INTRODUCTION

Parkinson’s disease (PD) is a common and progressive neurodegenerative disorder affecting about 10 million people worldwide (^{Ball et al., 2019}). The prevalence of the disease is predicted to increase as the elderly people have significantly increased in the recent years (^{Heemels, 2016}; ^{Renaud & Martinoli, 2019}). The disease results primarily from the loss of dopamine-producing neurons (dopaminergic) in the substantia nigra pars compacta (SNpc) (^{Khalifeh et al., 2019}; ^{Yao et al., 2019}) and subsequent deficiency of striatal dopamine (^{Poewe et al., 2017}). Parkinson’s disease as a motor disorder is characterized by a number of classic motor signs including rest tremors, bradykinesia, rigidity and stooping posture (^{Beitz, 2014}). Non-motor symptoms such as sleep disorders, cognitive/ neurobehavioral abnormalities, autonomic dysfunction, and sensory abnormalities such as anosmia, paraesthesia and pain are considered (^{Jankovic, 2008}).

However the exact etiology of PD is still unclear, two forms of the disease exist; familial and sporadic. Familial form accounts for ~ 10 - 15 % of all PD cases and is associated with seven causal genes including alpha-synuclein (SNCA), leucine-rich repeat Kinase 2 (LRRK2), glucocerebrosidase (GBA), vacuolar protein sortingassociated protein 35 (VPS35), parkin RBR E3 ubiquitin protein ligase (PARK2),phosphatase and tensing homologinduced Kinase 1 (PINK1), and Parkinson protein 7 (PARK7) (^{Verstraeten et al., 2015}; ^{Kalinderi et al., 2016}). Sporadic PD affects ~ 85 - 90 % of the cases and is believed to return to environmental exposures, most notably, chemicals, pesticides and heavy metals (^{Castillo et al., 2017}; ^{Pouchieu et al., 2018}).

Autophagy is a crucial catabolic process that keeps homeostasis inside cells and helps them to face stressors through delivering misfolded proteins and damaged organelles to lysosomes for degradation (Cerri & Blandini, 2018; ^{Kirkin, 2019}). Recently, increasing evidence from PD patients and PD experimental models has showed that autophagy plays an essential role in PD pathogenesis (^{Zhang et al., 2012}). In this context, ^{Dehay et al. (2010}) found that post-mortem PD brain samples have an increased number of undegraded autophagosomes and a lower number of autophagolysosomes. ^{Park et al. (2014}) observed that treatment of mice with MPP+ inhibited formation of autophagosome and increased expression of a-synuclein. ^{Parganlija et al. (2014}) showed that knock downing of PINK1 in SH-SY5Y cells resulted in downregulation of key autophagic genes such as Beclin, LC3 and LAMP-2.

Accordingly, the current study was designed to investigate the role of the autophagy-inhibitor 3methyladenine against rotenone-induced dopaminergic cell death in the primary mesencephalic cell culture.

MATERIAL AND METHOD

Preparation of primary mesencephalic cell culture. Pregnant OF1/SPF mice were cared and handled in accordance with the guidelines of the European Union Council (86/609/EU) for the use of laboratory animals. Cultures were prepared from OF1/SPF embryos on the 14th day of gestation according to ^{Radad et al. (2018}). Briefly, brains were released from skulls, and mesencephala were dissected and cut into small pieces in a drop of D-PBS (Invitrogen, Germany). The tissues were transferred into a Griener centrifuge tube containing 2 ml of 0.2 % trypsin solution (Invitrogen, Germany) and 2 ml of 0.02 % DNase I solution (Roche, Germany). After incubation in water bath at 37°C for 7 min, 2 ml of trypsin inhibitor (0.125mg/ ml) (Invitrogen, Germany) were added, the tissue was centrifuged at 100x g for 4 min and the supernatant was aspired. The tissue pellet was then mechanically triturated with a fire-polished Pasteur pipette. Dissociated cells were seeded at a density of 257.000 cells/cm2 in DMEM (Sigma, Germany) supplemented with 4 mM glutamine, 10 mM HEPES buffer, 30 mM glucose, 100 IU/ml penicillin, 0.1 mg/ml streptomycin and 10 % heat-inactivated fetal calf serum (Sigma, Germany). The medium was exchanged with the same medium on the 1st day in vitro (DIV) and on the 3rd DIV. On the 5th DIV, half of the medium was replaced by serum-free DMEM containing 0.02 ml of B27/ml (Invitrogen, Germany). Serum-free supplemented DMEM was used from the 6th DIV and subsequently replaced every 2nd day.

Treatment of cultures with 3-MA. A stock solution of 10 mM was prepared by dissolving 1.49 mg of 3-MA (Sigma-Aldrich, Germany) in 1 ml distilled water. Final concentrations were prepared in DMEM. On the 10th DIV, cultures were treated with 3-MA alone (1, 10, 100, 200 mM) for 48 h to investigate its effect on the survival of dopaminergic neurons and the release of lactate dehydrogenase (LDH) in the culture media.

Treatment of cultures with rotenone. A rotenone (SigmaAldrich, Germany) stock solution (10 mM) was prepared by dissolving 3.94 mg of rotenone in 1 ml DMSO. Final concentrations of rotenone were prepared in DMEM. Fresh rotenone solutions were used in each treatment to avoid its breakdown by storage. On the 10th DIV, cultures were treated with 20 nM of rotenone for 48 h.

Treatment of cultures with rotenone and 3-MA. Cultures were concomitantly treated with rotenone (20 nM) and 3MA (1, 10, 100, 200 mM) on the 10th DIV for 48 h to investigate the effect of autophagy inhibitor 3-MA against rotenone-induced dopaminergic cell damage.

Identification of dopaminergic neurons. Dopaminergic neurons were identified by tyrosine hydroxylase immunostaining. On the 12th DIV, cultured cells were rinsed carefully with phosphate buffered saline (PBS, pH 7.2) and fixed with histochoice for 15 min at room temperature. After permeabilization with 0.4 % Triton X-100 for 30 min at room temperature, non-specific binding sites were blocked with 5 % horse serum (Vectastain ABC Elite kite) for 90 min at room temperature. Then, cells were incubated with anti-TH primary antibody over night at 4 °C and biotinylated secondary antibody (Vectastain) and avidin-biotinhorseradish peroxidase complex (Vectastain) for 90 min at room temperature. The reaction product was developed in a solution of diaminobenzidine (1.4 mM) in PBS containing 3.3 mM hydrogen peroxide (H2O2). Dopaminergic neurons were counted with a Nikon inverted microscope in 10 randomly selected fields/well at magnification10´.

Measurement of LDH activity. Measuring LDH activity in the culture media is usually used to quantify cell damage. At the end of each treatment, culture media were collected and used to measure LDH activity with a cytotoxic detection kit according to the manufacturer’s instructions. In brief, NADH + H+ produced from NAD+ by LDH is transferred by diaphorase to the yellow tetrazolium salt 2-[4iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride (INT) resulting in red formazan formation. The latter was measured spectrophotometrically at 490 nm with a reference at 688 nm. Supplemented medium was used as a blank and subtracted as background.

Statistics. Data were obtained from three experimental repeats (4 wells/experiment) for each treatment condition and presented as mean ± standard error of mean (SEM). Comparisons between groups were made using ANOVA and post-hoc Duncan's test using IBM SPSS statistics 22. (p<0.05 was considered as statistically significant).

RESULTS

Effect of 3-MA on primary mesencephalic cell culture. Treatment of primary mesencephalic cell cultures with different concentrations of 3-MA (1, 10, 100, 200 mM) on the 10th DIV for 48 h did not affect the survival of dopaminergic neurons (Fig. 1A). 3-MA also had no effect on the morphology of dopaminergic neurons. Dopaminergic neurons in both untreated controls and 3-MA-treated cultures were many with long and branched neuritis (Fig. 1B). Moreover, 3-MA did not affect the release of LDH into the culture medium compared to untreated controls (Fig. 2).

Effect of concomitant treatment with 3-MA and rotenone on primary mesencephalic cell cultures. Administration of rotenone (20 nM) to primary mesencephalic cell cultures on the 10th DIV for 48 h significantly decreased the number of dopaminergic neurons by about 25 % compared to untreated controls (Fig. 3A). When cultures were concomitantly treated with 3-MA and rotenone on the 10th DIV for 48 h, 3-MA did not modulate rotenone’s effects on dopaminergic neurons compared to rotenone-treated cultures (Fig. 3A). Treatment of cultures with rotenone markedly injured dopaminergic neurons. Survived neurons appeared few with shortened and thickened neuritis (Fig. 3B). 3-MA also did not affect the morphology of survived dopaminergic neurons in rotenone-treated cultures (Fig. 3B). Also, rotenone was found to increase the release of LDH into the culture medium compared to untreated control cultures and 3-MA had no significant effect on LDH levels compared to rotenone-treated cultures (Fig. 4).

Fig. 1 Treatment of cultures with different concentrations of 3-MA (1, 10, 100, 200 mM) on the 10th DIV for 48 h. 100 % corresponds to the total number of dopaminergic neurons (the average is 27.43 cells/field) after 12 DIV in untreated control cultures. Values represent the mean±SEM of three independent experiments with four wells in each treatment (p=0.223). 

Fig. 2 Measurement of LDH in the culture medium following treatment of cultures with different concentrations of 3-MA (1, 10, 100, 200 mM) on the 10th DIV for 48 h. 100 % corresponds to the amount of LDH in the culture medium on the 12th DIV. Values represent the mean±SEM of three independent experiments with four wells in each treatment (p=0.548). 

Fig. 3 Effect of concomitant treatment of cultures with 3-MA (1, 10, 100, 200 mM) and rotenone (20 nM) on the 10th DIV for 48 h. A) Number of dopaminergic neurons. 100 % corresponds to the total number of dopaminergic neurons (the average is 29.67 cells/field) after 12 DIV in untreated control cultures. Values represent the mean±SEM of three independent experiments with four wells in each treatment. (*p<0.001, #p<0.184). B) Representative micrographs of dopaminergic neurons after 12 DIV. Untreated control culture shows many dopaminergic neurons with long and branched processes. Rotenonetreated culture shows lower number of dopaminergic neurons with few, shortened and thickened neurites. Concomitant treatment of cultures with rotenone and 3-MA did not alter the morphology of surviving dopaminergic neurons compared to rotenone-treated cultures. 

Fig. 4 Measurement of LDH in the culture medium following concomitant treatment of cultures with different concentrations of 3-MA (1, 10, 100, 200 mM) and rotenone (20 nM) on the 10th DIV for 48 h. 100 % corresponds to the amount of LDH in the culture medium on the 12th DIV. Values represent the mean±SEM of three independent experiments with four wells in each treatment (*p<0.001, #p=0.064). 

DISCUSSION

Dysregulated autophagy has been observed in the brains of PD patients, and animal and cellular models of PD indicating the distinct role of autophagy in the pathogenesis of the disease (^{Lynch-Day et al., 2012}; ^{Janda et al., 2012}).

Accordingly, the present study aimed to investigate whether inhibition of the autophagy process would modulate rotenone-induced dopaminergic cell damage in primary mesencephalic cell culture. We have previously showed that activation of autophagy by rapamycin protected dopaminergic neurons against rotenone-induced damage in primary mesencephalic cell culture.

Treatment of cultures with different concentrations of 3-MA (1, 10, 100, 200 mM) was shown to have no effect on both the survival of dopaminergic neurons and the release of LDH into the culture medium. This might occur because autophagic process is minimal and its inhibition was not effective in this well-enriched and short-lived cell culture type. In parallel, fluorescence staining of untreated primary mesencephalic cell culture with LysotrackerÒ deep red, a dye that retained in acidic subcellular organelles such as phagolysosomes, showed very low red fluorescence intensity (^{Radad et al., 2015}).

Administration of 20 nM rotenone to the cultures significantly decreased the number of dopaminergic neurons and increased the release of LDH into the culture medium compared to untreated control cultures. Similarly, rotenone’s neurotoxicity was shown in different in vitro and in vivo models since 2000 when ^{Betarbet and her colleagues described rotenone as a PD neurotoxin (Betarbet et al., 2000}). For example, ^{Radad et al. (2015}) showed that 20 nM rotenone significantly destroyed dopaminergic neurons and increased LDH release in the culture medium. ^{Im et al. (2013}), found that rotenone decreased the viability of PC12 cells. ^{Tapias et al. (2014}), observed that rotenone led to loss of dopaminergic neurons and nigrostriatal terminals in rats. This damaging effect of rotenone on cultured cells was associated with increased autophagic process as indicated by the intense red fluorescence of the LysotrackerÒ deep red compared to untreated control cultures (see Radad et al., 2015). Interestingly, concomitant treatment of cultures with rotenone and 3-MA did not modulate rotenone’s effects on both the survival of dopaminergic neurons and the release of LDH into the culture medium. However, inhibition of autophagy was reported to deleteriously affect aged and toxin-sensitized and tumor cells. For instance, ^{Stroikin et al. (2004}) found that treatment of growtharrested human fibroblasts, a classical model of cellular aging, with 3-MA resulted in moderate increase in mitochondria with lowered membrane potential. ^{Pickard et al. (2015}) showed that 3-MA increased autophagic vesicle formation and apoptotic cell death of LNCaP prostate cancer cells. This toxic effect of 3-MA was reported to be due to blocking of autophagosome formation via the inhibition of class III PI3K with subsequent accumulation of misfolded proteins and altered cell organelles leading to cell damage (^{Wu et al., 2010}).

In conclusion, 3-MA, an autophagy inhibitor, does not modulate rotenone-induced dopaminergic cell damage in primary mesencephalic cell culture.