α-Synuclein and mitochondrial bioenergetics regulate tetrahydrobiopterin levels in a human dopaminergic model of Parkinson disease

Abstract

Parkinson disease (PD) is a multifactorial disease resulting in preferential death of the dopaminergic neurons in the substantia nigra. Studies of PD-linked genes and toxin-induced models of PD have implicated mitochondrial dysfunction, oxidative stress, and the misfolding and aggregation of α-synuclein (α-syn) as key factors in disease initiation and progression. Many of these features of PD may be modeled in cells or animal models using the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)). Reducing oxidative stress and nitric oxide synthase (NOS) activity has been shown to be protective in cell or animal models of MPP(+) toxicity. We have previously demonstrated that siRNA-mediated knockdown of α-syn lowers the activity of both dopamine transporter and NOS activity and protects dopaminergic neuron-like cells from MPP(+) toxicity. Here, we demonstrate that α-syn knockdown and modulators of oxidative stress/NOS activation protect cells from MPP(+)-induced toxicity via postmitochondrial mechanisms rather than by a rescue of the decrease in mitochondrial oxidative phosphorylation caused by MPP(+) exposure. We demonstrate that MPP(+) significantly decreases the synthesis of the antioxidant and obligate cofactor of NOS and TH tetrahydrobiopterin (BH4) through decreased cellular GTP/ATP levels. Furthermore, we demonstrate that RNAi knockdown of α-syn results in a nearly twofold increase in GTP cyclohydrolase I activity and a concomitant increase in basal BH4 levels. Together, these results demonstrate that both mitochondrial activity and α-syn play roles in modulating cellular BH4 levels.

Keywords: Bioenergetics; DAT; Free radicals; GTPCH; MPP(+); Parkinson disease; Tetrahydrobiopterin; α-Synuclein.

Fig. 1

BE(2)-M17 cells are a dopaminergic cell line expressing markers of dopaminergic neurons. (a and b) Representative western blots showing (a) a comparison of relative DAT and TH expression levels in SH-SY5Y and BE(2)-M17 and (b) a number of proteins involved in dopamine synthesis and homeostasis and the dopaminergic marker GIRK2 in BE(2)-M17 cell lysate (5 µg). (c) Immunocytochemistry showing cellular localization of α-syn, DAT, VMAT2, and TH (green) of fixed BE(2)-M17. Cell nuclei were stained with DAPI (blue). (d) Representative HPLC chromatogram showing catecholamine content in BE(2)-M17 (solid line) and SH-SY5Y (dotted line). Cells were harvested in perchloric acid (100 mM) and separated by reverse-phase HPLC with electrochemical detection. Inset: quantification of noradrenalin, L-DOPA, and dopamine content in BE(2)-M17 and SH-SY5Y cells, normalized for protein content (n=3) (e) [³H]DA uptake in BE(2)-M17 cells in the presence or absence of the DAT inhibitor mazindol. Cells were incubated with [³H]DA (10 nM) for time periods from 0 to 15 min either with or without mazindol (10 μM). Uptake was quantified using scintillation counting and normalized to protein content in each sample (n= 3, ± SEM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 2

MPP⁺ rapidly induces mitochondrial impairment in BE(2)-M17 cells in a DAT-dependent manner. (a) Stress test of BE(2)-M17 cells using mitochondrial electron transport chain uncoupling agents, identifying minimum and maximal oxygen consumption rates in cells. The OCR was measured in BE(2)-M17 cells treated with 300 nM FCCP or 2 μM oligomycin (injection A) and then with 300 nM FCCP or 3 μM rotenone and antimycin A (injection B). OCR was normalized to the OCR in the well immediately before injection (OCR %). Data points represent means of at least six determinations. (b) MPP⁺ rapidly impairs the cellular OCR of BE(2)-M17 cells. Oxygen consumption rate was measured in cultured BE(2)-M17 cells (1.5 × 10⁴ cells/well) before the injection of 100 μM MPP⁺ or medium alone (control). The change in the oxygen consumption rate (OCR %) was monitored for 270 min post-injection of MPP⁺ in a minimum of six wells. Curves were fitted using nonlinear regression analysis: t_1/2 = 34.4 min for MPP⁺-treated cells, error bars represent ± SD (n = 3). (c) MPP⁺ induced mitochondrial impairment in a DAT-dependent manner. BE(2)-M17 cells were pretreated with dopamine (30 nM), reserpine (10 μM), or mazindol (100 μM) for 2 h before injection of MPP⁺. Cellular OCR was monitored for 180 min post-MPP⁺ injection (n = 3).

Fig. 3

MPP⁺-induced mitochondrial impairment is independent of α-synuclein knockdown or manipulation of oxidative stress and NOS activity. (a) siRNA transfection reduces α-syn expression in cells by up to 80%. Knockdown of α-synuclein was assessed by Western blot and expression levels were normalized to actin expression (n = 3). (b and c) Knockdown of α-syn protects cells from MPP⁺ toxicity. Cells were transfected with SNCA or control oligo for 36 h before treatment with MPP⁺ (100 μM) for 48 h. (b) Total cell numbers were assessed by bright-field microscopy. Bars represent mean cell death compared to non-MPP⁺-treated controls ( ± SEM; n = 3). (c) Necrotic cells were stained for 30 min with propidium iodide and counted by fluorescence microscopy. Bars represent the mean percentage of propidium iodide-positive cells (± SEM; n = 3). (d) OCR was measured in BE(2)-M17 cells transfected with an siRNA targeting α-synuclein or a control oligo for 48 h before treatment with MPP⁺ (100 μM). The effects of MPP on OCR were monitored for 70 min post-MPP⁺ treatment. Data points represent mean OCR values ± SD of triplicate values obtained from three independent transfections. (e) Attenuation of oxidative stress or NOS activation/uncoupling does not attenuate MPP⁺-induced mitochondrial impairment. BE(2)-M17 cells were preincubated with compounds known to attenuate MPP⁺-induced cell death by inhibiting oxidative stress or NOS activation/uncoupling. BE(2)-M17 cells were pretreated with L-NAME (1 mM), BH4 (10 μM), or ascorbic acid (100 μM) for 2 h before cellular OCR was monitored for 90 min in the presence/absence of MPP⁺ (100 μM; n = 3). * P < 0.05 vs control oligo, ^# P< 0.05 vs untransfected cells.

Fig. 4

MPP⁺ induces decreased cellular BH4 levels through reduction of BH4 synthesis. MPP⁺ treatment of cells results in decreased BH4 levels. BE(2)-M17 cells were treated with 50 or 100 μM MPP⁺ for 24/48 h before harvest. Intracellular levels of (a) BH4, (b) BH2, (c) biopterin, and (d) total BH4, BH2, and biopterin (BH_x) were measured by HPLC coupled with both electrochemical and fluorescence detection, and (e) BH4:(BH2 + B) was calculated. Bars represent the mean + SD of triplicate determinations (n = 3) *P < 0.05, **P < 0.01, ***P<0.001 vs untreated cells.

Fig. 5

Knockdown of α-synuclein increases basal cellular BH4 levels through increased GTPCH activity. 36 h post-siRNA transfection, cells were treated with 100 μM MPP⁺ for 48 h before harvest. (a) Cellular BH4 levels are significantly increased by α-syn knockdown as measured by HPLC with electrochemical detection (n = 3; + SEM). **P< 0.01 vs untransfected cells/control oligo, #P< 0.05 vs untreated cells, ##P< 0.01 vs untreated cells. (b) α-Syn knockdown increases basal GTPCH activity. Activity was assessed in cell lysates by incubation with GTP, as described under Materials and methods. Bars represent mean GTPCH activity normalized for protein content by BCA assay, normalized to untreated cells (n = 5; + SEM). *P< 0.05. (c and d) α-Syn knockdown and MPP⁺ treatment do not significantly affect GTPCH expression in cells. (c) Expression levels of GTPCH in cell lysate (10 μg), as assessed by Western blot. (d) Densitometric analysis of GTPCH expression relative to actin expression in cell lysates (10 μg), normalized to untreated cells. Bars represent mean band intensity, relative to actin intensity, normalized to untreated cells (n = 4; + SEM).

Fig. 6

MPP⁺ reduces cellular BH4 synthesis through depletion of cellular GTP/ATP levels. (a) Guanosine supplementation does not rescue MPP⁺ -induced BH4 decreases. Cells were treated with 100 μM MPP⁺ for 48 h with or without the addition of 10 μM guanosine for 20 h before harvest. Bars represent mean BH4 levels + SD (n = 3). (b–f) MPP⁺ treatment disrupts cellular bioenergetics independent of α-synuclein levels. (b) HPLC chromatogram showing elution of indicated phosphorylated nucleosides in untreated and MPP⁺ -treated cells, as detected by absorbance at 254 nm. Cellular levels of (c) GTP, (d) ATP, (e)GDP, and (f) GMP after 24 h MPP⁺ treatment in cells with and without knockdown of α-syn with SNCA oligo (n = 3; + SEM). **P<0.01, ***P<0.001 vs untreated cells.

Fig. 7

Schematic representation of the interplay between α-syn and tetrahydrobiopterin after MPP⁺ treatment. (1) MPP⁺ enters dopaminergic cells through the DAT transporter and (2) accumulates in mitochondria, inhibiting complex I. Although α-syn knockdown reduces DAT activity and MPP⁺ import, α-syn knockdown does not impair complex I inhibition. (3) Complex I inhibition induces both ROS production and ATP depletion. ATP depletion results in decreased BH4 synthesis, exacerbating oxidative stress and increasing cell death. α-Syn knockdown increases GTPCH activity under basal conditions resulting in increased BH4 levels, increasing antioxidant capacity and decreasing cell death.