The effect of epigallocatechin gallate on suppressing disease progression of ALS model mice 한양대학교 대학원 신경과전공 박사과정 이상목 가족성 근위축성측삭경화증 모델 동물의 질병경과 완화를 보인 epigallocatechin gallate (EGCG) 의 치료효과

근위축성 측삭경화증 (ALS) 이란 ? 정상 ALS 근위축성 ( A mytrophic) 전각세포 측방 뇌척수로 경화증 ( S clerosis) 측삭 ( L ateral) 신경계 퇴행성 질환, 원인규명이 않되며 치료 불가능, 운동신경계를 침범하는 질환임

Environmental influence & Aging Oxidative Stress Cell death Mitochondrial dysfunction Altered calcium homeostasis Genetic; SOD1, ALSIN Pathogenic mechanism of ALS Microglial activation and astrocytosis : Immune-inflammatoin Abnormal protein aggreation : Bunina body Tauropathies (Guam ALS/PDC) Glutamate toxicity Neurotrophic factor Modification Protein/Lipid/DNA

1) one of the major constituents of green tea 2) cytoprotective effects a. anti-apoptotic b. anti-cancer c. anti-mutagenic 3) mechanism a. mitogen-activated protein kinase (MEK) signaling b. phosphatydilinositol 3-kinase (PI3K)/Akt c. glycogen synthase kinase-3 (GSK-3) d. caspase-3 Koh SH, Kim SH, et al., Mol Brain Res 2003; 118: Neurotoxicology 2004; 25: Epigallocatechin gallate (EGCG)

4) Our recent data indicate that EGCG protects in vitro model of ALS motor neuron transfected with mutant SOD1. Koh SH, Kim SH, et al., Toxicology 2004; 202: ) Alteration of intracellular signals by treatment of EGCG DNA damage Caspase-3 Cytochrome C PARP Survival signal G93A mSOD 1 PI3K/Akt GSK-3 Apoptotic signal DNA damage Caspase-3 Cytochrome C PARP Survival signal PI3K/Akt GSK-3 Apoptotic signal G93A mSOD 1 + EGCG Epigallocatechin gallate (EGCG)

Purpose To evaluate. 1. whether treatment with EGCG could delay symptom onset and prevent disease progression of ALS. 2. the cytoprotective mechanism of EGCG on the PI3-K/Akt and GSK-3 pathway as well as the caspase-3 pathway.

Materials and methods 1) Mice; B6SJL-Tg(SOD1-G93A)/1Gur (Jackson Lab) 2) Treatment Regimen low Number Body weight (g) EGCG (μg/g) control medium 22.8± ± high ±1.9 0 (only 0.9% saline) Transgenic mice with human G93A mSOD1 gene EGCG

3) Evaluation of body weight and motor performance (1) Body weight ; 2 times/week (2) Clinical evaluation a. symptom onset - when mice is suspended on the air by its tail, shaking their limbs b. endpoint - When the mice could not right themselves within 30 seconds when placed on their sides on a flat surface

(3) Accelerating Rotarod test ( 1 rpm acceleration/ 8sec) - 1 week training period - measuring the time mouse could stay on rod without falling Definition of Rotarod failure : Unable to stay on the rotating bar > 10sec

Schedule days 53 Rotarod training endpoint-2 endpoint-1 EGCG (sigma) group from asymptomatic period Tg SOD1 endpoint Control group (Normal saline) 1.5 (μg/g) 2.9 (μg/g) 5.8 (μg/g)

Analysis of data Perfusion at the date of rotarod failure of control group Dissection Survival signals p85a PI3K (Sigma, 1:500) Phospho-Akt (Ser 473) (Cell Signaling, 1:500) Phospho-GSK-3  (Ser 9) (Santa Cruz, 1:500) Death signals Cytochrome c (Santa Cruz, 1:1000) Cleaved Caspase-3 (Asp 175) (Cell Signaling, 1:500) PARP (Cell Signaling, 1:1000) Western blot Statistical analysis Kaplan-Meier survival analysis (SPSS12.0; Chicago, IL) Duncan’s Multiple Range Test (SAS 9.0)

Results I. Comparison of symptom onset control EGCG (low, 1.5μg/g) EGCG (medium, high; 2.9, 5.8μg/g) Days

II. Comparison of endpoint control EGCG (low, 1.5μg/g) EGCG (medium, high; 2.9, 5.8μg/g) Days

Wild Control Normal Saline EGCG Comparison of motor weakness at the time of endpoint (control group)

III. Changes of neurological course

IV. Western blot

1.Treatment of EGCG delayed symptom onset and prolonged survival time in mSOD1 Tg mice. 2.Alteration of immunoreactivities in EGCG-treated group (compared with non-treated group) EGCG pGSK-3 β activated caspase-3 cytochrome C pAkt p85a PI3K Summaries cleaved PARP

Conclusions These results suggest that EGCG could delay symptom onset and prolong life span, by preserving more survival signals and attenuating death signals.. Therefore, Treatment of EGCG could be an potential neuroprotection strategy in ALS.

Role of PI3-K and GSK-3 activity in neuronal survival signaling PI3K ⓟ Akt Insulin VEGF GSK-3 β (inactive) - ⓟ -Ser9 GSK-3  (active) GSK-3  (active) Neuronal cell death Proapoptotic signal ↑ PI-3 kinase pathway is one of the important signal transduction pathway implicated in the survival of neurons, activated by Insulin or IGF-1, VEGF, - -. Koh SH, Kim SH, et al., Mol Brain Res 2003; 118: Neurotoxicology 2004; 25: Toxicology 2004; 202: Eur J Neurosci 2004; 20: Mol Brain Res 2005;133: Eur J Neurosci 2005; 22: The downstream effector of PI3-K is Akt (PKB). Akt phosphorylates and inhibits GSK-3 activity. GSK-3 (Glycogen Synthase Kinase) was initially described as a key enzyme involved in glycogen metabolism, but is now known to regulate diverse array of cell functions including cell survival and differentiation. β-catenin ↑ NF  B ↑ Transcription factor activation ; Cell survival ; Anti-apoptotic effect Glycogen synthesis CREB ↑ HSF1 ↑