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VPS26A–SNX27 Interaction-Dependent Mglur5 Recycling in Dorsal Horn Neurons Mediates Neuropathic Pain in Rats

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VPS26A–SNX27 Interaction-Dependent Mglur5 Recycling in Dorsal Horn Neurons Mediates Neuropathic Pain in Rats The Journal of Neuroscience, November 4, 2015 • 35(44):14943–14955 • 14943 Behavioral/Cognitive VPS26A–SNX27 Interaction-Dependent mGluR5 Recycling in Dorsal Horn Neurons Mediates Neuropathic Pain in Rats Tzer-Bin Lin,1,2,3 XCheng-Yuan Lai,4,5* Ming-Chun Hsieh,4,6 Hsueh-Hsiao Wang,4* Jen-Kun Cheng,4,7 Yat-Pang Chau,4 X Gin-Den Chen,8 and XHsien-Yu Peng4 1Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, 2Graduate Institute of Basic Medical Science, College of Medicine, China Medical University, Taichung, Taiwan, 3Department of Biotechnology, Asia University, Taichung, Taiwan, 4Department of Medicine, Mackay Medical College, New Taipei, Taiwan, 5Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan, 6Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan, 7Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan, and 8Department of Obstetrics and Gynecology, Chung-Shan Medical University Hospital, Chung-Shan Medical University, Taichung, Taiwan Retromer, which crucially contributes to endosomal sorting machinery through the retrieval and recycling of signaling receptors away from degradation, has been identified as a critical element for glutamatergic-receptor-dependent neural plasticity at excitatory synapses. We observed it accompanied by behavioral allodynia; neuropathic injury time-dependently enhanced VPS26A and SNX27 expression; VPS26A–SNX27 coprecipitation; and VPS26A-positive, SNX27-positive, and VPS26A–SNX27 double-labeled immunoreactivity in the dorsal horn of Sprague Dawley rats that were all sufficiently ameliorated through the focal knock-down of spinal VPS26A expression. Althoughtheknock-downofspinalSNX27expressionexhibitedsimilareffects,spinalnerveligation(SNL)-enhancedVPS26Aexpression remained unaffected. Moreover, SNL also increased membrane-bound and total mGluR5 abundance, VPS26A-bound SNX27 and mGluR5 and mGluR5-bound VPS26A and SNX27 coprecipitation, and mGluR5-positive and VPS26A/SNX27/mGluR5 triple-labeled immunoreactivity in the dorsal horn, and these effects were all attenuated through the focal knock-down of spinal VPS26A and SNX27 expression. Although administration with MPEP adequately ameliorated SNL-associated allodynia, mGluR5 expression, and membrane insertion, SNL-enhanced VPS26A and SNX27 expression were unaffected. Together, these results suggested a role of spinal VPS26A– SNX27-dependentmGluR5recyclinginthedevelopmentofneuropathicpain.Thisisthefirststudythatlinksretromer-associatedsorting machinery with the spinal plasticity underlying pain hypersensitivity and proposes the possible pathophysiological relevance of endo- cytic recycling in pain pathophysiology through the modification of glutamatergic mGluR5 recycling. Key words: retromer; SNX27; VPS26; mGluR5; neuropathic pain; allodynia Significance Statement VPS26A–SNX27-dependent mGluR5 recycling plays a role in the development of neuropathic pain. The regulation of the VPS26A– SNX27 interaction that modifies mGluR5 trafficking and expression in the dorsal horn provides a novel therapeutic strategy for pain relief. Introduction surface complement of receptors (Ehlers, 2000; Martin et al., Endocytic sorting of signaling receptors between recycling and 2004; Temkin et al., 2011). Retromer, a stable trimer composed of degradative pathways is a key cellular process that controls the vacuolar protein sorting-associated protein 26 (VPS26), VPS29, and VPS35 (Collins et al., 2005; Hierro et al., 2007; Gallon et al., 2015), has been identified as a critical element of the endo- Received July 8, 2015; revised Sept. 22, 2015; accepted Sept. 25, 2015. somal sorting machinery that initiates cargo sorting for re- Author contributions: H.-Y.P. designed research; T.-B.L., C.-Y.L., M.-C.H., H.-H.W., and H.-Y.P. performed re- search; C.-Y.L., M.-C.H., H.-H.W., J.-K.C., Y.-P.C., G.-D.C., and H.-Y.P. analyzed data; T.-B.L., M.-C.H., and H.-Y.P. wrote the paper. The authors declare no competing financial interests. This work was supported by the Ministry of Science and Technology, Taipei, Taiwan (Grants MOST 104-2320-B- *C.-Y.L. and H.-H.W. contributed equally to this work. 715-004-MY3, NSC 102-2628-B-715-001, and 101-2320-B-715-001-MY3 to H.-Y.P. and Grants NSC MOST 104- Correspondence should be addressed to Hsien-Yu Peng, Department of Medicine, Mackay Medical College, 2320-B-038-019-MY3, MOST 104-2320-B-038-027-MY3, 101-2320-B-039-013-MY3 to T.-B.L.), the Mackay No.46, Sec. 3, Zhongzheng Rd, Sanzhi Dist, New Taipei, Taiwan 25245. E-mail: [email protected]. Memorial Hospital (Grants MMH-MM-10206 and MMH-MM-10302 to H.-Y.P.), Taipei Medical University (Grant DOI:10.1523/JNEUROSCI.2587-15.2015 TMU102-AE1-B06 to T.-B.L.), and Saint Paul’s Hospital (Grant SPMRD-U1-6003 to T.-B.L.). Copyright © 2015 the authors 0270-6474/15/3514943-13$15.00/0 14944 • J. Neurosci., November 4, 2015 • 35(44):14943–14955 Lin et al. • VPS26–SNX27/mGluR5-Dependent Pain trieval and recycling to the plasma membrane or trans-Golgi showing neurological deficits after surgery were killed and excluded from network and therefore away from degradation (Cullen et al., statistical analyses. 2011). Mammalian-retromer-dependent recycling of the signal- Behavioral studies. Tactile sensitivity was assessed by measuring each ing receptor is associated with glutamatergic receptor-dependent rat’s paw-withdrawal threshold in response to probing with von Frey neural plasticity at excitatory synapses in multiple neuropa- monofilaments (Stoelting) according to the method of Scha¨fers et al. thologies, such as Alzheimer’s disease (Wen et al., 2011) and (2003). In brief, rats were placed individually in an opaque plastic cylin- der, which was placed on a wire mesh. Animals were habituated for 1 h to Down’s syndrome (Wang et al., 2013). However, the potential allow acclimatization to the test environment before each test. After ac- contribution of retromer-dependent cycling in spinal gluta- climatization, calibrated von Frey filaments (0.07–26.0 g) were applied to mate-receptor-associated pain hypersensitivity has not been the plantar surfaces of the hindpaws of rats. The withdrawal threshold established. was assayed 1 d before surgery to ensure that the animals had normal Sorting nexin (SNX)27, an endosome-associated cargo adap- tactile sensitivity. Because a previous study showed that the mean with- tor, is unique among SNXs because this protein possesses a PDZ drawal threshold of naive rats was ϳ15g(Kim and Chung, 1992), rats domain (Wang et al., 2013; Gallon et al., 2014). The direct inter- that displayed a threshold lower or higher than 15 g were excluded from action of the PDZ domain of SNX27 with the retromer subunit further study. Motor function was assessed using an accelerating Rotarod VPS26A is necessary for the recognition and recycling of PDZ apparatus (LE8500; Ugo Basile). For acclimatization, the animals were motif-containing cargo (Steinberg et al., 2013; Gallon et al., 2014) subjected to three training trials at 3–4 h intervals on 2 separate days. from the endosome toward the plasma membrane (Temkin et al., During the training sessions, the rod was set to accelerate from 3 to 30 2011; Gallon and Cullen, 2015). Notably, through a PDZ- rpm over a 180 s period. During the test session, the performance times of dependent mechanism, SNX27 promotes the recycling and rats were recorded up to a cutoff time of 180 s. Three measurements were obtained at intervals of 5 min and were averaged for each test. membrane insertion of the glutamatergic receptor (Wang et al., Western blotting. In brief, the dissected dorsal horn (L4–L5) sample 2013), a key player in pain-associated neural plasticity (Larsson et was homogenized in 25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% al., 2011; Peng et al., 2013; Luo et al., 2014). Therefore, the sodium deoxycholate, and 0.1% SDS with a complete protease inhibitor SNX27-VPS26A complex is emerging as a potential link between mixture (Roche). After incubation on ice (1 h), the lysates were centri- glutamatergic receptor recycling and the spinal mechanism un- fuged (14,000 rpm, 20 min, 4°C). All of the protein concentrations were derlying neuropathic pain development. determined using a bicinchoninic acid assay. The supernatant was sepa- Metabotropic glutamate receptor 5 (mGluR5) undergoes rated on an acrylamide gel and transferred to a polyvinylidene difluoride cargo cycling in HEK293 cells (Trivedi et al., 2012). Inflammatory membrane, which was then incubated (1 h, room temperature) in pain increases the abundance and membrane insertion of either rabbit anti-SNX27 (SNX27, 1:1000; Santa Cruz Biotechnology, mGluR5 in the dorsal horn (Pitcher et al., 2007). However, the Santa Cruz, CA), rabbit anti-VPS26A (1:1000; Abcam, Cambridge), potential contribution of cargo cycling in pain-enhanced rabbit anti-mGluR5 (1:4000; Abcam, Cambridge), or mouse anti- mGluR5 abundance/trafficking remains unclear. Moreover, al- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:4000; Santa Cruz Biotechnology, Santa Cruz, CA). The blots were washed and incu- though the role of the SNX27–retromer complex in the endo- bated (1 h, room temperature) in peroxidase-conjugated goat anti-rabbit somal recycling of mGluR5 has not been established, considering IgG (1:8000; Jackson ImmunoResearch), goat anti-mouse IgG (1:8000; that SNX27 contributes to the subcellular recycling of G-protein- Jackson ImmunoResearch) or rabbit anti-sheep
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