Analysis of pyruvate dehydrogenase expression in embryonic mouse brain: localization and developmental regulation

Det~elopmental Brain Research, 77 (1994) 63-76

63

© 1994 Elsevier Science B.V. All rights reserved 0165-3806/94/$07.00

B R E S D 51729

Analysis of pyruvate dehydrogenase expression in embryonic mouse brain: localization and developmental regulation Fumie Takakubo, Hans-Henrik M. Dahl

*

The Murdoch Institute for Research into Birth Defects, Royal Children ~"tfo.spital, Flemington Road, Parkt'ilh,, Victoria 3052, Melbourne, Australia (Accepted 3 August 1993)

Key words: Pyruvate dehydrogenase; Expression; Mouse embryo; Development of brain; Energy metabolism; Brain malformation; Pyruvate dehydrogenase deficiency; Central nervous system

Brain malformations and neurological dysfunctions are often seen in pyruvate dehydrogenase (PDH) deficient patients. To understand these clinical presentations, we have analyzed the localization and developmental expression of P D H in the embryonic mouse nervous system. Immunostaining was performed to localize P D H E l a protein. P D H activities were measured before and after activation. PDH E I ~ m R N A levels were quantitated by reverse transcriptase-polymerase chain reaction. A b u n d a n t PDH Elc~ protein was localized in the central nervous system and other neural tissues in embryos at embryonic day (E) 11 onwards. The P D H activity was very low in E0 brain and it increased continuously until the end of gestation. The proportion of active form of P D H increased significantly in El5 brain. Analysis of the P D H E1 c~ m R N A showed that only the X-linked form of the gene was transcribed. The overall m R N A level of E9 brain was approximately 9351 of the adult value. It decreased gradually during embryogenesis. A large increase took place at the end of gestation. The m R N A level of PDH was approximately 100 times higher than that of the acetoacetyl-CoA thiolase gene. These results suggest that PDH E I ~ transcripts of E9 brain are not translated at a high level. The appearance of P D H activity and its increase during E l l and El5 are mainly due to increased levels of translation and activation of PDH. Increased P D H activity at the end of gestation is attributed to an increase in transcription. Our data to a large extent explain pathological presentations in P D H E l a deficient patients with congenital brain disorders.

INTRODUCTION Pyruvate dehydrogenase (PDH) complex is a multienzyme complex with molecular weight of approximately 7 × 10 6. It consists of seven components; PDH El, which is a tetramer of two Elc~ subunits and two El/3 subunits, E2, E3, protein X, PDH specific phosphatase and P D H specific kinase 26'42'43"56. The PDH complex catalyses the oxidative decarboxylation of pyruvate to acetyl CoA within the mitochondrial matrix. This process is an essential, irreversible and rate limiting step in aerobic glucose metabolism. The PDH complex connects the glycolytic pathway and the citric acid cycle, and plays a central role in the regulation of carbon flux from glucose into the citric acid cycle. Energy metabolism in adult brain is highly acrobic with an energy source restricted to glucose under the normal metabolic status. The PDH complex is there-

* Corresponding author. Fax: (61) (3) 348-1391.

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fore a key enzyme in the energy production in adult brain. The postnatal development of the PDH complex has been described in rat brains 5"~3"34"5s. However, almost nothing is known about PDH in embryonic brains and only a few reports on energy metabolism in embryonic brains have been published~'5'22'3'( Various congenital brain malformations and neurological dysfunctions are observed in PDH Elc~ deficient patients at birth, including cerebral atrophy, microcephaly and abnormal development of the corpus callosum, medullary pyramids and inferior olives. In addition, cystic or necrotic lesions are often seen in the cerebral cortex, cerebellum, the basal ganglia or brain stem in PDH E l a deficient patients ~'t5"4~'44. These clinical observations strongly suggest the importance of the PDH complex in embryonic brains especially during the formation and differentiation of these brain regions. The purpose of this study was to examine developmental profiles of PDH in embryos especially in developing brains in order to (1) understand the pathogenesis of variable congenital brain malformations and neurological dysfunctions in PDH deficiency,

~4

(2) e l u c i d a t e r e g u l a t i o n p a t t e r n s of P D H activity d u r i n g the e m b r y o n i c b r a i n d e v e l o p m e n t a n d (3) o b t a i n p r o files of the e n e r g y m e t a b o l i s m in e m b r y o n i c brains. E m b r y o s a n d d e v e l o p i n g b r a i n s from early o r g a n o genesis stage ( e m b r y o n i c day (E) 9) up to the e n d of g e s t a t i o n ( E l 8 ) w e r e e x a m i n e d b e c a u s e the m a i n form a t i o n a n d d i f f e r e n t i a t i o n of v u l n e r a b l e b r a i n r e g i o n s in P D H deficiency occur d u r i n g t h e s e stages in n o r m a l b r a i n d e v e l o p m e n t . It is k n o w n that d u r i n g starvation the a d u l t b r a i n utilizes k e t o n e b o d i e s as an a d d i t i o n a l source of energy. In suckling pups, the b l o o d k e t o n e b o d y level i n c r e a s e s d u e to the high fat c o n t e n t o f m o t h e r ' s milk. D u r i n g this p e r i o d , k e t o n e b o d i e s a r e an i m p o r t a n t s o u r c e of the e n e r g y in b r a i n s of rat zg, dog 49 a n d h u m a n 4°. A c e t o a c e t y l - C o A thiolase (thiolase) g e n e r a t e s acetyl C o A from the k e t o n e s in k e t o n e b o d y m e t a b o l i s m . F o r t h e s e reasons, the d e v e l o p m e n tal p a t t e r n of the expression o f the thiolase g e n e was e x a m i n e d and c o m p a r e d with that of the P D H gene. In this study, we have f o c u s e d on the P D H E l a s u b u n i t b e c a u s e ; (1) a l m o s t all m u t a t i o n s in P D H deficiency i d e n t i f i e d so far are in the P D H E l a subunit, (2) the activity o f the w h o l e P D H c o m p l e x is r e g u l a t e d by p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n by P D H phosp h a t a s e a n d P D H kinase t h r o u g h t h r e e s e r i n e r e s i d u e s in the P D H Elc~ s u b u n i t 35'46'57'5° and (3) the Elc~ s u b u n i t also c o n t a i n s b o t h a p y r u v a t e b i n d i n g site a n d the cofactor, t h i a m i n e p y r o p h o s p h a t e ( T P P ) b i n d i n g site 3'~a3°'5~. T h e P D H c o m p l e x is inactive when phosp h o r y l a t e d a n d can be r e a c t i v a t e d by d e p h o s p h o r y l a tion. T h e level o f active, d e p h o s p h o r y l a t e d P D H complex is d e p e n d e n t on the m e t a b o l i c e n v i r o n m e n t a n d i n f l u e n c e d by insulin action. D a h l et al. ~6 f o u n d two isozymes o f the P D H E l a s u b u n i t which a r e e n c o d e d by two d i f f e r e n t genes. O n e of t h e isogenes is e x p r e s s e d only in s o m a t i c tissues a n d l o c a t e d on Xp22.1 r e g i o n in h u m a n s ~° a n d the F 3 - F 4 region o f t h e X - c h r o m o s o m e in mice II ( X - l i n k e d form of t h e P D H Elc~ gene). This form o f the P D H E l a g e n e in h u m a n s spans a p p r o x i m a t e l y 17 k i i o b a s e s (kb) a n d c o n t a i n s 10 i n t r o n s 37. T h e o t h e r P D H E l c~ g e n e a p p e a r s to b e specifically e x p r e s s e d in testis. This form o f the P D H E l a gene is intronless and localized on c h r o m o s o m e 4, q 2 2 - 2 3 r e g i o n in h u m a n s m'~6, a n d n e a r t h e c e n t r o m e r e o f c h r o m o s o m e 19 in mice ~'2°. Expression of the testis-specific P D H Elc~ g e n e is r e g u l a t e d d u r i n g s p e r m a t o g e n e s i s in mice, the level b e i n g highest at the p a c h y t e n e s p e r m a t o c y t e stage 2s'52. e D N A clones for the m o u s e P D H Elc~ s u b u n i t of b o t h the X - l i n k e d form a n d testis-specific form have b e e n i s o l a t e d a n d c h a r a c t e r i z e d e°. This has e n a b l e d us to identify the P D H E l c~ i s o g e n e e x p r e s s e d d u r i n g e m b r y o n i c b r a i n d e v e l o p m e n t in mice a n d q u a n t i t a t e m R N A levels of

the P D H Else gene. T h e e x p e r i m e n t s were p e r l 0 r m e d by reverse t r a n s c r i p t a s e - p o l y m e r a s e chain r e a c t i o n ( R T - P C R ) using specific p r i m e r s e i t h e r to the X-linked form o r to the testis-specific form. W e have e x a m i n e d the localization of P D H E l , p r o t e i n by i m m u n o s t a i n i n g . T h e P D H a n t i b o d y d o e s not distinguish b e t w e e n active and inactive P D H . W e have t h e r e f o r e m e a s u r e d P D H activity in e m b r y o n i c m o u s e b r a i n b e f o r e and after activation. In a d d i t i o n , levels Of P D H El~x m R N A w e r e q u a n t i t a t e d . Since the m a j o r i t y of the b r a i n cells are diploid is'el, we quantit a t e d D N A o f the b r a i n a n d the d a t a were used to express P D H activities a n d m R N A levels in r e l a t i o n to cell n u m b e r . T h e s e d a t a are i m p o r t a n t for u n d e r s t a n d ing the p a t h o g e n e s i s of P D H deficiency. T h e y are also useful to u n d e r s t a n d the d e v e l o p m e n t a n d m e t a b o l i s m of e m b r y o n i c brains.

M A T E R I A L S AND METHODS

Animals ARC mice, obtained from the Mouse Facility at the Royal Children's Hospital (Melbourne, Australia), were used. Male and female mice were placed together overnight. The morning that the vaginal plug was found was considered to be day 0 of gestation (E0). Embryos were dissected and brains were isolated at embryonic day 9 (E9), Ell, El5 and El8. Embryonic stages were assigned by counting somite numbers, measuring the crown-rump length and gross observation of embryos and foetuses. E9 embryos with 23-24 somites, E11 embryos with 30 in tail somite number, El5 embryos and El8 foetuses with normal development according to the definition by Theiler 53 were chosen for the study: The tissues were immediately frozen in liquid nitrogen and stored at -70°C until used.

Antibody staining E9, E l l and E15 embryos were freed from extra,embryonic tissues in ice-cold phosphate-buffered saline (PBS). El8 foetuses were dissected and El8 brains were isolated in ice-cold PBS. These samples were immediately fixed with Bouin's fixative for 2-4 h at room temperature. Samples were dehydrated in an ethanol series and embedded in paraffin. After deparaffinization and re-hydration, sections (5 mm) were immersed in 1% hydrogen peroxide solution in methanol for 5min to block endogenous peroxidase activity. Normal sheep serum (10%) in PBS was applied to the sections for 15 min to block non-specific binding. Immunoperoxidase staining was performed as described in a previous paper 52. Biotinylated donkey anti-rabbit lg (Amersham) diluted 1:200 and Streptavidin-biotinylated horseradish peroxidase complex (Amersham) diluted in 1 : 100 with 2% sheep serum in PI3S were used as a second and a third label respectively. For PDH Elot-antibody staining, rabbit antibody (IgG fraction) to bovine heart PDH E l a 7'1°'55 was used as a primary antibody. Equivalently diluted rabbit non-immune IgG fraction was applied to the sections for negative controls. To examine the localization of neurofilament polypeptides in embryonic brains, sections were incubated with rabbit antisera against bovine neurofilament polypeptides (150 kDa) (Chemicon International, Inc.) diluted 1:500 with 2% normal sheep serum in PBS. Equivalently diluted rabbit non-immune serum was applied to the sections for the negative control. The coloured reaction products were developed with diaminobenzidine (DAB) and hydrogen peroxide. The sections were counterstained with hematoxylin to stain the nucleus and mounted for photomicroscopy.

65

Estimation of cell number Fresh tissues (100-200 rag) were weighed and cut into small pieces. They were incubated with 0.1% collagenase (Sigma, Type I) and 300 U of bovine testicular hyaluronidase (Sigma, Type I-S) in PBS (Mg 2+ and Ca2+-free) for I h at 37°C with mechanical shaking. Trypsin (Flow laboratories) at a final concentration of 0.25`% was added and cells were further incubated for another 1 h at 37°C with mechanical shaking. Pipetting was performed occasionally during the incubation to dissociate cells completely. Cells were counted under the microscope with a N e u b a u e r haemocytometer. For E9 brains, brains from one litter were collected in one petri-dish and treated together as described above. Six litters each at stages of E9 and E11, 20 brains each at El5 and El8 were examined in total. Numbers of cells per brain were calculated for each developmental stage.

Acticity assay of PDHt. Homogenization buffer for the activity assay of PDHt was the same buffer used for P D H a except that the following modifications were made. In order to determine the activity of PDHt, P D H phosphatase was activated by (1) adjusting MgCI 2 concentrations to 10.24 m M and 2.5 m M in homogenization buffer and the assay mixture respectively, (2) adjusting CaCI 2 concentrations to 0.5 m M in the homogenization buffer and (3) leaving E D T A out of the homogenization buffer and the assay mixture. To avoid the inhibition of P D H phosphatase, no NaF was added to the homogenization buffer and the assay mixture. PDtt was activated by adding D C A to both homogenization buffer and the assay mixture. Homogenates were prepared as described above and supernatants were incubated for 15 rain at 37°C to activate all P D H complex by P D H phosphatase-mediated dephosphorylation. The incubation and the assay of radioactivity were performed as described above.

Estimation ~?/'DNA D N A determinations were performed according to the method described by Labarca et al. (1980) 31 using 1 m g / m l Hoechst H 33258 in phosphate-saline buffer (0.05 M Sodium p h o s p h a t e / 2 . 0 M Sodium chloride, pH 7.4) and calf thymus D N A as the standard. Three ml of phosphate-saline buffer was added to 5, 10, or 20 /xl of the 10% homogenates, along with 3 ml of the dye solution. The fluorescence was measured at excitation 356nm and emission 458nm. T h e homogenization buffers for the P D H enzyme activity assay and the guanidium chloride buffer to extract R N A were found not to interefere with the assay at the level used.

Protein assay

DNase digestion of total RNA Because the mouse testis-specific form of the PDH El~r gene is intronless 21~, contaminating genomic D N A in total R N A preparations will be amplified by P C R with primers specific to the testisspecific form of the PDH E1c~ gene (see below). In order to avoid PCR amplification of genomic DNA. 5 mg of total R N A was digested with 27 U of RNase free-DNase (Boehringer M a n n h e i m ) at 37°C for 2 h. The buffer consisted of 50 mM Tris-HCl buffer (pH 7.5), 10 m M MgCIz, 0.1 m M D T T and 50 U RNase-lnhibitor. The samples were then boiled at 95°C for 5 rain, chilled in ice and ethanol precipitated. DNase digested R N A (5 rag) were used for c D N A synthesis and PCR amplifications with testis isoform specific primers.

Protein contents of samples were determined by the method of Lowry 3~, using albumin protein (Sigma Diagnostics) as a standard.

Identification of mouse PDH Ela gene e~pressed during embryonic brain det,elopment RNA ~<~traction Frozen tissues were homogenized and total R N A was extracted with 4M guanidinium thiocyanate in 0.05 M Tris buffer (pH 7.6) containing 12.5 m M E D T A , 2`% sodium N-laurosarcosinate and 1% beta-mercaptoethanol 6`H. The homogenate was layered over 0.5 ml of cesium chloride (5.7 M CsC1 z / 0 . 0 1 M E D T A , p H 7.5). Total R N A was purified by a centrifugation at 50,000 rpm for 2.5 h at 6°C (Beckman TL-100 Ultracentrifuge, TLS-55 rotor), and followed by ethanol precipitation. R N A samples were stored at - 7 0 ° C until required, Estimation of total R N A was performed by measuring optical density (OD2e,o).

Pyrut,ate dehydrogenase actit,ity assay Activities of the active (dephosphorylated) form of pyruvate dehydrogenase (PDHa) and total pyruvate dehydrogenase (PDHt; the activity after dephoshorylation of inactive PDH) were determined by m e a s u r e m e n t s of t4CO2 production based on [1-taC]pyruvate decarboxylation, by modifying the method of Sheu et al. 46. Actit'ity assay of PDHa. The activity of P D H a was determined by (1) inactivating P D H phosphatase by chelating Mg 2+35 and Ca 2+ 17 by adding E D T A to both homogenization buffer and the assay mixture, (2) inhibiting P D H phosphatase by adding NaF 23 to both homogenization buffer and the assay mixture, and (3) inactivating P D H kinase by adding dichloroacetate (DCA) 54 to both homogenization buffer and the assay mixture. Frozen tissues were weighed and homogenized with ice-cold buffer containing 0.6 m M Na2SO4, 0.6 m M E D T A , 0.6 mM dithiothreitol, 15 m M NaF, 5 m M D C A and 40% glycerol. H o m o g e n a t e s were further frozen and thawed three times, then spun at 2,000 rpm for 10 rain at 4°C. Supernatants were immediately used for the enzyme activity assay. The assay mixture consisted of 40 m M potassium phosphate buffer (pH 7.4), 1.3 m M E D T A , 4 mg bovine serum albumin, 0.15 m M CoA, 1.6 mM NAD, 0.9 m M TPP, 1 m M Na2SO3, 1% rat serum, 5 m M DCA, 15 m M NaF, 0.28 mM[1-14C]pyruvate (specific activity: 2,065 d p m / n m o l e , NEN) and homogenate. The assay mixture was incubated for 30 rain at 37°C in the presence of 0.2 ml hyamine hydroxide. The reaction was stopped by injecting 0.5 ml of 50% acetic acid and the incubation was continued for 15 min. The radioactivity associated with hyamine hydroxide was counted in non-aqueous scintillant.

Reverse transcriptase (RT)-PCR experiments were performed to identify the isogene of the P D H E l a subunit expressed during embryonic brain development, c D N A was synthesized from 5 mg of total R N A by incubating samples at 42°C for I h with 25 U of Avian Myeloblastosis Virus Reverse Transcriptase (Boehringer Mannheim), 50 ng oligo dT and 300 tzM of each dATP, dCTP, d G T P and dTTP in the mixture of c D N A buffer (50 m M Tris-HCI, pH 8.3, 6 m M MgCI 2, 10 m M D T T and 100 mM NaCI) and 50 U of RNase-lnhibitot ( H u m a n placenta, Boehringer Mannheim). The following PCR was performed with specific primers to the X-linked form of the P D H Elc~ gene (pdha-1) or the testis-specific form of the P D H Elo~ gene. The X-linked isoform specific primers were PDH-12 (5'CCAGTGTGGAAGAACTAAAG-3') and mPDHS-3 (5'T T C A A G C C T T T T G T T G T C T G - 3 ' ) , which amplify 256 base pairs (bp) of the pdha-1. The conditions used for PCR amplification was 2 min at 95°C, 2 min at 57°C and 2 min at 72°C, and 35 cycles were conducted. The testis isoform specific primers were m P D H T - A (5'T T C G G G A G G C A A C C A A G T T T - 3 ' ) and m P D H T - 3 ( 5 ' - G A A G T T TCCTAGAGTACACC-3'), which amplify 452 bp of the testisspecific form of the mouse P D H gene. The conditions used for PCR amplification were 2 min at 95°C, 2 min at 59°C and 2 min at 72°C, and 35 cycles were conducted. PCR was performed with the PCR mixture consisting of 200 /xM of each deoxy nucleotides (dATP, dCTP, d G T P and dTTP), 50 mM Tris-HCl (pH 9.0), 20 m M (NH4)2S04, 1.5 m M MgCI z, oligonucleotide primers (1 /zg of each), and 1 unit Replinase (NEN). RT-PCR products were analyzed on a 2.5'% NuSieve gel ( F M C BioProducts).

E~'aluation of expression let'els qf pdha-l, 3-actin gene attd thiolase gctle To estimate m R N A levels of the X-linked form of the P D H E l a gene (pdha-1), /3-actin gene and thiolase gene, quantitative PCR experiments were performed. Total R N A was extracted from embryonic brains at E9, E l l , E l 5 and E l 8 and from adult brains, c D N A s were synthesized as described above. The following PCR amplification was performed in a tube containing the three sets of primers: pdha-1 specific primers were ms PDH-1; 5 ' - G C T G G T T G C T T C C C G T A A T T - 3 ' and ms PDH-A; 5 ' - A G G T G G T C C G T A G G G T T TAT-3', which amplify a region of 273bp; ,8-actin gene specific

primers were m/3A-l; 5 ' - A T G G G T C A G A A G G A C T C C T A G ' and m/3A-A; 5'-TCCATACCCAAGAAGGAAGG+3', which amplify 671 bp of the coding region; acetoacetyl-CoA thiolase specific primers were Th-l: 5'-GAGACCATGTCAAATGTCCC-3' and Th-A: 5'TCATGAGAACCACAGCAGCT-3', which amplify 430 bp of the coding region. PCR was performed in the PCR mixture consisting of 200 #M of each deoxy nucleotides (dATP, dCTP, dGTP and dTTP), 50 mM Tris-HCl (pH 9.0) and 4 mM MgCI 2, Ill #Ci ~2P-dCTP (NEN), 2U of Replinase (NEN) and three sets of oligonucleotide primers described above (0.4 /zg of each). Samples were amplified for 12-27 cycles of 2 min at 95°C, 2 rain at 60°C and 2 min at 72°C with total volume of 100 /zl. The other primer set specific Io the pdha-1 described before (PDH-12 and mPDHS-3) was not used in this experiment because it required the lower annealing temperature. Ten #1 of PCR products were electrophoresed in a 55~ acrytamide gel and bands with correct sizes were excised from the gel and quanlitated by liquid scintillation counting.

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RESULTS

De~,elopmental profile of embryonic brains An great increase in cell number per whole brain occurred during E9 and El5 in mouse embryos. The increase in cell number was about 4.9-fold between E9 and E l l , and 7.7-fold between E l l and El5. However, the increase in cell number appeared to be only 1.4-fold between El5 and El8 (Fig. la). Fig. lb shows that there is an increase in protein/DNA ratio of embryonic brain cells during E11 and El8. The level of the increase in protein/DNA ratio was greater during El5 and El8 than during E l l and E15. Fig. lc shows that during E l l and E15, R N A / D N A ratio remains almost constant and it increased greatly during El5 and E18. In E9 brain, the protein/DNA ratio was higher than in E l l . R N A / D N A ratio appeared to be 4-fold of the adult value in E9. From these data, we conclude that there are two phases in the development of mouse embryonic brains during E l l and El8. From E l l up until around E15, there is a great cell multiplication with a slight increase in protein content and with a proportional increase in RNA content. Between El5 and El8, the rate of cell multiplication slows down and cells develop by increasing in synthesis and accumulation of protein and RNA.

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Fig. 1. Developmental profiles of the brain growth in mouse embryos, a: number of cells per whole brains; six litters each at stages of E9 and E l l , 20 brains each at El5 and E18 were examined in total. Results of brains at the same developmental stage agreed in the range of _+ 10%. b: ~,g protein/ /~g DNA ratio; four litters each at stages of E9 and E l l , 12 brains each at E15 and El8 were examined in total. Results of brains at the same developmental stage agreed in the range of ± 10%. c: Izg RNA//zg DNA ratio; four litters each at stages of E9 and E l l , 12 brains each at El5 and El8 were examined in total. Results of brains at the same developmental stage agreed in the range of ± 10%.

Developmental profile of PDH activity in embryonic brains Developmental profiles of PDHa (PDH in active form before activation) and PDHt (PDH in active form after activation) in embryonic mouse brains are shown in Figs. 2a and 2b. As shown in Fig. lb, protein

contents of embryonic brain cells vary depending on developmental stages. In order to obtain precise developmental profiles, PDH activities are expressed as nmol Z4CO2 produced/mg protein/min in Fig. 2a and

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Fig. 3. Identification of P D H Elc~ isogene expressed in mouse embryonic brains and adult tissues, a: expression of the X-linked form of the P D H E l a gene (lanes 1-8): b: expression of the testis-specific form of the P D H E I ~ gene (lanes 1 9); M, 0X174 HaeIII marker; 1-3, adult tissues; 1, brain; 2, kidney; 3, testis; 4, E9 brain; 5, E l l brain; 6, El5 brain: 7, E l 8 brain; 8, no D N A control; 9, E9 whole-embryo. The bands show reverse transcriptase-PCR products from 5/zg of total R N A for each sample.

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activated. In E9 and E l l brains, PDHa accounted for 15% of PDHt, with an increase to 23% occurring between E l l and El5. A slight increase in this ratio was observed at El8. The developmental profile of PDH a c t i v i t y / D N A / m i n (Fig. 2b)was similar to that of PDH a c t i v i t y / p r o t e i n / m i n (Fig. 2a).

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Fig. 2. Activities of active P D H (PDH activity before activation) and total P D H (PDH activity after activation) in embryonic and adult mouse brains, a: the results were expressed as n tool 14C02 prod u c e d / m g p r o t e i n / m i n , b: the results were expressed as nmol 14C02 p r o d u c e d / g D N A / m i n , For each active P D H and total PDH, the values represent the mean of 12 brains from 4 different litters at E9, the mean of 6 brains from 4 different litters at E l l , the mean of 5 brains from 4 different litters at El5, the mean of 4 brains from 3 different litters at El8, and 3 adult brains. The values of brains at the same developmental stage agree within the range of ± 10%. *, active P D H activity; D, total P D H activity.

M

1

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4

5

6

7

bp 872 -

~ - 13-actin

603 -

*-- thiolase

310 -

nmo114C02 p r o d u c e d / g

D N A / m i n in Fig. 2b. Fig. 2a shows that the activity of P D H a increased as the brain developed. In E9 brains, the level of P D H a was approximately less than 1% of the adult value. A low PDHa activity (3%) was found in E l l brain, increasing significantly to 11% in El5 brain and to 15% in E l 8 brain. The activity of PDHt reflects the level of synthesis of the PDH complex. The developmental profile of PDHt activity was very similar to PDHa. The significant increase occurred during E l l and E15. The activity of P D H a was lower than PDHt, which indicates that the PDH complex in embryonic brains is not fully

281. 271

-,-- pdha-1 234

-

r ~

Fig. 4. Reverse transcriptase-PCR amplification of transcripts of X-linked form of the P D H E l a gene (pdha-1), /3-actin gene and acetoacetyl-CoA thiolase gene of mouse embryonic brains, adult brain, adult heart and adult kidney. M, 0X174 HaelI1 marker: 1, E9 brain; 2, E l l brain; 3, E l 5 brain; 4, E l 8 brain: 5, adult brain: 6. adult heart; 7, adult kidney. Total R N A (5 ~g) of each sample were reverse transcribed and amplified with pdha-I specific primers, the /3-actin gene specific primers and the thiolase gene specific primers. PCR products were separated on 5% acrylamide gel and visualized by autoradiography.

6~

PDH Elcr gene expressed during embryonic brain del:elopment Fig. 3a shows that the X-linked form of the PDH E l a gene (pdha-1) was expressed in embryonic brain development. The testis-specific form of the PDH E l a gene was not expressed during embryonic brain development from the early organogenesis stage until the end of gestation (Fig. 3b).

Quantitation of transcripts of pdha-1, ~-actin gene and thiolase gene Products of RT-PCR experiments were visualized by autoradiography as shown in Fig. 4. The data show specific amplifications of transcripts of pdha-1 (273

bp), thiolase gene (430 bp) and /3-actin gene (67l bp). The expression of the /3-actin gene was examined, in order to (1) compare the developmental profile with profiles of pdha-1 and thiolase gene and (2) check RT-PCR variability between experiments by examining ratios between pdha-1 signals and /3-actin signals. Linearity of RT-PCR experiments. To examine the linearity of RT-PCR experiments, the amounts of radioactivity recovered from the excised bands were plotted against the number of cycles on semi-log scale (Fig. 5). Regarding pdha-1, there is a linear relationship between cycle numbers and signals for all samples with the cycle number from 15 through to the cycle number 21 (Fig. 5a). This linear relationship shows the exponential increase in product with increasing cycle num-

a 100C0

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Fig. 5. PCR signal as a function of the number of cycles, a: pdha-I signal; b:/3-actin signal; c: thiolase signal; e, E9 brain; o , E l l brain; I , El5 brain; [3, El8 brain; A, adult brain. The results represent mean values of four sets of experiments. The values of each sample at each cycle point agreed within the range of ± 10%.

69

ber. With the/3-actin gene, the linear relationship was observed with the cycle number from 15 through to 24 (Fig. 5b). Therefore, we chose 18 cycles for the quantitation of PCR products from the pdha-1 or the/7-actin gene. With the thiolase gene, the linear relationship was observed with the cycle number from 21 through to 27 (Fig. 5c). We chose 24 cycles for the quantitation of PCR products from the thiolase gene. Fig. 6 shows standard curves for pdha-1 (Fig. 6a) and for/3-actin gene (Fig. 6b) obtained at the 18 cycle point, and for the thiolase gene (Fig. 6c) obtained at the 24 cycle point. Regarding pdha-1, a linear relationship was observed at concentrations (ng/100 /xl of PCR mixture) between 0.00005 and 0.01, with the signal range of 200dpm and 2,000dpm. As shown in Fig. 5a, signals of all samples at the 18 cycle point were in this range (approximately 200 dpm-600 dpm). The linear relationship for/3-actin was obtained at concentrations (ng/100 #1 of PCR mixture) between 0.01 and 1, with the signal range of 300 dpm and 5,000 dpm. From Fig. 5b, /3-actin signals of all samples at a 18 cycle point (approximately 2,000 dpm-4,500 dpm) were

within this range. The linear relationship for thiolase was obtained at concentrations (ng/100 ml of PCR mixture) between 0.5 x 10 -6 and 1 x 10 4, with the signal range of 100 dpm and 5,600 dpm. From Fig. 5c, thiolase signals of all samples at a 24 cycle point (approximately 130 dpm-400 dpm) were within this range. Variability between experiments. We have repeated RT-PCR experiments over a period of 2 months, using the same PCR machine. Standards were always included in each set of experiments. The data presented in this paper were the averages of at least four sets of experiments. Each sample was examined by duplication in each set of experiment. Their values agreed within the range of +10%. Ratios between pdha-1 signals and /3-actin signals in each sample were also checked.

Deuelopmental profiles of gene expressions of pdha-1, ¢3-actin gene and thiolase gene in embryonic brains Fig. 7 shows developmental profiles of pdha-1 (Fig. 7a), /3-actin (Fig. 7b) and thiolase (Fig. 7c) mRNA levels during embryonic mouse brain development. It is

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Fig. 6. P C R signal as a function of the amount of standard PCR products, a: pdha-1 signal; b: fl-actin signal; c: thiolase signal. The results represent m e a n values of four sets of experiments. T h e X axis represents the amount of standard P C R product (ng) in 100/zl of PCR mixture. The values of each sample agreed within the range of + 10%.

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Localization of PDH Ela protein in embryos

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In E9 brain, the m R N A level of pdha-I was approximately 93% of the adult value. The pdha-1 m R N A level gradually decreased up to El5. A dramatic increase in pdha-1 m R N A took place between El5 and El8. The level of pdha-I transcripts is approximately 0.014% of that of the /3-actin gene and approximately 100-fold of the thiolase gene during brain development. The /3-actin gene was expressed at the same level throughout the development except in E9 brain. The levels were approximately 4.8-fold of the adult value and approximately 7-fold in E9 brain. In E9 brain, cells contain RNA, the level being approximately 4-fold of the adult value measured by the R N A / D N A ratio (Fig. lc). This probably resulted in high levels of /3-actin, pdha-1 and thiolase m R N A s in E9 brain cells. In order to obtain precise developmental patterns of these gene expressions, the ratio of amounts of pdha-1 e D N A and amounts of /3 actin eDNA, and the ratio of amounts of thiolase c D N A and amounts of/3-actin e D N A were calculated. The results are shown in Fig. 7a and Fig. 7c respectively. With pdha-1, the level decreased in El5 brain and increased dramatically late in gestation. In contrast, the expression level of the thiolase gene increased gradually and constantly as brain developed.

i i i 4/ . . . . . . . . //, adult 9 11 15 18 Developmental stage (days of gestation)

0

Fig. 7. Developmental profiles of the transcriptional expression of pdha-1, /3-actin and thiolase genes in mouse embryonic and adult brains, a: expression pattern of pdha-1; b: expression pattern of /3-actin gene; c: expression of thiolase gene. The data are expressed as ng of eDNA per /zg of DNA (D). The results represent mean values of four sets of experiments. The bars indicate SE. Closed circles (e) represent ratios of PDH eDNA (ng/l~g DNA) and actin cDNA (ng//zg DNA) in Fig. 7a, and ratios of thiolase eDNA (ng//zg DNA) and actin eDNA (ng/~g DNA) in Fig. 7c.

not possible to measure m R N A and P D H activity in individual cells or brain structures. The results are therefore an average of P D H expression in the brain.

Localization of P D H E l a protein was examined by immunoperoxidase staining (Fig. 8). P D H shows as a brown staining. P D H Ela-immunoreactivity was observed in neural tissues and the heart in E9 embryos (data not shown). In the E l l embryo, the localization of the abundant P D H E l a protein was mainly limited to central and peripheral neural tissues and the heart (Fig. 8a,b,c). In the E15 embryo, P D H E l a protein was abundantly present in the central nervous system (Fig. 8d), ganglia and optic nerve (Fig. 8d). Low levels of the immunoreactivity for P D H E l a protein was observed in almost all tissues except for some negative cells in cartilages and bones (Fig. 8d). We detected variable levels of P D H E l a - i m munoreactivity in different regions of the developing brain as shown in Fig. le. The roof of diencephalon (Fig. 8e, white arrow 1) which is a presumptive epithalamus, the basal region of the mesencephalon (Fig. 8e, white arrow 2) which forms some motor neurons, and metencephalon (Fig. 8e, white arrow 3) which forms pons showed a higher level of P D H E l a protein relative to that of other regions in the E l l brain. The cerebral cortex of the frontal lobe in the E l 5 embryo (Fig. 8d, white arrows) showed a high level of P D H Ela-immunoreactivity. The frontal lobe regions in the

71

El5 embryo marked I and II in Fig. 8d were examined with the higher magnification and the results are presented in Fig. 8d-I and Fig. 8d-II respectively. It is clear that PDH E l a protein was present abundantly in

the cortical plate (Fig. 8d-I) comparing to the cortical neuroepithelium (Fig. 8d-II). Purkinje cells (Fig. 8g, arrows) of the cerebellum of the El8 embryo also showed strong staining.

Fig. 8. (legend on p. 72)

72

Fig. 8. Localization of PDH E l a protein and neurofilament polypeptides in mouse embryos. The brown colour indicates PDH E l a protein (in a, b, c, d, e and g), and neurofilament polypeptides (in D. Sections were counterstained with hematoxylin. White arrows indicate parts of the brain expressing high levels of PDH E l a protein (in d and e), and neurofilament polypeptides (in f). a: sagittal section of the E l l whole-embryo, b: saginal section of the E l l embryo, neck region; c: sagittal section of the E l l embryo, abdominal region; d: sagittal section of El5 head. The areas marked I and II are presented with the higher magnification in Fig. ld-I and Fig. ld-II respectively. Note that the abundant PDH E l a protein is observed in the cortical layer 1 and cortical plate (Fig. ld-I) comparing to that in the cortical neuroepithelium (Fig. ld-II); e,f: sagittal sections of E l l brains. White arrow 1, the roof of diencephalon; white arrow 2, the basal region of the mesencephalon; white arrow 3, metencephalon; g: sagittal sections of E18 cerebellum. Arrows indicate Purkinje cells. C1, cortical layer 1; Ce, cerebellum; Cx, cortical neuroepithelium; Cxp, cortical plate; Di, diencephalon; Fg, facial acoustic ganglion; Fn, facial nerve; G, ganglions; H, heart; Lv, lateral ventricle; Met, metencephalon; Mes, mesencephalon; Mn, mandibular nerve; Mye, myelencephalon; Op, optic nerve; Pk, Purkinje cells; Sc, spinal cord; Tel, telencephalon; Tg, trigeminal ganglion; Ver, cervical vertebrae; Vn, vagus nerve; 4V, 4th ventricle. Scale bar = 400 ~m in a and d; 100/zm in b, c, e and f; 40/zm in g; 10/zm in d-I and d-II.

T h e l o c a l i z a t i o n o f n e u r o f i l a m e n t p o l y p e p t i d e s (150 the same tissue preparation of Ell which

appeared

to h a v e

the

Ela-immunoreactivity

in E l l

b r a i n (Fig. 8e) w e r e also

protein on

p o s i t i v e by i m m u n o p e r o x i d a s e s t a i n i n g w i t h r a b b i t anti-

brains. T h e r e g i o n s

s e r a a g a i n s t n e u r o f i l a m e n t p o l y p e p t i d e s (150 k D a ) ( F i g .

kDa) was compared with that of PDH Ela highest

l e v e l of P D H

8f; b r o w n staining).

73 The results described above are based on the observation of series of sections of at least eight embryos from four different litters at each developmental stage. DISCUSSION

PDH E l a deficiency: clinical problems are related to deL~elopmental disturbance of the brain The clinical and pathological spectrum of P D H E l a deficiency is extremely broad. It ranges from fatal lactic acidosis in the newborn period to chronic neurological dysfunctions without severe lactic acidosis 7'9"44. Unlike many other inborn errors of metabolism, significant structural abnormalities in the central nervous system are observed in PDH E l a deficiency. In addition, these abnormalities are predominantly observed in females 9 and are usually present at birth. The cerebral malformations in PDH E l a deficiency are gross microcephaly and cerebral atrophy. Patients often have ventricular dilation, abnormally developed corpus callosum, absent or extremely hypoplastic pyramids, heterotopic inferior olives, focal cystic changes in the germinal matrix. Neurological dysfunctions lead to profound mental retardation, seizures, abnormalities of motor functions and blindness s'15'4~. The present study was conducted to understand the pathogenesis of PDH deficiency which leads to these structural brain malformations and dysfunctions. Due to difficulties and limitations in obtaining human tissues, mouse embryos were used in this study. Mouse is one of the 'non-precocial' species such as rat, dog and man, which are born in a relatively poor state of neurological development 5. In this sense, the results are relevant to discuss the pathogenesis of PDH deficiency. PDH E l a in embryos is encoded by the X-linked form of the gene Previously it was not known if PDH E l a in embryonic brain development is encoded by the testis-specific form or X-linked form of the PDH E l a gene. Our RT-PCR results clearly showed that P D H E l a protein in embryonic brains from E9 to E l 8 is indeed encoded by the X-linked form of the P D H E l a gene. Therefore, congenital brain disorders in P D H E l a deficiency in females can partly be explained by X chromosome inactivation'( However, this explanation is not applicable to male patients. PDH actiL'ity increases during brain deL,elopment, especially after mid organogenesis stage Among E9, E l l , El5 and E18 brains, the E l 8 brain showed the highest P D H a activity, which was approximately 15% (activity/protein) of the adult value. Kennedy et al. (1972) 2~ showed that the oxygen con-

sumption by the cerebrum remains at a low level at birth. Leong and Clark (1984) 34 examined the development of activities of pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, fumarase and D-3hydroxybutyrate dehydrogenase in rat cortex, cerebellem and medulla oblongata, from 2 days before birth to 60 days after birth. In all cases, great increases in enzyme activities were observed after birth and adult values were more than 2-fold of the values in embryonic brains. These results suggest that energy demands in embryonic brains are not as high as those required after birth. However, the mRNA level of pdha-1 was approximately 100 times higher than that of the acetylCoA thiolase gene which is a key enzyme in ketonebody metabolism. The data obtained by PDH activity assays suggest that an involvement of the PDH complex in embryonic brain development becomes more important after mid organogcnesis stage. Bennett et al. (1977) 4 observed increases in levels of activities of glycolytic and pentose phosphate shunt enzymes (hexokinase, phosphoglucomutase, phosphofructokinase, aldolase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) at 14 weeks of gestation in fetal human brains. The corpus callosum appears around 10 weeks 32 to 14 weeks 1'~ of gestation in human. In mouse, 15 days of gestation is a similar developmental stage to that of 14 weeks of gestation in human 2v. Angevine and Sidman 2 reported that the rapid proliferation of neural precursor cells within the neuroepithelium, which is accompanied by terminal differentiation, occur between El0 and E l 6 in mouse. Our results of cell number, p r o t e i n / D N A ratio and R N A / D N A ratio of whole brains support these findings. The /3-actin expression level observed between E l i and E l 8 was approximately 4.8-fold of the adult value. This probably reflects the dramatic brain development during this period, associated with the rapid proliferation and neurite extension of brain cells. These results suggests that PDH might be necessary to support the development of these neural precursor cells into neurons after E11. They also strongly suggest that this period will be a very critical period in brain development. A lack of PDH after mid organogenesis stage might cause malformations and dysfunctions of the brain, particularly in regions which develop mainly after mid organogenesis stage. These developmental profiles of PDH during embryonic brain development reflect embryonic energy metabolism. In mouse, the allantoic placenta is established between E9 and El0. After the establishment of allantoic circulation, the embryo is more dependent on aerobic energy production and the number of mito-

74 chondria increase. The increase of PDH protein after E l l might reflect an increased number of mitochondria. Late in gestation, after the completion of gross organogenesis, the level of transcription and the proportion of active PDH increase. This is likely to be a reflection of metabolic changes within the cells and the effect of insulin on PDH activity during and after organogenesis 33,45.

PDH Ela-immunoreactivity correlates with regions vulnerable to PDH deficiency It is clear that certain regions of the brain are often more severely affected than other regions in P D H deficiency 8"15'4l'44. This is not likely to be caused by local lactic acidosis, since accumulated lactic acid in female P D H deficient patients' cells can diffuse and be taken up by adjacent normal cells. Immunostaining showed that high levels of PDH E t a protein were localized in neural tissues in embryos, such as brain, spinal cord, ganglion and nerve during organogenesis. We detected variable levels of P D H Ela-immunoreactivity in different regions of the developing brain. The roof of diencephalon, basal region of the mesencephalon and metencephalon in E l l brain showed particularly high level of P D H E l a protein. These parts of the brain are often severely affected or malformed in PDH deficiency 15'44. The cerebral cortex of the frontal lobe in El5 brain and Purkinje cells in E l 8 cerebellum showed strong staining. These parts of the brain are often cystic and necrotic in P D H deficiency 15,44. It is known that levels of oxygen consumption vary in different anatomical areas of the brain 6°. Interestingly, Sokoloff et al. (1977) 48 have demonstrated that the cerebral cortex and the cerebellem in the rat consume 3-5 times more oxygen than do other regions. These results suggest the demands of P D H in these regions are higher and in the case of P D H deficiency, that might bring severer brain damages in these regions. We conclude that the parts of the embryonic brain with high levels of PDH E l a - i m munoreactivity are the most vulnerable to the effect of P D H deficiency. Immunostaining with E l l brain using an antisera against neurofilament polypeptides (150kD) revealed that neurofilament polypeptides were present in the regions expressing the highest level of P D H E l a - i m munoreactivity in E l l brain. These results suggest that neuroepithelial cells which differentiated into neurons in E l l brain produce PDH E l a protein at a high level. Regulation of PDH actiL,ity in the brain c'aries with embryonic stage The regulation of the activity of the P D H complex during embryonic brain development is as follows: de-

spite that pdha-I was transcribed, the mRNA level being more than 93% of the adult value in E9 brain. the activity of PDHt per DNA in E9 brain cells was only 1.2%. These results suggest that thc PDH E l a mRNA accumulates in brain cells, and it is not translated at a high level at the early organogenesis stage (E9). An increase of production of the PDH complex occurs between E l l and El5. The PDH complex is also functionally regulated at the post-translational level by activation of the PDHt. The proportion of active PDH increased in El5 and E l 8 brains. These ways of regulating PDH activity are presumably needed in order to respond to the rapid changes in embryonic energy metabolism. We have also analyzed activities of PDHa and PDHt per mg of protein in adult mouse brain and liver (data not shown). The proportion of PDHa to PDHt has been reported to vary widely in mammalian tissues. Our results in liver (33%; P D H a / PDHt = 3.97/11.87 ( n m o l / m g p r o t e i n / m i n ) × 100%) is consistent with results previously reported by Wieland et al. 56 and Siess and Wieland 47 (20-30% in rat liver). The data also indicate that the PDH complex in adult mouse brain is highly activated (49%; P D H a / PDHt = 19.64/39.87 ( n m o l / m g p r o t e i n / r a i n ) × 100%) compared to mouse liver (33%) and cultured human fibroblasts ( 5 - 1 0 % ) 46, and less activated than that in rat heart (approximately 60%) 24,38 and adipose tissues (approximately 60%) s°. In rats, the P D H activity per weight of brains increases approximately four fold during postnatal brain development, with the peak occurring at around 20 days. It then decreases to the adult value 5,34"58. Using rats, Cain et al. 1~ have shown that the magnitude of postnatal increase in PDH activity coincides with that of the PDH E l a back-titration phosphorylation increase. Our data show that 25% of PDH exists in the active dephosphorylated form at the end of gestation. In adult mouse brain, 50% of PDH exists in the active form. Increase in P D H protein and activity may enable the cells to respond to the rapid increase in demand for aerobic energy production at birth and during the postnatal period. CONCLUSION In conclusion, a high level of PDH protein exists in the central nervous system, other embryonic neural tissues and the heart in embryos at E l l and onwards. A detectable level of PDH activity is first found in E l l brain. The PDH activity increases as brain develops. The developmental expression is regulated mainly at translational level at the mid organogenesis stage, and post-translational and transcriptional levels later on.

75

The X-linked form of the PDH E l a gene is transcribed during embryonic brain development. The X chromosome location of the PDH E l a gene and brain's dependency on aerobic energy metabolism are impor. tant factors when trying to understand clinical variations in PDH deficient patients. It has become clear from our studies that cells with higher levels of PDH Elo~-immunoreactivity in normal brain development are especially vulnerable to decreased PDH activity. Our results are important to understand the pathogenesis of PDH deficiency, metabolic changes and energy requirements in the developing brain. The data are also relevant for understanding the pathogenesis of other disorders affecting the aerobic energy production in the embryonic brain. Acknowledgments. We gratefully acknowledge Miss E. Tsotsis (Murdoch Institute, Melbourne) for her technical assistance in determining enzyme activities. We thank Dr. G. Brown (Department of Biochemistry, University of Oxford) for providing a PDH E l a antibody, Miss D. Kirby (Murdoch Institute, Melbourne) for synthesizing the oligonucleotide primers, and Dr. D. Newgreen (Murdoch Institute, Melbourne) and Dr. D. Thorburn (Murdoch Institute, Melbourne) for their helpful advice and stimulating discussions. This work was partly supported by a Centre Block Grant from the National Health and Medical Research Council of Australia.

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