Mitochondrial DNA-related mitochondrial dysfunction in neurodegenerative disease. (Advances in the Science of Pathology).
These disorders share other similar features. Protein aggregations are typically observed on histopathologic survey. While some of these diseases are characterized by autosomal-dominant inheritance (Huntington disease, spinocerebellar degenerations) and rare autosomal-dominant forms exist for AD, PD, and ALS, in most cases these diseases present either sporadically or pseudosporadically. Mitochondrial dysfunction also tends to occur Since mitochondria contain their own genome and mitochondrial genetics do not adhere to mendelian principles, the concomitant existence of nonmendelian epidemiology and mitochondrial dysfunction suggested to some that mitochondrial genes might play a role in these diseases. (1) While it has been difficult to prove or disprove this hypothesis, substantial data exist that are consistent with it. This review focuses on the potential role of mitochondrial DNA (mtDNA)-related mitochondrial dysfunction in the late-onset, sporadic neurodegenerative diseases and describes the cybrid technique, a research tool useful for studying mtDNA genotype-phenotype relationships. First, a brief discussion of mitochondrial basics is in order.
REVIEW OF MITOCHONDRIAL STRUCTURE, PHYSIOLOGY, AND GENETICS
Mitochondria are organelles partially encircled by a cardiolipin-rich "inner" membrane and ultimately surrounded by an exterior "outer" membrane. The area within the relatively impermeable inner membrane is called the matrix. The inner membrane and matrix contain a multitude of enzymes, and the matrix also contains copies of a distinct mitochondrial genome, the mtDNA.
A series of enzyme complexes associate with the inner membrane to comprise the electron transport chain (ETC). There are 5 such complexes. Reduced nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) consists of 41 known protein subunits; succinate dehydrogenase (complex II) contains 5; cytochrome c reductase (complex III), 11; cytochrome c oxidase (complex IV), 13; and adenosine triphosphate (ATP) synthetase (complex V) contains 14 protein subunits. The process by which the ETC produces energy is explained by Mitchell's chemiosmotic hypothesis. (2) Briefly, NADH is oxidized at complex I or flavin adenine dinucleotide (reduced form) (FAD[H.sub.2]) is oxidized at complex II. The acquired electron is then shuttled by ubiquinone from either of these complexes to complex III. Complex III shuttles the electron to complex IV, and under controlled circumstances oxygen is reduced to form water Energy from the passage of electrons is used to transfer protons from the matrix to the intermembrane space. The matrix thus carries a net negative charge. At complex V, which forms a pore that permits proton passage, [H.sup.+] ions reaccess the matrix along their electrochemical gradient. This proton transfer is coupled to phosphorylation of adenosine diphosphate (ADP) to form ATP (Figure 1).
[FIGURE 1 OMITTED]
A number of other enzyme systems are partly or completely contained within the matrix. Among these are the Krebs tricarboxylic acid cycle, enzymes for the urea cycle, the heme (porphyrin) synthesis pathway, pyruvate dehydrogenase complex, enzymes involved in amino acid metabolism, detoxification enzymes, enzymes necessary for pyrimidine synthesis, and enzymes for fatty acid oxidation.
The mtDNA is essentially a modified plasmid. (3) It consists of approximately 16.5 kb arranged in a circular format. Except for an area called the "D loop," virtually the entire DNA is exon. It contains a total of 37 genes, 13 of which code for structural proteins of the ETC (7 complex I subunits, 1 complex III subunit, 3 complex IV subunits, and 2 complex V subunits). The other 24 mtDNA genes are "synthetic genes" that encode 22 transfer RNA (tRNA) and 2 ribosomal RNA molecules that are necessary for the translation of the mtDNA structural genes.
The number of mtDNA molecules per mitochondrion varies by tissue type, but often ranges between 5 and 10 copies. (4) As multiple mitochondria reside within most cell types, thousands of mtDNA copies can exist within a single cell. (5) For this reason, although it is only 16.5 kb, mtDNA accounts for as much as 1% of genomic DNA. While replication regulation of this cytoplasmic DNA is not entirely understood, it does appear that mtDNA replication is a stochastic process that relies on nuclear-encoded proteins. (6)
The rules of mitochondrial genetics differ from those of nuclear genetics in several important ways. (7) The mitochondrial correlate of nuclear heterozygosity is heteroplasmy. Heteroplasmy occurs when not all mtDNA copies within a given cell are identical. In fact, various ratios of wild type (wt) to non-wt mtDNA can coexist inside a cell and probably even within a single mitochondrion. Whether non-wt mtDNA that is present within a heteroplasmic cell carries a phenotypic consequence depends on the burden of the non-wt species. If the burden is high enough to surpass a defined threshold, then functional consequences result. Mitotic segregation further complicates the impact of a given heteroplasmy. Because mitochondria are dispersed from parent to daughter cells through cytoplasmic partition, cells in different tissues or organs and even different cells within a given tissue or organ can display heteroplasmic variation. Heteroplasmic thresholds may also vary among cell types, with the most aerobically active cells being the most likely to express an altered phenotype. Furthermore, heteroplasmic ratios can vary over time, particularly in postmitotic cells such as neurons, which are particularly sensitive to mitochondrial dysfunction. Lastly, as the vast majority of an individual's mitochondrial issue derives from the oocyte, mtDNA is essentially maternally inherited. It is important to point out, however, that although mtDNA is maternally inherited, for many reasons (including heteroplasmy, mitotic segregation, mtDNA drift, and environmental/nuclear gene product interactions) at least some diseases of mtDNA are more likely to present sporadically or pseudosporadically than within the framework of a recognizable matrilineal pedigree. (8,9)
The rest (majority) of matrix and membrane-associated mitochondrial proteins are encoded by nuclear genes and translated within the cytoplasm. These proteins are then transported to the mitochondria. Translocation to either the inner membrane or matrix space requires a number of transport proteins, such as the heat shock proteins. (10)
Improper or inadequate mitochondrial metabolic performance can disrupt a variety of crucial cell housekeeping or homeostatic processes. (11) Electron transport failure of the ETC can lead to depletion of ATP supplies. An inability to complete electron transfer to oxygen at complex IV may lead to the electron's escape from the ETC, with subsequent reaction with free oxygen to form reactive oxygen species, or free radicals. Mitochondria are leading production sites of free radicals and oxidative stress within cells. When ETC failure or oxidative stress is severe enough, matrix depolarization occurs. This depolarization renders mitochondria less able to sequester positively charged entities, such as calcium. Calcium efflux from mitochondria can perturb basal cytoplasmic calcium levels and diminish the ability of mitochondria to buffer calcium fluctuations, as may occur with calcium influx proceeding through activated N-methyl-D-aspartate (NMDA) receptor channels. Mitochondrial depolarization may also result in a process called permeability transition, in which a mitochondrial transition pore forms a channel through both mitochondrial membranes. Mitochondrial transition pore patency provides cytoplasmic passage to molecules that are usually concentrated within mitochondria, such as cytochrome c. Cytochrome c can trigger programmed cell death pathways by contributing to the activation of cysteine-utilizing, aspartate-cleaving (caspase) enzyme pathways. (12-14)
MITOCHONDRIA IN NEURODEGENERATIVE DISEASES
It is now firmly established that mitochondrial dysfunction can and does cause human disease. The first true "mitochondrial disease," Luft disease, was described in 1962. (15) Luft disease is an incredibly rare disorder of mitochondrial uncoupling that is clinically characterized by profound metabolic overdrive. The primary biochemical defects underlying a multitude of other principally metabolic disorders were subsequently localized to mitochondria, and by 1988 a review of mitochondrial diseases listed more than 120 specific disorders. (16) In that same year, specific human diseases (Leber hereditary optic neuropathy [LHON], mitochondrial myopathy, and Kearns-Sayre syndrome) were first linked to mtDNA mutation. (17-20) Since then, mtDNA mutations have been shown to be responsible for a continually increasing list of disorders. Indeed, an entire class of "mitochondrial encephalomyopathies" affecting muscle and the central nervous system are now defined. This disease category includes some relatively well-known disorders, including the mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes syndrome (MELAS) (21,22) and the myoclonic epilepsy with ragged-red fiber syndrome (MERRF). (23) A comprehensive listing and description of these disorders is beyond the scope of this article, and the interested reader may wish to consult some of the many literature (24-26) and Web-based reviews (27,28) of the subject.
Leber hereditary optic neuropathy is an mtDNA mutation-related mitochondrial cytopathy that suggests insights into how mitochondrial dysfunction might play a central role in classic neurodegenerative diseases and perhaps provides a precedence as well. (29-31) Leber hereditary optic neuropathy is characterized by an ultimately progressive degeneration of the optic nerves. (32) It is occasionally associated with a progressive movement disorder. (33,34) The epidemiology of LHON was particularly intriguing to those studying its etiology because kindreds demonstrating strict maternal transmission of the phenotype were known. In the 1970s, LHON was proposed to arise from a virus transmitted in utero from affected mothers to affected children. (35) As the field of mitochondrial medicine became established, the maternal inheritance aspect of the disease suggested to some the potential presence of mtDNA mutation as an etiologic cause. This contention was further supported by the demonstration of complex I deficiency in platelets of those with the disorder. (36) In 1988, Singh et al (37) discovered the presence of a nucleotide 11778 mutation in a maternal LHON kindred. Since then, a number of different causative mtDNA mutations have been recognized. (38) Genotyping for these mutations permits the clinician to confirm the diagnosis in patients with the appropriate clinical syndrome. Interestingly, the ability to render a molecular diagnosis reveals that about 85% of LHON cases present sporadically; recognizable matrilineal inheritance patterns are seen in only a minority of those afflicted. (8,9)
Observations that mitochondrial dysfunction occurs in common neurodegenerative diseases actually predate by several decades the realization that disorders such as MELAS, MERRF, and LHON are mitochondrial cytopathies. For example, in AD, Friede noted in 1965 that alterations of oxidative metabolism occurred in AD patients. (39) He further proposed this phenomenon might precede and drive amyloid deposition in the brains of affected subjects. In 1970, Johnson and Blum (40) noted that morphologically abnormal mitochondria exist around tangles and plaques, the histopathologic hallmarks of AD. Wisniewski et al (41) published in 1971 that mitochondrial distortion was an early histopathologic event in degenerating neurites of AD subjects. In the 1980s, functional neuroimaging data appeared to corroborate this finding. Ferris et al (42) reported in 1980 that AD subjects demonstrate reduced glucose metabolism on positron emission tomography. Multiple other investigators subsequently replicated this finding. (43-45) Hoyer et al (46) went on to suggest that metabolic decline on functional neuroimaging preceded the development of brain atrophy that is often detectable on structural neuroimaging studies. In 1987, Sims et al (47,48) found evidence of mitochondrial dysfunction in fibroblast cultures and brain of patients with AD. Parker et al (49,50) perhaps clarified this biochemical observation when they reported that activity of the ETC enzyme cytochrome oxidase (COX) was reduced in mitochondria derived from the platelets of AD subjects. It was subsequently demonstrated that COX activity is reduced in AD brain. (51-56) The Table provides a list of the different studies showing COX abnormalities in AD.
Mitochondrial dysfunction also occurs in PD. The first indications of this dysfunction came from observations that humans exposed to the pyridine molecule 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) developed a PD phenotype. (57,58) MPTP was later demonstrated to undergo metabolic conversion to a derivative, 1-methyl-4-phenylpyridine (MPP+), by monoamine oxidase in glial cells. (59) MPP+ is taken up by cells possessing dopamine reuptake sites and further concentrated within negatively charged mitochondrial matrices. (60-62) Inside mitochondria, MPP+ inhibits the ETC enzyme complex I, (63) which causes specific degeneration of catecholaminergic neurons in the substantia nigra and locus ceruleus. This reaction results in a constellation of clinical and histopathologic features reminiscent of idiopathic PD. (57,58,64,65)
Since MPTP-induced complex I inhibition can cause a PD-like syndrome, the question of whether complex I dysfunction occurs in idiopathic PD arose. In 1989, 4 independent laboratories reported complex I abnormalities in this disease. (66-69) Initially, controversy existed over whether this enzymatic defect was generalized throughout the body or else limited to the substantia nigra. (70) As multiple laboratories report complex I activity is abnormal in non-nigral brain, (68,71-73) muscle, (69,74-79) platelets, (66,80-83) lymphocytes, (80,84) and fibroblasts, (85) it appears that in PD complex I dysfunction is systemic.
Evidence of mitochondrial dysfunction exists for other neurodegenerative diseases as well. For, example, ETC defects are observed in both degenerating and nondegenerating tissues of ALS subjects. (86) There are data to suggest that these defects arise at the mtDNA level. (87-89) Mitochondrial dysfunction is also seen in progressive supranuclear palsy and multisystem atrophy. (77,90-92) In Huntington disease, an autosomal-dominant disorder which arises from mutation of the gene encoding the protein huntingtin on chromosome 4, (93) mitochondrial abnormalities occur in both degenerating and nondegenerating tissues. (94-100)
For the neurodegenerative diseases mentioned above, mitochondrial deficits were described despite a lack of genetic data to indicate their potential presence. Through leads provided by recent advances in molecular genetics, mitochondrial dysfunction is now recognized in an ever-increasing number of other degenerative disorders. Friedreich ataxia is an autosomal-recessive neurodegeneration that arises from an intronic triple repeat expansion and sometimes point mutation of the frataxin gene on chromosome 9. (101) Frataxin, although transcribed in the nucleus and translated cytoplasmically, is transported to mitochondria and can be considered a mitochondrial protein. (102) It appears to play a role in mitochondrial iron homeostasis, and frataxin mutation is associated with dysfunction of a number of iron-dependent mitochondrial enzymes. (103) In Wilson disease, a progressive disorder of movement and liver dysfunction, the basic defect involves autosomal-recessive mutation of a mitochondrially targeted protein that may regulate copper homeostasis of the organelle. (104-106) Some progressive hereditary spastic paraplegia subtypes are also clearly related to mitochondrial events. An autosomal-recessive form of hereditary spastic paraplegia occurs with mutation of the paraplegin gene on chromosome 16, which is also a mitochondrially targeted protein that appears to facilitate oxidative phosphorylation pathways. (107) Late-onset, progressive dystonia syndromes can arise both from mutation of mtDNA and from mutation of nuclear genes encoding mitochondrial transport proteins. (108-110) For the disorders listed above, and for additional ones as well, a better understanding of how primary mitochondrial defects specifically affect mitochondrial/ cellular physiology and further promote cell dysfunction or death is developing. To assist those interested in learning more about such specifics, a number of excellent reviews are available. (111-114)
One common feature of the neurodegenerative diseases is their tendency to accumulate protein aggregations. Perhaps relevant to this topic are observations that ETC dysfunction leads to aberrant processing of aggregable proteins. (115) Alternatively, aggregating proteins can induce ETC dysfunction in vitro. (116-120)
THE CYBRID TECHNIQUE AND ITS CONTRIBUTION TO NEURODEGENERATION RESEARCH
Cellular depletion of mtDNA occurs naturally under certain conditions. For instance, yeast cells are capable of switching from aerobic to anaerobic conditions, depending on their immediate environment. During this switch from aerobic to anaerobic states, endogenous levels of normal mtDNA are reduced. (121,122) Attempts at inducing mtDNA depletion experimentally in culturable cell lines eventually yielded cells containing no detectable mtDNA, called [[rho].sup.0] cells, (121-123) as mtDNA was initially referred to as "[rho]" DNA prior to its localization within mitochondria. (124,125) Such cells can survive if appropriate nutritional support is provided, such as supplementation of pyruvate (to regenerate NAD+ following its conversion to NADH in glycolysis) and uridine (to facilitate pyrimidine synthesis, which becomes ineffective under conditions of ETC failure). (126,127)
In 1989, King and Attardi (127) succeeded in repopulating a human [[rho].sup.0] cell line with mitochondrial genes from a donor source. This mtDNA transfer was accomplished by introduction of whole mitochondria from the donor to the recipient cells; mtDNA was essentially "along for the ride." Over time, the exogenous mitochondria (and the mtDNA they contained) repopulated the [[pho].sup.0] cells they were transferred to. As these mtDNA-reconstituted cells were cytoplasmic hybrids (arising through a combination of nucleated and nonnucleated cell preparations), (128) they met criteria for a previously created term, cybrids. (129)
One effective way of accomplishing mtDNA transfer is to isolate platelets from a small amount of blood (several milliliters), and to fuse these platelets with an aliquot of [[rho].sup.0] cells. (130) A diagram illustrating this method is shown in Figure 2. Platelets are a particularly appropriate source for donor mtDNA transfer because they contain mitochondria (and hence mtDNA) but are anuclear. A fusion of platelets harvested from 5 mL of blood and 1 x [10.sup.6] [[rho].sup.0] ceils typically yields up to several hundred transformed cybrid cells. By removing pyruvate and uridine supplementation from the culture media, untransformed cells are removed, while the transformed individual cybrid cells expand. The end result is a unique "cybrid cell line" that contains and expresses the nuclear genes of the original [[rho].sup.0] cell line and the mitochondrial genes of the platelet donor
[FIGURE 2 OMITTED]
During the initial weeks in which a newly synthesized cybrid line is undergoing expansion, multiple mitotic cycles occur, and transferred but nonreplicable entities degrade and dilute. Nuclear-encoded proteins (including nuclear-generated ETC subunits) contained within the transferred mitochondria are eventually replaced by protein transcribed from the host cell nucleus. The only persisting characteristics unique to the donor are those which derive from their transferred perpetuable mtDNA.
Cybrids were initially used to study issues of heteroplasmy and threshold for known diseases of mtDNA point mutation, such as MELAS and MERRF. (130-132) In the mid 1990s, they were first employed to screen for mtDNA aberration in late-onset, sporadic neurodegenerative diseases in which no mtDNA mutation was previously defined. (133,134) Potential explanations for mitochondrial dysfunction in these disorders included the hypothetical presence of mitochondrial toxins, production of mutant nuclear-encoded ETC subunits, or abnormal production of mitochondrially encoded ETC subunits. As cybrid methodology controls for environmental/toxic contributions (all cybrid cell lines are equivalently maintained) and nuclear background (the nuclear genes of individual cybrid cell lines originate from the same [[rho].sup.0] cell line and are clonal), the status of the transferred mtDNA remains the only variable. In other words, cybrid cell lines that express mitochondrial genes derived from different donors should differ only in the content of their mtDNA. Differences in ETC phenotype between these cell lines are most likely to arise from differences in their mtDNA genotype (Figure 3).
[FIGURE 3 OMITTED]
In 1997, it was shown that transferring mitochondrial genes from AD patient platelets to cells previously depleted of endogenous mtDNA ([[rho].sup.0] cells) yielded COX-deficient cybrid cell lines. (134) This result suggested that the COX defect of AD arises from aberration of mtDNA. (135,136) Cybrid cell lines that express AD patient mitochondrial genes also suffer from increased oxidative stress that is due to elevated reactive oxygen species generation, (135) perturbed static and dynamic calcium homeostasis, (137) and decreased mitochondrial membrane potential. (138) Alzheimer disease cybrids also exhibit oversecretion of [beta]-amyloid protein. (139)
Data from cybrid studies suggest mtDNA aberration is at least partly responsible for the complex I defect observed in PD. (134,140-142) Transfer of mitochondrial genes from platelets of PD subjects to culturable [[rho].sup.0] cells results in cybrid cell lines that manifest deficient complex I activity. (134) Moreover, PD transmitochondrial cybrid lines manifest oxidative stress due to overproduction of reactive oxygen species, (134,143) perturbed static and dynamic calcium homeostasis, (144) reduced mitochondrial membrane potentials, (145) decreased ability to survive MPP+ exposure, (134) altered mitochondrial morphology, (142) increased Bcl-2 and Bcl-[X.sub.L] expression, (146) increased basal nuclear factor-[kappa] B activation, (147) and abnormal protein aggregations. (148)
Cybrid cell lines expressing mitochondrial genes from ALS subjects demonstrate complex I dysfunction, oxidative stress, altered calcium homeostasis, and abnormal protein aggregations, (87) suggesting that mtDNA aberration is at least partly responsible for mitochondrial dysfunction in this disease. Cybrids that express mitochondrial genes from patients with progressive supranuclear palsy and multisystem atrophy manifest deficient complex I catalytic activity and (at least in the case of progressive supranuclear palsy) increased oxidative stress. (149,150) In Huntington disease, however, mitochondrial dysfunction that is apparent on direct tissue studies is not apparent in cybrid cell systems. (151) Correction of mitochondrial defects in Huntington disease cybrid lines supports the view that cybrid mitochondrial dysfunction, when present, truly reflects underlying mtDNA aberration. It is important to point out, however, that implication of mtDNA aberration in a particular disease by cybrid analysis does not indicate whether the underlying genetic defect is inherited or acquired (somatic).
Beyond the fact that cybrid biochemical defect screening for mtDNA mutations, when used in isolation, cannot distinguish between inherited or acquired mtDNA aberration in the mtDNA donor subject, several other limitations of the system are worth mentioning. The nuclear background of the [[rho].sup.0] cell line utilized may influence whether the consequences of a particular mtDNA lesion are phenotypically detectable by biochemical assay. (152) Furthermore, it is important to keep in mind that [[rho].sup.0] cells are derived from culturable tumor lines that continuously replicate in culture. Mitochondrial DNA diseases, however, tend to affect nonreplicating tissues, such as brain and muscle. Constant cell turnover within a cybrid line containing a heteroplasmic mtDNA defect, therefore, may theoretically dilute the magnitude of an mtDNA mutation-related phenotypic consequence or lead to a state in which the cells containing the greatest amount of mutation are "weeded out" of the culture over time. On the flip side of the coin, since the actual transfer of mtDNA from donor subjects to [[rho].sup.0] ceils actually involves transfer of intact mitochondrial organelles, it is conceivable that premature biochemical assay of cybrid lines could reveal mitochondrial dysfunction that is not actually related to the mtDNA contained within. Finally, one must consider whether the mtDNA donor tissue used (such as platelets) accurately reflects the status of the mtDNA or the degree of an mtDNA heteroplasmy in a disease-affected tissue. To circumvent this issue, some investigators have attempted to harvest mitochondria from autopsy brain material and use such mitochondria to accomplish [[rho].sup.0] cell transfection. Unfortunately, transfection efficiency with autopsy-derived brain mitochondria is exceptionally low, which creates its own set of limitations. (153,154)
The cybrid technique is therefore best used as a research tool. Because of the potential confounding issues described above, and because of the variability inherent to biochemical measurements of individual biologic samples, any conclusions reached from cybrid analysis of a single subject would have to be interpreted quite cautiously.
MITOCHONDRIA AND PROGRAMMED CELL DEATH
As discussed above, whether arising from mitochondrial genetic or other causes, acquired mitochondrial dysfunction may play a crucial role in neurodegeneration by initiating programmed cell death pathways. Current evidence suggests neuronal demise proceeds through a final common programmed cell death pathway in several neurodegenerative diseases. (155-160)
Events initiated in mitochondria can trigger programmed cell death. (14) For example, ETC failure leading to either overproduction of reactive oxygen species or ATP depletion leads to mitochondrial depolarization. Several additional consequences can arise from this situation. Dissipation of the mitochondrial membrane potential is associated with an efflux of positively charged calcium ions from the mitochondrial matrix, which leads to elevations of cytoplasmic calcium. Moreover, regulation of calcium accessing the cytoplasm through plasma membrane NMDA receptors becomes impaired. This impairment can result in both toxic cell signaling events and induction of certain forms of nitric oxide synthase. In an oxidative environment, nitric oxide synthase will generate nitric oxide radical (NO*), which contributes to further oxidative stress. (161) As mitochondrial depolarization deepens, a channel spanning the mitochondrial inner and outer membranes develops. This permeability transition represents the open conformation of the mitochondrial transition pore, through which large mitochondrially contained molecules can pass. (162,163) Mitochondrial transition pore formation allows the ETC component cytochrome c to enter the cell cytoplasm, where it activates caspase enzymes. (12,13,164,165) Initiation of the caspase cascade ultimately results in autodigestion of cell contents. A schematic emphasizing these relationships is shown in Figure 4.
[FIGURE 4 OMITTED]
CLINICAL EVALUATION OF mtDNA ABERRATION
In general, how should the clinician or pathologist proceed when evaluating a patient with suspected mtDNA-related disease? The most widely used noninvasive approach focuses on the use of laboratory assays to first screen for underlying mitochondrial biochemical dysfunction. It is currently common practice to measure serum lactate, as deficits of electron transport and pyruvate dehydrogenase complex can certainly result in elevations of this by-product of anaerobic metabolism. Pyruvate elevations may also occur when normal functioning of these enzyme pathways is abnormal. Pyruvate and Krebs cycle intermediates exist in equilibrium with amino acid pools, and elevation of certain amino acids, such as alanine, can occur in the presence of mitochondriopathy. Unfortunately, the sensitivity of these tests is poor For instance, although lactate elevations may reliably occur with tRNA gene mutation disorders such as MELAS, lactate levels in LHON commonly are normal. (166,167) In an effort to improve the sensitivity of lactate screening, some advocate measuring this metabolite under conditions of aerobic stress or glucose loading. (168-170) Beyond this approach, application of new technologies such as magnetic resonance spectroscopy will, in the future, hopefully advance our ability to noninvasively screen for mitochondrial dysfunction in vivo.
Direct tissue evaluation by the pathologist is still the gold standard for demonstrating the presence of mitochondriopathy. Tissue studies can include direct biochemical measurements of relevant enzyme activities or coenzyme levels, or structural anatomic assessments. Because multiple catabolic pathways ultimately converge on mitochondria, demonstrating abnormalities of cell lipid or carbohydrate storage at the light microscopy level can suggest the presence of mitochondrial pathology. Occasionally, mitochondrial dysfunction will result in demonstrable proliferation of the organelle. As with lactate screening, however, at the light microscopy level such proliferations may only reliably occur in the tRNA gene mutation mitochondrial cytopathies (ie, the "ragged red fibers" of MERRF). Electron microscopy is a particularly useful adjunct in the evaluation of suspected mitochondrial disease, and mitochondrial morphologic alterations and/or inclusions can sometimes help establish whether nonspecific abnormalities of a tissue are related to mitochondrial pathology. Indeed, reports of mitochondrial ultrastructural abnormalities in AD, PD, and ALS have existed in the literature for many years. (41,171,172)
Of course, demonstrating mitochondrial biochemical or structural abnormalities in a patient does not establish whether mtDNA mutation is the cause or whether mtDNA integrity is preserved. Advances in molecular neuroscience can help render focused, reliable diagnoses for some mtDNA disorders in which specific causative mutations are known, such as LHON, MELAS, MERRF, and KearnsSayre syndrome. Beyond this, while cybrid studies do provide important indirect evidence that mtDNA aberration does occur in neurodegenerative diseases, the nature of this aberration is unknown. This shortcoming limits our clinical ability to molecularly evaluate mtDNA in such disorders, although various strategies have been experimentally employed. For example, Brown et al (173) recently used a competitive PCR assay to demonstrate that levels of amplifiable mtDNA are reduced in AD brain.
Our understanding of how diseases of mitochondria and, specifically, mtDNA manifest at biochemical, histopathologic, ultrastructural, and molecular levels continues to evolve. Since we are still frequently discovering exceptions to many of the diagnostic rules that have been proposed, when working in the field of mitochondrial medicine quite possibly the clinician's, pathologist's, or investigator's greatest asset is an appreciation of just how limited our current knowledge of the field is.
Mitochondrial dysfunction is a common feature of several neurodegenerative disorders. While it is unclear whether this commonality represents primary pathology that etiologically drives neurodegeneration or secondary pathology that is driven by another more basic process, it is probably relevant to the neuronal demise that characterizes this disease category. Available data suggest mtDNA aberration is at least partially responsible for this dysfunction, and that mtDNA-derived mitochondrial dysfunction can result in or manifest as oxidative stress, intracellular signaling impairment, toxin vulnerability, protein aggregation, and induction of apoptotic pathways. Regardless of whether mtDNA aberration in persons with neurodegenerative diseases is inherited or acquired, defining mitochondrial pathogenesis in neurodegenerative disorders may contribute to future diagnostic and therapeutic advances. Application of advances in cybrid, DNA sequencing, gene expression screening, and other novel technologies (174) should help further resolve the role of mitochondria and mtDNA in this class of diseases.
This work was supported by grants from the National Institutes of Health (AG00800) and American Parkinson Disease Association (Cotzias Award).
Electron Transport Chain Abnormalities in Alzheimer Disease Source, y Defect(s) Sims et al, (48) 1987 Decreased oxygen metabolism, uncoupling Sims et al, (47) 1987 Decreased oxygen, glutamine [sup.14]C[O.sup.2] metabolism Parker et al, (49) 1990 Complex IV decreased 50% Kish et al, (51) 1992 Complex IV decreased 16%-26% Reichmann et al, (52) 1993 Complex II, III, IV decreased Mutisya et al, (54) 1994 Complex IV decreased 25%-30% Chandrasekaran et al, (55) 1994 Cytochrome oxidase I, III mRNA decreased 50% Parker et al, (53) 1994 Complex III, IV decreased 43% and 53%, respectively Parker et al, (50) 1994 Complex IV decreased 17% Chagnon et al, (56) 1995 Complex IV decreased 34%-38% Source, y Comments Sims et al, (48) 1987 Brain Sims et al, (47) 1987 Fibroblasts Parker et al, (49) 1990 Pure platelet mitochondria Kish et al, (51) 1992 Brain homogenates Reichmann et al, (52) 1993 Brain homogenates Mutisya et al, (54) 1994 Crude brain mitochondria Chandrasekaran et al, (55) 1994 Brain Parker et al, (53) 1994 Pure brain mitochondria Parker et al, (50) 1994 Platelet crude mitochondria Chagnon et al, (56) 1995 Brain homogenates
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Accepted for publication October 5, 2001.
From the Center for the Study of Neurodegenerative Diseases and the Department of Neurology, University of Virginia Health System, Charlottesville, Va.
Presented at the 10th Annual William Beaumont Hospital Seminar on Molecular Pathology, DNA Technology in the Clinical Laboratory, Royal Oak, Mich, March 8-10, 2001.
Reprints: Russell H. Swerdlow, MD, Box 394, Department of Neurology, University of Virginia Health System, Charlottesville, VA 22908 (e-mail: email@example.com).
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|Author:||Swerdlow, Russell H.|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Mar 1, 2002|
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