Neuroprotective Effect of PD-1 Extract in MPTP-lesioned Mouse Model of Parkinson's Disease

서 론

(dopamine)

,

, (substantia nigra pars compacta; SNpc)

1).

. ,

(tremor), (bradykinesia), (rigidity), (postural instability), (akinesia)

2,3). 이정욱, 정혜미, 서운교

동국대학교 한의과대학 내과학교실 Original Article

Neuroprotective Effect of PD-1 Extract in MPTP-lesioned Mouse Model of Parkinson’s Disease

Jung-Wook Lee, Hye-Mi Jung, Un-Kyo Seo

Dept. of Oriental Internal Medicine, College of Oriental Medicine, Dongguk University

Objectives: The aim of the present study was to explore the neuroprotective effect and the possible mechanism of the PD-1 extracts on 1-methyl-4-phenyl-1,2,3,6-tetrahydrophridine (MPTP)-lesioned C57BL/6 mouse model of Parkinson’s disease (PD).

Methods: The mice were supplemented (or not) with 50 or 100 mg/kg/day of PD-1 for 2 weeks, after which MPTP was injected intraperitoneally. We observed that daily administration of PD-1 prevented MPTP-induced depletion of striatal DA, and maintained striatal and nigral tyrosine hydroxylase (TH) protein levels.

Results: Our results demonstrated that mice treated with PD-1 prior to MPTP administration showed more abundant TH-immunopositive (TH-ir) fibers and neurons than mice given only MPTP, indicating that PD-1 protects dopaminergic striatal fibers and nigral neurons from MPTP insults. Possible neuroprotective effect of PD-1 was further studied by the detection of antiapoptotic protein (bcl-2) and proapoptotic protein (Bax). In this assay, MPTP elevated the Bax protein and decreased the bcl-2 protein, while these expressions were prevented by PD-1 pre-treatment.

Conclusions: The present results suggest that PD-1 is able to protect dopaminergic neurons from MPTP-induced neuronal injury with anti-apoptotic activity being one of the possible mechanisms.

Key Words : PD-1, Parkinson disease, 1-methyl-4-phenyl-1,2,3,6-tetrahydrophridine

Received：14 January 2009 Revised：21 May 2009 Accepted：26 May 2009

Correspondence to：서운교(Un-Kyo Seo)

경기도 성남시 분당구 수내3동 87-2 동국대학교 분당한방병원 2내과

Tel：+82-31-710-3751, Fax：+82-31-710-3780, E-mail：[email protected]

(oxidative

stress) (free radical species)

4), (β-amyloid)

5),

6),

microglia/macrophage ^7,8)

.

,

. , L-dopa (levodopa)

(dopamine receptor agonist)

1,9), pharma- cological agent

(loss) 5-6

, dyskinesia .

11).

(neur-

oprotective agents) .

17-22)

.

100

, ,

23,24)

.

.

.

PD-1 MPTP

.

재료 및 방법

1.

PD-1 Table 1 .

( ) ( , )

.

2)

1-methyl-4-phenyl-1,2,3,6-tetrahydrophridine(MPT P), 1-methyl-4-phenylpyridinium(MPP+), dimethyl sulfoxide(DMSO), diaminobenzidine(DAB), mouse anti-β-actin monoclonal antibody, skim milk

Sigma (St Louis, MO, USA) .

Improm-IITM reverse transcriptase PCR Taq- polymerase kit Promega (Madison, WI 53711, USA) , Affinity- purified rabbit anti-mouse TH polyclonal antibody Chemicon Internal Inc.(CA, USA) , Affinity- purified goat anti-bcl-2, bax, caspase-3 antibodies, anti-rabbit/anti- mouse IgG Santa Cruz Biote- chnology (Santa Cruz, CA, USA) , affinity-purified rabbit anti-mouse cytochrome c antibody BD Pharmingen (BD Biosciences, San Diego, CA, USA) , rabbit anti- mouse PARP antibody Cell Signaling Technology

avidin-biotin- peroxidase complex system Biosys (CA, USA) , nitrocellulose memb- rane, ECL Western detection reagent, protein assay solution, 30% acrylamide solution Amersham Bioscience (Piscataway, NJ, USA)

, X-ray film develop solution Kodak (Burnaby, British Columbia)

. 3)

Microplater Reader(Asys, Sunnyvale, CA, USA), Deep-freezer Advantage(Queue, USA), Rotary Evaporator(Eyela, Japan), Thermal Cycler (BioRad Labolatories Inc.), Orbital Shaker (Finemould Precision IN, Co, Gyeonggi-do, korea), BioDoc ItTM Imaging System(UVP, Cambridge, UK), Western Blot System(Bio-Rad Laboratories, Hercules, CA, USA), UV-VIS Spectrophotometer (Shimazu, Japan), CO2 incubator(Thermo Electron Corporation, NH, USA), fluorescence microscopy (Olympus Imaging America Inc., Center Valley, PA 18034-0610, USA), cryostat cryocut microtome(LEICA

CM. 3050S, Germany) .

4)

MPTP

15-17

20-30 g C57BL/6 .

12

. , NIH

(2000 ) .

2.

PD-1 1

10 2

(rotary vacuum evaporator)

, PD-1 . PD-1

315.5 g , (yield) 32.4% . PD-1

. 2)

藥材名 학명 생약명 조성비

白芍藥 Paeonia lactiflora PALL. Paeoniae Radix Alba 3

葛根 Pueraria thunbergiana BENTH. Puerariae Radix 2

桂枝 Cinnamomum cassia PRESL Cinnamomi Ramulus 2

天花粉 Trichosanthes kirilowii MAX. Trichosanthis Radix 1.5

麥門冬 Liriope platyphylla WANG et TANG Liriopis Tuber 1.5

甘草 Glycyrrhiza uralensis FISCH. Glycyrrhizae Radix 1

釣鉤藤 Uncaria rhynchophylla(MIQ) JACJS. Uncariae Ramulus Et Uncus 1

白疆蠶 Bombyx mori Linne. Bombyx Batryticatus 1

木瓜 Chaenomeles sinensis KOEHNE Chaenomelis Fructus 1

防風 Ledebouriella divaricata(TURCZ.) HIROE Ledebouriellae Radix 1

竹茹 Phyllostachys nigra(LODD.) MUNRO var. henonis(BEAN.) STAPF Bambusae Caulis In Taeniam 1

生薑 Zingiber officinale ROSC. Zingiberis Rizoma Recens 1.5

大棗 Zizyphus jujuba MILL. var. inermis REHDER Jujubae Fructus 1.5

Table 1.

C57BL/6 (24 ) 5 .

Group I (D-S; DW-Saline) :

19 9:00-10:00 intra-

gastric lavage DW(distilled water)

, 15 2

.

Group II (PS; PD-1 200 / -Saline) :

19 9:00-10:00 100 /

PD-1 , PBS

. Group III (DW-MPTP) :

19 9:00-10:00

, 15 2

4 MPTP doses (15 / ) .

Group IV (PD-1 50 mg/kg; MPTP) :

19 9:00-10:00 50 /

PD-1 , 15 2

4 MPTP doses (15 / ) .

Group V (PD-1 100 mg/kg; MPTP) :

19 9:00-10:00 1000

/ PD-1 , 15

2 4 MPTP doses (15 / )

.

, MPTP MPTP(calculated as free

base) 0.9% (saline) 15 /

0.5

, 2 4

(Fig. 1). MPTP

PD-1(50 / , 100 / )

. 0.5 0.9%

MPTP .

3) Tyrosine hydroxylase(TH)

PD-1

(SNpc) .

4% paraformaldehyde 2-3

30% sucrose 4

.

OCT(Optimal Cutting Temperature) compound

-70 cryostat

cryocut microtome (SNpc)

(striatum) 30

coronal sections .

(immunohistochemistry)

slide PBS 3 5%

normal goat serum 0.3% Triton X-100

PBS 1 blocking .

2% normal goat serum 0.15% Triton X-100

PBS 1,000 anti-TH antibody

4 overnight PBS 3

. biotinylated secondary antibody(goat

anti-rat, 1:1000) 1

PBS 3 avidine-biotin-horseradish per-

oxidase complex 1

PD-150 mg/kg/d or 100 mg/kg/d or vehicle

d15 d20

4 injections MPTP 15mg/kg or vehicle

Sacrifice

Fig. 1.

. slide 0.1M Tris buffer (TB, pH 7.4) 3 , 0.004% H2O2

0.05% DAB TB 2-5

, 100% alcohol xylene DAKO^® fluorescent mounting medium

mounting .

, .

, slide cresyl violet striatum .

slide PBS

3 5% normal goat serum 0.3%

Triton X-100 PBS 1

blocking . slide 2% normal goat

serum 0.15% Triton X-100 PBS

2,000 FITC-labeled anti-TH antibody

4 2 PBS 3

. slide 100% alcohol xylene

mounting medium

. SNpc

TH (TH-positive

cells) .

4)

SNpc TH PD-1

Western blot

. , basal ganglia, diencephalon, midbrain 1% sodium deoxycholate, 0.1%

sodium dodecyl sulfate (SDS), 0.15 NaCl, 1% NP- 40 50 mM Tris-HCl(pH 7.2)

homogenizer . 12,000 rpm

10 (microcentrifuge)

.

Bio-Rad Bradford

. 5) TH

30 2× sample buffer(100mM Tris- HCl, pH 6.8, 200mM dithiothreitol, 4% SDS, 0.2%

bromophenol blue, 20% glycerol) 100

3 10% SDS-polyacrylamide gel

. gel

nitrocellulose (NC) membrane 4 , 30V 16 transfer . Membrane 5% skim

milk 1 blocking , TH

actin 0.05% Tween

Tris-buffered saline(TBS-T) 1:1000

4 overnight . Membrane

TBS-T 3 anti-rabbit IgG

conjugated HRP (1:1,000) 1

ECL x-ray film

.

6) bcl-2 bax

mitochondria cytochrome

c pro-

apototic molecule bax anti-apoptotic

molecule bcl-2 PD-1

SDS-polyacrylamide gel electr- ophoresis (SDS-PAGE) Western blot

. 2.4

. Bradford protein assay

bovine serum albumin(BSA) , 30

12.5% polyacrylamide gel SDS-PAGE

, gel NC memb-

rane (transfer), 5% skim milk

1 membrane blocking . mem-

brane anti-bcl-2 antibody(1:500, monoclonal), anti- bax antibody(1:1000, monoclonal), anti-actin antibody (1:5000, polyclonal) 150 mM NaCl 0.5% Tween- 20 10 mM Tris-HCl(TBS-T)

4 . ,

membrane TBS-T 3 TBS-T

(1:5000) horseradish peroxidase-conjugated

1 .

TBS-T 3 enhanced chemilu-

minescence detection system X-ray film .

house keeping protein β-actin

. 7)

3 ±

(standard error of mean; SEM)

, SPSS program(version

13.0) posthoc LSD test program One-way

ANOVA(analysis of variance) P

0.05 .

결 과

1.

Striatal TH

대한 효과

TH rat-limiting enzyme

TH L-DOPA

.

(striatum) striatal dop-

amnergic fiber .

TH PD-1

, MPTP

TH (Fig. 2). PBS

(group I), PD-1 (group II)

striatal dopaminergic

fiber TH , MPTP

striatal TH

(group III). , PD-1 striatal TH

, 100 / PD-1 50

/ PD-1

(Fig. 2A).

, TH-positive

, group I 99±8 , group II 94±7 , group III 28±6 group IV

group V 39±4 62±7 PD-1

TH-positive

(Fig.

2B). PD-1 MPTP

striatal dopaminergic neuronal cells TH .

A A A

A BBBB CCCC

D D D

D EEEE

F F F F

TH-positive cells (numbers)

300000 250000 200000 150000 100000 50000 0

D-S P-S D-M L-M H-M

b b a

Fig. 2.

2.

SNPc TH

TH PD-1

MPTP

SNPc TH

(Fig. 3).

PBS , PD-1

(SNPc)

TH , MPTP

TH

. , PD-1 SNPc

TH , 100 /

PD-1 50 / PD-1

(Fig. 3A).

, SNPc TH-positive

, group I 100±6 , group II 95±5 , group III 28±4 group IV

group V 40±2 63±3 PD-1

TH-positive

(Fig. 3B).

PD-1 MPTP TH-

positive

.

3.

SNPc

TH

대한 효과

TH

Western blot (Fig. 4).

, group I PD-1

group II TH (60

kDa) , MPTP

(group III)

. , PD-1 50 / 100 / (groups IV, V) group III

TH .

4.

Caspase

25,26)

, caspase-3 PARP cleavage

bcl-2 family bax

cytochrome c

27). MPTP

A A A

A BBBB CCCC

D D D

D EEEE

FF FF

Cell counts

120 100 80 60 40

0

D-S P-S D-M L-M H-M

b

a b

20

Fig. 3.

caspase-3 PARP cleavage

mitochondria cytochrome c bax/

bcl-2 PD-1

SDS-PAGE Western blot (Fig. 5).

, cytochrome c

group I PD-1 group

II , MPTP (group

III) . MPTP

cytochrome c PD-1 50 / 100 / (groups IV, V) group III AA

AA BBBB

3.5 3 2.5 2 1.5 1 0.5 0

TH/actin intensity

TH (60kDa)

Actin

TH (60kDa)

Actin

D-S P-S D-M L-M H-M

a b

b

3 2.5 2 1.5 1 0.5 0

TH/actin intensity

D-S P-S D-M L-M H-M

a

b b

Fig. 4.

A A A A

1

D-S P-S D-M L-M H-M

a

b b

0.8 0.6 0.4 0.2 0

Cytochrome C

Actin

Cytochrome C/actin intensity

B BB B

D-S P-S D-M L-M H-M

a b b

2.5

Bcl-2

Bax

Actin

Active caspase-3

bcl-2/bax ratio

2 1.5 1 0.5 0

Fig. 5.

(Fig. 5A). , anti-apoptotic molecule bcl-2 group I group II

, MPTP ,

PD-1 (Fig.

5B). pro-apoptotic molecule bax

group I group II , MPTP

, PD-1 molecules

.

bax/bcl , PD-1

bcl-2

. caspase-3 group I

group II , MPTP

, PD-1 (Fig. 5B).

PD-1 MPTP SNPc

bax/

bcl , caspase ,

cytochrome c

.

고 찰

(SNPc) (striatum)

,

3).

, oxidative stress,

proteasomal impairment, mitochondrial dysfunction , excytotoxicity, ER stress, inflammation neurotrophic deficiency

12). mitochondrial

complex I ,

6).

glutathione , ,

28).

,

.

.

, “

, ,

” .

.

(agonist) ,

10,11)

.

24),

22), levodopa

19),

21) .

,

PD-1

.

PD-1 , ( ;

Paeoniae Radix Alba) , ,

, ( ; Puerariae Radix)

, , ( ; Cinnamomi Ram-

ulus) , . ,

( ; Trichosanthis Radix)

, ( ; Liriopis Tuber) ,

, ( ; Glycyrrhizae

Radix) .

( ; Uncariae Ramulus Et Uncus)

, , ( ; Bombyx

Batryticatus) , ( ;

Chaenomelis Fructus) ,

. , ( ; Ledebouriellae Radix)

, , , ( ; Bambusae

Caulis In Taeniam) ,

, ( ; Zingiberis Rizoma Recens)

, , , ( ;

Jujubae Fructus) , , ,

29). PD-1

.

,

6-OHDA MPTP

16). , toxin model

neurotoxin

(degeneration) MPTP, 6-hydr- oxydopamine (6-OHDA) rotenone

, paraquat MPP+

13,14)

. MPTP model mouse monkey

15,16)

.

acute intoxification Lewy body .

MPTP MPTP toxin

mitochondrial dysfunction

13). MPTP

monoamine oxidase(MAO)-B 1-methyl-4-phenyl pyridinium (MPP+) dopamine transporter

mitochondrial complex I(NADH-ubiquinone

oxidoreductase) inhibitor ,

oxidative stress ATP

11,12,27)

. , MPP+

mitochondrial complex I ATP

. MPP+

MPTP

. MPTP

. (ventral

tegmentum)

.

catalepsy

30).

.

free radicals superoxide radicals

31). gene mutation

,

course (slow or stop)

.

target molecule target

signaling pathway .

.

MPTP PD-1

in vivo .

tyrosine hydroxylase(TH) TH-positive

(Fig. 2, 3).

PD-1 MPTP TH-positive

.

, bcl-2 family

bcl-2 family

,

25,26,32)

. bcl-2

family pro-apoptotic member bax

apoptosis bax

33). bcl-2 family

anti-apoptotic group pro-apoptotic group , pro-apoptotic group bax, Bak, Box

, anti-apoptic group bcl-2, bcl-XL

. bcl-2 bax

,

34,35)

. PD-1 anti- apoptotic

bcl-2 ,

MPTP bax/bcl

(Fig. 4).

bax bcl-2

, bax

cytochrome c

. , bax

cytochrome c

35). Cytochrome c

100

polypeptide ,

c heme heme

. cytochrome c

Apaf1, procaspase-9 apoptosome

caspase

caspase-3 . caspase-3

DNA cleavage, nuclear condensation, cytop- lasmic bleb

27). PD-1 anti- apoptotic bcl-2

cytochrome c

(Fig. 5). PD-1 bcl-2

bax

cytochrome c (Fig. 4).

MPTP/MPP+ caspase-3

PARP bax

caspase-3

DNA caspase

32).

cytochrome c , caspase-3 PARP

, ,

3-6).

cytochrome c caspase

, PD-1 MPTP/MPP+

caspase-3 cytochrome c

(Fig. 5).

, PD-1 MPTP SNPc

bax/

bcl , caspase ,

cytochrome c

. , PD-1

,

.

결 론

PD-1 MPTP

.

1. PD-1 50, 100 / MPTP

(striatum) TH (the density of striatal TH-positive fibers)

.

(SNPc) TH (the number of SNPc TH-positive cells)

.

3. PD-1 50, 100 / MPTP

anti-apoptotic molecule bcl-2 bax/bcl

.

4. PD-1 50, 100 / MPTP

caspase-3 ,

cytochrome c .

, PD-1 in vivo

bax/bcl caspase

cytochrome c

.

참고문헌

1. , , . (I),

. ; 2006, p.

25-55.

2. . . : ; 1992,

p. 37-49, 77-83, 107-22, 137-9, 142-9.

3. Jenner P, Olanow C. Understanding cell death in Parkinson’s disease. Ann Neurol. 1998;3:S72 -S84.

4. Camins A, Pallas M, Silvestre JS. Apoptotic mechanisms involved in neurodegenerative dise- ases: experimental and therapeutic approaches.

Methods Find Exp Clin Pharmacol. 2008;30 (1):43-65.

5. Mancuso C, Scapagini G, Currò D, Giuffrida Stella AM, De Marco C, Butterfield DA et al.

Mitochondrial dysfunction, free radical genera- tion and cellular stress response in neurodegen- erative disorders. Front Biosci. 2007;12:1107-23.

Cassidy RM, Johansson CB et al. Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci USA. 2003;100(13):

7925-30.

7. Rock RB, Peterson PK. Microglia as a pharma- cological target in infectious and inflammatory diseases of the brain. J Neuroimmune Pharmacol.

2006;1(2):117-26.

8. Wilms H, Zecca L, Rosenstiel P, Sievers J, Deuschl G, Lucius R. Inflammation in Parki- nson’s diseases and other neurodegenerative diseases: cause and therapeutic implications.

Curr Pharm Des. 2007;13(18):1925-8.

9. Fox SH, Chuang R, Brotchie JM. Parkinson’s disease-opportunities for novel therapeutics to reduce the problems of levodopa therapy. Prog Brain Res. 2008;172:479-94.

10. Zhang X, Andren PE, Greengard P, Svenni- ngsson P. Evidence for a role of the 5-HT1B receptor and its adaptor protein, p11, in L-DOPA treatment of an animal model of Parkinsonism.

Proc Natl Acad Sci USA. 2008;105(6):2163-8.

11. Yang YL, Meng CH, Ding JH, He HR, Ellsworth K, Wu J et al. Iptakalim hydrochloride protects cells against neurotoxin-induced glutamate transporter dysfunction in vitro and in vivo models. Brain Res. 2005;1049:80-8.

12. Moore DJ, West AB, Dawson VL, Dawson TM.

Molecular pathophysiology of Parkinson’s disease.

Annu Rev Neurosci. 2005;28:57-87.

13. Nicklas WJ, Vyas I, Heikkila RE. Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4- phenyl-1,2,5,6-tetr- ahydropyridine. Life Sci. 1985;36(26):2503-8.

14. Liu HQ, Zhang WY, Luo XT, Ye Y, Zhu XZ.

Paeoniflorin attenuates neuro- inflammation and dopaminergic neurodegeneration in the MPTP model of Parkinson’s disease by activation of adenosine A1 receptor. Br J Pharmacol. 2006;

148:314-25.

15. Eslamboli A. Marmoset monkey models of Park- inson’s disease: which model, when and why?

Brain Res Bull. 2005;68(3):140-9.

16. Smeyne RJ, Jackson-Lewis V. The MPTP model of Parkinson’s disease. Brain Res Mol Brain Res.

2005;134(1):57-66.

17. Liu HQ, Zhang WY, Luo XT, Ye Y, Zhu XZ.

Paeoniflorin attenuates neuro- inflammation and dopaminergic neurodegeneration in the MPTP model of Parkinson’s disease by activation of adenosine A1 receptor. Br J Pharmacol. 2006;

148:314-25.

18. Van Kampen J, Robertson H, Hagg T, Drobitch R. Neuroprotective actions of the ginseng extract G115 in two rodent models of Parkinson’s disease. Exp Neurol. 2003;184(1):521-9.

19. Cao F, Sun S, Tong ET. Experimental study on inhibition of neuronal toxical effect of levodopa by ginkgo biloba extract on Parkinson disease in rats. J Huazhong Univ Sci Technolog Med Sci.

2003;23(2):151-3.

20. Li FQ, Cheng XX, Liang XB, Wang XH, Xue B, He QH et al. Neurotrophic and neuropr- otective effects of tripchlorolide, an extract of Chinese herb Tripterygium wilfordii Hook F, on dopaminergic neurons. Exp Neurol. 2003;179 (1):28-37.

21. Ramassamy C. Emerging role of polyphenolic compounds in the treatment of neurodegenerative diseases: a review of their intracellular targets.

Eur J Pharmacol. 2006;545(1):51-64.

22. Van Kampen J, Robertson H, Hagg T, Drobitch R. Neuroprotective actions of the ginseng extract G115 in two rodent models of Parkinson’s disease. Exp Neurol. 2003;184(1):521-9.

23. Chen LW, Wang YQ, Wei LC, Shi M, Chan YS.

Chinese herbs and herbal extracts for neurop- rotection of dopaminergic neurons and potential therapeutic treatment of Parkinson’s disease.

CNS Neurol Disord Drug Targets. 2007(4):273-81.

24. Li Q, Zhao D, Bezard E. Traditional Chinese medicine for Parkinson’s disease: a review of Chinese literature. Behav Pharmacol. 2006 Sep;

17(5-6):403-10.

25. Zhou J, Tang XC. Huperzine A attenuates apoptosis and mitochondria-dependent caspase-3 in rat cortical neurons. FEBS Lett. 2002;526:21-5.

26. Grammatopoulos TN, Morris K, Bachar C, Andres MS, Weyhenmeyer RJ. Angiotensin II attenuates chemical hypoxia-induced caspase-3 activation in primary cortical neuronal cultures.

Brain Res Bull. 2004;62:297-303.

27. Chang SH, Phelps PC, Berezesberger IK, Ebersberger ML Jr, Trump BF. Studies on the mechanisms and kinetics of apoptosis-induced by microinjection of cytochrome c in rat kidney tubule epithelial cells(NRK-52E). Am J Pathol.

2000;156:637-49.

28. Schapira AH. Progress in neuroprotection in Parkinson’s disease. Eur J Neurol. 2008;15:5-13.

29. . .

: ; 2007, p. 154-5, 163-4, 171-2, 184-5, 205-6, 315-6, 503-4, 540-4, 583-6, 634-5, 644-5.

30. Sedelis M, Hofele K, Auburger GW, Morgan S, Huston JP, Schwarting RK. MPTP susceptibility in the mouse: behavioral, neurochemical, and

histological analysis of gender and strain diffe- rences. Behav Genet. 2000;30(3):171-82.

31. Stack EC, Ferro JL, Kim J, Del Signore SJ, Goodrich S, Matson S et al. Therapeutic attenu- ation of mitochondrial dysfunction and oxidative stress in neurotoxin models of Parkinson’s disease. Biochim Biophys Acta. 2008;1782(3):

151-62.

32. Kaul S, Kanthasamy A, Kitazawa M, Anant- haram V, Kanthasamy AG. Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1- methyl-4-phenylpy- ridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration. Eur J Neurosci.

2003;18(6):1387-401.

33. Yuan J, Lipinski M, Degterev A. Diversity in the mechanisms of neuronal cell death. Neuron.

2003;40:401-413.

34. Harris CA, Johnson EM, Jr. BH3-only bcl-2 family members are coordinately regulated by the JNK pathway and require bax to induce apoptosis in neurons. J Biol Chem. 2001;37754 -60.

35. Gross A, McDonnell JM, Korsmeyer SJ. bcl-2 family members and the mitochondria in apoptosis.

Genes Dev. 1999;13:1899-911.