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Anomalies of Mitochondrial ATP Synthase Regulation in Four Different Types of Neuronal Ceroid Lipofuscinosis
Molecular Genetics and Metabolism 66, 349 –355 (1999) Article ID mgme.1999.2811, available online at http://www.idealibrary.com on
Anomalies of Mitochondrial ATP Synthase Regulation in Four Different Types of Neuronal Ceroid Lipofuscinosis A. M. Das, R. D. Jolly,* and A. Kohlschu¨tter Department of Paediatrics, University of Hamburg, D-20246 Hamburg, Germany; and *Faculty of Veterinary Science, Massey University, Palmerston North, New Zealand Received January 5, 1999, and in revised form January 15, 1999
Key Words: ATP synthase; mitochondrial energy metabolism; neurodegeneration; fibroblast culture.
Several neuronal ceroid lipofuscinoses (NCL) show storage of subunit c of mitochondrial ATP synthase. The neurodegenerative process, however, remains obscure. We previously reported a decreased basal ATP synthase activity in fibroblasts from late-infantile NCL (CLN2) and juvenile NCL (CLN3) patients. We have now extended the study of the ATP synthase system to an ovine NCL (a model for the late-infantile NCL variant, CLN6) and the infantile NCL (CLN1). In fibroblasts from healthy sheep, active regulation of ATP synthase in response to cellular energy demand was present similar to human cells: ATP synthase was down-regulated under conditions of anoxia or functional uncoupling and was up-regulated in response to calcium. In fibroblasts from NCL sheep, basal ATP synthase activity was slightly elevated and downregulation in response to anoxia or uncoupling of mitochondria also occurred. Calcium produced an unexpected down-regulation to 55% of basal activity. Activities of respiratory chain enzymes did not differ between healthy and NCL sheep. In fibroblasts from CLN1 patients, basal ATP synthase activity was reduced and regulation of the enzyme was absent. Activities of respiratory chain complexes II and IV were reduced. The defect of ATP synthase regulation found in fibroblasts from NCL sheep and infantile NCL patients is different from the ATP synthase deficiencies demonstrated in lateinfantile and juvenile NCL, but problems of mitochondrial energy production, if also expressed in brain, would be a common feature of several NCL forms. Deficient ATP supply could result in degeneration of neurons, especially in those with high energy requirements. © 1999 Academic Press
The neuronal ceroid lipofuscinoses (NCL) 1 are a genetically heterogeneous group of clinically related disorders which have in common a degeneration of neuronal structures leading to blindness, dementia, and epilepsy. They share the accumulation of characteristic lipopigments which are the traditional basis of diagnosis and classification (1). The pathogenesis of neuronal dysfunction and cell death in these disorders, however, remains unexplained. Some observations suggest mitochondrial involvement in the pathogenesis of NCL disorders: Structurally altered mitochondria have been described in canine and murine NCL models (2,3). Furthermore, loss of neurons in NCL has been reported to be most pronounced in pyramidal cell layers III, IV, and V (4,5). The affected layer IV shows high metabolic activity, mostly maintained by mitochondrial energy production (6). Thus, in NCL, brain areas depending particularly on mitochondrial metabolism seem to be most vulnerable to damage. Positron emission tomography studies revealed reduction of glucose oxidation in gray matter of NCL patients (7). The intralysosomal storage material in an ovine form of NCL has been shown to be composed of at least 50% subunit c of mitochondrial ATP synthase (complex V, EC 3.6.1.34) (8,9). Subsequently, sub1 Abbreviations used: NCL, neuronal ceroid lipofuscinosis; CLN1, infantile NCL; CLN2, late-infantile NCL; CLN3, juvenile NCL; CLN6, late-infantile NCL variant.
349 1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
350
¨ TTER DAS, JOLLY, AND KOHLSCHU
unit c was also found as a major component of the intralysosomal storage material in the human late-infantile and juvenile forms of NCL (10,11). Whether these findings have any bearing on the function of mitochondrial ATP synthase in the NCLs is unknown. In a previous study, we have therefore looked at the ATP synthase activity and its regulation in skin fibroblasts from patients with LINCL and JNCL and have found a decreased basal activity of the mitochondrial ATP synthase in both disorders (12). ATP synthase, as one of the key enzymes in mitochondrial energy production, is responsible for the bulk of ATP synthesis in aerobic tissues such as the brain. To explore the hypothesis that defective mitochondrial energy production is part of the general pathogenesis of various NCL forms, we now examined the activity and regulation of mitochondrial ATP synthase in skin fibroblasts from Southhampshire sheep affected with NCL. This disease maps to ovine chromosome 7q13-15, which is syntenic to human chromosome 15q21-23, the region which was recently defined as the locus of the gene mutated in the late-infantile variant disease CLN6 (13). If abnormalities of mitochondrial function play a role in the pathogenesis of NCL, mitochondrial enzyme activity should be also defective in NCL types without intralysosomal storage of subunit c, such as infantile NCL. We therefore examined respiratory chain complexes in fibroblasts from children with infantile NCL. MATERIALS AND METHODS Patients, animals and tissue culture. Cultured skin fibroblasts were studied from five children with infantile NCL (kindly supplied by Dr. J. Tyynela¨, Helsinki, Finland), from five healthy children, from five Southhampshire sheep affected with NCL, and from four normal sheep. This ovine NCL is a model for the human late-infantile NCL variant (13) and is described more closely elsewhere (14). Enzyme analysis. Human and sheep fibroblasts were cultured on petri dishes. Cells were washed twice with a Hepes buffer and then incubated for 15 min in this buffer with 10 mM glucose as substrate as described for human cells in detail previously (15). Fibroblasts and their mitochondria were then broken by sonication. A special low-ionic strength buffer was used to “freeze” the in vivo binding state of the naturally occurring inhibitor protein IF 1 (16) which is responsible for down-regulation of ATP syn-
thase (15). ATP synthase activity was assayed in the direction of ATP hydrolysis as oligomycin-sensitive ATPase (15). The ATP synthase reaction has previously been shown to be fully reversible (17). Thus, under substrate (ATP) saturation and absence of membrane potential across the inner mitochondrial membrane, measuring ATP hydrolysis is a convenient method of monitoring ATP synthase activity. Active regulation of ATP synthase in response to cellular energy demand was examined by adding different substances to the Hepes buffer used for incubation of cells (see above). A quantity of 1 mM sodium cyanide was added to inhibit cytochrome c oxidase, thereby mimicking anoxia, 2 mM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) was used to functionally uncouple the mitochondria, and 4 mM calcium was added to stimulate ATP synthase. Rotenone-sensitive NADH cytochrome c reductase (complex I 1 III, EC 1.6.5.3 1 EC 1.10.2.2) was measured using a slight modification of the method described for skeletal muscle mitochondria (18). Antimycin-sensitive succinate cytochrome c reductase activity (complex II 1 III, EC 1.3.5.1 1 EC 1.10.2.2) was measured spectrophotometrically (19). Cytochrome c oxidase activity (complex IV, EC 1.9.3.1) was measured in cell sonicates at 37°C as described elsewhere (20). Protein was determined according to Bensadoun and Weinstein (21). Materials. Falcon 3001 petri dishes were from Becton Dickinson (Heidelberg, Germany); all other tissue culture material, including fetal calf serum, was from Gibco (Eggenstein, Germany). ATP, lactic dehydrogenase, pyruvate kinase, phosphoenolpyruvate, and NADH for enzyme assays were purchased from Boehringer (Mannheim, Germany). Oligomycin, antimycin, rotenone, cytochrome c, FCCP, Hepes, and all other chemicals of the highest purity available were from Sigma (Deisenhofen, Germany). Statistical analysis. Data for ATP synthase activities in cells from healthy and diseased children and sheep under basal conditions and the other metabolic conditions and activities of respiratory chain enzymes were compared using Student’s t test. RESULTS On phase-contrast microscopy, there was no difference between the cells from healthy and diseased animals and children.
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ATP SYNTHASE IN NEURONAL CEROID LIPOFUSCINOSIS
FIG. 1. Regulation of mitochondrial ATP synthase in response to cellular energy demand in fibroblasts from four healthy sheep. Mean 1 SD; n:, number of petri dishes (four from each animal) incubated under the different metabolic conditions.
Several methods to break open mitochondria of sheep fibroblasts were tested. As in fibroblasts from humans (15) sonication for 2 3 10 s led to complete exposure of intramitochondrial enzymes in sheep. Breakage of mitochondria was judged by the addition of FCCP to the sonicate during the measurement of ATP synthase activity. Variation of duplicates from the same sonicate was 5%; variation between sonicates from petri dishes with cells from the same cell line incubated under the same conditions was typically less than 10% both for human and sheep cells. ATP synthase activity and its regulation in fibroblasts from healthy sheep are shown in Fig. 1. Basal activity was 110 nmol/min/mg protein. When cells were incubated in the presence of cyanide (mimicking anoxia) or functionally uncoupled by FCCP, ATP synthase activity was reduced to 76 and 79% of basal activity, respectively. Addition of 4 mM calcium to
the incubation medium resulted in activation of the ATP synthase to 145% of basal activity. This did not simply reflect the calcium optimum of the enzyme as addition of calcium to the sonicate in the measuring cuvette did not alter ATP synthase activity. The activities of respiratory chain complexes I 1 III, II 1 III, and IV (cytochrome c oxidase) are summarized in Table 1. ATP synthase regulation in response to cellular energy demand in fibroblasts from sheep affected with NCL is shown in Fig. 2. Basal activity in these cells was 185 nmol/min/mg protein. ATP synthase activity in response to cyanide and FCCP was reduced to 78 and 53% of basal activity, respectively. Down-regulation in response to uncoupling of mitochondria was significantly different (more pronounced) compared to cells from healthy animals. Addition of 4 mM calcium to the incubation medium resulted in down-regulation of ATP synthase to 55% of basal activity. As shown in Table 1, activities for the respiratory chain complexes I 1 III, II 1 III, and IV did not differ significantly from the values in cells from healthy animals. Active regulation of mitochondrial ATP synthase in response to cellular energy demand in fibroblasts from healthy children is shown in Fig. 3. Basal ATP synthase activity was 181 nmol/min/mg protein. Down-regulation occurred in the presence of sodium cyanide (mimicking anoxia) or FCCP (mitochondrial uncoupling). Up-regulation could be observed after preincubation of the cells with 4 mM calcium. Basal ATP synthase activity in fibroblasts from children with infantile NCL was 84 nmol/min/mg protein. Regulation of the enzyme was absent in cells from TABLE 1 Activities of Respiratory Chain Complexes in Fibroblasts from Four Healthy Sheep, Five Sheep Affected with NCL, Five Healthy Children, and Five Children with CLN1 Sheep Respiratory chain enzymes Complexes I 1 III Complexes II 1 III Complex IV (Cytochrome c oxidase)
Human
Healthy
NCL
Healthy
CLN1
58 6 21 46 1
57 6 23 66 3
105 6 40 11 6 4
151 6 72 2 6 2*
56 6 24
69 6 14
69 6 24
31 6 9*
Note. Two petri dishes were measured from each individual. Results are nmol/min/mg protein, mean 6 SD. * P , 0.05 vs healthy children.
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In fibroblasts from healthy sheep, we showed that active regulation of ATP synthase in response to cellular metabolic conditions was present. ATP synthase activity was down-regulated when the cells were incubated in the presence of cyanide (mimicking anoxia) or when the mitochondria were functionally uncoupled. Under these conditions, ATP synthase would work in reverse, thus hydrolyzing ATP. Down-regulation of ATP synthase, therefore, helps to conserve cellular ATP which is necessary to maintain structural and functional integrity of the cell. Up-regulation of ATP synthase could be demonstrated in the presence of 4 mM calcium in the incubation medium. Calcium is known to be an important second messenger and has previously been shown to be increased in situations of high cellular energy demand (e.g., beating of cardiomyocytes, action of thyroid hormones and adrenergic substances) (22). Similar active regulation of ATP synthase in response to cellular metabolic conditions as de-
FIG. 2. Regulation of mitochondrial ATP synthase in response to cellular energy demand in fibroblasts from five sheep affected with NCL. Mean 1 SD; n:, number of petri dishes incubated under the different metabolic conditions.
patients with infantile NCL (Fig. 4). Activities of respiratory chain complexes from patients with the infantile form of NCL were assayed (Table 1). While activity of complex I 1 III was found to be normal, activities of complex II 1 III and complex IV were significantly reduced. DISCUSSION ATP synthase is one of the key enzymes responsible for aerobic energy production and is subject to regulatory effects which depend on specific cellular metabolic conditions such as hypoxia, metabolic uncoupling of mitochondria, or calcium concentration. This study demonstrates that one of the characteristic regulatory features of ATP synthase, its upregulation by calcium, is lost in fibroblasts from sheep with NCL and children with infantile NCL.
FIG. 3. Regulation of mitochondrial ATP synthase in response to cellular energy demand in fibroblasts from five healthy children. Mean 1 SD; n:, number of petri dishes incubated under the different metabolic conditions.
ATP SYNTHASE IN NEURONAL CEROID LIPOFUSCINOSIS
FIG. 4. Regulation of mitochondrial ATP synthase in response to cellular energy demand in fibroblasts from five children affected with CLN 1. Mean 1 SD; n:, number of petri dishes incubated under the different metabolic conditions (four petri dishes from each cell line).
scribed here for healthy sheep fibroblasts has been reported for cultured cardiomyocytes from rat (23), canine myocardium (24), human skin fibroblasts (15), and human skeletal muscle (25). Activity of antimycin-sensitive succinate cytochrome c reductase as a mitochondrial marker enzyme remained unchanged under the different incubation conditions showing constant mitochondrial recovery (results not shown). Activities of respiratory chain complexes I, II, III, and IV did not differ significantly between healthy and diseased cells which shows that mitochondrial content and recovery were the same in both types of cells. Effects of an allosteric regulator mediating upand down-regulation of ATP synthase can be ruled out as regulated states were stable to dilution of mitochondria into large volumes of incubation and assay media. The most likely candidate for downregulation of ATP synthase is the naturally occur-
353
ring inhibitor protein IF 1. This protein has been shown to bind to ATP synthase when the membrane potential across the inner mitochondrial membrane is reduced (26,27) as in anoxia or when mitochondria are uncoupled. A calcium binding inhibitor (CaBi) has been described (28) which dissociates from ATP synthase at increased calcium levels. This regulatory protein could be responsible for the upregulation of ATP synthase seen in response to increased calcium levels in cells from healthy sheep and humans. In fibroblasts from NCL sheep, basal ATP synthase activity (i.e., under control conditions) was slightly elevated compared to cells from control animals. When fibroblasts from NCL animals were incubated in the presence of calcium, down-regulation of ATP synthase instead of up-regulation occurred. Such an abnormal regulatory response could lead to cellular ATP deficiency due to insufficient ATP production in situations with high energy requirements. Elevation of basal ATP synthase activity may be a compensatory mechanism to overcome this difficulty. Disturbances in the CaBi or IF 1 protein or a defect in the ATP synthase molecule itself may be responsible for the dysregulation. Defects in the activities of the other respiratory chain complexes could lead to cellular lack of energy as well. However, no significant differences in the activities of complexes I, II, III, and IV could be detected when healthy and diseased animals were compared. Basal activity of ATP synthase was reduced in fibroblasts from children with infantile NCL. In these cells no regulation of ATP synthase was observed. Thus, the capacity of these cells to produce energy is compromised, though no intralysosomal storage of subunit c of ATP synthase occurs in this form of NCL. Activities of complex II and complex IV were reduced compared to cells from healthy children. This may be due to intramitochondrial energy depletion caused by the reduced capacity of the mitochondrial ATP synthase. The defective regulation of ATP synthase described here in an ovine model of the human late infantile NCL variant (CLN6) (13) and in human infantile NCL (CLN1) is different from the deficiencies of basal ATP synthase activity demonstrated previously in late-infantile NCL (CLN2) and juvenile NCL (CLN3) (12). All ATP synthase abnormalities described in the four forms of NCL, however, can interfere with mitochondrial energy production and, if also expressed in brain, could play a crucial role in the pathogenesis of the four different NCL
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forms. In brain tissue from three of these NCL forms, intralysosomal storage of subunit c of mitochondrial ATP synthase has been reported (10,11) but is absent in infantile NCL. The neurodegenerative process does not seem to be directly related to the storage process. Deficient ATP supply could result in dysfunction and eventual death of neuronal cells, especially in those with high energy requirements. Those neuronal cells that are predominantly affected by the neurodegenerative process in several NCL disorders seem to have a particularly high energy demand (see introduction). Excitotoxicity has also been discussed in the pathogenesis of neurodegenerative diseases (29). Inhibitory GABAergic neurons rich in mitochondria are especially susceptible to compromised energy production. Loss of inhibition may lead to excitotoxicity in other brain cells with compromised energy production (3). Thus, compromised energy production may be a key to understanding the neurodegenerative process in several forms of NCL. The mechanism of dysfunction of the mitochondrial ATP synthase system in NCL cells is unknown. The dysregulation may be caused by a primary extramitochondrial defect leading to a secondary disturbance of mitochondrial energy metabolism. Alternatively, the primary defect could affect the mitochondrial ATP synthase more directly. ACKNOWLEDGMENTS We are indebted to Mrs. I. Wernicke for excellent technical assistance. This work was supported by the United States Institute of Neurological Disorders and Stroke (Grant NS 32348) and by “Freunde der Universita¨tskinderklinik Hamburg.”
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Anomalies of Mitochondrial ATP Synthase Regulation in Four Different Types of Neuronal Ceroid Lipofuscinosis A. M. Das, R. D. Jolly,* and A. Kohlschu¨tter Department of Paediatrics, University of Hamburg, D-20246 Hamburg, Germany; and *Faculty of Veterinary Science, Massey University, Palmerston North, New Zealand Received January 5, 1999, and in revised form January 15, 1999
Key Words: ATP synthase; mitochondrial energy metabolism; neurodegeneration; fibroblast culture.
Several neuronal ceroid lipofuscinoses (NCL) show storage of subunit c of mitochondrial ATP synthase. The neurodegenerative process, however, remains obscure. We previously reported a decreased basal ATP synthase activity in fibroblasts from late-infantile NCL (CLN2) and juvenile NCL (CLN3) patients. We have now extended the study of the ATP synthase system to an ovine NCL (a model for the late-infantile NCL variant, CLN6) and the infantile NCL (CLN1). In fibroblasts from healthy sheep, active regulation of ATP synthase in response to cellular energy demand was present similar to human cells: ATP synthase was down-regulated under conditions of anoxia or functional uncoupling and was up-regulated in response to calcium. In fibroblasts from NCL sheep, basal ATP synthase activity was slightly elevated and downregulation in response to anoxia or uncoupling of mitochondria also occurred. Calcium produced an unexpected down-regulation to 55% of basal activity. Activities of respiratory chain enzymes did not differ between healthy and NCL sheep. In fibroblasts from CLN1 patients, basal ATP synthase activity was reduced and regulation of the enzyme was absent. Activities of respiratory chain complexes II and IV were reduced. The defect of ATP synthase regulation found in fibroblasts from NCL sheep and infantile NCL patients is different from the ATP synthase deficiencies demonstrated in lateinfantile and juvenile NCL, but problems of mitochondrial energy production, if also expressed in brain, would be a common feature of several NCL forms. Deficient ATP supply could result in degeneration of neurons, especially in those with high energy requirements. © 1999 Academic Press
The neuronal ceroid lipofuscinoses (NCL) 1 are a genetically heterogeneous group of clinically related disorders which have in common a degeneration of neuronal structures leading to blindness, dementia, and epilepsy. They share the accumulation of characteristic lipopigments which are the traditional basis of diagnosis and classification (1). The pathogenesis of neuronal dysfunction and cell death in these disorders, however, remains unexplained. Some observations suggest mitochondrial involvement in the pathogenesis of NCL disorders: Structurally altered mitochondria have been described in canine and murine NCL models (2,3). Furthermore, loss of neurons in NCL has been reported to be most pronounced in pyramidal cell layers III, IV, and V (4,5). The affected layer IV shows high metabolic activity, mostly maintained by mitochondrial energy production (6). Thus, in NCL, brain areas depending particularly on mitochondrial metabolism seem to be most vulnerable to damage. Positron emission tomography studies revealed reduction of glucose oxidation in gray matter of NCL patients (7). The intralysosomal storage material in an ovine form of NCL has been shown to be composed of at least 50% subunit c of mitochondrial ATP synthase (complex V, EC 3.6.1.34) (8,9). Subsequently, sub1 Abbreviations used: NCL, neuronal ceroid lipofuscinosis; CLN1, infantile NCL; CLN2, late-infantile NCL; CLN3, juvenile NCL; CLN6, late-infantile NCL variant.
349 1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
350
¨ TTER DAS, JOLLY, AND KOHLSCHU
unit c was also found as a major component of the intralysosomal storage material in the human late-infantile and juvenile forms of NCL (10,11). Whether these findings have any bearing on the function of mitochondrial ATP synthase in the NCLs is unknown. In a previous study, we have therefore looked at the ATP synthase activity and its regulation in skin fibroblasts from patients with LINCL and JNCL and have found a decreased basal activity of the mitochondrial ATP synthase in both disorders (12). ATP synthase, as one of the key enzymes in mitochondrial energy production, is responsible for the bulk of ATP synthesis in aerobic tissues such as the brain. To explore the hypothesis that defective mitochondrial energy production is part of the general pathogenesis of various NCL forms, we now examined the activity and regulation of mitochondrial ATP synthase in skin fibroblasts from Southhampshire sheep affected with NCL. This disease maps to ovine chromosome 7q13-15, which is syntenic to human chromosome 15q21-23, the region which was recently defined as the locus of the gene mutated in the late-infantile variant disease CLN6 (13). If abnormalities of mitochondrial function play a role in the pathogenesis of NCL, mitochondrial enzyme activity should be also defective in NCL types without intralysosomal storage of subunit c, such as infantile NCL. We therefore examined respiratory chain complexes in fibroblasts from children with infantile NCL. MATERIALS AND METHODS Patients, animals and tissue culture. Cultured skin fibroblasts were studied from five children with infantile NCL (kindly supplied by Dr. J. Tyynela¨, Helsinki, Finland), from five healthy children, from five Southhampshire sheep affected with NCL, and from four normal sheep. This ovine NCL is a model for the human late-infantile NCL variant (13) and is described more closely elsewhere (14). Enzyme analysis. Human and sheep fibroblasts were cultured on petri dishes. Cells were washed twice with a Hepes buffer and then incubated for 15 min in this buffer with 10 mM glucose as substrate as described for human cells in detail previously (15). Fibroblasts and their mitochondria were then broken by sonication. A special low-ionic strength buffer was used to “freeze” the in vivo binding state of the naturally occurring inhibitor protein IF 1 (16) which is responsible for down-regulation of ATP syn-
thase (15). ATP synthase activity was assayed in the direction of ATP hydrolysis as oligomycin-sensitive ATPase (15). The ATP synthase reaction has previously been shown to be fully reversible (17). Thus, under substrate (ATP) saturation and absence of membrane potential across the inner mitochondrial membrane, measuring ATP hydrolysis is a convenient method of monitoring ATP synthase activity. Active regulation of ATP synthase in response to cellular energy demand was examined by adding different substances to the Hepes buffer used for incubation of cells (see above). A quantity of 1 mM sodium cyanide was added to inhibit cytochrome c oxidase, thereby mimicking anoxia, 2 mM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) was used to functionally uncouple the mitochondria, and 4 mM calcium was added to stimulate ATP synthase. Rotenone-sensitive NADH cytochrome c reductase (complex I 1 III, EC 1.6.5.3 1 EC 1.10.2.2) was measured using a slight modification of the method described for skeletal muscle mitochondria (18). Antimycin-sensitive succinate cytochrome c reductase activity (complex II 1 III, EC 1.3.5.1 1 EC 1.10.2.2) was measured spectrophotometrically (19). Cytochrome c oxidase activity (complex IV, EC 1.9.3.1) was measured in cell sonicates at 37°C as described elsewhere (20). Protein was determined according to Bensadoun and Weinstein (21). Materials. Falcon 3001 petri dishes were from Becton Dickinson (Heidelberg, Germany); all other tissue culture material, including fetal calf serum, was from Gibco (Eggenstein, Germany). ATP, lactic dehydrogenase, pyruvate kinase, phosphoenolpyruvate, and NADH for enzyme assays were purchased from Boehringer (Mannheim, Germany). Oligomycin, antimycin, rotenone, cytochrome c, FCCP, Hepes, and all other chemicals of the highest purity available were from Sigma (Deisenhofen, Germany). Statistical analysis. Data for ATP synthase activities in cells from healthy and diseased children and sheep under basal conditions and the other metabolic conditions and activities of respiratory chain enzymes were compared using Student’s t test. RESULTS On phase-contrast microscopy, there was no difference between the cells from healthy and diseased animals and children.
351
ATP SYNTHASE IN NEURONAL CEROID LIPOFUSCINOSIS
FIG. 1. Regulation of mitochondrial ATP synthase in response to cellular energy demand in fibroblasts from four healthy sheep. Mean 1 SD; n:, number of petri dishes (four from each animal) incubated under the different metabolic conditions.
Several methods to break open mitochondria of sheep fibroblasts were tested. As in fibroblasts from humans (15) sonication for 2 3 10 s led to complete exposure of intramitochondrial enzymes in sheep. Breakage of mitochondria was judged by the addition of FCCP to the sonicate during the measurement of ATP synthase activity. Variation of duplicates from the same sonicate was 5%; variation between sonicates from petri dishes with cells from the same cell line incubated under the same conditions was typically less than 10% both for human and sheep cells. ATP synthase activity and its regulation in fibroblasts from healthy sheep are shown in Fig. 1. Basal activity was 110 nmol/min/mg protein. When cells were incubated in the presence of cyanide (mimicking anoxia) or functionally uncoupled by FCCP, ATP synthase activity was reduced to 76 and 79% of basal activity, respectively. Addition of 4 mM calcium to
the incubation medium resulted in activation of the ATP synthase to 145% of basal activity. This did not simply reflect the calcium optimum of the enzyme as addition of calcium to the sonicate in the measuring cuvette did not alter ATP synthase activity. The activities of respiratory chain complexes I 1 III, II 1 III, and IV (cytochrome c oxidase) are summarized in Table 1. ATP synthase regulation in response to cellular energy demand in fibroblasts from sheep affected with NCL is shown in Fig. 2. Basal activity in these cells was 185 nmol/min/mg protein. ATP synthase activity in response to cyanide and FCCP was reduced to 78 and 53% of basal activity, respectively. Down-regulation in response to uncoupling of mitochondria was significantly different (more pronounced) compared to cells from healthy animals. Addition of 4 mM calcium to the incubation medium resulted in down-regulation of ATP synthase to 55% of basal activity. As shown in Table 1, activities for the respiratory chain complexes I 1 III, II 1 III, and IV did not differ significantly from the values in cells from healthy animals. Active regulation of mitochondrial ATP synthase in response to cellular energy demand in fibroblasts from healthy children is shown in Fig. 3. Basal ATP synthase activity was 181 nmol/min/mg protein. Down-regulation occurred in the presence of sodium cyanide (mimicking anoxia) or FCCP (mitochondrial uncoupling). Up-regulation could be observed after preincubation of the cells with 4 mM calcium. Basal ATP synthase activity in fibroblasts from children with infantile NCL was 84 nmol/min/mg protein. Regulation of the enzyme was absent in cells from TABLE 1 Activities of Respiratory Chain Complexes in Fibroblasts from Four Healthy Sheep, Five Sheep Affected with NCL, Five Healthy Children, and Five Children with CLN1 Sheep Respiratory chain enzymes Complexes I 1 III Complexes II 1 III Complex IV (Cytochrome c oxidase)
Human
Healthy
NCL
Healthy
CLN1
58 6 21 46 1
57 6 23 66 3
105 6 40 11 6 4
151 6 72 2 6 2*
56 6 24
69 6 14
69 6 24
31 6 9*
Note. Two petri dishes were measured from each individual. Results are nmol/min/mg protein, mean 6 SD. * P , 0.05 vs healthy children.
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In fibroblasts from healthy sheep, we showed that active regulation of ATP synthase in response to cellular metabolic conditions was present. ATP synthase activity was down-regulated when the cells were incubated in the presence of cyanide (mimicking anoxia) or when the mitochondria were functionally uncoupled. Under these conditions, ATP synthase would work in reverse, thus hydrolyzing ATP. Down-regulation of ATP synthase, therefore, helps to conserve cellular ATP which is necessary to maintain structural and functional integrity of the cell. Up-regulation of ATP synthase could be demonstrated in the presence of 4 mM calcium in the incubation medium. Calcium is known to be an important second messenger and has previously been shown to be increased in situations of high cellular energy demand (e.g., beating of cardiomyocytes, action of thyroid hormones and adrenergic substances) (22). Similar active regulation of ATP synthase in response to cellular metabolic conditions as de-
FIG. 2. Regulation of mitochondrial ATP synthase in response to cellular energy demand in fibroblasts from five sheep affected with NCL. Mean 1 SD; n:, number of petri dishes incubated under the different metabolic conditions.
patients with infantile NCL (Fig. 4). Activities of respiratory chain complexes from patients with the infantile form of NCL were assayed (Table 1). While activity of complex I 1 III was found to be normal, activities of complex II 1 III and complex IV were significantly reduced. DISCUSSION ATP synthase is one of the key enzymes responsible for aerobic energy production and is subject to regulatory effects which depend on specific cellular metabolic conditions such as hypoxia, metabolic uncoupling of mitochondria, or calcium concentration. This study demonstrates that one of the characteristic regulatory features of ATP synthase, its upregulation by calcium, is lost in fibroblasts from sheep with NCL and children with infantile NCL.
FIG. 3. Regulation of mitochondrial ATP synthase in response to cellular energy demand in fibroblasts from five healthy children. Mean 1 SD; n:, number of petri dishes incubated under the different metabolic conditions.
ATP SYNTHASE IN NEURONAL CEROID LIPOFUSCINOSIS
FIG. 4. Regulation of mitochondrial ATP synthase in response to cellular energy demand in fibroblasts from five children affected with CLN 1. Mean 1 SD; n:, number of petri dishes incubated under the different metabolic conditions (four petri dishes from each cell line).
scribed here for healthy sheep fibroblasts has been reported for cultured cardiomyocytes from rat (23), canine myocardium (24), human skin fibroblasts (15), and human skeletal muscle (25). Activity of antimycin-sensitive succinate cytochrome c reductase as a mitochondrial marker enzyme remained unchanged under the different incubation conditions showing constant mitochondrial recovery (results not shown). Activities of respiratory chain complexes I, II, III, and IV did not differ significantly between healthy and diseased cells which shows that mitochondrial content and recovery were the same in both types of cells. Effects of an allosteric regulator mediating upand down-regulation of ATP synthase can be ruled out as regulated states were stable to dilution of mitochondria into large volumes of incubation and assay media. The most likely candidate for downregulation of ATP synthase is the naturally occur-
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ring inhibitor protein IF 1. This protein has been shown to bind to ATP synthase when the membrane potential across the inner mitochondrial membrane is reduced (26,27) as in anoxia or when mitochondria are uncoupled. A calcium binding inhibitor (CaBi) has been described (28) which dissociates from ATP synthase at increased calcium levels. This regulatory protein could be responsible for the upregulation of ATP synthase seen in response to increased calcium levels in cells from healthy sheep and humans. In fibroblasts from NCL sheep, basal ATP synthase activity (i.e., under control conditions) was slightly elevated compared to cells from control animals. When fibroblasts from NCL animals were incubated in the presence of calcium, down-regulation of ATP synthase instead of up-regulation occurred. Such an abnormal regulatory response could lead to cellular ATP deficiency due to insufficient ATP production in situations with high energy requirements. Elevation of basal ATP synthase activity may be a compensatory mechanism to overcome this difficulty. Disturbances in the CaBi or IF 1 protein or a defect in the ATP synthase molecule itself may be responsible for the dysregulation. Defects in the activities of the other respiratory chain complexes could lead to cellular lack of energy as well. However, no significant differences in the activities of complexes I, II, III, and IV could be detected when healthy and diseased animals were compared. Basal activity of ATP synthase was reduced in fibroblasts from children with infantile NCL. In these cells no regulation of ATP synthase was observed. Thus, the capacity of these cells to produce energy is compromised, though no intralysosomal storage of subunit c of ATP synthase occurs in this form of NCL. Activities of complex II and complex IV were reduced compared to cells from healthy children. This may be due to intramitochondrial energy depletion caused by the reduced capacity of the mitochondrial ATP synthase. The defective regulation of ATP synthase described here in an ovine model of the human late infantile NCL variant (CLN6) (13) and in human infantile NCL (CLN1) is different from the deficiencies of basal ATP synthase activity demonstrated previously in late-infantile NCL (CLN2) and juvenile NCL (CLN3) (12). All ATP synthase abnormalities described in the four forms of NCL, however, can interfere with mitochondrial energy production and, if also expressed in brain, could play a crucial role in the pathogenesis of the four different NCL
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forms. In brain tissue from three of these NCL forms, intralysosomal storage of subunit c of mitochondrial ATP synthase has been reported (10,11) but is absent in infantile NCL. The neurodegenerative process does not seem to be directly related to the storage process. Deficient ATP supply could result in dysfunction and eventual death of neuronal cells, especially in those with high energy requirements. Those neuronal cells that are predominantly affected by the neurodegenerative process in several NCL disorders seem to have a particularly high energy demand (see introduction). Excitotoxicity has also been discussed in the pathogenesis of neurodegenerative diseases (29). Inhibitory GABAergic neurons rich in mitochondria are especially susceptible to compromised energy production. Loss of inhibition may lead to excitotoxicity in other brain cells with compromised energy production (3). Thus, compromised energy production may be a key to understanding the neurodegenerative process in several forms of NCL. The mechanism of dysfunction of the mitochondrial ATP synthase system in NCL cells is unknown. The dysregulation may be caused by a primary extramitochondrial defect leading to a secondary disturbance of mitochondrial energy metabolism. Alternatively, the primary defect could affect the mitochondrial ATP synthase more directly. ACKNOWLEDGMENTS We are indebted to Mrs. I. Wernicke for excellent technical assistance. This work was supported by the United States Institute of Neurological Disorders and Stroke (Grant NS 32348) and by “Freunde der Universita¨tskinderklinik Hamburg.”
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