Spinal nerve transection-induced upregulation of SAP97 via promoting membrane trafficking of GluA1-containing AMPA receptors in the dorsal horn contributes to the pathogenesis of neuropathic pain

Emerging evidence has implicated an important role of synapse-associated protein-97 (SAP97)-regulated GluA1-containing AMPARs membrane trafficking in cocaine restate and in contextual episodic memory of schizophrenia. Herein, we investigated the role of SAP97 in neuropathic pain following lumbar 5 spinal nerve transection (SNT) in rats. Our results showed that SNT led to upregulation of SAP97, enhanced the interaction between SAP97 and GluA1, and increased GluA1-containing AMPARs membrane trafficking in the dorsal horn. Microinjection of AAV-EGFP-SAP97 shRNA in lumbar 5 spinal dorsal horn inhibited SAP97 production, decreased SAP97-GluA1 interaction, reduced the membrane trafficking of GluA1-containing AMPARs, and partially attenuated neuropathic pain following SNT. Intrathecal injections of SAP97 siRNA or NASPM, an antagonist of GluA1-containing AMPARs, also partially reversed neuropathic pain on day 7, but not on day 14, after SNT. Spinal overexpression of SAP97 by AAV-EGFP-SAP97 enhanced SAP97-GluA1 interaction, increased the membrane insertion of GluA1-containing AMPARs, and induced abnormal pain in naïve rats. In addition, treatment with SAP97 siRNA or NASPM i.t. injection alleviated SNT-induced allodynia and hyperalgesia and exhibited a longer effect in female rats. Together, our results indicate that the SNT-induced upregulation of SAP97 via promoting GluA1-containing AMPARs membrane trafficking in the dorsal horn contributes to the pathogenesis of neuropathic pain. Targeting spinal SAP97 might be a promising therapeutic strategy to treatment of chronic pain.


Introduction
The lesion or disease of somatosensory nervous system often results in neuropathic pain, which has been described as 'the most terrible of all tortures.However, pharmacotherapy for this problem has achieved limited success (Costigan et al., 2009).Peripheral nerve injury leads to a persistent increase in pain-related synaptic transmission and maladaptive structural changes in the spinal dorsal horn, which is referred to as central sensitization.A growing body of evidence indicates that the insertion of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the plasma membrane is a key step in synaptic delivery of AMPARs during the maladaptive synaptic plasticity in the dorsal horn (Gangadharan et al., 2011;Rosner et al., 2023;Wu et al., 2022).AMPARs consists of four subunits (GluA1-GluA4).Modulation of synaptic plasticity by dynamic trafficking of GluA1-containing AMPARs has been shown to be important for chronic pain (Gangadharan et al., 2011;Tao et al., 2014).Therefore, molecules involved in trafficking and organization of glutamate receptors may play a key role in the Abbreviation: SNT, spinal nerve transection; SAP97, synapse-associated protein-97; AMPARs, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; PSD, postsynaptic density; MAGUKs, membrane-associated guanylate kinase homologs; NMDAR, N-methyl-D-aspartate receptor; PWT, paw withdrawal threshold; PWL, paw withdrawal latency; SPF, Specific Pathogen-Free.. regulation of pain-related synaptic transmission.
The electron-dense thickening of postsynaptic membrane in glutamatergic synapses is termed as postsynaptic density (PSD) in which a high concentration of glutamate receptors, associated signaling proteins, and cytoskeletal elements are assembled by synaptic scaffold proteins.Among these scaffolding proteins are the membrane-associated guanylate kinase homologs (MAGUKs) including synapse-associated protein-97 (SAP97), PSD95, PSD93, and SAP102 (Won et al., 2017).Previous studies have demonstrated the important role of PSD95 (Florio et al., 2009;Li et al., 2022;Peng et al., 2013) and PSD93 (Tao et al., 2003) in the pathogenesis of neuropathic and inflammatory pain through regulating AMPARs-and NMDARs-mediated synaptic plasticity in the spinal dorsal horn.SAP97 is the rat homologue of the Drosophila and human discs large tumor suppressor protein (hDlg), and is widely expressed throughout the brain and in multiple organs (Müller et al., 1995;Valtschanoff et al., 2000).It has been demonstrated that SAP97 is the only member of the MAGUK family to interact directly with the AMPARs subunit GluA1, which binds at the C-terminal PDZ and directs GluA1 forward trafficking from the Golgi network to the plasma membrane (Leonard et al., 1998;Nash et al., 2010;Wu et al., 2002).Previous studies have shown that overexpression of SAP97 in cultured hippocampal neurons causes the enlargement of dendritic spines, enhances surface expression of GluA1-containing AMPA receptors and synaptic transmission (Howard et al., 2010;Waites et al., 2009).However, the role of SAP97 in the trafficking and synaptic localization of AMPAR is still debated with conflicting results have been reported previously.These contradictory data may be in part due to earlier studies have relied on dissociated neuron cultures where synapses form between unknown neuron subtypes (Fourie et al., 2014).A recent study reveals that activation of GluA1-containing AMPARs in subregions of the nucleus accumbens reinstates cocaine seeking (Caffino et al., 2022;White et al., 2016).Knockdown of SAP97 protein in the medial accumbens shell attenuated reinstatement of cocaine seeking.Mutations of SAP97 are associated with the development of schizophrenia in humans (Xu et al., 2020;Xu et al., 2023).Inhibition of SAP97 expression specifically within the dentate gyrus disrupts contextual episodic memory processing in rats (Kay et al., 2022).These studies indicates that abnormal expression of SAP97 and subsequently mediated-dysfunction of AMPARs in brain is sufficient to causing some central nervous diseases.
Compelling evidence has implicated a critical role of GluA1 trafficking and membrane insertion of Ca 2+ permeable AMPA receptors in the pathogenesis of chronic pain (Chen et al., 2014;Chen et al., 2016;Gangadharan et al., 2011;Su et al., 2015).However, the role of SAP97 in the occurrence of neuropathic pain has yet to be studied.Therefore, the purpose of the current study was to investigating the role of spinal SAP97 in the pathogenesis of neuropathic pain and the underlying mechanisms.

Animals
The SPF-grade adult male Sprague-Dawley rats, weighing 200-250 g, were used.The animals were randomly assigned into each group and were housed in individual standard rat cages with free access to water and chows.The rats and standard rat chow were purchased from the Laboratory Animal Center of Zhengzhou University in China.The room temperature was maintained at 23 ± 2 • C and the humidity was kept at 50% to 60% under a 12:12-h light-dark cycle.The procedures in the current study conformed to the guidelines of the International Association for the Study of Pain and were approved by the Animal Care and Use Committee of Zhengzhou University in China (RRID: MGI:5651135).

Lumbar 5 spinal nerve transection (L5 SNT)
The model of neuropathic pain was prepared by a surgery of left L5 SNT following our published paper (Li et al., 2021).Briefly, an incision on the lower back midline was made after the animals were anesthetized with isoflurane.The L5 lumbar transverse process was identified and then removed with the rongeur.The underlying L5 spinal nerve was isolated and ligated with a 3-0 silk thread.The ligated nerve was then transected distal to the ligature.In sham operated rats, the left L5 spinal nerve was exposed, but without ligation.One day after surgery, all rats were subjected to locomotor function test.Three reflexes (placing, grasping, and righting) were tested following the previous described method (Park et al., 2009).Rats displayed any abnormal of these reflexes will be excluded from following experiment.

Intrathecal catheterization and drugs or SAP97 siRNA delivery
Drugs and SAP97 siRNA were delivered by intrathecal (i.t.) injection to rats.The intrathecal catheterization was performed as described previously (Xu et al., 2014) and refined following the method described by Yaksh (Hunt et al., 2024).In brief, a polyethylene-10 (OD, 0.61 mm; ID, 0.28 mm) catheter was inserted into the rat's subarachnoid space through the L5-L6 intervertebral space, and the tip of the catheter was located at the L5 spinal segmental level.The Ca 2+ permeable GluA1containing AMPARs antagonist NASPM (MedChemExpress, USA) was dissolved in sterile normal saline containing 10% DMSO.The i.t.injections of drug were performed half hour before surgery and once daily 30 min before behavioral test after SNT.The doses of NASPM (25, 50, 100 μg/10 μL) used in current experiment were based on a previous study (Chen et al., 2014).To specifically knockdown the expression of SAP97 in L4-6 spinal cord, three siRNAs targeting rat SAP97 mRNA were designed and synthesized by GenePharma Inc. (Shanghai, China).As control, a scrambled non-targeting oligo designed by Genepharma was used.To screen the effective SAP97 siRNA, the three siRNAs (numbered as 1074, 2057, and 3105) and the negative control scramble RNA (scRNA) were injected intrathecally in different groups half hour before SNT, and once daily thereafter for 3 days.The PWT and PWL were tested to assess the effect of the treatment on SNT-induced neuropathic pain.After the L4-6 spinal dorsal horns were harvested, the SAP97 protein and mRNA were examined to verify the effective siRNA.The nucleotide sequences of SAP97 siRNAs are listed in Table 1.

Pain-related behavioral tests
The behavioral tests were performed following our previous described methods (Xu et al., 2006).All rats were adapted to the testing environment for at least three days before baseline measurement.The paw withdrawal threshold (PWT) to assess mechanical sensitivity was determined by applying von Frey hairs to the plantar surface of the hind paw, and 50% PWT was defined using the up-down method (Chaplan et al., 1994).Heat hypersensitivity was evaluated by testing paw withdrawal latency (PWL) using a plantar analgesia tester (7370, Ugo Basile, Comeria, Italy) according to the method described by Hargreaves et al. (1988).The entire behavioral test was carried out blindly to the performer who did not know the experimental design.

Immunofluorescence staining
Immunofluorescence staining was done following our previous described methods (Qiao et al., 2023).Briefly, after defined survival times, control and nerve injured rats were terminally anesthetized and perfused through the ascending aorta with cold normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer.After perfusion, the L4-6 spinal cord were removed and post fixed in the same fixative for 3 h, which was then replaced by 30% of sucrose phosphate-buffered saline over two nights.Transverse spinal cord sections (25 μm) were cut on a cryostat (Leica, CM1950) and prepared for immunofluorescence staining.Sections were randomly selected and put into different wells of a 24well plate.After washing with phosphate-buffered saline (PBS), the sections were blocked with 5% goat serum in 0.3% Triton X-100 for 1 h at 37 • C, and incubated with primary antibody overnight at 4 • C. The following primary antibodies were used: rabbit anti-SAP97 (1:200, Invitrogen PA1-741) and rabbit anti-GluA1 (1:100, ABclonal A1826).
For double immunofluorescence staining, the sections were incubated with a mixture of goat anti-mouse FITC-(1:200, Jackson ImmunoResearch) and goat anti-rabbit Cy3-conjugated secondary antibody (1:400, Jackson ImmunoResearch) for 2 h at room temperature.The stained sections were mounted onto slides and examined with an Olympus BX53 (Olympus Optical, Tokyo, Japan) fluorescence microscope or by a high-resolution laser confocal fluorescence microscope (Nikon A1R MP+, Japan) and images were captured with a CCD spot camera.The computer-assisted imaging analysis system (ImageJ; NIH, USA) was used to quantify the intensity of immunofluorescence.An intensity threshold was set at the background level, firstly, to determine the total number of cells in a section.And then the threshold intensity was set above background level to identify structures with positive staining signals.In the spinal cord, 5-6 slides per animal were counted.An average percentage relative to the total number of cells was obtained for each animal across the different slides, and then the mean ± SE across animals was determined.The specificity of anti-SAP97 antibody was verified by an experiment in which the SAP97 expression was knocked down in the dorsal horn by microinjection of AAV-EGFP-SAP97 shRNA in the L5 spinal dorsal horn.The results showed that the SNTinduced enhanced SAP97-immunoreactivity in the dorsal horn was clearly reduced by the treatment of AAV-SAP97 shRNA (Suppl.Fig. 1).

RNA extraction and Real-time quantitative RT-PCR
Real-time quantitative RT-PCR (qPCR) was performed following the method described previously (Jiang et al., 2021).After the rats were sacrificed by decapitation at a designed time point, the lumbar L4-6 spinal dorsal horn was harvested for quantitative real-time RT-PCR.Total RNA was extracted via the Trizol method (Invitrogen/Thermo-Fisher Scientific).Reverse transcription was performed using oligo-dT primers and PrimeScriptII RTase (TAKARA) according to the manufacturer's protocol.Each sample was run in triplicate in a 20 μl reaction volume which contains 10 μM each of forward and reverse primers, 10 μl of SYBR Green qPCR Super Mix (Invitrogen) and 25 ng of cDNA.Reactions were performed in an Applied ABI QuantStudio 3 Real-Time PCR System.GAPDH was used as an internal control for normalization.The relative expression of SAP97, GluA1, and GluA2 mRNA to GAPDH mRNA in the spinal dorsal horn was quantified by the 2 − ΔΔCt method described previously (Li et al., 2021).The rat-specific primer sequences used to detect SAP97, GluA1, GluA2, and GAPDH mRNA in the qPCR analysis were listed in Table 2.
Then the beads separation was repeated 4 times by Magnetic Stand as above.Finally, the precipitated beads were added 20 μl 1× SDS-PAGE loading buffer and heated in 95 • C for 5 min to dissociating beads from binding protein.Then the beads were reseparated in Magnetic Stand.The supernatants were collected and were subjected to Western Blotting.The GluA1 (or SAP97 in another group) in the supernatants was detected by anti-GluA1 antibody or anti-SAP97 antibody.

Intraspinal cord microinjection
To specifically knock down SAP97 only in lumbar 5 spinal dorsal horn, the pAAV-U6-shRNA (SAP97)-CMV-EGFP-WPRE (AAV-EGFP-SAP97 shRNA) or negative control pAAV-U6-EGFP-WPRE-H1-shRNA NC (AAV-EGFP-SAP97 shRNA NC) (OBiO Technology, Shanghai) were microinjected into unilateral L5 spinal dorsal horn in adult rats following the procedures described previously (Li et al., 2021;Qiao et al., 2023).Briefly, under constant anesthesia with isoflurane, unilateral laminectomy of thoracic vertebra 12 was carried out.After the spinal cord was exposed, the rat was placed in the stereotaxic frame and the vertebral column was immobilized.The glass micropipette was positioned 200 μm lateral from the posterior median sulcus and 300 μm below the dorsal surface of the spinal cord at the level of L5 spinal cord under a stereomicroscope.The viral solution (2 μl, titer >1.2 × 10 13 /ml) was injected at a rate of 100 nl/min with the micropipette connected to a Hamilton syringe.The pipette was removed 10 min after injection.Two injection sites within a 1-mm interval along the posterior median sulcus were carried out.The wound was washed with saline and closed in layers (fascia and skin) with 3-0 silk thread.No impairment of motor function after intraspinal microinjection was observed.Four weeks later, The L5 SNT was carried out in the AAV injected side.To overexpression of SAP97 specifically in lumbar 5 spinal dorsal horn, the pAAV-CMV-Dlg1(SAP97)-3xFLAG-P2A-EGFP-tWPA (AAV-EGFP-SAP97) or negative control pAAV-CMV-3xFLAG-P2A-EGFP-tWPA (AAV-EGFP) (OBiO Technology, Shanghai) were microinjected into unilateral L5 spinal dorsal horn in adult rats as above.One week after the injection, the PWT and PWL were tested to observe the development of SAP97-induced abnormal pain.

Statistical analysis
All data are presented as the means ± SEM and analyzed with GraphPad Prism 8.2.1 (GraphPad, San Diego, CA, USA).Sample sizes of per group were predicted based on a power analysis (G*Power 3.0.10)and the experience of our previous studies (Li et al., 2021).All data were subjected to tests for normality by Shapiro-Wilk test.If the Data exhibit normal distribution, the differences were tested by One-way ANOVA followed Dunnett's multiple comparisons test or using Student's t-test if only two groups were applied.Otherwise, if the data do not exhibit a normal distribution, it was analyzed via a non-parametric Kruskal-Wallis or Mann-Whitney U test.Data were considered significant when p < 0.05, and were graphed using GraphPad Prism and plotted as mean ± SEM.
To identify the cell types that expressed SAP97 in the dorsal horn after SNT, we performed double immunofluorescence staining of SAP97 with three cell-specific markers: NeuN (a marker of the neuronal cell), GFAP (a marker of astrocyte), and OX-42 (a marker of microglia), respectively.The results showed that the SAP97 colocalized with NeuN (Fig. 2A-C), but not with GFAP (Fig. 2D-F) and OX-42 (Fig. 2G-I), on day 7 after SNT.

Spinal specific blocking GluA1-containing AMPARs attenuated the neuropathic pain following SNT
To further validate the role of SAP97-regulated membrane trafficking of GluA1-containing AMPARs in the pathogenesis of neuropathic pain, repeat i.t.injections of NASPM, an antagonist of Ca 2+ permeable GluA1-containing AMPARs, were performed 30 min before SNT and once daily thereafter for 3 days.Data of behavioral tests showed that compared with SNT plus vehicle group, NASPM treatment dosedependently prevented SNT-induced reductions of PWT (Mean ± SEM in the suppl Table S1 and P-value in Table S2, Fig. 7A) and PWL (Mean ± SEM in the suppl Table S1 and P-value in Table S2, Fig. 7B).To further observe the effect of NASPM treatment on the developed and established neuropathic pain, a single bolus i.t.injection of NASPM was performed on day 7 and day 14, respectively, after SNT.Results of behavioral tests showed that NASPM treatment on day 7 partially reversed the decrease of PWT and PWL after SNT.Compared with SNT plus vehicle group, the significant increase of PWT (Mean ± SEM in the suppl Table S1 and Pvalue in Table S2, Fig. 7C) and PWL (Mean ± SEM in the suppl Table S1 and P-value in Table S2, Fig. 7D) in SNT plus NASPM group occurred at 2 h, peaked at 4 h, and persisted to 6 h after injection of NASPM.However, treatment with NASPM on day 14 did not alleviated the SNTinduced mechanical allodynia (Mean ± SEM in the suppl Table S1 and Pvalue in Table S2, Fig. 7E) and thermal hyperalgesia (Mean ± SEM in the suppl Table S1 and P-value in Table S2, Fig. 7F).These results indicates that SNT-induced membrane trafficking of GluA1-containing AMPARs was involved in the development, but not the maintenance, of neuropathic pain.

Intrathecal injection of SAP97 siRNA or NASPM alleviated neuropathic pain in female rats
Because previous studies have shown some differences between male and female in the pathogenesis of chronic pain (Chen et al., 2018;Luo et al., 2021), the role of SAP97-regulated membrane trafficking of GluA1-containing AMPARs in the pathogenesis of neuropathic pain in female rats was also investigated in the current study.Our results showed that compared with SNT or SNT plus scRNA group, repeat i.t injections of SAP97 siRNA led to a significant increase of PWT (Mean ± SEM in the suppl Table S1 and P-value in Table S2, Fig. 8A) and PWL (Mean ± SEM in the suppl Table S1 and P-value in Table S2, Fig. 8B).In addition, repeat i.t.injections of NASPM (100 μg/10 μl) also resulted in increase of PWT (Mean ± SEM in the suppl Table S1 and P-value in Table S2, Fig. 8C) and PWL (Mean ± SEM in the suppl Table S1 and Pvalue in Table S2, Fig. 8D) when compared with SNT or SNT plus vehicle group.To further validating the role of GluA1-containing AMPARs in the developed neuropathic pain, a single bolus i.t.injection of NASPM was performed on day 7 after SNT.The results showed that the NASPM treatment partially reversed the SNT-induced reductions of PWT (Mean ± SEM in the suppl Table S1 and P-value in Table S2, Fig. 8E) and PWL (Mean ± SEM in the suppl Table S1 and P-value in Table S2, Fig. 8F).Compared to SNT plus vehicle group, the significant difference occurred at 4 h and persisted >8 h after the injection.It implies that SAP97-regulated membrane trafficking of GluA1-containing AMPARs may also be involved in the genesis of neuropathic pain in female rats.

Discussion
Compelling evidence has demonstrated the critical role of GluA1containing AMPARs membrane trafficking in the pathogenesis of chronic pain (Gangadharan et al., 2011).However, the mechanisms of regulating AMPARs trafficking are still incompletely understood.In the current study we found that the L5 SNT-induced upregulation of SAP97 via interacting directly with GluA1 subunit and subsequently promotedmembrane trafficking of GluA1-containing AMPARs in the spinal dorsal horn contributes to the pathogenesis of neuropathic pain.
Previous studies have shown that a reduction in Ca 2+ -permeable AMPARs in spinal PSD fraction is accompanied by a loss of nociceptive plasticity, whereas an increase in spinal Ca 2+ -permeable AMPARs facilitates nociceptive plasticity (Hartmann et al., 2004;Youn et al., 2008).It has been demonstrated that SAP97 is the only member of the MAGUK family to interact directly with the AMPARs subunit GluA1 (Leonard et al., 1998;Wu et al., 2002).However, the expression of SAP97 in the spinal dorsal horn has yet to be examined in chronic pain.
In the current study, we found that L5 SNT led to a significant increase in the expression of SAP97 mRNA and the production of SAP97 protein in the dorsal horn neurons.These increases exhibited a long-lasting pattern which occurred 3 days after SNT and persisted >2 weeks after surgery.Although previous studies have shown that the interaction of SAP97 with GluA1 is crucial for activity-dependent neuronal dendrite growth and branching in the spinal motor neurons in mice (Zhang et al., 2017;Zhou et al., 2008), our current observations provided new evidence for the first time that SAP97 also expressed in somatosensory neurons in the dorsal horn and may be involved in the synaptic plasticity of SNTinduced neuropathic pain.Moreover, our results also shewed an expression of SAP97 in the ventral horn.It suggests that SAP97 may play a role in the motor-related synaptic plasticity in ventral horn.
Because previous studies have implicated a critical role of SAP97 in the regulation in membrane trafficking of GluA1-containing AMPARs in brain (Howard et al., 2010;Schlüter et al., 2006), the expression of GluA1 and GluA2, and their membrane trafficking were examined in the dorsal horn following SNT in the current study.Our results showed that the SNT did not lead to the alteration of GluA1 and GluA2 expression, but caused a significant increased insertion of GluA1-containing AMPARs in PSD fraction and a decrease in cytosol in the dorsal horn.Although accumulating evidence has verified the critical role of increased membrane insertion of GluA1-containig MAPARs in the genesis of post-surgical pain (Kang et al., 2022;Zhang et al., 2018) and the persistence of inflammatory pain (Kopach et al., 2013;Lu et al., 2008;Peng et al., 2012;Tao et al., 2014), the spinal GluA1-containing AMPARs in neuropathic pain remains conflicting results between different laboratories (Lu et al., 2008;Zhao et al., 2006).Chen et al. observed a significant internalization of GluA2-containing AMPARs, but not increased membrane trafficking of GluA1-containing AMPARs in the dorsal horn, following peripheral nerve injury (Chen et al., 2019;Chen et al., 2013).While, the others studies reveal that the enhancement of membrane insertion of GluA1-containing AMPARs in the dorsal horn contributes to postsynaptic amplification of neuropathic pain after nerve injury (Lin et al., 2015;Liu et al., 2020;Miletic et al., 2015).This discrepancy may due to the different model and different procedure of membrane preparation were used.Using a specific PSD fraction preparation in current study, we observed a significant increase of GluA1containing AMPARs in the PSD fraction of spinal dorsal horn, which occurred on day 3, peaked on day 7, and persisted >10 days after SNT.Meanwhile, a clear reduction of GluA1 subunit was evidenced in cytosol following SNT.Unexpectedly, the GluA2-containing AMPARs in PSD fraction and in cytosol were not changed after SNT.Our results indicate that the increased membrane trafficking of GluA1-containing AMPARs, but not the internalization of GluA2-containing, in the dorsal horn might    contribute to the pathogenesis of neuropathic pain following SNT.A previous study reported that the nociceptor-specific deletion of GluA1 led to disruption of calcium permeability and reduced capsaicin-evoked activation of nociceptors (Gangadharan et al., 2011).Deletion of GluA1, but not GluA2, in the dorsal horn led to reduced mechanical hypersensitivity and sensitization in models of chronic inflammatory pain and arthritis (Gangadharan et al., 2011;Hartmann et al., 2004).These results agree with our present funding and imply that the GluA1containing AMPARs membrane trafficking might play more important role in chronic pain.
It has been demonstrated that SAP97 serve as a molecular link between GluA1 and the actin-dependent motor protein myosin VI during the dynamic translocation of AMPA receptors from Golgi network to postsynaptic membrane (Leonard et al., 1998).Therefore, the interaction between SAP97 and GluA1 was examined with Co-IP in the dorsal horn.Our results showed that SNT-induced an enhanced combination between SAP97 and GluA1.But this interaction was disrupted by microinjection of AAV-EGFP-SAP97 shRNA in L5 spinal dorsal horn.Moreover, the SNT-induced increase of membrane insertion of GluA1containing AMPARs in the PSD fraction of dorsal horn was also inhibited by the treatment of SAP97 shRNA.Conversely, overexpression of SAP97 by microinjection of AAV-EGFP-SAP97 in L5 spinal dorsal horn in naïve rats resulted in enhancement of combination of SAP97 with GluA1, and increased the membrane trafficking of GluA1-containing AMPARs in the dorsal horn.These results indicate that SNT-induced membrane trafficking of GluA1-containing AMPARs in the dorsal horn was regulated by SAP97.
Although previous studies have implicated a critical role of SAP97 in the regulation of membrane trafficking of GluA1-containing AMPARs in different region of brain (Kay et al., 2022;Rumbaugh et al., 2003;Waites et al., 2009;White et al., 2016), the role of SAP97 in the pathogenesis of chronic pain still remains unclear.In the current study, we found that spinal knockdown of SAP97 by AAV-EGFP-SAP97 shRNA attenuated mechanical allodynia and thermal hyperalgesia following L5 SNT.This effect accompanied a significant decrease of interaction between SAP97 and GluA1, and a reduction of membrane trafficking of GluA1containing AMPARs in the dorsal horn.Moreover, the developed neuropathic pain was partially reversed by repeat injections of SAP97 siRNA or NASPM, a specific antagonist of Ca 2+ permeable AMPARs, on day 7, but not on day 14 after SNT.To further validate the role of SAP97 in the genesis of pain, the spinal overexpression of SAP97 was developed by microinjection of AAV-EGFP-SAP97 in L5 spinal dorsal horn in naïve rats.Behavioral data showed that the rats exhibited abnormal pain at about 3 weeks after the injection and lasted >8 weeks.The interaction between SAP97 and GluA1, and the membrane trafficking of GluA1containing AMPARs in the dorsal horn were enhanced by this treatment.These results suggests that SNT-induced upregulation of SAP97 via facilitating GluA1-containing AMPARs membrane trafficking in the dorsal horn contributes to the occurrence of neuropathic pain.Although our present results and amounts of previous studies have shown that inhibition of SAP97 production impairs surface insertion of GluA1 (Fourie et al., 2014), some conflicting results are also reported from different laboratories.Kim et al. reported that a knock-in mouse lacking the final seven GluA1 C-terminal amino acids (the SAP97-binding site) has normal GluA1 synaptic localization, trafficking, and basal synaptic transmission (Kim et al., 2005).However, Zhou et al. find that deleting the C-terminal 7 amino acids of GluA1 does not influence trafficking, processing, or cell surface GluA1 expression but does prevent translocation of SAP97 from the cytosol to membranes (Zhou et al., 2008).These contrary results may be due to compensation by other MAGUKs in basal synaptic transmission.Our results also showed that spinal blockade of GluA1-containing AMPARs is more effective in relieving neuropathic pain than blocking SAP97.Spinal knockdown SAP97 only reduced 50% of neuropathic pain following SNT.It implies that has a compensate effect by other MAGUK family member when reduced the SAP97 activity.Because except SAP97, the membrane trafficking of GluA1-containing AMPARs were also regulated by other MAGUK family member, such as PSD95 and PSD93.In addition, interactions between SAP97 and GluA1 also promote dendritic branching in an activitydependent manner in spinal motor neurons (Jablonski and Kalb, 2013;Zhou et al., 2008).Given that structural plasticity is observed in the spinal dorsal horn following peripheral injury (Costigan et al., 2009), it is intriguing to speculate that SAP97-GluA1 may have a role in this process and deserve further study in future.Except AMPARs, SAP97 also regulates membrane trafficking of NMDA receptors (Lin et al., 2013).Amounts of studies have been demonstrated the critical role of NMDA receptor in the synaptic plasticity of chronic pain.Therefore, whether or not the SAP97-regulated NMDA receptor trafficking playing a role in neuropathic pain also needs to be studied in future.Because previous studies have observed difference reactions between male and female rats in the pathogenesis of chronic pain (Chen et al., 2018;Luo et al., 2021), the role of SAP97-regulated membrane trafficking of GluA1-containing AMPARs in the occurrence of neuropathic pain in female rats was also investigated.Our results showed that SNT-induced mechanical allodynia and thermal hyperalgesia were attenuated by the treatment of spinal knockdown SAP97 or blocking GluA1-containing AMPARs in female rats.Moreover, compared with male rats, the treatment with NASPM in female rats exhibited a longer effect, although the initiate effect had somewhat delay.It implies that SAP97 is required for the genesis of neuropathic pain in both the male and female rats.
In conclusion, the present study has implicated a critical role of SNTinduced upregulation of SAP97 in the spinal dorsal horn in neuropathic pain.The upregulated-SAP97 via interacting with GluA1 and subsequently promoting GluA1-containing AMPARs membrane trafficking in the dorsal horn contributed to the development of neuropathic pain following SNT.Therefore, targeting spinal SAP97 might be a promise therapeutic strategy to treatment of chronic pain.

Declaration of generative AI in scientific writing
Authors did not use AI and AI-assisted technologies in the writing process.

Fig. 1 .
Fig. 1.Lumbar 5 spinal nerve transection (SNT) induced neuropathic pain and led to upregulation of SAP97 in the spinal dorsal horn.(A,B) SNT caused mechanical allodynia (A: Kruskal-Wallis) and thermal hyperalgesia (B: One-way ANOVA) in the ipsilateral hind paw (n = 6 for each group, *P < 0.05, **P < 0.01, ***P < 0.001 vs. baseline or sham group).(C) SNT caused increase in the production of SAP97 protein in the ipsilateral dorsal horn.*P < 0.05, ***P < 0.001 vs. sham group (day 7 after sham operation), One-way ANOVA followed by the Dunnett's multiple comparisons test, n = 6 repeats from 6 rats for each group.(D) Results of qPCR indicating increase in the expression of SAP97 mRNA in the ipsilateral dorsal horn following SNT.*P < 0.05, ***P < 0.001 vs. sham group, One-way ANOVA followed by the Dunnett's multiple comparisons test, n = 5 repeats from 5 rats for each group.(E-J) Images of immunofluorescence staining showing clear increase of SAP97-positive staining cells in ipsilateral spinal dorsal horn after SNT.(K) Quantitative Result of immunofluorescence staining indicating increase of SAP97-positive staining cells.**P < 0.01, ***P < 0.001 vs. Naïve or sham group, One-way ANOVA followed by the Dunnett's multiple comparisons test, n = 3 for each group.(L) Representative image showing the increased SAP97-positive staining cells aggregated mainly in the ipsilateral spinal dorsal horn on day 7 after SNT.Scale bar: (E-J) = 100 μm, (K) = 500 μm.

Fig. 2 .
Fig. 2. The cell types expressing SAP97 in the spinal dorsal horn following SNT.(A-I) Representative images showing the results of double immunofluorescence staining in the L5 ipsilateral dorsal horn between SAP97 (red; B, E, and H) and NeuN, a neuronal marker (green; A); GFAP, an astrocyte marker (green; D); and OX-42, a microglia marker (green; G), indicating that SAP97 colocalized with NeuN (C), but not with GFAP (F) and OX-42 (I) 7 days after SNT.Scale bar: (A-I) = 100 μm.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 .
Fig. 5. Effect of SNT on the expression of GluA1, GluA2, and their membrane trafficking in the ipsilateral spinal dorsal horn.(A,B) SNT did not alter the production of GluA1 protein (A: n = 5 for each group) and the expression of GluA1 mRNA (B: n = 3 for each group), but increased the level of phosphorylated-GluA1 (p-GluA1) (A) in the dorsal horn.**P < 0.01, ***P < 0.001 vs. sham group, One-way ANOVA followed by the Dunnett's multiple comparisons test.(C,D) SNT led to increase of GluA1-containing AMPARs membrane trafficking (C: n = 6 for each group) and decrease of GluA1 in cytosol (D: n = 5 for each group) in the dorsal horn.*P < 0.05, **P < 0.01, ***P < 0.001 vs. sham group, One-way ANOVA followed by the Dunnett's multiple comparisons test.(E,F) SNT did not change the production of GluA2 protein, p-GluA2 (E: n = 5 for each group), and the expression of GluA2 mRNA (F: n = 3 for each group) in the dorsal horn.(G,H) SNT did not alter GluA2 protein in PSD fraction (G) and in cytosol (H) in the dorsal horn.One-way ANOVA followed by the Dunnett's multiple comparisons test, n = 5 for each group.(I-K) Images of double immunofluorescence staining showing that GluA1 colocalized with neuronal marker NeuN (I), but not with astrocytic marker GFAP (J) and microglial marker OX-42 (K) in the dorsal horn on day 7 after SNT.Scale bar: I-K = 100 μm.
Z.Liang et al.
Z.Liang et al.