MtDNA mutagenesis in the brain

mtDNA alteration are possibly associated with neurodegenerative diseases

The discovery that mtDNA mutations are of pathological importance (82,129,130), and that mitochondria play an important role in the mechanisms of aging ((131-133), reviewed in (2)) and cell death (7,134) shows the importance of mitochondria in pathological research. The spectrum of phenotypes has expanded from rare myopathies to multiple diseases representing virtually all branches of medicine. The possibility that some of the most common and devastating degenerative diseases seems to involve mitochondria implicates the importance of investigations of mitochondrial genetics and biochemical changes in these organelles. Although mitochondrial mutations are present at low levels (usually < 2%) in the whole tissue, it could be possible that mutations clonally expand within one cell and exceed a defined threshold which could cause defects of mitochondrial oxidative metabolism and may lead to cell death.

Although cells possess an intricate network of defense mechanisms to neutralize excess ROS and reduce oxidative stress, some tissues, especially the brain, are much more vulnerable to oxidative stress because of their elevated consumption of oxygen and the consequent generation of large amounts of ROS. For the same reason, the mtDNA of brain cells is highly susceptible to structural alterations resulting in mitochondrial dysfunction (135).

There are many reports on mitochondria and mtDNA and a possible involvement with neurological degenerative diseases (136-138), e.g. Parkinson's disease (PD) or Alzheimer's disease (AD) (139-146). Still, investigations are contradictory or not yet confirmed (147). Screening for a specific substitution did not reveal any differences between brains from normal elderly persons or patients with AD or PD (140,148). However, increasingly studies report correlations between certain neurological diseases and mtDNA mutagenesis. In this section, some of the diseases that are possibly connected to mitochon-drial mutagenesis and that are currently under excessive investigation will be presented briefly as an overview.

mtDNA maintenance is possibly connected with Parkinson's disease (PD)

PD is one of the most widespread age-associated neurodegenerative disease with motor abnormalities. PD is caused by a decrease in nerve cells in the substantia nigra. Mito-chondrial dysfunction in nigral neurons is supposed to be involved in its etiology and progression of the symptoms (146). Nigral cells of PD patients are intensely stained with anti-4-hydroxynonenal (HNE) antibodies, whereby HNE is used as a marker molecule for lipid peroxidation (147-149). Hence, it is suggested that in PD the brain is under increased oxidative stress. Matching these findings is the increased amount of 4977-bp deleted mtDNA molecules in PD patients compared to age-matched controls (150). Regarding 4977 bp as a marker molecule for oxidative stress to mtDNA, as already mentioned, this would imply that mtDNA in PD is more damaged, whereby this deterioration seems to be rather intense in nigral cells.

Friedreich's ataxia is caused by a depletion of the frataxin protein

Friedreich's ataxia (FRDA) is a rare neurodegenerative disease accompanied by cardiomyopathy and diabetes (151), causing same symptoms that are frequently observed in mitochondrial encephalomyopathy with mtDNA abnormalities. Meanwhile, it is known that mitochondrial dysfunction and oxidative stress cause FRDA. Thereby the causative gene is found and designated as frataxin (152). Its exact function is not yet completely understood, but most of the patients show a frataxin decrease. Since this protein is involved in the homeostasis of mitochondrial iron and/or assembling of iron-sulfur clusters, a frataxin decrease leads to an iron accumulation and decrease in the activities of mitochondrial respiratory chain complex enhancing mitochondrial oxidative stress and mtDNA damage (151). Confirming the correlation between ROS and FRDA is the finding that antioxidant therapy can slow the progression of Friedreich's ataxia (152,153).

The findings concerning the correlation between Alzheimer's disease (AD) and mtDNA mutagenesis are contradictory

AD is the most prevalent late-onset neurodegenerative disorder with an estimated 3-4 million affected individuals only in the US (145). Although there are a small number of families with autosomal-dominant AD, more than 90% of the cases are classified as sporadic in origin. The neuropathology is rather complex with abnormalities in many neuronal functions. In addition to the characteristic late-stage amyloid plaques and neu-rofibrillary tangles, there is considerable evidence for abnormal mitochondrial function and oxidative stress in affected patients (154). According to this, decreased mitochondrial COX in multiple tissues from AD patients, including autopsied brain samples was found (155,156).

However, the etiologic origin of the mitochondrial functional defect in AD - and its pathogenic significance - remains unclear (139,157) and controversial. Despite findings supporting mitochondrial involvement in experiments with cybrids, e.g. an increase of morphologically abnormal mitochondria, no candidate mtDNA mutations in such AD cybrids have been identified yet. With respect to the many studies on mitochondrial mutagenesis and AD, the experimental design might be crucial. Elson et al., for example, found no evidence that pathogenic mtDNA mutations play a major or dominant role in the development of AD, whereby they admit that it remained unsettled whether mtDNA mutations might have a pathogenic role in a small subset of patients or that a small subset of the population that does not develop AD carries a mutation that serve a protective role (158). Additionally, the existence of an mtDNA pseudogene might be troublesome for the detection of disease-related mtDNA mutations as already shown in 1998 (81).

Mitochondria seem to be involved in the motor neuron degeneration process found in patients with Amyotrophic lateral sclerosis (ALS)

ALS is a devastating neurodegenerative disease that affects the anterior horn cells of the spinal cord and cortical motor neurons, where accumulation of abnormal mitochondria has been observed (159). In 5-10% of the cases, ALS is inherited as an autosomal trait; the etiology of the remaining 90-95% of the sporadic (sALS) is currently undefined. Meanwhile, it is accepted that genetic susceptibility factors exist and should play a key role, interacting with environmental and toxic factors, in the etiopathogenesis of this disorder. Mitochondrial variations seem to contribute to the risk of ALS development in Caucasians as shown by Mancuso et al. (160). They had genotyped predefined European mtDNA haplogroups in Italian patients with sALS and matched controls and found a possible protective gene factor (mtDNA haplotype I) associated with ALS (161). Several other studies also suggested a potential role of mitochondrial dysfunction in the pathogenesis of ALS, e.g. respiratory chain deficiency in skeletal muscle of patients with late stage of ALS (162), a decrease in COX activity in motor neurons of patients with sALS (163), and an increased level of 4977-bp deleted mtDNA in brain and skeletal muscle of sALS patients (164).

Mitochondrial mutagenesis in the brain might be relevant for a forensic approach

More than 50% of all forensic autopsies give evidence of brain-induced functional arrest of the organ systems, i.e. in brain-caused death, which can be partly the result of mechanical injury, of chemical influences, or of the result of hypoxic/ischemic events. Death due to functional disturbances of the brain is suggested in a great number of these cases, i.e. in about 20-30% of all autopsies. In two-third of these cases, cerebral hypoxia and/or ischemia explains the functional failure of the central nervous system (CNS). The hypoxic/ischemic event might be caused by passing cardiac arrest, by asphyxia, by vessel's obstruction, and by traumatic or chemical mechanisms as well. Thus, questions concerning recognition of tissue and cell destruction and recovery, especially of the cause that is suspected to be induced by hypoxic/ischemic influences arise for the forensic neuropathologist. Therefore, in 10-20% of all autopsy cases a forensic pathologist is called for an expert's report on the pathophysiology and morphology of the CNS under hypoxic/ischemic conditions (165). On the subcellular level, acute ischemic neuronal injury is characterized by mitochondrial alteration. Opening of the mitochondrial membrane permeability transition pore (MPTP) can be triggered in postischemic neurons by a variety of accumulated free radicals. All of them are consequences of impairment of mitochondrial function and energy homeostasis of the neuron. Under ischemic conditions, mitochondria lose their capacity for OXPHOS. The resulting loss of energy leads to an electrolyte disturbance developing cellular edema, i.e. swelling of mitochondria. The sudden increase in extracellular K+ is associated with a rapid increase of intracellular Ca2+. Changes of intracellular Ca2+ play a crucial role in the destructive events of neurons, because abnormal levels of intramitochondrial Ca2+ alter the activity of the electron transport chain (ETC) complexes leading to the impairment of OXPHOS with reduced ATP levels. High levels of intramitochondrial Ca2+ can promote the release of cytochrome c, which can trigger the apoptic cascade as well as the generation of free radicals, that can damage the macromolecules, i.e. proteins, lipids, and DNA, of the cell (1,166).

The amount of 4977-bp dmtDNA increases region-specific in the human brain

Older individuals show an accumulation of a heterogeneous array of mtDNA rearrangements as well as heterogeneity of mutations between different brain regions within the same individual (92,167). This was confirmed in our investigations detecting the common deletion in five different brain regions obtained at autopsies (n = 26). Fragment specific for total and 4977-bp deleted mtDNA were amplified in a Duplex-PCR, detected, and relatively quantified after capillary electrophoresis in an ABI Prism 310 using the Gene Scan software 2.1 (Applied Biosystems). The amount of deleted mtDNA (dmtDNA) increased with advancing age in four investigated regions: caudate nucleus, substantia nigra, putamen, and parietal cortex, but not in cerebellar tissue. The strongest correlation with age and the highest amount of dmtDNA was found in caudate nucleus. In this tissue, the deletion-specific signal was detected in children aged six years and younger and the signal increased in this assay to more than fivefold of the dmtDNA-specific fragment in 73- and 84-year-old individuals. This strong correlation was followed by putamen, substantia nigra, and parietal cortex (112).

The cerebellum seems to be well protected against ROS

In this same study in cerebellar tissue, however, the 4977-bp deletion could only be detected in very low amounts in six out of 26 investigated persons independent of their age. Subsequent histological investigations of these cases revealed signs of prolonged ischemia (112). Thus, it might be hypothesized, that the 4977-bp deletion is an indicator for oxidative stress and that its accumulation correlates with oxidative damage sustained some weeks or more ago. Supporting the existence of protective mechanisms is the study by (35). They quantified multiple oxidized bases in nuclear and mtDNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age-matched controls (n = 8 per group) using gas chromatography/mass spectrometry with selective ion monitoring (GS/MS-SIM) and stable labeled internal standards. Levels of multiple oxidized bases in AD brain samples were significantly higher in frontal, parietal, and temporal lobes compared to control subjects whereas the cerebellum was only slightly affected (35). Similar findings were made in C57BL/6 mice treated with MPTP. The animals developed elevated tissue hydroxyl radical levels in striatum and ventral midbrain but not in the cerebellum (168). As already known, free radicals in the cell can be disarmed by antioxidative enzymes, e.g. the mangan-dependent superoxide dismutase (MnSOD), the Cu/Zn superoxide dismutase or the glutathione peroxidase. The overexpression of MnSOD reduces neuronal damage due to oxidation processes and elevates the tolerance against ischemia (169). The cerebellum seems to be well protected against ROS due to sufficient amount of antioxidative enzymes.

Single-cell PCR might reveal more insights into the distribution pattern of mtDNA alterations in the brain

The development of single-cell PCR can be useful to study mtDNA alterations and their direct influence on biochemical function in the mitochondria such as COX activity (80,170). To investigate whether certain cell characteristics underlie the mosaic-like distribution pattern of the 4977-bp deletion in the brain and to look for possible age-related changes, single-cell PCR analysis can provide more detailed information. In one study, two cell types - GFAP expressing astrocytes and MAP expressing neurons - were visualized and isolated in the caudate nucleus from five young and five older individuals. Tissues were obtained at autopsy and subjected to formalin fixation. After staining and histological examination, the cells were microdissected under microscopical control and mtDNA was analyzed in a single-cell PCR. For each of the ten individuals, at least 30 cells per cell type were collected in this way and subjected to PCR individually. Screening for the presence of the 4977 common deletion yielded no significant differences in relative distribution, neither between astrocytes and neurons, nor between healthy young and old individuals (171). These findings imply that cellular susceptibility to copy errors during mtDNA replication does not change as a function of age and that the mere passage of time is crucial for intracellular fixation and expansion of the 4977-bp deletion. Thereby the cellular origin of the aging process of mtDNA in the brain and the cellular specificity of hypoxic changes of the level of mtDNA is still unclear. However, a more quantitative approach could definitely lead to more reliable and reproducible insights into the occurrence of cell-type-specific mitochondrial mutagenesis. Thus, a reinvestigation using the same experimental design concerning the DNA isolation via microdissection but with a real-time PCR would certainly be interesting and promising.

Animal models provide excellent methods to study mitochondrial mutagenesis in the brain

The development of animal models led to more information on the genesis of specific diseases. For many diseases, a matching animal model exists and enables thorough experiments on genetic disposition, development or possible prevention of specific diseases. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), for example, is used to produce an experimental model of PD in primates, rats, and mice (172,173). After the intake of MPTP, animals show marked reductions in the number of dopaminergic cells in the substantia nigra pars compacta. In glial cells, MPTP is oxidized to MPP+ which is considered to be directly responsible for cell death and it is concentrated in mitochondria due to its lipophilic and cationic nature. Thus, MPP+ is an inhibitor of NADH-ubiquinone oxidore-ductase (complex I) in the mitochondrial respiratory chain. Studies on transgenic mouse models led to strong evidences that mitochondrial dysfunction results in neurodegeneration and may contribute to the pathogenesis of the diseases described in this chapter (139).

The employment of new methods seems to be a promising tool to elucidate mitochondrial mutagenesis in the brain

Improved detection methods, especially modifications of the PCR, led to better detection thresholds in recent years. They allow the detection of deletions in tissues, that usually contain only a very low amount of mutated DNA, e.g. skin (173), blood (22), or bone (174), or enables the detection of the common 4977-bp deletion in individuals much younger than 20 years of age or from less template DNA (110). Using real-time PCR, even a single molecule can reproducibly be detected and quantified (116) which can improve the detection limits for several mutation assays.

Single nucleotide polymorphism (SNP) analysis, for example, is another great new method to check for specific point mutations and is very common in forensic and also anthropological analysis to determine individual differences in the mitochondrial D-loop region (175) and to increase the power for identification also in the coding regions (176). This method also gained increasing importance for pathological research and is meanwhile used very often to check for possible disease-related haplotypes, e.g. for ALS, PD, or AD (51,92,147,177,178). These improved or newly developed methods, especially the development of designing animal models (in (179)) may contribute to more insights into the occurrence of certain mutations or the content of total mtDNA and their relation to degenerative and harmful processes in the organism.

Additionally, the possible role of mtDNA alterations in carcinogenesis processes (14,180,181) or other phenomena, such as sudden infant death syndrome (SIDS) (182) makes the investigation of human mitochondrial genetics even more important for pathological research with special regard to the human brain and neuropathology.

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