The cholesterol 24-hydroxylase CYP46A1 promotes α-synuclein pathology in Parkinson’s disease

CYP46A1 and 24-OHC are increased in PD patients and PD model mice

α-Syn pathology and its spread are significantly reduced after CYP46A1 removal in vivo

Elevated CYP46A1 promotes α-Syn aggregation by 24-OHC in vitro

24-OHC accelerates the seeding activity and neurotoxicity of α-Syn fibrils in vitro

The seeding activity of 24-OHC PFFs is greater than that of α-Syn PFFs in vivo

24-OHC PFFs induce more severe nigrostriatal dopaminergic degeneration and motor impairments than α-Syn PFFs

Exogenous 24-OHC facilitates the propagation of α-Syn pathology

24-OHC exacerbates the mitochondrial dysfunction induced by α-Syn PFFs

Intracerebroventricular injection of 24-OHC aggravates neurodegeneration in vivo

24-OHC activates the XBP1/LAG3 axis in vivo and in vitro

Activated XBP1 and LAG3 axis by 24-OHC enhances cell-to-cell transmission of α-Syn

Blocking XBP1 ameliorates 24-OHC-induced mitochondrial damage

Human plasma

Plasma samples were collected from PD patients and age-matched controls in Renmin Hospital of Wuhan University. Informed consent was obtained from the subjects. The study was approved by the ethical committee of Renmin Hospital, Wuhan University. The approval number is WDRY2024-K104. All participants provided oral informed consent prior to the start of the study. This study was conducted in strict adherence to the ethical principles outlined in the Declaration of Helsinki. PD was diagnosed according to the criteria of the Movement Disorders Society 2015. Disease severity was assessed with Hoehn and Yahr staging (H-Y stage). The plasma was centrifuged, aliquoted, and stored at −80°C.

Human tissue samples

Postmortem brain samples were dissected from the frozen brains of PD patients and age-matched control cases from the Emory Alzheimer’s Disease Research Center. Age- and sex-matched PD-free samples were used as controls. PD patients were clinically diagnosed and neuropathologically confirmed. The study was approved by the Biospecimen Committee.

Animals

CYP46A1-knockdown mice on the C57BL/6 background were generated by GemPharmatech (Nanjing, China). Briefly, CRISPR/Cas9 technology was used to modify the mouse Cyp46a1 gene. The Cyp46a1 gene has 4 transcripts: cyp46a1-201 (ENSMUST00000021684.5), 202 (ENSMUST00000221708.1), 203 (ENSMUST00000222240.1), and 204 (ENSMUST00000222902.1). According to the gene structure, exons 2–9 of the Cyp46a1-201 transcript were selected as the knockout region. The region contains a 788 bp coding sequence. Knocking out this region results in non-expression of protein. Wild-type (WT), CYP46A1 heterozygous (CYP46A1^+/−), and homozygous (CYP46A1^−/−) littermates were randomly assigned to different treatment groups. The human A53T variant α-syn transgenic line M83 was obtained from the Jackson Laboratory (stock number: 004479). All animal studies were performed in accordance with the Experimental Animal Management Criterion. Experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Renmin Hospital of Wuhan University with the IACUC issue number of WDRM animal (welfare) 20210609.

Antibodies and reagents

The following antibodies were used in this study: phospho-α-Syn (pSer129) (1:1,000, Biolegend, 825701), phospho-α-Syn (pSer129) (1: 1,000, Cell Signaling Technology, 23706), α-syn (1:1,000, syn211, Invitrogen, MA5-12272), α-syn (1:1,000, Cell Signaling Technology, 4179), MAP2 (1:1,000, Proteintech, 17490-1-AP), CYP46A1 (1:1,000, Proteintech, 12486-1-AP), XBP1 (1:1,000, Abcam, ab37152), LAG3 (1:1,000, Abcam, ab209236), LAG3 (1:500, Biolegend, C9B7W), and TH (1:2,000, Abcam, ab117112). Alexa Fluor 488-goat anti-mouse (1:500, Invitrogen, A11001), Alexa Fluor 488-goat anti-rabbit (1:500, Invitrogen, A11034), Alexa Fluor 594-goat anti-rabbit (1:500, Invitrogen, A11012), Alexa Fluor 594-goat anti-mouse (1:500, Invitrogen, A11005), anti-mouse IgG-HRP-conjugated detection antibody (1:3,000, Cell Signaling Technology, 7076S), and anti-rabbit IgG-HRP-conjugated detection antibody (1:3000, Cell Signaling Technology, 7074S). The following reagents were used in this study: DAPI (1 μg/ml, BioFroxx, 1155MG010). IHC Detection System Kit (ZSGB-BIO, PV-6001/PV-6002), DAB Kit (ZSGB-BIO, ZLI-9019), MitoTracker (Invitrogen, M7512), Toyocamycin (MedChemExpress, HY-103248), TMRE (Invitrogen, T669), Fluo-4 acetoxymethyl (Fluo-4 AM) ester (Solarbio, IF1500), Lipofectamine 2000 (Invitrogen, 11668019), B-27 (Invitrogen/Gibco, 17504044), L-glutamine (Invitrogen/Gibco, A1286001), penicillin and streptomycin (Invitrogen/Gibco, 10378016), fetal bovine serum (Invitrogen/Gibco, 10099–141), BCA Kit (Invitrogen, A55860), 24-OHC (MedChemExpress, HY-N2370), and α-Syn aggregate ELISA Kit (Biolegend, 449407).

Purification of recombinant α-Syn

Generation of α-Syn PFFs and 24-OHC PFFs

Transmission electron microscopy

To examine the morphological characteristics of different fibrils, 24-OHC PFFs and α-Syn PFFs were adsorbed onto glow-discharged 400 carbon-coated copper grids for 2 min. Following rapid double washing with Tris-HCl (50 mM, pH 7.4), the samples were floated on 2 drops of 0.75% uranyl formate for 30 s. The grids were subsequently air-dried and subjected to imaging via a Hitachi HT7800 transmission electron microscope operating at 80 kV. Image capture and digitization were conducted with an ER-80 charge-coupled device (8 megapixels) employing advanced microscopy techniques.

Transduction of α-Syn PFFs

Before transduction, the fibrils were sonicated with 60 pulses of 10% power (total of 30, 0.5 s on, 0.5 s off). α-Syn fibrils (140 ng/ml, final concentration) were transduced with Lipofectamine 2000. Briefly, α-Syn fibrils were combined with Opti-MEM (Gibco) to a final volume of 100 μl. Then, 96 μl of Opti-MEM and 4 μl of Lipofectamine 2000 (Invitrogen) were mixed in another tube. The 2 tubes were mixed and incubated for 20 min before being added to the culture medium.

Cell culture

Primary neuronal culture

Primary cortical neurons were prepared at embryonic day 16 and cultured in neurobasal media supplemented with B-27, 0.5 mM L-glutamine, penicillin, and streptomycin on tissue culture plates coated with poly-L-lysine. The neurons were cultured for 5 days in vitro; treated with α-Syn PFFs, 24-OHC (final concentration, 30 μm), control AAVs, or AAV-CYP46A1 (prepared by Brain VTA Co., Ltd., Wuhan, China), and incubated for 9 days. Then, the neurons were fixed and immunostained. Each experiment was performed in duplicate and repeated 3 to 6 times.

Microfluidic neuronal culture

Microfluidic devices (RD900) were obtained from Xona Microfluidic, LLC (Temecula, California, USA). Approximately 100,000 neurons were plated in each chamber. After being cultured for 5 days in vitro, 0.5 μg of α-syn PFFs was added to chamber 1 (C1), which was supplemented with 24-OHC (final concentration, 30 μm), the LAG3 antibody (50 μg/ml), or toyocamycin (final concentration, 40 nM). To control the direction of flow, a 50 μl difference in media volume was maintained between C1 and chamber 2 (C2). After treatment with α-syn PFFs for 9 days, the neurons were fixed with 4% paraformaldehyde (PFA) in PBS and then processed for immunofluorescence.

Quantification of the fluorescence intensity of the α-Syn inclusions

After treatment, the α-Syn-HEK293 cells were fixed with 4% PFA (40 mg/ml) for 15 min, after which the soluble protein was permeabilized with 1% TX-100 for 15 min at room temperature. After washing, the cells were stained with 1 μg/ml DAPI for 2 min and imaged via an Olympus inverted fluorescence microscope (Olympus TH4-200, Japan). The fluorescence intensities of the α-Syn inclusions were quantified via ImageJ software (version 2.1.0/1.53c).

Immunofluorescence

For immunofluorescence, cells and neurons were fixed with 4% PFA for 15 min, followed by permeabilization and blocking with 0.1% Triton X-100 (TX-100)/3% BSA at room temperature for 30 min. Samples were incubated with primary antibodies overnight at 4°C. After being washed, the cells were stained with Alexa Fluor 488/594-conjugated anti-mouse/rabbit secondary antibodies for 2 h at room temperature, followed by incubation with 1 μg/ml DAPI for 2 min. The cells were subsequently imaged via an Olympus inverted fluorescence microscope (Olympus TH4-200, Japan). ImageJ software (version 2.1.0/1.53c) was used for quantification of the signals.

Immunoblotting

The samples were prepared in loading buffer containing 4% SDS and 5% β-mercaptoethanol, boiled for 10 min at 100°C and then separated on 10% SDS–PAGE gels. The proteins were subsequently transferred to nitrocellulose membranes via a semidry system (Bio-Rad, Switzerland). The membranes were blocked with blocking buffer (5% (w/v) nonfat milk (50 mg/ml) in 1× TBS containing 0.05% (v/v) Tween-20 (TBST)) for 1 h at room temperature and then incubated overnight at 4°C with primary antibody diluted in blocking buffer. After being washed 3 times with TBST, the membranes were incubated with appropriate HRP-conjugated secondary antibodies in blocking buffer for 1 h at room temperature. After 3 washes with TBST, the blots were developed via Western light-enhanced chemiluminescence (ECL).

Immunohistochemistry

The mice were perfused with PBS and 4% PFA successively. The brain was postfixed in 4% PFA overnight at room temperature. The brain samples were dehydrated through a graded series of ethanol and then embedded in paraffin wax. Five micron-thick brain sections were mounted on glass slides, deparaffinized and then rehydrated via a graded series of ethanol. An IHC detection system kit was used for immunohistochemistry according to the manufacturer’s instructions. Briefly, the brain sections were deparaffinized, rehydrated, and then incubated with 3% (v/v) H₂O₂ for 25 min to quench endogenous peroxidase activity. Then, the slides were blocked by incubation in 5% (v/v) normal horse serum for 60 min at room temperature and incubated with primary antibodies overnight at 4°C. The signals were developed via the 3,3′-diaminobenzidine (DAB) peroxidase substrate. The brain sections were imaged via an Olympus inverted fluorescence microscope (Olympus TH4-200, Japan) with 20×, 40×, or 63× objectives.

LAG3 antibody blocking experiments and XBP1 inhibition

α-Syn inclusions colocalized with mitochondria

MitoTracker (final concentration of 500 nM, 37°C for 30 min) was used to stain the mitochondria. Live imaging was performed under a confocal microscope with an excitation wavelength of 579 nm and an emission wavelength of 599 nm.

Detection of mitochondrial morphology and fusion activity

Measurement of the mitochondrial membrane potential

The mitochondrial membrane potential was detected via tetramethylrhodamine ethyl ester (TMRE). After treatment with α-Syn PFFs in the presence or absence of 30 μm 24-OHC for 48 h, the SH-SY5Y cells were incubated with 150 nM TMRE at 37°C for 30 min. Then, the cells were washed 3 times with PBS. Live imaging was performed under a confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 575 nm. Images were analyzed via ImageJ software (version 2.1.0/1.53c).

Calcium imaging

The fluorescent calcium indicator Fluo-4 acetoxymethyl (Fluo-4 AM) ester (Solarbio, IF1500) was used to monitor the levels of intracellular calcium. After treatment, the SH-SY5Y cells were incubated with Fluo-4 AM for 30 min at a final concentration of 1 μm. After 3 washes with Hank’s balanced salt solution (HBSS) (with 2 mM calcium chloride), the cells were imaged under a confocal microscope. Live imaging was performed with an excitation wavelength of 492 nm and an emission wavelength of 514 nm. Images were acquired at 30-s intervals and analyzed via ImageJ software (version 2.1.0/1.53c).

Stereotaxic injection

Three-month-old mice were anesthetized. PBS containing recombinant α-syn PFFs or 24-OHC PFFs (5 μg/2 μl) was stereotactically delivered into the right striatum. The following reference coordinates for the dorsal neostriatum were used: + 2.0 mm medial–lateral (ML), + 2.6 mm dorsoventral (DV), and + 0.2 mm antero–posterior (AP) from bregma. Injections were performed via a 10 μl syringe (Hamilton, Reno, Nevada, USA) at a rate of 0.3 μl per minute. The needle was left in place for 8 min before being slowly removed. After surgery, the animals were monitored and postsurgical care was provided. The mice were euthanized for biochemical and histological analysis at 30 dpi, 90 dpi, and 180 dpi.

Intracerebroventricular injection of 24-OHC

Quantification of TH-positive neurons

Optical densitometry analysis

Sections through the whole striatum at 6 coronal levels of equal distance relative to the bregma were stained with TH. Optical densitometry was performed with ImageJ software (version 2.1.0/1.53c). The staining signal was calibrated by subtracting the baseline of the cortex.

Proteinase K (PK) digestion assays

For PK digestion, the brain sections were incubated with PK at a final concentration of 10 μg/ml at 37°C for 45 min. Digestion was terminated by the addition of a protease inhibitor cocktail. The sections were then analyzed via immunohistochemistry.

Behavioral analysis

To evaluate behavioral deficits, the mice were subjected to the pole test, grip test, balance beam test, and rotarod test 1 week prior to sacrifice. The experimenter was blinded to the treatment groups or genotypes for all behavioral tests. All tests were conducted between 10:00 and 16:00. In the pole test, a metal rod 45-cm long and 1 cm in diameter wrapped with bandage gauze was used as the pole. The mice were placed on the top of the pole facing head-up. The time taken to turn and the total time taken to reach the base of the pole were recorded. The end of the test was defined by the placement of all 4 paws on the base. Before the test, the mice were trained for 2 consecutive days, and each training session consisted of three test trials. The maximum cutoff time to stop the test was 60 s. The turn and total time (in seconds) were recorded. In the grip test, the metal grip was lightly shaken so that the mouse could grab the grip and then turn upside down. The latency of the mice to fall off the grid was recorded. Each training session consisted of 3 test trials. The maximum cutoff time to stop the test was 5 min. Each mouse was examined 3 times. In the rotarod test, the mice were placed on a rotating cylinder with uniform acceleration, rising from 4 rpm to a maximum of 40 rpm in 300 s. The latency of the mice to fall off the rotating cylinder was measured. The maximum cutoff time to stop the test and recording was 300 s. Each mouse was examined 3 times. The average time of the 3 trials was recorded. A narrow beam (2 × 100 cm) was used in the balance beam test. The mice walked along the beam. The time to cross the beam or drop off the beam was measured. Each mouse was examined 3 times. The average time of the 3 trials was recorded.

Detection of DA and DOPAC in the striatum

HPLC was used to measure the concentrations of biogenic amines. Briefly, the ipsilateral striatum was lysed and sonicated in ice-cold 0.01 mM perchloric acid containing 0.01% EDTA. Then, the lysates were centrifuged at 15,000 × rpm for 15 min. The supernatants were filtered through a 0.2 μm filter and analyzed by HPLC. Thirty microliters of the supernatant was analyzed on an HPLC column (4.6 mm × 150 mm, C-18 reversed-phase column, Acclaim TM Polar Advantage II, Thermo Scientific) by a dual channel coulochem III electrochemical detector (Model 5300, ESA, Inc. Chelmsford, Massachusetts, USA). A BCA protein assay kit was used to measure the protein concentration of the tissue homogenates. The data were normalized to protein concentrations and expressed in ng/mg protein.

Detection of 24-OHC and cholesterol levels in brain tissue and plasma

The concentrations of 24-OHC were determined by Sensichip Biotech (Sensichip Biotech Co., Ltd., Shanghai, China). The brain tissues were washed 3 times with PBS to remove contaminating blood. The brain and plasma samples were stored at −80°C before being analyzed. Briefly, brain tissues were mixed with 1.5 ml of chloroform/methanol (2/1, v/v) solution, swirled for 1 min, mixed with 0.5 ml of water, and ultrasonicated at 4°C for 30 min. The brain lysates were centrifuged at 12,000 × rpm and 4°C for 10 min. D4-24-hydroxycholesterol was used as an internal standard. The supernatants were used to detect 24-OHC and cholesterol levels via liquid chromatography–mass spectrometry (LC–MS) analysis. The quantification of 24-OHC and cholesterol was performed via the internal standard ratio method.

Lipidomic analysis

GST pull-down assay

To determine whether CYP46A1 directly interacts with α-Syn, HEK293 cells were co-transfected with GST-tagged α-Syn together with GFP-tagged tau or His-tagged CYP46A1 for 48 h. Then, the cells were collected and lysed with NP-40 for 30 min on ice. The lysates were incubated with glutathione agarose overnight at 4°C. The beads were subsequently washed 4 times with NP-40, boiled in SDS loading buffer, and analyzed by immunoblotting.

Statistical analysis

All statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., San Diego, California, United States, version 8.0) with a significance threshold of P = 0.05. Results were expressed as means ± SEM. One-way ANOVA was performed to confirm the significant main effects and differences among groups, followed by Tukey’s multiple comparisons for post hoc tests. P values of