Specific Delay in the Degradation of Mitochondrial ATP Synthase Subunit c in Late Infantile Neuronal Ceroid Lipofuscinosis Is Derived from Cellular Proteolytic Dysfunction Rather than Structural Alteration of Subunit c

Abstract: Previously we indicated that a specific delay in subunit c degradation causes the accumulation of mitochondrial ATP synthase subunit c in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (NCL). To explore the mechanism of lysosomal storage of subunit c in patient cells, we investigated the mechanism of the lysosomal accumulation of subunit c both in cultured normal fibroblasts and in in vitro cell-free incubation experiments. Addition of pepstatin to normal fibroblasts causes the marked lysosomal accumulation of subunit c and less accumulation of Mn²⁺-superoxide dismutase (SOD). In contrast, E-64-d stimulates greater lysosomal storage of Mn²⁺-SOD than of subunit c. Incubation of mitochondrial-lysosomal fractions from control and diseased cells at acidic pH leads to a much more rapid degradation of subunit c in control cells than in diseased cells, whereas other mitochondrial proteins, including Mn²⁺-SOD, β subunit of ATP synthase, and subunit IV of cytochrome oxidase, are degraded at similar rates in both control and patient cells. The proteolysis of subunit c in normal cell extracts is inhibited markedly by pepstatin and weakly by E-64-c, as in the cultured cell experiments. However, there are no differences in the lysosomal protease levels, including the levels of the pepstatin-sensitive aspartic protease cathepsin D between control and patient cells. The stable subunit c in mitochondrial-lysosomal fractions from patient cells is degraded on incubation with mitochondrial-lysosomal fractions from control cells. Exchange experiments using radiolabeled substrates and nonlabeled proteolytic sources from control and patient cells showed that proteolytic dysfunction, rather than structural alterations such as the posttranslational modification of subunit c, is responsible for the specific delay in the degradation of subunit c in the late infantile form of NCL.     

New insight into lysosomal protein storage disease: Delayed catabolism of ATP synthase subunit c in batten disease

Subunit c is normally present as an inner mitochondrial membrane component of the Fo sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. Mechanism for specific accumulation of subunit c in lysosomes is not known. The rate of degradation of subunit c as measured by pulsechase and immunoprecipitation showed a marked delay of degradation in patients fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes, through an autophagic process. There were no large differences of various lysosomal protease activities between control and patient cells. In patient cells sucrose loading caused a marked shift of lysosomal density, but did not a shift of subunit c containing storage body. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes.Special issue dedicated to Dr. Leon S. Wolfe.     

Tripeptidyl peptidase I, the late infantile neuronal ceroid lipofuscinosis gene product, initiates the lysosomal degradation of subunit c of ATP synthase

The specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN2 gene product, tripeptidyl peptidase I (TPP-I). The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c. The inclusion of a specific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c. In an in vitro incubation experiment the addition of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteolysis of subunit c, but not the b-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation of subunit c was markedly delayed through the specific inhibition of TPP-I in lysosomal extracts by AAF-CMK. The stable subunit c in the mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.     

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.     

Batten disease (ceroid-lipofuscinosis): The enigma of subunit c of mitochondrial ATP synthase accumulation

Ceroid-lipofuscinosis is an inherited neurodegenerative disease of human beings and domestic animals characterized by the accumulation in neurons and other cells of a fluorescent lipopigment. In the ovine form of disease, subunit c of mitochondrial ATP synthase is the dominant accumulated metabolite (>50%). It also accumulates significantly in the late infantile and juvenile forms of the human disease and several other animal forms. Evidence is accumulating that the underlying biochemical defect may be associated with mitochondria. The extreme hydrophobicity of subunit c and its propensity to aggregate with lipids into regular multilamellar arrays that cannot be catabolised may reflect an initial defect not necessarily associated with faulty proteolysis. This hypothesis extends an earlier one that subunit c accumulated due to a defect in its catabolic pathway.Special issues dedicated to Dr. Leon S. Wolfe.     

Batten disease fibroblasts in culture accumulate mitochondrial ATP synthase subunit 9.

Batten Disease is a lysosomal storage disease in which the major component that accumulates is subunit 9 of mitochondrial ATP synthase. Whether or not fibroblasts in culture exhibit this phenotype is controversial. We show that fibroblasts from a human Batten Disease patient and from a mouse model of this disease exhibit autofluorescent inclusion bodies. We also demonstrate that levels of ATP synthase subunit 9 are elevated in these diseased fibroblasts when compared to control cells. However, the exact growth state of the human fibroblasts was critical, and this factor probably accounts for discrepencies in the literature.     

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.     

Specific Delay of Degradation of Mitochondrial ATP Synthase Subunit c in Late Infantile Neuronal Ceroid Lipofuscinosis (Batten Disease)

Abstract: Subunit c is normally present as an inner mitochondrial membrane component of the F₀ section of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. To explore the mechanism of storage of subunit c, the rates of degradation and synthesis of subunit c were measured in fibroblast cell types from controls and patients with the late infantile form of NCL. The radiolabel from subunit c decreased with time in control cells, whereas no apparent loss of radioactivity of subunit c was found in patients' cells. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. A combination of pulse-chase and subcellular fractionation analysis showed that a delay of intramitochondrial loss from prelabeled subunit c was seen in all diseased cells tested. Lysosomal appearance of labeled subunit c could be detected after chase for more than 1 week and its radioactivities were variable among diseased cell types. The biosynthetic rate of subunit c was almost the same in both control and patient cells. Northern blotting analyses showed that mRNAs for P1 and P2 genes had no significant difference in lengths and amounts between control and patient cells. Results suggest a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. This is the first direct evidence to show a delay of subunit c degradation in the cells from the late infantile form of NCL.     

Preparative electrophoretic method for the purification of a hydrophobic membrane protein: subunit c of the mitochondrial ATP synthase from rat liver.

A method is described for the purification of subunit c of ATP synthase from rat liver mitochondria. After sample preparation and solvent extraction, the protein was purified to homogeneity by a single-step preparative electrophoretic procedure, using aqueous buffer and containing lithium dodecyl sulfate. The subunit is an extremely hydrophobic and insoluble protein and all solubilization attempts, using a variety of detergents, were unsuccessful except for lithium dodecyl sulfate. Buffer exchange and FPLC gel filtration removed the detergent from the purified sample, leaving the protein in a soluble form. The mammalian protein is composed of 75 amino acid residues, with a molecular mass of 7602 Da and is classified as a proteolipid. Subunit c accounts for 25 and 85% of the intralysosomal accumulation, within neurons, of storage material in juvenile and late-infantile forms of Batten's disease, respectively. This purification procedure allows access to a continuous supply of pure subunit c from a conventional source such as rat liver and preserves precious autopsy materials. The protein could be used as substrate in future proteolytic studies involving pepstatin-insensitive lysosomal proteases and for raising of more specific antibodies. The procedure could also be adapted/modified and used as a model for purifying other extremely insoluble proteins.     

Each yeast mitochondrial F1F0-ATP synthase complex contains a single copy of subunit 8

The stoichiometry of subunit 8 in yeast mitochondrial F(1)F(0)-ATP synthase (mtATPase) has been evaluated using an immunoprecipitation approach. Single HA or FLAG epitopes were introduced at the N-terminus of subunit 8. Expression of each tagged subunit 8 variant in yeast cells lacking endogenous subunit 8 restored a respiratory phenotype and had little measurable effect on ATP hydrolase activity of the isolated enzyme. Moreover, the two epitope-tagged subunit 8 variants could be stably co-expressed in the same host cells and both of HA-Y8 and FLAG-Y8 could be detected in ATP synthase complexes isolated by native gel electrophoresis. Mitochondria isolated from each yeast strain were solubilized to release ATP synthase complexes in either the monomeric or dimeric forms. In each case, monoclonal antibodies directed against either the FLAG or HA epitope could immunoprecipitate intact ATP synthase complexes. When both HA-Y8 and FLAG-Y8 were co-expressed in cells, monomeric ATP synthases contained only a single subunit 8 variant after immunoprecipitation, corresponding to the particular antibody used (HA or FLAG). By contrast, both subunit 8 variants were recovered in samples of immunoprecipitated dimeric ATP synthase complexes, irrespective of the antibody used. We conclude that each monomeric yeast mitochondrial ATP synthase complex contains a single copy of subunit 8.     

Apoptosis as the Mechanism of Neurodegeneration in Batten's Disease

Abstract: Batten's disease is a genetic neurodegenerative disease of childhood. Its hallmarks are retinitis pigmentosa and neuronal degeneration. As some types of photoreceptor death in mice are mediated by apoptosis, we investigated whether apoptosis is responsible for retinal and neuronal degeneration in the late infantile and juvenile forms of Batten's disease. Using the terminal dUDP nick end-labeling (TUNEL) staining method, we detected apoptotic neuronal cells in brain from patients and a canine model and in brain and retina from an ovine model for Batten's disease. We confirmed apoptosis by flow cytometry, electron microscopy, and DNA laddering. This is the first inherited neurodegenerative disease involving brain and retina in which apoptosis has been established as the mechanism of neuronal and photoreceptor cell death in both humans and animal models.     

Cytosolic subunits of ATP synthase are localized to the cortical endoplasmic reticulum‐rich domain of the ascidian egg myoplasm

Previously, we revealed that p58, one of the ascidian maternal factors, is identical to the alpha‐subunit of F1‐ATP synthase (ATPα), a protein complex of the inner mitochondrial membrane. In the current study, we used immunological probes for ascidian mitochondria components to show that the ascidian ATPα is ectopically localized to the cytosol. Virtually all mitochondrial components were localized to the mitochondria‐rich myoplasm. However, in detail, ATP synthase subunits and the matrix proteins showed different localization patterns. At least at the crescent stage, transmission electron microscopy (TEM) distinguished the mitochondria‐less, endoplasmic reticulum (ER)‐rich cortical region and the mitochondria‐rich internal region. ATPα was enriched in the cortical region and MnSOD was limited to the internal region. Using subcellular fractionation, although all of the mitochondria components were highly enriched in the mitochondria‐enriched fraction, a considerable amount of ATPα and F1‐ATP synthase beta‐subunit (ATPβ) were recovered in the insoluble cytoplasmic fraction. Even under these conditions, F1‐ATP synthase gamma‐subunit (ATPγ) and F0‐ATP synthase subunit b (ATPb) were not recovered in the insoluble cytoplasmic fraction. This result strongly supports the exomitochondrial localization of both ATPα and ATPβ. In addition, the detergent extraction of eggs supports the idea that these cytosolic ATP synthase subunits are associated with the egg cytoskeleton. These results suggest that the subunits of ATP synthase might play dual roles at different subcellular compartments during early development.     

Expression and processing of the TMEM70 protein

TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We have investigated import and processing of this factor in human cells using GFP- and FLAG-tagged forms of TMEM70 and specific antibodies. TMEM70 is synthesized as a 29kDa precursor protein that is processed to a 21kDa mature form. Immunocytochemical detection of TMEM70 showed mitochondrial colocalization with MitoTracker Red and ATP synthase. Western blot of subcellular fractions revealed the highest signal of TMEM70 in isolated mitochondria and mitochondrial location was confirmed by mass spectrometry analysis. Based on analysis of submitochondrial fractions, TMEM70 appears to be located in the inner mitochondrial membrane, in accordance with predicated transmembrane regions in the central part of the TMEM70 sequence. Two-dimensional electrophoretic analysis did not show direct interaction of TMEM70 with assembled ATP synthase but indicated the presence of dimeric form of TMEM70. No TMEM70 protein could be found in cells and isolated mitochondria from patients with ATP synthase deficiency due to TMEM70 c.317-2A>G mutation thus confirming that TMEM70 biosynthesis is prevented in these patients.     

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.     

N-acetylneuraminic acid accumulation in a buoyant lysosomal fraction of cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease

Cultured fibroblasts from control individuals and two patients affected with the infantile variant of generalized N-acetylneuraminic acid (NeuAc) storage disease were disrupted by nitrogen cavitation, and the post-nuclear supernatant fractions were subjected to subcellular fractionation on Percoll gradients. Accumulating NeuAc in affected fibroblasts (approx. 150 nmol/mg protein) co-localized with the lysosomal marker N-acetyl-beta-hexosaminidase (Hex), in a fraction with a mean density of 1.035 g/ml. In contrast, more than 70% of the Hex activity of control cells sedimented in comparable gradients with a density of more than 1.07 g/ml. The lysosomal localization of NeuAc accumulation in affected fibroblasts was confirmed by treatment of post-nuclear supernatant fractions with 0.5 mM Gly-Phe-beta-naphthylamide (20 min, 37 degrees C) prior to centrifugation, which resulted in the simultaneous loss of latency of Hex and free NeuAc, and their association with the soluble fraction on Percoll gradients. The results provide direct evidence for the accumulation of free NeuAc in a unique buoyant lysosomal fraction of affected fibroblasts.     

Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration

ClC-7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H(+)-ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC-7 or the H(+)-ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC-7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC-7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron-dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC-3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H(+)-ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H(+)-ATPase and ClC-7 can underlie human osteopetrosis.     

Altered Mitochondrial Function in Canine Ceroid-Lipofuscinosis

The neuronal ceroid-lipofuscinoses (NCL) are a group of autosomal recessively inherited neurodegenerative disorders characterized by progressive dementia, neuronal atrophy, and premature death. The late infantile and juvenile types of NCL show massive accumulation of mitochondrial ATP synthase subunit c protein in both mitochondria and lysosomes. The specific accumulation of this mitochondrial protein suggests that mitochondrial function may be impaired in the NCL diseases. Therefore, a study was conducted to determine whether oxidative phosphorylation is altered in liver mitochondria from English setters with NCL, an animal model in which there is also massive accumulation of the subunit c protein. The ADP/O ratios were significantly depressed in affected and carrier dogs, suggesting that the disease mutation led to a partial uncoupling of oxidative phosphorylation. On the other hand, ADP-stimulated respiration rates were higher than normal in both carriers and affected dogs. The increased respiration rates were highest in the carriers, and may reflect a compensatory response to the reduced efficiency of oxidative phosphorylation. Accompanying the increased respiration rates were elevations in mitochondrial ADP content with the elevation being greater in the carriers than in the affected dogs. This suggests that the increased respiration rates may be due, at least in part, to enhanced ADP uptake by the mitochondria. In the carriers, the enhanced respiration rate may be sufficient to offset the reduced efficiency of oxidative phosphorylation. In the affected animals, which had lower respiration rates than the carriers, the enhanced respiration rates may not be sufficient to offset the reduced efficiency of oxidative phosphorylation. Impaired mitochondrial function may therefore contribute to the disease pathology.     

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.     

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.