Dopaminochrome induces caspase‐independent apoptosis in the mesencephalic cell line, MN9D

Abstract

Parkinson’s disease (PD) is characterized by a deficiency in motor cortex modulation due to degeneration of pigmented dopaminergic neurons of the substantia nigra projecting to the striatum. These neurons are particularly susceptible to oxidative stress, perhaps because of their dopaminergic nature. Like all catecholamines, dopamine is easily oxidized, first to a quinone intermediate and then to dopaminochrome (DAC), a 5-dihydroxyindole tautomer, that is cytotoxic in an oxidative stress-dependent manner. Here we show, using the murine mesencephalic cell line MN9D, that DAC causes cell death by apoptosis, illustrated by membrane blebbing, Annexin V, and propidium iodide labeling within 3 h. In addition, DAC causes oxidative damage to DNA within 3 h, and positive TUNEL fluorescence by 24 h. DAC, however, does not induce caspase 3 activation and its cytotoxic actions are not prevented by the pan-caspase inhibitor, Z-VAD-fmk. DAC-induced cytotoxicity is limited by the PARP1 inhibitor, 5-aminoisoquinolinone, supporting a role for apoptosis inducing factor (AIF) in the apoptotic process. Indeed, AIF is detected in the nuclear fraction of MN9D cells 3h after DAC exposure. Taken together these results demonstrate that DAC induces cytotoxicity in MN9D cells in a caspase-independent apoptotic manner, likely triggered by oxidative damage to DNA, and involving the translocation of AIF from the mitochondria to the nucleus.

Metabolic Dysfunction in Parkinson’s Disease: Bioenergetics, Redox Homeostasis and Central Carbon Metabolism

“…NAD + is also consumed by poly (ADP-ribose) polymerase-1 (PARP-1) during DNA-damage repair (Figure 2.5). Contradictory reports exist regarding the role of PARP-1 genetic variants in PD [40, 140], but a number of studies have suggested that PARP-1 activation upon PD-related insults leads to energy depletion associated to the consumption of ATP by the NAD + salvage pathway, and subsequent neuronal cell loss with necrotic characteristics (Parthanatos) [143, 144, 164, 181, 185, 195, 211, 252, 338, 350]. Parthanatos might be a secondary or complementary cell death mechanism to apoptosis in PD [334].…”

Catecholamine autotoxicity. Implications for pharmacology and therapeutics of Parkinson disease and related disorders

“…Aminochrome has been shown to be toxic to murine mesencephalic MN9D cells at concentrations as low as 50 μM (Linsenbardt et al, 2009). In the mesencephalic cell line, MN9, aminochrome induces caspase-independent apoptosis (Linsenbardt et al, 2012). Aminochrome can also be formed from auto-oxidation of DA in the setting of hydrogen peroxide and a peroxidase, yielding a superoxide anion (Hastings, 1995), and activity of the peroxidase is increased in post-mortem midbrain tissue from PD patients (De Iuliis et al, 2002).…”

Direct intranigral injection of dopaminochrome causes degeneration of dopamine neurons

“…DAC is cytotoxic to mesencephalic cells in an oxidative stress-dependent manner [35, 36], inhibits mitochondrial respiration, and inactivates intracellular proteins [2, 13, 31, 57, 58, 62]. DAC is produced when DA is oxidized to quinone species that cyclize [21].…”

“…DA oxidation produces dopaminochrome (DAC) [10, 21, 42, 50, 56], a component of neuromelanin, the substance that pigments SNpc neurons [17, 60]. We have previously reported that DAC is cytotoxic to mesencephalic cells in an oxidative stress-dependent manner [35, 36]. …”