Chemical Compounds and Reagents

4-Phenylbutyric acid (4-PBA, purity > 99.98%, #HY-A0281), 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (#HY-15608), 1-methyl-4-phenyl-pyridine (MPP+) (#HY-W008719), Mito-Q (#HY-100116A), Cell Counting Kit-8 (#HY-K0301), Dihydroethidium (DHE) (#HY-D0079), MitoSOX Red (#HY-D1055), and Cell Counting Kit-8 (#HY-K0301) were obtained from MedChemExpress (MCE, New Jersey, USA). ER-Tracker Blue-White DPX (Ex/Em 374/430-640 nm, #40761ES50) and Annexin V-FITC/PI Apoptosis Detection Kit (#40302ES20) were obtained from Yeasen Biotechnology (Shanghai, China). ATP Assay Kit (#S0026), Mitochondrial Calcium Assay Kit with Rhod-2 AM (#S1062S), Mito-Tracker Deep Red FM (Ex/Em 644/665 nm, #C1032), Rhodamine 123 (Ex/Em 507/529 nm, #C2007) and Antifade Mounting Medium with DAPI (#P0131) were obtained from Beyotime Biotechnology (Shanghai, China). The bicinchoninic acid (BCA) protein assay kit (#UA276918) was obtained from Thermo Fisher Scientific (Massachusetts, USA).

Antibodies

The antibodies to the β-tubulin, Calnexin, LC3B (#ab51520), Miro1 (#ab211363), and Tyrosine Hydroxylase (#ab137869) were purchased from Abcam (Cambridge Science Park, UK). The antibodies to the GRP78 (#11587-1-AP), CCAAT-enhancer-binding protein homologous protein (CHOP) (#15204-1-AP), NDUFS1 (#12444-1-AP), SDHB (#10620-1-AP), Alpha Tubulin (#11224-1-AP), Parkin (#14060-1-ap) and PTEN induced putative kinase 1(PINK1) (#23274-1-ap) were purchased from Proteintech Group (Chicago, USA). The antibodies to the Bax Mouse mAb (#89477), Bcl2 Mouse mAb (#15071), and Cle-Caspase 3 Rabbit mAb (#Asp175) were obtained from Cell Signaling Technology (CST, Massachusetts, USA). The secondary antibodies were HRP Goat Anti-Rabbit IgG (H + L) (#AS014), HRP Goat Anti-Mouse IgG (H + L) (#AS003) and FITC Goat Anti-Rabbit IgG (H + L) (#AS011), all above were purchased from Abclonal (MA, USA). The antibody to the P62 (#HA721171) was purchased from HUABIO (Hangzhou, China). All antibodies were used at the manufacturer’s recommended dilutions.

Cell Culture and Drug Treatment

The SH-SY5Y cells were purchased from Shanghai Fuheng Biotechnology (Shanghai, China). The SH-SY5Y cells were cultured as described previously (Chen et al. 2020). Referring to previous reports (Li et al. 2004), cells were treated with or without 4-PBA (1 μM) for 30 min, followed by treatment with MPP+(1 mM) for 24 h.

Animal Models

C57BL/6 mice (male, 8–10 weeks) were purchased from the Laboratory Animal Center at the Fourth Military Medical University, Xi’an, China. All mice were housed in a temperature- and humidity-controlled environment under a 12-h light/dark cycle with free drinking water and food. After 1 week of acclimation, 48 mice were randomly assigned to four groups (each group contained 12 mice): Control group, MPTP group, MPTP + 4-PBA group, and 4-PBA group. PD mice received MPTP (dissolved in 0.9% saline) intraperitoneal injections at a dose of 30 mg/kg/day for five consecutive days. Referring to previous reports (Chen et al. 2021a), the MPTP + 4-PBA and 4-PBA groups were given 4-PBA (300 mg/kg/d) by intragastric administration, which was started 4 days before MPTP injection. Control group mice were intraperitoneally injected with an equal volume of 0.9% saline. The animal experimentation procedures adhered rigorously to the National Standards for Animal Experimentation, and the study obtained approval from the Medical Ethics Committee of Tangdu Hospital, the Fourth Military Medical University (approved on March 9, 2022, Grant No. 202203-074).

Cell Viability Assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK8) according to the manufacturer’s protocol (Delmotte et al. 2023). Briefly, the SH-SY5Y cells from different experimental groups were seeded in 96-well plates (1 × 104 cells/well) and then treated with a diluted CCK-8 solution for 1 h. Subsequently, the absorbance was quantified by a microplate reader (Spectra Max, M2, USA) at 450 nm.

Detection of Reactive Oxygen Species (ROS)

SH-SY5Y cells were seeded into confocal petri dishes and treated according to the experimental protocol. The cells were washed twice with PBS, and then 1 mL of dihydroethidium working solution (100 μM) was added to the cells, which were incubated at room temperature (RT) for 30 min and then observed under a confocal microscope (A1 Si, Nikon). For the detection of ROS in mouse brain tissue, frozen sections of mice SNpc were washed twice with PBS and then treated with Dihydroethidium working solution (100 μM) for 30 min at RT and examined under a confocal microscope (Zheng et al. 2020).

Mitochondrial Calcium Assay

Mitochondrial Calcium was detected using the Mitochondrial Calcium Assay Kit with Rhod-2 AM. SH-SY5Y cells were processed according to the experimental protocols. The cells were washed with PBS and then added 100 μL of Rhod-2 staining solution. After being incubated for 20 min at 37 °C, the cells were washed twice with PBS and detected with a fluorescent microplate reader.

ATP Level Detection

ATP levels were assessed with the ATP Assay Kit according to the manufacturer’s instructions. Briefly, SH-SY5Y cell protein extracts were assayed for luciferase activity in a luminometer (Berthold Systems), and results were normalized to total protein content (Li et al. 2022). The ATP concentration was converted to nanomoles per milligram of protein.

Endoplasmic Reticulum Staining

The ER-Tracker Blue-White DPX was used to assess ERS. After the cell treatment according to the experimental protocol, cells (seeded in confocal culture dishes) were washed with Hank’s Balanced Salt Solution (HBSS) and incubated with 0.1 μM ER-Tracker dissolved in HBSS for 20 min at 37 °C. Finally, the staining solution was replaced with HBSS and imaged with a confocal microscope (A1 Si, Nikon).

Cellular Immunofluorescence (IF) and Mito Tracker Staining

IF staining of the cells was performed as described previously (Han et al. 2021). Briefly, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton for 10 min. After blocking with 5% BSA for 30 min, cells were incubated with primary antibodies overnight at 4 °C, followed by fluorescent secondary antibody incubation for 1 h at RT. Mitochondria were labeled with Mito Tracker (red) according to the manufacturer’s instructions. Cells were stained with Mito Tracker working solution at 37 °C for 20 min. After DAPI counterstaining, images were acquired using a confocal microscope.

Cell Apoptosis Assay

To evaluate the effect of 4-PBA on MPP+-induced SH-SY5Y cell apoptosis, Flow cytometry was used to analyze the apoptosis rate with a FITC-Annexin V apoptosis detection kit, according to the manufacturer’s protocol (Chen et al. 2020). Finally, FlowJo_v10.8.1 (FlowJo Software, USA) was used to detect and analyze the data. The number of early and late apoptotic cells was counted as the basis for calculating the total apoptosis rate.

Western Blot Assay

The cells and the SNpc tissues of mice brains were lysed with pre-cold RIPA lysis buffer, respectively. After centrifugation (12,000 rpm) for 15 min at 4 °C, the protein concentration was measured with BCA Standard Solution. The denatured protein samples were analyzed by the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% skim milk for 2 h at RT and then incubated with primary antibody at 4 °C overnight. The membranes were incubated with HRP-conjugated secondary antibody (2 h, RT) and then washed three times. The protein bands were detected by Bio-Rad imaging system (Bio-Rad, Hercules, USA) and quantified with Image J software.

Transmission Electron Microscopy (TEM)Cell Samples

SH-SY5Y cells were processed according to established experimental protocols. After centrifugation, the cells were fixed with 2.5% glutaraldehyde overnight at 4 °C. The cells were then rinsed with PBS and further fixed with 1% osmium tetroxide for 2 h at 4 °C, and gradient dehydration was performed using ethanol and acetone, followed by resin embedding and oven polymerization. The resin block was sectioned on Leica EM UC7 ultramicrotome to a thickness of 70 nm and placed on a copper mesh (Chen et al. 2020). The sections were stained with 4% uranyl acetate and 1% lead citrate and observed under a TEM (HT7800, Hitachi, Japan).

Tissue Samples

Mice were anesthetized with sodium pentobarbital by intraperitoneal injection and the brain was extracted through the cervical dislocation euthanasia method (Zhang et al. 2017). The size of the mouse brain SNpc samples was no more than 1 mm3, and the rest of the treatment was the same as the cell samples.

Measurement of Mitochondrial Membrane Potential (MMP) and Mitochondrial ROS

Rhodamine 123 was used to assess MMP (He et al. 2020), and the experiments were performed according to the manufacturer’s protocol. Briefly, cells were seeded in confocal dishes, incubated in the dark with 5 μM concentration of Rhodamine 123 working solution at RT for 30 min, then observed by confocal microscopy. MitoSOX Red was used to assess mitochondrial ROS levels. Cells were stained with a 5 μM working solution of MitoSOX Red and subsequently observed using confocal microscopy. Quantitative IF analysis was performed using Image J software.

Brain Tissue IF StainingBrain Slices Preparation

Mice were anesthetized with pentobarbital sodium by intraperitoneal injection and transcardially perfused with PBS and 4% paraformaldehyde (Xu et al. 2022). Whole brain tissues were dissected from the skull, postfixed overnight with 4% paraformaldehyde at 4 °C, and then stored in gradient sucrose solution for dehydration. The brains were sectioned on a frozen microtome (CM1950, Leica, Germany) at a thickness of 25 μm. The SNpc regions were serially sectioned, samples were collected at intervals of four sections, and a total of 10 representative sections were selected (Liu et al. 2021).

IF Staining

The brain sections were blocked with QuickBlock™ Blocking Buffer (Beyotime, P0260, China) for 10 min. Afterward, sections were incubated at 4 °C with the primary antibody overnight. After washed three times, the sections were incubated with the secondary fluorescent antibody for 1 h at RT (Saha et al. 2023). The sections were mounted, and the images were taken with a confocal microscope. Image analysis was performed using Image J software. For each mouse, the total number of TH-positive cells across the SNpc was counted in 10 midbrain sections (Liu et al. 2017, 2021).

Behavioral TestsMorris Water Maze

The Morris water maze (MWM) test was performed to assess the cognitive function of mice, including spatial learning and memory function (Chen et al. 2015). Briefly, mice were trained in a maze pool (30 cm deep, 23 ± 1 °C) with a hidden platform in the first quadrant for 4 days, with a 60 s time limit to find it. If unsuccessful, they were guided to the platform for 10 s. On day 5, the platform was removed, and mice were placed in the opposite quadrant. Swimming paths were recorded for 1 min, and key metrics, including platform site crossings and time spent in the target quadrant, were analyzed. The final test was conducted 24 h after training, with crossings and quadrant time recorded automatically by a video camera.

Rotarod Test

The rotarod test was conducted utilizing an accelerating rotarod apparatus to assess motor coordination among mice. Each mouse was positioned on a rotating rod that gradually accelerated from 4 to 40 rotations per minute (rpm) over 5 min. Latency to fall was recorded as an index for locomotor ability, and subsequently, the average time across three consecutive trials was computed for accurate analysis.

Pole Test

The pole test served as a method to evaluate movement disorders among mice. In this test, the mice were placed on a vertical pole with a rough surface (50 cm in height). Mice were trained to climb down the pole into the cage for 2 days, with three sessions per day. The time taken by each mouse to flip and descend to the bottom of the pole was recorded on the testing day, and the average time across three consecutive trials was then computed for analysis.

Preparation of Small Interfering RNA, Overexpression Plasmid, and Adeno-Associated Virus

According to the manufacturer’s instruction, SH-SY5Y cells were transfected with the Miro1-specific siRNA at a final concentration of 20 μM using Lipofectamine™ 3000 reagent (Thermo Fisher Scientific, USA). An overexpressed plasmid was constructed to upregulate the Miro1 level (Sino Biological, Beijing). The transfection efficiency was evaluated by Western blot assay after transfection. The sequences of Miro1 siRNAs were as follows: sense sequence: 5′-GGAGGAUGUCAAGAAUGUAGU-3′, antisense sequence: 5′-UACAUUCUUGACAUCCUCCAG-3′).

Statistical Analysis

GraphPad Prism 9 (V10.1.2) was used to perform statistical analyses. A student’s t-test was used to compare two different conditions. For comparisons of multiple groups, one-way ANOVA with Tukey’s multiple comparison test was performed. Unless otherwise stated, experiments were performed independently with the sample number “n” indicated in the figure legend. Bars and errors represent the mean ± standard deviation (SD) of repeated measurements. Statistical significance is denoted as: ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.