HUMANA PRESS Methods in Molecular Biology TM Edited by William C. Copeland Mitochondrial DNA Methods and Protocols HUMANA PRESS Methods in Molecular Biology TM Edited by William C. Copeland Mitochondrial DNA Methods and Protocols VOLUME 197 Animal Models for Mitochondrial Disease 1 I METHODS FOR THE ANALYSIS OF MTDNA Animal Models for Mitochondrial Disease 3 3 From: Methods in Molecular Biology, vol. 197: Mitochondrial DNA: Methods and Protocols Edited by: W. C. Copeland © Humana Press Inc., Totowa, NJ 1 Animal Models for Mitochondrial Disease Douglas C. Wallace 1. Introduction Although a variety of degenerative diseases are now known to be caused by two mutations in mitochondrial genes, the pathophysiology of these diseases remains poorly understood. As a consequence, relatively little progress has been made in developing new therapies for mitochondrial diseases. What has been needed are animal models for these diseases that are amenable to detailed biochemical, physiological, and molecular analysis, and on which promising therapies can be tested. In the past 5 yr, this defi ciency has begun to be addressed by the construction of a number of mouse models of mitochon- drial disease. These have already revolutionized our understanding of the pathophysiology of mitochondrial disease and demonstrated the effi cacy of some new antioxidant drugs. 1.1. Mitochondrial Biology and Genetics The mitochondria generate much of the cellular energy through the process of oxidative phosphorylation (OXPHOS). As a byproduct, they produce most of the endogenous toxic reactive oxygen species (ROS). The mitochondrial are also the central regulator of apoptosis (programmed cell death), a process initiated by the activation of the mitochondrial permeability transition pore (mtPTP). These interrelated mitochondrial systems are assembled from roughly 1000 genes distributed between the two very different genetic systems of the mammalian cell: the nuclear genome and the mitochondrial genome. Hence, the complexities of mitochondrial disease refl ect the intricacies of both the physiology and the genetics of the mitochondrion. 4 Wallace 1.1.1. Mitochondrial Physiology To understand the pathophysiology of mitochondrial diseases, it is necessary to understand the physiology of OXPHOS. The mitochondria oxidize hydrogen derived from carbohydrates and fats to generate water and ATP (see Fig. 1). Reducing equivalents in the form of hydrogen are recovered from carbohydrates by the tricarboxylic acid (TCA) cycle, while those recovered from fats are collected through β-oxidation. The resulting electrons are transferred to NAD + , to give NADH + H + , or to fl avins located in iron–sulfur (Fe–S)-center- containing enzymes that interface with the electron transport chain (ETC). Electrons donated from NADH + H + to complex I (NADH dehydrogenase) or from succinate to complex II (succinate dehydrogenase, SDH) are passed sequentially to ubiquinone (coenzyme Q or CoQ) to give ubisemiquinone (CoQH • ) and then ubiquinol (CoQH 2 ). Ubiquinol transfers its electrons to complex III (ubiquinolϺcytochrome-c oxidoreductase) which transfers the electrons to cytochrome-c. From cytochrome-c, the electrons move to complex IV (cytochrome-c oxidase, COX) and fi nally to oxygen to give H 2 O. The energy released by this ETC is used to pump protons out of the mitochondrial inner membrane, creating the trans-membrane, electrochemical gradient (∆µ H+ ), Fig. 1. (see opposite page) Diagram showing the relationships of mitochondrial oxidative phosphorylation (OXPHOS) to (1) energy (ATP) production, (2) reactive oxygen species (ROS) production, and (3) initiation of apoptosis through the mito- chondrial permeability transition pore (mtPTP). The OXPHOS complexes, designed I to V, are as follows: complex I (NADH: ubiquinone oxidoreductase) encompassing a FMN and six Fe–S centers (designated with a cube); complex II (succinate: ubiquinone oxidoreductase) involving an FAD, three Fe–S centers, and a cytochrome-b; complex III (ubiquinol: cytochrome-c oxidoreductase) encompassing cytochromes-b and c1 and the Rieske Fe–S center; complex IV (cytochrome-c oxidase) encompassing cytochromes a+a 3 and CuA and CuB; and complex V (H + -translocating ATP synthase). Pyruvate from glucose enters the mitochondria via pyruvate dehydrogenase (PDH), generating acetyl CoA that enters the TCA cycle by combining with oxaloacetate (OAA). Cis-Aconitase converts citrate to isocitrate and contains an 4Fe–4S center. Lactate dehydrogenase (LDH) converts excess pyruvate plus NADH to lactate (1–3). Small molecules defuse through the outer membrane via the voltage-dependent anion channel (VDAC) or porin. The VDAC together with ANT, Bax, and the cyclophilin D (CD) protein are thought to come together at the mitochondrial inner and outer membrane contact points to create the mtPTP. The proapoptotic Bax of the mtPTP is thought to interact with the antiapoptotic Bcl2 and the benzodiazepine receptor (BD). The opening of the mtPTP is associated with the release of cytc, activating which activates Apaf-1 that then binds to and activates procaspase-9. The activated caspase-9 then initiates the proteolytic degradation of cellular proteins (4–7). Modifi ed from ref. 8 with permission from Science.) Animal Models for Mitochondrial Disease 5 Fig. 1. 6 Wallace {∆µ H+ = ∆ψ + ∆pH}. The potential energy stored in ∆µ H+ is used to condense ADP and Pi to make ATP via complex V (ATP synthase), driven by the movement of protons back through a complex V proton channel. Each of the ETC complexes incorporates multiple electron carriers. Com- plexes I, II, and III encompass several Fe–S centers, whereas complexes III and IV encompass the cytochromes. Mitochondrial aconitase also contains an Fe–S center (8–10). Matrix ATP is exchanged for cytosolic ADP by the adenine nucleotide translocator (ANT). ANT isoforms are derived from multiple genes. In humans, there are three tissue-specifi c isoforms (11): a heart-muscle-specifi c isoform (ANT1) located at the chromosome 4q35 locus (12–19), an inducible isoform (ANT2) located at Xq24 (13,20–23), and a systemic isoform (ANT3) located in the pseudoautosomal region at Xp22.3 (13,14,24,25). In mouse, there are only two ANT genes (Ant1 and Ant2), homologs of the human ANT1 and ANT2 proteins (26). Mouse Ant1 maps to chromosome 8, syntenic to human 4q35 (27,28), whereas mouse Ant2 maps to regions A-D of X chromosome, syntenic to human Xq24 (29). Because the ETC is coupled to ATP synthesis through ∆µ H+ mitochondrial oxygen consumption is regulated by ∆µ H+ and hence the matrix concentration of ADP. In the absence of ADP, oxygen consumption is slow (state IV respira- tion). However, when ADP is added and transported into the matrix by the ANT, ∆µ H+ falls. As the ATP synthase utilizes the proton gradient to phosphorylate the ADP back to ATP, oxygen consumption goes up as the ETC reconstitutes ∆µ H+ (state III respiration). The ratio of state III and state IV respiration is called the respiratory control ratio (RCR) and the amount of molecular oxygen consumed relative to the ADP phosphorylated is the P/O ratio. Addition of uncouplers such as 2,4-dinitrophenol (DNP) and FCCP collapses ∆µ H+ and permits the ETC and oxygen consumption to run at their maximum rates. Cells regulate ∆µ H+ through the uncoupling proteins, Ucp. These proteins form proton channels in the mitochondrial inner membrane. Mammals have three uncoupling proteins. Uncoupler protein 1 (Ucp1) is primarily associated with brown adipose tissue (BAT), where it functions in thermal regulation. It is strongly induced by cold stress through a β3-adenergic response pathway (30–33). Uncoupler protein 2 (Upc2) has 59% amino acid identity to Ucp1 and is widely expressed in adult human tissues with mRNA levels being highest in skeletal muscle. It is also upregulated in white fat in response to an increased fat diet. In mouse, it has been linked to quantitative trait locus for hyperinsulinemia and obesity (33). Uncoupler protein 3 (Ucp3) is 57% identical to Ucp1 and 73% identical to Ucp2. Ucp3 is widely expressed in adult tissues and at particularly high levels in skeletal muscle. Moreover, it is hormonally regulated, being induced in skeletal muscle by thyroid hormone, Animal Models for Mitochondrial Disease 7 in white fat by β3–adrenergic agonists, and also regulated by dexamethasone, leptin, and starvation. Ucp3 is located adjacent to Ucp2 in human chromosome 11q13 and mouse chromosome 7 (34–37). Superoxide anion (O 2 • – ) is generated from OXPHOS by the transfer of one electron from the ETC to O 2 (see Fig. 1). Ubisemiquinone, localized at the CoQ binding sites of complexes I, II, and III, appears to be the primary electron donor. Because the free-radical ubisemiquinone is the probable electron donor in the ETC, conditions that maximize the levels of ubisemiquinone should also maximize mitochondrial ROS production. This would occur when the ETC is primarily but not completely reduced. This might explain why mitochondrial ROS production is further increased when uncouplers are added to Antimycin A-inhibited mitochondria (38–41). The O 2 • – is converted to H 2 O 2 by Mn superoxide dismutase (MnSOD) or cytosolic Cu/ZnSOD, and the resulting H 2 O 2 is reduced to water by glutathione peroxidase (GPx1) or catalase. However, H 2 O 2 , in the presence of reduced transition metals, can be converted to the highly reactive hydroxyl radical ( – OH). Major targets of ROS reactivity are the Fe–S centers of the TCA cycle and the ETC. Hence, mitochondria are particularly sensitive to oxidative stress (8,42–45). Superoxide production and H 2 O 2 generation are highest when the ETC is more reduced (state IV respiration) and lowest when it is more oxidized (state III respiration) (46–50). Therefore, the blocking of electron fl ow through the ETC by drugs such as Antimycin A, which inhibits complex III, stimulates ROS production (38,46,48,50). The mitochondria are also the major regulators of apoptosis, which is initiated though the opening of mtPTP (see Fig. 1). The mtPTP is thought to be composed of the inner membrane ANT, the outer membrane voltage- dependent anion channel (VDAC) or porin, Bax, Bcl2, cyclophilin D, and the benzodiazepine receptor (4,51,52).When the mtPTP opens, ∆µ H+ collapses and ions equilibrate between the matrix and cytosol, causing the mitochondria to swell. Ultimately, this disrupts the outer membrane, releasing the contents of the intermembrane space into the cytosol. The intermembrane space contains a number of cell-death-promoting factors, including cytochrome-c, procaspases-2, -3, and -9, apoptosis-initiating factor (AIF), as well as the caspase-activated DNase (CAD) (5,53–56). On release, cytochrome-c interacts with the cytosolic Apaf-1 and procaspase-9 complex. This cleaves and activates procaspase-9. Caspase-9 then cleaves procaspase-3, which activates additional hydrolytic enzymes, destroying the cytoplasm. AIF and CAD are transported to the nucleus, where they degrade the chromatin (8). The mtPTP can be stimulated to open by uptake of excessive Ca 2+ ; increased oxidative stress, decreased mitochondrial ∆µ H+ , ADP, and ATP, and ANT 8 Wallace ligands such as atractyloside (4,5). Thus, disease states that inhibit OXPHOS and increase ROS production should also increase the propensity of cells to undergo apoptosis (4,6,7). There are two major apoptosis pathways, the “mitochondrial” or “cellular stress” pathway described earlier and the “death ligand/receptor” pathway. The “mitochondrial” pathway is initiated by cytochrome-c release from the mitochondrion and can be activated by multiple stress signals. These can include transfection with tBID (a caspase activated [BH3-domain-only] Bcl2 derivative) or treatment with staurosporine (a general kinase inhibitor), etoposide (topoisomerase II inhibitor), ultraviolet (UV) light, thapsigargin (inhibitor of the endoplasmic reticulum [ER] Ca 2+ ATPase), tunicamycin (inhibitor of ER N-linked glycosylation), or brefeldin A (inhibitor of ER–Golgi transport). The “death ligand/receptor” pathway is activated by the interaction of the Fas ligand on a lymphoid effector cell with the Fas-receptor target cell. Alternatively, tumor necrosis factor (TNF)-α plus cycloheximide (CHX) can also activate the “death receptor” pathway. These signals initiate a signal transduction pathway through FADD and caspase-8, leading to the activation of caspase-3, which is central to the maturation and function of the immune system (57,58). 1.1.2. Stress Response and the Mitochondria The mitochondria interact with the cellular stress response pathways to globally regulate cellular functions, survival, and proliferation. Two such stress-response proteins are the poly(ADP-ribose) polymerase (PARP) (59) and the histone deacetylase SIR2. The PARP protein is a nuclear DNA enzyme that is activated by fragments of DNA resulting from DNA damage. Utilizing NAD + as a substrate, it transfers 50 or more ADP-ribose moieties to nuclear proteins such as histones and PARP itself. Massive DNA damage results in excessive activation of PARP that leads to the depletion of NAD + . The resynthesis of NAD + from ATP then markedly depletes cellular ATP leading to death (60). Mice in which the PARP gene has been genetically inactivated show remarkable resistance to cellular stress such as cerebral ischemia (stroke) (61,62). and streptozotocin-induced diabetes (63). The nuclear protein p53 is also activated by DNA damage and can initiate programmed cell death. This pathway has been shown to be mediated through mitochondrial release of cytochrome-c, which, in turn, activates Apaf-1 and caspase-9. The p53 initiation of mitochondrial cytochrome-c release requires the intervention of proapoptotic protein Bax. Hence, DNA damage activates p53, which activates Bax, which causes mitochondrial cytochrome-c release, which initiates apoptosis (64). Animal Models for Mitochondrial Disease 9 Another nuclear protein, SIR2, uses NAD + as a cofactor to diacetylate histones. Diacetylated histones keep inactive genes, such as proto-oncogenes, silent (65). Degradation of NAD + inactivates SIR2, permitting the histones to be acetylated and silent genes to be illegitimately expressed. Cellular and DNA damage can be caused by ROS. NADPH oxidases reduce O 2 to generate superoxide anion in the cytosol. The best characterized of the NADPH oxidases is the macrophage “oxidative burst” complex involved in generating the O 2 • – to kill engulfed micro-organisms. However, an additional NADPH oxidase, Mox1, is a homolog of the gp91phox catalytic subunit of the phagocyte NADP oxidase. Mox1 generates O 2 • – . When Mox1 is overexpressed in NIH3T3, it increases the mitotic rate, cell transformation, and tumorgenicity of cells (66). This mitogenic activity of Mox1 is neutralized by overexpression of catalase, indicating that cell growth signal must be H 2 O 2 (67). The fact that H 2 O 2 is a mitogenic signal for the cell nucleus is of great importance for the mitochondria, as H 2 O 2 is the only mitochondrial ROS that it stable enough to defuse to the nucleus. Therefore, cellular H 2 O 2 levels can be affected by mitochondrial H 2 O2 production. Acting together, these various enzymes and molecules form an integrated metabolic network with the mitochondria. Inhibition of the mitochondrial ETC results in increased O 2 • – production that is converted to H 2 O 2 by mitochondrial MnSOD. Mitochondrial H 2 O 2 can diffuse to the nucleus, where, at low concentrations, it acts as a mitogen. However, excessive mitochondrial genera- tion of H 2 O 2 can overwhelm the antioxidant defenses of the cytosol (catalase, glutathione peroxidase, etc.) and cause DNA damage. DNA damage would mutagenize proto-oncogenes, the cause of their activation. Excessive DNA damage then activates PARP, which degrades NAD + . Depletion of NAD + blocks the transfer of reducing equivalents to the mitochondrial ETC, causing a depletion of ATP. Reduced NAD + would inactivate SIR2, causing inappropriate activation of genes, including proto-oncogenes. 1.1.3. Mitochondrial Genetics The mitochondrial OXPHOS complexes are composed of multiple polypep- tides, most encoded by the nDNA. However, 13 polypeptides are encoded by the closed circular, 16,569 base pairs (bp) mtDNA. The mtDNA also codes for the 12S and 16S rRNAs and 22 tRNAs necessary for mitochondrial protein synthesis. The 13 mtDNA polypeptides include 7 (ND1, 2, 3, 4, 4L, 5, 6) of the 43 subunits of complex I, 1 (cytb) of the 11 subunits of complex III, 3 (COI, II, III) of the 13 subunits of complex IV, and 2 (ATP6 and 8) of the 16 subunits of complex V. The mtDNA also contains an approx 1000-bp control region that encompasses the heavy (H)- and light (L)-strand promoters (P H and P L ) and 10 Wallace the H-strand origin of replication (O H ). The H-strand primer is generated by cleavage of the L-strand transcript by the nuclear-encoded RNase MRP at runs of G nucleotides in the conserved sequence blocks CSBIII, CSBII, and CSBI, primarily after CSBI (68–71). P H and P L are associated with mitochondrial transcription factor (Tfam) binding sites that are essential for the effective expression of these promoters (72–76). Whereas the P H is responsible for transcribing both of the rRNA genes and 12 of the protein coding genes, P L transcribes the ND6 protein gene and several tRNAs and generates the primers used for initiation of H-strand replication at O H . The L-strand origin of replication (O L ) is located two-thirds of the way around the circle from O H (70). All of the other genes necessary to assemble a mitochondrion are encoded by the nucleus (8). Each human cell contains hundreds of mitochondria and thousands of mtDNAs. The semiautonomous nature of the mitochondria has been demon- strated by showing that mitochondria and their resident mtDNAs can be transferred from one cell to another by enucleating the donor cell and fusing the mitochondria-containing cytoplast to a recipient cell. The feasibility of this cybrid transfer procedure was fi rst demonstrated using cells harboring a mtDNA mutation that imparts resistance to the mitochondrial ribosome inhibitor chloramphenicol (CAP) (77–79). This cybrid transfer process has been further refi ned by curing the recipient cell of its resident mtDNA by long-term growth in ethidium bromide or by treatment with the mitochondrial toxin rhodamine-6G (R6G). Cells lacking mtDNA, resulting from prolonged growth in ethidium bromide, have been designated ρ o cells. These cells require glucose as an energy source, uridine to compensate for the block in pyrimidine biosynthesis, and pyruvate to reoxidize the NADH generated during glycolysis, a combination called GUP medium. R6G-treated cells or mtDNA-defi cient ρ o cells are ideal recipients for transmitochondrial experiments, as the result- ing cybrids will not retain the recipient cells mtDNAs (80–83). CAP R was subsequently shown to result from single nucleotide substitutions in the 16S rRNA gene (84,85). The mtDNA is maternally inherited and has a very high mutation rate. When a new mtDNA mutation arises in a cell, a mixed intracellular population of mtDNAs is generated, a state termed heteroplasmy. As a heteroplasmic cell replicates, the mutant and normal molecules are randomly distributed into the daughter cells and the proportion of mutant mtDNAs drifts, a process called replicative segregation. As the percentage of mutant mtDNAs increases, the mitochondrial energetic capacity declines, ROS production increases, and the propensity for apoptosis increases. The tissues most sensitive to mitochondrial dysfunction are the brain, heart, skeletal muscle, endocrine system, and kidney (8). [...]... gene Tfam; (2) mutations in the mitochondrial bioenergetic genes Ant1 and Unc1–3; (3) mutations in the mitochondrial antioxidant genes GPx1 and Sod2 (MnSOD); and (4) mutations in the mitochondrial apoptosis genes cytochrome-c (cytc), Bax, Bak, Apaf1, and caspases 9 and 3 2.2.1 Mutations in the Mitochondrial Biosynthetic Gene Tfam Genetic inactivation of the nuclear-encoded mitochondrial transcription factor,... (44,169,171,172), reduces mtDNA damage (163), and reverses many of the changes seen in mitochondrial gene expression (164,165) Thus, the age-related decline in OXPHOS, the accumulation of oxidative damage and mtDNA mutations, and the compensatory induction of bioenergetic and stress-response gene expression are all linked in both mitochondrial diseases and in aging 1.2.4 Mitochondrial Defects in Diabetes Mellitus... peripheral neuropathy, hypertrophic cardiomyopathy, and diabetes, and it results from the inactivation of the frataxin gene on chromosome 9q3 Frataxin regulates free iron in the mitochondrial matrix, and its absence results in increased matrix iron that converts H2O2 to •OH and inactivates the mitochondrial Fe–S center enzymes (aconitase and complexes I, II, and III) (45,124,125) Autosomal dominant progressive... hyperpolarized, and NAD+ becomes progressively reduced to NADH + H+ The excess of reducing equivalents of NADH reduce the ETC, which stimulates the transfer of electrons to O2 to give O2•– The mitochondrial O2•–, along with mitochondrial NO production, reacts with and damages mitochondrial membranes, proteins, and DNA The increased O2•– is also converted to H2O2 by mitochondrial MnSOD, and the excess... external ophthalmoplegia (CPEO) and the Kearns–Sayre syndrome (KSS) are associated with ophthalmoplegia, ptosis, and mitochondrial myopathy, together with a variety of other symptoms, including seizures, cerebellar ataxia, deafness, diabetes, heart block, and so on (8,98) CPEO and KSS patients typically develop mitochondrial myopathy with RRF that encompass COX-negative and SDH-hyperreactive muscle fiber... Ucp2, and Ucp3 have been inactivates As expected, the Ant1 mutant reduced heart and muscle energy capacity and the Ucp mutants reduced proton leak and increased ∆µH+ Unexpectedly, Animal Models for Mitochondrial Disease 23 however, all of the mutants increased mitochondrial ROS production resulting a variety of phenotypic effects 2.2.2.1 ANT1-DEFICIENT MICE DEVELOP MYOPATHY, CARDIOMYOPATHY, AND MULTIPLE... cellular ATP, inhibiting the ETC, and increasing mitochondrial ROS production in the pathophysiology of mitochondrial disease ANT1-deficient [Ant1tm2Mgr (–/–)] mice are viable, although they develop classical mitochondrial myopathy and hypertrophic cardiomyopathy They also develop elevated serum lactate, alanine, succinate, and citrate, consistent with the inhibition of the ETC and the TCA cycle (27) The mouse... ROS production and oxidative stress The apparent importance of regulating ROS production clearly attests to its toxicity Thus, it follows that mice deficient in the mitochondrial antioxidant genes GPx1 and Sod2 (MnSOD) should have increased oxidative stress and develop mitochondrial disease symptoms To determine if this is true, mice deficient in GPx1 and MnSOD have been generated 2.2.3 Mitochondrial. .. driven by the endogenous GPx1 promoter and preparation of GPx1-specific antibodies and their use in Western blot analysis These studies revealed that GPx1 is strongly expressed in the liver, brain, and renal cortex, but very weakly expressed in the heart and skeletal muscle Furthermore, the GPx1 protein was found in both the cytosol and the mitochondrial of liver and kidney but only found in the cytosol... almost entirely inactivated in heart and brain Thus, the increased mitochondrial O2•– appears to have inactivated all of the mitochondrial Fe–Scenter-containing enzymes, thus blocking the TCA cycle and ETC chain (237) This would inhibit mitochondrial fatty acid oxidation, causing fat to accumulate in the liver and energy starvation in the heart, leading to dilation and failure Respiration studies on . HUMANA PRESS Methods in Molecular Biology TM Edited by William C. Copeland Mitochondrial DNA Methods and Protocols HUMANA PRESS Methods in Molecular Biology TM Edited by William C. Copeland Mitochondrial DNA Methods. Copeland Mitochondrial DNA Methods and Protocols VOLUME 197 Animal Models for Mitochondrial Disease 1 I METHODS FOR THE ANALYSIS OF MTDNA Animal Models for Mitochondrial Disease 3 3 From: Methods in Molecular. Methods in Molecular Biology, vol. 197: Mitochondrial DNA: Methods and Protocols Edited by: W. C. Copeland © Humana Press Inc., Totowa, NJ 1 Animal Models for Mitochondrial Disease Douglas C. Wallace 1.