- Email: [email protected]
ATP synthase activity in ovine ceroid lipofuscinosis [OCL 6]
doi: 10.1053/ejpn.2001.0449 available online at http://www.idealibrary.com on I | [ ~ ! " European Journal of Paediatric Neurology 2001 ; 5(Suppl. A): 131-134
ORIGINAL
ARTICLE
A TP synthase activity in ovine ceroid lipofuscinosis [OCL6] ROBERT D JOLLY, I SHERYL L BAYLISS, 1 A N I B H DAS, 2 D E L W Y N G COOKE, 3 S I M O N B R O W N 3 1Institute of Veterinary, Animal and Biomedical Science, Massey University, Palmerston North, New Zealand; 2Zentrum Kinderheilkunde und Humangenetik, Hannover, Germany; 3Institute of Fundamental Science, Massey University, Palmerston North, New Zealand
We measured ATP synthase activities in mitochondria isolated from livers of lambs with ceroid lipofuscinosis (OCL6) and compared them with those from similar isolations from obligate heterozygous and control lambs. Addition of excess Ca~+ to the incubation mixture resulted in an up-regulation of activity in mitochondria from control lambs but down-regulation in those from OCL6 affected lambs. The mean change in activity with Ca2+ for heterozygous animals was midway between those from control and affected groups being significantly different from control but not from affected. The change in ATP synthase activity to added Ca2÷ was also measured in isolated mitochondria from affected and control lambs from 3 days to 25 months of age. As above, there was down-regulation to the addition of Ca2+ in affected lambs. There was a fall in percentage change to Ca2+ with age in both affected and control lambs. This was not significantly different in affected lambs indicating it was not associated with the stage of disease. The above in vitro results, if extrapolated to neurons in vivo, imply a potential dysfunction of mitochondria in OCL6 lambs that could lead to calcium mediated neurotoxicity and neuron death due to production of free radicals as implicit in the energy-linked excitotoxic hypothesis. Keywords: Batten disease. Neurons. Mitochondria. ATP synthase. Calcium. Excitotoxicity.
Introduction It has been postulated that selective loss of neurons in ovine ceroid lipofuscinosis (OCL6), canine (English setter), and perhaps other forms of Batten disease is due to energy-linked excitotoxicity. 1-7 This would involve neurons that were most metabolically active and particularly those with abundant glutamate input. The hypothesis was supported in OCL6 lambs by the finding that in cultured fibroblasts there was an increase in basal ATP synthase activity relative to controls. In the latter, addition of Ca 2+ to the incubation mixture resulted in up-regulation in normal cells but paradoxically a down-regulation in OCL6 cells. 7 These observations suggested that mitochondria, at
least in this form of Batten disease, might be under metabolic stress and predispose to an inability of neurons to produce sufficient ATP to adequately repolarize the plasma membrane following glutamate stimulation of N-methyl-D-aspartate (NMDA) receptor-associated ion channels. This could lead to calcium induced neurodegeneration. The mitochondrial defect was further investigated directly in mitochondria isolated from livers of OCL6 affected, heterozygous and control lambs.
Materials and methods Lambs affected by OCL6 were bred in an experimental flock maintained by breeding homozygous
Correspondence: Robert D. Jolly, Institute of Veterinary, Animal and Biomedical Science, Massey University, Palmerston North, New Zealand e-mail: [email protected] 1090--3798/01/05/A131+4 $35.00
© 2001 European Paediatric Neurology Society
Original article: R D Jolly et al.
132 Basal + Ca 2+
~
Basal + Ca 2+
Basal + Ca 2+
15
4
a.
3
g ~
~
[]
0 |
n
~
Control
°~
OCL6
Heterozygous
Fig. 1. ATP synthase activity (measured as #moles ATP hydrolysed/mg protein/h) in mitochondria isolated from livers of individual control, heterozygous and control lambs before and with additional Ca2÷ in the incubation mixture.
affected male lambs with obligate heterozygous ewes. Affected individuals were diagnosed on the basis of histopathology of brain biopsies at 2.5-3 months of age, or in the two 25-month-old animals, on clinical signs. All diagnoses were retrospectively confirmed by gross a n d / o r histopathological examinations. Control lambs were New Zealand Romneys or their crosses. All animals were maintained under normal New Zealand animal husbandry conditions except for the two oldest animals which were maintained under hospital conditions for the last 5 months during the latter stages of the disease. Euthanasia was by shooting with a captive bolt followed by exsanguination. Livers were removed immediately and placed on ice. Mitochondria were isolated by homogenization and centrifugation8 and incubated for 15 minutes at 4°C in the absence or presence of additional 4mM of Ca 2+ [as CaCI2] in the incubation mixture and then sonicated ( 2 x l 0 s [0.3s on/0.7s off] at 20W) immediately before the assay. The ATP synthase activity was measured spectrophotometrically using the reverse hydrolysis method. 7 Each assay was done in triplicate and results were analysed by a series of t-tests between the relevant groups to be compared.
Results The ATP synthase activity was measured in isolated mitochondria in OCL6 affected, obligate
.
0
.
.
.
0
5
.
.
.
.
0
10
.
.
.
.
1
15
.
.
.
.
1
20
.
.
.
.
,
25
Fig. 2. Percentage change in ATP synthase activity in isolated mitochondria from the livers of OCL6 and control lambs relative to age; control values, • OCL6 values.
heterozygous and control lambs with or without additional Ca 2+ (Fig. 1). Basal values in affected lambs were higher than those of controls but not significantly so (p=0.087). There was an increase in mean ATP synthase activity in control mitochondria with additional Ca 2+ in the incubation mixture (p=0.003), but a decrease in those from OCL6 affected lambs (p=0.043). The differences in response to Ca 2+ between control and affected groups was highly significant (p=0.001). The basal ATP synthase activity values in heterozygous lambs were different from those of controls (p=0.044) and affected animals (p=0.005). The change in mean value to the addition of Ca 2+ in heterozygous lambs was midway between those of controls and OCL6 affected lambs with an overall decrease of activity. This change was not significantly different from affected values (p=0.086) but was significantly different from control values (p=0.012). Directional changes in ATP hydrolysis to the addition of Ca 2+ in heterozygous lambs appeared as a mixture of affected and control patterns. In a second experiment, age-dependant ATP synthase activity values were measured in mitochondria from control and affected lambs aged 3 days to 25 months with and without additional Ca 2+ in the incubation mixture. Results, expressed as percentage increase/decrease in activity with additional Ca 2+ are shown in Fig. 2. As in the previous experiment there was down-regulation of ATP synthase activity with Ca 2+ in affected lambs. The change in ATP synthase activity in the presence of Ca 2+, decreased at a similar rate with age in both groups as shown by linear regression lines. Similar declines occured in basal ATP synthase activities (data not shown).
Original article: ATP synthase in ovine ceroid lipofuscinoisis
Discussion The descriptor OCL6 is applied to ovine ceroid lipofuscinosis in New Zealand South Hampshire sheep because it is syntenic to CLN6 in humans. 9 It is thus a specific model for this form of Batten disease and a generic model for others. The ATP synthase activity was measured in the direction of reverse hydrolysis of ATP. This reaction has been shown to be fully reversible under substrate (ATP) saturation and, in the absence of membrane potential across the inner mitochondrial membrane, it is a convenient method of monitoring ATP synthase activity.7,1° Given the wide range of basal values recorded even in control animals, little emphasis is given to the differences between basal values between controls, affected and heterozygous groups. These could reflect differences in the purity of mitochondrial preparations. However, the change in ATP synthase activity to additional Ca 2+ is within aliquots of the same preparation and considered a more reliable indication of essential differences between mitochondria from different groups of animals. With the exception of one control animal, all changes to Ca 2+ were in the same direction within control (up) and affected animals (down). This difference was highly significant (p=0.001) and when taken in conjunction with the directional changes noted for individual OCL6 lambs, it allows the conclusion that Ca 2÷ caused down-regulation of ATP synthase activity in this group. The intermediate change and mixture of individual directional changes to Ca 2+ in the heterozygous lambs, suggested a variable genetic affect with no immediate explanation which deserves further investigation. In the second study, there was a general decrease in ATP synthase activity with age in both groups with and without additional Ca 2+ and this is in accord with other published data. n A comparison of the rate of decline showed no evidence that down-regulation of ATP synthase activity in affected lambs was related to the stage of the disease. It thus offers little insight in to how this effect is mediated. A decrease is known to occur due to the cumulative affect of mitochondrial DNA mutations with age 12,13 but most of these are likely to affect the electron transfer chain reactions as ATP synthase contains only two of 16 subunits that are coded by this form of DNA and this is true also for the sheep. 14 The electron transfer chain is unlikely to have an effect on the ATP synthase assay performed, as it was done under conditions where it was essentially isolated from its effects.
133
The results validate the fibroblast model; 7 they also further support the hypothesis that selective neuron death is associated with energy linked excitotoxicity as proposed above. In view of ethical, logistical and cost considerations, further studies, which should be extended to other forms of Batten disease in humans, should be done in fibroblasts. Chronic excitotoxicity has been postulated to account for selective neuronal death in a variety of neurodegenerative disorders due to age, inherited disorders or environmental toxins. 13,15-17 Neurons with suboptimal mitochondrial activity and receiving abundant excitatory glutamate input would be anticipated to be most at risk. It has been argued, that at least some of the populations of neurons preferentially lost in several forms of Batten disease, are of this category. 1-6 The major utilization of ATP in neurons is in maintenance of ATPase pumps to maintain polarization of the plasma membrane. TM Any defect in ATP production as implied by these results, could lead to failure of this homeostatic mechanism with failure of closure of the Mg 2+ block on NMDA receptor-associated ion channels and continued influx of Ca 2+ into the cell. Intracellular Ca 2+ concentration in unexcited cells is several orders of magnitude below that in the extracellular milieu. This gradient offers the chance to abruptly raise intracellular Ca 2+ for signalling purposes by opening Ca 2+ channels in the plasma membrane. The cell has buffering mechanisms mediated through proteins such as parvalbumin, smooth endoplasmic reticulum and mitochondria which regulate Ca 2+ homeostasis during recovery from a Ca 2+ influx. Ca 2+ can also regulate mitochondrial function by activating several important enzymes within the electron transfer chain as well as ATP synthase. 13 It has recently been shown that this latter may be modulated through phosphorylation of a small molecular weight peptide that appears to be subunit c of ATP synthase.19 A normal response to increased intramitochondrial Ca 2+ (that does not exceed a critical threshold), is up-regulation of ATP synthase with increased production of ATP to be used by the ATPase pumps to repolarize the cell membrane by excluding Na + and Ca 2+ ions. In OCL6 mitochondria, the response was in the opposite direction. Although ATP synthase activity was measured in vitro from mitochondria isolated from livers, the results if extrapolated to those of neurons in vivo, suggest a mechanism of neurodegeneration; i.e. a failure to restore Ca 2+ homeostasis would lead to calcium mediated neurotoxicity and cell death due to production of free radicals as implicit in the energy-linked excitotoxicity hypothesis. 13,15-17
Original article: R D Jolly et al.
134
Acknowledgements The research w a s d o n e w i t h the h e l p of grants f r o m the P a l m e r s t o n N o r t h Medical Research Foundation a n d the Batten Disease S u p p o r t a n d Research Association.
10
11
References 1 March PA, Wurzelmann S, Walkley SU. Morphological alterations in neocortical and cerebellar GABAergic neurons in a canine model of juvenile Batten disease. Am J Med Genet 1995; 55: 204-212. 2 Walkley SU, March PA, Schroeder CE et al. Pathogenesis of brain dysfunction in Batten disease. Am J Med Genet 1995; 55: 196-203. 3 Walkley SU, Siegal DA, Dobrenis K. A typical neuronal storage disease or a genetic neurodegenerative disorder characterised by excitotoxicity? In: Fiskum G (ed) Neurodegenerative Diseases. New York: Plenum Press, 1996: 217-224. 4 Walkley SU. Cellular pathology of lysosomal storage disorders. Brain Pathol 1998; 8: 175-193. 5 Jolly RD, Walkley SU. Ovine ceroid lipofuscinosis: Postulated mechanism of neurodegeneration. Mol Genet Metab 1999; 66: 376-380. 6 Jolly RD, Kohlsch~itter A, Palmer DN, Walkley SU. The neuronal ceroid lipofuscinoses (Batten disease). In: Mattson MP (ed) Genetic Aberrancies and Neurodegenerative Disorders. Stamford, Connecticut: JAI Press Inc. 1999: 391-420. 7 Das AM, Jolly RD, Kohlsch~itter A. Anomalies of mitochondrial ATP synthase regulation in four different types of neuronal ceroid-lipofuscinosis. Mol Genet Metab 1999; 66: 349-354. 8 Reid JC, Husbands DR. Oxidative metabolism of long chain fatty acids in mitochondria from sheep and rat liver. Biochem J 1985; 225: 233-237. 9 Broom MF, Zhou C, Broom J E e t al. Ovine ceroid lipofuscinosis: A large animal model syntenic with
12
13
14
15 16
17
18
19
the human neuronal ceroid-lipofuscinosis variant CLN6. J Med Genet 1998; 35: 717-721. Gresser M, Cardon J, Rosen G, Boyer PD. Demonstration and quantitation of catalytic and noncatalytic bound ATP in submitochondrial particles during oxidative phosphorylation. J Biol Chem 1979; 254:10 649-10 658. Papa S. Mitochondrial oxidative phosphorylation changes in the life span. Molecular aspects and physiopathological implications. Biochim Biophys Acta 1996; 1276: 87-105. Wallace DC, Shoffner JM, Trounce I e t al. Mitochondrial DNA mutations in human degenerative diseases and aging. Biochim Biophys Acta 1995; 1271: 141-151. Glazner GW. The role of mitochondrial genome mutations in neurodegenerative disease. In: Mattson MP (ed) Genetic Aberrancies and Neurodegenerative Disorders. Stamford, Conecticut: JAI Press Inc. 1999: 313-354. Hiendleder S, Lewalski H, Wassmuth R, Janke A. The complete mitochondrial DNA sequence of the domestic sheep (Ovies aries) and comparison with the other major ovine haplotype. J Mol Evolut 1998; 47: 441-448. Choi DW. Glutamate neurotoxicity and diseases of the central nervous system. Neuron 1988; 1: 623-634. Beal MF. Does impairment of energy metabolism result in exctitotoxic neuronal death in neurodegenerative disease? Ann Neurol 1992; 31: 119-130. Mattson MP. Genetic contributions to the pathogenesis of Alzheimer's disease. In: Mattson MP (ed) Genetic Aberrancies and Neurodegenerative Disorders. Stamford, Connecticut: JAI Press Inc. 1999: 1-31. Wong-Riley MTT. Cytochrome oxidase: an endogenous metabolic marker for neuronal activity. Trends Neurosci 1989; 12: 94-101. Azarashvily TS, Tyynela Y, Baumann M e t al. Ca2÷ modulated phosphorylation of a low-molecular mass polypeptide in rat liver mitochondria: Evidence that it is identical with subunit c of FoF1 ATPase. Biochem Biophys Res Commun 2000; 270: 741-744.
ORIGINAL
ARTICLE
A TP synthase activity in ovine ceroid lipofuscinosis [OCL6] ROBERT D JOLLY, I SHERYL L BAYLISS, 1 A N I B H DAS, 2 D E L W Y N G COOKE, 3 S I M O N B R O W N 3 1Institute of Veterinary, Animal and Biomedical Science, Massey University, Palmerston North, New Zealand; 2Zentrum Kinderheilkunde und Humangenetik, Hannover, Germany; 3Institute of Fundamental Science, Massey University, Palmerston North, New Zealand
We measured ATP synthase activities in mitochondria isolated from livers of lambs with ceroid lipofuscinosis (OCL6) and compared them with those from similar isolations from obligate heterozygous and control lambs. Addition of excess Ca~+ to the incubation mixture resulted in an up-regulation of activity in mitochondria from control lambs but down-regulation in those from OCL6 affected lambs. The mean change in activity with Ca2+ for heterozygous animals was midway between those from control and affected groups being significantly different from control but not from affected. The change in ATP synthase activity to added Ca2÷ was also measured in isolated mitochondria from affected and control lambs from 3 days to 25 months of age. As above, there was down-regulation to the addition of Ca2+ in affected lambs. There was a fall in percentage change to Ca2+ with age in both affected and control lambs. This was not significantly different in affected lambs indicating it was not associated with the stage of disease. The above in vitro results, if extrapolated to neurons in vivo, imply a potential dysfunction of mitochondria in OCL6 lambs that could lead to calcium mediated neurotoxicity and neuron death due to production of free radicals as implicit in the energy-linked excitotoxic hypothesis. Keywords: Batten disease. Neurons. Mitochondria. ATP synthase. Calcium. Excitotoxicity.
Introduction It has been postulated that selective loss of neurons in ovine ceroid lipofuscinosis (OCL6), canine (English setter), and perhaps other forms of Batten disease is due to energy-linked excitotoxicity. 1-7 This would involve neurons that were most metabolically active and particularly those with abundant glutamate input. The hypothesis was supported in OCL6 lambs by the finding that in cultured fibroblasts there was an increase in basal ATP synthase activity relative to controls. In the latter, addition of Ca 2+ to the incubation mixture resulted in up-regulation in normal cells but paradoxically a down-regulation in OCL6 cells. 7 These observations suggested that mitochondria, at
least in this form of Batten disease, might be under metabolic stress and predispose to an inability of neurons to produce sufficient ATP to adequately repolarize the plasma membrane following glutamate stimulation of N-methyl-D-aspartate (NMDA) receptor-associated ion channels. This could lead to calcium induced neurodegeneration. The mitochondrial defect was further investigated directly in mitochondria isolated from livers of OCL6 affected, heterozygous and control lambs.
Materials and methods Lambs affected by OCL6 were bred in an experimental flock maintained by breeding homozygous
Correspondence: Robert D. Jolly, Institute of Veterinary, Animal and Biomedical Science, Massey University, Palmerston North, New Zealand e-mail: [email protected] 1090--3798/01/05/A131+4 $35.00
© 2001 European Paediatric Neurology Society
Original article: R D Jolly et al.
132 Basal + Ca 2+
~
Basal + Ca 2+
Basal + Ca 2+
15
4
a.
3
g ~
~
[]
0 |
n
~
Control
°~
OCL6
Heterozygous
Fig. 1. ATP synthase activity (measured as #moles ATP hydrolysed/mg protein/h) in mitochondria isolated from livers of individual control, heterozygous and control lambs before and with additional Ca2÷ in the incubation mixture.
affected male lambs with obligate heterozygous ewes. Affected individuals were diagnosed on the basis of histopathology of brain biopsies at 2.5-3 months of age, or in the two 25-month-old animals, on clinical signs. All diagnoses were retrospectively confirmed by gross a n d / o r histopathological examinations. Control lambs were New Zealand Romneys or their crosses. All animals were maintained under normal New Zealand animal husbandry conditions except for the two oldest animals which were maintained under hospital conditions for the last 5 months during the latter stages of the disease. Euthanasia was by shooting with a captive bolt followed by exsanguination. Livers were removed immediately and placed on ice. Mitochondria were isolated by homogenization and centrifugation8 and incubated for 15 minutes at 4°C in the absence or presence of additional 4mM of Ca 2+ [as CaCI2] in the incubation mixture and then sonicated ( 2 x l 0 s [0.3s on/0.7s off] at 20W) immediately before the assay. The ATP synthase activity was measured spectrophotometrically using the reverse hydrolysis method. 7 Each assay was done in triplicate and results were analysed by a series of t-tests between the relevant groups to be compared.
Results The ATP synthase activity was measured in isolated mitochondria in OCL6 affected, obligate
.
0
.
.
.
0
5
.
.
.
.
0
10
.
.
.
.
1
15
.
.
.
.
1
20
.
.
.
.
,
25
Fig. 2. Percentage change in ATP synthase activity in isolated mitochondria from the livers of OCL6 and control lambs relative to age; control values, • OCL6 values.
heterozygous and control lambs with or without additional Ca 2+ (Fig. 1). Basal values in affected lambs were higher than those of controls but not significantly so (p=0.087). There was an increase in mean ATP synthase activity in control mitochondria with additional Ca 2+ in the incubation mixture (p=0.003), but a decrease in those from OCL6 affected lambs (p=0.043). The differences in response to Ca 2+ between control and affected groups was highly significant (p=0.001). The basal ATP synthase activity values in heterozygous lambs were different from those of controls (p=0.044) and affected animals (p=0.005). The change in mean value to the addition of Ca 2+ in heterozygous lambs was midway between those of controls and OCL6 affected lambs with an overall decrease of activity. This change was not significantly different from affected values (p=0.086) but was significantly different from control values (p=0.012). Directional changes in ATP hydrolysis to the addition of Ca 2+ in heterozygous lambs appeared as a mixture of affected and control patterns. In a second experiment, age-dependant ATP synthase activity values were measured in mitochondria from control and affected lambs aged 3 days to 25 months with and without additional Ca 2+ in the incubation mixture. Results, expressed as percentage increase/decrease in activity with additional Ca 2+ are shown in Fig. 2. As in the previous experiment there was down-regulation of ATP synthase activity with Ca 2+ in affected lambs. The change in ATP synthase activity in the presence of Ca 2+, decreased at a similar rate with age in both groups as shown by linear regression lines. Similar declines occured in basal ATP synthase activities (data not shown).
Original article: ATP synthase in ovine ceroid lipofuscinoisis
Discussion The descriptor OCL6 is applied to ovine ceroid lipofuscinosis in New Zealand South Hampshire sheep because it is syntenic to CLN6 in humans. 9 It is thus a specific model for this form of Batten disease and a generic model for others. The ATP synthase activity was measured in the direction of reverse hydrolysis of ATP. This reaction has been shown to be fully reversible under substrate (ATP) saturation and, in the absence of membrane potential across the inner mitochondrial membrane, it is a convenient method of monitoring ATP synthase activity.7,1° Given the wide range of basal values recorded even in control animals, little emphasis is given to the differences between basal values between controls, affected and heterozygous groups. These could reflect differences in the purity of mitochondrial preparations. However, the change in ATP synthase activity to additional Ca 2+ is within aliquots of the same preparation and considered a more reliable indication of essential differences between mitochondria from different groups of animals. With the exception of one control animal, all changes to Ca 2+ were in the same direction within control (up) and affected animals (down). This difference was highly significant (p=0.001) and when taken in conjunction with the directional changes noted for individual OCL6 lambs, it allows the conclusion that Ca 2÷ caused down-regulation of ATP synthase activity in this group. The intermediate change and mixture of individual directional changes to Ca 2+ in the heterozygous lambs, suggested a variable genetic affect with no immediate explanation which deserves further investigation. In the second study, there was a general decrease in ATP synthase activity with age in both groups with and without additional Ca 2+ and this is in accord with other published data. n A comparison of the rate of decline showed no evidence that down-regulation of ATP synthase activity in affected lambs was related to the stage of the disease. It thus offers little insight in to how this effect is mediated. A decrease is known to occur due to the cumulative affect of mitochondrial DNA mutations with age 12,13 but most of these are likely to affect the electron transfer chain reactions as ATP synthase contains only two of 16 subunits that are coded by this form of DNA and this is true also for the sheep. 14 The electron transfer chain is unlikely to have an effect on the ATP synthase assay performed, as it was done under conditions where it was essentially isolated from its effects.
133
The results validate the fibroblast model; 7 they also further support the hypothesis that selective neuron death is associated with energy linked excitotoxicity as proposed above. In view of ethical, logistical and cost considerations, further studies, which should be extended to other forms of Batten disease in humans, should be done in fibroblasts. Chronic excitotoxicity has been postulated to account for selective neuronal death in a variety of neurodegenerative disorders due to age, inherited disorders or environmental toxins. 13,15-17 Neurons with suboptimal mitochondrial activity and receiving abundant excitatory glutamate input would be anticipated to be most at risk. It has been argued, that at least some of the populations of neurons preferentially lost in several forms of Batten disease, are of this category. 1-6 The major utilization of ATP in neurons is in maintenance of ATPase pumps to maintain polarization of the plasma membrane. TM Any defect in ATP production as implied by these results, could lead to failure of this homeostatic mechanism with failure of closure of the Mg 2+ block on NMDA receptor-associated ion channels and continued influx of Ca 2+ into the cell. Intracellular Ca 2+ concentration in unexcited cells is several orders of magnitude below that in the extracellular milieu. This gradient offers the chance to abruptly raise intracellular Ca 2+ for signalling purposes by opening Ca 2+ channels in the plasma membrane. The cell has buffering mechanisms mediated through proteins such as parvalbumin, smooth endoplasmic reticulum and mitochondria which regulate Ca 2+ homeostasis during recovery from a Ca 2+ influx. Ca 2+ can also regulate mitochondrial function by activating several important enzymes within the electron transfer chain as well as ATP synthase. 13 It has recently been shown that this latter may be modulated through phosphorylation of a small molecular weight peptide that appears to be subunit c of ATP synthase.19 A normal response to increased intramitochondrial Ca 2+ (that does not exceed a critical threshold), is up-regulation of ATP synthase with increased production of ATP to be used by the ATPase pumps to repolarize the cell membrane by excluding Na + and Ca 2+ ions. In OCL6 mitochondria, the response was in the opposite direction. Although ATP synthase activity was measured in vitro from mitochondria isolated from livers, the results if extrapolated to those of neurons in vivo, suggest a mechanism of neurodegeneration; i.e. a failure to restore Ca 2+ homeostasis would lead to calcium mediated neurotoxicity and cell death due to production of free radicals as implicit in the energy-linked excitotoxicity hypothesis. 13,15-17
Original article: R D Jolly et al.
134
Acknowledgements The research w a s d o n e w i t h the h e l p of grants f r o m the P a l m e r s t o n N o r t h Medical Research Foundation a n d the Batten Disease S u p p o r t a n d Research Association.
10
11
References 1 March PA, Wurzelmann S, Walkley SU. Morphological alterations in neocortical and cerebellar GABAergic neurons in a canine model of juvenile Batten disease. Am J Med Genet 1995; 55: 204-212. 2 Walkley SU, March PA, Schroeder CE et al. Pathogenesis of brain dysfunction in Batten disease. Am J Med Genet 1995; 55: 196-203. 3 Walkley SU, Siegal DA, Dobrenis K. A typical neuronal storage disease or a genetic neurodegenerative disorder characterised by excitotoxicity? In: Fiskum G (ed) Neurodegenerative Diseases. New York: Plenum Press, 1996: 217-224. 4 Walkley SU. Cellular pathology of lysosomal storage disorders. Brain Pathol 1998; 8: 175-193. 5 Jolly RD, Walkley SU. Ovine ceroid lipofuscinosis: Postulated mechanism of neurodegeneration. Mol Genet Metab 1999; 66: 376-380. 6 Jolly RD, Kohlsch~itter A, Palmer DN, Walkley SU. The neuronal ceroid lipofuscinoses (Batten disease). In: Mattson MP (ed) Genetic Aberrancies and Neurodegenerative Disorders. Stamford, Connecticut: JAI Press Inc. 1999: 391-420. 7 Das AM, Jolly RD, Kohlsch~itter A. Anomalies of mitochondrial ATP synthase regulation in four different types of neuronal ceroid-lipofuscinosis. Mol Genet Metab 1999; 66: 349-354. 8 Reid JC, Husbands DR. Oxidative metabolism of long chain fatty acids in mitochondria from sheep and rat liver. Biochem J 1985; 225: 233-237. 9 Broom MF, Zhou C, Broom J E e t al. Ovine ceroid lipofuscinosis: A large animal model syntenic with
12
13
14
15 16
17
18
19
the human neuronal ceroid-lipofuscinosis variant CLN6. J Med Genet 1998; 35: 717-721. Gresser M, Cardon J, Rosen G, Boyer PD. Demonstration and quantitation of catalytic and noncatalytic bound ATP in submitochondrial particles during oxidative phosphorylation. J Biol Chem 1979; 254:10 649-10 658. Papa S. Mitochondrial oxidative phosphorylation changes in the life span. Molecular aspects and physiopathological implications. Biochim Biophys Acta 1996; 1276: 87-105. Wallace DC, Shoffner JM, Trounce I e t al. Mitochondrial DNA mutations in human degenerative diseases and aging. Biochim Biophys Acta 1995; 1271: 141-151. Glazner GW. The role of mitochondrial genome mutations in neurodegenerative disease. In: Mattson MP (ed) Genetic Aberrancies and Neurodegenerative Disorders. Stamford, Conecticut: JAI Press Inc. 1999: 313-354. Hiendleder S, Lewalski H, Wassmuth R, Janke A. The complete mitochondrial DNA sequence of the domestic sheep (Ovies aries) and comparison with the other major ovine haplotype. J Mol Evolut 1998; 47: 441-448. Choi DW. Glutamate neurotoxicity and diseases of the central nervous system. Neuron 1988; 1: 623-634. Beal MF. Does impairment of energy metabolism result in exctitotoxic neuronal death in neurodegenerative disease? Ann Neurol 1992; 31: 119-130. Mattson MP. Genetic contributions to the pathogenesis of Alzheimer's disease. In: Mattson MP (ed) Genetic Aberrancies and Neurodegenerative Disorders. Stamford, Connecticut: JAI Press Inc. 1999: 1-31. Wong-Riley MTT. Cytochrome oxidase: an endogenous metabolic marker for neuronal activity. Trends Neurosci 1989; 12: 94-101. Azarashvily TS, Tyynela Y, Baumann M e t al. Ca2÷ modulated phosphorylation of a low-molecular mass polypeptide in rat liver mitochondria: Evidence that it is identical with subunit c of FoF1 ATPase. Biochem Biophys Res Commun 2000; 270: 741-744.