Lipophilic Cationic Cyanines Are Potent Complex I Inhibitors and Specific in Vitro Dopaminergic Toxins with Mechanistic Similarities to Both Rotenone and MPP⁺

Abstract

We have recently reported that simple lipophilic cationic cyanines are specific and potent dopaminergic toxins with a mechanism of toxicity similar to the Parkinsonian toxin MPP+. In the present study a group of fluorescent lipophilic cyanines have been used to further exploit the structure-activity relationship of the specific dopaminergic toxicity of cyanines. Here we report that all cyanines tested were highly toxic to dopaminergic MN9D cells with IC50s in the range of 60–100 nM and not toxic to non-neuronal HepG2 cells parallel to the previously reported for 2,2’- and 4,4’-cyanines. All cyanines non-specifically accumulate in the mitochondria of both MN9D and HepG2 cells at high concentrations, inhibit the mitochondrial complex-I with the inhibition potencies similar to the potent complex I inhibitor, rotenone. They increase the reactive oxygen species (ROS) production specifically in dopaminergic cells causing apoptotic cell death. These and other findings suggest that the complex-I inhibition, the expression of low levels of antioxidant enzymes, and presence of high levels of oxidatively labile radical propagator, dopamine, could be responsible for the specific increase in ROS production in dopaminergic cells. Thus, the predisposition of dopaminergic cells to produce high levels of ROS in response to mitochondrial toxins together with their inherent greater demand for energy may contribute to their specific vulnerability towards these toxins. The novel finding that cyanines are an unusual class of potent mitochondrial toxins with specific dopaminergic toxicity suggest that their presence in the environment could contribute to the etiology of PD similar to MPP+ and rotenone.

N-Methyl-4-phenylpyridinium Scaffold-Containing Lipophilic Compounds Are Potent Complex I Inhibitors and Selective Dopaminergic Toxins

“…To evaluate the dopaminergic selectivity and structure activity relationship of the toxicity of PP-PP + derivatives relative to MPP + , a series of standard in vitro toxicity experiments were carried out with dopaminergic MN9D and SH-SH5Y, and liver HepG2 (control) cells. These experiments show that the parent PP-PP + derivative [1] (Scheme) is toxic to dopaminergic MN9D and SH-SH5Y cells with IC50s of ~23 and ~50 μM (Table 1), respectively and not significantly toxic to HepG2 cells under the same experimental conditions (Fig 1A ), parallel to the previously reported behavior of MPP + , 4′I-MPP + , and cationic cyanines (11)(12)(13)(14). In addition, these studies reveal that lipophilic substituents in the phenyl rings of the parent derivative [1] significantly increase the MN9D toxicity (e.g.…”

“…Our findings strongly support the notion that the high vulnerability of dopaminergic cells to these toxins is likely a consequence of their high inherent propensity to produce excessive ROS in response to effective complex I inhibition and associated downstream effects, in comparison to nondopaminergic neurons (6,13). Perhaps the most significant implication of these and our previous findings is the indication that MPP + is the simplest of a larger group of unidentified environmental dopaminergic toxins [for example cationic cyanines (11)(12)(13)], a possibility that may have great public health consequences related to the environmental causes of PD.…”

“…In addition, most of these derivatives were significantly more toxic to MN9D cells in comparison to MPP + (~10-20 fold), suggesting that the structure of MPP + is not unique in its selective dopaminergic toxicity, as generally believed (11,12).…”

Characteristics of the mitochondrial and cellular uptake of MPP+, as probed by the fluorescent mimic, 4'I-MPP+

“…These possibilities are in good agreement with the above suggestion that mitochondrial and cellular uptakes of MPP + and 4'I-MPP + are coupled. Since mitochondrial accumulation and the complex-I inhibition mediated increase of ROS production is intimately associated with the MPP + and 4'I-MPP + -mediated apoptotic MN9D cell death [ 19 , 54 ], the inhibition of mitochondrial uptake by CGP37157 was expected to protect MN9D cells from these toxins. As expected, CGP37157 effectively protected MN9D cells from both MPP + and 4'I-MPP + toxicities ( Fig 7 ).…”

Characteristics of the mitochondrial and cellular uptake of MPP+, as probed by the fluorescent mimic, 4′I-MPP+

“…These possibilities are in good agreement with the above suggestion that mitochondrial and cellular uptakes of MPP + and 4'I-MPP + are coupled. Since mitochondrial accumulation and the complex-I inhibition mediated increase of ROS production is intimately associated with the MPP + and 4'I-MPP + -mediated apoptotic MN9D cell death, [18,52] the inhibition of mitochondrial uptake by CGP37157 was expected to protect MN9D cells from of these toxins. As expected, CGP37157 effectively protected MN9D cells from both MPP + and 4'I-MPP + toxicities (Fig 7).…”