Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study
Publication: Environmental Health Perspectives
Volume 130, Issue 5
CID: 057004
Abstract
Background:
Paraquat (PQ) is a pesticide, exposure to which has been associated with an increased risk of Parkinson’s disease; however, PQ transport mechanisms in the brain are still unclear. Our previous studies indicated that the organic cation transporter 3 (OCT3) expressed on astrocytes could uptake PQ and protect the dopaminergic (DA) neurons from a higher level of extracellular PQ. At present, it is unknown how OCT3 levels are altered during chronic PQ exposure or aging, nor is it clear how the compensatory mechanisms are triggered by OCT3 deficiency. Dynamic related protein 1 (DRP1) was previously reported to ameliorate the loss of neurons during Parkinson’s disease. Nowadays, mounting studies have revealed the functions of astrocyte DRP1, prompting us to hypothesize that DRP1 could regulate the PQ transport capacity of astrocytes.
Objectives:
The present study aimed to further explore PQ transport mechanisms in the nigrostriatal system and identify pathways involved in extracellular PQ clearance.
Methods:
Models of PQ-induced neurodegeneration were established by intraperitoneal (i.p.) injection of PQ in wild-type (WT) and organic cation transporter-3–deficient () mice. DRP1 knockdown was achieved by viral tools in vivo and small interfering RNA (siRNA) in vitro. Extracellular PQ was detected by in vivo microdialysis. In vitro transport assays were used to directly observe the functions of different transporters. PQ-induced neurotoxicity was evaluated by tyrosine hydroxylase immunohistochemistry, in vivo microdialysis for striatal DA and behavior tests. Western blotting analysis or immunofluorescence was used to evaluate the expression levels and locations of proteins in vitro or in vivo.
Results:
Older mice and those chronically exposed to PQ had a lower expression of brain OCT3 and, following exposure to a i.p. loading dose, a higher concentration of extracellular PQ. DRP1 levels were higher in astrocytes and neurons of WT and mice after chronic exposure to PQ; this was supported by finding higher levels of DRP1 after PQ treatment of dopamine transporter-expressing neurons with and without OCT3 inhibition and in primary astrocytes of WT and mice. Selective astrocyte DRP1 knockdown ameliorated the neurotoxicity in mice but not in WT mice. GL261 astrocytes with siRNA-mediated DRP1 knockdown had a higher expression of alanine–serine–cysteine transporter 2 (ASCT2), and transport studies suggest that extracellular PQ was transported into astrocytes by ASCT2 when OCT3 was absent.
Discussion:
The present study mainly focused on the transport mechanisms of PQ between the dopaminergic neurons and astrocytes. Lower OCT3 levels were found in the older or chronically PQ-treated mice. Astrocytes with DRP1 inhibition (by viral tools or mitochondrial division inhibitor-1) had higher levels of ASCT2, which we hypothesize served as an alternative transporter to remove extracellular PQ when OCT3 was deficient. In summary, our data suggest that OCT3, ASCT2 located on astrocytes and the dopamine transporter located on DA terminals may function in a concerted manner to mediate striatal DA terminal damage in PQ-induced neurotoxicity. https://doi.org/10.1289/EHP9505
Introduction
Paraquat [1,1′-dimethyl-4,4′-bipyridinium dichloride (PQ)], as one of the widely used broad-spectrum cationic herbicides,1–3 has been associated with a variety of toxicities, including pulmonary,4 neuro-,5 and nephrotoxicity.6 Experimental and epidemiological studies have shown that PQ is closely related to Parkinson’s disease (PD) risk.7–9 In experimental studies, PQ has been reported to impact mitochondrial functions and to produce reactive oxygen species (ROS),10–13 causing loss of dopamine neurons, poor locomotor performance, and synuclein aggregation.14–17 As reported previously in mouse studies, induces apoptosis of dopaminergic (DA) neurons in the substantia nigra, but because of compensatory striatal sprouting in the remaining neurons, the striatum is spared.18–20 However, when organic cation transporter-3 (OCT3), a bidirectional transporter highly expressed in astrocytes, is deleted, could induce significant loss in both DA neurons and terminals in the nigrostriatal system.19,21
Interestingly, mouse and cell studies have reported that can be reduced to by enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase,19 and microglia in the brain have been suggested to be the critical cell type for PQ toxicity.22,23 In human and rodent studies, has been reported to be the substrate for both dopamine transporter (DAT) and OCT3.24,25 As reported in mouse and cell studies, because OCT3 has a higher affinity for than DAT, extracellular can be readily cleared by OCT3 located on astrocytes, leaving less PQ available for DAT-mediated transport into striatal terminals.19,21 Overall, these studies focused on the transport process of PQ in the rodent nigrostriatal system, where OCT3 serves as a critical regulator. However, little is known about the changes in transport functions of OCT3 during aging or chronic PQ exposure, nor is it clear whether compensatory mechanisms prevail during OCT3 dysfunction.
Dynamin-related protein 1 (DRP1) is acknowledged as an inducer of mitochondrial fragmentation,26 and neural DRP1 inhibition can ameliorate neurodegeneration in rodents.26–28 However, to the best of our knowledge, little attention has been paid to astrocyte DRP1. Recently, DRP1 inhibition has been found to impact astrocyte function in mouse and cell studies,29,30 which prompted us to hypothesize on the potential effect of astrocyte DRP1 on PQ-induced neurotoxicity, especially the clearance process of extracellular PQ mediated by astrocytes.
In the present study, neurodegeneration was modeled in C57BL/6 mice by intraperitoneal (i.p.) injection of , commonly used in other PD-related studies.18,31–34 To further understand the mechanisms of PQ-induced neurotoxicity, the loss of nigral DA neurons and striatal terminals, the ability of synapses to release DA, and animal behaviors in both and wild-type (WT) mice were evaluated by immunohistochemistry (IHC), in vivo microdialysis, and behavior tests, respectively. In vitro transporter assays were conducted to directly evaluate the transporting capacity of different PQ transporters, including OCT3 and ASCT2, another monovalent cation transporter expressed in astrocytes that serve as an alternative transporter to remove extracellular PQ when OCT3 is deficient. These results provide a better understanding of the transporting processes of PQ in the rodent brain.
Materials and Methods
Animals
Eight-week-old male C57BL/6 (WT) mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After arrival, the WT mice were raised for 2 wk without any treatment to adapt to the environment and avoid stress. mice were originally generated by Zwart et al.35 and kindly gifted from K. Tieu (Florida International University). The animals were housed at , humidity with a 12‐h light/dark cycle (lights on between 0700 and 1900 hours). Water and standard chow were provided ad libitum. mice were bred in an animal platform, at the Institute of Neurology, Huashan Hospital, Fudan University. Eight- to twelve-wk-old homozygous breeders were mated, and once the vaginal embolus was found, the pregnant female mice were raised separately from other mice, until they gave birth to the newborns and fed for 4 wk. Mouse tail tissue was taken for genotyping by quantitative polymerase chain reaction (qPCR). After genotyping, male mice were used for experiments. The study was performed with the approval of the Institutional Animal Care and Use Committee of Fudan University. The experiments were performed according to the National Institute of Health Guide for the Care and Use of Laboratory Animals.36
Animal Genotyping
Mouse tail tissue was cut and ground in Trizol (Takara; Cat. No. D9108A), for each sample, and the process of RNA isolation was conducted on ice. One-half milliliter of chloroform was added to each tube and the tube was violently shaken for 30 s, followed by allowing the sample to stand for 10 min, until the liquid was layered. The sample was centrifuged at at 4°C. The supernatant was collected into another tube and gently mixed with of isopropyl alcohol, followed by gentle shaking and then allowed to stand for 15 min. The tube was centrifuged again at at 4°C. The supernatant was removed and of 75% ethanol was added into the tube. After gentle shaking, the tube was centrifuged at for 10 min at 4°C. The pellet was collected and dissolved in of diethyl pyrocarbonate (DEPC) water. The sample was prepared for reverse transcription, and the Takara Reverse Transcription Kit (Cat. No. RR036A) was used. To each RNA sample, of RT Master Mix and an appropriate volume of DEPC water was mixed to yield . The thermal cycle parameters were as follows: . After reverse transcription, the samples were stored at 4°C and prepared for qPCR. The Takara qPCR kit (Cat. No. RR420A) was used. The system included of complementary DNA (cDNA), of TB Green, each of forward and backward primer, of DEPC water, and of ROX Reference Dye. The sequences of the oct3 primers were as follows: forward 5′-GCCCGGAGCTCTCTTAATCC-3′; reverse 5′-CTCAGCCACGGTATCCCTTC-3′. The samples were added into 96-well plates. The Thermo Fisher Quant Studio5 Real-Time PCR system was used, and the thermal cycle parameters were as follows: stage 1: 95°C 30 s, 1 cycle; stage 2: 95°C 30 s, 40 cycles; stage 3: 95°C 60 s, 1 cycle; stage 4: , continuous.
Preparations and Animal Treatments
(Chemical Abstracts Services No. 75365-73-0; Sigma-Aldrich; Cat. No. 36541) was diluted in phosphate-buffered saline (PBS) to a concentration, and both WT and mice received by i.p. injection. (The extracellular concentration was measured in this study and it could not be achieved by any other method, including inhalation or gavage.) The dose () was used in this study based on the National Health Commission of China standard, which indicated that the permissible concentration–time-weighted average (PC-TWA) allowed for PQ is . Considering that the daily respiratory volume of a person is estimated as , and the body weight of a person is estimated to be , of PQ was allowed for a human. The dose for a mouse might be according to the uncertainty coefficient (100). In the present study, a PQ dose was used.30
As shown in Figure S1, the mice were treated as follows: a) For the chronic treatment (), the mice received injection (i.p., ) every 2 d, with a total of 10 injections in 20 d. b) For the PBS treatment (PBS), the mice received the same volume of PBS (i.p.) every 2 d, with a total of 10 injections in 20 d. Then, the mice were allowed a 1-wk wash-out period. After the wash-out period, the mice were used for behavioral tests, followed by IHC ( mice per group) or in vivo microdialysis ( mice per group) to evaluate DA release (Figure S1A,B).
To evaluate the clearance capacity of the loading in the brains of the mice, a second cohort of WT and mice with or without adeno-associated virus (AAV) injection ( mice per group) underwent PBS or . After a 1-wk wash-out period, all groups received a loading dose injection of [i.p., ; loading treatment ()]. Twenty-four hours after , extracellular was detected by in vivo microdialysis (Figure S1C).
To assess the cerebral expression level of OCT3 in mice of different ages, WT mice were raised to 10, 24, or 48 wk of age before or PBS treatment ( mice per group). After the 1-wk wash-out period, all the mice underwent . Then 24 h later, in vivo microdialysis was used to evaluate the extracellular concentration in the brain. Subsequently, the mice were euthanized by isoflurane (RWD; Cat. No. R-510-22-16), and the brain tissues were harvested for OCT3 detection by Western blotting (Figure S1D).
Cell Culture
The HEK293 (Cat. No. h242), GL261 (Cat. No. m063), and Neuro-2a (; Cat. No. m040) cell lines were purchased from iCell Co., Ltd. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sangon Biotech; Cat. No. H531FA0004) mixed with 10% vol/vol fetal bovine serum (FBS; Gibco; Cat. No. 10100147) and 1% vol/vol penicillin–streptomycin (P/S; Gibco; Cat. No. 2289321) solution. The incubator environment was maintained at 37°C and 5% carbon dioxide (). The cells were subcultured until a confluence of 80% was attained. The cells were then maintained in dishes (Corning; Cat. No. 430167). The cells were plated into 6- (Corning; Cat. No. 3516) or 24-well (Corning; Cat. No. 3524) dishes for plasmid transferring, PQ transporting experiments, and Western blotting.
Construction of AAV Tools and Stereotactic Injection
Interfering microRNA (miRNA) to block DRP1 (sequence: 5′-CGTTGTCAACCTGACACTTGT-3′) was constructed and purchased from Shanghai Taitool Bioscience Co., Ltd.; Cat. No. DNM1L-RNAi 33461-1. Scramble miRNA (sequence: 5′-AAATGTACTGCGCGTGGAGAC-3′, Shanghai Taitool Bioscience Co., Ltd.; Cat. No. NO.1) was used as the negative control. Both miRNAs were stored as freeze-dried powder at and dissolved in DEPC water to yield a solution before use.
To test the knockdown efficiency, miRNA was co-transferred into HEK293 cells with Drp1-egfp (Shanghai Bioegene Co., Ltd.; Cat. No. BIOE-PL-OE19). In detail, of miRNA and of Drp1-egfp were mixed and dissolved into of DMEM. Three microliters of lipofectamine 2000 (Thermo; Cat. No. 11668019) was added into another of DMEM. After 5 min of equilibration, the above two solutions were mixed and allowed to stand for 20 min, after which the mixture was added onto HEK293 cells. The cells were cultured in 6-well plates, reaching a confluence of 50% before transferring. After 48 h of incubation, the cells were gently scraped off and resuspended into culture medium ( per well). One-half milliliter of cell suspension was added onto a cell-slide placed into a well of a 24-well plate. After 24 h, cells had become attached to the slide. Four percent paraformaldehyde (PFA) was then gently added onto the slide, and it was incubated for 20 min. Then the cells on the slide were observed under a fluorescence microscope (Olympus DP72) for detections of enhanced green fluorescent protein (eGFP) fluorescence intensity, which was analyzed using ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD). The other of the cell suspension was centrifuged at for 5 min at 4°C to harvest the pellet. One hundred microliters of radio-immunoprecipitation assay (RIPA) buffer was added onto the pellet. After quantifying the protein (as described below), the DRP1 level was detected by Western blotting (both experiments were repeated three times with three replicate wells each time).
Then, the miRNA was cloned onto an astrocyte-specific RNA interference (RNAi) vector [pAAV2-gfaABC1D-mCherry-miRNA-WPRE-pA; Cat. No. AI004; Shanghai Taitool Bioscience Co., Ltd. gfaABC1D: a truncated 681-bp glial fibrillary acidic protein (GFAP) promoter] and a neuron-specific RNAi vector [pAAV2-hSyn-mCherry-miRNA-WPRE-pA; Cat. No. AI014; Shanghai Taitool Bioscience Co., Ltd. (human synapsin1; hSyn)].
The vectors were cut with restriction endonucleases in a system containing of double-distilled water, of Buffer (NED; Cat. No. B7204), of purified vector DNA (), of AgeI (; NED; Cat. No. R0552), and of EcoRI (; NED; Cat. No. R0101). The mixture was incubated at 37°C for 3 h. Linearized vectors were harvested by agarose gel electrophoresis (Biowest Agarose; Cat. No. Q-0024796), using 120 V for 30 min. The double-enzyme-cut linearized vectors were ligated with miRNA by T4 DNA ligase (Thermo; Cat. No. EL0016) at 16°C overnight. The system contained of linearized vector (), of miRNA (), of T4 DNA ligase buffer (Thermo; Cat. No. B69), of T4 DNA ligase, and of DEPC water. Ten microliters of the reaction product was added to of -competent cells (TIANGEN; Cat. No. CB101), mixed by flicking the tube wall several times, and placed on ice for 30 min. The cells were heat shocked at 42°C for 90 s and then incubated in an ice-water bath for 2 min. Subsequently, of lysogeny broth (LB) medium was added, and the tube was placed on a shaker at 37°C for 1 h. The bacterial liquid was spread evenly on a plate containing puromycin (; Beyotime; Cat. No. ST551) and then inverted in a 37°C incubator for 16 h. The bacterial solution was transferred to of LB liquid medium containing puromycin and cultivated overnight at 37°C for plasmid extraction according to the manual of the Endotoxin-Free Plasmid Mini-Lifting Kit (TIANGEN; Cat. No. DP118-2). Briefly, bacterium-containing miRNA plasmids were harvested and ruptured by the lysate buffer and proteinase K in the kit. After adding the neutralization buffer, the protein precipitated and could be separated by centrifugation at (Thermo; Sorvall ST1 plus). The supernatant was collected into the adsorption column, followed by centrifugation at . The liquid under the column was discarded, and the column was washed by the eluent. After the sample dried, the plasmid sample was dissolved in of DEPC water.
Next, the plasmids containing miRNA were triple-transfected along with pHelper1 and pHelper2 (both helper plasmids for viral packaging purchased from Shanghai Taitool Bioscience Co., Ltd.; Cat. No. Helper 1.0 and Helper 2.0) into HEK-293 cells to package AAV2/5-gfaABC1D-mCherry-miRNA-WPRE-pA and AAV2/5-hsyn-mCherry-miRNA-WPRE-pA. The scramble AAV contained a negative control sequence of miRNA. In detail, of miRNA plasmids, of pHelper1, and of pHelper2 were mixed and co-transferred into HEK293 cells (to a confluence of 70% in dishes) using of lipofectamine 2000. The cells were incubated at 37°C for 72 h, after which the culture medium was collected and filtered using a filter. Next, the medium was centrifuged at at 4°C (Beckman; XE-90). The supernatant was discarded and the pellet containing the viral particles was collected and dissolved in 5% glycerin and stored at . The AAV vectors were quantified by qPCR. Primers were designed according to the specific sequence of woodchuck hepatitis virus posttranscriptional regulatory element (WPRE): forward 5′-CCTTTCCGGGACTTTCGCTT-3′; reverse 5′-GCAGAATCCAGGTGGCAACA-3′. Thermo QuantStudio 5 Real-Time PCR system models were used. The program was as follows: Segment 1: () Step 1: 95°C for 15s; Segment 2: () Step 1: 95°C for 5 s, Step 2: 60°C for 30 s data collection and real-time analysis enabled; Segment 3: () Step 1: 95°C for 60 s, Step 2: 60°C for 60 s, Step 3: for 30 s, . The copy number of the WPRE gene was calculated using the absolute quantitative method. Design and Analysis Software (version 2.4.3; QuantStudio 6/7 Pro) was used for quantification. The titer of viral particles was expressed as copy number of virus genome (V.G) per milliliter.
C57BL/6 male WT or mice ( old, mice per group) received bilateral supra-nigral and striatal stereotactic injections of AAV capsids ( V.G/mL, ). Avertin was used for anesthesia ( i.p.). The injection was performed with a Hamilton syringe and a 33-ga needle (Hamilton) at a rate of (substantia nigra, relative to bregma: caudal, lateral, and ventral; striatum, relative to bregma: caudal, lateral, and ventral). After the injection, the needle was maintained at the injection site for 5 min before removal. Triple antibiotic and lidocaine topical ointments were applied, and the mice were kept in a 37°C incubator for 30 min to recover. It took 4 wk for the AAVs to take effect and to prepare for the next treatments on mice (PBS or paraquat). The process of AAV injection is summarized in Figure S1B.
Mitochondrial Division Inhibitor-1 Preparations and Animal Treatments
Mitochondrial division inhibitor-1 [mdivi-1; 3-(2,4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone; MCE; Cat. No. HY-15886] was purchased from the MCE company. Mdivi-1 is an inhibitor of DRP1-GTPase that can pass through the blood–brain barrier and inhibit DRP1 nonselectively based on in vitro and mouse studies.37,38 Mdivi-1 was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Cat. No. D8418) of a stock solution. For the i.p. injection, mdivi-1 was diluted in sterile saline (1:100, vol/vol) and gently sonicated (model S3000; Sonicator; with tapered microtip; Misonix, Inc.) at a power level of 0.5–1 for 30 s before injection. The mice treated with mdivi-1 were used as a control in the present study (inhibiting DRP1-GTPase in both neuron and astrocyte). Another cohort of mice, rather than those treated by AAVs, received mdivi-1 i.p. injections (, twice a day, mice per group), starting on the day of the first injection and continuing until 7 d after the -CT (Figure S1B,C).
Analysis of Locomotor Activity
Baseline behavior tests (1) started at 4 wk after AAVs injection. Another series of behavior tests (2) started after the 1-wk wash-out period following or PBS. Difference values (“2” minus “1”) were calculated for statistical analysis; mice for each group. The time points of the behavior tests are summarized in Figure S1B. Between the trials, apparatuses were cleaned with 70% (vol/vol) ethanol and dried with a paper tower to avoid interference due to odor.
Rotarod
The TSE Rotarod System was used to perform rotarod tests. The apparatus was accelerated from 0 to 40 rpm over 5 min and maintained at a constant speed for an additional 1 min. The latency to fall (the time duration from the start of tests to when the mouse fell from the apparatus) was then recorded as an index of locomotor activity. Mice were trained for three consecutive days before the tests by putting the mice on the rotarod at a speed of for 3 min to teach them moving on the rotarod.
Gait Analysis
The Noldus catwalk system (catwalk XT) was used for the gait tests. The camera’s visual field was defined as , including at least five paw prints from each mouse. Mice were put at one side of the track, and an eligible run required a mouse going through the visual field with a uniform speed without any disturbance. A mouse had to achieve three eligible runs before the next mouse started its test. Before the formal tests, mice underwent a process of training for 3 d, during which the mice were put on one side of the track and let go through the track without disturbance, three times per day. Data were expressed as different values of stride length, duration, stand, and swing speed before and after drug treatment. Stride length was defined as the length of step taken by the limb in one continuous step; duration, the time required for the mice to travel from one end of the track to the other; stand, the time duration when the mouse’s limb touches the ground; and swing speed, the movement speed of the limb when it was raised in the air.
Constructions of Cells with Stable Expression of DAT
cells with stable expression of DAT () were built by transfection of lentivirus. The process of constructing lentivirus tools was similar to the process of constructing AAV tools. Briefly, full-length cDNA of Dat (BioeGene Co. Ltd.; Cat. No. BIOE-PL-4) was cloned into linearized vector (Genomeditech Co. Ltd.; Cat. No. PGMLV-4931), named pLVX-DAT. The vectors were cut with restriction endonucleases, including AgeI (; NED; Cat. No. R0552) and EcoRI (; NED; Cat. No. R0101) to be linearized, which were then ligated with Dat-cDNA by T4 DNA ligase. The plasmids were amplificated in bacterial liquid and extracted using the Endotoxin-Free Plasmid Mini-Lifting Kit.
HEK293T cells were then co-transfected with the pLVX-DAT vector together with packaging plasmids (psPAX2 and pMD2G). The cells were cultured in 6-well plates to a confluence of 50% before transfection. The process was similar to the process of constructing AAV tools. Briefly, lipofectamine 2000 was used in the co-transfection. Seventy-two hours after transfection, viral particles were isolated from the culture medium by centrifugation. Pellets were dissolved in 5% glycerin and stored at . The lentiviral particles were quantified by qPCR, during which two pairs of primers were used. Primer 1: forward 5′-CCTTTCCGGGACTTTCGCTT-3′; reverse 5′-GCAGAATCCAGGTGGCAACA-3′, to amplify viral gene (gene A); Primer 2: forward 5′-CCTGGTACATGCCACTGATG-3′; reverse 5′-AGTGTAGAGGGCAAGCCAGA-3′, to amplify HEK293 cellular gene (gene B). The titer of lentivirus was expressed as transduction units (TUs) per milliliter. TU was calculated by , where N is the number of cells in corresponding wells of a 24-well plate at the time of infection; C is the number of viral particles in each cell, which was calculated by ; and V, is the volume of the viral particles used in the corresponding wells.
cells were seeded into 6-well plates ( cells per well). The next day, the cells were incubated with the lentivirus containing the Dat plasmid carrying puromycin resistance gene []. After incubation for 72 h, the medium containing the lentivirus was replaced by a culture medium containing puromycin so that cells with a stable expression of DAT survived and were selected for further experiment.
Isolation and Culture of Primary Astrocytes
Primary astrocyte cultures were harvested from Day 1 postnatal C57BL/6 mice (purchased from JSJ Laboratory Animal Technology Co., Ltd.), as previously described.39 Briefly, brain tissues from postnatal mice were isolated intact and meninges were peeled off. The brain tissues were transferred into centrifuge tubes with of DMEM and homogenized by soft pipetting with Pasteur straws and tips. The supernatant containing the cell suspension was collected and replaced by new DMEM until the visible pellet disappeared. The collected cell suspension was filtered using a strainer and centrifuged at for 10 min at 4°C. The pellet was collected, resuspended, and plated in T-75 flasks. The cells were cultured in DMEM, 10% vol/vol FBS, and 1% P/S. Once the cells reached confluence, microglia was cleaned away using a CD11b positive selection kit (Stemcell; Cat. No. 18770) according to the manufacturer’s instructions. The incubator environment was maintained at 37°C and 5% .
Treatments and Endogenous DRP1 Examination
For primary astrocyte and DAT-expressing cell treatment, was diluted in culture medium to a working concentration of 200 or . Before being added to cells, sodium dithionite (SDT; Sinopharm; Cat. No. 80116718) was mixed with to a final working concentration of to reduce to . Especially to inhibit OCT3 function in DAT-expressing cells, Decynium 22 (D22; R&D; Cat. No. 977-96-8) was mixed with and SDT to a final concentration of , to block the transportation activity of OCT3. After incubation for 20 min, the cells were washed with PBS three times and changed to a normal culture medium for 48 h. Cells were lysed using RIPA buffer (Beyotime Cat. No. P0013B). One hundred microliters of buffer was added onto cells. The mixture of buffer and cells was allowed to react on ice for 30 min. Then the lysate was scraped off and collected for Western blotting to detect changes in DRP1 levels.
DRP1 Knockdown by Small Interfering RNA
GL261 cells were plated into 24-well plates with a confluence of 50%. Drp1 small interfering RNA (siRNA) (5′-GGCAAUUGAGCUAGCGUAUAU-3′, Shanghai Bioegene Co., Ltd.; Cat. No. BIOE-SI-KD-1) and Lipofectamine 2000 (Invitrogen; Cat. No. 2209775) were diluted with DMEM and mixed in equal volumes according to the manufacturer’s instruction. After incubation for 20 min at room temperature, the mixture was added into 24-well plates (25 pmol siRNA, Lipofectamine 2000 in of DMEM per well). After 6 h, the medium was replaced with fresh medium, and the cells were cultured for 48 h for further treatment.
Uptake Assay
The functions of different transporters were tested in GL261 cells, both in normal cells and Drp1-siRNA transfected cells. The cells were treated with in diverse concentrations varying from 200, 400, 600, to , with or without SDT. The experiments included triplicated trials each time, and average values were calculated for statistics. Six independent experiments were conducted in total. A total of cells per well were plated into a 24-well plate. To block OCT3, D22 was added with and SDT to GL261 cells in a final concentration of . To block ASCT2, benzylserine (Benser; Sigma; Cat. No. B7283) was added together with and SDT or in a final concentration of . After incubation with , SDT, and different inhibitors for 20 min, the cells were washed three times with PBS and lysed. Then the intracellular concentration was detected by high-performance liquid chromatography (HPLC).
HPLC
A four-channel CoulArray (ESA, Inc.) equipped with a highly sensitive amperometric microbore cell (model 5041; ESA, Inc.) was used to analyze the content of DA and in the dialysates after in vivo microdialysis. The cell potential was set at . In brief, samples were manually injected into a sample injector (with a sample loop) and eluted on a narrow-bore (ID: ) reverse-phase C18 column (MD-150; ESA, Inc.) using MD-TM (ESA, Inc.) mobile phase. DA and were detected using an ultraviolet detector (model 526; ESA, Inc.). Samples were manually injected and separated by a narrow-bore column (ID: ; Altima HP C18; Alltech Associates, Inc.) using mobile phases consisting of 80.5% monopotassium phosphate (Sigma; PRH1330) and 9.5% acetonitrile (Sigma; Cat. No. 34851), pH 3.2. The flow rate was set at for all measurements using a solvent delivery pump (model 585; ESA, Inc.).
Mdivi-1 Treatment in GL261 Cells
For cell treatment, the mdivi-1 stock solution was diluted in a culture medium to . To inhibit DRP1 levels in GL261 cells, the culture medium containing of mdivi-1 was added and incubated for 24 h before the treatment.
Isolation and Culture of Primary Neurons
Primary neurons were harvested as previously documented.40 Three-centimeter culture plates were coated with poly l-lysine (Sigma; Cat. No. P2636) for 24 h in 37°C incubators. The embryos were removed from pregnant female C57BL/6 mice (16.5 d; purchased from JSJ Laboratory Animal Technology Co., Ltd.) and placed into ice-cold Hank’s balanced salt solution (HBSS; R&D; Cat. No. B31250). The brain tissues were taken out and meninges were peeled off, followed by digesting in of 0.25% trypsin (Gibco; Cat. No. 25200056) at 37°C for 15 min, gently shaking at . Then of DMEM was added to stop the digestion. The digested tissues were collected in new tubes and disassociated by soft pipetting with Pasteur straws until the visible pellet disappeared. The supernatant containing cell suspension was collected and filtered using a strainer. After centrifugation at for 10 min at 4°C, The pellet was collected and resuspended in neurobasal medium (Gibco; Cat. No. 21103049) containing b27 supplement (Gibco; Cat. No. 17504044, added at a 1:50 ratio). Primary neurons were plated into coated dishes at per dish, culturing at 37°C in 5% .
Western Blotting Analysis
The protein concentrations of samples were evaluated according to the manual of the Pierce bicinchoninic acid assay for protein quantity kit (Thermo; Cat. No. 23225). Briefly, the protein standards were diluted into concentrations of 25, 50, 100, 250, 500, 1,000, 1,500 and . Reagents A and B in the kit were mixed at 50:1. Two hundred microliters of the AB mixture and of the sample or protein standards were mixed and added into a well of 96-well culture plates, followed by incubation at 37°C for 30 min. The absorbance value was detected at using an H1 Hybrid reader (Bio-Rad). Protein () was added to lanes in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690). After electrophoresis, the protein was transferred onto polyvinylidene difluoride (PVDF) membranes with a constant current of for 90 min. The membranes were blocked with 5% wt/vol bovine serum albumin (BSA) for 1 h and incubated in primary antibodies (1:1,000 vol/vol) of DRP1 (Cell Signaling Technology; Cat. No. 8570), ASCT2 (Cell Signaling Technology; Cat. No. 8057), SLC1A4 (Cell Signaling Technology; Cat. No. 8442), 4F2hc (Cell Signaling Technology; Cat. No. 47213), (Cell Signaling Pathway; Cat. No. 13E5), and OCT3 (Affinity; Cat. No. AF5358) separately at 4°C overnight. The next day, after extensive washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Yeasen; Cat. No. 33101ES60) (1:5,000, vol/vol) for 1 h. The blots were visualized using HRP substrate peroxide solution (Millipore; Cat. No. WBKLS0500). The relative optical density (ROD) of the protein of interest and were analyzed using ImageJ software.
In Vivo Microdialysis for Detections of DA Release and Cerebral Extracellular
In vivo microdialysis was processed as previously reported.21 Briefly, the mice were anesthetized by 2.5% (wt/vol) avertin at . Stereotactic implantation of the guide cannula (CMA; Harvard Bioscience, Inc.) into the striatum was performed using the following striatal coordinates relative to bregma: , lateral , and dorsal–ventral (from the surface of the brain). The next day, a microdialysis probe (CMA8 with membrane length; CMA; Harvard Bioscience, Inc.) was inserted into the guide cannula and connected to a low-torque dual channel swivel (Instech Laboratories, Inc.). The channel was connected by the PEEK tube (Cat. No. 8409501) to a syringe pump perfusing artificial cerebrospinal fluid (aCSF). aCSF was purchased from Phygene company (Cat. No. PH1851). After a 2-h equilibration period, dialysates were collected for all DA release studies and detections for extracellular in the brains of the mice. To observe DA release, dialysates were collected every 15 min. One baseline fraction was collected, after which the perfusate was switched to aCSF containing potassium chloride (KCl; with an equimolar reduction in sodium chloride to maintain osmolality) for 15 min to deliver a total of KCl, followed by a return to normal aCSF for 45 min. DA was measured in each sample simultaneously. To measure extracellular , the probe was continuously perfused with normal aCSF for 30 min and dialysates were collected. Probe efficiency was 8%, and flow rate, . DA and in the dialysates were measured by HPLC, as previously described.
Immunohistochemistry
The mice were anesthetized using 2.5% (wt/vol) avertin at (Sigma-Aldrich; Cat. No. T48402) and perfused with an ice-cold saline solution followed by 4% wt/vol PFA (Sangon Biotech; Cat. No. A500684). Brain tissues were post-fixed with 4% PFA for 12 h and then gradually dehydrated in 10%, 20%, and 30% wt/vol sucrose (dissolved in PBS, pH 7.2) for 72 h in total. After being embedded in optimal cutting temperature (O.C.T.) gel (SAKURA; Cat. No. 4583) and quick-frozen at , the brains were frontally sectioned using a freezing microtome, and sections were sequentially collected in four- (substantia nigra) or eight- (striatum) section series. Endogenous peroxidase activity was inhibited by bathing twice for 15 min in 1% (0.01 M, pH 7.4). After washing, sections were incubated for 2 h in blocking buffer [0.1% TX-100/10% normal goat serum PBS, pH 7.4]. The tyrosine hydroxylase (TH) staining process was performed according to the manufacturer of the Vectastain Elite ABC HRP Kit (Vector Laboratories; Cat. No. PK6100). Briefly, brain sections were incubated at 4°C overnight with anti-TH primary antibody (1:500 in PBS containing 2% NGS; Abcam Cat. No. ab112), followed by 2 h in biotinylated secondary antibodies (Vector BA-1000, diluted at 1:200 in PBS containing 2% NGS, vol/vol). Then 3,3′-diaminobenzidine (DAB) substrate (Peroxidase; Vector Laboratories) was used for color development. The sections were then placed on glass slides and subjected to Nissl staining, as described below. An Olympus camera (DP72; Olympus) was used to take images. The quantification of TH-positive cells was achieved by stereoscopic counting, as described below. As to detections of optical density, six to eight sections per mice were analyzed, with mice per group.
Nissl Staining
The brain sections on object-glass slides were incubated with Nissl staining solution (Solarbio; Cat. No. G1430) at 56°C for 1 h, then decolorized for 5 min at room temperature. Next, the sections were sequentially dehydrated by 75% and 100% ethanol and immersed into dimethylbenzene for 5 min; this procedure was repeated in triplicate. Finally, the microslides were covered with neutral gum (Solarbio; Cat. No. G8590) and cover slips. Nissl bodies in the soma of neurons were stained blue and counted stereoscopically.
Quantifications of TH-Positive and Nissl Neurons
TH-positive cells and Nissl bodies in the substantia nigra pars compacta (SNpc) were analyzed by stereoscopic counting using Stereo Investigator software (version 7; MicroBrightField) and a photomicroscope (Olympus BX53), as previously reported.41 Briefly, an area of SNpc in the substantia nigra was selected as the counting range, in which frame size was under magnification (set to be under magnification). TH-positive cells in each frame were counted manually, and the total cell numbers were calculated in the ipsilateral SNpc. Striatal TH fiber optical density was scanned in a high-resolution scanner and measured using ImageJ software.
Immunofluorescence Staining
The cerebral sections were incubated in 5% BSA (MP Biomedicals Cat. No. 02FC007791) for 2h. After three washes, the sections were incubated with rabbit anti-ASCT2 (1:100; Cell Signaling Technology; Cat. No. 8057), TH (1:1,000), DRP1 (1:500; Cell Signaling Technology; Cat. No. 8570), and chicken anti-GFAP (1:1,000; Abcam; Cat. No. ab4674) vol/vol primary antibodies at 4°C overnight. After three washes, the sections were incubated with goat anti-rabbit Alexflour488 (Yeasen; Cat. No. 33106ES60) and goat anti-chicken Alexflour647 (Abcam; Cat. No. ab150157) secondary antibodies at room temperature for 2 h (1:1,000 vol/vol). The sections were observed under a confocal microscope (Olympus FV-10). Six to eight sections were analyzed per mouse. Ex/Em spectra were as follows: Alexflour405, ; Alexflour488, ; Alexflour594, and Alexflour647, .
Statistical Analysis
Data were analyzed and graphed with GraphPad Prism 8 software (version 8.0.2). All the data from the mice are represented as , and data from cells are represented as . Different treatment groups were evaluated using an unpaired test, a one-way analysis of variance (ANOVA), or a two-way ANOVA. Post hoc tests were done for multiple pairwise comparisons after one- and two-way ANOVAs to determine differences among individual groups. Post hoc tests were corrected by the Bonferroni method. The null hypothesis was rejected when the -value was . */#, ; **/##, ; ***/###, .
Results
The Cerebral Expression Level of OCT3 in Mice of Different Ages with or without
It remained unknown whether the levels of OCT3 could change with the progression of aging or treatment. Thus, OCT3 levels in the brains of different-aged mice with or without chronic treatment () were analyzed. Brain OCT3 levels in the older mice were significantly lower than in younger ones. Furthermore, after stratifying mice based on their ages, the brain OCT3 levels were lower in the mice that received than in those that received PBS (Figure 1A,B). To assess the capacity of the brain to remove the extracellular , another loading dose of (; LT) was injected after a 1-wk washout period following the or PBS treatment. In vivo microdialysis was performed to detect the extracellular residues. The results suggested that consistent with the lower brain OCT3 expression levels, extracellular residues of the loading dose was higher in the older mice and in mice that received than in the younger ones and in PBS-treated mice (Figure 1C).
The Location and Expression of DRP1 after in WT or Mice
To date, the relationship between and DRP1, especially astrocyte DRP1, remains undetermined. We detected the DRP1 levels in the substantia nigra of WT and mice with or without . Our results showed DRP1 levels were higher in mice that received than in those treated with PBS, both in neurons and astrocytes (Figure 2A–C). Besides animal experiments, we also detected DRP1 changes in cultured neurons and primary astrocytes. To facilitate the entry of into cells, we treated the cells with and SDT. This reducing agent was used to donate an electron and hence convert to . In cells without Decynium 22 (D22; a blocker of OCT3) treatment, (reduced from by SDT) treatment induced a higher level of DRP1 (Figure 2D,F). In primary astrocytes from WT mice, treatment induced a higher level of DRP1 in a dose-dependent manner (Figure 2E,H). Although OCT3 has a limited expression on neurons, D22 was used to inhibit the potential transporting activity of OCT3. Compared with the cells treated with PBS, DRP1 levels were not influenced by D22 (Figure 2D,G). In primary astrocytes from mice, still induced a dose-dependent DRP1 up-regulation (Figure 2E,I).
The Effects of Selective Neural and Astrocyte DRP1 Knockdown on Neurotoxicity in WT and Mice
Higher DRP1 protein level in both neurons and astrocytes after treatment suggested that DRP1 could be involved in the toxic effects of . The present study tested the impact of DRP1 inhibition in a PD model with or without Oct3 deficiency. Mdivi-1, an inhibitor of DRP1-GTPase that can pass through the blood–brain barrier and inhibit DRP1 in both neurons and astrocytes42, was used as a control. Selective DRP1 knockdown was achieved by stereotactic injections of AAV, which had no effect on the death of TH-positive cells compared with PBS treatment (Figure S2A,B) but had considerable efficiency of DRP1 knockdown (Figure S3A,B) and selectivity of neurons or astrocytes with neural (hsyn) or astrocyte (gfaABC1D) promotor (Figure S3C). There were 10 mice/group and all of them went through the whole process of experiments. Their body weight was measured before the behavior tests (1 and 2) and during the process of (Figure S1B). No statistical difference was observed in the comparations among mice in different groups or at different time points (Figure S4A,B). Results revealed that the number of TH neurons, density of striatal TH terminals, and dopamine release were not influenced by selective DRP1 knockdown in either WT mice or mice without (Figures 3A–H and 4A–H). After , the number of TH-positive neurons was lower (Figure 3A,C), but the striatal TH terminals or dopamine release was spared in WT mice (Figure 3D,F–H). The lower number of nigral TH-positive cells was ameliorated in the mice that received neural DRP1 knockdown and mdivi-1 rather than DRP1 knockdown in astrocytes (Figure 3A–C,E). had no influence on the behavior performance of WT mice, neither did DRP1 inhibition in neurons, astrocytes, or both (Figure 3I,J; Figure S4C,E). In the mice, comparatively fewer DA neurons were observed after treatment with , with significantly less immunoreactivity of striatal TH. Similar to the WT mice, the neurotoxicity was relieved by selective neural DRP1 knockdown and mdivi-1 treatment; however, selective astrocyte DRP1 knockdown also attenuated the -induced pathological changes in the nigrostriatal system that had no significant difference in neural DRP1 knockdown (Figure 4A–E). To evaluate the DA synaptic function, we used in vivo microdialysis to assess depolarization-induced DA overflow in the striatum of freely moving mice. After PQ treatment, mice exhibited significantly less DA overflow; a restoration of evoked DA overflow was achieved by selective astrocyte DRP1 knockdown and was more effective than neural DRP1 knockdown (Figure 4F–H). The locomotor performance in mice were also improved by astrocyte DRP1 knockdown. (Figure 4I,J; Figure S4D,F).
The Capacity of the Nigrostriatal System to Clear Extracellular after Astrocyte DRP1 Inhibition
As reported above, selective astrocyte DRP1 knockdown ameliorated the neurodegeneration in the mice with Oct3 deficiency, and the mechanisms remained undemonstrated. We hypothesized that astrocyte DRP1 knockdown might enhance the clearance in astrocytes and that this would not occur in WT mice. To elucidate this hypothesis, a loading dose of () was injected and in vivo microdialysis was performed to detect extracellular in the striatum in both WT and mice after neural or astrocyte DRP1 knockdown. mice had significantly higher levels of extracellular than WT mice, and this difference was much less pronounced after selective astrocyte DRP1 knockdown in mice. By contrast, no differences in extracellular were observed between WT mice exposed to either empty AAV control, neuronal DRP knockdown by AAV, astrocyte DRP1 knockdown by AAV, or non-selective DRP1 knockdown using mdivi-1. (Figure 5A,B). To support the hypothesis that DRP1 knockdown might contribute to astrocytic clearance in mice, in vitro transport assays were conducted using a cultured astrocyte cell line. Regardless of whether Drp1 siRNA was added, astrocytes had very limited capacity to uptake (Figure 5C). After was converted to by SDT, the intracellular content of was dramatically higher, suggesting considerable uptake by cultured astrocytes. Accordingly, transport could be significantly inhibited by D22, indicating the critical role of OCT3 in the transport of extracellular to astrocyte. Furthermore, Drp1 siRNA partially reestablished the transport originally blocked by D22 (Figure 5D).
Expressions of Astrocytic Transporters after DRP1 Knockdown
To further clarify the mechanism of the greater uptake of in astrocytes after DRP1 knockdown, the differences in the expression levels of three major monovalent cation transporters, including SLC1A4, ASCT2, and 4F2hc, were examined in the cultured astrocyte cell line. The level of ASCT2 was significantly higher in cells treated with Drp1 siRNA and mdivi-1, whereas the levels of other two monovalent cation transporters did not differ between the groups (Figure 6A–H). Besides, itself had a limited impact on the expression of these transporters.
In vivo immunofluorescent staining was conducted to locate the distribution of highly expressed ASCT2. Confocal scanning showed that selective astrocyte DRP1 knockdown or mdivi-1-treated mice and WT mice had higher expression of ASCT2. The ASCT2 was mainly colocalized with GFAP, the marker of astrocytes (Figure 7A–E), but rarely distributed on neurons (Figure S5A–D).
The Comparation of Transportation Activity between ASCT2 and OCT3
Although higher expression of ASCT2 was observed in both WT and mice after astrocyte DRP1 knockdown, the greater clearance of extracellular only occurred in mice. As presented above, selective astrocyte DRP1 inhibition restored the transport capacity of astrocytes and relieved neurodegeneration in mice. This observation led us to hypothesize that OCT3 accumulated more than ASCT2 could, which may be related to the transporter expression level, affinity, and maximal transport rates. To compare the affinity of the two transporters for , we performed a transport study using cultured astrocyte cell lines. The uptake by astrocytes was limited and was not affected by Drp1 siRNA or transporter blockers (Figure 8A,B). When was converted to by reducing agent SDT, a higher uptake of into astrocytes was observed, which was mitigated by treatment with D22 (blocker of OCT3), but not Benser (blocker of ASCT2), indicating that OCT3 might have a higher affinity for than ASCT2 under physiological conditions (Figure 8C). When astrocyte DRP1 was knocked-down by siRNA (which led to higher protein expression of ASCT2 in GL261 astrocytes; Figure 6), Benser partially inhibited the uptake, but had a weaker inhibitory effect than D22. Of note, in astrocytes treated by Drp1 siRNA, when OCT3 was blocked by D22, Benser further reduced the uptake of PQ (Figure 8D).
To further test the complementary role of ASCT2 in extracellular clearance in vivo, we used in vivo microdialysis after and applied aCSF containing Benser to observe the role of ASCT2 in clearing extracellular in mice and WT mice. Benser had a limited impact on extracellular levels in WT mice (Figure 9A). mice demonstrated higher extracellular than WT mice, but treatment with selective astrocyte DRP1 knockdown or mdivi-1 resulted in lower extracellular concentrations. More importantly, Benser infusion mitigated the effect of DRP1 inhibition such that extracellular levels were significantly higher in mice treated with Astro-DRP1 KD and Mdivi-1 plus Benser than in the corresponding groups without Benser (Figure 9B).
Discussion
Epidemiological studies indicated that PD is a complex disease caused by the interaction of genetic and environmental factors.42,43 However, only 10% of PD cases can be attributed to the disease-causing gene. There are no definite pathological mutations in most PD patients,8 indicating that environmental factors play an important role in the pathogenesis of PD. Epidemiological and experimental studies reported that many substances have been found to increase the risk of PD including .9,16 has a similar chemical structure of organic cation to 1-methyl-4-phenylpyridinium (), the active form of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP),44 which is a selective dopaminergic pro-neurotoxin and an acknowledged inducer of PD phenotype in animal models.45 Both and have been documented by studies in mouse and cells to disrupt mitochondrial respiratory activities, inducing the overloading of reactive oxygen species (ROS), thus accelerating the death of dopaminergic neurons in the brains of mice.46,47 Furthermore, research in human and rodents reported that some compounds such as tetraisoquinolines and beta-carbolines present in our diets were linked to PD onset,48,49 and all of them structurally resembled organic cations. It was critical to discover how these cations were transported between cells in the brain, so that we could understand the pathogenic mechanisms of environmentally associated PD.
In experimental studies, has been used to model the pathology of PD, including the loss of TH-positive neurons and locomotor deficiency.50 When reaches the nigrostriatal system after systemic injection in mice, it is converted to , a substrate for both OCT3 and DAT,19 suggesting opportunities for crosstalk between glia and DA neurons in transportation. In the brain, OCT3 is mainly expressed on astrocytes, a finding based on experiments using astrocytes of human and rodents.51 Our previous experimental studies have revealed that neurodegeneration induced by chronic treatment was more severe in mice compared with WT ones, suggesting a protective effect of OCT3 in -induced neurodegeneration.21 However, it was unknown how OCT3 expression and functions would change during the process of neurodegeneration. In the present study, we found that OCT3 expression differed throughout the process of aging and neurodegeneration, as protein levels were lower with older mice and mice. The lower expression of OCT3 protein could be related to compromised clearance, which, we argue, should be a target of the disease-modifying therapy. A genome-wide association study about PD also has reported that the single nucleotide polymorphism (SNP) disturbing the expression of OCT3 was related to a higher risk of PD.52 This evidence indicates that OCT3 deficit is probably a clinical risk factor of PD onset that had not previously been recognized. To our best knowledge, the present study provides the first link between the transporting mechanisms of paraquat in mice and PD-related behavioral phenotype, which manifests as dyskinesia. In behavior tests, rotarod and gait analysis were used to assess the severity of movement disorders. Mice with dyskinesia tended to fall from the rotarod and thus had lower duration than normal mice. These mice also had abnormal gait, manifesting as shorter stride length, longer stand, and slower swing speed. It was proved that induces neuronal death through oxidative stress and ROS.9 The lack of an assay for ROS or oxidative defense is a limitation of this study; however, because we focused on the relationship between paraquat transportation and PD phenotype, we used the damages of TH-positive neurons and animal behavior phenotypes to bridge this gap and reflect the effects of paraquat.
The neurotoxic model used in the present study has been acknowledged in other in vivo studies on PD.19,30 During the process of chronic treatment, was injected i.p. into mice. We acknowledge that i.p. injection is not as close to the real-world exposure as other methods, including inhalation or gavage. However, an i.p. injection was the most feasible method to use the least dosage and ensure that each mouse received an equivalent dose of in such a way that extracellular measurements by in vivo microdialysis were comparable, given that we mainly focused on the transport mechanisms of in the brain. It was reported that inhalation and gavage of were not efficient enough to model the PD symptoms but, rather, caused pulmonary fibrosis in mice.53–56 The method of gavage has been usually used to model acute lung injury, pulmonary fibrosis and liver damage in mice.57–59 Only a few studies in rodents used gavage to model PD symptoms, but usually in a larger dose (such as for 28 d)60 or supplemented with another drug.61 These studies observed the pathology of PD in the brain but did not detect the cerebral extracellular level of , which could be influenced by absorption in the digestive system. Moreover, the dosage of in the present study has been commonly used in similar mouse studies about PD.18,30 In the real world, although epidemiological studies have revealed the relationship between PQ exposure and PD onset, the levels in the brains of exposed population are still unknown16 and difficult to detect. Thus, no “standard concentration” could be considered in experimental studies to mimic the PQ exposure in the real world. The dosage in the present study () was probably higher than in the real-world exposure, but it was lower than other methods used in experimental studies, such as gavage. Despite this “overload” exposure, the pathology of PD could only be mimicked subclinically, with loss of TH-positive neurons but sparing of striatal terminals. We assumed that human beings and rodents might have different sensitivities to PQ. It remained to be explored how to better mimic the real-world exposure of PQ in rodent models. The PQ dosages we used to treat the cells were used in previous in vitro studies.30,62 However, in the present study, cells were not continuously incubated in reported concentrations of PQ. A short incubation time of 20 min was adopted, after which the PQ was replaced by normal culture medium so that we could observe uptake activities during this period of time and the effects of PQ intake to the cells. The PQ taken up by the cells represented only a small portion of the PQ in the culture medium in the present study. DRP1 has been acknowledged as a key regulator of mitochondrial fission based on research on rodents and cells.26 Excessive elevation of DRP1 exacerbated mitochondrial fragmentation, accelerating the apoptosis of cells in a nonhuman primate.63 Recently, the roles of DRP1 in astrocytes have been gradually revealed. For example, DRP1 could regulate the activation of astrocytes and mediate the inflammatory reactions related to astrocytes in the brains of mice.29 In the present study, we found that astrocyte DRP1 protein expression could be stimulated by . Selective astrocyte DRP1 knockdown significantly relieved the neurotoxicity, but these protective effects were only observed in mice. As we discussed above, mice were less able transport extracellular into astrocytes,21 which led us to speculate whether DRP1 knockdown in astrocytes could restore a compromised process of PQ clearance. In vivo microdialysis showed that extracellular residue of the loading was lower in the striatum of mice receiving astrocyte DRP1 knockdown, suggesting restored clearance and transport in the nigrostriatal system of mice. Through in vitro experiments, we directly measured the higher levels of transport in astrocytes after treatment of Drp1 siRNA. Furthermore, increased transport induced by astrocyte DRP1 knockdown was related to the higher expression of ASCT2, one of the transporters mainly distributed in astrocytes.64 The function of this transporter was further evaluated by in vitro transport studies. We found that was a poor substrate for both OCT3 and ASCT2 until reduced to . Interestingly, OCT3 had a much higher affinity than ASCT2, combined with the low endogenous level of ASCT2, which might explain the limited functions of ASCT2 in WT mice under physiological conditions. However, when OCT3 protein levels were lower during aging or chronic toxic exposure, ASCT2 could function as an alternative transporter to compensate for its role, contributing to the continuous clearance of extracellular . Taken together, we established here the relationships between astrocyte DRP1 and neurotoxicity. In particular, we revealed a complementary pathway to restore PQ clearance in the brains of mice with OCT3 deficiency. In environmentally pathogenic PD, the function of glia surrounding DA neurons including astrocytes is crucial for the microenvironment of the nigrostriatal system and the survival of DA neurons. Interfering with the transporters located on these astrocytes may bring more benefits than just regulating neuronal function. Our research suggests that DRP1 could be one of the potential targets of modifying therapies for PD. In addition to the amelioration of mitochondrial fragmentation,65 astrocyte DRP1 inhibition was also involved in the regulation of toxin transport, one of the critical causes of environmentally pathogenic Parkinson’s disease.66 As discussed above, ASCT2 was up-regulated by selective astrocyte DRP1 inhibition. However, the underlying mechanisms require further elucidation. Mitochondrial retrograde signaling is suggested to be a potential pathway.67 The influence of DRP1 on mitochondrial functions could regulate the expression of nuclear-encoded proteins, enabling communication between mitochondrial activities and the intracellular protein expression system, which has been reported in studies of drosophila and animal cells.68,69 The modified nuclear-encoded proteins consist of different transporters on the cell membrane related to amino acid metabolism.70 Pathways including the mammalian target of rapamycin (mTOR) and nuclear factor kappa B () are involved in these responses.71,72 The results in the present study enrich our knowledge on astrocyte DRP1 and substantiate that cultivating mitochondrial fission is not the only function of DRP1.
In the present study, mdivi-1 was revealed to ameliorate neurodegeneration in both WT and mice. A similar protective effect of mdivi-1 in both genotypes could be related to its lipophilic and small molecular structure, making it possible to freely pass through the blood–brain barrier38 and nonselectively regulate mitochondrial fission/fusion in the neurons73 and astrocytes.74 Recently, ROS production and oxidative stress have also been reported to be the targets of mdivi-1 in vitro.75 This small molecule could be a promising molecular target for the disease-modifying treatment of environmentally pathogenic Parkinson’s disease.
In summary, the present study highlights the existence of a complementary pathway modulated by astrocyte DRP1 during neurodegeneration through coordinated interactions of astrocytes and DA terminals. Our study revealed that OCT3, ASCT2 located on astrocytes, and DAT located on DA terminals might function in a coordinated manner to mediate the susceptibility to toxicity induced by PQ. The conclusions of the present study are summarized in Figure 10. By extension, other monovalent cation toxicants might also share the same mechanisms; accordingly, this present study provides the basis for future studies investigating the effect of neurotoxicity on the nigrostriatal pathway.
Acknowledgments
M.C., Yx.Z, and Q.D. determined the subject direction and designed the research. S.H. and Y.F. completed experiments and wrote the manuscript. M.G. helped with the experiment design, figure preparation, data analysis, draft writing and manuscript revision. Y.H. and J.S. helped complete the in vivo experiments and modified the manuscript. Yc.Z. helped complete the in vitro experiments and analyzed data.
We thank K. Tieu (Florida International University) for providing us with the mice. The institute of Neurology of Fudan University provided the experimental platform for this study.
Funding for this work was provided by the National Natural Science Foundation of China (81971013 to M.C., 82071197 to Q.D., and 82101336 to M.G.) and by the Science and Technology Commission of Shanghai Municipality (20ZR1443500 to Yx.Z).
Article Notes
*
These authors contributed equally to this work.
The authors declare they have no actual or potential competing financial interests.
Supplementary Material
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EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted.
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Received: 15 April 2021
Revision received: 24 March 2022
Accepted: 31 March 2022
Published online: 5 May 2022
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