Correspondence: Hao Pang (E-mail: hpang@cmu.edu.cn) Xu Wu (E-mail: xwu@cmu.edu.cn)

Secretogranin III upregulation is involved in parkinsonian toxin-mediated astroglia activation

Xiaoni Zhan1, Gehua Wen4, Enzhu Jiang1, Fengrui Li2, Xu Wu3 and Hao Pang1

1Department of Forensic Genetics and Biology, School of Forensic Medicine, China Medical University, No.77 Puhe Road, Shenyang, China

2Baotou Medical University, No.31 Jianshe Road, Baotou, China3Department of Forensic Pathology, School of Forensic Medicine, China Medical University,

No.77 Puhe Road, Shenyang, China4Department of Forensic Clinical Medicine, School of Forensic Medicine, China Medical University,

No.77 Puhe Road, Shenyang, China

(Received December 23, 2019; Accepted March 4, 2020)

ABSTRACT — Environmental neurotoxins such as paraquat (PQ), manganese, and 1-1-methyl-4-phe-nyl-1,2,3,6-tetrahydropyridine (MPTP) are associated with a higher risk of Parkinson’s disease (PD). These parkinsonian toxins exert certain common toxicological effects on astroglia; however, their role in the regulatory functions of astroglial secretory proteins remains unclear. In a previous study, we observed that secretogranin II (SCG2) and secretogranin III (SCG3), which are important components of the regu-lated secretory pathway, were elevated in PQ-activated U118 astroglia. In the current study, we used the parkinsonian toxins dopamine (DA), active metabolite of MPTP (MPP+), MnCl2, and lipopolysaccharide (LPS) as inducers, and studied the potential regulation of SCG2 and SCG3. Our results showed that all the parkinsonian toxins except LPS affected astroglial viability but did not cause apoptosis. Exposure to DA, MPP+, and MnCl2 upregulated glial fibrillary acidic protein (GFAP), a marker for astrocyte activa-tion, and stimulated the levels of several astrocytic-derived factors. Further, DA, MPP+, and MnCl2 expo-sure impeded astroglial cell cycle progression. Moreover, the expression of SCG3 was elevated, while its exosecretion was inhibited in astroglia activated by parkinsonian toxins. The level of SCG2 remained unchanged. In combination with our previous findings, the results of this study indicate that SCG3 may act as a cofactor in astrocyte activation stimulated by various toxins, and the regulation of SCG3 could be involved in the toxicological mechanism by which parkinsonian toxins affect astroglia. Key words: Parkinsonian toxins, Astroglia activation, Secretogranin II, Secretogranin III,

Secretory granules

INTRODUCTION

Prolonged exposure to environmental toxins can increase the risk of various neurodegenerative disorders (Cox et al., 2016), including Parkinson’s disease (PD), which has been linked to exposure to neurotoxic com-pounds such as paraquat (PQ), rotenone, 1-methyl-4-phe-nyl-1,2,3,6-tetrahydropyridine (MPTP), and manganese. Inflammatory stimuli such as lipopolysaccharide (LPS) and catecholamines like dopamine (DA) and 6-hydroxy-dopamine (6-OHDA) are also used in chemical-induced models of PD (Goldman, 2014). Astroglia regulate neu-ronal metabolism and activity in the central nervous sys-

tem, and have important roles in regulating immune and nutritional support for neurons in the brain. External stimuli can cause astroglia activation and thereby accel-erate the production of various inflammatory mediators and gliotransmitters (Pekny and Pekna, 2014). The secre-tory machinery in astroglia is elaborate and complex, and ensures the sensitive and precise release of bioactive sub-stances (Verkhratsky et al., 2016). Parkinsonian toxins exert certain common toxicological effects on astroglia, including oxidative stress, mitochondrial impairment, dis-ruption of homeostatic Ca2+ signaling, and DNA dam-age (Alaimo et al., 2013; Jennings et al., 2017; Li et al., 2016; Sidoryk-Wegrzynowicz and Aschner, 2013; Yu et

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al., 2016). However, the effects of parkinsonian toxins on the astroglial secretory process remain unclear.

Dense-core vesicles (DCVs) are major vesicular com-partments that are largely responsible for the release of neuropeptides and hormones in neurons and neuroendo-crine cells (Kim et al., 2006). DCVs have been identi-fied in astrocytes from mouse primary culture and human brain tissue, and the secretory proteins secretogranin II (SCG2) and secretogranin III (SCG3) are targeted to DCVs (Hur et al., 2010; Paco et al., 2010; Prada et al., 2011). SCG2 and SCG3 are members of the “granin fam-ily”, which are relatively abundant acidic proteins local-ized in secretory vesicles. They play an essential role in regulating the biogenesis of secretory granules and are usually used as markers to study the regulated secre-tory pathway in endocrine and neuroendocrine cells (Bartolomucci et al., 2011). SCG2 plays a crucial role in the formation of secretory granule-like structures and packaging of neuropeptides into secretory vesicles. The endoproteolytic processed bioactive peptides secretone-urin (SN), EM66, and manserin play a prominent role in stimulating neurotransmitter and inflammatory fac-tor release, energy expenditure, and blood pressure main-tenance (Boutahricht et al., 2007; Troger et al., 2017). SCG3 is one of the least studied members of the granin family. It acts as a bridge that simultaneously binds to chromogranin A (CgA) in the cargo aggregates and the cholesterol-rich membrane, and interacts with carbox-ypeptidase E (CPE) in the membrane. This complex form enables SCG3 to participate in early peptide processing in the trans-Golgi network (TGN) and subsequent peptide secretion in DCVs (Hosaka and Watanabe, 2010). Recent studies have discovered that SCG3 is a novel retinopa-thy-selective angiogenic vascular and anti-leakage factor in diabetes (LeBlanc et al., 2017). Further, the differen-tial expression and secretion of SCG3 in various endo-crine tumors suggest that it could be a biological marker for tumor diagnosis (Portela-Gomes et al., 2010). How-ever, SCG3 function as an extracellular regulator in the nervous system is yet to be clarified.

In our previous studies, we used PQ to stimulate U118MG astrocytoma cells, and observed upregulation of SCG2 and SCG3 in PQ-activated astroglia. Further, de novo SCG2 and SCG3-positive DCVs, which were possi-bly involved in the trafficking of interleukin 6 (IL-6) and brain-derived neurotrophic factor (BDNF), were signifi-cantly accumulated in the perinuclear region (Zhan et al., 2018a, 2018b). These results suggest that secretogranins may act as biological indicators in toxin-induced astro-glia activation. In the current study, we selected four tox-ins used in PD models (DA, MPP+, MnCl2, and LPS) as

inducers, and studied their effect on the expression and secretion of SCG2 and SCG3.

MATERIALS AND METHODS

MaterialsDulbecco’s modified Eagle’s medium (DMEM), pen-

icillin, streptomycin, and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Dopamine (DA), Manganese(II) chloride tetrahydrate (MnCl2·4H2O), MPP+ iodide (MPP+ is active metabolite of MPTP), and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). CellTiter 96®Aqueous One Solu-tion Reagent was purchased from Promega (Promega, WI, USA), and the eBioscience™ Annexin V-FITC Apoptosis Detection Kit and Cell Cycle Detection Kit were obtained from Invitrogen (Thermo Fisher, Wa l t h a m , M A , U S A ) a n d K e y g e n ( K e y g e n Biotechnology, Jiangsu, China), respectively. The radi-oimmunoprecipitation assay (RIPA) reagent and BCA Protein Assay Kit were obtained from Beyotime Biotechnology (Beijing, China). The Easysee reagent was purchased from TransGen Biotech (Beijing, China). Anti-bodies against SCG3 (SC-50289), SCG2 (SC-50290), and β-actin (SC-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). RNAiso Plus, PrimeScript RT Reagent Kit, and SYBR Prime EX Taq were purchased from Takara Biotechnology (Shiga, Japan).

Cell culture and treatmentHuman glioblastoma U118 cells were purchased

from Shanghai Institutes for Biological Sciences (SIBS, Shanghai, China). Cells were grown in Dulbecco’s mod-ified Eagle’s medium (high glucose) containing 10% FBS and 1% antibiotic at 37°C in a humidified incubator containing 5% CO2. U118 cells were seeded in Nunc™ 60 mm culture dishes (Thermo Fisher) at a concentra-tion of 5 × 104 cells/cm2, and treated with only medium or medium containing specific concentrations of induc-ers after 24 hr. Treatments with toxins were performed in accordance with ISO 15190:2003 Medical Laboratories-Requirements for safety. Cell morphology was observed under the phase contrast mode of the DMi8 inverted fluo-rescence microscope (Leica, Heidelberg, Germany).

Cell Viability AssayThe effect of the four inducers on the viability of U118

astroglia was determined using CellTiter 96®Aqueous One Solution Reagent (MTS). Briefly, cells (1 × 104 per well) were seeded in 96-well plates and incubated in a humid-

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ified atmosphere containing 5% CO2. The cells were then treated with different concentrations of inducers for 24 or 48 hr, and subsequently incubated in MTS (reagent: medium=1:5) for 1 hr. Absorbance was then measured at 490 nm with a SpectraMax M2 plate reader (Molecular Devices, Sunnyvill, CA, USA).

Quantitative polymerase chain reaction (qPCR)Cells were collected using the RNAiso Plus kit, and

total RNA samples were prepared by chloroform extrac-tion according to manufacturer’s instructions. cDNA was synthesized with the PrimeScript™ RT Reagent Kit. Real-time PCR was conducted with an ABI 7500 Real-Time PCR System (Thermo Fisher) by using a SYBR Prime EX Taq Kit. The amount of target gene expression was calcu-lated using the 2–ΔΔCt method. All primer sequences were designed as described in our previous studies, and β-actin was used as internal reference (Zhan et al., 2018a).

Western blottingU118 astroglia were lysed in ice-cold RIPA lysis with

protease inhibitor for 30 min at 4°C. The lysates were cleared by centrifugation at 10,000 × g for 10 min. Con-ditioned medium (CM) was collected after centrifuga-tion at 600 × g for 10 min to remove cell debris. The protein samples (20-30 μg per lane) and CM (20 μL per lane) were separated in 10% SDS-polyacrylamide gel and then transferred to the PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked with a solution containing 5% nonfat milk powder in tris-buff-ered saline tween-20 (TBST) and then incubated with primary antibodies overnight at 4°C. Next, membranes were washed with TBST for 5 min three times and incu-bated with peroxidase-conjugated secondary antibodies for 90 min at room temperature. The results were detect-ed using an Easysee Western Blot Kit in a Tanon imager system (Shanghai, China). Densitometry was performed with Image J software (NIH, Bethesda, MD, USA) with β-actin as the loading control. Total protein level in the CM was monitored by staining of the gels with 0.5% Coomassie blue.

Annexin V-FITC/PI assayU118 cells from the control and treated groups were

digested with EDTA-free 0.25% trypsin, washed twice with pre-cooled PBS, and adjusted to a density of 1 × 105 cells/mL. Cells were centrifuged at 715 ×g for 5 min, and approximately 1 × 105 cells were resuspended in 200 µL binding buffer and stained with 10 µL Annex-in V-FITC and 5 µL propidium iodide (PI) at room tem-perature for 15 min in the dark. A total of 200 µL binding

buffer was then added, and cell apoptosis was detected using a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA) within 1 hr.

Cell cycle assayU118 astroglia were treated with different concentra-

tions of the four inducers for 24 or 48 hr prior to analy-sis. Cell cycle analysis was performed using a Cell Cycle Detection Test kit according the manufacturers’ instruc-tions. The astroglia were trypsinized, washed with ice-cold PBS twice, and fixed in 70% ice-cold ethanol over-night. After removing ethanol by centrifugation and washing with PBS, cells were treated with 100 µL RNase A at 37°C for 30 min, and finally stained with 400 µL PI in the dark at 4°C for 30 min. The analysis was conducted using a CytoFLEX cytometer, and the cell cycle was ana-lyzed using modfitLT software (Verity Software House, Topsham, ME, USA).

Statistical analysisStatistical analysis was performed using GraphPad

Prism Software 6.0 (San Diego, CA, USA). All data are representative of at least three independent experiments and the final results are shown as means ± standard devi-ation (SD). To determine differences between the experi-mental and control groups, Student’s t-tests were applied, and a P-value <0.05 was considered statistically signifi-cant.

RESULTS

DA, MPP+, and MnCl2 showed cytotoxic effects in

U118 astrogliaCell viability of U118 astroglia treated with DA,

MnCl2, MPP+, and LPS was assessed using MTS assay. As shown in Fig. 1A, DA, MPP+, and MnCl2 induced a concentration-dependent decrease of cell viability, where-as LPS did not. Based on the results, the doses that lead to approximately 50% decrease in cell viability were 200 μM DA, 250 μM MnCl2, and 500 μM MPP+. These doses and the highest concentration of LPS (100 μg/mL) were selected as the treatment dosages for subsequent experiments. Data were presented as percentage of cell viability in the control group, and the incubation times at which a decrease in viability of approximately 50% was observed on the time-viability curves were select-ed for subsequent experiments (Fig. 1B). The incubation times selected were: 24 hr for DA and MnCl2 and 48 hr for MPP+. We did not observe significant changes in cell survival after LPS (0.01–100 μg/mL) exposure for 60 hr. Phase contrast microscopy showed that the cell numbers

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decreased in the DA, MnCl2, and MPP+ treated groups. Exposure to MnCl2 induced body shrinkage and loss of astroglial processes, and DA exposure led to shrunken cell bodies and thin and long processes. MPP+ treatment appeared to cause cell process retraction but barely affect-ed the cell body of U118 astroglia. MTS results showed that LPS treatment did not have a significant effect on morphology (Fig. 1C). Therefore, LPS was not used as an inducer in subsequent experiments.

DA, MPP+, and MnCl2 induced U118 astroglial cell

cycle arrest without increasing apoptosisThe apoptosis-inducing effects of the different induc-

ers in U118 cells was determined by double staining with Annexin V-FITC and PI and flow cytometric anal-ysis. Dot plots were analyzed and the percentage of cells in each quadrant were quantified (Fig. 2A and B). Expo-sure of cells to DA, MnCl2, or MPP+ did not cause sig-nificant early or late apoptosis compared with the control group. However, the cell cycle was affected to varying degrees by DA, MnCl2, and MPP+ treatment (Fig. 2C and D). All cells were arrested in the G1 and S phages after DA treatment, and the percentage of cells in the S phage was increased. Similarly, exposure to MPP+ decreased the number of cells in the G2/M stage, and slightly increased the number of cells in G1. In contrast, the percentage of

Fig. 1. Effect of parkinsonian toxins on the viability and morphology of U118 astroglia. (A) U118 astroglia cultures were stimulat-ed with the four parkinsonian toxins DA (24 hr), MnCl2 (24 hr), LPS (48 hr), and MPP+ (48 hr) at different concentrations, and the cell viability was estimated by MTS assay. (B) Astroglia cultures were incubated with each inducer at the selective concentrations (DA 200 μM, MnCl2 250 μM, LPS 100 μg/mL, and MPP+ 500 μM) for 0-60 hr, and the cell viability was es-timated by MTS assay. Values are expressed as the mean ± SD of three independent experiments. (C) Phase-contrast images showing morphological changes in the astroglia after treatment with the inducers. Scale bar: 50 μm.

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cells in the in G2/M stage was significantly increased, and that in the G1 phase decreased, when astroglia were treated with MnCl2. Thus, the toxic effects of DA, MnCl2, and MPP+ were likely not related to apoptosis, but affect-ed cell cycle progression.

U118 astroglia was activated under the treatments of different parkinsonian toxins

To determine the effects of the different treatments on astroglia, the expressions of astrocyte-derived factors in U118 astroglia, including astrocytic marker proteins, pan-reactive astrocyte markers, neurotrophic factors, and inflammatory cytokines, were assessed by qPCR. As shown in Fig. 3, glial fibrillary acidic protein (GFAP), a

Fig. 2. Parkinsonian toxins induce U118 astroglial cell cycle arrest without increasing apoptosis. (A) Effects of the four inducers on apoptosis of U118 astroglia was determined using annexin V and PI double staining. The cells were gated into three quadrants: living cells (Annexin V-, PI-); early apoptotic cells (Annexin +, PI-), and late apoptotic/necrotic cells (Annexin +, PI+). (B) Proportion of apoptotic cells after different treatments. Data are expressed as the mean ± SD of three independ-ent experiments. (C) Effects of the four inducers on cell cycle progression. (D) Cell cycle distributions before and after the different treatments. Values are expressed as the mean ± SD of three independent experiments. *P < 0.05.

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marker for activated astrocytes, was remarkably upregu-lated after treatment with DA, MnCl2, or MPP+. In addi-tion, pan-reactive astrocyte markers that have been reported in LPS-treated and middle cerebral artery occlu-sion (MCAO) animal models, including sphingosine-1-phosphate receptor 3 (S1PR3), heat shock factor bind-ing protein 1 (HSBP1), TIMP metallopeptidase inhibitor 1 (TIMP1), and vimentin (VIM), were upregulated. Fur-thermore, tumor necrosis factor-α (TNF-α) and glial cell-derived neurotrophic factor (GDNF) expression increased after treatment with DA (Fig. 3A), and MnCl2 stimu-lated the expression of interleukin 1 β (IL-1β) and IL-6 (Fig. 3B). In U118 astroglia treated with MPP+, we observed that glutamate transporter-1 (GLT-1), BDNF, IL-1β, and IL-6 were upregulated (Fig. 3C). These results suggested that astroglia were activated after exposure to DA, MnCl2, and MPP+, and that the parkinsonian toxins affected the expression of various cellular factors.

Scg3 was upregulated and its exosecretion was inhibited after U118 astroglia activation

Results of our previous studies suggested that secre-togranins, which anchor in DCVs, might be an indica-tor of PQ-induced astroglia activation. We therefore stud-ied the expression and secretion of SCG2 and SCG3 in U118 astroglia activated by other parkinsonian toxins. As shown in Fig. 4A, there was a significant increase in SCG3 mRNA when astroglia were activated by DA, MnCl2, or MPP+, while upregulation of SCG2 was only observed after MPP+ exposure. In addition, the protein expression of SCG3 was clearly elevated, and its exose-cretion in the CM inhibited in the activated astroglia com-pared to the control (Fig. 4B and C). Unlike SCG3, SCG2 was barely secreted. The level of total protein for each

line was verified by Coomassie blue staining (Fig. 4D). Thus, DA, MnCl2, and MPP+ influence the expression of SCG3 rather than SCG2 in activated astroglia, and SCG3 was upregulated but its release was inhibited after treat-ment with DA, MnCl2, or MPP+.

DISCUSSION

Parkinsonian toxin-induced astroglia activation has been observed in animal models and postmortem (Hao et al., 2016; Kim et al., 2016; Sai et al., 2013). However, the changes in astrocyte functions, and the underlying molec-ular mechanisms, caused by toxin-induced astroglia acti-vation, have not been completely characterized. Several neuroinflammatory factors were upregulated after astro-glia activation induced by different toxins (Pekny and Pekna, 2014). In Mn2+-activated astroglia, the transcript levels of several proinflammatory chemokines, includ-ing chemokine (C-X-C motif) ligands, interleukins IL-12A and IL-7, were upregulated, and the IFN-γ signaling path-way was activated (Sengupta et al., 2007). MPTP caused translocation of the nuclear factor NF-kB, and increased phosphorylated p38 mitogen activated protein kinase (P-p38 MAPK) levels, significantly increasing IL-1β and TNF-α expression in C6 astroglia (Niranjan et al., 2010). Acute MPTP treatment induced astroglia activation and increased IL-10, IL-12, IL-13, and IFN-γ levels (Yasuda et al., 2008). In the current study, we initially used the parkinsonian toxins MPP+, MnCl2, DA, and LPS to stim-ulate U118 astroglia. Interestingly, exposure to high con-centrations of LPS did not significantly affect viabili-ty in U118 astroglia. TLR4 receptor complex proteins are crucial for astroglia to react to LPS. Previous study has proved that human astrocytes lack CD14, which is

Fig. 3. Real-time PCR analysis of the expression of astrocytic-derived factors. U118 astroglia cultures were stimulated with (A) DA (200 μM, 24 hr), (B) MnCl2 (250 μM, 24 hr) or (C) MPP+ (500 μM, 48 hr). Real-time PCR was performed to iden-tify expression of astrocyte-derived factors, including astrocytic markers (GFAP, GLT-1, S100 calcium binding protein β [S100β], and glutamine synthetase [GS]), pan-reactive astrocyte markers (S1PR3, VIM, HSPB1, and TIMP1) inflammatory genes (TNFα, IL-1β, and IL-6), and neurotrophic genes (BDNF and GDNF). Values are normalized to the control group (control = 1) and expressed as the mean ± SD of three independent experiments. *P < 0.05.

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an important downstream regulator of TLR4 (Tarassishin et al., 2014), which could explain the lack of significant LPS-mediated activation and metabolic changes in U118 cells. We observed a significant increase in GFAP after MPP+, MnCl2, or DA exposure. Some factors that were previously selected as pan-reactive markers in LPS and MCAO-induced reactive astrocytes by microfluidic qPCR were also assessed in the current study (Liddelow et al.,

2017). We found that most of these pan-reactive markers were upregulated after toxin treatment. These results sug-gested that all three parkinsonian toxins were able to acti-vate astroglia to varying degrees. Our results could facil-itate the development of efficient cell models to study toxin-induced astroglia activation in vitro. We therefore examined the expressions of selected representative astro-cytic factors, including astrocytic markers, cytokines, and

Fig. 4. Expression and exosecretion of SCG2 and SCG3 in astroglia induced by parkinsonian toxins. (A) Real-time PCR was per-formed to study the mRNA expression of SCG2 and SCG3 in U118 astroglia after treatment with DA (200 μM, 24 hr), MnCl2 (250 μM, 24 hr), or MPP+ (500 μM, 48 hr). (B) Immunoblotting was performed to determine the post-nuclear protein levels of SCG2 and SCG3 (expression) and SCG2 and SCG3 protein levels in conditioned medium (CM; exosecretion). (C) Relative SCG2 and SCG3 protein expression and SCG3 secretion levels normalized to β-actin. (D) The level of total protein in the CM was monitored using Coomassie blue staining. Values are normalized to the control group (control = 1) and ex-pressed as the mean ± SD of three independent experiments. *P < 0.05.

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neurotrophic factors. As reported previously (Zhan et al., 2018a, 2018b), increases in TNFα, IL-1β, and IL-6 were the most prominent besides GFAP. Activated astroglia are mainly identified by morphological changes and the over-expression of GFAP, which is the most commonly used marker (Ben Haim et al., 2015). Although inflammation plays an important role in activated astroglia (Liddelow et al., 2017), there is currently no known related indicator of astroglia activation. Our results suggest that inflammatory chemokines lack specificity and might not be good mark-ers of toxin-induced astroglia activation.

Cell cycle interference has been reported in astroglia exposed to different toxins. Cholera toxin preferential-ly affected the G0/G1stage of astroglia, while Shiga tox-in bound maximally at G2 to telophase (Majoul et al., 2002). Acrylamide (ACR) treatment increased the expres-sion of DNA damage-associated and checkpoint-relat-ed signaling molecules in astroglia, in a time- and dose-dependent manner, resulting in significant increases in G0/G1-arrested cells (Chen et al., 2010). However, pre-vious studies on parkinsonian toxins in astroglia have focused more on apoptosis than cell cycle changes. In the current study, treatment with MPP+, MnCl2, or DA did not induce significant apoptosis but led to S or G2/M arrest in U118 astroglia. These findings suggested that DNA repair and protein synthesis were affected in astroglia activated by parkinsonian toxins (data not shown). MnCl2 caused a significant accumulation of astroglia in the G2/M stage. These results were different from the findings of Deng et al. (2011) and Sengupta et al. (2007), who reported that Mn2+ exposure led to concentration-dependent apoptosis and G0/G1 phase cell cycle arrest in rat astrocytes, and S phase arrest in primary human astrocytes, respectively. As no significant apoptosis was observed in the current study, we consider that the inconsistency may result from the difference in MnCl2 dosage and the state of astroglia after stimulation. Thus, alternation of the cell cycle like-ly has a role in astroglia activation; however, it could be influenced by toxicity as well as dosage of the inducers. Our finding demonstrated that inflammatory stimulation and cell cycle interference could be two conserved mech-anisms, but might not be good indicators of parkinsonian-induced astroglia activation.

Secretogranins are important components of the regu-lated secretory pathway, which target to DCVs. As with the classic members CgA and chromogranin B (CgB), the roles of SCG2 and SCG3 in peptide hormone sequestra-tion and neurotransmitter sorting in the nervous system have attracted much attention in recent years (Dominguez et al., 2018). In addition, secretogranin imbalanced avail-ability has been identified in several neurodegenerative

disorders (Brinkmalm et al., 2018; Teunissen et al., 2011). In this study, after astroglial exposure to DA, MPP+, or MnCl2, the expression of SCG2 remained unchanged, and basic and stimulated secretion were low. However, the mRNA and protein expression of SCG3 increased while the levels in the medium slightly decreased. SCG3 upreg-ulation has been previously identified in reactive astro-cytes induced by perforating injury and in plaque asso-ciate-reactive astrocytes in Alzheimer’s disease (AD) cortices (Paco et al., 2010; Plá et al., 2013). As observed in PQ-activated astroglia in our previous study (Zhan et al., 2018b), SCG3 levels increased in astroglia exposed to other parkinsonian toxins. These results indicate that SCG3 expression is influenced by different extracellu-lar stimuli, and could be an indicator of astrocyte acti-vation. In addition, the release of SCG3 in U118 astro-glia was inhibited by DA, MPP+, and MnCl2. Impaired SCG3 secretion has been observed in the cerebral cortex of patients with AD and transgenic mice models of AD, in which SCG3 accumulated in dystrophic neurites sur-rounding amyloid plaques. The basal and Ca2+-regulated secretion of endogenously produced SCG3 was dramati-cally impaired by amyloid-β (Aβ1-42) in cultured neurons and astrocytes, suggesting that SCG3-mediated dysregu-lation of peptidergic transmission could play a role in the pathogenesis of neurological disorders (Plá et al., 2017). However, our current results are not in agreement with the results of our previous study, wherein elevated lev-els of SCG3 in CM was observed in PQ-activated astro-glia. This could be because PQ induced different toxico-logical mechanisms than those induced by other toxins. We hypothesized that the mechanism of action of these parkinsonian toxins could include impairment of SCG3 in peptidergic transmission and loss of SCG3-mediated reg-ulatory effects. SCG2 plays an important role in the bio-genesis of DCVs. Depletion of SCG2 expression in PC12 cells led to a decrease in vesicle number and size (Courel et al., 2010). However, in the current study, we did not observe significant changes in SCG2 expression. SCG3 exerts important neuropeptide sorting and processing functions by interacting with CgA and CPE. There are no known sorting and processing functions of SCG2. Further, the accurate targeting of SCG2 to secretory granules may depend on its binding with the TGN membrane-anchored SCG3 (Hotta et al., 2009). Therefore, the increased levels of SCG3 and unaltered SCG2 could suggest an enhanced process of neuropeptide processing rather than biogen-esis, and increased SCG3 might promote the sorting of SCG2 to DCVs in activated astroglia induced by parkin-sonian toxins.

In summary, the parkinsonian toxins DA, MPP+, and

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MnCl2 significantly activated U118 astroglia, and stim-ulated various astrocytic-derived factors, especially cytokines. In toxin-activated astroglia, the expression of the regulated secretory pathway component SCG3 was elevated, but its release was inhibited. Disruption of the regulated secretory pathway could be a toxicological effect of the parkinsonian toxins, and SCG3 may act as a sensitive indicator of the secretory system during astro-cyte activation. However, the cargos processed by SCG3 that are affected by parkinsonian toxins require further study.

ACKNOWLEDGMENTS

XZ wrote the manuscript. XZ, GW and EJ conduct-ed the experiments. XZ and FL analyzed the results and modified the manuscript. HP and XW made substantial contributions to conception and design, and also revised the manuscript critically for important intellectual con-tent. All authors read and approved the final manuscript.

This work was supported by grants from the Nation-al Natural Science Foundation of China (81901434) and China Postal Science Foundation 2018M641743.

The authors thank Elixigen company for comments on the manuscript and for language editing.

Conflict of interest---- The authors declare that there is no conflict of interest.

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