DT-Diaphorase Prevents Aminochrome-Induced Lysosome Dysfunction in SH-SY5Y Cells

Abstract

Aminochrome has been reported to induce lysosomal dysfunction by inhibiting the vacuolar H-type ATPase localized in lysosome membrane. DT-diaphorase has been proposed to prevent aminochrome neurotoxicity but it is unknown whether this enzyme prevents aminochrome-induced lysosomal dysfunction. In the present study, we tested the protective role of DT-diaphorase in lysosomal dysfunction by generating a cell line (SH-SY5YsiNQ7) with a stable expression of a siRNA against DT-diaphorase with only 10% expression of mRNA enzyme. The cells differentiated with retinoic acid and 12-o-tetradecanoylphorbol-13-acetate show a significant increase in the expression of tyrosine hydroxylase, vesicular monoamine transporter-2, and dopamine transporter. The incubation of SH-SY5YsiNQ7 cells with 10 μM aminochrome resulted in a significant decrease of lysosome pH determined by using acridine orange, while aminochrome has no effect on SH-SY5Y cells. These results support the proposed protective role of DT-diaphorase against aminochrome-induced lysosomal dysfunction.

NQO1: A target for the treatment of cancer and neurological diseases, and a model to understand loss of function disease mechanisms

“…Similarly, aminochromeinduced disruption of actin and α-and β-tubulin was enhanced in the presence of dicoumarol [94,95]. Dopaminergic cell lines with silenced expression of NQO1 show lysosomal dysfunction and increase in cell death in response to aminochrome [96,97].…”

Neuroprotection against Aminochrome Neurotoxicity: Glutathione Transferase M2-2 and DT-Diaphorase

“…The most neurotoxic of these ortho-quinone is aminochrome, which induces (i) oxidative stress by being reduced with a single electron to leukoaminochrome o -semiquinone radical [ 73 ]; (ii) the formation of neurotoxic oligomers of alpha-synuclein [ 60 ]; (iii) mitochondrial damage including membrane impairment and inhibition of complex I that ultimately leads to mitochondrial dysfunction [ 73 , 74 , 75 , 76 , 77 ]; (iv) neuroinflammation [ 78 , 79 ]; (v) stress of the endoplasmic reticulum [ 80 ]; and (vi) impairment of protein degradation of both lysosomal and proteasomal systems [ 81 , 82 ]. Aminochrome is not a stable molecule that can be secreted from dopaminergic neurons to neighboring neurons, generating a propagative neurotoxic effect, since aminochrome immediately after its formation can be reduced by flavoenzymes or form adducts with proteins such as mitochondrial complex I, alpha-synuclein, actin, alpha- and beta-tubulin in cytoskeleton, and lysosomal vacuolar-type H + -ATPase, among other proteins [ 60 , 74 , 83 , 84 ].…”

“…Microtubules play an important role in neuron cytoskeleton structure, as well as an essential role in axonal transport of proteins and neurotransmitter vesicles that is strongly decreased in animals treated with aminochrome [ 90 ] ( Figure 3 ). Furthermore, microtubules are involved in the fusion between lysosomes and autophagosomes that it is crucial for autophagy-dependent protein degradation, and DT-diaphorase prevents tubulin aggregation caused by aminochrome [ 82 ].…”

On the Role of Aminochrome in Mitochondrial Dysfunction and Endoplasmic Reticulum Stress in Parkinson's Disease

“…Aminochrome is the most stable and studied o -quinone formed during dopamine oxidation into neuromelanin. Paradoxically, aminochrome under certain conditions can be neurotoxic as a result of inducing mitochondrial dysfunction (Arriagada et al, 2004; Paris et al, 2011; Aguirre et al, 2012; Huenchuguala et al, 2017; Segura-Aguilar and Huenchuguala, 2018), endoplasmic reticulum stress (Xiong et al, 2014), the formation of neurotoxic SNCA oligomers (Muñoz et al, 2015; Muñoz and Segura-Aguilar, 2017), proteasome dysfunction (Zafar et al, 2006; Zhou and Lim, 2009), autophagy dysfunction (Muñoz et al, 2012; Huenchuguala et al, 2014), lysosome dysfunction (Meléndez et al, 2018), neuroinflammation (Santos et al, 2017; de Araújo et al, 2018), cytoskeleton architecture disruption (Paris et al, 2010; Briceño et al, 2016) and oxidative stress (Arriagada et al, 2004). Aminochrome in vivo induces neuronal dysfunction as a consequence of mitochondrial dysfunction, decreased axonal transport resulting in a significant decrease in the number of synaptic monoaminergic vesicles, reduced dopamine release accompanied by an increase in GABA levels, and a dramatic change in the neurons' morphology characterized as cell shrinkage (Herrera et al, 2016).…”

“…DT-diaphorase is expressed in dopaminergic neurons and astrocytes and catalyzes the two-electron reduction of aminochrome into leukoaminochrome, preventing aminochrome one-electron reduction into the leukoaminochrome o -semiquinone radical, catalyzed by flavoenzymes that transfer one electron and use NADH or NADPH. DT-diaphorase prevents aminochrome-induced cell death (Lozano et al, 2010), mitochondrial dysfunction (Arriagada et al, 2004; Paris et al, 2011; Muñoz et al, 2012), cytoskeleton architecture disruption (Paris et al, 2010), lysosomal dysfunction (Meléndez et al, 2018), the formation of neurotoxic SNCA oligomers (Muñoz et al, 2015; Muñoz and Segura-Aguilar, 2017), oxidative stress (Arriagada et al, 2004); dopaminergic neurons' degeneration in vivo (Herrera-Soto et al, 2017) and astrocytes dell death (Huenchuguala et al, 2016). GSTM2 catalyzes the GSH conjugation of aminochrome into 4-S-glutathionyl-5,6-dihydroxyindoline, which is resistant to biological oxidizing agents such as oxygen, hydrogen peroxide, and superoxide (Segura-Aguilar et al, 1997).…”