Brain Research, 126 (1977) 295-307
295
,~) Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
I M P A I R M E N T OF D N A SYNTHESIS IN G U N N RAT C E R E B E L L U M
NORIKO YAMADA, YOSHIO SAWASAKI and HIROSHI NAKAJIMA
Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental Universit)', Tokyo 113 (Japan) (Accepted September 3rd, 1976)
SUMMARY
Brain DNA synthesis was developmentally investigated in Gunn rat with marked cerebellar hypoplasia due to hereditary hyperbilirubinemia. In this mutant rat, the Purkinje cell was nearly selectively affected in the cerebellar cortex by bilirubin. The impaired D N A synthesis was observed in homozygous (jj) Gunn rat cerebellum, in which the DNA content and [~H]thymidine incorporation rate into DNA decreased after 10 days of age compared to those in the heterozygous (Jj) littermate. In contrast, these impairments were not found in the non-cerebellar parts of the brain and liver of j / G u n n rat. The activity of cerebellar thymidine kinase in jj Gunn rat decreased from a very early stage, being 80 ~ of J j r a t at 6 days, and 50 ~ at l0 days of age. The enzyme activity was not affected in the non-cerebellar parts of the brain. Although bilirubin competitively inhibited cerebellar thymidine kinase activity in vitro (15 ~ at 10-5 M), such bilirubin level was found to be about 1000-fold that in vivo. Moreover, photodegradation of bilirubin in jj cerebellum exhibited no improvement in thymidine kinase activity, and the presence of an enzyme inactivator was not suggested in jj cerebellum. These results seem to indicate that the induction of thymidine kinase might be affected in jj Gunn rat cerebellum. The possibility that the impaired DNA synthesis in the external granular cells in jj cerebellum may be due to Purkinje cell damage is discussed.
INTRODUCTION
Gunn rat is a mutant of Wistar rat with autosomal recessive hereditary hyperbilirubinemia 1°. Its genetic defect has been reported to be in the hepatic uridine 5'diphosphate (UDP) glucuronyltransferase~6, zl. In the homozygous (jj) Gunn rat, marked cerebellar hypoplasia has been observed, and the Purkinje cell was found to be affected nearly selectively in cerebellar cortex19,2°, ')z. This hypoplasia seems to be
296 due to hyperbilirubinemia, since treatments such as hemolysis or administration t~f novobiocin which increase serum bilirubin produced cerebellar hypoplasia in the neonate rat aa. Conversely, therapeutic attempts for hyperbilirubinemJa such as phototherapy resulted in a less affected cerebellum injj Gunn rat te,e'~,:~. However, the pathogenesis of this cerebellar abnormality has not been investigated. In the previous studies19, "°, it was reported that the weight of~j cerebellum did not increase after 10 days of age, and the cerebellar lobuli which developed earlier were less affected i n j j cerebellum. The explanation for such a development-dependency of cerebellar lesion in Ii/Gunn rat was suggested to be in the characteristic nature of rat cerebellar development, and not in the brain bilirubin level, because brain bilirubin level injj Gunn rat decreased after birth and no selective accunaulation of bilirubin was observed in./j cerebellum e~. It is well known that the rat cerebellum develops by postnatal neurogenesis, showing highly active cell proliferation and differentiation after birth" ~,~L Therefore, it seems reasonable to consider that bilirubin might interfere with cerebettar DNA synthesis either directly or indirectly, resulting in cerebellar hypoplasia. Thus we focused the investigation on the possibility that cerebellar DNA replication might be impaired in the./'j Gunn rat. In the present paper, the changes of DNA content and [aH]thymidine incorporation rate into brain D N A during the postnatal development ofjj and heterozygous (Jj) Gunn rats were investigated. Next, our investigation was extended to the activity of brain thymidine kinase, which is presumed to play an important role in DNA synthesis~--s, "~7.The possible regulatory mechanism in cerebellar cell proliferation by Purkinje cell is also discussed. Preliminary communications of part of tiffs work have been presenteda°,aL MATERIALS AND METHODS
Animals Male jj and Jj Gunn rat littermates born from Jj mothers and .jj fathers were sacrificed by decapitation. The brain was divided into cerebellum and non-cerebellar parts. Six animals were used in each study unless otherwise mentioned.
Extraction and estimation of D NA Extraction of D N A was carried out by the modified Schmidt-Thannhauser procedure 29. DNA was estimated spectrophotometrically by absorbance at 260 nm, using calf thymus DNA (BDH) as a standard. Incorporation of [6-3H]thymidine into D N A was studied in rats which had received a subcutaneous injection of 2 #Ci/g body weight of [6-3H]thymidine (14.5 Ci/mmole, Daiichi Pure Chemicals). Two hours later, the animals were decapitated and the cerebellum and non-cerebellar parts of the brain and liver were removed. After the extraction and the estimation of DNA according to the above method, the incorporation of [6-3H]thymidine into DNA was measured in an Aloka scintillation spectrometer using toluene-Triton X-100 scintillation fluid (PPO, 0.5 '~, dPOPOP, 0.03 ~).
297
Estimation of thymidine kinase activity The activity of thymidine kinase was assayed by the method of Taylor et al. ~7. Cerebellum and non-cerebellar parts of the brain were homogenized with 5 vol. 0.02 M Tris • HCI buffer, pH 7.5, containing 1 m M EDTA and 2 m M mercaptoethanol. The homogenate was centrifuged at 105,000 × g for 60 rain. The supernatant was used as a crude enzyme preparation. The reaction mixture consisted of 5 m M MgCl2, 10 m M ATP, 2,uM [6-3H]thymidine with specific radioactivity described above, 105,000 x g supernatant, and 0.1 M Tris • HCI buffer, pH 7.5, to attain a final volume of 200/~1. Incubation was carried out at 30 °C for 15 rain, and the reaction was stopped by boiling for 3 rain. One hundred/~1 of reaction mixture was spotted on 1.8 sq.cm of DEAE-paper (Toyo Filter Co.). The paper was washed by suspending in 1 m M ammonium formate for four 15 rain periods, then washed with methanol, dried, and counted with toluene scintillation fluid (PPO, 0.5 °//,,, dPOPOP, 0.03 ~), in a liquid scintillation spectrometer. Protein was determined by biuret reaction with bovine serum albumin as a standard. Enzyme activity was presented as counts/rain/rag protein. At 2 and 4 days of age, the tissues of two animals were combined for the estimation of thymidine kinase activity.
Effect of bilirubin on the cerebellar thymidine kinase activity Bilirubin solution was prepared immediately before use by dissolution in 0.1 M Na2CO3 and dilution with 0.1 M Tris • HCI buffer, pH 7.5. In the experiment of bilirubin inhibition, 10/zl of bilirubin solution was added to the reaction mixture, bringing the final volume to 200 #1. The final pH remained at 7.5. The cerebella of 12-day-old Jj rats were used for enzyme preparation. Bilirubin levels of brain and liver subcellular fractions were determined by the method previously reported z0. Twelve day-oldjj rats were anesthetized with ether and perfused with 0.25 M sucrose. The removed brains and livers were homogenized with 5 vol of 0.25 M sucrose, centrifuged at 105,000 × g for 60 rain. Bilirubin contents of homogenates, 105,000 x g supernatants, and pellets suspended in 0.25 M sucrose were determined. The effect o f j j cerebellar cytosol on thymidine kinase activity in Jj cerebellar cytosol was studied using 12-day-old Jj a n d j j Gunn rats in which the effect of photoirradiation on thymidine kinase activity in jj cerebellum was also investigated. The 105,000 × g supernatants of Jj and jj cerebellum were irradiated by fluorescent lamp for phototherapy (FL 20BW-NU, National) for 1 h at 4 °C. In such a situation, bilirubin in the,/.'/sample was completely degraded. RESULTS
Developmental change in DNA synthesis in Gunn rat brain The postnatal change in total DNA content of Gunn rat brain is shown in Fig. 1. In the non-cerebellar parts of the brain, DNA content of both Jj a n d j j rats showed the same developmental pattern, and rose gradually until 12 days after birth and then leveled off. Cerebellar DNA content in both Jj and jj rats increased linearly during the first 10 days, and Jj cerebellar DNA content increased continuously thereafter,
298
1.0
I
non
--
05
Jl
4
"
i
0.0
~-
~ ..... :....
f ... ~ _.
8
a _ _ _ x __ ---Joo 16 '
12
Age in Days Fig. 1. P o s t n a t a l c h a n g e in t o t a l D N A c o n t e n t of G u n n ra t brain. H e t e r o z y g o u s (
295
,~) Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
I M P A I R M E N T OF D N A SYNTHESIS IN G U N N RAT C E R E B E L L U M
NORIKO YAMADA, YOSHIO SAWASAKI and HIROSHI NAKAJIMA
Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental Universit)', Tokyo 113 (Japan) (Accepted September 3rd, 1976)
SUMMARY
Brain DNA synthesis was developmentally investigated in Gunn rat with marked cerebellar hypoplasia due to hereditary hyperbilirubinemia. In this mutant rat, the Purkinje cell was nearly selectively affected in the cerebellar cortex by bilirubin. The impaired D N A synthesis was observed in homozygous (jj) Gunn rat cerebellum, in which the DNA content and [~H]thymidine incorporation rate into DNA decreased after 10 days of age compared to those in the heterozygous (Jj) littermate. In contrast, these impairments were not found in the non-cerebellar parts of the brain and liver of j / G u n n rat. The activity of cerebellar thymidine kinase in jj Gunn rat decreased from a very early stage, being 80 ~ of J j r a t at 6 days, and 50 ~ at l0 days of age. The enzyme activity was not affected in the non-cerebellar parts of the brain. Although bilirubin competitively inhibited cerebellar thymidine kinase activity in vitro (15 ~ at 10-5 M), such bilirubin level was found to be about 1000-fold that in vivo. Moreover, photodegradation of bilirubin in jj cerebellum exhibited no improvement in thymidine kinase activity, and the presence of an enzyme inactivator was not suggested in jj cerebellum. These results seem to indicate that the induction of thymidine kinase might be affected in jj Gunn rat cerebellum. The possibility that the impaired DNA synthesis in the external granular cells in jj cerebellum may be due to Purkinje cell damage is discussed.
INTRODUCTION
Gunn rat is a mutant of Wistar rat with autosomal recessive hereditary hyperbilirubinemia 1°. Its genetic defect has been reported to be in the hepatic uridine 5'diphosphate (UDP) glucuronyltransferase~6, zl. In the homozygous (jj) Gunn rat, marked cerebellar hypoplasia has been observed, and the Purkinje cell was found to be affected nearly selectively in cerebellar cortex19,2°, ')z. This hypoplasia seems to be
296 due to hyperbilirubinemia, since treatments such as hemolysis or administration t~f novobiocin which increase serum bilirubin produced cerebellar hypoplasia in the neonate rat aa. Conversely, therapeutic attempts for hyperbilirubinemJa such as phototherapy resulted in a less affected cerebellum injj Gunn rat te,e'~,:~. However, the pathogenesis of this cerebellar abnormality has not been investigated. In the previous studies19, "°, it was reported that the weight of~j cerebellum did not increase after 10 days of age, and the cerebellar lobuli which developed earlier were less affected i n j j cerebellum. The explanation for such a development-dependency of cerebellar lesion in Ii/Gunn rat was suggested to be in the characteristic nature of rat cerebellar development, and not in the brain bilirubin level, because brain bilirubin level injj Gunn rat decreased after birth and no selective accunaulation of bilirubin was observed in./j cerebellum e~. It is well known that the rat cerebellum develops by postnatal neurogenesis, showing highly active cell proliferation and differentiation after birth" ~,~L Therefore, it seems reasonable to consider that bilirubin might interfere with cerebettar DNA synthesis either directly or indirectly, resulting in cerebellar hypoplasia. Thus we focused the investigation on the possibility that cerebellar DNA replication might be impaired in the./'j Gunn rat. In the present paper, the changes of DNA content and [aH]thymidine incorporation rate into brain D N A during the postnatal development ofjj and heterozygous (Jj) Gunn rats were investigated. Next, our investigation was extended to the activity of brain thymidine kinase, which is presumed to play an important role in DNA synthesis~--s, "~7.The possible regulatory mechanism in cerebellar cell proliferation by Purkinje cell is also discussed. Preliminary communications of part of tiffs work have been presenteda°,aL MATERIALS AND METHODS
Animals Male jj and Jj Gunn rat littermates born from Jj mothers and .jj fathers were sacrificed by decapitation. The brain was divided into cerebellum and non-cerebellar parts. Six animals were used in each study unless otherwise mentioned.
Extraction and estimation of D NA Extraction of D N A was carried out by the modified Schmidt-Thannhauser procedure 29. DNA was estimated spectrophotometrically by absorbance at 260 nm, using calf thymus DNA (BDH) as a standard. Incorporation of [6-3H]thymidine into D N A was studied in rats which had received a subcutaneous injection of 2 #Ci/g body weight of [6-3H]thymidine (14.5 Ci/mmole, Daiichi Pure Chemicals). Two hours later, the animals were decapitated and the cerebellum and non-cerebellar parts of the brain and liver were removed. After the extraction and the estimation of DNA according to the above method, the incorporation of [6-3H]thymidine into DNA was measured in an Aloka scintillation spectrometer using toluene-Triton X-100 scintillation fluid (PPO, 0.5 '~, dPOPOP, 0.03 ~).
297
Estimation of thymidine kinase activity The activity of thymidine kinase was assayed by the method of Taylor et al. ~7. Cerebellum and non-cerebellar parts of the brain were homogenized with 5 vol. 0.02 M Tris • HCI buffer, pH 7.5, containing 1 m M EDTA and 2 m M mercaptoethanol. The homogenate was centrifuged at 105,000 × g for 60 rain. The supernatant was used as a crude enzyme preparation. The reaction mixture consisted of 5 m M MgCl2, 10 m M ATP, 2,uM [6-3H]thymidine with specific radioactivity described above, 105,000 x g supernatant, and 0.1 M Tris • HCI buffer, pH 7.5, to attain a final volume of 200/~1. Incubation was carried out at 30 °C for 15 rain, and the reaction was stopped by boiling for 3 rain. One hundred/~1 of reaction mixture was spotted on 1.8 sq.cm of DEAE-paper (Toyo Filter Co.). The paper was washed by suspending in 1 m M ammonium formate for four 15 rain periods, then washed with methanol, dried, and counted with toluene scintillation fluid (PPO, 0.5 °//,,, dPOPOP, 0.03 ~), in a liquid scintillation spectrometer. Protein was determined by biuret reaction with bovine serum albumin as a standard. Enzyme activity was presented as counts/rain/rag protein. At 2 and 4 days of age, the tissues of two animals were combined for the estimation of thymidine kinase activity.
Effect of bilirubin on the cerebellar thymidine kinase activity Bilirubin solution was prepared immediately before use by dissolution in 0.1 M Na2CO3 and dilution with 0.1 M Tris • HCI buffer, pH 7.5. In the experiment of bilirubin inhibition, 10/zl of bilirubin solution was added to the reaction mixture, bringing the final volume to 200 #1. The final pH remained at 7.5. The cerebella of 12-day-old Jj rats were used for enzyme preparation. Bilirubin levels of brain and liver subcellular fractions were determined by the method previously reported z0. Twelve day-oldjj rats were anesthetized with ether and perfused with 0.25 M sucrose. The removed brains and livers were homogenized with 5 vol of 0.25 M sucrose, centrifuged at 105,000 × g for 60 rain. Bilirubin contents of homogenates, 105,000 x g supernatants, and pellets suspended in 0.25 M sucrose were determined. The effect o f j j cerebellar cytosol on thymidine kinase activity in Jj cerebellar cytosol was studied using 12-day-old Jj a n d j j Gunn rats in which the effect of photoirradiation on thymidine kinase activity in jj cerebellum was also investigated. The 105,000 × g supernatants of Jj and jj cerebellum were irradiated by fluorescent lamp for phototherapy (FL 20BW-NU, National) for 1 h at 4 °C. In such a situation, bilirubin in the,/.'/sample was completely degraded. RESULTS
Developmental change in DNA synthesis in Gunn rat brain The postnatal change in total DNA content of Gunn rat brain is shown in Fig. 1. In the non-cerebellar parts of the brain, DNA content of both Jj a n d j j rats showed the same developmental pattern, and rose gradually until 12 days after birth and then leveled off. Cerebellar DNA content in both Jj and jj rats increased linearly during the first 10 days, and Jj cerebellar DNA content increased continuously thereafter,
298
1.0
I
non
--
05
Jl
4
"
i
0.0
~-
~ ..... :....
f ... ~ _.
8
a _ _ _ x __ ---Joo 16 '
12
Age in Days Fig. 1. P o s t n a t a l c h a n g e in t o t a l D N A c o n t e n t of G u n n ra t brain. H e t e r o z y g o u s (
