Lipid profiles in the cerebrospinal fluid of rats with 6-hydroxydopamine-induced lesions as a model of Parkinson's disease

doi:10.21203/rs.3.rs-2098905/v1

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Lipid profiles in the cerebrospinal fluid of rats with 6-hydroxydopamine-induced lesions as a model of Parkinson's disease

https://doi.org/10.21203/rs.3.rs-2098905/v1

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Background

Parkinson's disease (PD) is a progressive neurodegenerative disease with characteristic pathological abnormalities, including the loss of dopaminergic (DA) neurons, a dopamine-depleted striatum, and microglial activation. Lipid accumulation exhibits a close relationship with these pathologies in PD. Few studies on PD have focused on the lipid profile of cerebrospinal fluid (CSF), which may represent an ideal biomarker for disease diagnosis.

Methods

Here, 6-hydroxydopamine (6-OHDA) was used to construct a rat model of PD, and the lipid profile in CSF obtained from model rats was analyzed using lipidomic approaches.

Results

Establishment of this PD model was confirmed by apomorphine-induced rotation behaviors, loss of DA neurons, depletion of dopamine in the striatum, and microglial activation after 6-OHDA-induced lesion generation. Unsupervised and supervised methods were employed for lipid analysis. A total of 172 lipid species were identified in CSF and subsequently classified into 18 lipid families. Lipid families, including eicosanoids, triglyceride (TG), and free fatty acid (FFA), and 10 lipid species exhibited significantly altered profiles 2 weeks after 6-OHDA administration, and significant changes in eicosanoids, TG, CAR, and 3 lipid species were noted 5 weeks after 6-OHDA administration. During the period of 6-OHDA-induced lesion formation, the lipid families and species showed concentration fluctuations related to the recovery of behavior and nigrostriatal abnormalities. Correlation analysis showed that the levels of eicosanoids, TG families and TG (16:0_20:0_18:1) exhibited positive relationships with apomorphine-induced rotation behaviors and negative relationships with tyrosine hydroxylase (TH) expression in the midbrain.

Conclusions

These results revealed that nonprogressive nigrostriatal degeneration induced by 6-OHDA promotes the expression of an impairment-related lipidomic signature in CSF that may function as a biomarker to aid in PD diagnosis, and the level of eicosanoids, TG families and TG (16:0_20:0_18:1) in CSF may reveal pathological changes in the midbrain of PD patients.

Lipid profiles

Cerebrospinal fluid

6-hydroxydopamine

Parkinson's disease

Parkinson's disease (PD) is characterized by pathological progressive degeneration involving massive loss of dopaminergic (DA) neurons in the substantia nigra (SN), dopamine-depleted striatum, microglial activation, and accumulation of α-synuclein (α-syn)¹. Gene‒environment interactions may contribute to the development of PD¹. As an ideal environmental toxin, 6-hydroxydopamine (6-OHDA) is used to construct unilateral lesions in the SN of rats, cats and primates². The 6-OHDA-induced PD model recapitulates most pathological abnormalities except α-syn accumulation and alters dopamine-related behaviors, such as apomorphine-induced rotation². However, 6-OHDA exposure represents a nonprogressive insult, and the resulting pathological and behavioral abnormalities can be reversed by drugs, which differs from PD treatment responses observed in the clinic^{2, 3}. This special characteristic of the 6-OHDA model may provide an advantage in exploring sensitive biomarkers for nigrostriatal function.

Lipid and lipoprotein metabolism play essential roles in the occurrence and development of neurodegenerative disorders⁴. Apolipoprotein E, as a component of lipoprotein, may disrupt the blood‒brain barrier (BBB) and increase the risk of Alzheimer’s disease (AD)^{5, 6}. Some mutations in the glucosylceramidase-beta (GBA) gene encoding the lysosomal hydrolase glucocerebrosidase (GCase) involved in membrane lipid composition represent high risk factors for PD and dementia with Lewy bodies^{7, 8}. PD patients with GBA mutations exhibit dramatic changes in lipid concentrations in serum, including increased ceramide and sphingomyelin levels and decreased phosphatidylic acid levels⁹. Lipid disorders are also closely related to 6-OHDA-related pathological abnormalities, such as the loss of DA neurons^{10, 11}. 15-Hydroperoxy-arachidonoyl-phosphatidylethanolamine, as a characteristic product of lipid peroxidation, can activate the ferroptotic death signal of DA neurons in PD¹². Regarding dopamine depletion, diets rich in saturated fat may change dopamine precursor availability related to dopamine concentrations in the midbrains of humans¹³. Several anionic and zwitterionic lipids bind to triggering receptor expressed on myeloid cells 2 (TREM₂) receptors in microglia involved with the recognition of abnormally deposited proteins, such as Aβ and α-syn, in neurodegenerative diseases¹⁴. Thus, lipid metabolism exhibits a close relationship with PD pathology and is considered to be an ideal biomarker for diagnosis.

In many studies on clinical PD, the search for biomarkers focuses on serum. Due to BBB blockade, biomarkers in cerebrospinal fluid (CSF) exhibit better accuracy and sensitivity to reflect the pathological changes of nigrostriatal in PD¹⁵. Thus, this study aims to explore lipid metabolism in CSF obtained from rats with 6-OHDA lesions and evaluate the association between lipids in CSF and PD-related abnormalities to explore ideal biomarkers for PD diagnosis.

Animals

Adult male Sprague‒Dawley rats (4 months old, weighing 350-490 × g) were purchased from Jiangsu Aniphe Biolaboratory (Jiangsu, China) and were maintained under specific-pathogen-free (SPF) conditions with free access to food and water and a 12-h light/12-h dark cycle in the Laboratory Animal Center (Guangzhou Medical University, Guangzhou, China). All procedures used in the present study were performed in accordance with the guidelines on animal care. All experiments were approved by the Animal Experiments and Ethics Committee of Guangzhou Medical University (No. 2020143, 21 August 2020). The rats were assigned randomly to five groups: the control group without injection, the sham group with injection of vehicle only, and the 3 6-OHDA groups injected with 6-OHDA for observation times of 2, 3 and 5 weeks, separately.

6-OHDA lesions

The neurotoxic rat model of PD was constructed by a unilateral injection of 6-OHDA into the medial forebrain bundle (MFB) as reported previously¹⁶. Using the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 2007) as a guide, 6-OHDA (Sigma‒Aldrich, Shanghai, China) was injected into the MFB based on the following coordinates: anterior-posterior, -4.4 mm; medial-lateral, -1.2 mm; dorsal-ventral, -7.8 mm relative to bregma. The rats were placed in a stereotaxic device under 1% pentobarbital sodium anesthesia. Here, 6-OHDA was dissolved at 2 μg per 1 μL in vehicle solution that contained 0.2% ascorbic acid in an equal volume of 0.9% NaCl. In the PD rats, 4 μL of the 6-OHDA solution was administered at a rate of 400 nl/min using a Hamilton syringe equipped with a 30-gauge needle. The needle was left after injection for an additional 10 min and withdrawn at a speed of 1 mm/min. The same coordinates were used in rats undergoing sham operation, but only vehicle was delivered.

Apomorphine-induced rotation test

To evaluate the presence of 6-OHDA lesions, the apomorphine-induced turning rate was assessed according to a previously described protocol (0.5 mg/kg, i.p. apomorphine) ¹⁶. At 2, 3, and 5 weeks after 6-OHDA injection, the rats were injected with 0.5 mg/kg apomorphine (Sigma‒Aldrich, Shanghai, China) at 0.5 mg/ml dissolved in NaCl by intraperitoneal injection. Then, the rats were transferred individually to a transparent plastic cylinder, and the rotations were recorded for 30 min in a self-constructed rotometer according to a previously reported method¹⁶^,¹⁷. The total number of complete 360° anti-clockwise rotations was counted, and the mean number per minute was calculated and expressed in the Results section.

Immunohistochemical staining (ICH)

Rats were anesthetized with chloral hydrate and then perfusion-fixed with precooled 4% paraformaldehyde (Sigma‒Aldrich, Shanghai, China) followed by 0.9% NaCl. The brains were separated, postfixed in 4% PFA for 24 h and cryoprotected in 30% w/v sucrose in PBS for dehydration. After embedding in OCT medium (Tissue-Tek, Sakura Finetek, USA), the brains were sectioned at 15 or 20 μm using a freezing microtome. The brain sections were incubated with primary antibodies against tyrosine hydroxylase (TH, Millipore, MAB318, 1:400) and Iba1 (Proteintech, 10904, 1:200). For immunofluorescence staining, the sections were counterstained with DAPI. The morphometric analyses were performed using the ImageJ program (NIH, Bethesda, MA).

The number of TH⁺ neurons in the substantia nigra pars compacta (SNpc) was counted using an optical fractionator. Every three sections from a total of 9-12 sections per animal throughout the entire SNpc were counted in the lesion side under a ×20 objective view. The SNpc was identified by defined anatomic landmarks. TH⁺ cells with optimally visualized nuclei in one view were considered a valid count. To count the number of Iba1⁺ cells based on immunofluorescence staining, three different sections containing 1,200 µm × 1,400 µm standardized areas in the SNpc were selected randomly as described previously¹⁸. The total area of Iba1⁺ cells was calculated as the whole image fluorescence. After the mean background fluorescence was subtracted, the program ImageJ was used to identify and count the number of Iba1⁺ cells. The density of Iba1⁺ cells was determined based on the number of cells and area. Three sections were quantified per rat.

Quantification of TH expression in the striatum was performed using the Fiji image analysis system in ImageJ as previously described¹⁶^,¹⁸. The whole striatum region in both hemispheres was identified as a region of interest (ROI). TH immunoreactivity was measured in both hemispheres. After the sections were scanned, the images were converted to binary mode. All the rats shared the same threshold. The percentage was considered as the TH intensity of the lesion side compared with the contralateral side in the same section. A total of 4-5 striatal sections were assessed per rat.

Western Blot (WB)

The midbrain was separated, and its proteins were extracted as previously reported¹⁶^,¹⁸. Protein concentrations were estimated using a Nanodrop™ 8,000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The protein extracts were run on a 4-12% SDS‒PAGE gel and then transferred to PVDF membranes. The membranes were then incubated with primary antibodies against TH (Millipore, MAB318, 1:2000) or β-actin (Bioworld, AP0060, 1:5000). After washing with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with HRP-conjugated secondary antibodies. An enhanced chemiluminescence system (ECL, Millipore) was used for visualization. The blots were assessed using the Image Lab program, and the results were normalized to the intensity of β-actin.

UPLC‒MS/MS analysis of CSF

After anesthesia with 1% pentobarbital sodium (40 mg/kg, i.p.), the rats were placed in a stereotaxic device. The head was position at a 135 degree angle to the body. After shaving and disinfection, a 2-cm longitudinal incision along the back midline in the occipital was generated to expose the foramen magnum. The insulin needle was used directly to puncture the dura mater to the cerebellomedullary cistern. Then, the CSF was drawn for UPLC‒MS/MS analysis.

Lipid extraction from CSF was performed as previously reported¹⁹. Fifty microliters of CSF sample was mixed with methyl tert-butyl ether and lipid standards to 1 ml. After incubation in an ultrasonic water bath with a frequency of 40 kHz and a power of 100 W for 5 min, 500 μl Milli-Q water was added, and the sample was centrifuged at 12,000 r/min at 10 °C. Then, 500 μl supernatant was collected and mixed with 100 μl mobile phase B. Then, 20 µl of each sample was added to a pool as a quality control. The detection system included a Shim-pack UFLC SHIMADZU CBM30A ultra-performance liquid chromatograph (UPLC) (SHIMADZU, JAPAN) coupled with a QTRAP5500 tandem mass spectrometer (MS/MS) (SCIEX, USA). Chromatographic separation was performed on a Thermo Accucore™ C30 column (2.1 mm × 100 mm, 2.6 μm). The column temperature was maintained at 45 °C. Mobile Phases A (60/40 acetonitrile/water) and B (10/90 acetonitrile/water) both contained 0.1% acetic acid and 10 mmol/L ammonium formate. The injection volume was 2.0 μL, and the flow rate was 0.35 mL/min. The gradient elution program was set as follows: 20% B at the beginning; 30-60% B between 2 to 9 min, 85-90% B between 9 to 15.5 min, 95% B between 15.5 to 17.5 min; 20% B for 17.5 to 20 min. The desolvation temperature was set as 500 °C. The MS voltage in positive and negative modes was set to 5500 V and -4500 V, respectively. The pressures of the curtain gas, gas II and ion source gas were 35 psi, 55 psi, and 45 psi, respectively. The ion pairs were detected by optimized collision energy and declustering potential in the triple quadrupole LC‒MS/MS.

Lipidomic analysis in CSF

The UPLC‒MS/MS analysis used a widely targeted metabolome method based on the Metware database (Metware Biotechnology Co., Ltd, Wuhan, Hubei, China)²⁰. The retention time, ion pair information, and secondary spectrum data were used for qualitative analysis. The quantitative analysis was performed by the multiple reaction monitoring modes (MRM) of triple quadrupole mass spectrometry, and the data were analyzed using Analyst 1.6.3 software (AB SCIEX, Framingham, MA, USA) and MultiQuant (version 3.0, AB SCIEX). For peak area determinations, an individual area of the same metabolite was normalized to the integrated area of all peaks. All sample extracts were mixed as the quality control (QC), and one QC sample was inserted into every ten samples. Total ion flow diagrams of various QC samples were used to evaluate the repeatability of metabolite extraction and detection. The lipid species were classified as eicosanoids, triglycerides (TGs), phosphatidylcholine (PC), sphingomyelin (SM), free fatty acids (FFAs), diglyceride (DG), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), ceramide (Cer), carnitine (CAR), monoglyceride (MG), cholesterol ester (CE), phosphatidylserine (PS), hexosylceramide (HexCer), sphingomyelin (SPH), butyric acid (BA), and phosphatidylglycerol (PG).

Unsupervised and supervised methods were used to evaluate whether the lipidomic signature in CSF could be determined after the induction of lesions using 6-OHDA. In the comparison between the early stage after 6-OHDA treatment and the control, the PCA results showed that PC-1 and PC-2 accounted for 26.2% and 16.5%, respectively (Supplement data 1 A). The PLS-DA results indicate that 22% of the total variance can be explained by component-1, and 12% of the total variance can be explained by component-2 (Supplement data 1 B). Using the OPLS-DA method, the orthogonal T score and T score were 22.4% and 9.3%, respectively (Supplement data 1 C). OPLS-DA data were subject to S-plot analysis (Supplement data 1 D). The Q² intercept value of - 0.237 in the permutation test (n = 200) suggested that the OPLS-DA model had statistically effective quality and robustness (Supplement data 1 E). The lipids with the top 15 VIP values were identified and are shown in the VIP score plot (Supplement data 1 F).

The same methods were selected to compare the control with early- and late-stage 6-OHDA lesions. The PCA results showed that PC-1 and PC-2 accounted for 23.8% and 14.9%, respectively, in the comparison of the late stage with the control (Supplement data 2 A, Supplement data 3 A) and 30.7% and 18.5%, respectively, in the comparison between the early and late stages. The PLS-DA results showed that 15.1% of the total variance can be explained component-1 and 14.2% of the total variance can be explained by component-2 in the comparison of the late stage with the control. These values were 20.1% for component-1 and 19.3% for component-2 in the comparison of the two stages (Supplement data 2 B, 3 B). Using the OPLS-DA method, the orthogonal T score and T score were 22.1% and 6% as well as 25.7% and 13.7% in the two comparisons, respectively (Supplement data 2 C, 3 C). The S-plot and permutation test were performed, and the Q² intercept values were - 0.301 and -0.275 in two comparisons (Supplement data 2 D, E; 3 D, E). The lipids with the top 15 VIP values are shown in the VIP score plot (Supplement data 2 F; 3 F).

Statistical analysis

The data are presented as the means ± standard errors of the means. Comparisons between two groups accepted the unpaired t test or Mann‒Whitney test after the normality test. Comparisons among multiple groups were analyzed by one-way ANOVA followed by the LSD post hoc test. Data analyses were performed using SPSS 16.0 (IBM, USA), and p < 0.05 indicated a statistically significant difference.

In lipidomic analysis, SIMCA version 15 software (Umetrics, Umeå, Sweden) and MetaboAnalyst software (Version 5.0, https://www.metaboanalyst.ca/) were used for the principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and orthogonal partial least-squares discrimination analysis (OPLS-DA). The volcano plot, box plot and heatmap were drawn using GraphPad Prism (GraphPad Prism® Software version 8.0.2 for Windows; La Jolla, CA) and Origin software (Version 2022, OriginLab Inc., USA). The metabolites with variable importance in the projection (VIP) ≥ 1 and absolute log₂Fold change ≤ 1 were used for cluster analysis with Origin software (Version 2022, OriginLab Inc., USA). Violin plots were generated using Hiplot (https://hiplot.com.cn). Pearson correlations were analyzed by Origin software (Version 2022, OriginLab Inc., USA). In the Pearson correlation and linear mixed-effects model, the significance of each variable was assessed at a level of 0.05.

Nonprogressive nigrostriatal degeneration in the 6-OHDA model of PD

Rats with nigrostriatal degeneration induced by 6-OHDA, as an animal model of PD, were assessed using apomorphine before sacrifice. The timepoints of assessment were 2, 3 and 5 weeks after 6-OHDA administration. At these 3 time points, the number of DA neurons in the SN and TH-positive area in the striatum both decreased significantly in PD rats compared with the sham group as assessed by ICH and WB (Figure 1 B). A-D, Figure 2 A, B). These effects were accompanied by an upregulated density of microglia in the SN as assessed by immunofluorescence staining and dramatically increased apomorphine-induced rotations (Supplement data 1, Figure 1 E). Interestingly, at 5 weeks after lesion induction, apomorphine-induced rotations, the number of DA neurons and the density of microglia in the SN all markedly recovered compared to that noted 2 weeks after lesion induction, and the TH-positive area in the striatum also showed a recovery trend without significance. These data suggest that functional recovery or compensatory nigrostriatal impairment may occur in the 6-OHDA model, and this nonprogressive characteristic may help to explore and evaluate ideal biomarkers of nigrostriatal lesions.

Lipid metabolism in CSF at an early stage of 6-OHDA lesions

The lipidomic signature in CSF may be a considerable biomarker that is sensitive to nigrostriatal degeneration in PD²¹. Based on the results of TH expression and rotation tests, abnormal performances are observed at an early stage approximately 2 weeks after 6-OHDA lesion. At this time point, the global lipidomic differences in CSF were detected and analyzed. A total of 172 lipid species were found and classified into 18 families. The lipid families included 2 eicosanoids, 9 FFAs, 6 Bas, 1 PS, 1 PI, 1 SPH, 1 HexCer, 1 PG, 8 CARs, 5 CEs, 2 CERs, 21 DGs, 4 LPCs, 6 MGs, 29 PCs, 10 PEs, 14 SMs and 51 TGs. According to the percentage of expression, the DG, MG, TG and PC families accounted for 51%, 24%, 11%, and 6% in the control group and 50%, 23%, 13%, and 6% in the 6-OHDA group, respectively (Figure 3 A). The overview of lipid families was provided by hierarchical clustering analysis (Figure 3 B), and their changed expressions were provided more details by box plot (Figure 3 C). Among them, the eicosanoid, TG and FFA families exhibited 1.13-, 1.21- and 0.93-fold changes in expression in the 6-OHDA group compared with the control, respectively. The eicosanoid, TG and FFA families had significantly changed expression (p = 0.0003, 0.0043, and 0.0287, respectively, Figure 4).

A complete overview of 172 lipid species was provided by hierarchical clustering (Figure 5 A, B). The 11 lipid species met the selection criteria of VIP > 1 and |log₂FC| > 1 (Figure 5 B). The characterized lipidomic signature in CSF including these 11 lipid species was drawn for PD (Figure 5 C), and their expression details are presented by box plot (Figure 5 D). Compared with the control, the following lipid species exhibited significantly upregulated levels: TG (16:0_16:0_18:1) with p = 0.0031, TG (18:1_18:1_18:1) with p = 0.0015, TG (16:0_16:1_20:1) with p = 0.0024, TG (18:0_18:1_18:2) with p = 0.0015, TG (16:0_18:1_18:1) with p = 0.0010, TG (16:0_20:0_18:1) with p = 0.0017, TG (16:0_18:0_18:2) with p = 0.0018, TG (16:0_18:0_18:1) with p = 0.0015, TG (17:0_18:0_18:1) with p = 0.0013, TG (18:0_18:0_18:1) with p = 0.0020, and TG (18:0_18:1_18:1) with p= 0.027 (Figure 6).

Lipid metabolism in CSF at the late stage of 6-OHDA lesions

The above results showed that functional recovery or compensatory nigrostriatal degeneration occurred at the late stage approximately 5 weeks after 6-OHDA-induced lesion formation. At this time point, global lipidomic differences in CSF were detected and analyzed. Some lipid species and families at the late stage showed similar expression at the early stage. The DG, MG, TG and PC families accounted for 51%, 24%, 12%, and 5% in the 5-week 6-OHDA group, respectively (Figure 7 A, B). Hierarchical clustering analysis and box plots were also generated (Figure 7 C, D). Among them, the eicosanoid, CAR and TG families exhibited 1.11-, 1.04- and 1.14-fold changes in expression in the 5-week 6-OHDA group compared with the control, respectively. The eicosanoid, CAR and TG families exhibited significantly upregulated expression (p = 0.005, 0.041, and 0.042, respectively) (Figure 8). An overview of the lipid signature was provided by hierarchical clustering (Figure 9 A). Eleven lipid species met the selection criteria of VIP > 1 and |log₂FC| > 1 (Figure 9 B) and were selected to build the characterized lipidomic signature (Figure 9 C). The expression data are presented by box plot (Figure 9 D). Compared with the control, lipid species that exhibited significantly upregulated levels included carnitine C5-OH with p = 0.0344, carnitine C4:1-2OH with p = 0.0301 and TG (16:0_20:0_18:1) with p = 0.0295 (Figure 10).

Lipid metabolism in CSF follows the recovery of nigrostriatal impairment

Lipid metabolism was compared among different stages of 6-OHDA lesion formation. The proportion of lipids in CSF at the late stage (5 weeks) differed from that noted at the early stage (2 weeks) (Figure 11 A, B). Hierarchical clustering analysis and box plots were also performed (Figure 11 C, D). Among them, CAR expression exhibited a 1.10-fold change in the late stage compared to the early stage, and the difference was significant (p = 0.0381) (Figure 11 D, E).

An overview of the lipid signature was provided by hierarchical clustering (Figure 12 A). Ten lipid species met the selection criteria of VIP > 1 and |log₂FC| > 1 (Figure 12 B) and were selected to build the characterized lipidomic signature (Figure 12 C). Their expression details are presented by box plot (Figure 12 D). Compared with the early-stage group, carnitine C4:1-2OH (p = 0.001) was significantly upregulated in the late-stage group. In contrast, the following lipid species were downregulated: TG (16:0_18:0_18:2) with p = 0.013, TG (18:0_18:1_18:1) with p = 0.024, TG (18:0_18:0_18:1) with p = 0.039, TG (16:0_18:0_18:1) with p = 0.039, TG (17:0_18:0_18:1) with p = 0.026, TG (16:0_18:1_18:1) with p = 0.038, TG (18:0_18:1_18:2) with p = 0.046, TG (16:0_16:1_20:1) with p = 0.043, and TG (16:0_16:0_18:1) with p = 0.034 (Figure 13).

During the period of 6-OHDA lesion formation, 6 lipid families showed upregulated trends, whereas 12 lipid families exhibited downregulated trends at the early stage of 2 weeks. Among them, eicosanoids, TG and FFA had significantly different expression levels. Over the next 3 weeks, CAR levels increased significantly. At the late stage of 5 weeks, eicosanoids and TG and CAR exhibited a partial recovery, but their levels remained higher than those of the control, and FFA almost recovered to normal levels (Figure 14 A). All the lipid species with significantly changed concentrations after 6-OHDA lesion formation were included in the characteristic signature (Figure 14 B). At the early stage of 2 weeks, 11 lipid species were upregulated. Over the next 3 weeks, 8 of the 11 species almost recovered to normal concentrations (Figure 14 C). Only TG (16:0_20:0_18:1) maintained an increased level compared with the control, and the lipids carnitine C5-OH and carnitine C4:1-2OH were upregulated at the late stage of 5 weeks (Figure 14 D).

Relationship of lipid composition in CSF and 6-OHDA lesions

Correlation analysis was used to evaluate the lipids in CSF as biomarkers for nigrostriatal degeneration in the 6-OHDA model. The rotation induced by apomorphine, as a behavior detection, can help to estimate the lesion of 6-OHDA in SN. Eicosanoids, TG and DG were selected by their changed expression at 5 weeks after 6-OHDA lesion. The levels of eicosanoids and TG had a positive relationship with rotation behavior with P values of < 0.001 and 0.006, respectively (Figure 15 A). The negative relationship of eicosanoids and TG with TH expression in the SN also exhibit different levels of significance with P values of <0.001 and 0.005, respectively (Figure 15 B). CAR exhibited a positive relationship with TH expression with a P value < 0.001 but an unclear relationship with rotation behavior with a P value of 0.558. Regarding lipid species, carnitine C5-OH, carnitine C4:1-2OH and TG (16:0_20:0_18:1) were also selected based on late-stage changes in expression. All of the lipid species exhibited significant positive relationships with rotation behavior, including carnitine C5-OH with a P value of 0.279, carnitine C4:1-2OH with a P value of 0.280, and TG (16:0_20:0_18:1) with a P value of 0.011 (Figure 16 A). TG (16:0_20:0_18:1) exhibited a negative relationship with TH expression in the SN with a P value of 0.011. Both carnitine C5-OH and C4:1-2OH had generally positive relationships with TH expression (Figure 16 B). These associations suggested that the eicosanoids, TG families and TG (16:0_20:0_18:1) may be sensitive biomarkers of nigrostriatal degeneration in PD.

Clinical and preclinical diagnosis remains a major challenge for PD. When PD patients exhibit motor symptoms, up to 50–70% of DA neurons in the SN have been lost²². Our study aims to explore biomarkers of PD that help realize early diagnosis and intervention for disease. Here, we used lipidomic approaches to develop a special lipid signature expressed in 6-OHDA-induced PD rats and found changes in the expression of lipid families and species in CSF that followed the progression of 6-OHDA lesions. We suggest that eicosanoids, TG and TG (16:0_20:0_18:1), which are related to 6-OHDA-induced pathological and behavior abnormalities, may represent suitable biomarkers for PD.

The lipid families in CSF were classified into eicosanoids, FFA, BA, PS, PI, SPH, HexCer, PG, CAR, CE, CER, DG, LPC, MG, PC, PE, SM and TG in this study. Among them, eicosanoids, DG and TG were significantly upregulated compared with the control. Many studies have investigated to the role of TG in serum associated with ischemic cerebrovascular disease, but few studies have focused on the function of TG in neurodegenerative diseases²³. In nondemented aging persons, TG levels in serum are inversely correlated with executive function²⁴. Some studies suggest that increased serum TG exhibits a close relationship with poor cognition²⁵, whereas other studies report either no relation²⁶ or suggest the opposite conclusion²⁷. Circulating TG can increase the activity of the reward circuit in brain function²⁸ and may be related to smaller cortical thickness in older persons with cerebral Aβ burden²⁹. TG was proposed to cross the BBB and induce special central receptor resistance, including leptin and insulin, which resulted in functional changes in feeding and cognition^{30, 31}. TG metabolism also affects DA release, which plays multiple pivotal roles in reward systems, associative learning, motor planning, and decision-making^32–34. In PD patients, the concentration of TG in the SN is correlated with inflammation-attenuating signaling³⁵. TG in the diet can affect the function of DA neurons to modulate dopamine-dependent behaviors and promote microglial polarization in the SN^{28, 36}. Some studies hold the opinion that a high-fat diet includes neurotoxins and accelerates the loss of DA neurons in PD^{37, 38}. Regarding TG in CSF, relative reports on PD are rare. This study showed that the TG family and TG (16:0_20:0_18:1) exhibited significantly upregulated levels at 2 weeks after 6-OHDA-induced lesion formation. These upregulated levels partly recovered in the subsequent 3 weeks but were still higher than those noted in the control at 5 weeks. The pathological and behavioral abnormalities in the 6-OHDA model were most serious at 2 weeks, and their severity was consistent with the change in TG levels in the CSF. The above evidence indicated that the levels of the TG family members and TG (16:0_20:0_18:1) in CSF may be indicative of the presence of SN lesions in PD.

Eicosanoids and FFA are other lipid families that exhibit significantly changed expression in CSF at 2 weeks after 6-OHDA-induced lesion formation in this study. Eicosanoids, as amphipathic molecules, are derived from the oxidation of arachidonic acid and polyunsaturated fatty acids³⁹. Its subfamilies include prostaglandins, leukotrienes, thromboxanes, resolvins, lipoxins, protectins, isoprostanes, maresins, endocannabinoids, and others³⁹. Eicosanoids in the CNS regulate neuronal functions, including memory and learning, cerebral blood flow, sleep, and neuroinflammation^40–42. In aging mice, altered eicosanoid profiles in the hippocampus exhibit a close relationship with inflammation, synaptic dysfunction and cognitive impairment⁴³. The eicosanoid lipidome was activated in the brains of AD patients and was especially increased in carriers of the APOE4 allele⁴⁰. Regarding PD, relevant reports are lacking. This PD study showed that significantly upregulated levels at both 2 and 5 weeks after 6-OHDA-induced lesion formation were found in the eicosanoid family but not its lipids. The changed expression of the eicosanoid family potentially included all the small differences in its lipids to achieve significance. This finding suggests that the eicosanoid family may be more suitable as a biomarker for PD than its lipids.

Regarding FFA, its accumulation in the brain is associated with some inherited neurological diseases, including Refsum disease and X-linked adrenoleukodystrophy⁴⁴. In AD patients, lower FFA levels were noted in APOE4 carriers compared with noncarriers⁴⁰. FFA may prevent the assembly of amyloid and tau filaments^{45, 46}. However, the roles of eicosanoids and FFA in nigrostriatal function and the pathogenesis of PD remain unclear. In this study, significantly upregulated FFA levels were exclusively noted 2 weeks after 6-OHDA-induced lesion formation but not at 5 weeks. This result suggested that FFAs in CSF may be released by damaged DA neurons or other cells, which were present in limited numbers at 5 weeks. Thus, alterations in FFA expression in CSF may serve as a useful biomarker that helps to identify pathological changes in the early stage of PD. More details regarding the function and expression of eicosanoids and FFA in PD are still needed.

Carnitine C5-OH and carnitine C4:1-2OH exhibited a special expression profile in which upregulated levels were found only at 5 weeks after 6-OHDA-induced lesion formation. Acetyl-L-carnitine has been a research hotspot in AD⁴⁷. Some studies have suggested that acetyl-L-carnitine can protect against inflammatory processes, revert the neurodegeneration caused by excitotoxicity, and play a preventive role in AD^47–49. The role of acetyl-L-carnitine in PD remains unclear. We found upregulated acetyl-L-carnitine expression at the late stage of 6-OHDA-induced lesion formation with improved pathological and behavioral abnormalities in a rat model. This finding suggested that carnitine C5-OH and carnitine C4:1-2OH may be related to the recovery of nigrostriatal lesions. It seems that different lipid profiles were found at different stages of 6-OHDA-induced lesion formation. A single lipid family or species is not sufficient to depict the complete picture of 6-OHDA-induced lesion formation in the brain. Different expression patterns involving multiple lipid families and species exhibit more sensitivity and accuracy for evaluating PD progression.

In conclusion, 6-OHDA-induced lesions cause changes in the lipidomic profile of CSF, and these changes exhibit potential value as a potential diagnostic tool for PD. Characterization of lipid metabolism profiles that are altered following 6-OHDA-induced lesion formation, especially eicosanoids, TG families and TG (16:0_20:0_18:1), may open a new window for the evaluation of nigrostriatal impairment in PD and help to develop new anti-PD drugs.

Acknowledgements

We thank Juan Lin for the excellent technical assistance.

Funding

This work was supported by National Natural Science Foundation of China (81701254, 82071444), General Project of Basic and Applied Basic Research of Guangzhou Bureau of Science and Technology (2060206), Yang-cheng Scholar Project of Guangzhou Municipal Bureau of Education (202032790), General Project of Natural Science Foundation of Guangdong Province (2021A1515011043) and Guangzhou key medical discipline grant (2021-2023).

Availability of data and materials

All data generated and analyzed in the current study were collected from distinct subjects and are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

All procedures involving animals were approved and carried out according to the guidelines of the Animal Experiments and Ethics Committee of Guangzhou Medical University (No. 2020143, 21 August 2020).

Consent for publication

Not applicable.

Competing interests

None of the authors report any disclosures.

Author information

Author notes

These authors contributed equally: Jiewen Qiu, Guoyou Peng, Yuting Tang.

Authors and Affiliations

Department of Neurology, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China: Jiewen Qiu, Yuting Tang, Lijian Wei, Guoyou Peng, Yuwan Lin, Wei Dai, Zhiling Zhang, Yilin Su, Xiang Chen, Hanqun Liu, Liuyan Ding, Wenyuan Guo, Pingyi Xu, Mingshu Mo

Department of Physiology, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, China: Xiaoqin Zhu

Department of internal medicine, Huilai people's Hospital, Jieyang, China: Mingshu Mo

Corresponding authors

Correspondence to Mingshu Mo, Pingyi Xu or Xiaoqin Zhu.

Contributions

Mingshu Mo, Xiaoqin Zhu, and Pingyi Xu conceived and designed the study and interpreted experiments. Yuting Tang, Lijian Wei, Yilin Su, and Jiewen Qiu performed the study and prepared the initial draft of the manuscript. Mingshu Mo, Pingyi Xu, Guoyou Peng, and Yuting Tang supervised the project. Wei Dai, Jiewen Qiu, Yuwan Lin, Wenyuan Guo, Zhilin Zhang, Liuyan Ding, Hanqun Liu and Xiang Chen provided the clinical samples and data. All authors read and approved the final submission.

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Lipid profiles in the cerebrospinal fluid of rats with 6-hydroxydopamine-induced lesions as a model of Parkinson's disease

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