Mitochondrial targeting of cyclosporin A enables selective inhibition of cyclophilin-D and enhanced cytoprotection after glucose and oxygen deprivation

Article abstract of DOI:10.1042/BJ20090332

CsA (cyclosporin A) is a hydrophobic undecapeptide that inhibits CyPs (cyclophilins), a family of PPIases (peptidylprolyl cis-trans isomerases). In some experimental models, CsA offers partial protection against lethal cell injury brought about by transient ischaemia; this is believed to reflect inhibition of CyPD, a mitochondrial isoform that facilitates formation of the permeability transition pore in the mitochondrial innermembrane. To evaluate this further, we have targeted CsA to mitochondria so that it becomes selective for CyP-D in cells. This was achieved by conjugating the inhibitor to the lipophilic triphenylphosphonium cation, enabling its accumulation in mitochondria due to the inner membrane potential. In a cell-free system and in B50 neuroblastoma cells the novel reagent (but not CsA itself) preferentially inhibited CyP-D over extramitochondrial CyPA. In hippocampal neurons, mitochondrial targeting markedly enhanced the capacity of CsA to prevent cell necrosis brought about by oxygen and glucose deprivation, but largely abolished its capacity to inhibit glutamate-induced cell death. It is concluded that CyP-D has a major pathogenic role in 'energy failure', but not in glutamate excitotoxicity, where cytoprotection primarily reflects CsA interaction with extramitochondrial CyPs and calcineurin. Moreover, the therapeutic potential of CsA against ischaemia/reperfusion injuries not involving glutamate may be improved by mitochondrial targeting. The Authors Journal compilation

Full text of DOI:10.1042/BJ20090332

Biochem. J. (2010) 425, 137–148 (Printed in Great Britain) doi:10.1042/BJ20090332
137
Mitochondrial targeting of cyclosporin A enables selective inhibition
of cyclophilin-D and enhanced cytoprotection after glucose and
oxygen deprivation
1
Sylvanie MALOUITRE*1, Henry DUBE† , David SELWOOD† and Martin CROMPTON*2
*Research Department of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K., and †Wolfson Institute for Biomedical Research,
University College London, Gower Street, London WC1E 6BT, U.K.
CsA (cyclosporin A) is a hydrophobic undecapeptide that inhibits
CyPs (cyclophilins), a family of PPIases (peptidylprolyl cis–
trans isomerases). In some experimental models, CsA offers
partial protection against lethal cell injury brought about by
transient ischaemia; this is believed to reflect inhibition of CyP-
D, a mitochondrial isoform that facilitates formation of the
permeability transition pore in the mitochondrial inner membrane.
To evaluate this further, we have targeted CsA to mitochondria so
that it becomes selective for CyP-D in cells. This was achieved by
conjugating the inhibitor to the lipophilic triphenylphosphonium
cation, enabling its accumulation in mitochondria due to the
inner membrane potential. In a cell-free system and in B50
neuroblastoma cells the novel reagent (but not CsA itself)
preferentially inhibited CyP-D over extramitochondrial CyP-
A. In hippocampal neurons, mitochondrial targeting markedly
enhanced the capacity of CsA to prevent cell necrosis brought
about by oxygen and glucose deprivation, but largely abolished
its capacity to inhibit glutamate-induced cell death. It is concluded
that CyP-D has a major pathogenic role in ‘energy failure’, but
not in glutamate excitotoxicity, where cytoprotection primarily
reflects CsA interaction with extramitochondrial CyPs and
calcineurin. Moreover, the therapeutic potential of CsA against
ischaemia/reperfusion injuries not involving glutamate may be
improved by mitochondrial targeting.
Key words: cyclophilin (CyP), cyclosporin A (CsA), glutamate
toxicity, hippocampal neuron, ischaemia, necrosis.
I/R injury is supported by whole organ and animal studies. In
particular, CsA can attenuate reperfusion-induced cell necrosis
in heart [7], brain [8] and other tissues. Use of 2-deoxyglucose
[9] and calcein [10] reveals that the mitochondrial inner membrane
does become non-selectively permeable on reperfusion, and that
CsA can prevent the permeability changes. Genetic ablation of
CyP (cyclophilin)-D, a PT pore component and a target of CsA
(see below), markedly reduces infarct size in heart [11] and brain
[12] following transient ischaemia.
CyP-D is a PPIase (peptidylprolyl cis–trans isomerase) that
is located in the mitochondrial matrix [13] and is inhibited by
CsA. CyP-D overexpression promotes PT pore formation [14],
whereas CyP-D ablation attenuates it [15]. It is believed that,
under pathological conditions, CyP-D catalyses or stabilizes a
proline-dependent conformational switch in an inner membrane
protein to form the non-selective PT pore. Conceivably, the CyP-
D-sensitive protein is a transport protein that loses its selective
transport properties when exposed to high Ca2+ concentrations
and oxidative stress. Reconstitution studies indicate that the
adenine nucleotide translocase can deform into a non-selective
pore under the influence of Ca2+ and CyP-D [16,17], but
mitochondria from mice lacking the two translocase isoforms do
display PT pore activity, albeit with much reduced Ca2+ sensitivity
[18], and additional (or possibly alternative) inner membrane
INTRODUCTION
Ischaemic diseases, notably myocardial infarction and stroke,
are the leading cause of death and disability throughout the
world. In general, early restoration of blood flow is essential to
restrict tissue damage. Paradoxically, however, as the duration of
ischaemia increases, cells become adversely sensitive to restored
blood supply, giving rise to additional damage on reperfusion
(reperfusion injury). Resolving the critical cellular changes that
underlie I/R (ischaemia/reperfusion) injury is a major goal in
developing effective therapies.
A mitochondrial pore model for I/R injury was derived from
two lines of evidence: first, the commonality between factors
that induce formation of the PT (permeability transition) pore
and recognized cellular changes in I/R, principally cellular Ca2+
overload, oxidative stress and depleted adenine nucleotides [1,2];
secondly, inhibition of both pore formation and cell necrosis
under pseudo-I/R by CsA (cyclosporin A) [3–5]. The non-
specific PT pore [6], with an estimated internal diameter of
2 nm [1], allows free flow of low Mr solutes across the inner
membrane. Under these conditions, mitochondria carry out ATP
hydrolysis, rather than synthesis. Once ATP hydrolysis exceeds
the glycolytic capacity of the cell to maintain ATP, irreversible
injury leading to cell necrosis ensues. The PT pore model of
Abbreviations used: CsA, cyclosporin A; CyP, cyclophilin; CyP-D+, cell line overexpressing CyP-D; DCM, dichloromethane; DMEM, Dulbecco’s
minimal essential medium; DMF, dimethylformamide; ESI–MS, electrospray ionization MS; FBS, fetal bovine serum; Fmoc, fluoren-9-ylmethoxycarbonyl;
HBSS, Hanks balanced salt solution; I/R, ischaemia/reperfusion; LDA, lithium diisopropylamide; L-NAME, NG-nitro-L-arginine-methyl ester; mtCsA,
mitochondrially targeted CsA; NBA, Neurobasal A; NBQX, 2,3-dihydro-6-nitro-7-sulfamoylbenzoquinoxaline; NMDA, N-methyl-D-aspartate; OGD, oxygen
and glucose deprivation; PPIase, peptidylprolyl cis–trans isomerase; PT, permeability transition; PyBOP, benzotriazol-1-yl-tris-pyrrolidinophosphonium
hexafluorophosphate; SMBz-CsA, [sarcosine-3(4-methylbenzoate)]-CsA; THF, tetrahydrofuran; TMRE, tetramethylrhodamine ethyl ester; TPP+,
triphenylphosphonium.
1
These authors contributed equally to this work.
To whom correspondence should be addressed (email m.crompton@ucl.ac.uk).
2
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

138
S. Malouitre and others
proteins that can interact with CyP-D, e.g. the phosphate carrier
[19], have been proposed. Irrespective of the identity of the pore
protein itself, however, there is little doubt that CsA inhibits PT
pore formation by inhibiting CyP-D.
However, despite this evidence that CyP-D has a key role in I/R
injury, cytoprotection by CsA in experimental models is highly
variable (and frequently marginal) and, in recent pilot trials, CsA
yielded modest protection [20]. This suggests that CyP-D has a
restricted pathogenic function and/or that CsA sensitivity is not
a precise indicator of CyP-D involvement in cell death, possibly
due to interaction with other CyPs ; the cytosolic CyP-A and
the endoplasmic reticulum CyP-B also have known links to cell
viability (see the Discussion section).
In order to shed light on these issues, we have developed
a novel CsA derivative that is effectively CyP-D selective in
cells because it accumulates in the mitochondrial compartment.
Application of the mtCsA (mitochondrially targeted CsA) to
hippocampal neurons indicates that CyP-D has a major pathogenic
role in energy failure, but only a minor role in glutamate
excitotoxicity. Moreover, the cytoprotective capacity of CsA
against the adverse consequences of energy failure is markedly
improved by mitochondrial targeting.
{1−(vi/vo)}, where vi and vo are the reaction velocities in the
presence and absence of inhibitor respectively. The Km value
for the cis-peptide used (980 μM; [21]) is much higher than its
concentration in the assay (<35 μM). As Km>>[S], the equation
may be simplified:
Io
P
1
=
• Ki + Eo
(1 − P)
and plots of Io/P against 1/(1−P) are linear with slope equal to
Ki.
Interaction of CyP–cyclosporin complexes with calcineurin
[also known as PPP3CA (protein phosphatase 3, catalytic subunit,
α isoform)] was evaluated from inhibition of the phosphatase
activity of calcineurin as measured by the release of inorganic
phosphate from the RII phosphopeptide (Biomol International).
Experiments with isolated mitochondria
Mitochondria were isolated from rat livers as described previously
[1]. All animal experimentation conformed to U.K. Home
Office legislation. PT pore opening was monitored by the
associated swelling of the mitochondria [9] as measured by
the decrease in absorbance at 540 nm. Mitochondria (2 mg of
protein) were suspended in 3 ml of buffer (10 mM Hepes, pH 7.2,
containing 120 mM KCl, 2 mM KH2PO4, 3 mM succinate, 1 μM
rotenone and 5 μM EGTA) with the recombinant CyP-A and test
cyclosporins, and maintained under continuous stirring at 25◦C.
After 5 min, calcium chloride was slowly infused (at 10 μM/min)
to a final concentration of 50 μM. Changes in preincubation time
with CsA or mtCsA from 5 min to 2 min or 10 min did not
change the degree of PT pore inhibition. In a parallel incubation,
mitochondria were sedimented at the time indicated and the CyP-
A activity of the supernatant determined (0.5 ml of supernatant
per ml of reaction volume).
EXPERIMENTAL
Preparation of recombinant CyP-D and CyP-A
Recombinant rat CyP-D was prepared and purified as described
previously [14]. For CyP-A, the coding sequence in rat was PCR-
amplified with the addition of BamH1 and EcoR1 restriction sites,
and cloned between the same sites of pGEX-4T-1 in Escherichia
coli DH5α cells. Transformed cells were grown for 5 h at
21◦C. The GST (glutathione transferase)–CyP-A fusion protein
was extracted, purified on a GSH–Sepharose column (Sigma–
Aldrich), and then cleaved with thrombin to release CyP-A. The
CyP-A was purified on a cation exchange column (Mono S®;
Amersham Pharmacia) and by a gel filtration column (Superdex-
75; Amersham Pharmacia) to give a single band on SDS/PAGE.
Neuronal cultures and assays
Interactions of cyclosporins with CyPs and calcineurin
B50 cells from a rat neuronal cell line and a clone stably
overexpressing CyP-D were cultured on coverslips in DMEM
(Dulbecco’s minimal essential medium) containing 10% (v/v)
FBS (fetal bovine serum) as described previously [14]. Uptake
of TMRE (tetramethylrhodamine ethyl ester) was measured
by incubating the cells at 25◦C in basic medium (24 mM
Hepes, pH 7.4, containing 140 mM NaCl, 4 mM KCl, 1 mM
MgSO4, 1 mM CaCl2, 1 mM KH2PO4 and 11 mM glucose)
containing 50 nM TMRE. Fluorescence images were obtained
at an excitation wavelength of 530 nm and emission wavelength
of >595 nm with an Olympus IX-70 fluorescence microscope
with a ×60 oil objective, Micromax 1401E CCD (charge-
coupled-device) camera and Metamorph software (Molecular
Devices). For nitroprusside treatment, cells were incubated in
basic medium containing 100 μM sodium nitroprusside for
40 min and then returned to DMEM. After 5 h, cells were
extracted and the extracts were assayed for caspase-3 activity
using the fluorescent derivative of the caspase-3/7 selective
substrate, Ac-DEVD (N-acetyl-Asp-Glu-Val-Asp-)-AFC (7-
amino-4-trifluoromethylcoumarin), as described previously [23].
For antisense suppression of CyP-A, cells were incubated with
1 μM phosphorothioate oligodeoxynucleotide (5ꢁ-CATGGCT-
TCCACAATGCT) for 48 h [23].
Dissociation constants for CyP–cyclosporin interactions were
measured as inhibitor constants, Ki. PPIase assays were conducted
at 15◦C in 20 mM Hepes, pH 7.5, containing 100 mM NaCl using
N-succinyl-alanyl-alanyl-prolyl-4-nitroanilide as the test peptide
as described previously [14]. The peptide contains a mixture of
cis- and trans-alanine–proline isomers, of which only the trans-
conformer is hydrolysed by chymotrypsin at the C-terminal amide
bond to release chromophore. Existing trans-isomer is cleaved
within the mixing time, hence further cleavage requires cis–
trans isomerization, which is measured. CyPs were preincubated
with cyclosporins for 5 min before addition of chymotrypsin and
60 μM peptide (containing approx. 35 μM cis-peptide) to start
the reaction. Cyclosporins inhibit by competing at the active site
with substrate [21]. Accordingly, kinetic data were analysed by
the Henderson equation for a tight-binding, competitive inhibitor
[22], which can be written:
ꢀ
ꢁ
Io
P
1
[S]
=
• Ki • 1 +
+ Eo
(1 − P)
Km
where Eo and Io are the total concentrations of enzyme and
inhibitor (cyclosporin) respectively, Ki is the enzyme inhibitor
dissociation constant, Km is the Michaelis constant and [S] is the
substrate concentration. P is the fractional inhibition, equal to
Hippocampal neurons were prepared from 2–4-day-old
Sprague–Dawley rats as mixed cultures with glial cells. Dissected
hippocampi were incubated in HBSS (Hanks balanced salt
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

Mitochondrially targeted cyclosporin A
139
Figure 1 Chemical synthesis of the CsA analogues, and their binding by CyPs
The reagents used in each step are: i) lithium diisopropylamide, trimethylsilyl chloride, 4-bromomethylbenzoate; ii) LiOH, methanol; iii) Fmoc-diaminohexane, PyBOP; iv) piperidine, DMF; and v)
5-(carboxypentyl)triphenylphosphonium bromide, PyBOP. Compound (1) is CsA, compound (2) SMBz-CsA and compound (4) mtCsA. The lower table shows the dissociation constants [determined
as inhibitor constants (Ki) as described for Figure 2] for the binding of CsA and the CsA analogues to CyP-D and CyP-A.
solution) containing 0.1% trypsin for 5 min at 37◦C, and were
of glia nuclei) from the Hoechst fluorescence. Necrosis was
quantified from the nuclear staining by ethidium homodimer. For
treatment with glutamate, cultures were incubated under 95%
air/5% CO2 in a solution of Locke’s medium (5 mM Hepes,
150 mM NaCl, 5 mM KCl, 25 mM NaHCO3, 2.3 mM CaCl2
and 6 mM glucose) with the cyclosporins as indicated. After
10 min, 1 mM glutamate was added. After a further period, as
indicated, cells were returned to NBA medium containing Hoechst
33342 and ethidium homodimer, and necrosis was quantified
15 min later. Further inhibitors and antagonists [FK506, L-NAME
(NG-nitro-L-arginine methyl ester), MK801, or NBQX (2,3-
dihydro-6-nitro-7-sulfamoylbenzoquinoxaline); Sigma–Aldrich]
were added as stated in the legend of Figure 8.
washed twice in HBSS. Hippocampi were then dissociated
in HBSS containing 1 mg/ml BSA, 5% (v/v) FBS and
8 mM MgCl2. Dissociated cells were sedimented, suspended
in NBA (Neurobasal A) medium (Gibco) supplemented with
0.5 mM glutamine, 2% (v/v) B27 supplement (Gibco) and
5% (v/v) FBS, seeded onto coverslips and incubated under
95% air/5% CO2 in the same medium with an antimitotics
mix (1 μM of each of 5-fluor-2ꢁ-deoxyuridine, uridine and 1-β-
D-arabinofuranosylcytosine). Medium without antimitotics was
introduced after 3 days.
For OGD (oxygen and glucose deprivation) experiments,
coverslips with hippocampal neurons were seated to form the
base of a small, capped chamber mounted on the microscope stage.
The chamber contained an inlet and outlet for continuous gassing,
input and output tubes for changing the incubation medium, and a
heating element to maintain the temperature at 36◦C. Pseudo-
ischaemic conditions were imposed by omitting glucose and
displacing air with nitrogen in the experimental chamber. Cells
were incubated under 95% N2/5% CO2 with a pregassed solution
of 145 mM NaCl, 26 mM NaHCO3, 5 mM KCl, 1.8 mM CaCl2,
0.8 mM MgCl2, 4 μM ethidium homodimer, 2 μM Hoechst 33342
and the cyclosporins as indicated. After 30 min the gassing was
switched to 95% air/5% CO2 and the medium replaced with NBA
medium containing 4 μM ethidium homodimer. Hippocampal
neurons were identified under brightfield illumination and then
correlated with their respective nuclei (just above the focal plane
Statistical analyses were made using a one-way ANOVA
test with a post-test of Dunnett and results are presented as
+
means S.E.M.
−
Synthesis of cyclosporin analogues
A scheme of the syntheses is given in Figure 1.
Synthesis of compound (2), SMBz [Sarcosine-3(4-methylbenzoate)]-CsA
To a stirred solution of CsA (1.00g, 0.83 mmol) in dry THF
(tetrahydrofuran, 25 ml) under nitrogen at 0◦C was added
dropwise fresh LDA (lithium diisopropylamide; 4.6 mmol, 2.3 ml,
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

140
S. Malouitre and others
2 M in THF). To the resultant deep-brown suspension was added,
dropwise, trimethylsilylchloride (0.83 mmol, 0.1 ml) to give a
clear brown solution. The mixture was stirred at 0◦C for 10 min.
Then further LDA (7.1 mmol, 3.5 ml, 2 M in THF) was added
dropwise and the reaction stirred for 30 min at 0◦C. A solution
of 4-bromomethylbenzoate (1.3 g, 5.8 mmol) in dry THF (10 ml)
was added dropwise to give a pale yellow solution which was
stirred for a further 1 h at room temperature (22◦C). The reaction
was quenched with saturated NH4Cl (aq) (10 ml) followed by 2 M
HCl and then diluted with DCM (dichloromethane) (20 ml). The
separated aqueous layer was extracted with DCM (2 × 20 ml).
The combined organic phases were washed with 2 M HCl (aq)
(2 × 20 ml), saturated NH4Cl (aq) (2 × 20 ml) and saturated NaCl
(2 × 20 ml) and then dried (MgSO4). The volatiles were removed
in vacuo to leave a dark brown oil residue. Purification by flash
chromatography eluting with 6% (v/v) methanol in DCM gave a
yellow solid residue (0.930 g) as a mixture of the unreacted CsA
and the alkylated ester product. This was used in the next reaction
without further purification.
Saponification was next performed. To a stirred solution of the
yellow solid residue (0.93 g) in THF/methanol (1:1, 20 ml) at
0◦C was added dropwise a solution of LiOH · H2O (500 mg) in
water (10 ml). The reaction was allowed to gradually warm-up to
room temperature over 18 h. Then DCM (20 ml) was added. The
resultant solution was acidified with 2 M HCl (aq) (pH 3). The
separated aqueous layer was extracted with DCM (3 × 30 ml).
The combined organic extracts were washed with saturated 2 M
HCl (aq) (2 × 30 ml) and saturated NaCl (2 × 30 ml) and then
dried (MgSO4). The volatiles were removed in vacuo to leave a
solid residue as a mixture of the acid [Figure 1, compound (2)]
and unreacted CsA. The acid was separated from the CsA by flash
column chromatography through an amine column eluting with
a mixture of methanol/DCM/NH3 (aq) (1:8:1) to give the acid as
a salt. After stirring the salt in DCM (20 ml) and 2 M HCl (aq)
(20 ml) for 10 min, extraction by DCM (3 × 20 ml), concentration,
and purification by flash column chromatography gave the title
compound, (2) (0.30 g, 0.24 mmol, 27%) as a yellow solid.
Fast atom bombardment MS in positive ionization mode:
calculated m/z C70H117N11O14 (M + Na) 1358.86787; found m/z
(M + Na) 1358.86447.
ESI–MS (electrospray ionization MS) in positive ionization
mode: calculated m/z C76H131N13O13 (M + 1) 1435.00, (M + 2)
718; found m/z (M + 1) 1435.53, (M + 2) 718.76.
Synthesis of compound (4), mtCsA
To a stirred solution of the amine compound (3) (65 mg, 0.05
mmol) in dry THF (3 ml) under argon at room temperature
was added in one portion PyBOP (35.5 mg, 0.07 mmol),
5-(carboxypentyl)triphenylphosphonium bromide (32 mg, 0.07
mmol) and triethylamine (0.15 mmol, 0.05 ml) and the resultant
mixture stirred for 24 h at room temperature. The volatiles were
removed al vacuo to leave a yellow oil. The oil was purified by
flash column chromatography on silica gel eluting with 6% (v/v)
methanol in DCM followed by methanol/DCM/NH3 (aq) (1:8:1)
to afford the title compound mtCsA, compound (4) (55 mg, 0.03
mmol, 70%) as a white solid.
ESI–MS in positive ionization mode: calculated m/z
C100H155N13O14P+ (M + 1) 1793.8199; found m/z (M + 1)
1794.8270, (M + 2) 898.
RESULTS
Synthesis of CsA derivatives and their interactions with CyPs and
calcineurin
CsA is a lipophilic cyclic undecapeptide (Figure 1). The
highly conserved nature of the CsA-binding site in CyPs
offers little scope for CyP-D-selective cyclosporins (see the
Discussion). Therefore we investigated the possibility of
rendering CsA effectively CyP-D-selective in cells by targeting
it to mitochondria, where CyP-D is located. The strategy was
to conjugate CsA to the lipophilic TPP+ (triphenylphosphonium)
cation so that the positively charged conjugate is accumulated
electrophoretically by mitochondria in response to the negative-
inside inner membrane potential. This principle has been widely
applied in targeting antioxidants to mitochondria [24]. Seebach
et al. [25] demonstrated that CsA may be specifically alkylated at
the sarcosine-3 position if several equivalents of a strong base are
used to generate a polyanion, and the same method was utilized
here, as shown in Figure 1. Briefly, CsA [Figure 1; compound (1)]
was treated with LDA/trimethylsilyl chloride and then with LDA
to generate the sarcosine-3 anion. This was alkylated with 4-
bromomethylbenzoate and the crude product saponified with
lithium hydroxide to give the acid [Figure 1; compound (2)]. The
side chain was elongated by coupling with Fmoc-diaminohexane
and Fmoc cleavage yielded the amine [Figure 1; compound (3)].
This was coupled with 5-(carboxypentyl)triphenylphosphonium
bromide to give the target triphenylphosphonium salt, mtCsA
[Figure 1; compound (4)].
The effects of these modifications of CsA on interactions
with CyP-D and CyP-A were investigated by measuring PPIase
inhibition. Approx. 7 nM (total) CsA yielded 50% inhibition
of CyP-D (Figure 2A). However, this underestimated the true
CsA-binding affinity as the assays had a similar concentration
of CyP-D, at 8 nM. Accordingly, inhibition was analysed using
the Henderson equation for a tight-binding inhibitor (see the
Experimental section), which gave an inhibitory (dissociation)
constant for CsA and CyP-D of 3 nM (Figure 2A, inset). Additions
to position-3 impaired binding slightly for SMBz-CsA (Figure 1),
but strongly for mtCsA, where the binding affinity to CyP-D was
approx. 30-fold lower than for CsA (Ki of 93 nM; Figure 2B).
The binding affinities of CsA, SMBz-CsA and mtCsA to CyP-A
were similar to those with CyP-D (Figure 1).
Synthesis of compound (3)
To a stirred solution of the acid compound (2) (109 mg,
0.08 mmol) in dry THF (3.0 ml) was added N-Fmoc (fluoren-
9-ylmethoxycarbonyl)-1,6-diaminohexane hydrobromide (68.5
mg, 0.16 mmol), PyBOP (benzotriazol-1-yl-tris-pyrroli-
dinophosphonium hexafluorophosphate; 84.5 mg, 0.16 mmol)
and triethylamine (0.25 mmol, 0.4 ml) under nitrogen at room
temperature and the resultant mixture was stirred for 24 h. Then
DCM (5 ml) followed by saturated NH4Cl (aq) (5 ml) were
added. The mixture was extracted with DCM (2 × 3 ml) and
dried (MgSO4). The volatiles were removed in vacuo to leave
a brown oil residue. Purification by chromatography gave the
Fmoc-protected derivative (100 mg) as a yellow solid. This was
used without further purification.
Fmoc-deprotection was next performed. A solution of the Fmoc
compound (100 mg) was stirred in 20% piperidine in DMF
(dimethylformamide; 4 ml) under argon for 24 h. The volatiles
were removed al vacuo to leave a yellow oil. The oil was purified
by flash column chromatography on silica gel eluting with 6%
methanol in DCM followed by methanol/DCM/NH3 (aq) (1:8:1)
to afford the title compound (3) (70 mg, 0.05 mmol, 85%) as a
yellow solid.
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

Mitochondrially targeted cyclosporin A
141
Figure 3 Complexes of CyP-A with SMBz-CsA and mtCsA do not inhibit
calcineurin
The phosphatase activity of calcineurin was measured in the presence and absence of CyP-A,
CsA, SMBz-CsA and mtCsA at concentrations to give 720 nM complexes (see the Experimental
section).
influx into mitochondria is electrophoretic, the inner membrane
potential, ꢀϕM, becomes dissipated during rapid Ca2+ uptake
{and then restored when uptake is complete (e.g. see [14])}.
As dissipation of ꢀϕM would compromise accumulation of the
positively charged mtCsA in mitochondria, Ca2+ was infused
slowly into the test incubations to limit the rate of Ca2+ uptake and
thereby avoid membrane depolarization (this was confirmed using
a tetraphenylphosphonium electrode, as described in Li et al. [14],
and CsA to block PT pore opening; results not shown). CsA and
its derivatives inhibited pore opening (Figures 4A, 4C and 4E).
Estimation of the degrees of inhibition, assessed by the decreases
in absorbance (at the time marked by the broken lines in Figure
4) indicated that approx. 0.1 μM CsA and 0.4 μM mtCsA gave
50% inhibition of PT pore opening (Figures 4B and 4D, closed
symbols). In the same system, CsA inhibited extramitochondrial
CyP-A with a concentration profile similar to that of the PT pore
(Figure 4B); this was expected given that CyP-D and CyP-A have
similar binding affinities for CsA (Figure 1) and, being uncharged,
CsA should equilibrate to the same free concentrations on either
side of the inner membrane. In contrast, mtCsA inhibited PT pore
formation considerably better than it inhibited CyP-A (Figure
4D), even though it binds to CyP-A and CyP-D with similar
affinities (Figure 1). This indicates that mtCsA was accumulated
in the mitochondrial matrix, where CyP-D is located, in preference
to the external medium, containing CyP-A. SMBz-CsA gave
similar inhibition profiles for the PT pore and CyP-A, indicating a
similar free-SMBz-CsA concentration across the inner membrane
(Figure 4F).
Figure 2 CyP-D inhibition by CsA and mtCsA
The PPIase activity of recombinant CyP-D was determined in the presence and absence of
the given concentrations of (A) CsA and (B) mtCsA. Each data point is the average of two
determinations differing by <7%. Insets show results when replotted according to the simplified
Henderson equation as described in the Experimental section yielding values for Ki (slope).
In addition to inhibiting CyPs, CsA forms a complex with
CyP-A that, in turn, inhibits the Ca2+/calmodulin-dependent
serine/threonine protein phosphatase, calcineurin [26], thereby
enlarging considerably CsA’s sphere of action [27]. It was
important therefore to establish how the position-3 modification
affected the capacity of the complex to inhibit calcineurin.
To enable comparisons, the concentrations of CsA, SMBz-
CsA and mtCsA (at 0.9, 1 and 4 μM respectively) in the test
incubations were chosen to establish the same concentrations
(720 nM) of their respective complexes with CyP-A (calculated
using the Ki values of CyP-A with CsA, SMBz-CsA and
mtCsA; Figure 1). Figure 3 shows that, whereas CyP-A alone
produced a small (20%) activation of calcineurin, the CsA–
CyP-A complex inhibited calcineurin by approx. 70%. In
contrast, the SMBz-CsA–CyP-A and mtCsA–CyP-A complexes
produced no inhibition. Conjugation to position-3 of the CsA ring
evidently prevents formation of the ternary CyP–cyclosporin–
calcineurin complex, which is known to require interactions
between calcineurin and positions 3–7 of the ring (see the
Discussion).
Evaluating the CyP-D selectivity of mtCsA in a mixed in vitro system
Evaluating the CyP-D selectivity of mtCsA in intact cells
We first investigated whether mtCsA would select for intramito-
chondrial CyP-D and the PT pore, rather than extramitochondrial
CyPs, using a test system comprising isolated mitochondria and
externally added, recombinant, CyP-A. PT pore opening was
induced by addition of a high concentration of Ca2+, and was
monitored by the resultant mitochondrial swelling, as the inner
membrane became freely permeable to low-Mr solutes. As Ca2+
Selectivity of mtCsA for CyP-D in intact cells was investigated
using rat B50 neuroblastoma cells and a clone in which
CyP-D is overexpressed by approx. 10-fold (CyP-D+). CyP-
D overexpression did not affect CyP-A (Figure 5F). As shown
previously [14], CyP-D+ cells maintain a relatively low ꢀϕM,
indicative of transient PT pore opening. As the lowering of ꢀϕM
is caused by excessive CyP-D, restoration of ꢀϕM to wild-type
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

142
S. Malouitre and others
Figure 4 mtCsA preferentially inhibits intramitochondrial CyP-D, rather than extramitochondrial CyP-A, in a mixed in vitro system
(A, C and E) Isolated liver mitochondria were incubated with added recombinant CyP-A. After preincubation with CsA, mtCsA or SMBz-CsA, as indicated, PT pore opening was initiated by infusion
of Ca2+ and monitored by recording the decrease in A540 due to mitochondrial swelling. (B, D and F) The degree of PT pore opening was compared by using the absorbance decreases attained at
the time indicated by the broken lines in (A, C and E) and replotted as percentage activity (black symbols). In parallel incubations, mitochondria were sedimented immediately after Ca2+ addition
and CyP-A activity in the supernatant was determined (white symbols).
values provides an unequivocal measure of CyP-D inhibition.
Changes in ꢀϕM were monitored from the uptake of TMRE,
a fluorescent, lipophilic cation accumulated by mitochondria
according to the magnitude of the potential. Figure 5(A) shows
typical images of TMRE accumulated within the mitochondria
of these cells. Mitochondria of the CyP-D+ clone accumulated
considerably less TMRE than wild-type cells, but the difference
was negated by CsA, which promoted uptake by the CyP-D+
cells. Maximal restoration of TMRE uptake by CyP-D+ cells was
obtained with approx. 0.8 μM CsA (Figure 5C), 2.4 μM mtCsA
(Figure 5D) and 2 μM SMBz-CsA (results not shown). The same
concentrations did not affect TMRE uptake by wild-type cells
[Figure 5B; also confirmed for SMBz-CsA (results not shown)].
It may be concluded that approx. 0.8 μM CsA, 2 μM SMBz-
CsA and 2.4 μM mtCsA are sufficient to inhibit CyP-D in B50
cells.
To investigate whether mtCsA inhibited CyP-D selectively, i.e.
without appreciable inhibition of CyP-A, we needed a marker
of CyP-A activity. Previously, we reported that nitroprusside-
induced caspase activation in B50 cells is largely prevented by
CsA and by antisense suppression of CyP-A. This indicates
that CsA inhibits caspase activation in this model by inhibiting
CyP-A [23]. Although the specific site of action of CyP-A is
unresolved, this system offers a measure of CyP-A activity.
Antisense treatment decreased CyP-A expression by >85%
without affecting CyP-D (Figure 5F), and antisense treatment,
CsA and SMBz-CsA all reduced nitroprusside-induced activation
of caspase-3 (Figure 5E). Unlike CsA and SMBz-CsA, however,
2.4 μM mtCsA (which was sufficient to inhibit mitochondrial
CyP-D; Figure 4D), had no significanteffect on caspase activation,
indicating that it was not accumulated in the cytosol, where CyP-A
is located.
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

Mitochondrially targeted cyclosporin A
143
Figure 5 mtCsA preferentially inhibits CyP-D, rather than CyP-A, in B50 neuronal cells
(A) Wild-type B50 cells (WT), or a CyP-D+ clone overexpressing CyP-D (CLONE) were incubated with 50 nM TMRE for 30 min (panels 1 and 3) after which 1 μM CsA was added and the cells
incubated for a further 30 min (panels 2 and 4). (B–D) Wild-type (WT) or CyP-D+ cells were incubated in the presence or absence of the indicated concentrations of CsA and mtCsA. TMRE uptake
+
+
was measured by whole cell fluorescence determined at intervals. The results are the means S.E.M. for four experiments. (E) CyP-D cells pretreated with CyP-A antisense oligodeoxynucleotide
−
(AS; 42 h), 0.8 μM CsA, 2 μM SMBz-CsA or 2.4 μM mtCsA (for 10 min) were incubated with or without 100 μM nitroprusside (for 40 min) and extracts were assayed for caspase-3. *P<0.05,
comparing test nitroprusside data with control (no addition) nitroprusside data over four experiments. (F) Immunoblots showing that CyP-D overexpression (CyP-D+) does not change CyP-A, and
that CyP-A knockdown (AS CyP-A) does not change CyP-D.
Following OGD, approx. 60% of neurons became necrotic
within 90 min (Figure 6A), but mortality was approximately
halved in the presence of mtCsA. Maximal protection was given
with >0.8 μM mtCsA (Figure 6B). CsA was less protective
(Figure 6B). There was a relatively small protection with
0.1 μM CsA, but this was reversed at higher CsA concentrations,
suggesting the existence of secondary CsA targets outside
mitochondria that counteracted the protection. Thus restricting
the action of CsA to mitochondria using mtCsA improves its
protective capacity against cell necrosis brought about by a
period of OGD, indicating that CyP-D and the PT pore are major
contributors to this form of injury.
I/R and glutamate-induced injury in hippocampal neurons
I/R was mimicked by incubating hippocampal neurons under
OGD conditions for 30 min, after which glucose and oxygen
were restored. To estimate the time period of OGD needed
to remove oxygen sufficiently for impairment of mitochondrial
electron transport, we followed TMRE loss from mitochondria of
preloaded cells as an index of ꢀϕM dissipation. TMRE was lost
after approx. 5 min OGD, indicating respiratory inhibition at this
time (results not shown). At the outset of each experiment, a group
of hippocampal neurons were distinguished from underlying glial
cells and the same neurons were imaged at intervals thereafter.
The susceptibility of neuronal cells (but not glial cells) to OGD-
induced necrosis increased with the number of days in culture,
and results were obtained after culture for 24–28 days.
In vivo, a major component of I/R-induced neuronal injury is
believed to be initiated by prolonged exposure to extracellular
glutamate and overactivation of ionotropic glutamate receptors.
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

144
S. Malouitre and others
Figure 7 CsA, but not mtCsA, strongly reduces glutamate toxicity in
hippocampal neurons
Figure 6 Mitochondrial targeting enhances protection of hippocampal
neurons by CsA following transient oxygen and glucose deprivation
(A) Hippocampal neurons were incubated in Locke’s medium with (᭹) and without (᭺) 1 mM
◦
glutamate at 37 C and cell viability was determined from nuclear staining with ethidium
(A) Hippocampal neurons were preincubated under OGD for 30 min, and then returned to
glucose-replete normoxia (OGD). The experiment was repeated with mtCsA included in both
anoxic and normoxic media (OGD + mtCsA). The lower curve is without an OGD step (No
ODG). Necrotic cells were quantified from nuclear staining with ethidium homodimer. (B) Cells
were incubated with CsA or mtCsA and necrosis determined after 30 min OGD and 90 min
homodimer. Results from a single representative preparation. (B) Neurons were incubated in
Locke’s medium with CsA (᭺) or mtCsA (᭹). After 10 min, 1 mM glutamate was added and
+
cell necrosis determined 3 hours later. Results are means S.E.M. of five cell preparations with
−
>20 cells per preparation.
+
glucose-replete normoxia. Results are means S.E.M. for five cell preparations with >20 cells
−
both protective (glutamate) and counterprotective (OGD), might
involve inhibition of calcineurin by the CsA–CyP-A complex,
we used FK506 (an immunosuppressant macrolide) which, as the
FK506–FKBP (FK506-binding protein) complex, also inhibits
calcineurin. As shown in Figure 8(B), FK506 partially reversed
mtCsA-protection against OGD-induced necrosis, indicating that
calcineurin inhibition is counterprotective. In contrast, FK506
partially prevented glutamate-induced necrosis. Thus calcineurin
appears to have opposing roles in OGD-induced and glutamate-
induced cell death.
Calcineurin dephosphorylates, and thereby activates, neuronal
NO synthase, and this activation is believed to contribute to
glutamate toxicity [27]. However, the NO synthase inhibitor L-
NAME did not yield significant protection. Indeed, it is notable
that both L-NAME and FK506 were less effective than CsA (Fig-
ure 8A). To test whether the acute effectiveness of CsA against
glutamate toxicity reflected a dual action in inhibiting both
calcineurin and CyP-D (and the PT pore), we applied FK506 and
mtCsA together; however this was no more effective than FK506
alone. This confirms that the inhibition of glutamate excitotoxicity
by CsA is not due to inhibition of CyP-D.
per preparation.
To investigate PT pore involvement in glutamate toxicity, neurons
were exposed to added glutamate under normoxic conditions in
the presence of glucose. Following a short lag-period, this led
to progressive necrosis (Figure 7A). Surprisingly, the relative
protective capacities of CsA and mtCsA were now reversed with
respect to OGD. Whereas CsA yielded good protection, mtCsA
was ineffective (Figure 7B). In a further series of experiments
(Figure 8A), mtCsA did reduce necrosis to a small extent (24%,
but not significant) but, again, CsA was far more effective
with 88% protection. Thus restricting the action of CsA to
mitochondria with mtCsA largely removes protection by CsA,
indicating that CsA attenuates glutamate toxicity by interacting
with extramitochondrial CyPs, rather than intramitochondrial
CyP-D.
As expected, glutamate-induced necrosis was prevented
by glutamate antagonists, i.e. by MK-801, an NMDA (N-
methyl-D-aspartate) receptor antagonist, and by NBQX, an
AMPA (amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid)
receptor antagonist (Figure 8A). However, glutamate antagonists
had relatively little effect on OGD-induced necrosis (Figure
8B), indicating negligible contribution of glutamate toxicity
under this protocol (any glutamate released from the cells
during anoxia was presumably quickly diluted in the incubation
medium). Thus the involvement of extramitochondrial CyPs
in CsA protection correlates with glutamate toxicity, whereas
their involvement in CsA counterprotection correlates with
OGD-induced necrosis. To investigate whether these effects,
The greater effectiveness of CsA compared with mtCsA plus
FK506 in inhibiting glutamate-induced necrosis (Figure 8A)
suggests that CsA-mediated inhibition of extramitochondrial
CyPs (PPIase activity) may also be protective. This was further
investigated using SMBz-CsA, which inhibits CyP PPIase activity
(Figure 1), but not calcineurin when in complex with CyP-A (Fig-
ure 3). Similar to CsA, SMBz-CsA permeates into mitochondria
and inhibits the PT pore (Figure 4E), but is not accumulated by
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

Mitochondrially targeted cyclosporin A
145
Figure 9 Cytoprotection by SMBz-CsA
Glutamate-induced and OGD-induced necrosis in hippocampal neurons was determined as
in Figure 8 in the presence of 0.3–2.0 μM CsA, SMBz-CsA and mtCsA, as indicated. In the
+
+
absence of CsA and derivatives, necrosis was 34 5% (glutamate) and 64 6% (OGD). Results
−
−
+
are means S.E.M. of five cell preparations with >20 cells per preparation. *P<0.05 for
−
SMBz-CsA compared with mtCsA.
DISCUSSION
CsA is a lipophilic, cyclic undecapeptide which half-inserts, edge-
on, into a cleft on the CyP surface, occluding the active PPIase site
and forming interactions with 15 amino acids [28]. The binding
region in CyPs is highly conserved. In particular, apart from a
single conservative substitution (lysine for arginine), the binding
residues in CyP-A are all present in CyP-D, and a high resolution
structure of CyP-D has confirmed that the same residues as in CyP-
A make contact with CsA [29]. Although differences in adjacent
amino acids might offer potential for selective inhibition, the CsA-
binding affinities of CyP-D and CyP-A are about the same (Figure
1). It is therefore difficult to envisage CyP-D selectivity based on
binding affinities alone.
To address this problem, CsA was conjugated to the lipophilic
cation TPP+ in order to allow electrophoretic accumulation
within mitochondria. The CyP-binding amino acids in CsA
have been identified as residues 9–11 and 1–3 [28]. However,
substitution at the sarcosine in position-3 does allow retention
of the tight binding to CyPs [30], and the same position was
utilized in the present study for conjugation to TPP+. In the
ternary CyP–CsA–calcineurin complex, positions 3–7 of the CsA
ring form contacts with calcineurin [26,28]. As expected the
position-3 conjugates, SMBz-CsA and mtCsA, therefore were
unable to bind to calcineurin (Figure 3). The position-3 addition
also reduced the binding affinity to CyPs (Figure 1). This
reduction was marginal for SMBz-CsA, presumably reflecting
steric factors, but was marked in the case of mtCsA. A possible
explanation lies in the fact that the predominant conformation
of free CsA is quite different from that bound to CyPs [28] and
modifications that stabilize the unbound conformations would
effectively lower the binding affinity for CyPs. In addition, mtCsA
would be more lipophilic than CsA itself given the nature of the
linking region. The TPP+ moiety, however, would not increase
lipophilicity (the octanol/buffered saline partition coefficient of
triphenylmethylphosphonium is 0.35 [31]). In this connection,
analyses of radiolabelled CsA binding to mitochondria reveal
that there are components besides CyP-D that bind CsA [32];
these components bind CsA far more weakly than CyP-D, but are
present in excess. For mtCsA, which bound with a 30-fold lower
Figure 8 Protection against glutamate-induced and OGD-induced necrosis
in hippocampal neurons
(A) Glutamate-induced necrosis. Hippocampal neurons were incubated in Locke’s medium
containing either no addition (−), 1 μM CsA, 1.3 μM mtCsA, 1 μM FK506, 250 μM L-NAME,
1 μM FK506 and 1.3 μM mtCsA (mtCsA + FK506), 50 μM MK801 or 100 μM NBQX. After
10 min, 1 mM glutamate was added and cell necrosis was determined 3 h later. (B) OGD-induced
necrosis. Neurons were subjected to oxygen and glucose deprivation for 30 min, and then
glucose-replete normoxia for 90 min, before necrosis was quantified. Inhibitors were added to
+
both anoxic and normoxic media at concentrations as in (A). Results are means S.E.M. of
−
five cell preparations with >20 cells per preparation. *P<0.05; **P<0.01 with respect to no
addition.
mitochondria (Figure 4F) and it enters cells where it inhibits
CyP-A (Figure 5E). As shown in Figure 9, SMBz-CsA was
more cytoprotective than mtCsA, consistent with some protection
due to inhibition of extramitochondrial CyPs. Figure 9 also
shows that SMBz-CsA was a better cytoprotectant than CsA
following OGD and that, unlike CsA (Figure 6), protection was
not reversed with an increased SMBz-CsA concentration. This
may reflect the lack of calcineurin inhibition by SMBz-CsA, as
calcineurin inhibition appears to be counterprotective in OGD-
induced necrosis (Figure 8B, mtCsA compared with mtCsA plus
FK506). However, SMBz-CsA yielded less cytoprotection than
mtCsA, suggesting that inhibition of extramitochondrial CyPs
might also be counterprotective in OGD-induced necrosis (Figure
9). This is consistent with the fact that FK506 does not fully
reverse cytoprotection by mtCsA (Figure 8B). In summary, the
involvement of CyPs and calcineurin appears to differ between
the two forms of injury; CyP-D inhibition is more protective after
OGD than after glutamate, whereas inhibition of calcineurin and
extramitochondrial CyP(s) is more protective after glutamate than
after OGD (when these interventions actually reverse protection).
These differences point to different pathogenic mechanisms.
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

146
S. Malouitre and others
conditions causes excessive Ca2+ influx into the cell, depletion
of ATP and ADP, and oxidative stress [40,44]. Moreover, there
is evidence that glutamate toxicity requires mitochondrial Ca2+
uptake and induces mitochondrial dysfunction (ꢀϕM dissipation)
[40,45], consistent with PT pore formation. Accordingly, it might
be expected that the PT pore would contribute similarly to
injury resulting from transient energy failure alone and from
glutamate toxicity. Surprisingly, however, comparisons of the
cytoprotective capacities of CsA and mtCsA reveal markedly
differing contributions of the PT pore in the two forms of
pathogenesis.
Although CsA-mediated inhibition of glutamate toxicity is
frequently ascribed to inhibition of CyP-D and prevention of the
PT, we obtained no indication for this, as the inhibition of
glutamate-induced necrosis by CsA was largely abolished by
mitochondrial targeting. This mirrors a previous report showing
that genetic ablation of CyP-D had no effect on glutamate toxicity
in hippocampal neurons [45]. It appears that the major site of
CsA-mediated protection against glutamate toxicity is extramito-
chondrial. In agreement, and in line with other studies [27,40], we
obtained evidence that calcineurin plays a major part in the patho-
genesis of cell necrosis induced by glutamate; in particular, FK506
was protective (Figure 8A) and SMBz-CsA was less protective
than CsA (Figure 9). This may involve calcineurin-mediated
activation of neuronal NO synthase. Neuronal NO synthase
is a Ca2+/calmodulin-dependent enzyme, and activation of the
synthase is believed to exert a prominent role in glutamate toxicity,
aided by being ‘scaffolded’ to the NMDA receptor mediating Ca2+
influx [46]. Our results are consistent with this. However, as CsA
was a better inhibitor of glutamate-induced necrosis than either
FK506 (and hence calcineurin inhibition) or L-NAME (and hence
NO synthase inhibition), and the extra inhibition is not explained
by CyP-D inhibition, it appears that inhibition of extramitochon-
drial CyPs may also play a role in CsA-mediated protection
against glutamate-induced necrosis. Consistent with this, SMBz-
CsA was a better cytoprotectant than mtCsA (Figure 9).
In contrast, the major site of CsA-mediated protection against
OGD-induced necrosis appears to be mitochondrial. Thus
necrosis resulting from energy failure alone was substantially
prevented by mtCsA. CsA itself yielded relatively poor protection
against OGD-induced necrosis, with a narrow concentration range
of effectiveness, similar to that found in cardiomyocytes subjected
to transient OGD [4] and in perfused heart [7]. Secondary,
extramitochondrial targets of CsA presumably negate protection
gained by PT inhibition as the CsA concentration is increased.
These counterprotective targets may include calcineurin, as
mtCsA-mediated protection was decreased by FK506 (Figure
8B), and SMBz-CsA was a better cytoprotectant than CsA
(Figure 9). Other extramitochondrial counterprotective targets
cannot be ruled out, as mtCsA protection was only partially
reversed by FK506 (Figure 8B) and mtCsA was a better
cytoprotectant than SMBz-CsA (Figure 9). The augmented
protective capacity of CsA against OGD-induced necrosis
obtained by mitochondrial targeting cannot be explained by
increased inhibition of mitochondrial CyP-D, as CsA was
tested at concentrations (1 μM) sufficient to inhibit CyP-
D completely (in B50 cells, 0.8 μM total CsA completely
reversed the PT activation by CyP-D overexpression; Figure
5C). Rather, enhanced protection by mtCsA must reflect the
relatively low mtCsA concentration outside mitochondria so
that non-mitochondrial, counterprotective effects of CsA are
minimized. Overall, enhanced protection by mtCsA indicates that
it protects against energy-failure-induced necrosis by inhibiting
mitochondrial CyP-D and supports the applicability of the PT
pore model of pathogenesis to this form of injury.
affinity to CyP-D compared with CsA, the matrix free-mtCsA
concentration needed to block the PT will be 30-fold higher
than for CsA, and unspecific binding to other components will
be correspondingly greater. Thus the combination of decreased
CyP-D-binding affinity, increased partitioning into membrane
phospholipids, and unspecific binding may explain why more
mtCsA than CsA was needed to inhibit the PT pore (Figures
4B and 4D). Nevertheless, mtCsA displayed a good selectivity
for intramitochondrial CyP-D over extramitochondrial CyP-A
when tested in a mixed in vitro system (Figure 4D) and in
intact B50 cells (Figure 5). The latter made use of the fact
that nitroprusside-induced apoptosis in B50 cells is promoted by
cytosolic CyP-A [23], butnot by CyP-D [14]. In addition, although
apoptosis in this system involves the release of cytochrome c from
mitochondria [M. Capano (University College London, U.K.) and
M. Crompton, unpublished work], CyP-D and the PT pore do not
appear to be involved in this process [33]. Thus the fact that
mitochondrial targeting of CsA largely removes its capacity to
inhibit apoptosis in this system (Figure 5E) is consistent with
mitochondrial accumulation of the conjugate out of the cytosol.
CyPs have both positive and negative roles in cell viability;
CyP-D is linked to the PT, CyP-A (cytosol) participates in
the translocation of AIF (apoptosis inducing factor) to the
nucleus [34], in caspase activation [23] and in protection
against oxidative stress [35] and CyP-B (endoplasmic reticulum)
suppresses apoptosis associated with oxidative stress and altered
Ca2+ metabolism [36]. In addition, the CyP-A–CsA complex
inhibits calcineurin, which dephosphorylates pro-apoptotic BAD
(Bcl-2/Bcl-XL-antagonist, causing cell death) [37] and the L-
type Ca2+ channel [38] in neurons. The effects on calcineurin
are avoided with some CsA derivatives, e.g. NIM811 and Debio-
025 [39], as with SMBz-CsA (Figure 3), but these derivatives
remain general CyP inhibitors and their overall effects can be
difficult to interpret. For example, SMBz-CsA protected against
both glutamate- and OGD-induced necrosis (Figure 9). At face
value, this could be taken to indicate similar roles of CyP-D and
the PT pore in the two cases. However, use of the CyP-D-specific
mtCsA reveals that this is not the case.
The extent to which the CyP-D-dependent PT pore is the root
cause of ischaemic cell death was addressed in neuronal cells,
where the issue appears complex. Many models of transient
ischaemia show that protection of hippocampal and other neuronal
cells by CsA is equalled by FK506, suggesting that CsA inhibition
of calcineurin-mediated processes, rather than the PT, is critical
(reviewed in [39]). In contrast, other studies point to a primary
role of the PT [8,40,41]. Thus CsA inhibition of both calcineurin
and the PT may yield cytoprotection in different proportions
depending on experimental conditions. To address this issue, we
have taken account of the fact that hippocampal and other neuronal
cells are subjected to two forms of pathogenic insult during I/R. In
common with other kinds of cell, the normally high cellular energy
state (the phosphorylation potential) becomes compromised in
ischaemia as glycolysis and the tricarboxylic acid cycle are
inhibited due to lack of glucose and oxygen, leading to ‘energy
failure’. In addition, and unique to neuronal tissue, active transport
systems for glutamate uptake in astrocytes and neurons (normally
responsible for termination of synaptic transmission) operate
in reverse during energy failure leading to high extracellular
glutamate and over-activation of neuronal glutamate receptors
[42]. Both types of insult produce qualitatively similar changes
in critical factors needed for PT pore formation. Energy failure in
ischaemia leads to losses of ATP and total adenine nucleotides and
to a progressive increase in resting cytosolic Ca2+, and reperfusion
generates ROS (reactive oxygen species) (reviewed in [43]).
Similarly, addition of high exogenous glutamate under aerobic
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

Mitochondrially targeted cyclosporin A
147
12 Schinzel, A. C., Takeuchi, O., Huang, Z., Fisher, J. K., Zhou, Z., Rubens, J., Hetz, C.,
Danial, N. N., Moskowitz, M. A. and Korsmeyer, S. J. (2005) Cyclophilin D is a
component of mitochondrial permeability transition and mediates neuronal
cell death after focal cerebral ischaemia. Proc. Natl. Acad. Sci. U.S.A. 102, 12005–12010
13 Johnson, N., Khan, A., Virji, S., Ward, J. M. and Crompton, M. (1999) Import and
processing of heart mitochondrial cyclophilin D. Eur. J. Biochem. 263, 353–359
14 Li, Y., Johnson, N., Capano, M., Edwards, M. and Crompton, M. (2004) Cyclophilin-D
promotes the mitochondrial permeability transition but has opposite effects on apoptosis
and necrosis. Biochem. J. 383, 101–109
15 Basso, E., Fante, L., Fowlkes, J., Petronilli, V., Forte, M. A. and Bernardi, P. (2005)
Properties of the mitochondrial permeability transition pore in mitochondria devoid of
cyclophilin D. J. Biol. Chem. 280, 18558–18561
16 Crompton, M., Virji, S. and Ward, J. M. (1998) Cyclophilin D binds strongly to
complexes of the voltage dependent anion channel and the adenine nucleotide
translocase to form the permeability transition pore. Eur. J. Biochem. 258, 729–753
17 Brustovetsky, N., Tropschug, M., Heimpel, S., Heidkaemoer, D. and Klingenberg, M.
(2002) A large Ca2+-dependent channel formed by recombinant ADP/ATP carrier from
Neurospora crassa resembles the mitochondrial permeability transition pore.
Biochemistry 41, 11804–11811
In conclusion, we have developed a novel, mitochondrially
targeted CsA and shown that it selectively inhibits the
mitochondrial CyP-D-dependent PT pore compared with
extramitochondrial CyP-A in intact cells. Application of this
targeted CsA to hippocampal neurons exposes a marked
difference between the contribution of CyP-D to necrosis induced
by glutamate alone (a low contribution) and that arising simply
from energy failure (a high contribution). The latter protocol may
be representative of I/R injury to cells in general when glutamate
does not contribute. In these cases, as selective targeting of CsA to
mitochondria produces better protection against transient OGD-
induced cell necrosis than CsA itself, the therapeutic potential of
CsA in limiting reperfusion injury, recently tested in pilot trials
[20], may be improved by mitochondrial targeting.
AUTHOR CONTRIBUTION
18 Kokoszka, J. E., Waymire, K. G., Levy, S. E., Sligh, J. E., Cal, J., Jones, D. P., MacGregor,
G. R. and Wallace, D. C. (2004) The ADP/ATP translocator is not essential for the
mitochondrial permeability transition pore. Nature 427, 461–465
19 Leung, A. W. C., Varayuwatana, P. and Halestrap, A. P. (2008) The mitochondrial
phosphate carrier interacts with cyclophilin D and may play a key role in the permeability
transition. J. Biol. Chem. 283, 26312–26323
Experimental work was carried out by Henry Dube (chemical syntheses) and Sylvanie
Malouitre (biological assays), both of whom also provided intellectual input throughout
the course of the study. The project was conceived and directed by David Selwood
(chemical aspects) and Martin Crompton (biological aspects). All authors contributed to
writing this article.
20 Piot, C., Croisille, P., Staat, P., Thibault, M. D., Rioufol, G., Mewton, N., Elbelghiti, R.,
Cung, T. T., Bonnefoy, E., Angoulvant, D. et al. (2008) Effect of cyclosporine on
reperfusion injury in acute myocardial infarction. New England J. Med. 359, 473–481
21 Kofron, J. L., Kuzmic, P., Kishore, V., Colon-Bonilla, E. and Rich, D. H. (1991)
Determination of kinetic constants of peptidylprolyl cis–trans-isomerases by an improved
spectrophotometric assay. Biochemistry 30, 6127–6134
22 Henderson, P. J. F. (1972) A linear equation that describes the steady-state kinetics of
enzymes and subcellular particles interacting with tightly bound inhibitors. Biochem. J.
127, 321–333
23 Capano, M., Virji, S. and Crompton, M. (2002) Cyclophilin-A is involved in
excitotoxin-induced caspase activation in rat neuronal B50 cells. Biochem. J. 363, 29–36
24 Murphy, M. P. and Smith, R. A. J. (2007) Targeting antioxidants to mitochondria by
conjugation to lipophilic cations. Ann. Rev. Pharm. Toxicol. 47, 629–656
25 Seebach, D., Beck, A. K., Bossler, H. G., Gerber, C., Ko, S. Y., Murtiashaw, C., Naef, R.,
Shoda, S., Thaler, A., Krieger, M. et al. (1993) Modification of cyclosporin A: generation of
an enolate at the sarcosine residue and reactions with electrophiles. Helv. Chim. Acta 76,
1564–1590
26 Jin, L. and Harrison, S. C. (2002) Crystal structure of human calcineurin complexed with
cyclosporin A and human calcineurin. Proc. Natl. Acad. Sci. U.S.A. 99, 13522–13526
27 Morioka, M., Hamada, J. I., Ushio, Y. and Miyamoto, E. (1999) Potential role of
calcineurin for brain ischaemia and traumatic injury. Progr. Neurobiol. 58, 1–30
28 Taylor, P. T., Husi, H., Kontopidis, G. and Walkinshaw, M. D. (1997) Structures of
cyclophilin-ligand complexes. Progr. Biophys. Molec. Biol. 67, 155–181
29 Kajitani, K., Fujihashi, M., Kobayashi, Y., Shimizu, S., Tsujimoto, Y. and Miki, K. (2008)
The crystal structure of human cyclophilin D in complex with its inhibitor cyclosporin A at
0.96 angstrom resolution. Proteins 70, 1635–1639
30 Baumgrass, R., Zhang, Y., Erdmann, F., Thiel, A., Weiwad, M., Radbruch, A. and Fischer,
G. (2004) Substitution in position 3 of cyclosporin A abolishes the cyclophilin-mediated
gain of function mechanism but not immunosuppression. J. Biol. Chem. 279, 2470–2479
31 Asin-Cayuela, A., Manas, A. B., James, A. M., Smith, R. A. J. and Murphy, M. P. (2004)
Fine-tuning the hydrophobicity of a mitochondria-targeted antioxidant. FEBS Lett. 571,
9–16
32 McGuinness, O., Yafei, N., Costi, A. and Crompton, M. (1990) The presence of two
classes of high affinity cyclosporin binding sites in mitochondria. Evidence that the minor
component is involved in the opening of an inner-membrane, Ca2+-dependent pore. Eur.
J. Biochem. 194, 671–679
33 Gillick K, Crompton M (2008) Evaluating cytochrome c diffusion in the intermembrane
spaces of mitochondria during cytochrome c release. J. Cell. Sci. 121, 618–626
34 Zhu, C., Wang, X., Deinum, J., Huang, Z., Gao, J., Modjtahedi, N., Neagu, M. R., Nilsson,
M., Eriksson, P. S., Hagberg, H. et al. (2007) Cyclophilin A participates in the nuclear
translocation of apoptosis inducing factor in neurons after cerebral hypoxia-ischaemia. J.
Exp. Med. 204, 1741–1748
ACKNOWLEDGEMENTS
We thank the following colleagues at University College London: Dr Philip Thomas for
significant help in the culture of hippocampal neurons; Michela Capano for help with the
CyP-A studies; and Dr Mina Edwards for providing the B50 cell cultures.
FUNDING
This work was supported by the Wellcome Trust [grant number 077357].
REFERENCES
1
Crompton, M. and Costi, A. (1988) Kinetic evidence for a heart mitochondrial pore
activated by Ca2+, inorganic phosphate, and oxidative stress. A potential mechanism for
mitochondrial dysfunction during cellular Ca2+ overload. Eur. J. Biochem. 178, 489–501
Crompton, M. and Andreeva, L. (1993) On the involvement of a mitochondrial pore in
reperfusion injury. Basic Res. Cardiol. 88, 513–523
Crompton, M., Ellinger, H. and Costi, A. (1988) Inhibition by cyclosporin A of a
Ca2+-dependent mitochondrial pore activated by inorganic phosphate and oxidative
stress. Biochem. J. 255, 357–360
2
3
4
5
Nazareth, W., Yafei, N. and Crompton, M. (1991) Inhibition of anoxia-induced injury in
heart myocytes by cyclosporin A. J. Mol. Cell. Cardiol. 23, 1351–1354
Duchen, M. R., McGuinness, O., Brown, L. A. and Crompton, M. (1993) On the
involvement of a cyclosporin A sensitive mitochondrial pore in myocardial reperfusion
injury. Cardiovasc. Res. 27, 1790–1794
6
7
Haworth, R. A. and Hunter, P. R. (1979) The Ca2+-induced membrane transition in
mitochondria III transitional Ca2+ release. Arch. Biochem. Biophys. 195, 468–477
Griffiths, E. J. and Halestrap, A. P. (1993) Protection by cyclosporin A of
ischaemia-reperfusion induced damage in isolated rat hearts. J. Mol. Cell. Cardiol. 25,
1461–1469
8
9
Uchino, H., Minamikawa-Tachino, R., Kristian, T., Perkins, G., Narazaki, M., Siesjo, B. K.
and Shibasaki, F (2002) Differential neuroprotection by cyclosporin A and FK506
following ischaemia correlates with differing abilities to inhibit calcineurin and the
mitochondrial permeability transition. Neurobiol. Dis. 10, 219–233
Javadov, S. A., Lim, K. H. H., Kerr, P. M., Suleiman, M. S. and Halestrap, A. P. (2000)
Protection of hearts from reperfusion injury by propofol is associated with inhibition of
the permeability transition. Cardiovasc. Res. 45, 360–369
10 Kim, J. S., Jin, Y. G. and Lemasters, J.J. (2006) Reactive oxygen species, but not Ca2+
overloading, trigger pH- and mitochondrial permeability transition-dependent death of
adult rat myocytes after ischaemia-reperfusion. Am. J. Physiol. 290, H2024–H2034
11 Baines, C. P., Kaiser, R. A., Purcell, N. H., Blair, N. S., Osinska, H., Hambleton, M. A.,
Brunskill, E. W., Sayen, R. M., Gottlieb, R. A., Dorn, G. W. et al. (2005) Loss of cyclophilin
D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434,
658–662
35 Doyle, V., Virji, S. and Crompton, M. (1999) Evidence that cyclophilin-A protects cells
against oxidative stress. Biochem. J. 341, 127–132
36 Kim, J., Choi, T. G. and Ding, Y. (2008) Overexpressed cyclophilin-B suppresses
apoptosis associated with ROS and Ca2+ homeostasis after endoplasmic reticulum
stress. J. Cell. Sci. 121, 3636–3648
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society

148
S. Malouitre and others
37 Yang, L., Omori, K., Suzukawa, J. and Inagaki, C. (2004) Calcineurin-mediated BAD
Ser155 dephosphorylation in ammonia-induced apoptosis of cultured rat hippocampal
neurons. Neurosc. Lett. 357, 73–75
38 Norris, C. M., Blalock, E. M., Chen, K. C., Porter, N. M. and Landfield, P. W. (2002)
Calcineurin enhances L-type Ca2+ channel activity in hippocampal neurons: increased
effect with age in culture. Neuroscience 110, 213–225
39 Waldmeier, P. C., Zimmerman, K., Qian, T., Tintelnot-Blomley, M and Lemasters, J. J.
(2003) Cyclophilin D as a drug target. Curr. Med. Chem. 10, 1485–1506
40 Ruiz, F., Alvarez, G., Ramos, M., Hernandez, M., Bogonez, E. and Satrastegui, J. (2000)
Cyclosporin targets involved in protection against glutamate toxicity. Eur. J. Pharm. 404,
29–39
42 Bonde, C., Noraberg, J., Noer, H. and Zimmer, J. (2005) Ionotropic glutamate receptors
and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen
and glucose deprivation of hippocampal slice cultures. Neuroscience 136, 779–794
43 Crompton, M. (1999) The mitochondrial permeability transition pore and its role in cell
death. Biochem. J. 341, 233–249
44 Reynolds, I. J. and Hastings, T. G. (1995) Glutamate induces the formation of reactive
oxygen species in cultured forebrain neurons following NMDA receptor activation. J.
Neurosci. 15, 3318–3327
45 Abramov, A. Y. and Duchen, M. R. (2008) Mechanisms underlying the loss of
mitochondrial membrane potential in glutamate toxicity. Biochim. Biophys. Acta 1777,
953–964
41 Rytter, A., Cardoso, C. M. P., Johanssohn, P., Cronberg, T., Hanssohn, M. J., Mattiasson,
G., Elmer, E. and Wieloch, T. (2005) The temperature dependence and involvement
of the mitochondrial permeability transition and caspase activation in damage to
organotypic hippocampal slices following in vitro ischaemia. J. Neurochem. 95,
1108–1117
46 Ishii, H., Shibuya, K., Ohta, H., Mukai, H., Uchino, S., Takata, N., Rose, J. A. and Kawato,
S. (2006) Enhancement of nitric oxide production by association of nitric oxide synthase
with N-methyl-D-aspartate receptors via postsynaptic density 95 in genetically engineered
Chinese hamster ovary cells: real-time fluorescence imaging using nitric oxide sensitive
dye. J. Neurochem. 96, 1531–1539
Received 25 February 2009/2 October 2009; accepted 15 October 2009
Published as BJ Immediate Publication 15 October 2009, doi:10.1042/BJ20090332
ꢀ
c
ꢀ
c
The Authors Journal compilation 2010 Biochemical Society