Oxidative Stress and JNK Activation Cause Mitochondrial

Dysfunction after VDAC Opening

Diana FangClemson University

Mentors: Dr. John J. Lemasters and Dr. Eduardo N. Maldonado

Department of Drug Discovery & Biomedical Sciences

ΔΨ

Mitochondrial Bioenergetics: Non-proliferating Cells

Maldonado & Lemasters, Mitochondrion 2014

ΔΨ

Warburg Metabolism

Mitochondrial Bioenergetics: Proliferating Cells

Maldonado & Lemasters, Mitochondrion 2014

Erastin & X1 Cause Hyperpolarization and Subsequent Depolarization of Mitochondrial Membrane in HepG2 Cells

Baseline

120 min 240 min

Erastin

X1Baseline

60 min 120 minMaldonado & DeHart et al., unpublished

VDAC Opening ?

VDAC Opening ?

JNK P

c-Jun N-terminal Kinase (JNK)

Mitochondria

ROS

MKK4MKK7

MEKK 1,4MLK3ASK1

JNK1,2,3

Stress(ie. ROS)

ROS

ROS

Translocation

Mitochondrial Dysfunction & Cell Death

ΔΨ

HypothesisWe hypothesize that VDAC-Tubulin antagonist, X1, promotes hyperpolarization and reactive oxygen species (ROS) formation which leads to activation of JNK and cause mitochondrial depolarization and mitochondrial dysfunction.

AimTo evaluate the effects of decreasing ROS and JNK inhibition on late depolarization induced by X1.

JNK Inhibition with SP600125 Delays Mitochondrial Dysfunction after X1

15 min 30 min 60 min

+ SP600125 + X1

0 min 15 min 30 min 60 min

+ X1 0

255

0 min 15 min 30 min 60 min

+ JNKi VIII + X1

+ X1 0

255

JNK Inhibition with JNK Inhibitor VIII Delays Mitochondrial Dysfunction after X1

N-Acetylcysteine does not prevent hyperpolarization but does prevent depolarization of ∆Ψ

+ NAC + X1

0 min 15 min 30 min 60 min0 min 15 min 30 min 60 min

+ X1 0

255

ROS Promotes Mitochondrial Dysfunction after X1

Time (min)

0 30 60

Nuc

lear

TM

RM

Flu

ores

cenc

e (%

of B

asel

ine)

0

500

1000

1500

X1JNKi VIII + X1SP600125 + X1NAC + X1Vehicle

****

****

***

Summary • JNK Inhibitors do not prevent X1 induced hyperpolarization but does

attenuate subsequent depolarization • NAC does not prevent the hyperpolarization of the mitochondrial

membrane but does prevent the late depolarization after X1, blocking mitochondrial dysfunction

ConclusionThe late depolarization following the hyperpolarization induced by X1 is mediated by oxidative stress and JNK activation.

Acknowledgements Lab Members • John J. Lemasters, MD,

PhD• Eduardo N. Maldonado,

DVM, PhD• Monika Gooz, MD, PhD• David N. DeHart, soon PhD • Kareem Heslop• David Johnson

MUSC Summer Undergraduate Program

• Stephanie Brown-Guion, MSM

Questions?

Downstream of JNKKey Players:

- Sab: sequesters SHP-1 (phosphatase) and Binds with JNK

- SHP-1: Tyrosine Phosphatase

- c-Src: Tyrosine Kinase that activates SHP-1, complexes I and II

- DOK-4: Docking protein that is necessary for SHP-1 to dephosphorylate c-Src

Info from Win et al., Hepatology 2016

Compounds used

SP600125 JNK Inhibitor VIII N-Acetylcysteine (NAC)

NAC Prevents ∆

Time (min)

0 30 60

Nuc

lear

TM

RM

Flu

ores

cenc

e (A

U)

0

25

50X1JNKi VIII + X1SP600125 + X1NAC + X1Vehicle