Point mutations of mitochondrial genome in Parkinson's disease

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 28 (1995) 281-295

Research report

Point mutations of mitochondrial genome in Parkinson's disease Shin-ichiro Ikebe a, Masashi Tanaka b, Takayuki Ozawa b,, a Department of Neurology, Juntendo University School of Medicine, Tokyo 113, Japan b Department of Biomedical Chemistry, Faculty of Medicine, University of Nagoya, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan Accepted 6 September 1994

Abstract

Oxidative stress and subsequent energy crisis have been proposed as the cause of nigral neuronal cell death in Parkinson's disease. We have reported defects in the mitochondrial respiratory chain and increased amount of deleted mitochondrial genome in the nigrostriatal system of patients with Parkinson's disease. Deletion in mitochondrial DNA could be ascribed to somatically acquired premature aging leading to cell death. To elucidate the contribution of maternally transmitted point mutations in mitochondriai DNA to the premature DNA damages, we employed a direct sequencing system and analyzed the total nucleotide sequences of mitochondrial DNA in the brains of five patients with idiopathic Parkinson's disease. There were no predominant point mutations among the patients in contrast to some neuromuscular diseases. However, each patient had several point mutations that would result in a significant change in the gene products. Some of these mutations may be involved either in the increased production of oxygen radicals from the mitochondrial respiratory chain or in the increased susceptibility of the respiratory chain components to oxidative damage. We propose that some of these mutations can be regarded as one of the risk factors accelerating degeneration of nigrostriatal pathway in Parkinson's disease.

Keywords: Parkinson's disease; Mitochondrion; Mitochondrial DNA; Nigrostriatal pathway; Oxidative damage; Point mutation; Complex I; NADH dehydrogenase

I. Introduction

Parkinson's disease (PD) is characterized by bradykinesia, tremor, rigidity of muscles, and postural instability that are based on the degeneration of nigrostriatal dopaminergic neurons. Abnormality of the mitochondrial electron transfer chain in the substantia nigra, skeletal muscle, and platelets of patients with PD has been reported by several workers [4,38,39,44,45] suggesting its etiological role in the pathogenesis of PD. In a recent study, we have shown that the proportion of nigral neurons with reduced immunostaining for Complex I was significantly larger in patients with PD than in control [15]. We have observed decreased amounts of Complex I subunits in the striatal mitochondria of parkinsonian patients using Western blotting [29]. Thus, mitochondrial dysfunction and energy

* Corresponding author. Fax: (81) 52-731-0293. 0169-328X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 9 - 3 2 8 X ( 9 4 ) 0 0 2 0 9 - 6

crisis are supposed to be important contributors to the pathogenesis of PD. It is actually documented that the amount of deleted mitochondrial D N A (mtDNA) was markedly increased in the striatum of patients with PD as compared with normal controls [18]. M t D N A is replicated with a high mutation rate [27] and without an efficient D N A repair system [10]. M t D N A is vulnerable to attack by reactive oxygen species and free radicals, because m t D N A is not bound to histones and is located just beneath the inner m i t o c h o n d r i a l m e m b r a n e w h e r e oxygen matabolism is actively carried out. Soong and associates reported that the level of deleted m t D N A was significantly higher in dopaminergic neurons than in other part of the brain [49]. Mann and associates reported that the accumulation of deleted m t D N A seemed to be an age-dependent phenomenon [28]. Although the deleted m t D N A was also detectable in the tissue of aged controls [18,26,50,58], kinetic PCR analysis demonstrated that the amount of deleted m t D N A was distinctly larger in a PD patient than in an

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age-matched control [33]. Therefore, we interpreted that the increased amount of deleted mtDNA in the parkinsonian striatum resulted from a premature aging process. The premature aging process in PD may be attributable to heritable point mutations in mtDNA. In a recent study, it is reported that 28% of patients with PD have a positive family history [25]. This finding could be explained by autosomal dominant inheritance with low penetrance, but it is also possible that some of these cases of familial PD are maternally inherited as in the case of chronic progressive external ophthalmoplegia [36]. Thus, we can hypothesize that some point mutations of mtDNA are involved in the defects in the respiratory chain in PD. In addition, the mutant gene products might impair molecular assembly of the mitochondrial respiratory chain, leading to increased leakage of reactive oxygen species from the chain. This increased leakage of free radicals from the respiratory chain may be responsible for the accumulation of oxidative damage to mtDNA itself, causing large deletions of mtDNA [16,17] and random point mutations at the time of replication [23]. The increased leakage of radicals would also cause oxidative damage to other cellular components, leading to degeneration of the nigral neurons. Alternatively, some of the amino acid replacements may render the mitochondrial gene products susceptible to oxidative damage. In the present study, the entire nucleotide sequences of mtDNA isolated from the striatum of patients with PD were analyzed by using a fluorescencebased automated direct sequencing system. The data presented here will provide an important clue to elucidate the role of point mutations resulting in significant changes in the gene products as risk factors for nigrostriatal degeneration both among patients with PD.

2. Materials and methods

2.1. DNA extraction Total DNA was extracted from 20 mg of the striatum as previous reported [18].

2.2. Oligonucleotide primers Primers were synthesized with a model 381A DNA synthesizer (Applied Biosystems, Foster City, CA) and then purified on oligonucleotide purification cartridges from Applied Biosystems according to the manufacturer's instruction. Our nomenclature system for primers is as follows. Primers L and H are 20-mer oligonucleotides possessing sequences specific for the light (L) strand and the heavy (H) strand of mtDNA, respectively. The 5' end of primer L(n) corresponds to

nucleotide position (10n + 1) and its 3' end to nucleotide position (10n + 20). Similarly, the 3' end of primer H(n) corresponds to nucleotide position (10n + I) and its 5' end to nucleotide position (10n + 20). For example, the primer L1641 is a 20-mer oligonucleotide 5'-CGTGAAATCAATATCCCGCA-3' corresponding to the light strand sequence from position 16411 to 16430; and the primer H136 is a 20-mer oligonucleotide 5'-CTGGGGTAGAAAATGTAGCC-3' corresponding to the heavy strand sequence from position 1361 to 1380. These sequences were selected so that no other homologous sequences (higher than 65-70% homology) of each primer were found in the entire mtDNA. Primers FL are 38-mer oligonucleotides possessing both the M13mpl8 forward universal sequence of 18 nucleotides (-21M13, 5'-TGTAAAACGACGGCCAGT-3') on the 5' side and a sequence of 20 nucleotides specific for the L strand of mtDNA at the 3' side [54]. For example, FL4 has the following sequence:

5'-TGTAAAACGACGGCCAGTCTCCATGCATTTGGTATTTT-3'. 2.3. First PCR reaction (symmetric) Thirty PCR cycles were performed with 5 ng of total DNA, 50 pmol of a pair of L and H primers, 200 tzM of each deoxyribonucleoside triphosphate (dNTP), 1.25 units of Taq DNA polymerase (Promega, Madison, WI) in 10 mM Tris-HCl, pH 8.3, 50 mM KCI, 1.5 mM MgC12, and 0.01% gelatin, in a total volume of 50 tzl. They were performed using a thermal cycler (PerkinElmer/Cetus. Norwalk, CT) with a cycle of denaturation at 94°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 40 s.

2.4. Second PCR reaction (asymmetric) Thirty PCR cycles were performed with 0.5 tzl of the first PCR product, 0.5 pmol of FL primer and 50 pmol of H primer, 20 txM of each dNTP, 1.25 units of Taq DNA polymerase in 10 mM Tris-HCl, pH 8.3, 50 mM KCI, 1.5 mM MgCI 2, and 0.01% gelatin in a total volume of 50 Izl with a cycle of denaturation at 94°C for 15 s, annealing at 55°C for 15 s and, extension at 72°C for 40 s. The amplified single-strand DNA template was stored at -20°C for 10 min after the addition of 5/~1 of 3 M sodium acetate, pH 7.4, and 100/xl of ethanol, and then centrifuged at 13,000 x g for 10 min. The precipitate was rinsed with 150 ~1 of 70% ethanol and then centrifuged at 13,000 x g for 5 min, dried in a vacuum chamber for 10 min, and then re-suspended in 8 #1 of distilled water. Combinations of primers for the first and second PCR amplifications are shown in Table 1. Ten overlapping fragments of 2-5 kb in length (fragments A - J in Table 1 and Fig. 1) were amplified in the first PCR

S-i. Ikebe et al. / Molecular Brain Research 28 (1995) 281-295

283

Table 1 List of primer pairs for sequencing of mtDNA First PCR

Second PCR

Analyzable region *

L Primer

H Primer

Fragment Name

FLPrimer

H Primer

L1641

H136

A

FL4 FL25 FL32

H60 H82 H82

1 2 3

61 271 341

411 621 691

L61

H339

B

FL61 FL87 FLll6 FL146 FL173 FL201 FL231

H136 H136 H280 H280 H280 H280 H280

4 5 6 7 8 9 10

631 891 1181 1481 1751 2031 2331

981 1241 1531 1831 2101 2381 2681

L23

H454

C

FL260 FL288 FL317 FL344

H339 H339 H394 H426

11 12 13 14

2621 2901 3191 3461

2971 3251 3541 3811

1_372

H559

D

FL372 FL403 FL434 FIA62

H483 H454 H509 H559

15 16 17 18

3741 4051 4361 4641

4091 4401 4711 4991

L488

H702

E

FL488 FL515 FL540 FL568 FL596 FL625 FL651

H559 H617 H594 H617 H650 H702 H702

19 20 21 22 23 24 25

4901 5171 5421 5701 5981 6271 6531

5251 5521 5771 6051 6331 6621 6881

L680

H929

F

FL680 FL704 FL731 FL761 FL790 FL820

H784 H815 H815 H854 H884 H884

26 27 28 29 30 31

6821 7061 7331 7631 7921 8221

7171 7411 7681 7981 8271 8571

L853

H1338

G

FL853 FL881 FL909 FL932 FL962 FL989 FL1019 FLI047 FL1076 FLll08 FLl138 FLl167 FLl192 FL1222

H929 H982 H982 H1014 H1043 H1043 Hl103 Hll0 Hl163 Hl189 H1219 H1219 H1275 H1308

32 33 34 35 36 37 38 39 40 41 42 43 44 45

8551 8831 9111 9341 9641 9911 10211 10091 10781 11101 11401 11691 11941 12241

8901 9181 9461 9691 9991 10261 10561 10841 11131 11451 11751 12041 12291 12591

L1250

H1534

H

FL1250 FL1280 FL1310 FL1341 FL1371 FL1396 FL1396 FL1429 FL1451

H1308 H1363 H1363 H1420 H1420 H1450 H1479 H1479 H1506

46 47 48 49 50 51 52 53 54

12521 12821 13121 13431 13731 13981 13981 14311 14531

12871 13171 13471 13781 14081 14331 14331 14661 14881

Fragment No.

From

To

284

S-i. lkebe et al. / Molecular Brain Research 28 (1995) 281-295

Table 1 First PCR

Second PCR

Analyzable region *

L Primer

H Primer

Fragment Name

FL Primer

H Primer

Fragment No.

From

To

L1482

H1619

I

FL1482 FL1512 FL1543 FL1569

H1534 H1560 H1619 H1619

55 56 57 58

14841 15141 15451 15711

15191 15491 15801 16061

L1594

H60

J

FL1594 FL1619 FL1641 FH60 * *

H60 H60 H60 L1594

59 60 61 62

15961 16211 16431 600

16311 16561 212 250

* Nucleotide position is according to Anderson et al. [1]. * * FH60 is useful for sequencing the C rich region from position 303 to 315.

step. Then the single strand DNA fragments were amplified with appropriate pairs of FL primers and H primers, the distance between which was 0.5-1.6 kb. Sequence of approximately 350-450 bp was analyzed from each template (fragments 1-62 in Table 1 and Fig. 1). Since the FL primers were synthesized approximately every 300 bp, the sequences were analyzed with overlapping of approximately 100 bp.

different concentrations of the template DNA. The sequencing results obtained from the Parkinsonian patients were compared with those obtained from controls using the template fragments prepared with the same primer pairs. The DNA sequences and the translated amino acid sequences were analyzed using a genetic information processing software GenetyxMac/CD, version 4.0.0 (Software Development Co., Tokyo, Japan).

2.5. Fluorescence-based direct sequencing 2.6. Clinical subjects A Taq sequencing kit was obtained from Applied Biosystems. The entire sequences of mtDNA were determined by a fluorescence-based automated direct sequencing technique using a model 373A DNA sequencer (Applied Biosystems) as described previously [54]. When there were some ambiguities in the sequencing data, sequencing was repeated using several

Clinical subjects consist of five patients with PD. All the PD patients studied had been treated with both levodopa and a peripheral dopa decarboxylase inhibitor until their deaths. Trihexyphenidyl and bromocriptine were used in Patients 1, 2 and in Patients 1, 3, 4, respectively, except for agonal periods.

Mitochondrial DNA 4000

8000

12000

I

ATPase8/6

A

C I ~

E

11 ~

2~ 3---]~

4 -----}~

22 . i ~

15

5~ 7 8

~--~ ~.~

9.-,-.--~I0 ---~-

17 ~ 18

~-

28 ~ 29

37 ~ >-38

42 ~ ~

5S

43 ~

56

3 4 ~ 39 ----]1~ 4 4 ~ 35 ~ 40 ~ 45 ~ 36 ---~1~ 41 ~ H

25~

F 26 27

>-

16 ~

6

33

21 ---lb.

14 ~ ,D

B

I

3 2 ~

20 - - - - ~ 4 ~

13 ------]D-

I

ND3

G

19----~3~

12 ~

16000

~

57 ,----]~ 58

j

46~

51 ~

47 ~r 30 ~

31 ~

48 ~ 49

~--52-----}~ 53 ~

59

60 61 ~

)~

~__62

50 ._~,$4 ---~,,-

Fig. 1. Strategy for sequencing of mtDNA. Bars (A to J) show the ten fragments amplified in the first PCR step. Arrows (1 to 62) indicate the second PCR products that are used for the templates for sequencing.

S-i. Ikebe et al. / Molecular Brain Research 28 (1995) 281-295

285

Table 2 List of individuals analyzed No.

Case 2

Age

Sex

Diagnosis

Reference

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

HCM1 HCM2 HCM3 HCM4 HCM5 HCM6 HCM7 HCM8 HCM9 ACM DCM1 DCM2 MELAS1 MELAS2 FICM FICMM Leberl CIVD MERRF CID2 CID1 CLA Pearson DM LGS Contl Cont2 Cont3 Cont4 Cont5

21 45 25 20 53 54 65 18 21 37 73 40 18 13 1 28 30 6 21 0 0.2 0 1 39 24 55 28 48 83 99

M M M M M F M F M M M M F M M M M M F F M F M F M M F F M M

Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy Apical hypertrophic cardiomyopathy Dilated cardiomyopathy Dilated cardiomyopathy Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes Fatal infantile cardiomyopathy Control Leber's hereditary optic neuropathy Myopathy with Complex IV deficiency Myoclonus epilepsy with ragged-red fibers Complex I deficiency Complex I deficiency Congenital lactic acidosis Pearson's syndrome Diabetes mellitus Limb-girdle syndrome Control (gastric cancer) Control (pulmonary embolism) Control (pneumonia) Control (hepatic failure) Control (unknown)

[31] [31] [31] Unpublished [31] [31] [31] [32] [32] Unpublished [35] [35] [20,53] [20,53] [31,52] Unpublished Unpublished Unpublished [59] Unpublished Unpublished Unpublished [43] Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished Unpublished

Table 3A Nucleotide changes in mtDNA of Patient 1 Position

Gene

Nucleotidechange

Amino acid change

Protein-coding genes (replacement of amino acid) 4824 ND2 A~ G Thr ~ Ala 8794 ATP6 C~ T His ~ Tyr

CD

Con or Non

X

P

S

D

Ch

R

M

B

H

Pt

Frequency

58 83

Con Con

T Y

T Y

L Y

M Y

L Y

T H

T H

T H

T H

A Y

1/30 1/30

Protein-coding genes (no replacement of amino acid) 3411 ND1 A --* G Lys 4248 ND1 T~ C lle 4655 ND2 G ~A Tyr 8563 ATP8 A~ G Pro 10730 ND4L A~ G Leu 11647 ND4 C~T Leu rRNA genes 663 12S rRNA 1736 16SrRNA 2151 16S rRNA

A~G A~G InsertionA

Major non-coding region 16290 C~ T 16319 G~ A The summary of nucleotide changes in mtDNA of Patients 1-5 (Table 3A-E). Con, conserved among human, bovine, rat, and mouse; Non, non-conserved amino acid; CD, chemical distance between the two amino acids investigated by using the procedure of Grantham [14], Position, nucleotide position of mitochondrial DNA sequence reported by Anderson et al. [1]; X, Xenopus laevis; P, Paracentrotus licidus; R, rat; S, Stongylocentrotus purpuratus; M, mouse; D, Drosophila yakuba; B, bovine; Ch, chicken; H, human. Amino acids are indicated with ~ingle-letter codes.

S-i. Ikebe et al. /Molecular Brain Research 28 (1995) 281-295

286

Table 3B Nucleotide substitutions in mtDNA of Patient 2 Position

Gene

Nucleotide change

Amino acid change

CD

Con or Non

R

M

B

H

Patient

Frequency

194

Non

I

1

V

Y

C

1/30

Protein-coding genes (replacement of amino acid) 14180

ND6

T~C

Tyr~C~s

Protein-coding genes(no replacement of amino acid) 3447 8020 8450 10181 13827 15217 15440 15805

ND1 CO2 ATP8 ND3 ND5 Cytb Cytb Cytb

A~G G~A T~C C~T A~G G~A T~C A ~G

tRNATM

A~G

Gin

Pro Leu Phe Gly Gly

Leu Val

tRNAgenes 15951

The histological diagnosis in all the PD cases examined was confirmed by the Departments of Pathology of Jichi Medical School or Juntendo University School of Medicine. All the patients fulfilled the pathological criteria of PD. Patient 1, a 51-year-old male, had an ll-year history of PD. He died of pneumonia. Microscopically, the lateral part of substantia nigra showed severe neuronal loss, but the neurons in the medial part of the substantia nigra was relatively preserved. Patient 2, a 65-yearold female, had a 5-year history of PD. She died of asphyxia caused by an intrabronchial mucous clot. The neurons in the medial part of the substantia nigra was relatively preserved compared with the lateral part. Patient 3, a 72-year-old male, had a 25-year history of

PD. He died of pneumonia. Microscopically, the neuronal loss was also dominantly found in the lateral part of the substantia nigra. The number of remaining neurons was very small. Patient 4, a 73-year-old female, had a 9-year history of PD. She developed disseminated intravascular coagulation and died of pneumonia. Microscopically, the nigral neuronal loss was extensive in both the lateral and the medial part. Patient 5, a 77-year-old male, had a 7-year history of PD. He died of pneumonia. The neuronal loss was severe in all parts of the substantia nigra. The m t D N A sequences of two patients (Patients 3 and 5 in the present study) were previously reported by Ozawa et al. [34] as PD2 and PD1, respectively. The profiles of control patients are summarized in

Table 3C Nucleotide changes in mtDNA of Patient 3 Position

Gene

Nucleotide change

Amino acid change

Protein-coding genes (replacement of amino acid) 5301 ND2 A ~ G lie --, Val 12026 ND4 A --, G lie ~ Val

CD

Con or Non

R

M

B

H

Patient

Frequency

29 29

Non Non

L M

M M

L I

1 1

V V

2/30 0/30

Protein-coding genes (no replacement of amino acid) 8071 CO2 A ~ G Ser 9180 ATP6 A ~ G Val 10397 ND3 A ~ G Trp 11944 ND4 T -~ C Val r R N A genes * 752 12S r R N A 1107 12S r R N A 1310 12S r R N A

C ~ T T -, C C -, T

Major non-coding region 43.1 16092 16164 16266

Insertion C T ~ C A -, G C -~ T

* 1438 A ~ G transition in the 12S r R N A gene, which was commonly observed in Patients 1, 2, 4 and 5 as well as in the controls, was not observed in Patient 3.

287

S-i. Ikebe et al. / Molecular Brain Research 28 (1995) 281-295

Table 3D Nucleotide substitutions in mtDNA of Patient 4 Position Gene Sequence change Amino acid change

CD

Con or Non

R

M

B

H

Patient

Frequency

Protein-coding genes (replacement of amino acid) 4602 ND 2 A~ G Thr -~ Ala 12358 ND 5 A -~ G Thr -~ Ala

58 58

Non Non

A I

I I

M S

T T

A A

0/30 0/30

Protein-coding genes (no replacement of amino acid) 5231 ND 2 G~ A Leu 5417 ND 2 G~ A Gin 12007 ND 4 G~ A Trp 12372 ND 5 G~ A Leu tRNA genes 4386 tRNAGIn

T~ C

Major non-coding region 16111 C -~ T 16257 C~ A 16261 C~ T T a b l e 2. N o n e of t h e c o n t r o l p a t i e n t s h a d any nigral or striatal lesions.

3. Results 3.1. N u c l e o t i d e substitutions in controls

We examined the profiles of the mtDNA sequences o f t h e five P D p a t i e n t s a n d t h o s e o f thirty d i s e a s e a n d n o r m a l controls. C o m p a r e d with t h e s e q u e n c e r e p o r t e d by A n d e r s o n et al. ( C a m b r i d g e s e q u e n c e ) [1] a n d with t h e s e q u e n c e s f o u n d in t h e d i s e a s e a n d n o r m a l controls, we e x c l u d e d n u c l e o t i d e s u b s t i t u t i o n s w h i c h h a d an i n c i d e n c e o f 5 a n d h i g h e r a m o n g 30 controls. N u c l e o t i d e s u b s t i t u t i o n s with i n c i d e n c e less t h a n 5 / 3 0 a r e listed in T a b l e s 3 A - E . C o m p a r e d with t h e C a m b r i d g e s e q u e n c e , s e v e r a l n u c l e o t i d e s u b s t i t u t i o n s (such as 73 A~G, 263 A ~ G , 750 A - ~ G , 2706 A ~ G , 3423 G ~ T, 4769 A ~ G, 4985 G ~ A , 7028 C ~ T , 8860 A ~ G, 9559 G ~ C, 11335 T -~ C, 11719 G ~ A , 13702

G~C, 14199 G ~ T , 14272 G ~ C , 14365 G ~ C , 14368 G ~ C, 15326 A ~ G ) w e r e f o u n d n o t only in t h e five P D p a t i e n t s a n a l y z e d b u t also in all t h e controls studied. A d e l e t i o n o f C at 3106 was also f o u n d in all t h e subjects analyzed. A n A ~ G t r a n s i t i o n at 1438, which was c o m m o n l y o b s e r v e d in P a t i e n t s 1, 2, 4, a n d 5 as well as in all t h e controls, was n o t f o u n d in P a t i e n t 3. T h e s e n u c l e o t i d e s u b s t i t u t i o n s w e r e n e g l e c t e d f r o m t h e p r e s e n t d a t a analysis. A l l o f t h e n u c l e o t i d e substitutions i d e n t i f i e d in this study w e r e a p p a r e n t l y in a h o m o p l a s m i c state, j u d g i n g f r o m t h e signal p a t t e r n s o f s e q u e n c i n g results. B e c a u s e we e m p l o y e d t h e d i r e c t s e q u e n c i n g m e t h o d using t h e P C R p r o d u c t s as the t e m p l a t e s for s e q u e n c i n g , t h e s e q u e n c e s o f m t D N A r e p o r t e d h e r e r e p r e s e n t t h e m a j o r p o p u l a t i o n of m t D N A in e a c h p a t i e n t . 3.2. Nucleotide changes in Patient 1

T h i r t e e n n u c l e o t i d e c h a n g e s w e r e f o u n d in P a t i e n t 1 ( T a b l e 3A). T w o n u c l e o t i d e s u b s t i t u t i o n s w e r e f o u n d in

Table 3E Nucleotide substitutions in mtDNA of Patient 5 Position

Gene

Sequence change

Amino acid change

Protein-coding genes (replacement of amino acid) 12358 ND5 A~ G Thr ~ Ala 15071 Cytb T~ C Tyr ~ His Protein-coding genes (no replacement of amino acid) 5231 ND2 G --* A Leu 8071 CO2 A~ G Ser 12372 ND5 G~ A Leu 14968 Cytb T~ C Phe Major non-coding region 16172 T~ C 16257 C~ A 16261 C -~ T 16304 T --, C 16497 A --* G

CD

Con or Non

R

M

B

H

Patient

Frequency

58 83

Non Non

I F

I F

S F

T Y

A H

0/30 1/30

288

S-i. lkebe et aL /Molecular Brain Research 28 (1995) 281-295

=01Vo'li "O

.E =,.. O.

V

"O "r

Pt.l 119Thr~ 3.0 1

la(4824A-~

)

I

i

i

1

51

101

151

201

251

Amino Acid Position Fig. 2. Hydropathy profiles of the h u m a n mitochondrial ND2 gene. The hydropathic index was calculated by using the procedure of Kyte and Doolittle [24]. The arrow indicates the change of hydrophobicity in Patient 1 that was caused by the replacement of 119 Thr to Ala in the hydrophilic region.

the non-coding region (C-to-T transition at 16,290 and G-to-A transition at 16,319). Eight nucleotide substitutions were found in the protein-coding region. Six were silent nucleotide substitutions causing no replacement of amino acids. Two nucleotide substitutions were nonsynonymous. The A-to-G transition at 4,824 replaces 119 Thr to Ala in the ND2. This threonine is conserved among four mammalian species (rat [13] mouse [3], bovine [2], and human [1]), Xenopus [42], and Paracentrotus lividus [7] but is not conserved in chicken [11], Strongylocentrotus purpuratus [21] or Drosophila [9]. The chemical distance (D) according to Grantham [14] between Thr and Ala was relatively large (D = 58). Computer analyses suggest that this amino acid replacement causes a change in the hydropathy profile at the third hydrophilic domain of the ND2 gene product (Fig. 2), and that this replacement also causes a change in the secondary structure of the gene product from the /3-sheet structure to a coil structure (Fig. 3). The C-to-T transition at 8,794 in the ATP6 gene replaces 90 His to Tyr with a large D value (D = 83). This His is conserved among four mammalian species but Tyr is

Normal ND2

]~

~27

!3-sheet | 119 Thr -~ Ala (4842 A -~ G) ~

,/9.~.).~. ~

/

g g g g"

~

¥

Coil

Pt.1 ND2

~

3~a

]~-1

Fig. 3. Secondary structure of the ND2 gene product that was calculated by the G E N E T Y X - M a c Ver 4.0.0 using the procedure of Chou and Fasman [8]. The structure of the /?-sheet at position 117 was transformed to a coil structure.

found in Xenopus, sea urchins, and chicken. The replacement of this amino acid also changes the hydropathy profile of this gene product (data not shown). The secondary structure analysis suggests that the coil structure at position 89 to 91 is transformed into a /3-sheet structure (data not shown). Three nucleotide changes were observed in rRNA genes: an A-to-G transition at position 663 in the 12S rRNA gene (Fig. 4), an A-to-G transition at position 1,736 in the 16S rRNA gene, and an insertion of A at position 2,151 in the 16S rRNA gene. Two transition nucleotide substitutions (C-to-T at 16,290 and G-to-A at 16,319) were found in the major non-coding region.

3.3. Nucleotide substitutions in Patient 2 Ten nucleotide substitutions were found in the mtDNA of Patient 2 (Table 3B). Nine nucleotide substitutions were found in the protein-coding region. Eight nucleotide substitutions were silent causing no replacement of amino acid. One nonsynonymous mutation (transition from T to C at position 14,180) was found in the ND6 gene. This T-to-C transition causes replacement of Tyr to Cys at position 164 with a very large D value (D = 194). This amino acid was n o t conserved among the species but Cys at position 164 was not observed in four mammalian species (human, rat, mouse, or bovine). This replacement of amino acid caused a change in the hydropathy profile at the last hydrophobic domain (Fig. 5), but caused little change in the secondary structure at this mutation site (data not shown). One point mutation (A-to-G transition at position 15,951) was found in the tRNA Thr gene. Fig. 6 represents the site of the mutation at the aminoacyl acceptor stem in tRNAXhL As shown in Table 4, base pairing of either T = A or C - G at this site is conserved among nine species (Drosophila to human).

289

S-L Ikebe et al. // Molecular Brain Research 28 (1995) 281-295 A C: A 1000

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3.4. Nucleotide changes in Patient 3 Thirteen n u c l e o t i d e changes found in Patient 3 are shown in Table 3C. In the protein-coding regions, four

synonymous nucleotide substitutions and two nonsynonymous nucleotide substitutions were found. Both of the two nonsynonymous mutations (A-to-G transitions at position 5301 in the N D 2 gene, at position 12,026 in

S-i. lkebe et al. / Molecular Brain Research 28 (/995) 281-295

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the ND4 gene) caused replacement from Ile to Val with a small D value ( D = 29). Both of the two lle residues are not conserved among four mammalian species (human, bovine, rat, and mouse). Neither the hydropathy profile nor the secondary structure were significantly changed by these two substitutions. Three nucleotide substitutions were observed in the 12S r R N A gene, which were transitions C-to-T at position 752, T-to-C at position 1,107, and C-to-T at position 1,310 (Fig. 4). Four nucleotide substitutions were also found in the major non-coding region. 3.5. Nucleotide substitutions in Patient 4

The nucleotide substitutions found in m t D N A of Patient 4 are shown in Table 3D. Six nucleotide substitutions were found in the protein-coding region: four of them were synonymous mutations; and two were nonsynonymous mutations. Both of these two nonsynonymous nucleotide substitutions (A-to-G transitions

at position 4,602 in the ND2 gene, at position 12,358 in the ND5 gene) caused replacements from Thr to Ala with D value of 58. The replacement of 45 Thr to Ala in the ND2 gene product did not cause significant change in hydropathy profile (not shown) but the secondary structure o f / 3 - s h e e t (at position 25 to 46) and coil (at 47 to 51) were transformed into an a-helix (at 46 to 49). The replacement of 8 Thr to AIa in the ND5 caused a small change in the hydropathy profile (not shown) with little change in the secondary structure of the ND5 gene product. Neither of these two Thr residues were conserved among mammalian species. A T-to-C transition was observed at position 4,386 in the t R N A ci~" gene (Fig. 7), but this substitution was found in four of thirty disease controls. The site of this substitution was located in D H U loop (Fig. 7) but the nucleotide at this position was not conserved among species (data not shown). Three nucleotide substitutions were found in the major non-coding region. 3.6. Nucleotide substitutions in Patient 5

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S-i. lkebe et al. / Molecular Brain Research 28 (1995) 281-295

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tions were found in the protein-coding region: four of them were synonymous mutations; and two were nonsynonymous mutations. An A-to-G transition at 12,358 in the ND5 gene causing a replacement from Thr to Ala was the same mutation that was found in Patient 4. An A-to-G transition at 15,701 in the Cytb gene caused a replacement from Tyr to His. Although the chemical distance between Tyr and His was large (D = 83), this Tyr residue is not conserved among four mammalian species. The hydropathy profile and the secondary structure are changed by this replacement from Tyr to His. The hydropathy profile was slightly changed by this replacement at the junction between a hydrophilic region and the third transmembrane domain of Cytb (data not shown). The structure of/3-sheet (at position 107 to 110) was transformed into a coil (Fig. 8). Five point nucleotide substitutions were found in the major non-coding region.

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When PCR products are cloned and sequenced, it is difficult to exclude the contribution of PCR-induced errors and the damaged template to the sequencing results. To overcome this problem, we employed the PCR-direct sequencing method [19,54]. In this method, the majority of the D N A fragments that are amplified and directly used as the templates represents the sequence of the original mtDNA. Thus, the point mutations identified in this study are not ascribed to the changes due to age of subjects or length of storage of brain specimens. Although we could find no predominant point mutations of m t D N A among the patients with PD in contrast to some neuromuscular diseases [46,53,57,60], each patient had several point mutations that would result in a significant change in the gene products. Some of these m t D N A point mutations are potential risk factors that may induce nigral degeneration when an additional insult, even if it is a rather minor one, is given to the substantia nigra.

4.1. Frequency of nonsynonymous substitutions Nonsynonymous substitutions found in the present patients with PD include four replacements from Thr to Ala, two replacements from Ile to Val, and one each of replacements from His to Tyr, Tyr to Cys, and Tyr to His. The A-to-G transition at 12,358 causing a Thr-to-Ala replacement in the ND5 gene product was found in both Patients 4 and 5, but this substitution was not found in 30 non-parkinsonian controls. The frequencies of other nonsynonymous substitutions were also low among non-parkinsonian controls. Recent restriction endonuclease analysis showed that several m t D N A variants were more frequently found in patients with Alzheimer disease and Parkinson disease than in the general Caucasian controls [45]. They found an A-to-G transition at nucleotide 4336 of the tRNA 6In gene, an A-to-G transition at nucleotide 3397 in the ND1 gene causing a Met-to-Val replacement, insertion of cytosines between 956 and 965 in the 12S r R N A gene, and a C-to-T transition at 3196 in the 16S r R N A gene, but these variations were not found in the present patients. In Patient 5 of the present study harbored a T-to-C transition at 16,304. Shoffner et al. [45] found clustering of patients with Alzheimer disease and Parkinson disease in a lineage characterized by both the T-to-C transition at 16304 and the T-to-C transition at 4336 in the L-strand sequence (the A-to-G transition of the t R N A GI" gene). Because the nucleotide sequence variations of the mitochondrial genome among human individuals are frequent, further studies are necessary to see whether the incidences of these variations are significantly

292

S-i. lkebe et aL / Molecular Brain Research 28 (1995) 281-295

higher in parkinsonian patients than in controls not only in the Japanese population but also in various racial groups. 4.2. Complex I and mtDNA mutations Each of the patients had at least one nonsynonymous substitution in the genes for Complex I subunits. Although these amino acid replacements occur at sites that are not highly conserved among species, some of the replacements have been demonstrated to exhibit significant effects on the hydropathy profile and the secondary structure of subunits in the respiratory chain. Therefore, these replacements might explain partly the decreased activity [4,38,39,44,47] and decreased amounts of subunits [29] of Complex I of patients with PD. Brown et al. [6] have proposed, on the basis of sequence analysis of mtDNA from patients with Leber's hereditary optic neuropathy, that additive effects of various combinations of mtDNA mutations with different risks as well as environmental insults are responsible for the increase in the possibility of blindness in this disease. In the case of PD, it is also possible that combination of the mutations in the genes for Complex I subunits and those in the genes for other complexes can be one of the intrinsic genetic risk factors that accelerate premature aging of nigral neurons. 4.3. mtDNA mutation and t,,ulnerability to oxygen free radicals When we analyze amino acid sequences of proteins including the mtDNA-encoded proteins, cysteinyl residues are frequently located at the sites that are conserved among mammalian species. This fact suggests that most of these cysteinyl residues play functionally important roles either as ligands for prosthetic groups or for formation of disulfide bonds. Conversely, cysteinyl residues, which are susceptible to the oxygen free radical damage, are rarely located at sites where these residues are not functionally necessary. A mutant gene product containing a cysteinyl residue at an unnecessary position would be easily damaged by oxygen free radicals, and therefore the protein would be inactivated or degraded at a higher rate than the gene product without the oxygen free radical-susceptible residue. From this stand point, the amino acid replacement Tyr --* Cys in the ND6 subunit of Patient 2 may render this gene product more susceptible to the oxygen free radical damage than the wild-type gene product. 4.4. mtDNA mutation and susceptibility to neurotoxins In experimental models using 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), it has been demonstrated that the MPTP-induced energy crisis leading to specific neuronal cell death results from inhibition of

Complex 1 activity by 1-methyl-4-phenylpyridine (MPP+), a metabolite of MPTP, that is specifically accumulated in the mitochondria of nigral neurons [30,40,41]. Similar mechanisms of neurotoxicity are proposed for MPTP-Iike endogenous substances, such as tetrahydroisoquinoline (TIQ) [61], tetrahydropapaverine (THPV), and tetrahydropapaveroline (THP) [51]. It seems possible that the amino acid replacements that are found in PD patients alter the affinity of Complex I for these inhibitors. According to this hypothesis, variation in the genotypes of mtDNA among individuals might explain different vulnerability of nigral neurons of each individual to putative extrinsic and intrinsic neurotoxins. It remains to be examined whether the incidence of these mutations is significantly higher in PD than in controls. 4.5. Nucleotide substitution and deletion of mtDNA Since various neurodegenerative diseases, including PD, occur age-dependently, aging seems to be an important factor in the mechanism of neuronal cell death. In parkinsonian patients, we have demonstrated the increased amount of deleted mtDNA in the striatum by the PCR method, but deleted mtDNA was also detectable in the control brain tissues [18], suggesting that the accumulation of deleted mtDNA occurs agedependently. Occurrence of deleted mitochondrial DNA in normally aged brain tissues is suported by other researchers [5,48]. Linnane et al. examined and found an age-related 5 kb deletion in the mtDNA from deceased human subjects with no known mitochondrial disease, aged from birth to 87 years [26]. They suggested that a consequence of the accumulation of this deletion could be a progressive decrease with age of bioenergetic capacity which in turn could influence the rate of ageing and predispose to age-associated degenerative diseases [26]. We have quantitatively demonstrated that the population of mutant mtDNA with 4,977-bp deletion is increased in the striatum of patients with PD and suggested that age-related accumulation of deleted mtDNA is focally accelerated in the striatum of PD and that PD can be considered as a phenotype of premature ageing [33]. In yeast m i t mutants, small and discrete mutations, are almost always associated with a significant rise in the rate of deletion [27]. It seems possible that the nucleotide substitutions found in the present patients are one of the genetic risk factors causing significant changes in the gene products. These structural alterations might affect the stability of these gene products leading to accelerated degradation. Alternatively, these changes might deteriorate the molecular assembly of the respiratory complexes, resulting in increased leakage of reactive oxygen species from the respiratory chain. This could explain the premature accumulation of deleted

S-i. lkebe et al. / Molecular Brain Research 28 (1995) 281-295

mtDNA in the nigrostriatal neurons of PD [18] leading to mitochondrial dysfunction and to oxygen free radical-mediated cell death, apoptosis. Therefore, it should be further elucidated whether the point mutations identified in this study exacerbate the accumulation of deleted mtDNA, leading to the focal neuronal cell death in PD.

4.6. Oxidative damage of mtDNA It is plausible that oxidative stress is an important factor in the mechanism of selective neuronal death in the substantia nigra. The increased amounts of iron [12,22,25] and copper [37] in the presence of catecholamines have been implicated to be responsible for the oxidative stress of nigral neurons. Because mitochondria are the site where more than 95% of molecular oxygen is metabolized in each cell and because ca. 5% of the oxygen consumed by the mitochondrial respiratory chain is converted into oxygen free radicals, these organelles is the major source of reactive oxygen species. The nucleotide substitutions identified in the present study might be responsible for accelerated production of reactive oxygen species from mitochondria. Not only the protein components of the respiratory chain but also mtDNA molecules that are located in the vicinity of the inner mitochondrial membrane can be damaged by these free radicals. In association with decreases in the mitochondrial respiratory enzyme activities [55] age-dependent accumulation of 8-hydroxydeoxyguanosine, a hydroxy-radical adduct of deoxyguanosine, in mtDNA has been observed in the diaphragm [17,56] and in the heart [16] concomitantly with the increase in the amount of deleted mtDNA. The oxidative damages of mtDNA will lead to deterioration of its gene products, resulting in further increase in the leakage of reactive oxygen species from the respiratory chain. The accumulation of the oxidative damages would result in a long stretch of singlestranded DNA that is prerequisite for the large deletion [16]. The mutant gene products resulted from nucleotide substitutions as well as the deletions of mitochondrial DNA would increase the leakage of reactive oxygen species from mitochondria. We have proposed that accumulation of mtDNA mutations is an important contributor to aging process and degenerative diseases [27]. Our assertion should be further confirmed by analyzing the putative accumulation of nucleotide substitutions resulting from oxidative DNA damage in the nigrostriatal system both in the normal aging process and in PD.

Acknowledgements We are grateful to Prof. Yoshikuni Mizuno for suggestions and encouragement throughout this work.

293

This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas to MT and TO, and Grants-in-Aid for General Scientific Research to MT and TO from the Ministry of Education, Science and Culture of Japan.

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