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Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism responsible for anti-apoptotic effects of melatonin ¹

SHAIDA A. ANDRABI^*, IQBAL SAYEED, DETLEF SIEMEN, GERALD WOLF^* and THOMAS F. W. HORN^*,2

^* Institute for Medical Neurobiology and
Department of Neurology Otto-von-Guericke University, Magdeburg, Germany

²Correspondence: Leipziger St. 44, Haus 36, Magdeburg, D-39120 Germany. E-mail: thomas.horn{at}medizin.uni-magdeburg.de

SPECIFIC AIM

We investigate whether the pineal hormone melatonin directly inhibits potentially proapoptotic mitochondrial permeability transition pore (mtPTP) activity at single-channel and cellular levels. We examined whether such mtPTP inhibition by melatonin leads to decreased NMDA receptor-induced increase in cytosolic calcium ([Ca²⁺]_c).

PRINCIPAL FINDINGS

1. Melatonin reduces sustained [Ca²⁺]_c increase in primary neuronal cultures exposed to NMDA
Upon stimulation of striatal neurons with 200 µM NMDA, we observed a fast increase in the fluo-4 fluorescence from a baseline intensity of 103.4 ± 4.3 (average of single cell arbitrary intensity values ± SE, n=5 cultures) to 282.3 ± 22.5 immediately after exposure to NMDA (Fig. 1 ). The increased fluorescence, indicating an increase in [Ca²⁺]_c, was still high (284.3±16.2) 18 min after NMDA application.

View larger version (41K): [in this window] [in a new window]   Figure 1. Neuronal cultures were loaded with the Ca2+-sensitive fluorochrome fluo-4 and a series of confocal images were recorded to monitor the fluorescence intensities. NMDA was applied alone (200 µM, blue line) or together with CsA (2 µM, green line) or melatonin (100 µM, red line). A) Average fluorescence intensity traces of the different treatment groups reflecting the [Ca2+]c levels. NMDA alone shows a rise in [Ca2+]c in neurons that sustained at a plateau level throughout the continuous NMDA application. Melatonin and CsA decreased the sustained rise in [Ca2+]c in NMDA-stimulated neurons. Normalized data are expressed as mean ± SE, n = 5 cultures for each group. B) Representative images of fluo-4-loaded cultures. Left column depicts baseline fluorescence before NMDA application; the two other columns show the elevated fluorescence during the NMDA exposure. Upper row (NMDA alone) shows the sustained rise in [Ca2+]c after NMDA application. The middle lower row (NMDA+Mel) indicates that although these neurons displayed an initial high [Ca2+]c level after the NMDA exposure onset, fluorescence decreased in the presence of melatonin.

When cyclosporine (CsA, 2 µM) was added to the NMDA-containing superfusion solution, neurons exhibited a fast initial increase in fluorescence, peaking at 297.3 ± 7.2 (n=5 cultures). After 5 min of CSA+NMDA application, a significant decrease in NMDA-induced sustained fluorescence was observed (207.7±15.2, P<0.05); at 18 min values declined to 188.8 ± 23.0 (P<0.05). When melatonin (100 µM) was added, the fluorescence plateau started to decline again in a pattern similar to that with CsA (Fig. 1) . At 5 min of melatonin+NMDA application, fluorescence values decreased from 281.9 ± 12.6 to 230.3 ± 14.7 (P<0.05) and at 18 min of melatonin application to 181.5 ± 11.9 (P<0.05).

2. Melatonin prevents the loss of mitochondrial membrane potential
Tetramethylrhodamine ethylester (TMRM) was applied after OGD insult to cultures with and without drug treatment to monitor mitochondrial membrane potential (_m) in the presence or absence of melatonin. In vehicle-treated OGD-subjected cultures, little or no TMRM fluorescence was observed (Fig. 2 D), indicating that the ability of cells to take up TMRM into the mitochondria was lost. When melatonin (100 µM) or CsA (2 µM) was present in the culture medium during OGD, mitochondria retained the ability to accumulate TMRM, reflected by a strong fluorescence of mitochondria-like structures within the cells (Fig. 2D ).

View larger version (49K): [in this window] [in a new window]   Figure 2. A) Representative traces of mtPTP activity with a 1 µM melatonin application. Baseline mtPTP activity (control, 1st trace) was blocked by 1 µM melatonin (2nd and 3rd trace, at 10 and 72 s, respectively, after switching to melatonin), which was reversible (4th trace, after switching to control). Holding potential (Eh): +20 mV. B) Representative traces of mtPTP activity (control, 1st trace), with CsA application (2nd and 3rd traces) and after switching back to control solution (4th trace). C) Concentration-response curve for the normalized open probability (Po) under the influence of melatonin at Eh = +20 mV: Po was estimated by all-point analysis of single-channel data in 1 min segments from each experiment starting 1 min after addition of melatonin. Mean Po were calculated from 2 independent experiments at each concentration. Data are shown as mean ± SE D) Melatonin preserves the rate of TMRM uptake after OGD: representative pictures showing an overlay of phase contrast and TMRM fluorescence. Red shows the TMRM-loaded mitochondria, indicating an intact m. The images were obtained after the cultures were subjected to 3 h OGD, followed by 30 min oxygen-glucose resupply. TMRM was added to culture medium at the time of oxygen-glucose resupply. a) Control culture with strongly stained mitochondria-like structures. b) An OGD-treated culture has almost completely lost the ability to take up TMRM into the mitochondria indicating a loss of m. OGD-subjected cultures (c) that contained melatonin (100 µM) in the medium, in contrast, retained the capacity to load TMRM. Melatonin alone (d) did not alter the TMRM uptake in control cultures. Analogous to melatonin, treatment with 2 µM CsA (e) also prevented the loss of m as indicated by the mitochondrial uptake of TMRM in some cells.

3. Melatonin inhibits mtPTP directly
mtPTP channel currents were recorded from patches of the inner mitochondrial membrane. Recordings displayed characteristic activity of the mtPTP with a large single-channel conductance of >1 nS and a large variety of subconductance states that could be reversibly blocked by 1 µM CsA (Fig. 2B ). Melatonin inhibited the open probability (P_o) of the mtPTP (Fig. 2A ) in a dose-dependent manner from 250 to 100 µM (Fig. 2C ). The best fit by the Hill equation was calculated with an IC₅₀ of 0.8 µM and a Hill coefficient of 1. A maximum decrease in the P_o by only 80% reflects melatonin’s effect even at higher concentration as gradual. This effect was reversible upon washout in the control solution (Fig. 2A , 4th trace).

4. Melatonin prevents the release of cytochrome c (cyt c)release from mitochondria
A 2 h middle cerebral artery occlusion (MCAO) followed by reperfusion caused a pronounced increase in cytosolic cyt c immunoreactivity 4 and 24 h after reperfusion. At 4 h the cytosol was strongly stained for cyt c; 24 h after reperfusion, cyt c immunoreactivity extended to the intercellular space. Sham-operated rats at 4 and 24 h showed a profile of cyt c staining indicative of intact mitochondria. When MCAO-subjected animals were treated with melatonin, the cytosolic and intercellular cyt c immunostaining signals decreased at 4 and 24 h.

5. Melatonin prevents caspase-3 activation
4 h after occlusion, vehicle-treated MCAO rats showed a massive activation of caspase-3 in the ischemic cortex compared with sham rats that was still detectable 24 h after reperfusion, but not localized in well-defined cellular compartments. Upon melatonin treatment, caspase-3 activation was strongly reduced in MCAO rats at both 4 and 24 h.

6. Melatonin reduces apoptotic DNA fragmentation
In vehicle-treated MCAO rats, the Apostain label was observed in the nuclei of a large cell population. Such Apostain-positive cells showed a characteristic morphology of apoptotic cells with shrunken structures. The number of Apostain-positive cells was reduced in melatonin treated MCAO rats, showing an anti-apoptotic effect of melatonin.

7. Melatonin reduces the MCAO-induced brain damage
Decrease in the infarct volume
TTC staining of brain slices obtained from vehicle-treated MCAO rats showed reproducible and readily detectable lesions in the areas supplied by MCA 3 days after the reperfusion. The lesions were present in the lateral striatum and the overlying cortex.

Melatonin (10 mg/kg, i.p.), when given in MCAO rats at the onset of occlusion and reperfusion, significantly reduced the infarct volume to 147.0 ± 10.4 mm³, (P<0.05, n=10) compared with vehicle-treated MCAO rats (290.0±13.5 mm³, n=10).

Prevention of the loss of MAP-2 and NeuN staining in the ischemic tissue
In vehicle-treated MCAO rats, MAP-2 staining was characterized by a uniform strong fluorescence of dendrites and soma. After 24 h of reperfusion, a pronounced loss of MAP-2 staining was observed in vehicle-treated MCAO rats. After melatonin treatment, MAP-2 staining was preserved in the dendritic arbor and soma of the neurons in the ischemic cortex.

NeuN-positive cells were almost completely lost from the ischemic cortex in vehicle-treated MCAO rats 24 h after reperfusion, indicating massive neuronal degeneration. Melatonin treatment prevented the loss of NeuN-positive cells in MCAO rats.

CONCLUSIONS AND SIGNIFICANCE

Our initial observation that melatonin decreased the NMDA-induced sustained cytosolic Ca²⁺ [Ca²⁺]_c plateaus in a pattern similar to that of the mtPTP inhibitor CsA prompted us to consider a direct action of melatonin on the mtPTP. Mitochondria act as Ca²⁺ buffers by sequestering excess Ca²⁺ from the cytosol. [Ca²⁺]_c continues to rise when NMDA receptors are continuously stimulated, causing Ca²⁺ uptake into the mitochondria that, upon reaching a threshold level leads to mtPTP opening, which in turn produces Ca²⁺-induced Ca²⁺ release. Indeed, our experiments using CsA at a low concentration, known to block the mtPTP, lowered the NMDA-induced [Ca²⁺]_c levels, indicating the presence of a mtPTP-mediated Ca²⁺ release that contributes to the overall [Ca²⁺]_c rise.

Melatonin reduced NMDA-induced [Ca²⁺]_c levels in an almost identical pattern as seen with CsA, pointing to a direct effect of melatonin on the mPTP or mechanisms upstream. We show that melatonin directly inhibits the mtPTP, as observed in patch-clamp recordings on the inner mitochondrial membrane. The patches were prepared at high Ca²⁺ concentrations and fully open mtPTPs were mostly detected. Hence, the dose-dependent reduction in the pore currents upon bath application of melatonin is due solely to a direct interaction of melatonin with the channel. Our data show that the efficacy of melatonin in inhibiting the mtPTP is high, with an IC₅₀ of 0.8 µM.

Previous studies using mtPTP-blocking agents show that in pathological conditions an excessive loading of Ca²⁺ into mitochondria induces apoptosis by stimulating the release of apoptosis-promoting factors like cyt c, AIF, Smac/DiaBLO, and procaspases from the mitochondrial intermembrane space into the cytoplasm via permeability transition mechanisms. One would assume that melatonin, being an mtPTP blocker, may preserve _m in ischemic conditions. To test this assumption, we used an OGD model of neuronal cultures in conjunction with live cell imaging of the fluorescent dye TMRM. We found that the TMRM uptake in OGD-subjected cultures was strongly compromised compared with control cultures, indicating that the noxious OGD stimulus leads to a decrease of _m as previously reported. The protection against the OGD-induced loss of TMRM uptake by CsA in our model indicates involvement of the mtPTP in the mitochondrial depolarization.

We used the mitochondrial cyt c release as a marker for mtPTP activation in our MCAO model. It has been shown that such ischemia-induced cyt c release is blocked by CsA, indicating a mtPTP-dependent mechanism. We also observed high levels of cytosolic cyt c immunoreactivity in the ischemic brain region of MCAO-subjected rats at 4 and 24 h after the onset of reperfusion, suggesting lower mtPTP activity in the presence of the drug.

We followed the cascade of events extending downstream from mtPTP-mediated cyt c release by examining how melatonin affects the caspase-3 activation and subsequent DNA fragmentation. Our in vivo data do not rule out an indirect effect of melatonin on the mtPTP activation by removing reactive nitrogen or oxygen species from the tissue. The action of melatonin directly on the mtPTP may instead contribute to the overall outcome of its protective effect in in vivo stroke models. The finding of a reduction in the infarct volume in our studies agrees with previous results obtained by different authors that melatonin reduces the infarct size after cerebral ischemia and serves as a control for effectiveness of the MCAO insult.

Our results demonstrate for the first time that melatonin directly inhibits the mtPTP and that this may contribute to the anti-apoptotic properties of melatonin. Therapeutic use of melatonin may provide a strategy for the treatment of stroke and neurodegenerative disorders that involve the mitochondrial apoptotic pathway.

View larger version (60K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1031fje;

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