Dopamine-induced Apoptosis In Human Melanocytes Involves Dysfunction Of Mitofilin: A Possible Cause For Melanocytes Loss In Vitiligo

Vitiligo is a chronic acquired depigmentation disorder characterized by generalized or circumscribed depigmented macules resulting from the loss of functional melanocytes. Theories concerning the cause of vitiligo have focused on several different mechanisms: autoimmune, biochemical, neural , self-destructive and genetic hypotheses, etc. In recent years, the relationship between oxidative stress and vitiligo has been extensively studied and oxidative stress now is recognized as an integral part contributing to vitiligo. Dopamine oxidation is one of special oxidative form in melanocytes and dopaminergic neurons. Several studies document that dopamine oxidation can alter mitochondrial respiration and induce permeability transition in brain mitochondria, which involves the down-regulation/dysfunction of mitofilin.The critical role of mitochondria for cellular survival is well known. Meanwhile, increasing reports indicate that mitochondria are the key in the regulation of apoptosis during oxidative stress. On one hand, ROS were mainly generated during mitochondrial redox process under physiological conditions. On the other hand, mitochondria may be the most sensitive primary cellular targets of oxidative stress. Numerous studies have indicated that mitochondria are the major source of oxidative damage in degenerative diseases. Dysfunction of mitochondria will lead to the abnormal of mitochondrial transmembrane potential and the release of cytochrome c, which will cause caspases activation and apoptosis.Several studies from gene polymorphism to dopamine metabolism have revealed that dopamine oxidation may play an important role in the disease. Our group has observed that there are an increasing level of mitofilin autoantibody in vitiligo patients, which indicates that dysfunction of mitofilin may play a crucial role in the pathogenesis of vitiligo.Though the exact role of inner mitochondrial membrane protein mitofilin has not been evaluated, the presence of mitofilin has been shown to be critical for maintenance of mitochondrial cristae structure. A recent study suggested that mitofilin forms a complex with PARP-1, which plays a role in mtDNA damage signaling and/or repair. It was reported that an ultrastructural study of the melanocytes mitochondria in the vitiligo perilesional skin appeared alterations of the cristae, reflecting dysfunction of mitofilin.As melanocyte is derived from neural crest cells, it shares several characteristic with neural cells. Here, we postulate that inner mitochondrial membrane protein mitofilin is one of the critical protein targets to the toxicity induced by dopamine .The down-regulation /dysfunction of mitofilin will induce disorder of mitochondria, increasing ROS generation and cell apoptosis, which may be a possible cause for melanocytes loss in vitiligo. PartⅠ: Identification of mitofilin as a vitiligo associated antigen1. Main methodsImmunoprecipitation experiment was carried out using vitiligo serum and membrane proteins which were extracted from cultured A375 cells (human melanoma cell lines), and positive control (using mitofilin antibody) were performed meanwhile. The present of mitofilin was checked by western blot analysis, respectively.2. Main resultsCompared with positive control, there were positive bands near 90kDa in western blot analysis.3. Main conclusionsPatients with vitiligo have circulating antibodies to mitofilin, and mitofilin is documented to be a vitiligo-associated antigen.PartⅡ: Dopamine induces melanocytes apoptosis involving dysfunction of mitofilin and mitochondria1. Main methodsImmortalized human epidermal melanocytes cell line PIG1 was treated with different concentration of dopamine for 24h or 48h, proliferating activity, apoptosis and mitofilin were assessed with crystal violet assay, flow cytometry and western blot. After treated with dopamine, mitochondrial membrane potential and ultrastructural analysis of melanocytes were investigated. The data was analyzed by unpaired two-tailed Student t test.2. Main resultsCompared with negative controls, dopamine significantly inhibited the proliferation of PIG1 cells in a dose dependent manner; meanwhile the apoptosis rate of the cells was obviously increased. With no difference in the expression of internal standardβ-actin, mitofilin expression was remarkably decreased in cells treated with dopamine. Mitochondrial transmembrane potential is significantly damaged in dopamine treated melanocytes. Ultrastructural study of the mitochondria of treated cells appeared very large, reflecting swelling of the mitochondrial cristae.3. Main conclusionsInner mitochondrial membrane protein mitofilin is one of the critical protein targets of dopamine oxidation. Dopamine oxidation significantly damages the cristae structure and mitochondrial transmembrane potential of melanocytes though down regulation of mitofilin.Part III: Dopamine induced melanocytes apoptosis involving activation of endoplasmic reticulum and mitochondria pathway.1. Main methodsAfter PIG1 cells were treated with 500μM dopamine for 1h, 3h, 6h or 12h, GRP78, PKR, P-PKR, P-eIF2α, Caspase3, Caspase9, Bax, and Bcl-2 were assessed with western blot analysis.2. Main resultsCompared with negative controls with no difference in the expression of internal standardβ-actin, GRP78, P-PKR, P-eIF2α, Caspase3, Caspase9, and Bax expression was remarkably increased in cells treated with dopamine. Meanwhile, Bcl-2 was significant decreased, and PKR was no difference in dopamine treated cells.3. Main conclusionsDopamine induced melanocytes apoptosis involving activation of endoplasmic reticulum and mitochondria pathway.The study documents that vitiligo patients have circulating auto-antibodies to mitofilin, and reveals that mitofilin is one of the critical protein targets to the toxicity induced by DA. The dysfunction/loss of mitofilin induce disorder of mitochondria, increasing generation of ROS and apoptosis of melanocytes, which involving the activation of endoplasmic reticulum and mitochondria pathway.We postulate that there exists a vicious circle in vitiligo. Excessive ROS, which partly produced by metabolic disorder of dopamine, disturbs normal pathways such as antioxidant system, pigmentation, synthesis/recycling of the essential cofactor by oxidizing proteins, DNA, and membranes. Some of these products, such as MDA and dopaquinone, are unstable and can react with mitochondrial molecules in the cell, which induce the disorder of mitochondria. Dysfunction pathways lead to compromised ability to remove ROS and abnormal endogenous ROS generation. Dopamine mediated oxidative stress not only causes decreased mitofilin expression but also deactivates mitofilin. The consequences of it include swelling of the mitochondrial cristae, losing of mitochondrial membrane potential and an increasing of intracellular ROS level. All those further exacerbate oxidative stress and ultimately result in loss of function, even melanocytes death. And the nonenzymatic oxidative modification of proteins and the subsequent accumulation of the modified proteins, such as mitofilin, will released to serum, which will break down the tolerance to self proteins and lead to autoimmune.