Rate of Accumulation of Luxol Fast Blue Staining Material and Mitochondrial ATP Synthase Subunit 9 in Motor Neuron Degeneration Mice

The rate of accumulation of Luxol Fast Blue staining material in the hippocampus of motor neuron degeneration (mnd/mnd) mice, a model of Batten Disease, was quantitated. Stained material increased linearly up to 8 months of age. A quantitative immunoassay was used to measure levels of mitochondrial ATP synthase subunit 9 in brain and liver of mnd/mnd mice. Levels of subunit 9 increased progressively throughout the lifespan of mnd/mnd mice reaching levels approximately 5-fold higher than in control animals. The rate of accumulation of subunit 9 is not consistent with any simple complete or partial degradation defect that is constant throughout the animal's life. Two more complicated models are discussed which are consistent with the observed accumulation rate of subunit 9.     

Lysine 43 is trimethylated in subunit C from bovine mitochondrial ATP synthase and in storage bodies associated with batten disease

The hydrophobic membrane protein, subunit c, has been isolated from ATP synthase purified from bovine heart mitochondria. It has also been obtained from lysosomal storage bodies associated with ceroid lipofuscinosis from ovine liver and from human brain tissue of a victim of Batten disease. It is likely that the lysosomal protein has originated from the mitochondrion. These samples have been characterized by mass spectrometric methods. Irrespective of its source, subunit c has an intact molecular mass of 7650 Da, 42 Da greater than the value calculated from the amino acid sequence, and the protein has been modified post-translationally. In all three samples, the modification is associated with lysine 43, which lies in a polar loop region linking the two transmembrane alpha-helices of the protein. This residue is conserved throughout vertebrate sequences. The additional mass arises from trimethylation and not acetylation at the epsilon-N-position of the residue. These experiments show that the post-translational modification of subunit c is not, as has been suggested, an abnormal phenomenon associated with the etiology of Batten disease and ceroid lipofucinoses. Evidently, it occurs either before or during import of the protein into mitochondria or at a mitochondrial location after completion of the import process. The function of the trimethyllysine residue in the assembled ATP synthase complex is obscure. The residue and the modification are not conserved in all ATP synthases, and their role in the assembly and (or) functioning of the enzyme appear to be confined to higher organisms.     

NCL disease mechanisms

Despite the identification of a large number of disease-causing genes in recent years, it is still unclear what disease mechanisms operate in the neuronal ceroid lipofuscinoses (NCLs, Batten disease). As a group they are defined by the specific accumulation of protein, either subunit c of mitochondrial ATP synthase or SAPs A and D in lysosome-derived organelles, and regionally specific neurodegeneration. Evidence from biochemical and cell biology studies indicates related lesions in intracellular vesicle trafficking and lysosomal function. There is also extensive immunohistological evidence of a causative role of disease associated neuroinflammation. However the nature of these lesions is not clear nor is it clear why they lead to the defining pathology. Several different theories have proposed a range of potential mechanisms, but it remains to be determined which are central to pathogenesis, and whether there is a mechanism consistent across the group, or if it differs between disease forms. This review summarises the evidence that is currently available and the progress that has been made in understanding these profoundly disabling disorders. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.     

Mitochondrial dysfunction in the neuronal ceroid-lipofuscinoses (Batten disease)

There are at least eight genetic entities known as the ceroid-lipofuscinoses in humans which share clinical and pathological features that have caused them to be grouped together under the eponym of Batten disease. They present pathologically as lysosomal storage diseases but are also characterised by severe neurodegeneration. Although the biochemical defects appear primarily centred on lysosomes and defects in proteolysis, the link between this and pathogenesis of neuronal death is poorly understood.The pathogenesis of neurodegeneration has been studied particularly in two animal models these being the English setter dog and the New Zealand Southhampshire sheep (OCL6). In these, and some of the human entities, there is evidence of mitochondrial dysfunction. This includes the accumulation of subunit c of ATP synthase as a component of storage material in at least six of eight genetic forms of the disease; structural abnormalities of mitochondria and selective loss of neurons in areas of the brain that are particularly metabolically active. Direct evidence of dysfunction comes from mitochondrial function tests in fibroblasts and, in animal models, isolated liver mitochondria. Supporting evidence of mitochondrial dysfunction was shown by disturbances in proportions of energy-rich phosphates in fibroblasts in some of these diseases. If these various defects were reflected in neurons, then it would support the hypothesis that neuron death was associated with energy-linked excitotoxicity.     

Specific storage of subunit c of mitochondrial ATP synthase in lysosomes of neuronal ceroid lipofuscinosis (Batten's disease).

Immunochemical studies demonstrated the specific accumulation of subunit c of mitochondrial ATP synthase in the brain homogenates of late infantile and juvenile forms of Batten's disease. It is not stored in the infantile form. Storage of subunit alpha of mitochondrial ATP synthase and cytochrome c oxidase subunit IV, an inner membrane protein of mitochondria was not detected in the brains. There was also no difference in the levels of cathepsin B between the two forms of Batten's disease and controls. In cultured skin fibroblasts subunit c accumulates in the late infantile form, whereas it does not in other lysosomal storage diseases. Crude mitochondrial lysosomal preparations of control fibroblasts were separated into high-density fractions rich in a lysosomal marker and low-density fractions rich in a mitochondrial marker on Percoll density gradients. Subunit c was mostly recovered in low-density mitochondrial fractions, but in cells from the late infantile disease a part of subunit c was recovered in the high-density lysosomal fractions. Immunolocalization studies demonstrated a dot-like staining of storage materials for subunit c in the cells from late infantile patients and the staining pattern of subunit c is similar to that of a lysosomal membrane marker, lgp120. Immunostaining failed to detect subunit c in control cells. These results indicate a specific accumulation of subunit c in lysosomes, and suggest that the two forms of Batten's disease are caused by a specific failure in the degradation of subunit c.     

Biosynthesis of human cystathionine beta-synthase in cultured fibroblasts

A single specific radiolabeled polypeptide with an apparent Mr = 63,000 was recovered when cystathionine beta-synthase (EC 4.2.1.22) was precipitated from extracts of radiolabeled cultured human fibroblasts with an antiserum raised against pure human liver synthase, and the immunocomplexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial proteolysis of this fibroblast subunit and of the subunit of pure human liver synthase (Mr = 48,000) produced similar peptide patterns. Pulse-chase experiments, however, did not provide any evidence for post-translational modification of the fibroblast synthase subunit into a smaller "hepatic" form. Immunoprecipitation of polypeptides synthesized in vitro from human fibroblast mRNA revealed a polypeptide with the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the synthase subunit found in whole cell extracts. We conclude that the Mr = 63,000 subunit is the primary translational product of the gene for cystathionine beta-synthase in human fibroblasts.     

Lithium rescues the impaired autophagy process in CbCln3(Δex7/8/Δex7/8) cerebellar cells and reduces neuronal vulnerability to cell death via IMPase inhibition

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a neurodegenerative disorder caused by mutation in CLN3. Defective autophagy and concomitant accumulation of autofluorescence enriched with mitochondrial ATP synthase subunit c were previously discovered in Cln3 mutant knock-in mice. In this study, we show that treatment with lithium reduces numbers of LC3-positive autophagosomes and accumulation of LC3-II in Cln3 mutant knock-in cerebellar cells (CbCln3(Δex7/8/Δex7/8) ). Lithium, an inhibitor of GSK3 and IMPase, reduces the accumulation of mitochondrial ATP synthase subunit c and autofluorescence in CbCln3(Δex7/8/Δex7/8) cells, and mitigates the abnormal subcellular distribution of acidic vesicles in the cells. L690,330, an IMPase inhibitor, is as effective as lithium in restoring autophagy in CbCln3(Δex7/8/Δex7/8) cells. Moreover, lithium or down-regulation of IMPase expression protects CbCln3(Δex7/8/Δex7/8) cells from cell death induced by amino acid deprivation. These results suggest that lithium overcomes the autophagic defect in CbCln3(Δex7/8/Δex7/8) cerebellar cells probably through IMPase, thereby reducing their vulnerability to cell death.     

Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene

Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome. To enable analysis of this mutation in control nuclear backgrounds, two different cell lines were transformed with mitochondria carrying NARP mutant mitochondrial DNA. Transformant cell lines had decreased ATP synthesis capacity, and many also had abnormally high levels of two ATP synthase sub-complexes, one of which was F(1)-ATPase. A combination of metabolic labeling and immunoblotting experiments indicated that assembly of ATP synthase was slowed and that the assembled holoenzyme was unstable in cells carrying NARP mutant mitochondrial DNA compared with control cells. These findings indicate that altered assembly and stability of ATP synthase are underlying molecular defects associated with the NARP mutation in subunit 6 of ATP synthase, yet intrinsic enzyme activity is also compromised.     

Glutathione s-transferase omega 1 activity is sufficient to suppress neurodegeneration in a Drosophila model of Parkinson disease

A loss-of-function mutation in the gene parkin causes a common neurodegenerative disease that may be caused by mitochondrial dysfunction. Glutathione S-transferase Omega (GSTO) is involved in cell defense mechanisms, but little is known about the role of GSTO in the progression of Parkinson disease. Here, we report that restoration of Drosophila GSTO1 (DmGSTO1), which is down-regulated in parkin mutants, alleviates some of the parkin pathogenic phenotypes and that the loss of DmGSTO1 function enhances parkin mutant phenotypes. We further identified the ATP synthase β subunit as a novel in vivo target of DmGSTO1. We found that glutathionylation of the ATP synthase β subunit is rescued by DmGSTO1 and that the expression of DmGSTO1 partially restores the activity and assembly of the mitochondrial F(1)F(0)-ATP synthase in parkin mutants. Our results suggest a novel mechanism for the protective role of DmGSTO1 in parkin mutants, through the regulation of ATP synthase activity, and provide insight into potential therapies for Parkinson disease neurodegeneration.     

A "petite obligate" mutant of Saccharomyces cerevisiae: functional mtDNA is lethal in cells lacking the delta subunit of mitochondrial F1-ATPase

Within the mitochondrial F(1)F(0)-ATP synthase, the nucleus-encoded delta-F(1) subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the delta subunit gene (Deltadelta) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% rho(-)/rho degrees petites (i.e. cells missing a large portion (>50%) of the mtDNA (rho(-)) or totally devoid of mtDNA (rho degrees )). Previous work has suggested that the absence of complete mtDNA (rho(+)) in Deltadelta yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8(m)) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Deltadelta yeast. We further show that combined with a nuclear mutation (Deltaatp4) preventing the ATP synthase proton channel assembly, a lack of delta subunit fails to destabilize the mtDNA, and rho(+) Deltadelta cells become viable. We conclude that Deltadelta yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the rho(-)/rho degrees mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae.     

Interaction with monomeric subunit c drives insertion of ATP synthase subunit a into the membrane and primes a-c complex formation

Subunit a is the main part of the membrane stator of the ATP synthase molecular turbine. Subunit c is the building block of the membrane rotor. We have generated two molecular fusions of a and c subunits with different orientations of the helical hairpin of subunit c. The a/c fusion protein with correct orientation of transmembrane helices was inserted into the membrane, and co-incorporated into the F(0) complex of ATP synthase with wild type subunit c. The fused c subunit was incorporated into the c-ring tethering the ATP synthase rotor to the stator. The a/c fusion with incorrect orientation of the c-helices required wild type subunit c for insertion into the membrane. In this case, the fused c subunit remained on the periphery of the c-ring and did not interfere with rotor movement. Wild type subunit a inserted into the membrane equally well with wild type subunit c and c-ring assembly mutants that remained monomeric in the membrane. These results show that interaction with monomeric subunit c triggers insertion of subunit a into the membrane, and initiates formation of the a-c complex, the ion-translocating module of the ATP synthase. Correct assembly of the ATP synthase incorporating topologically correct fusion of subunits a and c validates using this model protein for high resolution structural studies of the ATP synthase proton channel.     

Identification of subunit g of yeast mitochondrial F1F0-ATP synthase, a protein required for maximal activity of cytochrome c oxidase.

By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bovine mitochondrial F1F0-ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function. Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well-characterized subunits of the yeast complex. Whilst mitochondria isolated from the atp20-null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20-null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate. The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.     

Ovine ceroid lipofuscinosis. The major lipopigment protein and the lipid-binding subunit of mitochondrial ATP synthase have the same NH2-terminal sequence

Previous studies on lipopigment isolated from sheep affected with ceroid lipofuscinosis (Batten's disease) showed that the disease is a lysosomal proteinosis, involving specific storage of peptide(s) that migrate in dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 3500. This band is the dominant contributor to the lipopigment mass. When purified total lipopigment proteins were loaded onto a protein sequencer, a dominant sequence was found, identical to the NH2 terminus of the lipid-binding subunit of protein translocating mitochondrial ATP synthase. This sequence was determined to 40 residues and a minimum estimate of 40% made for its contribution to the lipopigment protein mass. The full lipid-binding subunit has physical and chemical properties similar to those of the specifically stored low Mr peptide, which may be the full protein or a large NH2-terminal fragment of it. Lipopigments in the human ceroid lipofuscinoses also contain a major component with similar physical and chemical properties. These and previous results indicate that the genetic lesion in ovine ceroid lipofuscinosis causes an abnormal accumulation of this peptide in lysosomes, i.e. the disease is a proteolipid proteinosis, specifically a lysosomal mitochondrial ATP synthase lipid-binding subunit proteinosis. The analogous human diseases are likely to reflect storage of the same or similar peptides.     

The F0 subunits of bovine mitochondrial ATP synthase complex: purification, antibody production, and interspecies cross-immunoreactivity.

The known subunits of the membrane sector F0 of the bovine mitochondrial ATP synthase complex are subunits b, d, 6, F6, OSCP (oligomycin sensitivity-conferring protein), the DCCD (dicyclohexylcarbodiimide) binding proteolipid, and A6L. The first six subunits were purified from SMP or preparations of the ATP synthase complex, and monospecific antibodies were raised against each. The antisera were shown to be competent for immuno-blotting, and each antiserum recognized a single polypeptide of the expected Mr in preparations of the ATP synthase complex. Immunoblots utilizing antibodies to OSCP and subunits d and 6, which exhibit the same Mr on dodecyl sulfate-polyacrylamide gels, showed clearly that these polypeptides are immunologically distinct. Immunological cross-reactivity was demonstrated between bovine, human, rat, Saccharomyces cerevisiae, Paracoccus denitrificans, and Escherichia coli for subunit 6; between bovine, human, and rat for subunits b, d, OSCP, and F6; and between bovine and rat for the DCCD binding proteolipid. Anti-subunit 6 antiserum, before or after immunopurification against the ATP synthase complex, recognized a single polypeptide in the bovine ATP synthase complex and S. cerevisiae mitochondria, but two polypeptides of different Mr in bovine SMP, human, and rat mitochondria, and Paracoccus and E. coli membranes.     

Effect of selenite on basic mitochondrial function in human osteosarcoma cells with chronic mitochondrial stress

Mitochondrial chronic stress that originates from defective mitochondria is implicated in a growing list of human diseases. To enhance understanding of pathophysiology of chronic mitochondrial dysfunction we investigated human osteosarcoma cells with 2 types of chronic stress: corresponding to the mutation in ATP synthase subunit 6 encoded by mtDNA (NARP syndrome-mild stress) and to a total lack of mtDNA (Rho0 cells-heavy stress). We previously found that selenium influenced mitochondrial stress response and lowered ROS production. Therefore, in this study effect of selenite on other mitochondrial parameters was investigated. We showed that presence of selenium improved survival of starved cells, modified organization of mitochondrial network in NARP cybrids and decreased cytosolic calcium level in NARP and Rho0 cells. Selenium did not affect mitochondrial membrane potential, ATP level, activity of ATP synthase and activity of complex II of the respiratory chain.     

A Lysosomal Proteinase, the Late Infantile Neuronal Ceroid Lipofuscinosis Gene (CLN2) Product, Is Essential for Degradation of a Hydrophobic Protein, the Subunit c of ATP Synthase

The specific accumulation of the hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa mature protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high-density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.     

Significance of the epsilon subunit in the thiol modulation of chloroplast ATP synthase

To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity.     

Topological and functional study of subunit h of the F1Fo ATP synthase complex in yeast Saccharomyces cerevisiae

Subunit h, a 92-residue-long, hydrophilic, acidic protein, is a component of the yeast mitochondrial F1Fo ATP synthase. This subunit, homologous to the mammalian factor F6, is essential for the correct assembly and/or functioning of this enzyme since yeast cells lacking it are not able to grow on nonfermentable carbon sources. Chemical cross-links between subunit h and subunit 4 have previously been shown, suggesting that subunit h is a component of the peripheral stalk of the F1Fo ATP synthase. The construction of cysteine-containing subunit h mutants and the use of bismaleimide reagents provided insights into its environment. Cross-links were obtained between subunit h and subunits alpha, f, d, and 4. These results and secondary structure predictions allowed us to build a structural model and to propose that this subunit occupies a central place in the peripheral stalk between the F1 sector and the membrane. In addition, subunit h was found to have a stoichiometry of one in the F1Fo ATP synthase complex and to be in close proximity to another subunit h belonging to another F1Fo ATP synthase in the inner mitochondrial membrane. Finally, functional characterization of mitochondria from mutants expressing different C-terminal shortened subunit h suggested that its C-terminal part is not essential for the assembly of a functional F1Fo ATP synthase.