**4. Discussion**

266 Bioenergetics

AV-6-93 concentrations of up to 100 µM for 3 min before energisation with succinate prevented total depolarisation of mitochondria (Fig. 3A), the increase in respiratory state 4 (Fig. 3B), and the release of mitochondrial Ca2+ (Fig. 3C), suggesting that this compound has a high ability to protect mitochondria against MPT induction. Incubation of mitochondria with 0.75 nmol/mg protein, CsA, a specific inhibitor of MPT (Broekemeier et al., 1989), for 2 min before energising with succinate, either in the absence or presence of 1 μM AV-6-93, completely blocked mitochondrial depolarisation (Fig. 3A), the increase in respiratory state 4 (Fig. 3B), and the Ca2+-induced release of mitochondrial Ca2+ (Fig. 3C). These data show that these effects had been induced by MPT. In contrast to AV-6-93, diflurone, in the same concentration range, did not prevent either the depolarisation of mitochondria or the release of mitochondrial Ca2+ (results not shown), indicating that this compound did not protect

The effects of AV-6-93 and diflurone on mitochondrial oxidative damage were assessed by detecting the mitochondrial membrane lipid peroxidation induced by the pro-oxidant pair ADP/Fe2+. Lipid peroxidation was evaluated by measuring oxygen consumption (Fig. 4) and TBARs formation (Table 3). In the absence of AV-6-93 and after the addition of the prooxidant pair, it is possible to distinguish two-phase kinetics in oxygen consumption: an initial lag phase, characterized by slow oxygen consumption lasting about 2 min, is followed by a rapid oxygen consumption phase. The lag phase is probably related with the time required for the generation of a sufficient amount of the perferryl ion complex (ADP-Fe2+- O2 ADP-Fe3+-O2-), which has been suggested to be responsible for the initiation of lipid peroxidation. The rapid oxygen consumption phase is probably due to the oxidation of the polyunsaturated fatty acid acyl chain of membrane phospholipids by reactive oxygen species (ROS) and, consequently, due to the propagation phase of lipid peroxidation (Sassa et al., 1990). AV-6-93 concentrations up to 100 M enlarged the lag phase of slow oxygen consumption before the oxygen uptake burst induced by the ADP/Fe2+ complex and increased the rate of the rapid oxygen consumption phase (Fig. 4), suggesting that the compounds affected both the initiation and the propagation of lipid peroxidation of

These results agree with the quantitative evaluation of TBARs formation performed to confirm the protective effects of AV-6-93. The data in Table 3 show that the kinetics of TBARs formation induced by ADP/Fe2+ are similar to that observed for oxygen consumption. The same range of AV-6-93 concentrations used in the oxygen consumption assays also affected TBARs formation. TBARs formation in the absence of ADP/Fe2+ was negligible (0.44 ± 0.25 nmol/mg of protein). In contrast to AV-6-93, diflurone, in the same concentration range, did not affect oxygen consumption induced by the ADP/Fe2+ complex or TBARs formation (results not shown), indicating that this compound has no capacity to protect mitochondria against the lipid peroxidation induced by the pro-oxidant pair

Lipid peroxidation was evaluated by oxygen consumption and initiated by adding 1 mM ADP/0.1 mM Fe2+ to mitochondrial suspensions (Fig. 4). The traces represent typical direct oxygen consumption recordings of three experiments obtained from different mitochondrial preparations; controls in the absence of ADP/Fe2+ (–ADP/Fe2+); assays in the presence of

AV-6-93 at the concentrations 1, 10, 20, 50, 100 μM (1, 10, 20, 50, 100).

**3.4 Effects of AV-6-93 and diflurone on mitochondrial oxidative stress**

mitochondria against MPT.

mitochondrial membranes.

ADP/Fe2+.

Studies examining the importance of mitochondrial pathophysiology in neurodegeneration provide a target for additional treatments with agents that improve mitochondrial function, protect MPT, and/or exert antioxidant activity (Petrozzi et al., 2007). These studies lead to novel approaches in the treatment of neurodegenerative diseases, such as Parkinson's disease, with disease-modifying drugs.

The aim of the present study was to examine the abilities of two novel adamantanecontaining DHP analogues, AV-6-93 and diflurone, to protect against cell death induced by mitochondrial toxin MPP+ and beneficially influence mitochondrial processes in an attempt to identify putative antiparkinsonian drugs.

First, we examined how both compounds acted in primary cortical cultures in response to MPP+. AV-6-93, at concentrations of 1 and 10 µM, significantly protected against MPP+ induced cell death by 75% and 56%, respectively, whereas diflurone protected against cell death by 35% at a concentration of 10 µM. Neither AV-6-93 nor diflurone, added without MPP+, changed cell viability.

Targeting the Mitochondria

**5. Conclusion** 

Parkinson's disease.

**7. References** 

**6. Acknowledgment** 

ISSN 0006-8993

0096-5316

302-310, ISSN 0076-6879

a one adamantine ring-containing DHP.

may be initiated by DHP compounds to increase cell survival.

by Novel Adamantane-Containing 1,4-Dihydropyridine Compounds 269

To address why both adamantane-containing compounds showed very distinct effects on mitochondrial damage induced by both Ca2+ and ADP/Fe2+, one may suggest that the molecular "volume" of AV-6-93 (one adamantane ring-containing DHP) is more optimal than that of diflurone (two adamantane ring-containing DHP) for mitochondrial protection. The two adamantane rings in the diflurone molecule probably generate a steric hindrance that prevents or delays the chemical reaction, which can easily occur in the case of AV-6-93,

Based on the results obtained in primary cortical cultures, the two-adamantane DHP structure is not as crucial as it is in isolated rat liver mitochondria because diflurone has not lost its activity to prevent cell death caused by MPP+ (a toxin focused on mitochondrial complex I). However, the activity of diflurone was lower than that of AV-6-93. One could suggest that, in addition to the protection of complex I, other cellular signalling mechanisms

The novel one-adamantane 1,4-dihydropyridine compound AV-6-93 is capable of regulating cell survival processes with regards to mitochondrial processes, such as inhibition of the induction of the permeability transition pore and prevention of oxidative stress. The effectiveness of AV-6-93 can be considered to be very promising in the treatment of neurodegenerative diseases associated with compromised mitochondrial processes, e.g.,

ESF project No. 2009/0217/1DP/1.1.1.2.0/09/APIA/VIAA/031; Latvian Science Council grant: No.10.0030. Center for Neuroscience and Cell Biology (CNC), and Center for Marine

Alho, H., Ferrarese, C., Vicini, S. & Vaccarino, F. (1988) Subsets of GABAergic neurons in

Becker, C., Jick, S.S. & Meier, C.R. (2008) Use of antihypertensives and the risk of Parkinson disease. *Neurology,* Vol.15, No.70, (April 2008), pp. 1438-1444, ISSN 0028-3878 Broekemeier, K. M., Dempsey, M. E. & Pfeiffer, D. R. (1989) Cyclosporin A is a potent inhibitor of

*Biological Chemistry,* Vol.264, No.14, (May 1989), pp. 7826-7830, ISSN 0021-9258 Buege, J.A. & Aust, S.D. (1978) Microsomal lipid peroxidation. *Methods in Enzymology*, Vol.52, pp.

Chance, B. & Williams, G. R. (1956) The respiratory chain and oxidative phosphorylation.

Costantini, P., Chernyak, B.V., Petronilli, V., Bernardi, P. (1996) Modulation of the mitochondrial

dissociated cell cultures of neonatal rat cerebral cortex show co-localization with specific modulator peptides. *Brain Research,* Vol.467, No.2, (April 1988), pp. 193-204,

the inner membrane permeability transition in heart mitochondria. *The Journal of* 

*Advances in Enzymology and Related Subjects of Biochemistry*, Vol.17, pp. 65-134, ISSN

permeability transition pore by pyridine nucleotides and dithiol oxidation at two

and Environmental Research (IMAR-CMA) of the University of Coimbra, Portugal.

A larger difference between the compounds' activities was observed in isolated rat liver mitochondria by the assessment of their ability to affect both the Ca2+-induced mitochondrial permeability transition (MPT) and lipid peroxidation. To assess the Ca2+ induced MPT, the evaluation of the drop of ∆, the increase in mitochondrial respiration associated with Ca2+ accumulation in the mitochondrial matrix, and the mitochondrial Ca2+ fluxes were carried out. Changes in these parameters help us to conclude whether the compound protects mitochondria against MPT induction and, consequently, to discern whether the compound alters mitochondrial Ca2+ homeostasis. AV-6-93, at a concentration of 10 µM, significantly protected mitochondria against MTP induction and provided complete protection at 100 µM, as revealed by its ability to prevent the depolarisation of mitochondria, the increase in mitochondrial respiration and mitochondrial Ca2+ release. These effects were comparable with that of CsA (0.75 nmol/mg protein), a specific inhibitor of the mitochondrial permeability transition pore. Diflurone was ineffective in these tests. The effectiveness of AV-6-93 can be considered to be very promising because it indicates the ability of this compound to halt mitochondrial swelling and cell death, both consequences of the induction of the permeability transition pore.

A critical factor for induction of MPT is the oxidation of thiol groups of the MPT complex, creating diethyl cross-links (Costantini et al., 1996; 1998, Halestrap et al., 1997; McStay et al., 2002). Therefore, the most plausible hypothesis to explain the partial MPT protection induced by AV-6-93 is that changes in the redox-state of thiol groups of the MPT complex is provided via avoiding of diethyl cross-links. This hypothesis is supported by the observation that AV-6-93 protected mitochondria against oxidative stress. Oxidative stress was assessed by evaluating the extent of lipid peroxidation by measuring oxygen consumption and TBARs formation. Alterations of these parameters may reveal whether the compound protects mitochondria against oxidative stress, i.e., whether the compound acts as an antioxidant. AV-6-93, at concentrations up to 100 µM, protected (by about a half) mitochondria against membrane lipid peroxidation, as inferred by its ability to inhibit both oxygen consumption and TBARs formation induced by the pro-oxidant pair ADP/Fe2+. These data suggest that this compound may act as antioxidant because it can avoid both the initiation and the propagation of the oxidation of polyunsaturated fatty acid acyl chains of membrane phospholipids induced by the perferryl ion complex ADP-Fe3+-O2 -, a mechanism suggested to be responsible for lipid peroxidation (Sassa et al., 1990). In contrast to AV-6-93, diflurone, under the same conditions, had no capacity to protect mitochondria against oxidative damage induced by the pro-oxidant pair ADP/Fe2+.

The only common feature of both compounds was a lack of influence on mitochondrial bioenergetics, which was assessed by analysing several mitochondrial functioning parameters of the respiratory chain (respiration states 2, 3, 4, FCCP-stimulated respiration, the RCR, and the ADP/O ratio) and the oxidative phosphorylation system (∆ and phosphorylation time), using both glutamate/malate and succinate as respiratory substrates. According to the mitochondrial parameters affected, it is possible to assess how the compound interferes with mitochondrial bioenergetics: by perturbing the permeability (integrity) of the inner mitochondrial membrane (stimulation of respiration states 2 and 4), by impairing the respiratory chain (inhibition of FCCP-stimulated respiration), and/or by acting at the level of the phosphorylation system (affecting respiration state 3). Both AV-6-93 and diflurone, at concentrations of up to 100 µM, failed to significantly affect liver mitochondrial bioenergetics, as shown by the lack of effects on both glutamate/malate- and succinate-supported respiration in state 2, state 3, state 4, FCCP-stimulated respiration, RCR and ADP/O ratios, ∆ and phosphorylation time.

To address why both adamantane-containing compounds showed very distinct effects on mitochondrial damage induced by both Ca2+ and ADP/Fe2+, one may suggest that the molecular "volume" of AV-6-93 (one adamantane ring-containing DHP) is more optimal than that of diflurone (two adamantane ring-containing DHP) for mitochondrial protection. The two adamantane rings in the diflurone molecule probably generate a steric hindrance that prevents or delays the chemical reaction, which can easily occur in the case of AV-6-93, a one adamantine ring-containing DHP.

Based on the results obtained in primary cortical cultures, the two-adamantane DHP structure is not as crucial as it is in isolated rat liver mitochondria because diflurone has not lost its activity to prevent cell death caused by MPP+ (a toxin focused on mitochondrial complex I). However, the activity of diflurone was lower than that of AV-6-93. One could suggest that, in addition to the protection of complex I, other cellular signalling mechanisms may be initiated by DHP compounds to increase cell survival.
