The human TAZ gene complements mitochondrial dysfunction in the yeast taz1Delta mutant. Implications for Barth syndrome

Barth syndrome is a genetic disorder that is caused by different mutations in the TAZ gene G4.5. The yeast gene TAZ1 is highly homologous to human TAZ, and the taz1Delta mutant has phospholipid defects similar to those observed in Barth syndrome cells, including aberrant cardiolipin species and decreased cardiolipin levels. Subcellular fractionation studies revealed that Taz1p is localized exclusively in mitochondria, which supports the theory that tafazzins are involved in cardiolipin remodeling. Because cardiolipin plays an important role in respiratory function, we measured the energy transformation and osmotic properties of isolated mitochondria from the taz1Delta mutant. Energy coupling in taz1Delta mitochondria was dependent on the rate of oxidative phosphorylation, as coupling was diminished when NADH was used as a respiratory substrate but was unaffected when ethanol was the substrate. Membrane stability was compromised in taz1Delta mitochondria exposed to increased temperature and hypotonic conditions. Mitochondria from taz1Delta also displayed decreased swelling in response to ATP, which induces the yeast mitochondrial unspecific channel, and to alamethicin, a membrane-disrupting agent. Coupling was measured in taz1Delta cells containing different splice variants of the human TAZ gene. Only the variant that restores wild type cardiolipin synthesis (lacking exon 5) restored coupling in hypotonic conditions and at elevated temperature. These findings may shed light on the mitochondrial deficiencies observed in Barth syndrome.     

The role of mitochondrial cardiolipin in heart function and its implication in cardiac disease

Mitochondria play an essential role in the energy metabolism of the heart. Many of the essential functions are associated with mitochondrial membranes and oxidative phosphorylation driven by the respiratory chain. Mitochondrial membranes are unique in the cell as they contain the phospholipid cardiolipin. The important role of cardiolipin in cardiovascular health is highlighted by several cardiac diseases, in which cardiolipin plays a fundamental role. Barth syndrome, Sengers syndrome, and Dilated cardiomyopathy with ataxia (DCMA) are genetic disorders, which affect cardiolipin biosynthesis. Other cardiovascular diseases including ischemia/reperfusion injury and heart failure are also associated with changes in the cardiolipin pool. Here, we summarize molecular functions of cardiolipin in mitochondrial biogenesis and morphology. We highlight the role of cardiolipin for the respiratory chain, metabolite carriers, and mitochondrial metabolism and describe links to apoptosis and mitochondria specific autophagy (mitophagy) with possible implications in cardiac disease.     

Cardiolipin remodeling by ALCAT1 links mitochondrial dysfunction to Parkinson’s diseases

Cardiolipin (CL) is a mitochondrial signature phospholipid that is required for membrane structure, respiration, dynamics, and mitophagy. Oxidative damage of CL by reactive oxygen species is implicated in the pathogenesis of Parkinson's disease (PD), but the underlying cause remains elusive. This work investigated the role of ALCAT1, an acyltransferase that catalyzes pathological remodeling of CL in various aging‐related diseases, in a mouse model of PD induced by 1‐methyl‐4‐phenyl‐1,2,4,6‐tetrahydropyridine (MPTP). We show that MPTP treatment caused oxidative stress, mtDNA mutations, and mitochondrial dysfunction in the midbrain. In contrast, ablation of the ALCAT1 gene or pharmacological inhibition of ALCAT1 prevented MPTP‐induced neurotoxicity, apoptosis, and motor deficits. ALCAT1 deficiency also mitigated mitochondrial dysfunction by modulating DRP1 translocation to the mitochondria. Moreover, pharmacological inhibition of ALCAT1 significantly improved mitophagy by promoting the recruitment of Parkin to dysfunctional mitochondria. Finally, ALCAT1 expression was upregulated by MPTP and by α‐synucleinopathy, a key hallmark of PD, whereas ALCAT1 deficiency prevented α‐synuclein oligomerization and S‐129 phosphorylation, implicating a key role of ALCAT1 in the etiology of mouse models of PD. Together, these findings identify ALCAT1 as a novel drug target for the treatment of PD.     

The Mitochondrial Quality Control Protein Yme1 Is Necessary to Prevent Defective Mitophagy in a Yeast Model of Barth Syndrome

The Saccharomyces cerevisiae TAZ1 gene is an orthologue of human TAZ; both encode the protein tafazzin. Tafazzin is a transacylase that transfers acyl chains with unsaturated fatty acids from phospholipids to monolysocardiolipin to generate cardiolipin with unsaturated fatty acids. Mutations in human TAZ cause Barth syndrome, a fatal childhood cardiomyopathy biochemically characterized by reduced cardiolipin mass and increased monolysocardiolipin levels. To uncover cellular processes that require tafazzin to maintain cell health, we performed a synthetic genetic array screen using taz1Δ yeast cells to identify genes whose deletion aggravated its fitness. The synthetic genetic array screen uncovered several mitochondrial cellular processes that require tafazzin. Focusing on the i-AAA protease Yme1, a mitochondrial quality control protein that degrades misfolded proteins, we determined that in cells lacking both Yme1 and Taz1 function, there were substantive mitochondrial ultrastructural defects, ineffective superoxide scavenging, and a severe defect in mitophagy. We identify an important role for the mitochondrial protease Yme1 in the ability of cells that lack tafazzin function to maintain mitochondrial structural integrity and mitochondrial quality control and to undergo mitophagy.     

Aberrant cardiolipin metabolism is associated with cognitive deficiency and hippocampal alteration in tafazzin knockdown mice

Cardiolipin (CL) is a key mitochondrial phospholipid essential for mitochondrial energy production. CL is remodeled from monolysocardiolipin (MLCL) by the enzyme tafazzin (TAZ). Loss-of-function mutations in the gene which encodes TAZ results in a rare X-linked disorder called Barth Syndrome (BTHS). The mutated TAZ is unable to maintain the physiological CL:MLCL ratio, thus reducing CL levels and affecting mitochondrial function. BTHS is best known as a cardiac disease, but has been acknowledged as a multi-syndrome disorder, including cognitive deficits. Since reduced CL levels has also been reported in numerous neurodegenerative disorders, we examined how TAZ-deficiency impacts cognitive abilities, brain mitochondrial respiration and the function of hippocampal neurons and glia in TAZ knockdown (TAZ kd) mice. We have identified for the first time the profile of changes that occur in brain phospholipid content and composition of TAZ kd mice. The brain of TAZ kd mice exhibited reduced TAZ protein expression, reduced total CL levels and a 19-fold accumulation of MLCL compared to wild-type littermate controls. TAZ kd brain exhibited a markedly distinct profile of CL and MLCL molecular species. In mitochondria, the activity of complex I was significantly elevated in the monomeric and supercomplex forms with TAZ-deficiency. This corresponded with elevated mitochondrial state I respiration and attenuated spare capacity. Furthermore, the production of reactive oxygen species was significantly elevated in TAZ kd brain mitochondria. While motor function remained normal in TAZ kd mice, they showed significant memory deficiency based on novel object recognition test. These results correlated with reduced synaptophysin protein levels and derangement of the neuronal CA1 layer in hippocampus. Finally, TAZ kd mice had elevated activation of brain immune cells, microglia compared to littermate controls. Collectively, our findings demonstrate that TAZ-mediated remodeling of CL contributes significantly to the expansive distribution of CL molecular species in the brain, plays a key role in mitochondria respiratory activity, maintains normal cognitive function, and identifies the hippocampus as a potential therapeutic target for BTHS.     

Barth syndrome cells display widespread remodeling of mitochondrial complexes without affecting metabolic flux distribution

Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by cardiac and skeletal myopathy, neutropenia and growth abnormalities. The disease is caused by mutations in the tafazzin (TAZ) gene encoding an enzyme involved in the acyl chain remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, this leads to decreased levels of mature CL and accumulation of the intermediate monolysocardiolipin (MLCL). At a cellular level, this causes mitochondrial fragmentation and reduced stability of the respiratory chain supercomplexes. However, the exact mechanism through which tafazzin deficiency leads to disease development remains unclear. We therefore aimed to elucidate the pathways affected in BTHS cells by employing proteomic and metabolic profiling assays. Complexome profiling of patient skin fibroblasts revealed significant effects for about 200 different mitochondrial proteins. Prominently, we found a specific destabilization of higher order oxidative phosphorylation (OXPHOS) supercomplexes, as well as changes in complexes involved in cristae organization and CL trafficking. Moreover, the key metabolic complexes 2-oxoglutarate dehydrogenase (OGDH) and branched-chain ketoacid dehydrogenase (BCKD) were profoundly destabilized in BTHS patient samples. Surprisingly, metabolic flux distribution assays using stable isotope tracer-based metabolomics did not show reduced flux through the TCA cycle. Overall, insights from analyzing the impact of TAZ mutations on the mitochondrial complexome provided a better understanding of the resulting functional and structural consequences and thus the pathological mechanisms leading to Barth syndrome.     

Ablation of ALCAT1 Mitigates Hypertrophic Cardiomyopathy through Effects on Oxidative Stress and Mitophagy

Oxidative stress causes mitochondrial dysfunction and heart failure through unknown mechanisms. Cardiolipin (CL), a mitochondrial membrane phospholipid required for oxidative phosphorylation, plays a pivotal role in cardiac function. The onset of age-related heart diseases is characterized by aberrant CL acyl composition that is highly sensitive to oxidative damage, leading to CL peroxidation and mitochondrial dysfunction. Here we report a key role of ALCAT1, a lysocardiolipin acyltransferase that catalyzes the synthesis of CL with a high peroxidation index, in mitochondrial dysfunction associated with hypertrophic cardiomyopathy. We show that ALCAT1 expression was potently upregulated by the onset of hyperthyroid cardiomyopathy, leading to oxidative stress and mitochondrial dysfunction. Accordingly, overexpression of ALCAT1 in H9c2 cardiac cells caused severe oxidative stress, lipid peroxidation, and mitochondrial DNA (mtDNA) depletion. Conversely, ablation of ALCAT1 prevented the onset of T4-induced cardiomyopathy and cardiac dysfunction. ALCAT1 deficiency also mitigated oxidative stress, insulin resistance, and mitochondrial dysfunction by improving mitochondrial quality control through upregulation of PINK1, a mitochondrial GTPase required for mitochondrial autophagy. Together, these findings implicate a key role of ALCAT1 as the missing link between oxidative stress and mitochondrial dysfunction in the etiology of age-related heart diseases.     

Cardiolipin remodeling in Barth syndrome and other hereditary cardiomyopathies

Mitochondria play a prominent role in cardiac energy metabolism, and their function is critically dependent on the integrity of mitochondrial membranes. Disorders characterized by mitochondrial dysfunction are commonly associated with cardiac disease. The mitochondrial phospholipid cardiolipin directly interacts with a number of essential protein complexes in the mitochondrial membranes including the respiratory chain, mitochondrial metabolite carriers, and proteins critical for mitochondrial morphology. Barth syndrome is an X-linked disorder caused by an inherited defect in the biogenesis of the mitochondrial phospholipid cardiolipin. How cardiolipin deficiency impacts on mitochondrial function and how mitochondrial dysfunction causes cardiomyopathy has been intensively studied in cellular and animal models of Barth syndrome. These findings may also have implications for the molecular mechanisms underlying other inherited disorders associated with defects in cardiolipin, such as Sengers syndrome and dilated cardiomyopathy with ataxia (DCMA).     

Role of cardiolipin alterations in mitochondrial dysfunction and disease

Cardiolipin (CL) is a structurally unique dimeric phospholipid localized in the inner mitochondrial membrane where it is required for optimal mitochondrial function. In addition to its role in maintaining membrane potential and architecture, CL is known to provide essential structural and functional support to several proteins involved in mitochondrial bioenergetics. A loss of CL content, alterations in its acyl chain composition, and/or CL peroxidation have been associated with mitochondrial dysfunction in multiple tissues in a variety of pathological conditions, including ischemia, hypothyroidism, aging, and heart failure. Recently, aberrations in CL metabolism have been implicated as a primary causative factor in the cardioskeletal myopathy known as Barth syndrome, underscoring an important role of CL in human health and disease. The purpose of this review is to provide an overview of evidence that has linked changes in the CL profile to mitochondrial dysfunction in various pathological conditions. In addition, a brief overview of CL function and biosynthesis, and a discussion of methods used to examine CL in biological tissues are provided. phospholipid; metabolism; heart failure; aging; hypothyroidism; lipid peroxidation; oxidative stress; diet; ischemia     

NAD supplementation improves mitochondrial performance of cardiolipin mutants

Cardiolipin (CL) deficiency causes mitochondrial dysfunction and aberrant metabolism that are associated in humans with the severe disease Barth syndrome (BTHS). Several metabolic abnormalities are observed in BTHS patients and model systems, including decreased oxidative phosphorylation, reduced tricarboxylic acid (TCA) cycle flux, and accumulated lactate and D-β-hydroxybutyrate, which strongly suggests that nicotinamide adenine dinucleotide (NAD) redox metabolism may be altered in CL-deficient cells. In this study, we identified abnormal NAD⁺ metabolism in multiple BTHS model systems and demonstrate that supplementation of NAD⁺ precursors such as nicotinamide mononucleotide (NMN) improves mitochondrial function. Improved mitochondrial function in the Drosophila model was associated with restored exercise endurance, which suggests a potential therapeutic benefit of NAD⁺ precursor supplementation in the management of BTHS patients.     

Unremodeled and Remodeled Cardiolipin Are Functionally Indistinguishable in Yeast

After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogeneous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence, cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities, and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here, we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.     

Mitochondrial respiratory chain supercomplexes are destabilized in Barth Syndrome patients

Mutations in the human TAZ gene are associated with Barth Syndrome, an often fatal X-linked disorder that presents with cardiomyopathy and neutropenia. The TAZ gene encodes Tafazzin, a putative phospholipid acyltranferase that is involved in the remodeling of cardiolipin, a phospholipid unique to the inner mitochondrial membrane. It has been shown that the disruption of the Tafazzin gene in yeast (Taz1) affects the assembly and stability of respiratory chain Complex IV and its supercomplex forms. However, the implications of these results for Barth Syndrome are restricted due to the additional presence of Complex I in humans that forms a supercomplex with Complexes III and IV. Here, we investigated the effects of Tafazzin, and hence cardiolipin deficiency in lymphoblasts from patients with Barth Syndrome, using blue-native polyacrylamide gel electrophoresis. Digitonin extraction revealed a more labile Complex I/III(2)/IV supercomplex in mitochondria from Barth Syndrome cells, with Complex IV dissociating more readily from the supercomplex. The interaction between Complexes I and III was also less stable, with decreased levels of the Complex I/III(2) supercomplex. Reduction of Complex I holoenzyme levels was observed also in the Barth Syndrome patients, with a corresponding decrease in steady-state subunit levels. We propose that the loss of mature cardiolipin species in Barth Syndrome results in unstable respiratory chain supercomplexes, thereby affecting Complex I biogenesis, respiratory activities and subsequent pathology.     

Phospholipid methylation controls Atg32‐mediated mitophagy and Atg8 recycling

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy‐inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria‐anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8–PMME, a mitophagy‐incompetent lipid conjugate of the autophagy‐related ubiquitin‐like modifier. Amelioration of Atg32 expression and attenuation of Atg8–PMME conjugation markedly rescue mitophagy in opi3‐null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32‐mediated mitophagy.     

Defective remodeling of cardiolipin and phosphatidylglycerol in Barth syndrome

Cardiolipin (CL) and phosphatidylglycerol (PG) are the major polyglycerophospholipids observed in mammalian tissues. CL is exclusively found in the inner mitochondrial membrane and is required for optimal function of many of the respiratory and ATP-synthesizing enzymes. The role of CL in oxidative phosphorylation is, however, not fully understood and although reduced CL content leads to aberrant cell function, no human disorders with a primary defect in cardiolipin metabolism have been described. In this paper we present evidence that patients with the rare disorder X-linked cardioskeletal myopathy and neutropenia (Barth syndrome, MIM 302060) have a primary defect in CL and PG remodeling. We investigated phospholipid metabolism in cultured skin fibroblasts of patients and show that the biosynthesis rate of PG and CL is normal but that the CL pool size is 75% reduced, indicating accelerated degradation. Moreover, the incorporation of linoleic acid, which is the characteristic acyl side chain found in mammalian CL, into both PG and CL is significantly reduced, whereas the incorporation of other fatty acids into these phospholipids is normal. We show that this defect was only observed in Barth syndrome patients' cells and not in cells obtained from patients with primary defects in the respiratory chain, demonstrating that the observed defect is not secondary to respiratory chain dysfunction. These results imply that the G4.5 gene product, which is mutated in Barth syndrome patients, is specifically involved in the remodeling of PG and CL and for the first time identify an essential factor in this important cellular process.     

Deacylation on the matrix side of the mitochondrial inner membrane regulates cardiolipin remodeling

The mitochondrial-specific lipid cardiolipin (CL) is required for numerous processes therein. After its synthesis on the matrix-facing leaflet of the inner membrane (IM), CL undergoes acyl chain remodeling to achieve its final form. In yeast, this process is completed by the transacylase tafazzin, which associates with intermembrane space (IMS)-facing membrane leaflets. Mutations in TAZ1 result in the X-linked cardiomyopathy Barth syndrome. Amazingly, despite this clear pathophysiological association, the physiological importance of CL remodeling is unresolved. In this paper, we show that the lipase initiating CL remodeling, Cld1p, is associated with the matrix-facing leaflet of the mitochondrial IM. Thus monolysocardiolipin generated by Cld1p must be transported to IMS-facing membrane leaflets to gain access to tafazzin, identifying a previously unknown step required for CL remodeling. Additionally, we show that Cld1p is the major site of regulation in CL remodeling; and that, like CL biosynthesis, CL remodeling is augmented in growth conditions requiring mitochondrially produced energy. However, unlike CL biosynthesis, dissipation of the mitochondrial membrane potential stimulates CL remodeling, identifying a novel feedback mechanism linking CL remodeling to oxidative phosphorylation capacity.