Coskun PE, Beal MF, Wallace DC.
Alzheimer's brains harbor somatic mtDNA control-region mutations that suppress mitochondrial transcription and replication.
Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10726-31.
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This is a remarkable paper. Coskun, Beal and Wallace demonstrate that many mutations to mtDNA occur near the PL region, an important regulatory region for replication of the genome. Therefore, this damage could ultimately block mitochondrial reproduction. In support of this possibility, they found a 50 percent reduction in mtDNA copy number in AD samples. Thus, their results partially explain the mitochondrial deficiencies that occur in AD.
Mitochondria constantly undergo fission and fusion, are replicated throughout the life of the cell, and autophagocytosed when their time has come. However, unlike their host, mitochondria possess multiple copies of their DNA, perhaps four or five. So, damage to one copy does not necessarily compromise the organelle, if it can be removed and disposed. That’s the catch-if the mitochondrion does not get rid of this DNA, eventually it may accrue more damaged copies.
The mitochondrial theory of aging states that enlarged and functionally disabled mitochondria gradually displace normal ones during senescence. These changes are most pronounced in postmitotic cells, such as neurons. The large size hints that fission is impaired, limiting the effectiveness of mitochondrial turnover. These mitochondria may incur further oxidative damage, resulting in decreased energy production and increased generation of reactive oxygen specie. Disabled mitochondria gradually displace normal ones, and the neuron eventually dies. The Wallace paper did not determine the cause of the damage to mtDNA. Oxidative stress is the most likely culprit, since all patients suffered from late-onset AD, the disease probably results from damage that builds up slowly over time.
Could a similar scenario explain the etiology of early onset, familial forms of AD? Mitochondria have fewer DNA repair mechanisms than the nucleus, thus cells-especially post-mitotic neurons-may have evolved an alternate response to this damage. It may be that neurons deal with intractable mtDNA damage by deleting the entire copy. Perhaps individual copies of mtDNA are targeted for disposal through association with the mitochondrial permeability transition pore. The oxidative stress that follows mtDNA damage would trigger the opening of the pore, and this complex could subsequently be removed by fission. The APP is involved in intracellular trafficking, and found in the mitochondrial membrane associated with the mitochondrial permeability transition pore, intriguingly, in a non-glycosylated form (Anandatheerthavarada et al. 2003). Could APP and the presenilins direct transport vesicles containing damaged mtDNA (and other mitochondrial components) to lysosomes? Thus, mutations that affect APP processing could affect vesicular transport or targeting, or perhaps even the initiation of this process. The damaged mtDNA would gradually increase over time to disable the neuron as previously described.
The disposal or recycling of fragments of this organelle has not yet been described, although autophagocytosis of mitochondria has been reported for many models of neurodegeneration. However, we have unpublished evidence that supports the existence of such a process. In our unpublished study, cerebellar granule cells were transfected with wild-type or the Swedish mutant APP, as well as β-lactosidase (Lac) as a control. Cultures were then treated with kainate to stimulate AMPA receptors and fixed for electron microscopy at 0-30 min. All treated cerebellar granule cells showed evidence for mitochondrial membrane breakdown, however, it was more robust for the wild-type, and less so for the Swedish mutant transfected-neurons. These were usually observed near to the microtubule organizing center (see the RAB27-/- frame in Figure 2 of Stinchcombe et al. (2004) for an example of this). Curiously, we observed several lysosomes undergoing exocytosis, as well as numerous extracellular lysosomes and multivesicular bodies among the fibers. This process appears to peak 2 min following kainate treatment, which could explain why it has not been previously observed, as no study has examined neuronal ultrastructure at similar time intervals following glutamate receptor stimulation.
There are reports that present data which support this hypothesis. One such study demonstrated that neurons showing increased oxidative damage in AD have a significant increase in mtDNA and cytochrome oxidase (Hirai et al. 2001). In this study, much of the mtDNA and cytochrome oxidase was found in the neuronal cytoplasm. Curiously, vacuoles associated with lipofuscin immunostained for mtDNA, while morphometric analysis showed that mitochondria were significantly reduced in AD, consistent with perturbed mitochondrial turnover. In our study, kainate treatment generated vacuoles that originated from mitochondria and appeared to contain lipofuscin, and were virtually identical in size and appearance to those that immunostained for mtDNA in the study by Smith’s group.
The APP is not functionally constrained to the mitochondria in this model. APP targeted to the plasma membrane is fully glycosylated, while as noted above, the protein found in the mitochondria is not. One function of APP may be to retrieve DNA, lipids and proteins damaged following oxidative stress, and direct these to the Golgi and lysosomes for recycling or disposal. Further studies addressing the localization of mtDNA as well as vesicular targeting following glutamate receptor stimulation are in order.
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J Cell Biol. 2003 Apr 14;161(1):41-54.
Coskun PE, Beal MF, Wallace DC.
Alzheimer's brains harbor somatic mtDNA control-region mutations that suppress mitochondrial transcription and replication.
Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10726-31.
Hirai K, Aliev G, Nunomura A, Fujioka H, Russell RL, Atwood CS, Johnson AB, Kress Y, Vinters HV, Tabaton M, Shimohama S, Cash AD, Siedlak SL, Harris PL, Jones PK, Petersen RB, Perry G, Smith MA.
Mitochondrial abnormalities in Alzheimer's disease.
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Stinchcombe J, Bossi G, Griffiths GM.
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A possible role for abnormal mitochondrial biology in the pathogenesis of Alzheimer’s disease (AD) has been an omnipresent theme over the last decades. Regional hypometabolism, documented by positron emission tomography and abnormalities in mitochondrial respiratory chain complexes—most consistently diminished activity of cytochrome c oxidase (COX), have been noted for some time (1-2). Furthermore, an important role for reactive oxygen species (ROS), which originate, at least in part, from mitochondria, in amyloid-β peptide (Aβ)-induced cellular perturbation has been indicated by multiple in vitro studies. In this regard, an attractive concept to consider is that of “retrograde signaling,” whereby mitochondrial generation of ROS diminishes calcium uptake by this organelle, resulting in increased oxidant stress and cytosolic-free calcium ([Ca2+]c), and subsequent activation of kinases, and activation of nuclear genes etc (3). Another close link between cellular perturbation, apoptosis and mitochondrial function is that a 75 kDa subunit of respiratory complex I (NDUFS1) is a functionally significant caspase substrate (4).
What are the mechanisms through which Aβ targets mitochondria? On the one hand, there could be specific mitochondrial components/macromolecules which are rendered dysfunctional either directly and/or indirectly by Aβ. On the other hand, there might be a genetic propensity towards mitochondrial dysfunction (due to mutations in either nuclear or mitochondrial encoded genes) which renders cells especially vulnerable to Aβ-induced cell stress. Of course, these are not mutually exclusive possibilities.
The paper from the laboratory of Dr. Wallace provides intriguing insights into these issues as pertains to genetic determinants of mitochondrial function. First, they show that there are certain mutations in the mitochondrial (mt) DNA control region (CR; a region of mtDNA that encodes essential regulatory binding sites) in AD brains that are not found in nondemented controls. Second, there were more mutations in the mtDNA CR of AD patients than controls. Interestingly, these mutations occurred at sites of known mtDNA regulatory elements, and would be expected to be associated with functional consequences for mitochondrial biology. Third, there was a decrease in mtDNA (along with ND6 mRNA) that would be likely be associated with reduced brain oxidative phosphorylation. Two distinct patterns of the mutations were especially notable. In one pattern, AD brains from patients of 74-83 years of age had a lower number of CR mutations, but they were present in a high percentage of the brain mtDNAs (70-80%). In the other pattern, AD patients over 83 years of age had many more CR mutations in frontal cortex mitochondria, but these were present in a lower percentage (We would like to suggest an expansion of this hypothesis and make a prediction. Certain patients have mtDNA mutations (presumably in, but not limited to, the control region), which render neurons vulnerable to oxidant stress. This might be considered a “first hit,” in a “two hit” model of cellular perturbation (see schematic figure below). However, the genetic defect encoding this “first hit” (grey shading) is not associated with a deleterious phenotype until a superimposed “second hit,” (red shading and red arrows) the latter associated with oxidant stress, occurs. The “second hit” could be an excitotoxic stimulus, regional cerebral ischemia or Aβ-induced pathology. If this concept is valid, one might expect that such mtDNA mutations (constituting the “first hit”) were distributed not only in frontal cortex (and, presumably, other AD-affected brain subregions), but also in areas of the brain spared in AD (for example, cerebellum). The occurrence of neuronal stress would require a localized oxidant stress (the “second hit,” due to an Aβ-rich environment, excitotoxic stimulus or ischemic insult), to bring out the phenotype caused by the mtDNA mutation(s). Alternatively, if Aβ-induced oxidant stress were the primary driver of mitochondrial dysfunction (in this case, the “first hit” would not be an important factor), one might expect mtDNA mutations to be focused only in AD-affected regions, to be multiple and locally (not widely) distributed. Of course, a combination of these hypotheses would be possible in which underlying mtDNA mutations occurred early in the disease, rendering neurons vulnerable to Aβ-induced cell stress, and this would provide a basis for additional mtDNA mutations at later times.
Another level of Aβ-induced mitochondrial dysfunction relates to a role for specific mitochondrial molecular targets of the amyloid peptide. In this context, our group has identified ABAD, a member of the short-chain dehydrogenase reductase family present in mitochondrial matrix, as a molecule capable of binding Aβ (5). ABAD-Aβ complex appears to promote mitochondrial generation of ROS and dysfunction. Presumably, ABAD-Aβ-induced mitochondrial dysfunction could promote mutagenesis of mtDNA, thereby leading to further mitochondrial dysfunction.
In summary, the paper by Coskun and colleagues highlights an increasingly recognized potential role of mitochondrial dysfunction in the pathogenesis of AD. It provides testable hypotheses to further investigate the contribution of mutations in the mtDNA CR to neuronal dysfunction.
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Etiology of late-onset Alzheimer’s disease (AD) is unknown; the paper of Coskun et al.  lends further support to an exciting hypothesis that it may be caused by somatic mutations in mtDNA. Mitochondria have long been known as a major source of reactive oxygen species (ROS) in actively metabolic cells; AD appears to most strongly affect brain regions with the highest metabolic rate and highest expression of mitochondrial enzymes. Thus, defects in mitochondrial oxidative phosphorylation and increased generation of ROS by defective mitochondria might mediate the relationship between late-onset AD and mtDNA mutations.
At a first glance, this mechanism is only relevant to the development of late-onset AD and seems to be unrelated to early onset forms of the disease which are caused by point mutations in APP and presenilins; such mutations result in accelerated production of amyloid-β (Aβ). Thus, the question of major importance remains, how can we combine late-onset and early onset AD within such mitochondrial hypothesis? Increased production of Aβ is a probable answer.
Several years ago, we showed that at low-nanomolar concentrations (i.e., those circulating in CSF and plasma), Aβ is monomeric and functions as an antioxidant [2, 3]. Mechanistically, the antioxidative activity of Aβ is related to its strong capacity to bind transition metal ions ; Aβ may therefore function as a metal-chelating, preventive antioxidant under normal physiologic conditions. This conclusion is consistent with results of other studies demonstrating that at low-nanomolar concentrations, Aβ has beneficial effects on neuron survival, axonal length, and neurite outgrowth (see [5, 6] for review).
Various stress conditions are known to increase Aβ production. Importantly, generation of Aβ increases under oxidative stress induced by different mechanisms (H2O2, UV irradiation, etc.) [5, 6]. Available data strongly suggest that Aβ behaves as a positive acute-phase reactant; antioxidant metal-chelating properties of Aβ may provide a rationale for this phenomenon. Indeed, an increase in Aβ production may be aimed at chelating potentially harmful transition metal ions which can be released, e.g., from metal-binding proteins, during abnormal cellular metabolism and otherwise catalyze adverse oxidation of biomolecules. This mechanism implies that transition metal ions become abnormally sequestrated and need to be chelated in a redox-inactive form by Aβ; indeed, metabolism of transition metals is heavily impaired in AD brains . Oxidative damage to neurons is one of the earliest pathological events in AD ; accelerated lipid peroxidation precedes accumulation of Aβ in AD transgenic mice . These data suggest that increased ROS production by defective mitochondria in AD brains may lead to increased generation of Aβ as a compensatory protective response.
Historically, Aβ has been long considered as a key pro-oxidant in AD . However, to accelerate oxidation, Aβ must be present in concentrations greatly exceeding those normally measured in biological fluids (i.e., micromolar vs. nanomolar; see [2, 5]). In addition, Aβ must be aggregated to fibrils by transition metals; fibrillated Aβ is highly toxic for neurons and other cells . We have therefore hypothesized that Aβ may become a pro-oxidant from an antioxidant, if its concentration increases enough to induce its substantial aggregation and if transition metal ions are available to catalyze this process . This is exactly what Coskun et al. propose in their excellent paper ; unfortunately, it does not contain a reference to our work. Furthermore, we have postulated that increased production of Aβ as a result of elevated production of ROS by defective mitochondria with subsequent chelation of transition metal ions, accumulation of toxic Aβ-metal complexes, production of ROS, and neurotoxicity form the temporal sequence of events in the development of late-onset AD .
According to this view, development of AD equally occurs along the pathway of increased Aβ production in early onset forms of the disease; the major difference between the early and late-onset forms lies in the elevated rate of Aβ production in early onset AD due to the presence of genetic defects in APP and presenilins, which should accelerate formation of toxic Aβ-metal complexes and the development of the disease. Targeting a pathologically increased formation of such complexes (i.e. by metal chelators ) therefore remains a promising therapeutic strategy in both early and late-onset AD; since Aβ appears to represent an important protective molecule, great caution must be however exercised about this approach, which may not target monomeric Aβ (cf. studies of AD vaccine).
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Mitochondria and Alzheimer’s Disease: A Complex Interrelationship
Recently, Coskun and colleagues (2004) reported that brains from patients with Alzheimer's disease (AD) present somatic mtDNA control-region mutations, especially in individuals older than 80 years, which supports the mitochondrial hypothesis for AD pathophysiology. However, the doubt remains whether these somatic mtDNA mutations are the cause or consequence of AD pathophysiology. If somatic mutations of mtDNA are a cause of AD, these mutations should appear in the vulnerable brain regions affected by the disease. However, this same pattern will be also observed if the somatic mutation of mtDNA is a consequence of AD pathophysiology. So, how and when do the somatic mutations of mtDNA accumulate in specific regions of brain?
A recent study suggests that DNA damage, recognized by the formation of 8-hydroxyguanosine (8OHG), a marker of nucleic acid oxidation, is markedly increased in the promoters of genes whose expression is decreased in the aged human cortex (Lu et al., 2004). Since modifications of nucleic acid bases cause mutations, this study also suggests that oxidative stress can cause mutations in gene promoters even in aged control brains.
Coskun et al. (2004) also observed that the number of mtDNA is decreased in brains of AD patients. It is known that the nucleus regulates the replication of mitochondria according to the metabolic needs of the cell. When cells possess a large amount of mitochondria harboring damaged mtDNA, they cannot function properly to maintain normal energetic metabolism. In that case, dysfunctional mitochondria are responsible for an increased leakage of free electrons that causes the production of harmful reactive oxygen species (ROS). In such situations, the suppression of the replication of abnormal mitochondria seems to be effective against excess ROS production.
A previous study from our laboratory showed the opposite results with mtDNA (Hirai et al., 2001). Using in-situ hybridization, we observed that brains from AD patients present a striking increase in mtDNA. When we analyzed mtDNA changes by PCR, we found only small differences between AD and age-matched controls, even in cases where the in-situ hybridization technique showed a fourfold mtDNA increase. Judging from our observations that damaged mtDNA is restricted to neurons vulnerable in AD, we should be cautious in the interpretation of the PCR results, since they include all populations of cells that reside in the brain and may include highly damaged DNA.
Although controversial findings exist, this study emphasizes the importance of the mitochondrial hypothesis in AD pathophysiology. More studies must be done to clarify the role and the place of mitochondria in the complex scenario of AD.
Lu T, Pan Y, Kao SY, Li C, Kohane I, Chan J, Yankner BA.
Gene regulation and DNA damage in the ageing human brain.
Nature. 2004 Jun 24;429(6994):883-91.
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