Mitochondrial genome changes and neurodegenerative diseases^☆

1.1. The mtDNA

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 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 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 tRNA^Leu(UUR) gene, whereas 10% of cases carry the T3271C mutation in the same tRNA^Leu(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 tRNA^Lys, [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 tRNA^Lys [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, 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 nDNA-encoded 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 infantile-onset 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].

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 postnatally, 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 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 mitomiceΔ 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 PD-like phenotype with progressive motor defect and loss of dopaminergic 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].