Dopamine induces TNFalpha and TNF-R1 expression in SH-SY5Y human neuroblastoma cells.

Cytotoxic concentrations of dopamine (100-500 microM DA) induce expression of tumour necrosis factor receptor-1 (TNF-R1) and tumour necrosis factor-alpha (TNFalpha) in SH-SY5Y human neuroblastoma cells. TNFalpha expression is dose-dependent and can also be detected after 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium iodide (MPP) treatment. The expression of TNF-R1 is also dose-dependent, but was not observed in 6-OHDA or MPP-treatment. Cells not expressing TNF-R1 were insensitive to TNFalpha, whereas those treated with DA showed a further decrease in viability when subsequently treated with TNFalpha. Thus, DA treatment confers sensitivity to TNFalpha. The decrease of cell viability caused by DA was in part prevented by neutralizing TNFalpha with anti-TNFalpha. As TNF-R1 is increased in substantia nigra of Parkinsonian brains, we suggest that nonvesiculated DA might also play a role in inducing TNF-R1 expression and predispose the neuron to the action of cytokines released in a microglia-mediated inflammatory response.

Intranigral dopamine toxicity and alpha-synuclein response in rats.

There is increasing evidence that, in addition to its function as the main neurotransmitter in the nigrostriatal pathway, dopamine (DA) may be neurotoxic in certain conditions. In this study, the toxicity of DA was assessed by direct injection into the substantia nigra of anaesthetised rats, and its effects were compared with those of 6-hydroxydopamine. Brains were removed 1, 2 and 3 weeks after the lesion for histological or neurochemical analysis. DA caused a significant loss of 35% of tyrosine hydroxylase-positive neurons in the pars compacta of substantia nigra and a 40% reduction of striatal DA content. Cells with signs compatible with both apoptosis and autophagy were observed. GADD153, a parameter of endoplasmic reticulum stress, was strongly induced by 6-hydroxydopamine but not by DA. DA increased the alpha-synuclein content 1 week after the lesion (but not at the later times analyzed) in tyrosine hydroxylase-positive and in non-dopaminergic fibers of pars reticulata. The alpha-synuclein increase may be a physiological temporal response to DA accumulation and/or to cell damage, but the simultaneous presence of alpha-synuclein and DA in the cell cytoplasm at concentration higher than normal is not exempt from risk. In fact, their incubation in a free cell system gives a stable dimerized form of alpha-synuclein that has been described as the critical rate-limiting step for its abnormal fibrillation.

Induction of C/EBP beta and GADD153 expression by dopamine in human neuroblastoma cells. Relationship with alpha-synuclein increase and cell damage.

Expression of CCAAT/enhancer-binding protein beta (C/EBP beta) and growth-arrest DNA damage-inducible 153/C/EBP beta homology protein (GADD153/CHOP) increased after incubation of human neuroblastoma SH-SY5Y cells with a range of dopamine concentrations. Dopamine (100 microM) caused an increase in C/EBP beta expression between 2 and 12 h of treatment, with no evident intracellular morphological changes. Dopamine (500 microM) led to the appearance of autophagic-like vacuoles and a marked increase in GADD153/CHOP between 6 and 24 h of treatment. The expression of alpha-synuclein, the main protein of Lewy bodies in Parkinson's disease and other neurological disorders, increased with a profile similar to C/EBP beta. In addition, overexpression of C/EBP beta caused a concomitant increase in the expression of alpha-synuclein but not of GADD153. In contrast, the overexpression of GADD153 did not alter the expression of alpha-synuclein. Inhibition of JNK by SP600125 reduced increases in C/EBP beta and alpha-synuclein expression, whereas inhibition of both JNK and p38MAPK (with SB203580) blocked the increase in GADD153 expression. We conclude that dopamine, through a mechanism driven by stress-activated MAPKs, triggers C/EBP beta and GADD153 expression in a dose-dependent way. Given that the promoter region of the alpha-synuclein gene contains distinct zones that are susceptible to regulation by C/EBP beta, this factor could be involved in the increased expression of alpha-synuclein after dopamine-induced cell stress. GADD153 increase seems to be related with the endoplasmic reticulum stress, autophagy and cell death observed at high dopamine concentrations.

Dopamine induces autophagic cell death and alpha-synuclein increase in human neuroblastoma SH-SY5Y cells.

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.

TGF-beta1 increases tyrosine hydroxylase expression by a mechanism blocked by BMP-2 in human neuroblastoma SH-SY5Y cells.

Human neuroblastoma SH-SY5Y cells were used to study the effects of transforming growth factor beta1 (TGF-beta1) and bone morphogenetic protein 2 (BMP-2) on neuronal differentiation and acquisition of a catecholaminergic phenotype. SH-SY5Y cells express the intracellular factors activated through the receptors of the TGFbeta superfamily members, Smad1 and Smad4, as in basal conditions or after differentiation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid (RA). TGF-beta1 and BMP-2 induce differentiation in SH-SY5Y cells by different pathways: the effect of TGF-beta1 is potentiated by TPA and the effect of BMP-2 is blocked by RA. Cell differentiation due to TGF-beta1 treatment is accompanied by an increase in tyrosine hydroxylase (TH) expression, more pronounced in the presence of TPA or RA and counteracted by BMP-2. BMP-2 and RA both induce noncatecholaminergic cell differentiation, and together they may induce choline acetyltransferase expression in serum-cultured cells. In conclusion, our results suggest that TGF-beta1 and BMP-2 may contribute, in opposite ways, to regulation of the neuronal catecholaminergic phenotype.

Citicoline increases glutathione redox ratio and reduces caspase-3 activation and cell death in staurosporine-treated SH-SY5Y human neuroblastoma cells.

Citicoline, or CDP-choline, is an essential endogenous intermediate in the biosynthesis of phosphatidylcholine that may act as a neuroprotector in several models of neurodegeneration. The present study analyses the effects of citicoline in the paradigm of staurosporine-induced cell death in human SH-SY5Y neuroblastoma cells. Citicoline reduces apoptosis induced by 100 nM staurosporine for 12 h in SH-SY5Y cells. This effect is higher with pre-treatment of 60 mM citicoline for 24 h after staurosporine challenge. Moreover, citicoline treatment restores glutathione redox ratio diminished after staurosporine challenge. Finally, citicoline also reduces the expression levels of active caspase-3 and specific PARP-cleaved products of 89 kDa resulting from staurosporine exposure when citicoline is added to the culture medium 24 h before staurosporine. These findings demonstrate that citicoline affects the staurosporine-induced apoptosis cell-signalling pathway by interacting with the glutathione system and by inhibiting caspase-3 in SH-SY5Y human neuroblastoma cells.

MPP+ increases alpha-synuclein expression and ERK/MAP-kinase phosphorylation in human neuroblastoma SH-SY5Y cells.

Alpha-synuclein is a brain presynaptic protein that is linked to familiar early onset Parkinson's disease and it is also a major component of Lewy bodies in sporadic Parkinson's disease and other neurodegenerative disorders. Alpha-synuclein expression increases in substantia nigra of both MPTP-treated rodents and non-human primates, used as animal models of parkinsonism. Here we describe an increase in alpha-synuclein expression in a human neuroblastoma cell line, SH-SY5Y, caused by 5-100 microM MPP+, the active metabolite of MPTP, which induces apoptosis in SH-SY5Y cells after a 4-day treatment. We also analysed the activation of the MAPK family, which is involved in several cellular responses to toxins and stressing conditions. Parallel to the increase in alpha-synuclein expression we observed activation of MEK1,2 and ERK/MAPK but not of SAPK/JNK or p38 kinase. The inhibition of the ERK/MAPK pathway with U0126, however, did not affect the increase in alpha-synuclein. The highest increase in alpha-synuclein (more than threefold) in 4-day cultures was found in adherent cells treated with low concentrations of MPP+ (5 microM). Inhibition of ERK/MAPK reduced the damage caused by MPP+. We suggest that alpha-synuclein increase and ERK/MAPK activation have a prominent role in the cell mechanisms of rescue and damage, respectively, after MPP+ -treatment.