Dopamine induces autophagic cell death and α‐synuclein increase in human neuroblastoma SH‐SY5Y cells

Abstract

Free cytoplasmic dopamine may be involved in the genesis of neuronal degeneration in Parkinson's disease and other such diseases. We used SH-SY5Y human neuroblastoma cells to study the effect of dopamine on cell death, activation of stress-induced pathways, and expression of alpha-synuclein, the characteristic protein accumulated in Lewy bodies. We show that 100 and 500 microM dopamine causes a 40% and 60% decrease of viability, respectively, and triggers autophagy after 24 hr of exposure, characterized by the presence of numerous cytoplasmic vacuoles with inclusions. Dopamine causes mitochondrial aggregation in adherent cells prior to the loss of functionality. Plasma membrane and nucleus also maintain their integrity. Cell viability is protected by the dopamine transporter blocker nomifensine and the antioxidants N-acetylcysteine and ascorbic acid. Dopamine activates the stress-response kinases, SAPK/JNK and p38, but not ERK/MAPK or MEK, and increases alpha-synuclein expression. Both cell viability and the increase in alpha-synuclein expression are prevented by antioxidants; by the specific inhibitors of p38 and SAPK/JNK, SB203580 and SP600125, respectively; and by the inhibitor of autophagy 3-methyladenine. This indicates that oxidative stress, stress-activated kinases, and factors involved in autophagy up-regulate alpha-synuclein content. The results show that nonapoptotic death pathways are triggered by dopamine, leading to autophagy. These findings should be taken into account in the search for strategies to protect dopaminergic neurons from degeneration.

Superoxide is the major reactive oxygen species regulating autophagy

“…Several reports have suggested that starvation-induced autophagy is mediated by ROS. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] In this study, we investigated the generation of O 2 KÀ and H 2 O 2 under two starvation conditions: starvation of glucose, L-glutamine, pyruvate, and serum (GP), and starvation of amino acids and serum (AA). Cellular levels of O 2 KÀ and H 2 O 2 were measured by flow cytometry using specific fluorescent dyes (see Materials and Methods).…”

“…We and others have found that these inducers of autophagy require ROS production. 8,13,12,18 Indeed, we have previously found that rotenone and TTFA induce O 2 KÀ production and that overexpressing SOD2 blocks autophagy. 17 This was reversed by knocking down SOD2 by siRNA.…”

“…When another autophagy inhibitor wortmannin (Supplementary Figure S4bi) was used in HeLa cells, the levels of both O 2 KÀ and H 2 O 2 induced by GP or AA starvation were not significantly affected (Figure 8b). In addition, beclin-1 and atg-7 siRNAs knockdown failed to significantly affect GP or AA starvation- demonstrated in many studies, [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] KÀ are also reduced through a chain reaction. 2,3 This illustrates the importance for measuring all forms of ROS.…”

“…Starvation-induced autophagy is associated with cell survival whereas autophagy induced by various other stimuli (including dopamine, 8 sodium selenite, 13 mETC inhibitors rotenone and TFFA, 17 SOD2 inhibitor 2-ME, 18 and exogenous H 2 O 2 12,18 ) is associated with cell death. We and others have found that these inducers of autophagy require ROS production.…”

Cell death in the nervous system

“…On the one hand, this may alert us to the possibility that anti-apoptotic therapies carry the potential risk of inducing non-apoptotic PCD; on the other hand, it may argue that therapeutics directed at multiple cell-death pathways will be required for optimal efficacy in diseases that involve PCD. Because cell death associated with some neurodegenerative diseases (or diseaseassociated mutants, such as α-synuclein) is associated with an autophagic morphology, these implications are potentially important for the development of effective therapies to prevent or ameliorate neurodegenerative disorders 36 .…”