ABSTRACT
Title
MPP+ modulates hyperpolarization-activated current (Ih) in dopaminergic neurons of the substantia nigra pars compacta.
Authors
A. Masi1, R. Narducci1, F. Moroni1, G. Mannaioni1
1Dept. of Pharmacology “Mario Aiazzi Mancini”, Università degli Studi di Firenze, Firenze, Italy
1Dept. of Pharmacology “Mario Aiazzi Mancini”, Università degli Studi di Firenze, Firenze, Italy
Abstract
Parkinson’s disease (PD) is a neurodegenerative and progressive movement disorder of the central nervous system. 1-methyl-4-phenylpyridinium (MPP+), a mitochondrial complex I inhibitor derived from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) metabolism, has long been used to induce a syndrome that recapitulates many of the pathological features of the human PD. MPP+ damages dopaminergic (DA) cells by impairing mitochondrial adenosine triphosphate (ATP) production and by increasing the rate of toxic reactive oxygen radicals (ROS) generation (Abou-Sleiman PM et al., 2006). However, a recent report has showed that complex I-defective transgenic mice injected with MPP+ still develop PD (Choi et al., 2008), indicating that the pathogenic mechanisms underlying this pathology need to be better investigated. We sought to address this issue by focusing on the early electrophysiological effects of MPP+administration on DA neurons of substantia nigra pars compacta (SNc) in acute midbrain slices by using whole cell patch clamp recordings.
Bath application of MPP+ (50-100 µM) failed to produce an effect in the holding current (Vh = -60 mV) and to induce significant changes in the spontaneous firing rate in SNc DA neurons. On the other hand, 50 and 100 µM MPP+ reduced hyperpolarization-activated current (Ih) amplitude by 20.5%±0.9 (mean±SEM, n=3, p<0.05) and 30.2%±4.6 (n=6, p<0.01) respectively. Similarly, in current clamp configuration, this effect was detected as an attenuation of the typical “sag” potential upon injection of negative current (-200 pA) and as a change in the shape of the inter-spike depolarizing phase in spontaneously firing cells. MPP+ dependent Ih reduction was ATP independent, as ATP was always present (4 mM) in our pipette solution. To test whether this effect was dependent on complex I inhibition-related ROS generation, we challenged our preparations with rotenone (10-100 µM). Rotenone (10 µM and 100 µM) did not replicate MPP+ dependent Ih reduction (4.1% ± 2.1, n = 3 cells, p>0.05 and 11.5% ± 2.5, n=3, p>0.05, respectively), suggesting that the observed effect was not dependent on complex I inhibition. Since several reports have associated MPTP-induced PD with PolyADP-RibosePolymerase (PARP) activation (Dawson et al., 1999, Yokoyama et al., 2010), we tried to prevent MPP+ induced effects with PJ34 (50 µM) a potent, water-soluble PARP inhibitor (Ki =0.5 µM). No significant reversion of MPP+ induced Ih reduction was observed (28.5% ± 2.8, n=5, p<0.01) when slices were challenged with 100 µM MPP+ after 5 minutes of pre-incubation with PJ34.
In summary, MPP+-dependent Ih modulation seems to be complex I, ATP and PARP1 independent. These findings, although unexpected, are consistent with the ability of MPTP/MPP+ to induce PD in a mitochondrial complex I independent manner.
Abou-Sleiman PM et al. (2006). Nat Rev. Neurosci. 7:207-219.
Choi et al. (2008). Proc Natl Acad Sci U S A. 105(39):15136-41.
Dawson et al. (1999). Proc Natl Acad Sci U S A. 96(10):5774-9.
Yokoyama et al. (2010). J Neurosci Res. 88:1522-1536.
Bath application of MPP+ (50-100 µM) failed to produce an effect in the holding current (Vh = -60 mV) and to induce significant changes in the spontaneous firing rate in SNc DA neurons. On the other hand, 50 and 100 µM MPP+ reduced hyperpolarization-activated current (Ih) amplitude by 20.5%±0.9 (mean±SEM, n=3, p<0.05) and 30.2%±4.6 (n=6, p<0.01) respectively. Similarly, in current clamp configuration, this effect was detected as an attenuation of the typical “sag” potential upon injection of negative current (-200 pA) and as a change in the shape of the inter-spike depolarizing phase in spontaneously firing cells. MPP+ dependent Ih reduction was ATP independent, as ATP was always present (4 mM) in our pipette solution. To test whether this effect was dependent on complex I inhibition-related ROS generation, we challenged our preparations with rotenone (10-100 µM). Rotenone (10 µM and 100 µM) did not replicate MPP+ dependent Ih reduction (4.1% ± 2.1, n = 3 cells, p>0.05 and 11.5% ± 2.5, n=3, p>0.05, respectively), suggesting that the observed effect was not dependent on complex I inhibition. Since several reports have associated MPTP-induced PD with PolyADP-RibosePolymerase (PARP) activation (Dawson et al., 1999, Yokoyama et al., 2010), we tried to prevent MPP+ induced effects with PJ34 (50 µM) a potent, water-soluble PARP inhibitor (Ki =0.5 µM). No significant reversion of MPP+ induced Ih reduction was observed (28.5% ± 2.8, n=5, p<0.01) when slices were challenged with 100 µM MPP+ after 5 minutes of pre-incubation with PJ34.
In summary, MPP+-dependent Ih modulation seems to be complex I, ATP and PARP1 independent. These findings, although unexpected, are consistent with the ability of MPTP/MPP+ to induce PD in a mitochondrial complex I independent manner.
Abou-Sleiman PM et al. (2006). Nat Rev. Neurosci. 7:207-219.
Choi et al. (2008). Proc Natl Acad Sci U S A. 105(39):15136-41.
Dawson et al. (1999). Proc Natl Acad Sci U S A. 96(10):5774-9.
Yokoyama et al. (2010). J Neurosci Res. 88:1522-1536.