS cases are caused by a very common A3243G mutation in the mitochondrial tRNALeu(UUR) gene, whereas 10% of cases carry the T3271C mutation in the same tRNALeu(UUR) (Fig. 1), although other mtDNA point mutations have been also associated with this phenotype (MITOMAP). As the vast majority of the mitochondrial diseases, this is a multisystemic disorder with symptoms varying depending on the heteroplasmy status and on the age of onset. Other than mitochondrial myopathy, encephalomyopathy, lactic acidosis and stroke-like symptoms, patients can also show deafness, diabetes, migraines, gut immobility and seizures [39]. Multiple strokes affect the patients, mainly in the cerebral cortex or

in the subcortical white matter, causing multifocal necrosis with lesions that do not respect vascular territories and are often accompanied by profound neuronal cell loss, neuronal eosinophilia, astrogliosis and spongiform degeneration [40]. There is also a loss of Purkinje cells causing cerebellar degeneration and a particularly prominent calcification in the basal ganglia. Depending on the portions of the brain affected by the ischemic lesions, symptoms may vary from focal status epilepticus and epilepsia partialis continua to just occipital migraines and a visual aura. A3243G is also the most frequent mutation associated with MIDD (maternally inherited diabetes and deafness) [41] in fact, patients diagnosed with this disease may also have typical symptoms of MELAS including myopathy, cardiomyopathy, neuropsychiatric symptoms, and renal disease and patients belonging to the same family can have MELAS or MIDD. The level of heteroplasmy can explain in part the different clinical outcome [42]. The neurodegeneration that typically causes the sensorineural deafness involves the auditory nerve or the brain, with defect in oxidative phosphorylation in the beta cells that sense glucose and diminished ATP production by strial marginal cells of the inner ear [43]. In 90% of the cases of MERRF, the mutation responsible is an A8344G transition, in the tRNALys, [44] (Fig. 1). The myoclonic epilepsy is the main symptom associated with this disease together with the presence of clumps of diseased mitochondria accumulation in the sub-sarcolemmal region of the muscle fiber called “Ragged Red Fibers.” Other symptoms like ataxia, neuropathy, and cardiac abnormalities can be present. Curiously, many patients with the A8344G mutation also show multiple lipomas in the back region [45]. The main neuropathological signs involve the olivocerebellar pathway, with severe neuron loss from the inferior olivary nucleus, Purkinje cells, and dentate nucleus. Neurodegeneration is present also in the gracile and cuneate nuclei, moreover there are signs of demyelination of the spinal cord [46]. In the surviving neurons there are evidences of respiratory chain deficiency and presence of enlarged mitochondria containing inclusions [47]. No clear correlation has been identified between genotype or heteroplasmy and clinical phenotype, nor it is clear why typical MERRF is associated with mutations in tRNALys [48]. Non-syndromic hearing loss, that means severe and profound deafness not associated to other neurologic or systemic diseases, can be caused by mutations in mitochondrial tRNAs like A3243G, A7443/4/5G, and in rRNAs like 961delT, and A1555G [49] (Fig. 1). NARP is associated mainly with the mutation T8993G (or T8993C) in the mtDNA encoding for the MTATP6 gene, even though a family with a G8989C mutation has also been described [50] (Fig. 1). This disease is characterized by sensory or sensorimotor axonal neuropathy, neurogenic muscle weakness, ataxia, cerebral or cerebellar atrophy, and retinitis pigmentosa. Other non-typical neurological symptoms,


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including seizures, learning problems, hearing loss, progressive external ophthalmoplegia, and anxiety can be present. 3.3. Mitochondrial diseases caused by mutations in nDNA genes affecting mtDNA stability MtDNA changes can be a consequence of mutations in nDNAencoded genes involved in the maintenance of mtDNA integrity and mtDNA copy number. The most common mutations affect POLG, the gene encoding for the catalytic subunit of the mitochondrial DNA polymerase gamma, and PEO1 which encodes for the DNA helicase Twinkle [51,52], both genes involved in mtDNA replication. Mutations in these genes provoke an accumulation of mtDNA point mutations and deletions eventually leading to a different clinical manifestation. Over 200 mutations in POLG associated with mitochondrial diseases have been identified, causing a plethora of heterogeneous disorders involving different tissues, time of onset and severity. At least five major phenotypes can be distinguished: Alpers–Huttenlocher syndrome, childhood myocerebrohepatopathy spectrum, myoclonic epilepsy myopathy sensory ataxia, the ataxia neuropathy spectrum, and progressive external ophthalmoplegia (PEO) with or without sensory ataxic neuropathy and dysarthria [53–56]. Patients with mutations in POLG also show defects in complex I and complex IV in dopaminergic neurons of the substantia nigra and neuronal cell loss [57,58]. Recessive mutations in Twinkle protein cause severe, early-onset disorders due also to defects in mtDNA maintenance, such as infantileonset spinocerebellar ataxia and a hepatocerebral mtDNA depletion disorder with severe epilepsy, migraine, and psychiatric symptoms [55]. Also TYMP, that encodes for thymidine phosphorylase and is essential in the nucleotide salvage pathway for mtDNA replication, is typically mutated (multiple deletions and single base changes) in mitochondrial neurogastrointestinal encephalomyopathy [59,60]. Mutations in OPA1, a protein involved in the mitochondrial fusion process, cause autosomal dominant optic atrophy with loss of retinal ganglion cells and progressive optic nerve degeneration with cases of more severe neuromuscular complications [61,62]. RRM2B encodes a ribonucleotide reductase and is mutated in cases of autosomal dominant OPA [63,64]. Mutations in both genes cause mtDNA instability and multiple deletions. Changes in mtDNA copy number are also associated with clinical syndromes. The MDS (mtDNA depletion syndromes) are autosomal recessive disorders caused by mtDNA depletion in clinically affected tissues [65]. Most of them derive from a mutation in nDNA genes involved in mtDNA replication machinery (POLG, Twinkle) or in different mitochondrial functions like nucleotide metabolism (TYMP, TK2, DGUOK and MPV17) or fission–fusion machinery (Mfn2) [66–70]. Phenotypically they are very heterogeneous with myopathic, encephalomyopathic, hepatocerebral or neurogastrointestinal manifestations, with a common neurological involvement [69,71]. 4. mtDNA changes in age-related neurodegenerative diseases MtDNA changes have been hypothesized to have a role also in agerelated neurodegenerative diseases like Parkinson's (PD), Alzheimer's (AD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). PD is the second most common progressive neurodegenerative disorder, mainly characterized by motor (resting tremor, postural instability, rigidity and bradykinesia) and non-motor symptoms (fatigue, depression, anxiety, sleep disturbances, autonomic disturbances, decreased motivation, apathy and a decline in cognition, dementia). The cause of the motor symptoms is a depletion of dopamine in the striatum, derived from a specific degeneration of dopaminergic neurons located in the midbrain and forming the substantia nigra (SN). The etiology of this disorder is still unknown, but a role of mitochondrial dysfunction, and in particular of Complexes I and IV of the electron transport chain, has been proposed in the last decades and extensively reviewed [72–75].

Different hypotheses have been proposed to explain the high susceptibility of SN to mitochondrial stress and to mtDNA damage, and the anatomical characteristics of these neurons, that have particularly long unmyelinated axons, seem to be the most accredited [76,77]. More specifically, different groups have also analyzed mtDNA changes in patients' post-mortem brain: high levels of mutations and deletions are found within the neurons of the SN both in patients with PD and during normal aging [78,79]. If mtDNA changes are sufficient to confer PD symptoms is still controversial, studies on animal models [80–82] and the fact that patients with POLG mutations also show Parkinsonism similar to cases of juvenile PD [57,83] suggest that they play a role in the pathogenesis. Moreover, it has also been suggested that different mtDNA haplotypes can confer higher or lower risk factors to develop PD, because different polymorphisms can show subtle differences of the respiratory chain activity and ROS production. In particular, the super-haplogroup JT seems to confer a reduced risk of PD while the super-haplogroup HV seems to be associated with an increased risk [84]. AD is the most common late-onset progressive neurodegenerative disease, clinically characterized by memory loss, impairment of cognitive functions and changes in behavior and personality. The neurodegeneration that occurs in these patients affect mostly the cortex and the hippocampus. Here there is an accumulation of senile plaques, composed mainly by beta-amyloid peptide, and intraneuronal tau deposition as neurofibrillary tangles. The most studied pathogenetic model for this disease is the “beta-amyloid cascade”, where an unbalance in the cleavage sequence of APP (amyloid precursor protein) leads to an accumulation of toxic Aβ fragment, cytotoxic plaques, and consequent neurodegeneration. Mitochondrial dysfunctions, in particular cytochrome c oxidase defects, have also been implicated in the development and progression of AD [85,86]. Aβ fragments negatively affect mitochondrial function, suggesting that mitochondrial dysfunction is a consequence of the Aβ toxicity, moreover studies in AD mouse models show also that Aβ oligomerization impairs mitochondrial function [87–90]. Moreover, the γ-secretase activity and so the APP cleavage, occurs predominantly in the MAM (mitochondria-associated ER membranes), a compartment of the endoplasmic reticulum physically and biochemically connected to mitochondria. MAM function and ER–mitochondrial connectivity are increased in AD, so that AD is also proposed to be an ER–mitochondrial communication and MAM dysfunction disease [91,92]. This hypothesis, other than the β-amyloid cascade one, would explain also other biochemical changes occurring in the disease like mitochondrial dysfunction, elevated levels of cholesterol, altered metabolism of fatty acids and phospholipids, and aberrant calcium homeostasis [93]. The presence of increased mutations in mtDNA of AD patients is still a controversial subject: different laboratories with various groups of patients and dissimilar techniques have obtained debatable results [94–98]. It has also been suggested that inherited haplogroups may influence AD risk but to date, no clear result has been found [99]. ALS is a motor neuron disease, characterized by rapidly progressive weakness, muscle atrophy and fasciculation, muscle spasticity, dysarthria, dysphagia, dyspnea caused by the degeneration of the upper and lower motor neurons. Increased mtDNA deletions have been found in muscle and brain of ALS patients, although the levels are still relatively low and of unknown consequence [100–102]. Moreover in some cases of patients with mtDNA mutations, an ALS phenotype has been diagnosed [103–106]. Also in this case the association of haplogroup with increased risk factor is still controversial [107]. MS is a chronic inflammatory autoimmune disease caused by loss of myelin and gliosis. The etiology of this disease is largely unknown and hypothesis on genetic, environmental and infective agents have been analyzed without a clear response. A mitochondrial role has been proposed since changes in the expression of TFAM, PGC1α and nuclear respiratory factor 1 (NRF1) have been found in cortical neurons of MS patients [108,109]. Moreover in patients' post-mortem brains, there is



a reduction of Complex I and Complex III activity [110]. Different mtDNA mutations have been reported in patients with MS [111] even though the direct role of mtDNA single mutation or deletion in the pathogenesis of this disease is also still controversial [112]. 5. Mouse models of mtDNA alterations in neurodegenerative conditions As described in the previous paragraphs, mtDNA changes have been implicated in a vast group of diseases and possibly normal aging. In order to study the mechanisms behind these pathologies, the use of mouse models is compelling. Unfortunately, engineering and integrating specific mtDNA mutations in mice, in particular pathogenic mutations, is technically challenging, most of all because of the “bottleneck effect” [113]. The specific mutation in mice will be diluted in the progeny so that to date, mouse models with pathological mtDNA mutations are rare [114] and, if not lethal, kept in homoplasmy [115]. As detailed below, the modeling of mtDNA changes has been approached in two alternative ways: by engineering nuclear genes or by direct manipulation of mtDNA [116]. On one side, mouse models with mtDNA mutations have been created by genetic modification of nDNA encoded genes involved in mtDNA maintenance like POLG, Tfam and Twinkle, on the other hand specific deletions have been created in the mtDNA by the introduction of defective mitochondria into mouse zygotes or by the expression of restriction mitochondria-targeted endonucleases. 5.1. Manipulation of nDNA encoded genes that affect mtDNA One of the first nDNA-encoded genes to be targeted in order to induce an indirect damage to mtDNA was TFAM. It is a transcriptional activator that binds mtDNA promoters and activates transcription. It is also necessary for mtDNA replication since it provides the RNA primers for initiation and it has an important histone-like role in mtDNA maintenance since it binds to mtDNA coating it [117]. Tfam KO mice completely lack mtDNA and die during embryogenesis. Tissue-specific disruption of Tfam shows different clinical evidences, in particular neurodegeneration is present when Tfam is knocked out in forebrain neurons (MILON mice) and in dopaminergic cells (MitoPark mouse). In MILON mice, Tfam was deleted post-natally, showing no sign of disease until 6–8 months of age, when deteriorating conditions and death appeared in 2–3 weeks caused by significant mtDNA depletion [118]. In Mito-Park mice, Tfam was deleted in dopaminergic neurons, causing a Parkinson-like phenotype with dopamine depletion, neurodegeneration of dopaminergic cells of substantia nigra and motor deficits [119]. POLG is also essential for mtDNA replication, so that POLG KO mice are embryonically lethal [120]. In order to induce mtDNA damage in different tissues, Polg gene has been manipulated to impair its proofreading activity. Two different groups created the “mutator mouse” by knocking-in PolgA gene carrying two different mutations: the D257A mutation [121] or the AC → CT substitution in positions 1054 and 1055 [122]. These mutations provoked an accumulation of mtDNA point mutations in different tissues [123], causing two very similar clinical phenotypes. The mutator mice showed signs typical of premature aging, with reduced life span, kyphosis, alopecia, weight loss, reduced fat content, osteoporosis, thymic involution, testicular atrophy, loss of intestinal crypts, progressive decrease in circulating red blood cells, hearing loss and sarcopenia. Surprisingly these mice did not show striking signs of neurodegeneration [124]. Nonetheless, detailed studies of brain mtDNA by NextGen sequencing show that brain of mutator mice accumulate mtDNA species with abnormal D-loop structures. These control region multimers (CRMs) may reflect disrupted replication events, although their functional consequences are unknown [123]. As previously described, Twinkle has a helicase activity essential for mtDNA replication and maintenance. The Twinkle A360T mutation and

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aa 353–365 duplication are typical of PEO so that Twinkle mice have been generated knocking in the mutated gene [125] resulting in an accumulation of multiple large mtDNA deletions mostly in brain and muscle. These transgenic mice show progressive mitochondrial myopathy and mitochondrial impairment in cerebellar Purkinje cells and hippocampal CA2 pyramidal neurons. Other nDNA-encoded genes have been manipulated to indirectly affect mtDNA stability. Among them, TYMP/UP KO mice, who lack thymidine phosphorylase activity, showed partial mtDNA depletion in brain, late-onset vacuoles in cerebral and cerebellar white matter without demyelination or axonal loss [126]. MPV17 KO mice showed mtDNA depletion in different tissues including brain [127] TK2 KO [128], mutant TK2 knock in mice [129] and RRM2B KO mice [64] also showed mtDNA depletion. A novel mouse model is also expressing a mutated version of UNG1 in neurons (that removes thymine, in addition to uracil from mtDNA), showing signs of neurodegeneration and impaired behavior [130].

5.2. Direct damage to mtDNA The “mito-mice” [131] have been generated by introducing exogenous mitochondria carrying mutated mtDNA into mouse zygotes. Three mito-mice have been generated so far, a ΔmtDNA (carrying a 4696 bp deletion), mtDNA-COI (carrying T6589C mutation in mtDNA gene encoding cytochrome c oxidase subunit I) and mtDNA-ND6 (with A13997G mutation in mtDNA gene encoding NADH dehydrogenase 6) [132–134]. ΔmtDNA mice show various percentage of heteroplasmy in different tissues and clinical defects resembling the ones of mitochondrial diseases like low body weight, lactic acidosis, systemic ischemia, hearing loss, renal failures, and male infertility. Mice with more than 60% ΔmtDNA show mitochondrial respiration defects in brain and a downregulation of Calmodulin-dependent Protein Kinase II subunit alpha (CamKIIa) leading to impairment of spatial remote memory. MtDNA-COI and mtDNA-ND6 are homoplasmic and show typical signs of COXI or Complex I deficiencies [135]. The disadvantage of the use of these mice is the colony maintenance: ΔmtDNA is inherited by progeny for only about three pregnancies, since the amount of ΔmtDNA in eggs decreases with the aging of the females. Moreover it is difficult to obtain a large population of mito-miceΔ with the same ΔmtDNA load. Recently, Lin et al. created a novel animal model of LHON by introducing the homoplasmic mtDNA ND6P25L mutation into the mouse [136]. Mice with this mutation exhibited reduction in retinal function, decline in optic nerve fibers, neuronal accumulation of abnormal mitochondria, axonal swelling, and demyelination. The mito-PstI mouse expresses a mitochondria-targeted restriction endonuclease, mito-PstI, in an inducible and tissue-specific way, depending on the promoter that controls the expression of the transgene [137]. Mito-PstI cleaves the mtDNA in two different sizes, leading to the accumulation of multiple deletions and eventual mtDNA depletion. When expressed in neurons (under the control of CamKIIα promoter), it causes different phenotype, depending on the level and timing of expression. On a high expression from birth, the mice show an acute neurodegeneration with an abnormal ‘limb-clasping behavior’ at 2 months of age. With a high expression starting at P21, mice progressively become less active and died before 100-day-old with a severe reduction of COX activity in the forebrain. When mito-PstI is expressed at lower levels, mice show no differences in lifespan up to 16 months of age with no obvious phenotypic abnormalities until 6–8 months, when they displayed abnormal and progressively worse motor behavior. This phenotype is caused by a progressive neurodegeneration particularly severe in the striatum and cortex due to a decreased COXI activity [138,139]. Moreover, when expressed selectively in dopaminergic neurons (under the control of DAT promoter), mito-PstI causes a PDlike phenotype with progressive motor defect and loss of dopaminergic


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neurons [81], providing further evidences of the role of mtDNA mutations in the pathogenesis of this disorder. Mito-PstI mice have also been crossed with an AD mouse model that shows plaque formation in cortex and hippocampus. When mtDNA double strand breaks are induced in these mice for two months starting at 6 months of age, there is an unexpected reduction of amyloid plaques, probably because of a misbalance in the secretases activity [140]. The accumulation of plaques has been associated with oxidative stress, and these data indicate that inducing mtDNA damage does not necessarily increase oxidative damage. When the levels of mtDNA are reduced, also the steady-state levels of OXPHOS complexes decrease, which can lead to a decrease in ROS formation [140].

6. Conclusions Mitochondria are essential organelles that provide energy thanks to the oxidative phosphorylation process. Since different proteins of the electron transport chain are encoded by the mtDNA, its stability is necessary for the health of the entire cell and tissue. Changes in mtDNA can derive from direct mutations and deletions (somatic or inherited), or from mutations in nDNA encoded genes involved in the maintenance, stability and replication of the mtDNA. When pathogenic mutations occur, post mitotic tissues are most vulnerable because of their biological characteristics, so that muscle and central nervous systems are often affected in mitochondrial diseases. Different forms of neurodegeneration are common features of mitochondrial diseases. Although low levels of mtDNA changes have been described in age-related neurodegenerative disorders like Parkinson's or Alzheimer's diseases, their contribution to the diseases process remains poorly understood. Thanks to the useful tools developed in the last decades such as different techniques to investigate the mtDNA sequence (including NextGen [123]) and the creation of mouse models, we have now a better idea of the spectra of mtDNA mutations observed in aged tissues [141] and the consequences and molecular features of inducing mtDNA damage in animal models [118–136]. Our knowledge about the role of mtDNA alterations leading to neurodegeneration is likely to increase at an accelerated rate in the next few years.

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Misfolded proteins, mitochondrial dysfunction, and neurodegenerative diseases.

p53 and mitochondrial dysfunction: novel insight of neurodegenerative diseases.

The role of mitochondrial DNA mutation on neurodegenerative diseases.

Mitochondrial quality control: decommissioning power plants in neurodegenerative diseases.

Leber's hereditary optic neuropathy: a model for mitochondrial neurodegenerative diseases.

Mitochondrial dysfunctions in neurodegenerative diseases: relevance to Alzheimer's disease.

Dysfunctional Mitochondrial Dynamics in the Pathophysiology of Neurodegenerative Diseases.

Exploring mitochondrial system properties of neurodegenerative diseases through interactome mapping.

Selenium, selenoproteins and neurodegenerative diseases.

Ubiquitin, lysosomes, and neurodegenerative diseases.

Protein misfolding and neurodegenerative diseases.

Scrapie and human neurodegenerative diseases.

Compromised autophagy and neurodegenerative diseases.

Ubiquitin, lysosomes and neurodegenerative diseases.

Genetics of neurodegenerative diseases.

Optogenetics for neurodegenerative diseases.

Spermidine on neurodegenerative diseases.

Cytoprotective pyridinol antioxidants as potential therapeutic agents for neurodegenerative and mitochondrial diseases.

Human transmissible neurodegenerative diseases (prion diseases).

Mitochondrial dysfunction and cell death in neurodegenerative diseases through nitroxidative stress.

Mitochondrial biogenesis: a therapeutic target for neurodevelopmental disorders and neurodegenerative diseases.

Emotional dysfunctions in neurodegenerative diseases.

Gene therapy for neurodegenerative diseases.

DNA damage in neurodegenerative diseases.