The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene, a potent tumor suppressor, is frequently mutated in human malignancies.9,54,55 The protein it expresses is a dual phosphatase with both protein and lipid phosphatase activities. Since the first discovery and identification in 1997,30 PTEN has been studied extensively in cancers, including solid tumors and lymphoid malignancies. Biochemically, PTEN plays 2 distinct roles depending on whether it is in the cytoplasm or nucleus.37,55 In the cytoplasm, PTEN antagonizes phosphoinositide 3-kinase (PI3K) and dephosphorylates (3, 4, 5) phosphatidylinositol (PIP3) by the phosphatase domain and further inhibits the threonine kinase (AKT)/mammalian rapamycin target protein (mTOR) pathway (AKT/mTOR pathway). When in the nucleus, PTEN functions through the Jun-N-terminal kinase (JNK) pathway. In addition, PTEN plays a crucial role in fundamental cellular processes, including the regulation of DNA repair, cellular senescence, and cell migration, etc.5,25,49,55
To date, a few reports have suggested the role of PTEN in neuropathic pain. The research by Huang et al. revealed that neuropathic pain induced by nerve injury was correlated with PTEN and the AKT/mTOR pathway in the spinal cord and intrathecal administration of PTEN mediated by adenovirus mitigated nociceptive sensitization.19 Another study showed that PTEN and JAK2 were involved in trigeminal neuropathic pain.31 Moreover, in vivo inhibition of PTEN increased the percentage of δ-opioid receptors expressed on the surface of trigeminal ganglion neurons and allowed efficient δR-mediated antihyperalgesia in mice.44 Although these studies confirm the role of PTEN in nociceptive signaling, they provide little information about the specific mechanism of PTEN in neuropathic pain.
Cholesterol or lipid rafts, the main lipids in the mammalian plasma membrane, are responsible for the resilience of the biological membrane and related to cellular function and viability.45,57 Cholesterol and sphingomyelin form the raft-like domains involved in the trafficking of surface proteins and the formation of dynamic signaling platforms. These raft-like domains have been demonstrated as a regulating switch in nociceptive ion channels, such as Nav1.9 and TRPV1, in neuropathic pain and inflammatory hyperalgesia.1,36,43
In this study, we characterized the specific mechanisms of PTEN's contribution to neuropathic pain that produced the following findings: (1) PTEN was expressed mainly in astrocytes and was dramatically downregulated in the spinal cord after chronic constriction injury (CCI) surgery; (2) spinal upregulation of astrocytic PTEN effectively alleviated CCI-induced pain and reduced glial activation, neuronal activity, and neuroinflammation; (3) PTEN regulated cholesterol biosynthesis mainly by interacting with enzymes involved in cholesterol biosynthesis processing; and (4) cholesterol replenishment attenuated CCI-induced pain hypersensitivity and, importantly, suppressed neuronal activity and glial activation. These results imply that disturbance of PTEN-regulated endogenous cholesterol biosynthesis in spinal astrocytes may underlie the development of pain after peripheral nerve injury, and recovery of PTEN or cholesterol might be an effective therapeutic strategy for neuropathic pain.
2. Materials and methods
2.1. Animals and surgery
Adult Sprague–Dawley rats (specific pathogen-free [SPF], female, 180-220 g) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Ptenfl/fl mice were a gift from Professor Hongbing Zhang at the Chinese Academy of Medical Sciences. All animal procedures performed in this study were approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences, Institute of Basic Medical Sciences (approval number #211-2014). Rats and mice were kept in the Laboratory Animal Center of School of Basic Medicine, Peking Union Medical College, in a temperature-controlled and humidity-controlled environment with an automatic 12-hour light–dark cycle and access to food and water ad libitum. The rat model of CCI (n = 72) was produced according to the description by Bennett and Xie6 and our previous study.56 In brief, animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p., Sigma-Aldrich, St. Louis, MO), and the right sciatic nerve was exposed to place 4 loose ligatures (4-0 chromic gut) around the nerve at 1-mm intervals and then to suture muscle and skin incisions. For sham operations, the surgery was performed only to expose the right sciatic nerve but without ligation.
2.2. Drugs and administration
The inhibitor of PTEN, SF1670, was purchased from Selleckchem (Houston, TX). Indole-3-carbinol (I3C), a potent PTEN protector,10 was purchased from Sigma-Aldrich. These 2 reagents were dissolved in dimethylsulfoxide (DMSO) first and diluted in saline solution. SF1670 was injected intrathecally (0.3 mg/kg) in naive rats32 to observe the nociceptive effect of PTEN deficiency, whereas I3C was injected in CCI rats (14 d after surgery) intrathecally (2 mg/kg)29 to verify the analgesic effects under prevention of PTEN degradation. AAV2/9-GFAP-r-PTEN-3×Flag (gPTEN), AAV2/9-GFAP-r-HMGCR-3×Flag (gHMGCR), AAV2/9-GFAP-Null (gNull), and AAV2/9-GFAP-Cre-EGFP (AAV-gCre) were synthesized by Hanbio Biotechnology (Shanghai, China). Intrathecal injection of AAV (10 µL) was performed 1 week before CCI surgery in rats. Intrathecal injection of AAV-gCre (5 µL) into Ptenfl/fl mice was conducted to generate astrocyte-specific Pten knockout (Pten CKO) mice. Water-soluble cholesterol (Sigma-Aldrich) was administered intrathecally at a concentration of 0.5 or 1 mM. All intrathecal injections were made with a 30-G needle between the L5 and L6 intervertebral spaces under isoflurane (2%) anesthesia.20
2.3. Behavioral test
Rats and mice were acclimatized to the testing environment for at least 2 consecutive days before baseline testing and were tested for mechanical and thermal hyperalgesia in a blind manner. Before each test, habituation was performed for 30 minutes, as described in our previous studies.33,34,56
For the Hargreaves test,15 rats (n = 48) were placed individually in a transparent plexiglass box (10 × 20 × 20 cm) placed on a 2-mm-thick glass plate, and the plantar surface of the hind paw was exposed to a beam of radiant heat from a thermal stimulator (BME-410C Plantar Test Apparatus, China). A cutoff time of 20 seconds was setup to avoid potential damage, and the test time interval between the tests of 2 feet was 5 minutes. The average of 3 recorded test times was considered the paw withdrawal thermal latency (PWL, s). For the von Frey test, rats (n = 48) were placed in a Plexiglas chamber (10 × 20 × 20 cm) placed on a wire grid floor, and the plantar surface of the hind paw was stimulated using an electronic von Frey anesthesiometer (Electronic von Frey 2390, IITC Life Science, Woodland Hills, CA). The maximum force (g) was automatically recorded, and the average of 3 successive force (g) readings was used as the paw withdrawal mechanical threshold (PWT, g). For the mouse von Frey test, the up–down method by Dixon was used.11 In brief, the plantar surface of the hind paw was simulated with a series of von Frey hairs (0.02-2.56 g, Stoelting, Wood Dale, IL). If a mouse responded to a given filament, the next lowest filament was used, or the next highest filament was used. Thirty-two mice were used for the von Frey test. For the mouse tail immersion test, the water temperature was set at 48, 50, or 52°C. The lower tail sections of mice were soaked in water, and the time of tail withdrawal from water was determined as the reaction time. The cutoff time was set at 10 seconds to avoid potential injury. Sixteen mice were used for this test.
2.4. Quantitative proteomics
Rats (n = 24) were deeply anesthetized with sodium pentobarbital, and protein samples were obtained from the L5 spinal dorsal horn in rats of 4 groups (6 rats per group): naive rats, CCI 21d rats, AAV-gNull rats, and AAV-gPTEN rats. After dissolving in 8 M urea and measuring the concentration using a NanoDrop 2000 (Thermo Fisher Scientific, MA), equal amounts of 6 samples from each group were mixed to make total protein at 100 µg. Equal amounts of proteins from each sample were reduced with dithiothreitol and alkylated with iodoacetamide (IAA) before dilution in 1 M urea. Then, the samples were sent to Beijing Qinglian Biotech Co, Ltd for further analysis, including tandem mass tag (TMT) labeling, peptide fractionation with high-performance liquid chromatography, liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) analysis, and subsequent data analysis.
For TMT labeling, 100 mg of protein from the 4 groups diluted in 1 M urea was reduced with dithiothreitol (Little Chalfont, Bucks, United Kingdom) and alkylated with iodoacetamide (IAA, Little Chalfont). After digestion in trypsin/Lys-C (Promega, WI) for 12 hours and termination by heating at 60°C, the proteins were then labeled using the TMT Mass Tagging Kit (Thermo Fisher Scientific) following the manufacturer's instruction. Four TMT labels were used to label the different groups: TMT-129 was used for the AAV-gPTEN group, TMT-128 for the AAV-gNull group, TMT-127 for the CCI 21d group, and TMT-126 for the naive group. After labeling, samples were dissolved in 0.1% trifluoroacetic acid (TFA). For high-performance liquid chromatography separation, TMT-labeled peptides (100 μL in 0.1% TFA) were mixed equally and desalted by peptide desalting spin columns (89852, Thermo Fisher Scientific) and then fractionated using a C18 column (Waters BEH C18 4.6 × 250 mm, 5 μm) on the UltiMate 3000 UHPLC (Thermo Fisher Scientific) with a flow rate of 1 mL/min. The column oven was set at 50°C. Mobile phases A (2% acetonitrile, pH 10.0) and B (98% acetonitrile, pH 10.0) were used to develop a gradient elution. Then, fractions were dried and resolved in 0.1% TFA for mass spectrometry analysis. For LC–MS/MS analysis, peptides were separated by a 120-minute gradient elution at a flow rate of 0.300 μL/min with the EASY-nLC 1000 system, which was directly interfaced with the Thermo Orbitrap Fusion mass spectrometer. The analytical column was purchased from Thermo Fisher (75 μm ID, 150 mm length; packed with C-18 resin). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The Orbitrap Fusion mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 3.0 software, and the scan cycle consisted of a single full-scan mass spectrum in the Orbitrap (350-1550 m/z, 120,000 resolution) followed by 3 seconds data-dependent MS/MS scans in an ion routing multipole at 38% normalized collision energy HCD (higher energy collisional dissociation). The MS/MS spectra from each LC–MS/MS run were searched against the selected database using the Proteome Discovery searching algorithm (version 2.1). The MS/MS spectra from each LC–MS/MS run were searched against rat fasta from UniProt using an in-house Proteome Discoverer (version PD1.4, Thermo Fisher Scientific). The search criteria were as follows: full tryptic specificity was required, 2 missed cleavages were allowed, carbamidomethylation (C) was set as the fixed modifications, oxidation (M) and TMT 6-plex were set as the variable modifications, precursor ion mass tolerances were set at 15 ppm for all MS acquired in an Orbitrap mass analyzer, and fragment ion mass tolerance was set at 0.02 Da for all MS2 spectra acquired. The peptide false discovery rate (FDR) was calculated using the Target Decoy PSM Validator provided by PD. When the q value was smaller than 1%, the peptide-spectrum match (PSM) was considered to be correct. FDR was determined based on PSMs when searched against the reverse decoy database. Peptides only assigned to a given protein group were considered unique. The FDR was also set to 0.01 for protein identification.
Relative protein quantification was performed using Proteome Discoverer software (version 2.1) according to the manufacturer's instructions on the 4 reporter ion intensities per peptide. Quantitation was performed only for proteins with 1 or more unique peptide matches. Protein ratios were calculated as the median of all peptide hits belonging to a protein. Quantitative precision was expressed as protein ratio variability.
2.5. Bioinformatic analysis
Relative protein abundances are presented as ratios of TMT-127/126 (CCI 21d/naive) and TMT-129/128 (AAV-gPTEN/AAV-gNull). A log2 (127/126) ratio ≥ 0.298 and a log2 (129/128) ratio ≥ 0.383 were set as upregulated proteins, and a log2 (131/128) ratio ≤ −0.25 and a log2 (129/128) ratio ≤ −0.177 were set as downregulated proteins according to a 95% prediction interval (Supplemental Fig. 1A and B, available at https://links.lww.com/PAIN/B645). For hierarchical clustering analysis, the MS differentially expressed peptide (DEP) data (Supplemental Table S1, available at https://links.lww.com/PAIN/B645) were input into JMP Pro, and a heatmap was then constructed using hierarchical clustering. Gene Ontology (GO) analysis was performed using DAVID bioinformatics resources (https://david.ncifcrf.gov). The Wiki pathway was displayed with Cytoscape (version 3.7). Protein–protein interaction networks of DEPs were constructed with an online STRING database (http://string-db.org). “Experimental” and “database” evidence was set at a medium (0.4) confidence level.
For coimmunoprecipitation (Co-IP), protein samples from the spinal dorsal horn were harvested, lysed in NP-40–containing lysis buffer supplemented with protease inhibitor cocktail (CWBio, Beijing, China), and then immunoprecipitated as instructed by the protocol of the Pierce Crosslink Magnetic IP/Co-IP Kit (Thermo Fisher Scientific, #88805) with the indicated antibodies.
2.7. Western blotting
After transcardial perfusion with PBS, the spinal cord was harvested and homogenized in lysis buffer (CWBio, Beijing, China) containing a protease inhibitor cocktail and a phosphate inhibitor cocktail (CWBio). Protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL). The homogenates (20 µg total protein) were separated on 12% SDS–PAGE gels and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk in TBST, the membrane was incubated overnight at 4°C with primary antibodies. The antibodies were as follows: PTEN (rabbit, 1:1000, Abcam, ab170941), FLAG (mouse, 1:3000, Sigma-Aldrich, F3165), HMGCR (mouse, 1:500, Santa Cruz, sc-271595), HMGCS1 (mouse, 1:500, Santa Cruz, sc-166763), MVD (mouse, 1:500, Santa Cruz, sc-376975), TNF-α (rabbit, 1:1000 Proteintech Group, 17590-1-AP), IL-1β (rabbit, 1:1000, Abcam, ab283818), IL-6 (rabbit, 1:1000, Abcam, ab6672), and GAPDH (rabbit, 1:3000, BBI Life Sciences, 110016). The membrane was further incubated with an HRP-conjugated secondary antibody for 1 hour at room temperature. Bands were detected using an eECL Kit and scanned by the ChemiDoc XRS system (Bio-Rad, Hercules, CA). The intensity of the selected bands was analyzed using ImageJ software.
2.8. Cholesterol quantitation assay
For dorsal horn total cholesterol (free cholesterol and cholesteryl esters) content determination, a Cholesterol/Cholesteryl Ester Quantitation Assay Kit (ab65359, Abcam) was used. Equal tissues (1 g) were taken from the ipsilateral and contralateral spinal cord of each rat (naive rats, CCI 21d rats, SF1670-treated or I3C-treated rats, and AAV-gNull or AAV-gPTEN or AAV-gHMGCR rats) or mouse (littermate, Ptenfl/fl, or Pten CKO). Then, the tissue was immediately processed for lipid extraction and colorimetric assay according to the manufacturer's instructions.
Rats and mice were deeply anesthetized with sodium pentobarbital and transcardially perfused with 0.01 M PBS, followed by 4% paraformaldehyde in 0.01 M PBS. The L5 spinal cord segment was isolated and used for immunohistochemistry as described previously.24,35 In brief, the tissue sections were first permeabilized in 0.1% Triton X-100 in PBS and then blocked with 10% donkey serum for 2 hours at room temperature, followed by overnight incubation at 4°C with primary antibodies. The following primary antibodies were used: PTEN (rabbit, 1:400, Cayman Chemical, 10005059), glial fibrillary acidic protein (GFAP, mouse, 1:400, Abcam, ab4648), NeuN (mouse, 1:400, Abcam, ab104224), CD11b/c (mouse, 1:400, Abcam, ab1211), c-fos (rabbit, 1:400, CST, 2250S), anti-FLAG (DDDDK tag, rabbit, 1:400, Abcam, ab205606), and calcitonin gene-related peptide (rabbit, 1:400, CST, 14959T). Sections were then incubated with the proper secondary antibodies (Alexa Flour 488-conjugated donkey anti-mouse, 1:400; Alexa Flour 594-conjugated donkey anti-mouse, 1:400; Alexa Flour 488-conjugated donkey anti-rabbit, 1:400; and Alexa Flour 594-conjugated donkey anti-rabbit, 1:400, Jackson ImmunoResearch Laboratories) for 1 hour at room temperature. Alexa Fluor 594-conjugated IB4 (1:400, Invitrogen) was added as a secondary antibody. After washed with 0.01 M PBS and coverslipped with VECTASHIELD mounting medium with DAPI (ZSGB-BIO, China), the stained sections were examined using a laser confocal microscopic imaging system (FV1000 and Olympus FluoView software, Olympus, Japan). Images were analyzed with ImageJ software.
2.10. Statistical analysis
Data values are presented as the mean ± SEM. SPSS software (version 17.0) was used for statistical analysis. The Student t test was used to test the difference between 2 groups. To determine statistical comparisons of differences among 3 or more groups in western blotting and immunohistochemistry experiments, we used one-way analysis of variance followed by the Bonferroni post hoc test. Two-way analysis of variance followed by the Bonferroni post hoc test was used to determine significant differences in behavioral tests. A statistically significant difference was defined as a 2-sided P value < 0.05.
3.1. Chronic constriction injury dramatically downregulated the expression of phosphatase and tensin homolog deleted on chromosome 10 in spinal astrocytes
To determine the expression and distribution of PTEN in the spinal cord, we performed western blotting to compare protein levels between naive, sham, CCI 7d, CCI 14d, and CCI 21d rats. The results revealed that PTEN expression in the spinal cord was reduced significantly at postoperative days 14 and 21 in the CCI group compared with naive animals (P < 0.05 vs naive group; Fig. 1A), whereas CCI 7d group rats and animals receiving the sham operation showed no significant difference (P > 0.05 vs naive group; Fig. 1A). We then performed a spinal immunofluorescence analysis using antibodies against PTEN. Compared with the sham group (Fig. 1B), immunofluorescence assessment of the ipsilateral dorsal horn at 7 d and 14 d after injury revealed significant decreases in PTEN (Fig. 1B), similar to the findings in a previous study.19 Quantification of the fluorescence intensity further confirmed the downregulation of PTEN (P < 0.01 vs ipsilateral in the sham group or contralateral in the same group; Fig. 1B) 7 and 14 days after CCI surgery.
To clarify the specificity of PTEN expression in cells, double immunofluorescence staining was applied using different cell-type markers. The results showed that in naive rats, PTEN was almost expressed in GFAP-positive astrocytes but not in NeuN-positive neurons or CD11b/c-positive microglia (Fig. 1C). Thus, PTEN is expressed in astrocytes in the rat spinal cord.
3.2. Spinal astrocytic phosphatase and tensin homolog deleted on chromosome 10 suppresses neuropathic pain after chronic constriction injury
After CCI, neuropathic pain symptoms such as mechanical allodynia and heat hyperalgesia developed on day 3 and sustained for many weeks.56 To assess the role of PTEN in neuropathic pain, the PTEN inhibitor SF1670 was intrathecally (i.t.) injected into naive rats. Then, we checked the time course of pain behavior 6 hours, 12 hours, 1 day, and 2 days after injection. The i.t. administration of SF1670 (0.3 mg/kg, 15 μL) significantly induced thermal hyperalgesia (PWL) and mechanical allodynia (PWT) from 6 hours to 1 day postinjection compared with that of i.t. injection of DMSO (P < 0.01 vs DMSO injection group; Figs. 2A–C). Rats returned to baseline levels after 2 days of injection.
We further examined the antinociceptive effect of indole-3-carbinol (I3C) in CCI rats. I3C is a derivative of glucobrassicin in cruciferous vegetables, which inhibits WWP1-mediated PTEN polyubiquitination and ultimately reactivates PTEN.10 Consistent with previous reports,2,10,19 CCI surgery–induced mechanical allodynia and thermal hyperalgesia were sustained for several weeks. After we intrathecally injected I3C (2 mg/kg, 15 μL) on day 14 after CCI surgery, thermal hyperalgesia induced by CCI operation was alleviated at 6 hours, 12 hours, and 3 days after I3C injection (P < 0.01; Fig. 2D). Meanwhile, mechanical allodynia was also markedly attenuated. The antiallodynic effect became evident as early as 6 hours after the injection and was maintained for 4 days (P < 0.01; Fig. 2E).
To further investigate the function of PTEN in spinal astrocytes, injection (i.t.) of AAV9-mediated astrocytic PTEN (AAV-gPTEN) was conducted to overexpress spinal PTEN in naive rats 7 days before CCI injury. Then, we tested the rat behavior until 21 days after surgery. In contrast with vehicle injection (AAV-gNull), i.t. injection of AAV-gPTEN significantly attenuated CCI-induced thermal hyperalgesia at days 7, 14, and 21 after CCI (P < 0.001 or 0.0001; Fig. 2F) and mechanical allodynia from 7 to 21 days after CCI (P < 0.001 or 0.0001; Fig. 2G). After behavior testing, we harvested the L4-6 spinal cord on day 30 after AAV injection. Further immunofluorescence and western blot assessment of the spinal cord indicated that FLAG and PTEN expression levels were upregulated by more than 2-fold compared with vehicle injection (Figs. 3A and B) and that FLAG and PTEN were expressed specific in astrocytes (Figs. 3C–E). Together, these results suggested that the expression level of PTEN in spinal astrocytes was successfully upregulated by i.t. AAV-gPTEN administration.
3.3. Astrocytic PTEN is involved in the cholesterol biosynthesis pathway of the spinal dorsal horn
To figure out the potential downstream regulatory target of PTEN in the spinal dorsal horn of CCI rats, TMT-based quantitative proteomics was performed using samples from the dorsal horn of 4 groups: naive rats, CCI 21d rats, AAV-gNull rats, and AAV-gPTEN rats. A total of 4697 proteins (Score SEQUEST HT > 0) were identified, 221 of which were differentially expressed (Supplemental Table S1, available at https://links.lww.com/PAIN/B645). The DEPs were then grouped into 4 clusters based on the protein expression ratio and are shown in the heatmap (Fig. 4A). The DEPs of the CCI model in cluster 2 (upregulated proteins) and cluster 3 (downregulated proteins) were reversed by PTEN overexpression. The next GO classification indicated that DEPs in cluster 3 were enriched in the cholesterol biosynthesis process, isoprenoid biosynthetic process, intracellular protein transport, and leucine metabolic process (Fig. 4B, details in Supplemental Table S2, available at https://links.lww.com/PAIN/B645). Next, the proteins in cluster 2 and cluster 3 were then analyzed with STRING, respectively. The protein–protein interaction results of cluster 3 (Fig. 4C) showed that 7 enzymes catalyze cholesterol biosynthesis, including HMGCR, HMGCS1, MVD, MVK, acetoacetyl-CoA synthetase, phosphatidate cytidylyltransferase 2, and PCYT1B. These enzymes were downregulated in the CCI group and upregulated by PTEN overexpression. These data suggest that PTEN regulates enzymes of the cholesterol biosynthesis process and may ultimately affect cholesterol content in CCI rats.
Next, we checked the protein expression of 3 enzymes (HMGCR, HMGCS1, and MVD) catalyzing cholesterol biosynthesis and cholesterol content according to the results of quantitative proteomics. Western blotting showed that the protein levels of HMGCR, HMGCS1, and MVD were significantly downregulated in CCI rats at 7 d and 21 d (P < 0.05 or P < 0.01 vs sham or naive) and were reversed by pretreatment with AAV-gPTEN (P < 0.01 vs AAV-gNull group; Fig. 4D). We then explored how PTEN regulates the expression level of enzymes in the cholesterol biosynthesis pathway. Because PTEN is a phosphatase with a dephosphorylation function, we detected its interactions with HMGCR, HMGCS1, and MVD through Co-IP experiments using spinal dorsal horn tissues in naive rats. As shown in Figure 4E, immunoprecipitation with commercially available anti-PTEN antibodies resulted in coprecipitation of HMGCR, HMGCS1, and MVD. Immunoprecipitation with anti-IgG antibody was used as a negative control. To further confirm the specificity of this interaction, immunoprecipitation with commercially available anti-HMGCR, HMGCS1, or MVD antibodies was performed in spinal dorsal horn tissue. Similarly, immunoprecipitation of these 3 enzymes resulted in coprecipitation of PTEN (Fig. 4E). These results demonstrate a physical interaction between PTEN and enzymes (HMGCR, HMGCS1, and MVD).
We then quantified the total cholesterol (free cholesterol and cholesteryl esters) contents of weighed ipsilateral dorsal horn tissue (1 g) from SF1670-injected rats, I3C-injected rats, and AAV-treated rats. In contrast with injection (i.t.) of an equal volume of DMSO, SF1670 administration significantly (13 ± 3.2%) decreased cholesterol levels in naive rats, whereas I3C injection (i.t.) exhibited a 12 ± 4.2% increase in cholesterol levels (Fig. 4F) in CCI 14d rats. In addition, CCI surgery (CCI 21d) induced a 12 ± 4.2% reduction in cholesterol levels, whereas pretreatment with AAV-gPTEN and AAV-gHMGCR (7 d before surgery) reversed this decreasing trend (Fig. 4G). The present data indicate that peripheral nerve injury decreases the cholesterol level in the spinal dorsal horn and that PTEN accelerates cholesterol synthesis.
Together, the above results indicated that PTEN may directly interact with enzymes catalyzing cholesterol biosynthesis, further intervening in cholesterol levels and ultimately regulating pain (Fig. 4H).
3.4. Astrocytic HMGCR overexpression relieves neuropathic pain after chronic constriction injury
HMGCR is the rate-limiting enzyme in cholesterol biosynthesis and close coupling with PTEN. Therefore, we tested whether HMGCR, like PTEN, is also essential for CCI-induced pain. Injection (i.t.) of AAV9-mediated astrocytic HMGCR (AAV-gHMGCR) was conducted to overexpress spinal HMGCR in naive rats 7 days before CCI injury. The western blot of HMGCR expression level and immunofluorescence of FLAG checked successful injection of AAV and showed that FLAG was almost expressed in GFAP+ astrocytes (Figs. 5A–E). The subsequent cholesterol content determination verified that the total cholesterol level increased in the HMGCR overexpression group (Fig. 5F).
Then, we tested the pain-like behavior of rats until 21 days after CCI. In contrast with vehicle injection (AAV-gNull), i.t. injection of AAV-gHMGCR significantly attenuated CCI-induced thermal hyperalgesia at day 7 after CCI (P < 0.05, Fig. 5G) and mechanical allodynia from 7 to 21 days after CCI (P < 0.05 or 0.0001; Fig. 5H). Thus, these data indicate that astrocytic HMGCR is essential for CCI-induced pain behavior.
3.5. Cholesterol replenishment alleviates chronic constriction injury–induced neuropathic pain
We then tested the effect of cholesterol replenishment in vivo on CCI-induced neuropathic pain. Water-soluble cholesterol was intrathecally injected into rats (injection of saline solution as a control) on postoperative day 14. We observed that both thermal hyperalgesia and mechanical allodynia induced by CCI surgery were significantly attenuated for at least 5 hours after 1 mM cholesterol injection (P < 0.05 or P < 0.01 vs saline; Fig. 6A and B). Cholesterol injection (0.5 mM) (i.t.) also alleviated thermal hyperalgesia in CCI rats for 4 hours (P < 0.001 vs saline; Fig. 6A) but showed no significant inhibitory effect on mechanical allodynia (P > 0.05 vs saline; Fig. 6B). Thus, our data show an antalgic effect of cholesterol supply on neuropathic pain.
3.6. Phosphatase and tensin homolog deleted on chromosome 10–HMGCR–cholesterol pathway suppresses glial activation and the release of proinflammatory cytokines
Activation of spinal glial cells, such as microglia and astrocytes, leads to central sensitization and eventually increases neuronal excitability, which has been demonstrated to play a pivotal role in the pathogenesis of neuropathic pain.22,23,59 We first investigated the activation of glial cells in CCI rats using antibodies against GFAP and CD11b/c. Spinal immunofluorescence analysis showed obvious activation of astrocytes and microglia (∼2-fold increase compared with the sham group; Supplemental Fig. 2A–H, available at https://links.lww.com/PAIN/B645) in the ipsilateral spinal cords 7 and 14 days after CCI. However, the immunoreactivity of NeuN, a neuronal-specific nuclear protein, did not show a significant difference (P > 0.05, vs sham, Supplemental Fig. 2I–L, available at https://links.lww.com/PAIN/B645) in CCI rats.
Next, we explored whether PTEN affected glial activation after CCI. Both astrocyte (GFAP+) and microglial (CD11b/c+) activation were significantly reduced in the ipsilateral dorsal horn in AAV-gPTEN rats (P < 0.01 vs AAV-gNull group; Fig. 6C). By contrast, the contralateral dorsal horn did not exhibit a significant difference in glial activation after AAV-gPTEN injection (Fig. 6C). We then checked the expression level of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). In the ipsilateral dorsal spinal cord, all 3 of them were markedly increased at postoperative day 21 in CCI rats but decreased after the injection of AAV-aPTEN (P < 0.01 or 0.001 vs sham or AAV-null group Supplemental Fig. 3A, available at https://links.lww.com/PAIN/B645). These data indicate that PTEN plays an essential role in the activation of spinal astrocytes and microglia after CCI.
We also observed the effect of cholesterol replenishment on glial activation. The immunostaining results revealed that both microglia (CD11b/c+) and astrocyte (GFAP+) activation were significantly reduced in the ipsilateral dorsal horn in cholesterol-treated rats (P < 0.01 or P < 0.05 vs ipsilateral dorsal horn in the vehicle group; Fig. 6D). By contrast, the contralateral dorsal horn did not exhibit a difference in glial activation after cholesterol supply (Fig. 6D). These data suggest the potential mechanism of cholesterol involvement in nociceptive behavior. Next, we found that both astrocyte (GFAP+) and microglial (CD11b/c+) activation were significantly reduced in the ipsilateral dorsal horn in AAV-gHMGCR rats (P < 0.05 vs AAV-gNull group; Fig. 6E and F). Overall, the above results suggest that the PTEN–HMGCR–cholesterol pathway mediates nociception at least partly by reducing central sensitization. Moreover, the expression level of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in the ipsilateral dorsal spinal cord were markedly increased at postoperative day 21 in CCI rats but decreased after the injection of AAV-gHMGCR (P < 0.05 or 0.01 vs sham or AAV-null group Supplemental Fig. 3B, available at https://links.lww.com/PAIN/B645). Thus, these data indicate that astrocytic HMGCR is essential for CCI-induced central sensitization.
Next, we confirmed the activation of pain signals at the cellular level by examining the expression of neuronal activity marker, c-fos. The immunohistofluorescence analysis showed abundant expression of c-fos protein in ipsilateral superficial laminae of the spinal dorsal horn 21 d after CCI (Supplemental Fig. 3C, available at https://links.lww.com/PAIN/B645). i.t. injection of AAV-gPTEN significantly reversed the activation of c-fos (P < 0.001 vs ipsilateral dorsal horn in the AAV-gNull group; Supplemental Fig. 3D, available at https://links.lww.com/PAIN/B645). The following western bolt result verified that the expression level of c-fos in the ipsilateral dorsal horn was markedly increased at postoperative day 21 in CCI rats but decreased after the injection of AAV-aPTEN (Supplemental Fig. 3E, available at https://links.lww.com/PAIN/B645). In addition, i.t. injection of cholesterol also reversed the activation of c-fos (P < 0.05 vs ipsilateral dorsal horn in the vehicle group; Supplemental Fig. 3F, available at https://links.lww.com/PAIN/B645).
Overall, the above results suggest that the PTEN–HMGCR–cholesterol pathway is involved in both the neuronal activity and central sensitization that results in neuropathic pain.
3.7. HMGCR overexpression reverses conditional knockout of astrocytic PTEN-induced pain in mice
We conducted intrathecal injection of AAV-gCre (5 µL) into Ptenfl/fl mice to generate astrocyte-specific Pten knockout (Pten CKO) mice (Fig. 7A). The GFP was expressed only in GFAP-positive astrocytes, and spinal PTEN expression was specifically lost in astrocytes in Pten CKO mice, as shown by immunofluorescence staining and western bolting (Figs. 7B–F, Supplemental Fig. 4A–D, available at https://links.lww.com/PAIN/B645), indicating the successful injection of AAV and Pten knockout. Pten CKO mice showed naive gross anatomy (Supplemental Fig. 4E, available at https://links.lww.com/PAIN/B645). Pten CKO mice also showed normal distribution patterns in the dorsal horn of the cell marker NeuN (Fig. 7G) and normal innervations of calcitonin gene-related peptide (Supplemental Fig. 4F, available at https://links.lww.com/PAIN/B645) and IB4 (Supplemental Fig. 4G, available at https://links.lww.com/PAIN/B645).
Total cholesterol quantification first revealed that cholesterol levels decreased in the Pten CKO mice (P < 0.01 or P < 0.05 vs littermates or Ptenfl/fl mice, Fig. 7H). Acute pain behaviors were then checked in Ptenfl/fl and Pten CKO mice. Acute thermal sensitivity, as assessed by tail immersion, was indistinguishable in Ptenfl/fl and Pten CKO mice (P > 0.05, Fig. 7I). Notably, i.t. injection of AAV-gCre induced bilateral mechanical allodynia in Ptenfl/fl mice from 7 days to 21 days after injection (P < 0.001, Fig. 7J). Next, immunostaining showed that both GFAP-positive astrocytes and CD11b/c-positive microglia were activated in Pten CKO mice (P < 0.01 or P < 0.05; Fig. 7K and L). Then, AAV-gHMGCR was intrathecally injected into Pten CKO mice. The western bolting showed that the expression level of HMGCR was increased after injection (Fig. 8A). i.t. injection of AAV-gHMGCR significantly attenuated mechanical allodynia from 14 to 21 days postinjection and also reduced glial activation 21 days postinjection, in contrast with AAV-gNull injection (P < 0.05; Fig. 8B–E). These data confirmed the role of the PTEN–HMGCR–cholesterol axis in pain development.
The loss of PTEN was originally found in various human cancers, including those of the brain, breast, and prostate.7,18,30 In addition to its antitumor effect, it has become evident that PTEN is also implicated in several neurological and psychiatric disorders, such as Alzheimer disease8,27 and autism spectrum disorder,39 and diverse neuropathic processes, such as spinal cord injury8 and axon regeneration.14,48 Recent studies have also revealed the role of PTEN in inflammatory pain36 and neuropathic pain.19 Despite such recent progress, the role of PTEN and its underlying mechanism in pain, especially in neuropathic pain, remains obscure. To the best of our knowledge, this is the first study revealing that PTEN may mitigate CCI-induced neuropathic pain by regulating cholesterol biosynthesis in the spinal dorsal horn. Our data indicate that dorsal horn PTEN functions as a painkiller in connection with endogenous cholesterol biosynthesis in neuropathic pain.
Neuropathic pain is refractory and stubborn because its underlying mechanism is quite complicated, which involves both peripheral and central sensitization.40,50 Recently, PTEN in the spinal cord has been found to play an anti-inflammatory effect in neuropathic pain, and CCI could elicit downregulation of ipsilateral dorsal spinal PTEN and upregulation of phospho-PTEN, the inactivated formation of PTEN.19 Consistent with this study, both the present western blotting and immunofluorescence data indicate the downregulation of PTEN in the spinal dorsal horn of CCI rats. In particular, the reduction of PTEN is concurrent with the progression of both the early acute and chronic pain phases. This demonstrates that PTEN is involved in both the occurrence and development of pain. In the central nervous system, PTEN loss of function in neurons increases excitatory synaptic connectivity and changes synaptic transmission.4,13 In our study, PTEN was highly coexpressed with GFAP (an astrocyte marker) in the spinal dorsal horn, indicating that PTEN may be chiefly synthesized by astrocytes. It is quite different from the expression profile in the rat brain, which is preferentially expressed in neurons.12 However, the expression level of c-fos was reduced after AAV-gPTEN injection, which supported that PTEN is also involved in regulating spinal neuronal activity. Our behavioral data further show that inhibiting PTEN by injecting the PTEN inhibitor SF1670 caused mechanical allodynia and thermal hyperalgesia in naive rats. Upregulation of PTEN by I3C or AAV alleviated CCI-induced mechanical allodynia and thermal hyperalgesia, suggesting that spinal PTEN may play an important role in neuropathic pain. Subsequently, astrocyte-specific Pten knockout (Pten CKO) mice showed nociceptive sensitization, and AAV-mediated overexpression of astrocytic PTEN showed an analgesic effect in CCI rats, indicating the leading effects of astrocytic PTEN in neuropathic pain.
Mounting evidence suggests that glia, both spinal microglia and astrocytes, become activated with changing morphology, increasing their number and expressing various genes in neuropathic pain conditions.17,21,52 Activated microglia and astrocytes are involved in neuropathic pain mainly through releasing a variety of proinflammatory cytokines and chemokines, such as TNF-α, IL-1, IL-6, C–C motif chemokine 2, and CXCL13, which have been known to be responsible for the development and maintenance of central sensitization.23 It has been reported that PTEN may inhibit the astrocytic inflammatory response,53 which partly explains its analgesic mechanism. This study shows that CCI surgery induced glial (astrocytic and microglial) activation but did not affect neuron numbers. Moreover, intrathecal injection of AAV-gPTEN reduced glial activation and downregulated the expression levels of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in CCI rats. This provides strong evidence that the prevention of neuropathic pain by overexpression of PTEN is associated with its inhibiting effect on astrocyte activation and also relates to the glial cell–associated inflammatory response.
Over the past years, the mechanism of PTEN in tumor suppression has been summarized into phosphatase-dependent and phosphatase-independent roles. In general, PTEN blocks the phosphatidylinositol 3 kinase (PI3K)/threonine kinase (AKT) signaling pathway (PI3K/AKT pathway), thereby inhibiting cell survival, growth, and proliferation.55 In addition, PTEN seems to participate in specific forms of synaptic plasticity leading to neuronal dysfunction and cognitive impairment.27 Previous studies also showed that spinal AKT/mTOR pathway–mediated cell apoptosis was involved in CCI-induced neuropathic pain and complete Freund adjuvant–induced inflammatory pain.3,19 PTEN is an upstream inhibitory mediator of mTOR and liberates the apoptosis process, which is deterred by neuropathy.46 To determine the potential downstream mechanism of PTEN in regulating pain, quantitative proteomic analysis was conducted. Intriguingly, the data indicated the link between PTEN and cholesterol biosynthesis, isoprenoid biosynthetic process, intracellular protein transport, and leucine metabolic process. As compelling evidence suggests the possibility of cholesterol in controlling pain transmission, which reported serum cholesterol associated with tendon pain,51 low back pain,16 and chest pain,45 we focused on PTEN's regulation of the cholesterol biosynthesis pathway. Further results of western blotting and cholesterol determination showed that i.t. injection of AAV-gPTEN reversed the expression level of enzymes (HMGCR, HMGCS1, and MVD) and spinal total cholesterol content. These results demonstrate that PTEN is involved in neuropathic pain through the regulation of cholesterol biosynthesis.
Theoretically, PTEN may inhibit transcription factors (eg, SREBP or PPARα)26,58 through the AKT/mTOR pathway to regulate cholesterol biosynthesis, or it can directly contribute to inhibiting the activity of enzymes (HMGCR or HMGCS1) in cholesterol biosynthesis and then regulate cholesterol biosynthesis. Previous studies have shown that the reductase activity of HMGCR can be downregulated by phosphorylation.28 In addition, PTEN can improve the expression of HMGCR in the liver for cholesterol synthesis.42 Thus, we hypothesize that depression of PTEN in CCI weakens its dephosphorylation of enzymes in the cholesterol biosynthesis pathway, therefore inhibiting the reductase activity of these enzymes and ultimately reducing cholesterol synthesis in the spinal dorsal horn. Our coimmunoprecipitation experiments identified the interaction between PTEN and enzymes (HMGCR, HMGCS1, and MVD), but the exact phosphorylation site of these enzymes and the specific mechanism remain to be further studied.
Regarding the relationship between cholesterol and pain, recent research has shown that cholesterol levels in skin tissue and sensory DRG are lowered in an inflammatory model and that cholesterol supply (transcutaneous delivery) alleviates acute and chronic inflammatory pain.1 In the central nervous system, it has been shown that ApoE, produced mostly by astrocytes, is critical in redistributing cholesterol and phospholipids for membrane repair and remodeling and is further associated with Alzheimer disease and other neurodegenerative disorders.38 Ulteriorly, cholesterol has been found to play a role in regulating TRPV1 and TRPA1 channels,36,47 and sodium channel Nav1.9 activity is also associated with cholesterol of nociceptive nerves in inflammatory conditions. Moreover, lipid rafts in glial cells also regulate neuroinflammation and pain processing.41 All these studies suggest that the improvement of abnormal neuron activity or inhibition of neuroinflammation is the possible mechanism by which cholesterol controls neuropathic pain. In this study, CCI surgery reduced cholesterol content in the spinal dorsal horn. Moreover, cholesterol replenishment also suppressed glial activation in CCI rats, indicating the involvement of glial-associated inflammatory responses in cholesterol-mediated analgesic effects. A schematic diagram for the potential involvement of the spinal dorsal horn PTEN-regulated cholesterol biosynthesis pathway in neuropathic pain after peripheral nerve injury is shown in Figure 8F.
In conclusion, our results suggest that peripheral nerve injury induces the downregulation of PTEN in the spinal dorsal horn, which further results in cholesterol deletion and glial activation. Treatments targeting PTEN or cholesterol supply might serve as a potential therapeutic strategy for the alleviation of neuropathic pain.
Conflict of interest statement
The authors have no conflicts of interest to declare.
Appendix A. Supplemental digital content
Supplemental digital content associated with this article can be found online at https://links.lww.com/PAIN/B645.
Supplemental video content
A video abstract associated with this article can be found at https://links.lww.com/PAIN/B646.
The authors thank Dr. Wenyin Qiu, Dr. Xiaojing Qian, and Dr. Yongmei Chen in the Department of Anatomy, Histology, and Embryology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, for their technical assistance in immunohistochemistry. The authors also thank Beijing Qinglian Biotech Co, Ltd for their assistance in quantitative proteomics.
Author contributions: Y. Fang, H. Cui, and F. Liu conducted experiments and analyzed the data. F. Liu, Y. Xie, and C. Ma designed the study. All authors participated in writing the manuscript and gave approval before submission.
Ethics approval: All animal procedures performed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College (Beijing, China), and were conducted in accordance with the guidelines of the International Association for the Study of Pain.
Data availability: There are no data, software, databases, or application/tools available apart from those reported in the present study. All data are provided in the manuscript and supplementary data section.
This work was supported by grants from the National Natural Science Foundation of China [#81771205 and #82050004 (C. Ma) and #81801114 (F. Liu)] and the CAMS Innovation Fund for Medical Sciences (CIFMS #2021-1-I2M-025).
. Amsalem M, Poilbout C, Ferracci G, Delmas P, Padilla F. Membrane cholesterol depletion as a trigger of Nav
1.9 channel-mediated inflammatory pain. EMBO J 2018;37:e97349.
. Austin PJ, Wu A, Moalem-Taylor G. Chronic constriction of the sciatic nerve and pain hypersensitivity testing in rats. J Vis Exp 2012;13:3393.
. Baniasadi M, Manaheji H, Maghsoudi N, Danyali S, Zakeri Z, Maghsoudi A, Zaringhalam J. Microglial-induced apoptosis is potentially responsible for hyperalgesia variations during CFA-induced inflammation. Inflammopharmacology 2020;28:475–85.
. Barrows CM, McCabe MP, Chen H, Swann JW, Weston MC. PTEN loss increases the connectivity of fast synaptic motifs and functional connectivity in a developing hippocampal network. J Neurosci 2017;37:8595–611.
. Bassi C, Ho J, Srikumar T, Dowling RJO, Gorrini C, Miller SJ, Mak TW, Neel BG, Raught B, Stambolic V. Nuclear PTEN controls DNA repair and sensitivity to genotoxic stress. Science (New York, NY) 2013;341:395–9.
. Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. PAIN 1988;33:87–107.
. Cai J, Li R, Xu X, Zhang L, Lian R, Fang L, Huang Y, Feng X, Liu X, Li X, Zhu X, Zhang H, Wu J, Zeng M, Song E, He Y, Yin Y, Li J, Li M. CK1α suppresses lung tumour growth by stabilizing PTEN and inducing autophagy. Nat Cell Biol 2018;20:465–78.
. Chang N, El-Hayek YH, Gomez E, Wan Q. Phosphatase PTEN in neuronal injury and brain disorders. Trends Neurosci 2007;30:581–6.
. Chen CY, Chen J, He L, Stiles BL. PTEN: tumor suppressor and metabolic regulator. Front Endocrinol 2018;9:338.
. Choi SR, Beitz AJ, Lee JH. Inhibition of cytochrome P450c17 reduces spinal astrocyte activation in a mouse model of neuropathic pain via regulation of p38 MAPK phosphorylation. Biomed Pharmacother 2019;118:109299.
. Dixon WJ. Efficient analysis of experimental observations. Annu Rev Pharmacol Toxicol 1980;20:441–62.
. Fang EF, Hou Y, Palikaras K, Adriaanse BA, Kerr JS, Yang B, Lautrup S, Hasan-Olive MM, Caponio D, Dan X, Rocktäschel P, Croteau DL, Akbari M, Greig NH, Fladby T, Nilsen H, Cader MZ, Mattson MP, Tavernarakis N, Bohr VA. Mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of Alzheimer's disease. Nat Neurosci 2019;22:401–12.
. Fraser MM, Bayazitov IT, Zakharenko SS, Baker SJ. Phosphatase and tensin homolog, deleted on chromosome 10 deficiency in brain causes defects in synaptic structure, transmission and plasticity, and myelination abnormalities. Neuroscience 2008;151:476–88.
. Geoffroy CG, Lorenzana AO, Kwan JP, Lin K, Ghassemi O, Ma A, Xu N, Creger D, Liu K, He Z, Zheng B. Effects of PTEN and nogo codeletion on corticospinal axon sprouting and regeneration in mice. J Neurosci 2015;35:6413–28.
. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. PAIN 1988;32:77–88.
. Heuch I, Heuch I, Hagen K, Zwart JA. Associations between serum lipid levels and chronic low back pain. Epidemiology (Cambridge, MA) 2010;21:837–41.
. Hoogland ICM, Houbolt C, van Westerloo DJ, van Gool WA, van de Beek D. Systemic inflammation and microglial activation: systematic review of animal experiments. J Neuroinflammation 2015;12:114.
. Hopkins BD, Hodakoski C, Barrows D, Mense SM, Parsons RE. PTEN function: the long and the short of it. Trends Biochem Sci 2014;39:183–90.
. Huang SY, Sung CS, Chen WF, Chen CH, Feng CW, Yang SN, Hung HC, Chen NF, Lin PR, Chen SC, Wang HMD, Chu TH, Tai MH, Wen ZH. Involvement of phosphatase and tensin homolog deleted from chromosome 10 in rodent model of neuropathic pain. J Neuroinflammation 2015;12:59.
. Hylden JL, Wilcox GL. Intrathecal morphine in mice: a new technique. Eur J Pharmacol 1980;67:313–16.
. Inoue K, Tsuda M. Microglia in neuropathic pain: cellular and molecular mechanisms and therapeutic potential. Nat Rev Neurosci 2018;19:138–52.
. Ji RR, Nackley A, Huh Y, Terrando N, Maixner W. Neuroinflammation and central sensitization in chronic and widespread pain. Anesthesiology 2018;129:343–66.
. Jiang BC, Cao DL, Zhang X, Zhang ZJ, He LN, Li CH, Zhang WW, Wu XB, Berta T, Ji RR, Gao YJ. CXCL13 drives spinal astrocyte activation and neuropathic pain via CXCR5. J Clin Invest 2016;126:745–61.
. Jiang H, Cui H, Wang T, Shimada SG, Sun R, Tan Z, Ma C, LaMotte RH. CCL2/CCR2 signaling elicits itch- and pain-like behavior in a murine model of allergic contact dermatitis. Brain Behav Immun 2019;80:464–73.
. Jung SH, Hwang HJ, Kang D, Park HA, Lee HC, Jeong D, Lee K, Park HJ, Ko YG, Lee JS. mTOR kinase leads to PTEN-loss-induced cellular senescence by phosphorylating p53. Oncogene 2019;38:1639–50.
. Kitagishi Y, Matsuda S. Diets involved in PPAR and PI3K/AKT/PTEN pathway may contribute to neuroprotection in a traumatic brain injury. Alzheimer's Res Ther 2013;5:42.
. Knafo S, Esteban JA. PTEN: local and global modulation of neuronal function in health and disease. Trends Neurosci 2017;40:83–91.
. Kuijk LM, Beekman JM, Koster J, Waterham HR, Frenkel J, Coffer PJ. HMG-CoA reductase inhibition induces IL-1beta release through Rac1/PI3K/PKB-dependent caspase-1 activation. Blood 2008;112:3563–73.
. Lee YR, Chen M, Lee JD, Zhang J, Lin SY, Fu TM, Chen H, Ishikawa T, Chiang SY, Katon J, Zhang Y, Shulga YV, Bester AC, Fung J, Monteleone E, Wan L, Shen C, Hsu CH, Papa A, Clohessy JG, Teruya-Feldstein J, Jain S, Wu H, Matesic L, Chen RH, Wei W, Pandolfi PP. Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway. Science (New York, NY) 2019;364:eaau0159.
. Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science (New York, NY) 1997;275:1943–7.
. Li L, Yao L, Wang F, Zhang Z. Knock-down of JAK2 and PTEN on pain behavior in rat model of trigeminal neuropathic pain. Gene 2019;719:144080.
. Li Y, Prasad A, Jia Y, Roy SG, Loison F, Mondal S, Kocjan P, Silberstein LE, Ding S, Luo HR. Pretreatment with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670 augments the efficacy of granulocyte transfusion in a clinically relevant mouse model. Blood 2011;117:6702–13.
. Liu F, Shen X, Su S, Cui H, Fang Y, Wang T, Zhang L, Huang Y, Ma C. Fcγ receptor I-coupled signaling in peripheral nociceptors mediates joint pain in a rat model of rheumatoid arthritis. Arthritis Rheumatol (Hoboken, NJ) 2020;72:1668–78.
. Liu F, Wang Z, Qiu Y, Wei M, Li C, Xie Y, Shen L, Huang Y, Ma C. Suppression of MyD88-dependent signaling alleviates neuropathic pain induced by peripheral nerve injury in the rat. J Neuroinflammation 2017;14:70.
. Liu F, Xu L, Chen N, Zhou M, Li C, Yang Q, Xie Y, Huang Y, Ma C. Neuronal Fc-epsilon receptor I contributes to antigen-evoked pruritus in a murine model of ocular allergy. Brain Behav Immun 2017;61:165–75.
. Liu M, Huang W, Wu D, Priestley JV. TRPV1, but not P2X, requires cholesterol for its function and membrane expression in rat nociceptors. Eur J Neurosci 2006;24:1–6.
. Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 1998;273:13375–8.
. Mahley RW. Central nervous system lipoproteins: ApoE and regulation of cholesterol metabolism. Arterioscler Thromb Vasc Biol 2016;36:1305–15.
. Mahmood U, Ahn S, Yang EJ, Choi M, Kim H, Regan P, Cho K, Kim HS. Dendritic spine anomalies and PTEN alterations in a mouse model of VPA-induced autism spectrum disorder. Pharmacol Res 2018;128:110–21.
. Meacham K, Shepherd A, Mohapatra DP, Haroutounian S. Neuropathic pain: central vs. Peripheral mechanisms. Curr Pain Headache Rep 2017;21:28.
. Miller YI, Navia-Pelaez JM, Corr M, Yaksh TL. Lipid rafts in glial cells: role in neuroinflammation and pain processing. J Lipid Res 2020;61:655–66.
. Parker RA, Miller SJ, Gibson DM. Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver. Impact on activity and degradation of the enzyme. J Biol Chem 1989;264:4877–87.
. Sághy É, Szőke É, Payrits M, Helyes Z, Börzsei R, Erostyák J, Jánosi TZ, Sétáló G Jr, Szolcsányi J. Evidence for the role of lipid rafts and sphingomyelin in Ca2+
-gating of Transient Receptor Potential channels in trigeminal sensory neurons and peripheral nerve terminals. Pharmacol Res 2015;100:101–16.
. Shiwarski DJ, Tipton A, Giraldo MD, Schmidt BF, Gold MS, Pradhan AA, Puthenveedu MA. A PTEN-regulated checkpoint controls surface delivery of δ opioid receptors. J Neurosci 2017;37:3741–52.
. Simons K, Ehehalt R. Cholesterol, lipid rafts, and disease. J Clin Invest 2002;110:597–603.
. Song MS, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol 2012;13:283–96.
. Startek JB, Boonen B, López-Requena A, Talavera A, Alpizar YA, Ghosh D, Van Ranst N, Nilius B, Voets T, Talavera K. Mouse TRPA1 function and membrane localization are modulated by direct interactions with cholesterol. eLife 2019;8:e46084.
. Sun F, Park KK, Belin S, Wang D, Lu T, Chen G, Zhang K, Yeung C, Feng G, Yankner BA, He Z. Sustained axon regeneration induced by co-deletion of PTEN and SOCS3. Nature 2011;480:372–5.
. Suo HB, Zhang KC, Zhao J. MiR-200a promotes cell invasion and migration of ovarian carcinoma by targeting PTEN. Eur Rev Med Pharmacoll Sci 2018;22:4080–9.
. Szok D, Tajti J, Nyári A, Vécsei L. Therapeutic approaches for peripheral and central neuropathic pain. Behav Neurol 2019;2019:8685954.
. Tilley BJ, Cook JL, Docking SI, Gaida JE. Is higher serum cholesterol associated with altered tendon structure or tendon pain? A systematic review. Br J Sports Med 2015;49:1504–9.
. Tozaki-Saitoh H, Tsuda M. Microglia-neuron interactions in the models of neuropathic pain. Biochem Pharmacol 2019;169:113614.
. Vergadi E, Ieronymaki E, Lyroni K, Vaporidi K, Tsatsanis C. Akt signaling pathway in macrophage activation and M1/M2 polarization. J Immunol (Baltimore, MD) 2017;198:1006–14.
. Wang SI, Puc J, Li J, Bruce JN, Cairns P, Sidransky D, Parsons R. Somatic mutations of PTEN in glioblastoma multiforme. Cancer Res 1997;57:4183–6.
. Wang X, Huang H, Young KH. The PTEN tumor suppressor gene and its role in lymphoma pathogenesis. Aging 2015;7:1032–49.
. Wang Z, Liu F, Wei M, Qiu Y, Ma C, Shen L, Huang Y. Chronic constriction injury-induced microRNA-146a-5p alleviates neuropathic pain through suppression of IRAK1/TRAF6 signaling pathway. J Neuroinflammation 2018;15:179.
. Wood WG, Igbavboa U, Müller WE, Eckert GP. Cholesterol asymmetry in synaptic plasma membranes. J Neurochem 2011;116:684–9.
. Yi J, Zhu J, Wu J, Thompson CB, Jiang X. Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis. Proc Natl Acad Sci U S A 2020;117:31189–97.
. Zhao B, Pan Y, Xu H, Song X. Kindlin-1 regulates astrocyte activation and pain sensitivity in rats with neuropathic pain. Reg Anesth Pain Med 2018;43:547–53.