Neurorescue by a ROS Decomposition Catalyst

Article abstract of DOI:10.1021/acschemneuro.6b00144

The effect of the bis-sulfonated iron(III) corrole (1-Fe), a potent decomposition catalyst of reactive oxygen species, on rescuing SN4741 cells that were damaged by 6-hydroxydopamine (6-OHDA) was investigated as an in vitro model system for studying cell death of dopaminergic neurons in the substantia nigra. Important findings that accompanied the ability to rescue dopaminergic neurons were increased expression of phenotypic dopaminergic proteins, such as tyrosine hydroxylase (TH) and dopamine transporter (DAT), which were significantly depleted upon 6-OHDA-mediated damage. 1-Fe also elevated expression levels of aldehyde dehydrogenase 1 (ALDH-1), previously disclosed as a cardinal protein in the pathogenesis of Parkinson's disease. Since these findings suggested that 1-Fe affects quite a wide range of intracellular mechanisms, vital intracellular pathways that involve neuroplasticity, growth, differentiation and survival of neurons, were examined. Phosphatidylinositol 3-kinase (PI3K) and protein kinase c (PKC) were found to be involved, as pharmacological inhibitors of these kinases abolished the neurorescue effect of 1-Fe. 1-Fe also elevated the expression of antiapoptotic protein Bcl-2, which is essential for proper mitochondrial function and cellular survival. The overall conclusion is that 1-Fe is capable of rescuing already damaged neuronal cells by a variety of mechanisms that are beyond its antioxidant activity.

Full text of DOI:10.1021/acschemneuro.6b00144

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Article
Neurorescue by a ROS Decomposition Catalyst
Matan Soll, Orit Bar am, Atif Mahammed, Irena Saltsman,
Silvia Mandel, Moussa B.H. Youdim, and Zeev Gross
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00144 • Publication Date (Web): 21 Jul 2016
Downloaded from http://pubs.acs.org on July 22, 2016
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ACS Chemical Neuroscience
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Neurorescue by a ROS Decomposition Catalyst
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Matan Solla, Orit Bar Amb, Atif Mahammeda, Irena Saltsmana, Silvia Mandelb, Moussa B.H. Youdimb and Zeev Gross*a
a) Schulich Faculty of Chemistry, Technion – Israel Institute of Technology, Haifa 32,000, Israel
b) Ruth & Bruce Rappaport Faculty of Medecine, Technion – Israel Institute of Technology, Haifa 32,000, Israel
Keywords: Parkinson's disease, catalytic anti oxidants, redox signaling, neurodegeneration, neurorescue
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ABSTRACT: The effect of the bisꢀsulfonated iron(III) corrole (1-Fe), a potent decomposition catalyst of reactive oxygen species,
on rescuing SN4741 cells that were damaged by 6ꢀhydroxydopamine (6ꢀOHDA) was investigated as an in vitro model system for
studying cell death of dopaminergic neurons in the substantia nigra. Important findings that accompanied the ability to rescue doꢀ
paminergic neurons were increased expression of phenotypic dopaminergic proteins, such as tyrosine hydroxylase (TH) and dopaꢀ
mine transporter (DAT), which were significantly depleted upon 6ꢀOHDAꢀmediated damage. 1-Fe also elevated expression levels
of aldehyde dehydrogenase 1 (ALDHꢀ1), previously disclosed as a cardinal protein in the pathogenesis of Parkinson's disease.
Since these findings suggested that 1-Fe affects quite a wide range of intracellular mechanisms, vital intracellular pathways that
involve neuroplasticity, growth, differentiation and survival of neurons, were examined. Phosphatidylinositol 3ꢀkinase (PI3K) and
protein kinase c (PKC) were found to be involved, as pharmacological inhibitors of these kinases abolished the neurorescue effect
of 1-Fe. 1-Fe also elevated the expression of antiꢀapoptotic protein Bclꢀ2, which is essential for proper mitochondrial function and
cellular survival. The overall conclusion is that 1-Fe is capable of rescuing already damaged neuronal cells by a variety of mechaꢀ
nisms that are beyond its antioxidant activity.
INTRODUCTION
Parkinson's disease (PD) is a multiꢀfactorial degenerative disease of the central nervous system with a prevalence rate of 1 in 300.1
Clinical features include abnormally slow voluntary movements, stiffness and asymmetric tremor. These arise from the loss of doꢀ
paminergic neurons (which release the neurotransmitter dopamine) in the substantia nigra pars compacta.2 Strong evidence suggests
that the common cause of death of these neurons involves mitochondrial dysfunction and dopamineꢀdependent oxidative stress.3ꢀ7
Vulnerability of mitochondrial complex I leads to the formation of reactive oxygen species (ROS) like superoxide anion radical
(O2ꢀ), which in the presence of nitric oxide (NO) forms peroxynitrite ( ONOOH), hydrogen peroxide (H2O2), and hydroxyl radical (
·OH ).
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We have recently introduced the iron(III) complex of bisꢀsulfonated corrole (1-Fe) (Figure 1A) as an excellent decomposition cataꢀ
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lyst of O2 , H2O2 and HOONO in purely chemical systems.8,9 The relevance of these findings to the prevention of biological malꢀ
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functions was demonstrated by using 1-Fe and other metallocorroles for defeating oxidative stress and its consequential deleterious
effects in several cellular and animalꢀbased models of cardiovascular, metabolic, and neurodegenerative diseases.10ꢀ 12 The investiꢀ
gations included the neuroblastoma cell lines SHꢀSY5Y and NSCꢀ34, challenged by the above mentioned toxins, as well as by the
neurotoxin, 6ꢀhydroxydopamine (6ꢀOHDA).11 The latter is frequently used to selectively destroy dopaminergic and noradrenergic
neurons in the brain and has quite a large array of pharmacological actions.13 Although 6ꢀOHDA was originally developed as a
neurotoxic synthetic compound, it has actually found to be formed endogenously in patients suffering from PD.14 Since PD is diagꢀ
nosed only at a stage where about 50%ꢀ70% of the neurons are already damaged, we have now focused on a cellular model that
mimics that situation. The SN4741 cell line was selected in this study, because it is considered an excellent in vitro model system
for studying cell death of dopaminergic neurons in the substantia nigra.15 Administration of 6ꢀOHDA induces apoptosis by a disꢀ
tinct mechanism through caspaseꢀ3 cleavage, decrease of the Bclꢀ2/Bax ratio, activation of cꢀJun Nꢀtermianl kinase (JNK), and the
formation of reactive oxygen species and nitrosative stress. Prophylactic administration of SOD mimetic compounds or peroxyniꢀ
trite decomposition catalysts has been reported to attenuate the 6ꢀOHDA induced apoptotic process.16,17 We have reported that 1-Fe
prevents the cellular death of SHꢀSY5Y cultured cells not only when present before exposure to 6ꢀOHDA but even when adminisꢀ
trated significantly later.11 This suggested that 1-Fe might not only prevent, but also rescue cells and recover their neuronal activity.
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We now focused on elucidating possible mechanisms by which 1-Fe might protect against or even rescue from neuronal damage,
by checking how it interferes with apoptotic pathways. This was done by checking its effects on SN4741 cells that were: a) notꢀ
challenged by any toxin; b) treated with the 6ꢀHODA neurotoxin after incubation with 1-Fe; and c) treated with 1-Fe only after the
damage by 6ꢀOHDA has been induced. New and quite surprising results that were obtained by this approach include the alleviation
of the antiꢀapoptotic Bclꢀ2 protein and remarkable differences in the expression of phenotypic dopaminergic proteins and cardinal
enzymes involved in the pathogenesis of PD. A number of intracellular pathways that involve neuroplasticity, growth, differentiaꢀ
tion and survival of neurons were examined as well. Altogether, the findings led to the conclusion that 1-Fe affects quite a wide
range of intracellular mechanisms, much beyond the expected antioxidant effect.
RESULTS
The first sets of experiments were performed on SN4741 cells that were not challenged by any toxin, as to evaluate cellular uptake
of 1-Fe and its possible effect on proteins involved in apoptosis. Live optical imaging assays were performed with 5,10,15ꢀ
tris(pentafluorophenyl)ꢀ2,17ꢀbis(sulfonato)corrole aluminum(III) (1-Al) (Figure 1A), the intensively fluorescent structural analog
of 1-Fe. Figure 1Cb discloses that 1-Al clearly enters the SN4741 cells, but not into the nucleus; and its appearance as distinct
granules suggests association to a specific organelle.
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(C)
Figure 1. (A) The structure of the iron(III) and aluminum(III) chelates of 5,10,15ꢀtris(pentafluorophenyl)ꢀ2,17ꢀbis(sulfonato) corrole. (B)
Elevation of basal expression levels of BCLꢀ2 and Bclꢀ2/Bax ratio by 1-Fe, (a) Representative western blots of Bclꢀ2 and Bax in SN4741
cells. (b) Graphs represent densitometry quantification of the lanes normalized to βꢀtubulin. Data are expressed as mean ±SEM (n=3) and
repeated twice with similar results. *p<0.05 vs. vehicle treated cells; #p<0.01 vs. vehicle treated cells. (C) Live imaging of SN4741 subꢀ
stantia nigra derived cells coꢀincubated with 1-Al (4µM) and mitotracker green (120nM) (MTG) for 30 min: (a) under conditions specific
for detection of MTG fluorescence; (b) 1-Al fluorescence data, (c) superimposed image of (a), (b) and (d); and (d) phase contrast microsꢀ
copy. Fluorescence was recorded using a X40 objective and a LSM700 confocal system supported with Zen software. Samples were excitꢀ
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ed at 488 nm (3%) for MTG detection and at 405 nm (10%) for 1-Al detection. Representative images of 12 separate fields, repeated 4
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The absorption and emission maxima of 1-Al are 422 and 633 nm, respectively, which allows for its utilization in the presence of
Mitotracker green (MTG) whose absorption and emission maxima are 490 and 516 nm, respectively. Coꢀincubation of 1-Al with
MTG, which selectively stains the mitochondria of living cells by a green color, was successfully separated from the color induced
by the corroles with the aid of specific filters (Figure 1Ca). The information obtained from the superimposed images is that 1-Al
did accumulate in the mitochondria, as signaled by the yellow color in Figure 1Cc, but with low selectivity. The much more nonꢀ
mitochondrial staining (the red color) indicates that 1-Al is dispersed between other organelles and the cytosol as well.
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Given the evidence that the corrole accumulates to some extent in the mitochondria, raised the interest in examining the effect of 1-
Fe on the ratio of antiꢀ/proꢀ apoptotic proteins. Our focus was on the antiꢀapoptotic Bclꢀ2 and the proꢀapoptotic Bax, since the senꢀ
sitivity of cells to apoptotic stimuli such as oxidative stress depends on their relative abundance: cells are more resistant when there
is an excess of antiꢀapoptotic proteins. To check if 1-Fe may affect the expression of antiꢀapoptotic factors and signals in native
(i.e., non 6ꢀOHDAꢀinjured) SN4741 cells, the cells were treated with two doses of 1-Fe. Levels of Bclꢀ2 protein were recorded
using western blot analysis. Bclꢀ2 levels were 200% and 250% in the presence of 10 µM and 20 µM 1-Fe, respectively, compared
to Bclꢀ2 protein levels in the control cells (Figure 1B). The treatment with 10 and 20 µM 1-Fe also slightly reduced the levels of
Bax to 90% and 80%, respectively, relative to the control cells. Consequently, the Bclꢀ2/Bax protein ratios were elevated by a facꢀ
tor of 2.5 and 3 for 10 µM 1-Fe and 20 µM 1-Fe, respectively (Figure 1B).
The next step was to check if 1-Fe exerts a substantial neuroprotective effect in the SN4741 model of PD. SN4741 cells were preꢀ
treated without or with 1-Fe (0.1 ꢀ 20 µM) 30 min before exposure to 6ꢀOHDA (50 µM); and cell viability was evaluated by the
MTT test 24 h later. Cell survival in the absence of 1-Fe was as low as 5% relative to control, while it was increased up to 80% in
cells incubated with 20 µM of 1-Fe (Figure 2A). Photoꢀmicrographic examination revealed that cells exposed to 6ꢀOHDA exhibitꢀ
ed retracted cell bodies and loss of neurite outgrowth branched morphology; and that both phenomena were significantly prevented
by 1-Fe treatment at doses of 10ꢀ20 µM (Figure 2B).
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Figure 2. (A) Neuroprotective effect of corrole 1-Fe against 6ꢀOHDA induced neurotoxicity. SN4741 cells were pretreated without or
with corrole 1-Fe (0.1ꢀ20 µM), 30 min before exposure to 6ꢀOHDA (50 µM). Cell viability was evaluated by the MTT test 24 h later. Data
are expressed as mean ±SEM (n=4). # p<0.01 vs. vehicle treated cells. (B) Morphological characteristics visualized by phaseꢀcontrast miꢀ
croscope. SN4741 cells treated without (a), or with 50 µM 6ꢀOHDA (bꢀd), and preꢀtreated with either 10 µM 1-Fe (c) or 20 µM 1-Fe (d)
30 min prior to the treatment with 50 µM 6ꢀOHDA. Pictures were obtained by using an inverted microscope connected to a digital camera
(10 x objective), after a 24 h incubation period.
The above results, obtained via treatment of the cell line prior to 6ꢀOHDAꢀinduced neurotoxicity, disclose a profound protective
effect of 1-Fe. This kind of neuroprotection may be considered as preventive medicine (secondary prevention), i.e., a situation
where a patient at risk has been given a therapy in order to prevent development of the disease. PD is a progressive disease, characꢀ
terized by gradual dopaminergic (DAergic) neuronal cell death in the SNpc, which eventually results in severe motor deficits.
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Slowing or attenuating the neurodegeneration process must hence be initiated before too much damage has occurred. The other
strategy is neurorescue, which is the repair and rehabilitation of already damaged cells undergoing progressive ongoing insult. In
the case of PD, elucidating a neurorescue effect of a therapeutic agent is essential since the disease is diagnosed when already half
the population of DAergic neurons is lost, and the rest are already under progressive damage and degeneration. One aspect that
encouraged us to pursue the prospect of 1-Fe as a neuroꢀrestorative agent was the substantial elevation of Bclꢀ2 in 1-Fe treated
SN4741 cells.
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Treatment of the SN4741 cell line with 6ꢀOHDA (50µM) starts a cascade of toxic events the moment it is added to the cells. It inꢀ
hibits mitochondrial complex I, with consequential production of toxic ROS; and apoptotic signaling is initiated within 1h after 6ꢀ
OHDA addition to the cells.13 We have now tested if 1-Fe displays neurorescue activity by treating SN4741 cells with 50 µM 6ꢀ
OHDA, followed by addition of 1-Fe (10 µM and 20 µM) 30, 60, or 120 min later. Examination by the MTT test after a further 24
h incubation revealed that only 5% (relative to control) of the cells that did not receive 1-Fe survived (Figure 3A). In contrast, a
survival of up to 40% was obtained for cells that received 1-Fe. Moreover, 20 µM of 1-Fe attenuated 6ꢀOHDAꢀinduced cell death
of the SN4741 cells even when added up to 2 h postꢀ6ꢀOHDA treatment. This point towards a more profound mechanism of celluꢀ
lar protection by 1-Fe, beyond its well established antioxidant activity. This hypothesis was accentuated by results obtained from
two neuronal markers, neuronꢀspecific class III betaꢀtubulin (Tujꢀ1) and growthꢀassociated protein 43 (Gapꢀ43). Figure 3Ba shows
the immunofluorescence obtained by utilizing a specific antibody against Tujꢀ1 together with 4',6ꢀdiamidinoꢀ2ꢀphenylindole
(DAPI) staining. Similar to earlier presented phaseꢀcontrasting results, postꢀdamage administration of 1-Fe (10 µM and 20 µM)
improved cell morphology and promoted neurite outgrowth. Quantitative analysis of the fluorescence intensity disclosed that the
levels of Tujꢀ1 in SN4741 cells incubated with 6ꢀHODA (50 µM) decreased to 40%, while treatment with 1-Fe (10 µM and 20 µM)
30 min after 6ꢀOHDA exposure increased its levels to 130% (Figure 3Bb), both relative to control (i.e., untreated cells). Western
blot analysis was used for the detection of Gapꢀ43, revealing an about 70% decrease in cells treated with 6ꢀOHDA (50 µM). Supꢀ
plement of 10 µM 1-Fe 30 minutes after 6ꢀOHDA restored Gapꢀ43 levels up to 76% relative to the control (untreated) cells (Figure
3Bd), while 20 µM of 1-Fe was less effective in preventing the 6ꢀOHDA initiated depletion of Gapꢀ43 (50% relative to control
cells). The substantial loss of these markers in 6ꢀOHDA treated cells indicates that they may not be considered vital neu ronal cells
any more. On the other hand, the treatment of already damaged SN4741 cells with 1-Fe apparently restored neuronal structure,
based on Tujꢀ1 immunofluorescence and Gapꢀ43 western blot analysis. Furthermore, tujꢀ1 expression in 1-Fe treated cells was
higher by 30% relative to control cells (Figure 3Bb) and neurite structures appeared to be more abundant. Taken together, these
results suggest that treatment with 1-Fe has induced both neuronal growth and structural robustness.
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Figure 3. (A) Neurorescue effect of corrole 1-Fe against 6ꢀOHDA induced neurotoxicity. SN4741 cells were initially treated with 6ꢀ
OHDA (50 µM), followed by addition of corrole 1-Fe (10 µM and 20 µM) after 30, 60, or 120 min incubation with 6ꢀOHDA. Cell viabilꢀ
ity was evaluated by the MTT test. Data are expressed as mean ±SEM (n=4) # p<0.01 vs. vehicle treated cells; * p<0.05 vs. vehicle treated
cells. High variability in 10 µM treated cells at t = 2h indicates insignificance of the result. (B) Neurorescue effect of corrole 1-Fe against
6ꢀOHDAꢀinjured dopaminergic SN4741 cells, as evaluated by restoration of the neuronal markers, Tujꢀ1 and GAPꢀ43. SN4741 cells were
initially treated with 6ꢀOHDA (50 µM) for 30 min, followed by the addition of 1-Fe or vehicle for 24 h incubation. Cells were fixed and
immunostained, using specific antibody against Tujꢀ1 conjugated to Alexa fluor 488. Immunofluorescence was recorded using a X40 obꢀ
jective and a LSM700 confocal system supported with Zen software. (a) Representative fields and the (b) graph represents values of 12
separate fields, expressed as mean ±SEM (n=3). # p<0.01 vs. vehicle treated cells; * p<0.05 vs. vehicle treated cells. (c) Representative
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western blots Gapꢀ43 in SN4741. (d) Graphs represent densitometry quantification of the lanes normalized to βꢀtubulin expressed as mean
±SEM (n=3) and repeated three times with similar results. * p<0.05 vs. vehicle treated cells.
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Figure 4. Neurorescue effect of 1-Fe against 6ꢀOHDAꢀinjured dopaminergic SN4741 cells, as evaluated by restoration of (A) TH and (B)
DAT. SN4741 cells were initially treated with 6ꢀOHDA (50 µM) for 30 min, followed by the addition of 1-Fe and 24 h incubation. Cells
were fixed and immunostained, using specific antibody against TH and DAT respectively followed by Fitcꢀconjugat AP132F conjugated
secondary antibody. Immunofluorescence was recorded using a X40 objective and a LSM700 confocal system supported with Zen softꢀ
ware. No fluorescence was detected in merely secondary antibody stained samples. (a) Representative fields and the (b) graph represents
values of 12 separate fields, expressed as mean ±SEM (n=3). # p<0.01 vs. control untreated cells; * p<0.05 vs. control untreated cells.
There is a significant impairment of dopamine (DA) neurotransmission in PD. As neurodegeneration progresses, there is a gradual
loss of DAergic specific phenotypic markers of DAergic neurons. DAT is used as a surrogate marker for the progression of early
phase PD.18 Another biomarker relevant for PD is TH, which was found to be downꢀregulated in the SNpc DAergic neurons of PD
patients.19 We have hence decided to check the effect of 6ꢀOHDA treatment on both TH and DAT, as well as that of postꢀinjury
added 1-Fe. Immunofluorescence analysis of TH and DAT in SN4741 cells disclosed a substantial depletion of the fluorescent sigꢀ
nal upon 6ꢀOHDA (50 µM) exposure (30% and 60% of the controlꢀuntreated cells respectively; Figure 4A, 4B). The presence of
10 µM and 20 µM 1-Fe in the 6ꢀOHDAꢀinjured dopaminergic SN4741 cells doseꢀdependently elevated the levels of the TH immuꢀ
nofluorescent signal, by up to 120% and 150% relative to control, respectively (Figure 4A). Immunostaining for DAT of 6ꢀ
OHDAꢀinjured SN4741 cells that were treated with 1-Fe (10 µM and 20 µM) disclosed an elevated signal of up to 120% relative to
control cells (Figure 4B). Our results disclose that treatment of the SN4741 cells with 6ꢀOHDA (50µM) substantially diminish the
levels of TH and DAT in the SN4741 DAergic cell line, indicating the loss of DAergic activity. Treatment of the damaged cells
with 1-Fe (10µM and 20µM) restored basal/normal levels of TH and DAT displayed in the SN4741 control cells. In most cases,
levels of DAergic biomarkers even surpassed the levels displayed by the control cells. This may be indicative of an active induction
of expression or translation of these DAergic biomarkers by 1-Fe, a hypothesis that needs further investigation for validation. The
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ability of 1-Fe to restore DAergic activity of the neurons is of prime importance considering the process of PD, since DAergic imꢀ
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While DA has long been implicated in the pathogenesis of PD, very high DA concentrations are required for observing toxicity in
vitro and in vivo.29 One hypothesis is that autoꢀoxidation of DA leads to an array of toxic metabolites25ꢀ27 and generation of
ROS.23,28 Other indications point towards a DA metabolite, such as 3,4ꢀdihydroxyphenylacetaldehyde (DOPAL) formed via the
monoamine oxidase (MAO) catalyzed reaction with DA, as the deliverable leading to neurotoxicity.21,30,31 This is consistent with an
emerging key protein in the pathogenesis of PD, ALDHꢀ1,21ꢀ24 whose role in the brain, specifically in the dopaminergic neuron, is to
metabolize and disarm toxic aldehydes such DOPAL.24 ALDHꢀ1 was found to be expressed highly and specifically in DAergic
cells of the SNpc and ventral tegmental area (VTA), but markedly reduced in SNpc DAergic neurons of PD brains (but not in their
VTA).21 It is further important to emphasize that inhibition of complex I of the mitochondria by hydroxyl radicals or inhibitors such
as 6ꢀOHDA reduce the ability of ALDHꢀ1 to remove DOPAL by lowering the availability of NAD+, a cofactor required for aldeꢀ
hyde dehydrogenase activity.32
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Consistent with the above, assessing the protein levels of ALDHꢀ1 in the SNpc derived SN4741 cell line using western blot analyꢀ
sis, disclosed a substantial decrease in the levels of ALDHꢀ1 upon 6ꢀOHDA (50 µM) exposure (40% relative to control cells) (Fig-
ure 5). In contrast, treatment of the 6ꢀOHDAꢀinjured cells with 10 µM and 20 µM 1-Fe elevated ALDHꢀ1 levels by up to 140%
and 120%, respectively. The consecutive treatment of 6ꢀOHDAꢀinjured neurons with 1-Fe increased the levels of ALDHꢀ1 quite
dramatically, back to the levels measured in the control cells. In fact, the elevation of ALDHꢀ1 by 1-Fe even exceeded the levels of
ALDHꢀ1 recorded in the control cells. Since the main goal of this research was to determine if 1-Fe may be used as a therapeutic
agent for the attenuation of the neurodegeneration process in PD, it may very well be that the elevation of ALDHꢀ1 is one of the
mechanisms by which 1-Fe executes its neurorescue effect on the SN4741 cell line.
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Figure 5. Neurorescue effect of 1-Fe against 6ꢀOHDAꢀinjured dopaminergic SN4741 cells, as evaluated by restoration of the dopaminerꢀ
gic pivotal protein ALDHꢀ1. Cells were initially treated with 6ꢀOHDA 50 µM for 30 min, followed by the addition of the compound 1-Fe
or vehicle and 24 h incubation. (a) Representative western blots of ALDHꢀ1 in SN4741. (b) Graphs represent densitometry quantification
of the lanes normalized to βꢀtubulin. Data are expressed as mean ±SEM (n=3) and repeated three times with similar results. * p<0.05 vs.
vehicle treated cells.
Figure 6. (A) Neurorescue effect of corrole 1-Fe against 6ꢀOHDAꢀinjured dopaminergic SN4741 cells, as evaluated by elevation of the
antiꢀapoptotic protein Bclꢀ2. SN4741 cells were initially treated with 6ꢀOHDA (50 µM) for 30 min, followed by the addition of the comꢀ
pound 1-Fe or vehicle and 24h incubation. (a) Representative western blots of Bclꢀ2 and Bax in SN4741 cells (b) Graphs represent densiꢀ
tometry quantification of the lanes normalized to βꢀtubulin and the Bclꢀ2/Bax ratio. Data are expressed as mean ±SEM (n=3) and repeated
three times with similar results. * p<0.05 vs. vehicle treated cells. (B) Neurorescue effect of 1-Fe on cytochrome c release from the mitoꢀ
chondria. SN4741 cells were initially treated with 6ꢀOHDA (50 µM) for 30 min, followed by the addition of the compound 1-Fe and 24 h
incubation. Cells were fixed and immunostained, using specific antibody against cytochrome c followed by Fitcꢀconjugat AP132F conjuꢀ
gated secondary antibody. Immunofluorescence was recorded using a X40 objective and a LSM510 confocal system supported with Zen
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software. No fluorescence was detected in merely secondary antibody stained samples. (a) Representative fields and the (b) graph repreꢀ
sents values of 12 separate fields, expressed as mean ±SEM (n=3) and repeated three times with similar results. # p<0.01 vs. control, unꢀ
treated cells.
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Assessing the apoptotic pathway under 6ꢀOHDA exposure, immunoblotting of Bclꢀ2 disclosed that treatment of the SN4741 cells
with 6ꢀOHDA (50 µM) decreased the Bclꢀ2 protein levels to about 15% relative to the control cells (Figure 6A). Postꢀtreatment of
the 6ꢀOHDAꢀinjured cells with 1-Fe (10 µM,) attenuated the excessive depletion of Bclꢀ2 protein levels, to 40% relative to the conꢀ
trol cells. An immunohistochemistry assay of cytochrome c disclosed a substantial decrease in the levels of cytochrome c in the
SN4741 cells that were treated with 6ꢀOHDA (50 µM) (Figure 6B). This was reflected in a 30% decrease of fluorescence intensity
relative to control (untreated cells). Cellular nucleus staining by the fluorescent dye DAPI revealed that the nuclei are still intact, a
common feature in cells undergoing apoptosis. In addition, the fluorescent signal from cytochrome c showed a morphologic
smeared pattern around the nucleus, as opposed to granular and dispersed in the control cells. This indicates a translocation of cytoꢀ
chrome c from the interꢀmembrane space of the mitochondria into the cytosol. Post 6ꢀOHDA ꢀinjured SN4741 cells that were treatꢀ
ed with 1-Fe (10 µM and 20 µM) displayed cytochrome c morphologic signals similar to untreated cells. The fluorescent signal was
granular and strong (100% relative to control), indicating a localization of cytochrome c within the interꢀmembrane space of the
mitochondria.
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Further clues regarding the general mechanism and intracellular signal pathways that might be traced back to the apparent neuroꢀ
rescue capabilities of 1-Fe were obtained by using a reversible specific PI3K inhibitor (LY294002), a reversible specific MEK inꢀ
hibitor (U120), and a specific broadꢀspectrum PKC inhibitor (GF109203X). The PI3K, PKC and MEK pathways are well identified
and characterized pathways in the survival, growth and differentiation of neurons, and we hence investigated their possible releꢀ
vance in the neurorescue effect of 1-Fe. Treatment of the SN4741 cells with 6ꢀOHDA (50 µM) depleted cell viability down to 10%
relative to the control cells (Figure 7), while 1-Fe (20 µM) strongly affected the outcome of the SN4741 6ꢀOHDAꢀinjured cells, as
reflected in a 60% cellular viability. However, preꢀincubation with LY294002 (20 µM) significantly attenuated the neurorescue
effect of 1-Fe (20 µM) (Figure 7a): the cellular viability was only 35% relative to control cells in that case. Pretreatment of the
SN4741 cells with GF109203X, a PKC inhibitor, revealed a similar trend. Cells treated with 6ꢀOHDA+1-Fe showed a 60% viabilꢀ
ity compared to control cells, but GF109203X (2.5 µM)ꢀpretreated cells showed a significant decline in cellular viability compared
to cells that were treated with 6ꢀOHDA+1-Fe only (30% viability, Figure 7b). SN4741 cells that were exposed to U120 (20 µM), a
MEK/MAPK pathway inhibitor, revealed a mild inhibition of the neurorescue effect of 1-Fe (20 µM) on the SN4741 6ꢀOHDAꢀ
injured cells (Figure 7c). Cells that were treated with 6ꢀOHDA+1-Fe showed a 50% viability compared to control cells, while cells
treated with 6ꢀOHDA+1-Fe+U120 displayed 40% viability compared to control cells (Figure 7c).
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Taken together, the neurorescue effect of 1-Fe was markedly depleted in the presence of PI3K or PKC and somewhat less for
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MEK/MAPK inhibitors, thus pointing towards a partial involvement of these signaling pathways in the process.
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Figure 7. Involvement of the (a) PI3K, (b) PKC and (c) MEK/MAPK pathways in the neurorescue effect of 1-Fe against 6ꢀOHDA induced
neurotoxicity. SN4741 cells were preꢀincubated with the apropriate kinase inhibitor for 20 min, followed by incubation of the SN4741
cells with 6ꢀOHDA (50 µM) for 30 min. SN4741 cells were then treated with 1-Fe (20 µM) and further incubated for 24 h. Cell viability
was evaluated by the MTT test. Data are expressed as mean ±SEM (n=4). * p<0.05 vs. 6ꢀOHDA treated group; # p<0.05 vs. 6ꢀOHDA+1-
Fe group.
DISCUSSION
The current concept regarding PD and other neurodegenerative disorders, considers them diseases of multiple etiological nature
where several mechanisms are implicated in a cascade/s of events involving many biochemical and signaling pathways.33 These
include the impairment in mitochondrial activity, a failure of the ubiquitinꢀproteasome system (UPS) to adequately remove abnorꢀ
mal proteins and a general environment of oxidative stress34ꢀ36, that might cause a derangement in cell cycle control. The presence
of ROS would exacerbate protein misfolding and the demand for their disposition by the UPS, which would result in the propagaꢀ
tion of a vicious cycle that may be selfꢀsustaining. The use of truly potent catalytic antioxidants may have a myriad of beneficial
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effects on its own when looking in to a therapeutic agent for PD, though therapy for PD will require a multimodal drug that will
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treat several phenomena that lead to the neurodegenerative process. 1-Fe appears to meet such requirements.
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Studies on the regulation of programmed cell death revealed that 1-Fe elevates the levels of the antiꢀapoptotic protein Bclꢀ2, and
that it restores the localization of the proꢀapoptotic protein cytochrome c to the mitochondria in 6ꢀOHDAꢀinjured SN4741 cells.
Given the known activity of PKCεꢀ it increases the expression of the protein Bclꢀ2 and PKCα activates it by phosphorylation37– and
the fact that PKC's are involved in the neurorescue effect of 1-Fe (we used a PKC α, β, γ, ε, δ inhibitor), it is reasonable to deduce
that 1-Fe elevates the levels of Bclꢀ2 through the PKC pathway.
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It is also known that PKCδ activates the MEK/MAPK pathway38, which in turn activates the expression of Bclꢀ2 and CREB. Our
results imply that the MEK/MAPK pathway is only partially involved in the neurorescue effect of 1-Fe, while the PI3K pathway
plays a pivotal role therein. PI3K is known to activate Akt/PKB, which deactivates (by phosphorylation) the proꢀapoptotic protein
Bad and also inhibits the translocation of Bax from the cytosol to the mitochondria where it facilitates its proꢀapoptotic activity.39,40
Previous work on the PI3K pathway showed that overexpression of SOD1, in addition to reduced oxidative stress, significantly
promoted activation of the PI3KꢀAkt/PKB pathway in a focal cerebral ischemia model.41,42 Baring this in mind, 1-Fe may mediate
its effect on the PI3KꢀAkt/PKB pathway simply by its catalytic antioxidant activity. Further work must be done on downstream and
upstream factors of the pathways under discussion in order to further substantiate their relative significance in the neurorescue efꢀ
fect of 1-Fe.
We conclude by stating that the neurorescue capability of 1-Fe strongly suggests that there is more to its therapeutic function than
merely antioxidative action. The initial effect of adding 1-Fe to the 6ꢀOHDA treated cells is likely to be ROS/ reactive nitrogen
species (RNS) scavenging. But since a substantial amount of damage has already been inflicted upon the cells at the 30 min period
they were incubated with 6ꢀOHDA only, radical scavenging alone is simply not enough to account for the apparent neurorescue
effect. This is reminiscent of work described by BatinicꢀHaberle et al., where manganese porphyrins and other SOD mimetics were
shown to have a direct or indirect (i.e., through ROS signaling mitigation) interaction with signaling proteins in cellular and in vivo
models of ischemic reperfusion, cancer, diabetes and stroke.43,44 The current research discloses several phenomena that are of very
strong relevance to progressing PD (Scheme 1): restoration of neuronal markers Gapꢀ43 and Tujꢀ1 which are essential for neuronal
structure; the elevation and restoration of DAergic markers TH and DAT, which are of prime importance in DA metabolism and
storage; elevation of key detoxifying enzyme ALDHꢀ1 levels back to normal; and elevation of antiꢀapoptotic protein Bclꢀ2 while
preventing the release of cytochrome c to the cytosol. The accumulated results from wellꢀdefined biomarkers, together with the
indications for the involvement of PI3K, PKC and MEK/MAPK pathways, apparently point towards a neurotrophic characteristic
of 1-Fe.
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Scheme 1. Summary of the neurorescue effects of 1-Fe on 6ꢀOHDAꢀtreated SN4741 cells.
MATERIALS and METHODS
Corrole synthesis
2,17ꢀbisꢀsulfonatoꢀ5,10,15ꢀtris(pentafluorophenyl)corrolato iron(III) (1-Fe) was prepared as previously described.45
Synthesis of 2,17ꢀbisꢀsulfonatoꢀ5,10,15ꢀtris(pentafluorophenyl)corrolato aluminum(III) (1-Al):
Portions of 1 mL of 2 M trimethylaluminum in toluene were added, by intervals of 30 min, to a solution of 20 mL of pyridine that
contained 60 mg (60 ꢀmol) of 2,17ꢀbisꢀsulfonatoꢀ5,10,15ꢀtris(pentafluorophenyl)corrole, under N2 and at reflux temperature. The
reaction was monitored by UVꢀvisible spectra measurement in 0.2 M sodium carbonate in water. After the consumption of all the
starting material (about three portions of trimethylaluminum were needed) the solution was cooled to RT and drops of water were
added carefully in order to destroy the excess of trimethylaluminum. 200 mL of 0.2 sodium carbonate in water were added and the
solution was filtrated. 200 mL of ethanol were added to the filtrate solution which precipitated most of the inorganic salts. The soluꢀ
tion was filtrated again and evaporated. The product was purified on silica gel column using ethanol as eluent. 1-Al was obtained in
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82% yield (50 mg, 49 ꢀmol). 1H NMR (CD3OD): δ = 9.90 (s, 1H), 8.79 (s, 1H), 8.70 (d, 3J(H,H) 4.6 Hz, 1H), 8.60 (d, 3J(H,H) = 4.6
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Hz, 1H), 8.50 (d, J(H,H) = 4.6 Hz, 1H), 8.45 (d, J(H,H) = 4.6 Hz, 1H). 19F NMR (CD3OD): δ= ꢀ134.9 (d, J(F,F) = 19.6 Hz, 2F,
orthoꢀF), ꢀ136.3 (d, 3J(F,F) = 18.0 Hz, 4F, orthoꢀF), ꢀ154.4 (t, 3J(F,F) = 20.3 Hz, 1F, paraꢀF), ꢀ155.0 (t, 3J(F,F) = 20.3 Hz, 1F, paraꢀ
3
F), ꢀ156.9 (t, J(F,F) = 20.3 Hz, 1F, paraꢀF), ꢀ162.7 (m, 4F, metaꢀF), ꢀ165.7 (m, 2F, metaꢀF). UV/vis (water) λmax [ε(M)ꢀ1 cmꢀ1)] =
420 (93 000), 584 (16 000), 600 (16 000). MS (electrospray): m/z 498.0 [(M + OH)/2]ꢀ.
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Antibodies
Goat polyclonal ALDHꢀ1 antibody, (#NB100ꢀ787, 1:500, Novus Biologicals, Littleton, CO, USA), rabbit polyclonal Bclꢀ2 antiꢀ
body (#2876, 1:1000, Cell Signaling; Beverly, MA, USA), rabbit polyclonal Bax antibody (#2772, 1:1000, Cell Signaling; Beverly,
MA, USA), rabbit polyclonal Gapꢀ43 antibody (#AB5220, 1:800, Millipore, Billerica, MA, USA), mouse monoclonal βꢀtubulin
(Sigma, St Louis, MO, USA), rabbit polyclonal antiꢀTH antibody (#AB152, 1:150, Millipore, Billerica, MA, USA), rabbit polycloꢀ
nal antiꢀDAT antibody (#AB159, 1:100, Millipore, Billerica, MA, USA), mouse monoclonal antiꢀTujꢀ1 antibody (#MMS435P,
1:500, Covance, Emeryville, CA, USA), mouse monoclonal antiꢀCytochrome C antibody (#556433, 1:350, BD, Franklin Lakes, NJ,
USA). Secondary antiꢀ goat and antiꢀrabbit IgG antibodies (1:10000 and 1:2000, respectively; Cell Signaling, Beverly, MA, USA)
and antiꢀmouse IgG peroxidase conjugate (1:15000, Sigma, St. Louis, Mo, USA). Secondary antibodies donkey antiꢀrabbit IgG Far
Redꢀconjugated and donkey antiꢀmouse IgGꢀ tetraꢀmethylꢀrhodamine (TMR)ꢀconjugated antibodies (1:750,Jackson Immuꢀ
noResearch laboratories Inc., Baltimore, MD, USA).
Cell cultures
SN4741, a mouse embryonic SNꢀderived cell line carrying the temperatureꢀsensitive mutant from the oncogene SV40TagꢀtsA58,
was grown at 33 °C in 5% CO2 humidified atmosphere in Dulbecco's modified Eagle's medium (DMEM) containing high glucose
(4.5 mg/ml) supplemented with 2 mM Lꢀglutamine, penicillin/streptomycin/nystatin (100 U/mL) and 10% heatꢀinactivated fetal
calf serum (FCS).
Cell Viability assay
Neuronal cell injury was evaluated by a colorimetric assay for mitochondrial function using the MTT test. SN4741 cells were
placed in 96ꢀwells microtiter plates at a density of 1x104 cells/well and allowed to attach for 24 h before treatment. Neuronal cell
death was promoted by exposure to 6ꢀOHDA (50 µM) under 2% FCS for 24h, following a treatment of the respective cells with 1-
Fe (10 µM and 20 µM) 30 min prior (neuroprotection) or post (neurorescue) the addition of 6ꢀOHDA. In the kinase inhibitor analyꢀ
sis, inhibitors (20 µM LY294002, 2.5 µM GF109203X and 20 µM U120) were added 20 min prior to 6ꢀOHDA and 1-Fe treatment.
MTT [3ꢀ(4, 5ꢀdimethylꢀthiazolꢀ2ꢀyl) 2, 5ꢀdiphenylꢀtetrazolium bromide] was then added at final concentration of 0.5 mg/mL for 24
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h at 37 °C. After this incubation period, purple formazan salts crystals were formed by a NADP/NADPH dependent process, from
metabolically active cells. These salt crystals are insoluble in aqueous solution, but were solubilized by adding a solubilizing soluꢀ
tion (0.01M HCl, 10% SDS) and incubating the plates for at least 6 h in humidified atmosphere (33 °C, 5% CO2). The absorption
was determined in a Tecan Sunrise ElizaꢀReader (Switzerland) at 570/650 nm after automatic subtraction of background readings.
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Immunoblotting Analysis
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SN4741 cells were plated in sixꢀwell culture dishes (~3x104 cells/well) in DMEM medium containing 2% fetal calf serum (FCS)
and allowed to attach for 24 h. Subsequently, after experimental assay (as described in the MTT assay), cells were placed on ice and
the medium was aspirated. After washing with cold phosphateꢀbuffered saline (PBS), cold lysis buffer containing of 50 mM Trisꢀ
HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton Xꢀ100 and protease and phosphtase inhibitors was added, and the cells were
left on ice for 25 min until solubilization. Whole cell homogenates were prepared by centrifuging the solubilized cells at 14,000 x g
for 10 min, at 4 °C. The supernatant was then transferred into clean 1.5 ml tubes and stored at ꢀ70 °C until use. Protein concentraꢀ
tion in the supernatant was determined by the Bradford reagent (BioꢀRad, Hercules,CA, USA), with monitoring at 595 nm. Samples
were mixed 1:1 (v/v) with loading buffer and boiled for 5 min at 95 °C. Equivalent amount of proteins were separated by SDS–
polyacrylamide gel electrophoresis (PAGE) using NuPAGE 4–12% bisꢀtris electrophoresis gel (Invitrogen, Groningen, The Netherꢀ
lands). Separated proteins were then transferred onto Protan nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany)
using the Xcell IITM Blot Module electrotransfer apparatus (Invitrogen, Groningen, The Netherlands). Proteins of relevance were
identified using relevant antibody diluted in Trisꢀbuffered saline, 0.1% Tweenꢀ20 (TBST) with 5% bovine serum albumin (BSA) or
with 10% milk accordingly. Bands were detected by enhanced chemiluminesence using the detection reagents, Acridan based ECL
(Thermo Scientific, Rockford, IL, USA). Film results were pictured using Biocapt software and Autoradiograms were quantified by
scanning laser densitometry (Imager, Bio1d software; Vilber Lourmat, Marne La Vallée, France). The autoradiogram bands were
quantified by scanning laser densitometryvalues normalized to βꢀtubulin.
Immunocytochemistry and confocal microscopy
Cells were grown on polylysineꢀcoated glass coverslips under conditions as described above up to 48 h and rinsed three times in
cold PBS, fixed in 4% paraformaldehyde in PBS, containing 4% sucrose, at 25 °C for 30 min, washed three times with PBS and
then permeabilized on ice with Triton Xꢀ100 (0.1%) in sodium citrate (0.1%) for 2 min. Cells were blocked with PBS containing
10% donkey serum at 37° C for 1 h and incubated with mouse monoclonal antibody against suitable antibody diluted in PBS conꢀ
taining 1% donkey serum and 0.05% Triton Xꢀ100 at 4 °C overnight. Wells were aspirated and rinsed twice with PBS for 10 min
before the addition of secondary antibodies at room temperature in the dark for 1 h. Immunofluorescence was observed using a
40× objective (NA 1.4) and a LSM700 and LSM500 (for Cyrotchrome C) confocal system (Zeiss, Oberkochen, Germany) supportꢀ
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ed with Zen software. Quantification of signals was calculated with Image J 1.47 imaging software (Rasband, W.S., ImageJ, U. S.
National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997ꢀ2012).
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Live imaging and confocal microscopy
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SN4741 cells were plated in D x H 35mm x 10mm culture dishes (~3x104 cells/well) in serum containing DMEM (free of phenol
red) and allowed to attach for 24 h. 1-Al (4µM) was added to the medium together with Mitotracker Green (MTG) (Invitrogen,
Grand Island, NY 14072, USA) (120nM). Fluorescence was observed using a 40× objective (NA 1.4) and a LSM700 confocal sysꢀ
tem (Zeiss, Oberkochen, Germany) supported with Zen software. Samples were excited at 488 nm for MTG detection and at 405
nm for 1-Al detection (MBS 405/488/555/639; PMTꢀ1 (MTG) excitationꢀ 488 nm (3%), splitꢀ SP 524 nm; PMTꢀ2 (1-Al) excitaꢀ
tionꢀ 405 nm (10%), splitꢀ LP 525 nm).
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Statistical analysis
Data were expressed as the mean ± S.E.M and were compared between experimental groups with the use of oneꢀway analysis of
variance followed by Tukey's post hoc test unless otherwise specified (Analyzeꢀit software for Windows Excel, Leeds, UK). Probaꢀ
bility values of p < 0.05 or less were considered statistically significant.
References
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8. Mahammed, A. and Gross, Z. Iron and Manganese Corroles Are Potent Catalysts for the Decomposition of Peroxynitrite. An-
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peroxide dismutase activity of corrole metal complexes. Dalton Trans., 7879–7882, (2009)
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10. Okun, Z., Kupershmidt, L., Amit, T., Mandel, S., BarꢀAm, O., Youdim, M. B. H., Gross, Z. Manganese Corroles Prevent Inꢀ
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Neurorescue
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Treated
Neuronal markers:
Gap-43 , Tuj-1
Dopaminergic markers:
DAT, TH, ALDH-1
Untreated
Anti/pro-apoptotic markers:
Bcl-2, Bax, Cytosolic Cyt C
Signaling pathways:
Involvement of PKCα,β,δ and
PI3K cascades
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