mtDNA T8993G Mutation-Induced Mitochondrial Complex V Inhibition Augments Cardiolipin-Dependent Alterations of Mitochondrial Dynamics During Oxidative, Ca2+ and Lipid Insults in NARP Cybrids: A Potential Therapeutic Target


Sunday, February 26, 2012 163a
mitochondrial membranes which result in the dramatic increase of their perme-
ability. This increase in permeability during necrosis is caused by the opening
of a Permeability Transition Pore - PTP in mitochondrial inner membrane. On
the other hand apoptosis induces opening of a large non-selective channel (Mito-
chondrial Apoptotic Channel - MAC) in the mitochondrial outer membrane. We
used Atomic force microscopy to study the structure and organization of native
mitochondrial membranes isolated from necrotic and apoptotic cells. We com-
plement our AFM imaging with nanopatricle-based immnunoassays, where mi-
tochondrial membranes are pretreated with nanoparticle-conjugated antibodies
for recognition of specific proteins. We will present our data on morphological
studies of these membranes and will discuss feasibility of this approach for visu-
alization of mPTP and MAC channels in the native mitochondrial inner mem-
branes (MIM) and mitochondrial outer membranes (MOM) using AFM methods.

808-Pos Board B594
Heterogeneity of Cardiolipin Regulation
Yi-Fan Lin, Mei-Jie Jou.
Department of Physiology and Pharmacology, Chang Gung University,
Tao-Yuan, Taiwan.
Cardiolipin (CL), is a protective phospholipid located exclusively on the mito-
chondrial inner membrane for stabilizing individual complex of mitochondrial
respiratory chain for energy production, mitochondrial Ca2þ (mCa2þ) regula-
tion and reactive oxygen spices formation. Deficiency of CL has been associated
with multiple mitochondrial dysfunctions upon pathological condition, diseases
and aging. Using fluorescence digital imaging microscopy this study investi-
gated how CL is preciously regulated in rat brain astrocytes RBA-1. Results
demonstrate that at the resting stage, the distribution of CL is rather heteroge-
neous within single cell. The perinulear mitochondria show lower CL than mi-
tochondria from the peripheral area. Heterogeneity of CL was observed even in
single mitochondrion, which may affects mitochondrial fission and fusion
mechanism. CL was found to be higher at the cross point area of mitochondrial
network. Thread-like mitochondria and granular-like mitochondria, however,
contain similar level of CL. Some of the thread-like mitochondria even contain
lower CL than granular-like mitochondria. Upon localized oxidative stress in-
duced by 488 nm laser irradiation, heterogeneous depletion of CL was observed
among mitochondria populations within single cell. Distribution of CL altered
also mitochondrial dynamics that depletion of CL always occurred before fis-
sion. Intriguingly, although complete depletion of CL is irreversible upon
high strength of laser irradiation, depleted CL recovered during lower strength
of laser irradiation and occurred heterogeneously within mitochondria popula-
tions. Condensation of CL was observed upon various stresses including oxida-
tive stress induced by 488 nm laser irradiation, ionomycin-induced mCa2þ
stress and arachidonic acid-induced lipid stress which crucially enhances pres-
ervation of CL and its resistance to stress which may be of significance for mi-
tochondrial protection and survival. Unveiling pathophysiological regulation of
CL thus may contribute significantly the prevention and treatment of CL asso-
ciated pathological condition, diseases and aging.

809-Pos Board B595
mtDNA T8993G Mutation-Induced Mitochondrial Complex V Inhibition
Augments Cardiolipin-Dependent Alterations of Mitochondrial Dynamics
During Oxidative, Ca2D and Lipid Insults in NARP Cybrids: A Potential
Therapeutic Target
Tsung-I Peng1, Chia-Wei Hsiao2, Mei-Jie Jou3.
1Keelung Chang Gung Memorial hospital, Tao-Yuan, Taiwan,
2National Tsing Hua University, Tao-Yuan, Taiwan, 3Chang Gung
University, Tao-Yuan, Taiwan.
Mitochondrial dynamics including morphological fission and mitochondrial
movement are essential to normal mitochondrial and cellular physiology. This
study investigated how mtDNA T8993G (NARP)-induced inhibition of mito-
chondrial complex V altered mitochondrial dynamics in association with a pro-
tective mitochondrial phospholipid, cardiolipin (CL), as a potential therapeutic
target. NARP cybrids harboring 98% of mtDNA T8993G genes and its parental
osteosarcoma 143B cells were studied for comparison, and protection provided
by melatonin, a potent mitochondrial protector, was explored. We demonstrate
for the first time that NARP mutation significantly enhances apoptotic death as
a result of three distinct lethal mitochondrial apoptotic insults including oxida-
tive, Ca2þ and lipidstress. In addition, NARPsignificantly augmented patholog-
ical depletion of CL. NARP-augmented depletion of CL results in enhanced
retardation of mitochondrial movement and fission and later swelling of mito-
chondria during all insults. These results suggest thatCL is a common and crucial
pathological target formitochondrial apoptoticinsults. Furthermore, CL possibly
plays a central role in regulating mitochondrial dynamics that are associated with
NARP-augmented mitochondrial pathologies. Intriguingly, melatonin, by differ-
entially preserving CL during various stresses (oxidation > Ca2þ > lipid), res-
cuesdifferentially CL-altered mitochondrial dynamics and cell death (oxidation >
Ca2þ > lipid). Thus, melatonin, in addition to being a mitochondrial antioxidant
to antagonize mitochondrial oxidative stress, a mitochondrial permeability tran-
sition modulator to antagonize mitochondrial Ca2þ stress, may stabilize directly
CL to prevent its oxidization and/or depletion and, therefore, it exerts great po-
tential in rescuing CL-dependent mitochondrial dynamics-associated mitochon-
drial pathologies for treatment of NARP-induced pathologies and diseases.

810-Pos Board B596
Shuttling of Membrane-Bound Cytochrome B5 between Mitochondria
and ER
David Weaver1, Veronica Eisner1, Peter Varnai2, Gyorgy Hajnoczky1.
1
Thomas Jefferson University, Philadelphia, PA, USA,

2
Semmelweis Medical University, Budapest, Hungary.
Cytochrome b5 type B has long been described as a tail-anchored protein of the
outer mitochondrial membrane (cb5-mito). Using a fusion protein consisting of
photoactivatable GFP fused to the C-terminus membrane-targeting segment of
cb5-mito, we have found that while this construct concentrates in the mitochon-
drial membrane, it is continuously exchanged between the membranes of the
mitochondrion and the endoplasmic reticulum. This effect does not depend
on the presence of mitofusin 1 or 2, as it occurs in Mfn-null MEFs as well as
in wild type cells. Interestingly, a point mutation in the hydrophobic,
membrane-targeting sequence of cb5-mito that was recently reported to prevent
its association with autophagosome membranes, also eliminates its exchange
with the ER. While lipids and viral proteins have been shown to traffic from
ER to mitochondria, this is, to our knowledge, the first demonstration of such
a back-and-forth shuttling, representing both a new aspect of the ER-
mitochondrial interface and an Mfn-independent pathway for the exchange
of an outer mitochondrial membrane protein.

811-Pos Board B597
Dichotomy between Mitochondrial Ca2D Uptake and Martix [Ca2D] in
MICU1 Deficient Cells
Tunde Golenar1, Gyorgy Csordas1, Fabiana Perocchi2, Vamsi K. Mootha2,
Gyorgy Hajnoczky1.
1Thomas Jefferson University, Philadelphia, PA, USA, 2Harvard Medical
School and Massachusetts General Hospital, Boston, MA, USA.
Mitochondrial calcium uptake is central to cell physiology, but the underlying
molecular mechanism remains elusive. Recently, MICU1 was identified as a mi-
tochondrial protein required for Ca2þ uptake but dispensible for mitochondrial
respiration or membrane potential generation. To study the specific role of
MICU1 in mitochondrial Ca2þ uptake, we used wild type HeLa cells (Ctrl),
MICU1 knockdown by 2 distinct shRNAs (KD) and MICU1 rescued cells (Res-
cue). First, we monitored the cytoplasmic and mitochondrial matrix [Ca2þ]
([Ca2þ]c and [Ca2þ]m, respectively) during store-operated Ca2þ entry. Specif-
ically, cells were preincubated in a Ca2þ-free buffer containing thapsigargin
(Tg), to deplete the ER Ca2þ store, and then Ca2þ was added back and the en-
suing [Ca2þ]c (fura2) and [Ca2þ]m (mitochondria-targeted pericams) changes
were measured. In both Ctrl and KD a [Ca2þ]c increase occurred that was asso-
ciated with a [Ca2þ]m rise only in Ctrl. The Rescue behaved similarly to the Ctrl.
Also, when Ca2þ was added to permeabilized cells, no [Ca2þ]m increase ap-
peared in the KD. These results confirm that the shortage of MICU1 causes sup-
pression of the [Ca2þ]m signal. Surprisingly, when the Ca2þ entry was followed
byuncoupler additiontoreleasethemitochondrial Ca2þ content, ahuge[Ca2þ]c
increase appeared in KD, However, when the uncoupler was added prior to Ca2þ
entry, no [Ca2þ]c increase occurred. Thus, mitochondria in the KD also accumu-
lated Ca2þ during Ca2þ entry. Mitochondrial Ca2þ uptake evoked by Ca2þ ad-
dition was also documented in permeabilized KD by measurement of ruthenium
red sensitive 45Ca uptake and cytoplasmic Ca2þ clearance (fura2). Thus, Ca2þ
is accumulated by MICU1 deficient mitochondria but it fails to result an increase
in[Ca2þ]m.Weare currentlyinvestigating, whether suppression ofthe [Ca2þ]m
rise in the KD is due to enhanced matrix Ca2þ-buffering.
812-Pos Board B598
MICU1 Serves as a Ca2D-Controlled Gatekeeper for the Mitochondrial
Ca2D Uniporter
György Csordás1, Tünde Golenár1, Erin L. Seifert1, Fabiana Perocchi2,
Vamsi K. Mootha2, György Hajnózky1.
1
Thomas Jefferson University, Philadelphia, PA, USA,

2
Department of

Systems Biology, Harvard Medical School, Boston, MA, USA.
MICU1 serves as a Ca2þ-controlled gatekeeper for the mitochondrial Ca2þ

uniporter Mitochondria are important targets and relay points of calcium
signaling. Ca

2þ
is driven to the mitochondrial matrix via a low-affinity

Ca
2þ

channel, the Ca
2þ

uniporter (MCU) that exhibits time-dependent


	Heterogeneity of Cardiolipin Regulation
	mtDNA T8993G Mutation-Induced Mitochondrial Complex V Inhibition Augments Cardiolipin-Dependent Alterations of Mitochondria ...
	Shuttling of Membrane-Bound Cytochrome B5 between Mitochondria and ER
	Dichotomy between Mitochondrial Ca2+ Uptake and Martix [Ca2+] in MICU1 Deficient Cells
	MICU1 Serves as a Ca2+-Controlled Gatekeeper for the Mitochondrial Ca2+ Uniporter