Central Mechanisms Mediating Thrombospondin-4-induced Pain States

Abstract

Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Ca_vα₂δ₁ subunit (Ca_vα₂δ₁), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Ca_vα₂δ₁ in the spinal cord in vivo and that TSP4/Ca_vα₂δ₁-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Ca_vα₂δ₁-dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory post-synaptic currents in second-order neurons as well as increased VGlut₂- and PSD95-positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Ca_vα₂δ₁-dependent processes with Ca_vα₂δ₁ ligand gabapentin or genetic Ca_vα₂δ₁ knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Ca_vα₂δ₁. Importantly, TSP4/Ca_vα₂δ₁-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Ca_vα₂δ₁-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.

Introduction

Neuropathic pain due to peripheral nerve injury is associated with up-regulation of expression of thrombospondin-4 (TSP4)⁵ in spinal cord and dorsal root ganglia (DRG) that induces increased frequency of miniature excitatory post-synaptic currents (mEPSC) in dorsal spinal cord and neuropathic pain states (1, 2). Details about the mechanisms remain to be defined however. TSP4 belongs to a five-member thrombospondin superfamily of oligomeric, extracellular matrix glycoproteins (TSP1–5) that can be subdivided into groups A (TSP1/2) and B (TSP3/4/5) based on structure and functional domain similarities (3). TSP proteins are important in mediating cell-to-cell and cell-to-matrix interactions (3, 4). TSP4 is expressed in multiple sites, and its functions are not well defined (5) although there is evidence that TSP4 promotes neurite outgrowth (6).

Recently, TSPs have been shown to regulate early excitatory synaptogenesis in the brain by interacting with its receptor, the voltage-gated calcium channel α₂δ₁ subunit (Ca_vα₂δ₁) proteins (7, 8). The Ca_vα₂δ subunit family of the voltage-gated calcium channels includes four Ca_vα₂δ subunits (Ca_vα₂δ_1–4) encoded by different genes (9–11). Ca_vα₂δ functions include trafficking and stabilizing voltage-gated calcium channel to the plasma membrane and pre-synaptic terminals (12–14), fine-tuning channel functions, and gating properties (13, 15, 16).

Importantly, Ca_vα₂δ₁ and Ca_vα₂δ₂ subunits are binding sites of gabapentinoids (17, 18), which have anti-neuropathic pain efficacy in patients (19–22) and animal models (23–26). Ca_vα₂δ₁, but not Ca_vα₂δ₂, is up-regulated in sensory neurons after peripheral nerve injury (27, 28), leading to increased Ca_vα₂δ₁ axonal transport to the central presynaptic terminals of sensory neurons in dorsal spinal cord (27, 29). Similar to injury-induced TSP4, injury-induced Ca_vα₂δ₁ also has been shown to increase mEPSC frequency in neurons of dorsal spinal cord, which may contribute to central sensitization and neuropathic pain states (24, 25, 27, 29–31, 33).

Collectively, these observations suggest the following intriguing hypothetical mechanistic model: 1) peripheral nerve injury up-regulates Ca_vα₂δ₁ in peripheral sensory neurons and its central terminals; 2) peripheral nerve injury also triggers increased synthesis and release of TSP4 in spinal cord and DRG; 3) increased TSP4 interacts directly with its receptor Ca_vα₂δ₁ on the central terminals of sensory neurons to increase excitatory synaptogenesis and synaptic neurotransmission; 4) increased excitatory transmission in dorsal spinal cord contributes to central sensitization and neuropathic pain development. This model leads to several predictions. First, there should be direct molecular interactions between TSP4 and Ca_vα₂δ₁ in the adult spinal cord in vivo. Second, if TSP4 and Ca_vα₂δ₁ are mechanistically linked, then manipulations of one protein should alter the development of pain states induced by manipulations of the other protein. Here, we tested critical aspects of this model and the underlying mechanisms.

Materials and Methods

Mouse Genetics

The Ca_vα₂δ₁ overexpressing transgenic (TG) mice were generated as described previously (34). TSP4 knock-out (KO) mice were from The Jackson Laboratory (Bar Harbor, ME). The Ca_vα₂δ₁ TG/TSP4 KO double mutant mice and their control littermates were bred internally. The Ca_vα₂δ₁ conditional knock-out (CKO) mice were generated by floxing exon 6 of the Ca_vα₂δ₁ gene (MGI (Mouse Genomics Informatics) ID 88295) with loxP sites. Homozygous Ca_vα₂δ₁ CKO mice were crossed with the Advillin-Cre mice with Cre recombinase expression only in Advillin-positive sensory neurons (35) to generate heterozygous CKO^Adv-Cre+/− mice, which were used to generate homozygous CKO^Adv-Cre+/− mice for experiments. Mouse genotyping was performed by TransnetYX, Inc. (Cordova, TN). All the mice appeared normal with respect to grooming, social interactions, and feeding and showed no signs of abnormality or any obvious motor defects, tremor, seizure, or ataxia. Only adult male mice were used for the experiments. All animal care and experiments were performed according to protocols approved by the Institutional Animal Care Committee of the University of California, Irvine.

Expression and Purification of Recombinant TSP4

Human embryonic kidney cell line 293-EBNA (Invitrogen) in DMEM/F-12 medium (Mediatech, Manassas, VA) was transfected with recombinant rat TSP4 with N-terminal His tag using the calcium phosphate transfection method, then transfected cells were selected with 0.5 μg/ml puromycin. Secreted TSP4 was confirmed by Western blots using anti-Tetra-His monoclonal antibodies (catalog #34670, negligible cross-reactivity with mammalian and other species cell lysates validated by Qiagen, Valencia, CA). The His-tagged proteins were purified using a nickel-nitrilotriacetic acid column based on the manufacturer's instructions (Invitrogen), concentrated and buffer-exchanged to PBS with Amicon Ultra-4 Centrifugal Filter Unit (50K molecular weight cutoff; Millipore, Billerica, MA), aliquoted, and stored at −80 °C until use.

Immunoprecipitation

The spinal cord tissues from adult male mice and adult male Harlan Sprague-Dawley rats were collected by hydraulic extrusion from animals deeply anesthetized with isoflurane and lysed in protein extraction buffer (50 mm Tris buffer, pH 8.0, 0.1% Triton X-100, 150 mm NaCl, 1 mm EDTA, and protease inhibitors). The cell lysate was then incubated on ice for 15 min, centrifuged ×20,000 × g for 20 min at 4 °C. The supernatant was incubated with anti-TSP4 polyclonal antibody (guinea pig, 1:750, validated previously; Ref. 36) overnight at 4 °C. Protein A/G-agarose beads (Thermo, Waltham, MA) were then added, incubated for 2 h at 4°C, and washed with protein extraction buffer. The antibody-captured proteins were eluted in non-reducing condition with low pH elution buffer (Thermo, Waltham, MA) at room temperature, and the same volume of control supernatant or immunocomplex samples was analyzed by Western blots under non-reducing conditions.

Solid-phase Binding

Briefly, FLAG-Ca_vα₂δ₁cDNA was transiently transfected into the tsA-201 cell line stably expressing Cav2.2 and Cavβ3 (a gift from Dr. D. Lipscombe from Brown University (37) by Lipofectamine 2000 (Invitrogen). The transfected cells were washed and extracted in protein extraction buffer (50 mm Tris, 150 mm NaCl, 1 mm EDTA, 0.1% Triton-X, pH 7.4) in 2–3 days. The cell lysate was incubated on ice for 15 min, then centrifuged ×13,000 × g at 4 °C for 20 min. The supernatant was rotating-incubated with anti-FLAG M2 agarose affinity resin (Sigma) for 2 h at 4 °C and washed with protein extraction buffer. FLAG-Ca_vα₂δ₁ was eluted in elution buffer (0.1 m glycine, pH 3.5) and stored at −20 °C until use.

The reagents for solid-phase binding were from Invitrogen. Recombinant TSP4 proteins (80 μg/ml) were immobilized onto a 96-well polystyrene plates (Thermo, Waltham, MA) overnight at 4 °C in coating buffer A. All further incubations were carried out at room temperature for 1 h, and proteins or antibodies were diluted in assay buffer containing bovine serum albumin (BSA). After washing and blocking, the plates were incubated with FLAG-Ca_vα₂δ₁, washed, then incubated with mouse monoclonal anti-FLAG antibodies (1:1000; catalog #F1804, validated against FLAG fusion proteins by Sigma) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. The bound FLAG-Ca_vα₂δ₁ complexes were detected by measuring a color reaction at 450 nm after adding tetramethylbenzidine for 15 min followed by adding sulfuric acid to stop the reaction.

Surface Plasmon Resonance Binding (38)

All experiments were carried out using BIAcore 3000 and CM5 Sensor Chip (GE Healthcare) at 25 °C. Ca_vα₂δ₁antibody (mouse, catalog #D219, Sigma) was coupled to the dextran matrix of a CM5 sensor chip using Amine Coupling kit as described (39). The antibody specificity for Ca_vα₂δ₁ was confirmed with tissue samples from Ca_vα₂δ₁knock-out mice (Fig. 4C). Excess reactive esters were quenched by injection of 1.0 m ethanolamine-hydrochloride, pH 8.5. The binding assays were performed using HBS-P buffer (0.01 m HEPES, pH 7.4, 0.15 m NaCl, 0.005%, and surfactant P20) as running buffer. Purified TSP4 proteins and Ca_vα₂δ₁ protein extracts from tsA-201 cells stably expressing Ca_V2.2e (Δ24a, 31a), Ca_Vβ₃, and Ca_vα₂δ₁ as described (40) were diluted in HBS-P buffer (GE Healthcare). Ca_vα₂δ₁ protein extracts were injected at a flow rate of 10 μl/min over the immobilized Ca_vα₂δ₁ antibody flow cells followed by injection of purified TSP4 proteins at a flow rate of 20 μl/min. Nonspecific binding of TSP4 to the flow cell without immobilized Ca_vα₂δ₁ antibody was subtracted from all binding curves using BIAevaluation software (version 3.0, GE Healthcare) and plotted using Graphpad Prism (Graphpad Software, San Diego, CA).

FIGURE 4.

Ca_vα₂δ₁ ablation from Advillin⁺ DRG neurons blocked TSP4-induced excitatory synaptogenesis. A, Ca_vα₂δ₁ gene structure. B, a strategy diagram of generating the Ca_vα₂δ₁ conditional knock-out mice. C, a representative Western blot (from n = 3) showing null Ca_vα₂δ₁ expression (KO) in brain lysates from homozygous Ca_vα₂δ₁ KO mice crossed with germ line Cre mice (FVB/N-Tg(ACTB-cre)^2Mrt/J (The Jackson Laboratory). D, validation of Ca_vα₂δ₁ conditional knock-out from Advillin-Cre expressing DRG neurons in 2-week SNL WT CKO control or CKO^Adv-Cre mice with Ca_vα₂δ₁ antibody immunofluorescent staining (red). Contra., non-injury; Ipsi., injury side. Summarized total intensity (D1) and surface area (D2) data are presented as the means ± S.E. from 12 sections, three mice in each group. *, p < 0.05 compared with non-injury (contra.) side in each group with paired Student's t test. Scale bar: 125 μm for all panels. E, a representative L4 dorsal spinal cord image showing random sampling (white squares) of fluorescent immunoreactivity from superficial dorsal horn for data analysis. Scale bar: 63 μm. F, representative images from co-immunostaining in thin sections of L4 dorsal spinal cord from WT CKO or CKO^Adv-Cre mice 4-days post-i.t. saline (vehicle) or TSP4 (5 μg/5 μl/mouse) injection. VGlut₂ (green), excitatory presynaptic marker; PSD95 (red), post-synaptic marker; yellow, colocalized immunoreactivity. Scale bar: 5 μm for all panels. Each of these images was enlarged to show detailed structure from a small area (similar to the white box in G) of a sampling image. G, a representative sampling image showing the area (in the white box) that was enlarged to show detailed structure above. Scale bar: 10 μm. H, summarized ratio of VGlut₂⁺/PSD95⁺ over VGlut₂⁺/PSD95⁻ immunoreactive puncta. Data are the means ± S.E., 36 sections from 3 mice each. ***, p < 0.001 versus vehicle injected WT CKO mice by one-way ANOVA with Dunnett's post hoc test.

Spinal Nerve Ligation (SNL) (41)

Briefly, the left L4 spinal nerve of mice, which is equivalent to L5 spinal nerve in rats (42), was exposed in isoflurane-anesthetized animals and tightly ligated between the DRG and their conjunction to form the sciatic nerve with a silk suture. Sham procedures were done in the same way without spinal nerve ligation. Behavioral tests were performed at designated times before collection of tissue samples, which were either processed immediately for biochemical studies or kept at −80 °C until use.

Ca_vα₂δ₁ shRNA design and delivery via an adeno-associated viral vector (scAAV). A cDNA encoding the complete coding sequence of the mouse Ca_vα₂δ₁ subunit was obtained from Open Biosystems (IMAGE: 40061614), then cloned into a mammalian expression vector (pYFP-C1, Clontech). Candidate shRNAs were designed using publicly available web tools (Invitrogen Block-it and Genscript). These shRNAs were imbedded in a mir30 backbone using opposing BsmBI sites to insert complementary oligonucleotides encoding the shRNAs without altering the miR sequence (43, 44). The shRNAs were cloned into a human H1 promoter amplified from pLVUTH (Addgene clone 11650). We engineered a novel scAAV using the pLVUTH backbone and the δ-ITR sequences described by McCarty et al. (45). This vector uses a CMV enhanced human synapsin-1 promoter (46) to drive the expression of mCherry (provided by Dr. Roger Y. Tsien, UCSD) that was modified by adding the C-terminal ER export signal (FCYENE) from Kir2.1 (47). Four shRNAs were screened for target knockdown after expression in HEK-293 cells. Knockdown efficiency was measured using quantitative PCR with primers that encompassed the shRNA binding site (48). The shRNA with the highest knockdown efficacy (80%) relative to a scrambled control was AD1: AACTGGACAAGTGCCTTAGAT. CSRH1AD1 scAAV particles pseudotyped with serotype 8 were purified by the University of North Carolina Vector Core (titer 2 × 10¹² virus molecules/ml).

Intrathecal Injection

Intrathecal injections between lumbar L5/6 regions for rats or L4/5 regions for mice were performed under light isoflurane anesthesia through a 30-gauge needle connected to a microinjector (Tritech Research, Inc., Los Angeles, CA). A total volume of 10 μl per rat or 5 μl per mouse was injected.

Behavioral Test

Testing was performed in a blinded fashion. Behavioral test values between left and right hind paws from the SNL groups were recorded separately and used for statistical analysis, and that from non-SNL groups were averaged and used for statistical analysis.

Tactile Allodynia

Hind paw sensitivities to von Frey filament stimulation were tested for tactile allodynia as described previously (2, 49, 50). After acclimatization in wire mesh-floored transparent enclosures, the animals were accessed for the 50% paw withdrawal thresholds (PWT) to von Frey filament (Stoelting Wood Dale) stimulation using the up-down method (51). Briefly, the plantar surface of the hind paw was contacted perpendicularly with the first filament (2.0 g for rats or 0.41 g for mice) until it was slightly bent. A positive response of paw lifting within 5 s led to the use of the next lighter filament, and a negative response led to the use of the next heavier filament until a total of six measurements had been made, starting from the one before any change in the behavioral response. A score of 15 g for rats or 3 g for mice was assigned if five consecutive negative responses had occurred or a score of 0.25 g for rats or 0.01 g for mice was assigned if four consecutive positive responses had occurred. The data were then used to determine the 50% response threshold described previously (25).

Thermal Hyperalgesia

Reduced hind paw withdrawal latency to thermal stimuli was measured using a Hargreaves (hot box) apparatus (University of California San Diego, CA) (52) as the indication of thermal hyperalgesia. After acclimatization for at least 30 min on a glass floor maintained at 30 ± 0.1 °C in transparent enclosures, the hind paw plantar surface of a free-moving animal was stimulated by radiant heat projecting from a high intensity light bulb through a small aperture below the glass surface. When the animal moved the paw away from the thermal stimulus, motion detectors on the apparatus turned off the heating light automatically. The paw withdrawal latency was recorded as the time between thermal stimulus application and hind paw withdrawal. 20 s were set as the cutoff time to prevent thermal injury or skin sensitization. Two readings per paw were averaged for statistical analysis.

Mechanical Hyperalgesia

After acclimatization for 1 week to human holding and touch, rats were tested for mechanical hyperalgesia (Randall-Selitto Test (53) using a Paw Pressure Analgesymeter (Ugo Basil North America)). Briefly, a rat hind paw was placed between a blunt pointer and a flat surface and subjected to an escalating force (16 g/s) until paw withdrawal by the animal. The recorded hind paw withdrawal inducing force was used as the paw pressure withdrawal thresholds.

Locomotor Function Tests

After acclimatization daily for 1 week to human handling and the open field test apparatus, mice were tested for locomotor function by a blinded observer using scores of 0–9 arranging from no ankle movement (0) to frequent or consistent coordinated plantar stepping, normal trunk stability, and tail up position (9) as described by Basso et al. (54).

Western Blots

Briefly, equal amounts of proteins were separated in 3–8% NuPAGE Tris acetate gels (Invitrogen) by electrophoresis, then transferred to polyvinylidene difluoride membranes electrophoretically. After blocking nonspecific binding sites with 5% low fat milk (in PBS-T containing 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na₂HPO₄ 1.4 mm KH₂PO₄, 0.1% Tween 20, pH 7.4), the membranes were cut into sections containing different target proteins and incubated with primary antibodies against Ca_vα₂δ₁ (mouse, 1:1000, catalog #D219, Sigma), TSP4 (rabbit, 1:1000, custom made and validated against purified TSP4 proteins, Genscript, Piscataway, NJ), β-actin (mouse, 1:10,000, catalog #MAB8929, validated against various β-actin expressing cell lines, Novus Biologicals, LLC, Littleton, CO) overnight at 4 °C followed by horseradish peroxidase-conjugated secondary antibody (1:2000, Cell Signaling, Danvers, MA) for 1 h at room temperature. After a brief incubation with chemiluminescent reagents (Thermo Scientific, Waltham, MA), the band densities were quantified by either imaging quantification (Kodak Image Station 2000MM) or densitometry within the linear range of the film sensitivity curve. The Ca_vα₂δ₁ band detected by the mouse Ca_vα₂δ₁ antibodies reflected the Ca_vα₂ protein only (≈150 kDa) as the Ca_vδ₁ peptide separates from the Ca_vα₂ protein under reducing conditions in Western blots (55). For quantification, band density ratios for the protein of interest over that of β-actin (≈42 kDa) were calculated within each sample first for normalization of total protein loading before cross-sample comparisons. Band density variations for the proteins of interest in the contralateral (non-injury) side were determined by comparing each band density with the mean of that from at least two different control samples in the same Western blot after taking the ratios to β-actin band densities.

Spinal Cord Slice Recording

α-Amino-3-hydroxyl-5-methylisoxazole-4-propionic acid(AMPA) receptor-mediated mEPSC and evoked excitatory post-synaptic currents (eEPSC) were recorded from lumbar spinal cord transverse slices (300 μm). Briefly, spinal cord slices were prepared and transferred to the recording chamber as described previously (30, 33). The patch electrode had a resistance of 5–7 megaohms when filled with pipette solution that contained 135 mm potassium gluconate, 5 mm KCl, 5 mm EGTA, 0.5 mm CaCl₂, 10 mm HEPES, 2 mm MgATP, and 0.1 mm GTP, pH 7.2, with an osmolarity 300 mosmol/liter. Superficial dorsal horn neurons were visualized with an upright microscope (Eclipse FN1, Nikon, Japan) and near-infrared illumination based on the gelatinous (semi-transparent) appearance of lamina II (substantia gelatinosa). Although neurons in superficial dorsal horn (including lamina I, lamina II outer or II_o, and lamina II inner or II_i) are heterogeneous (56, 57), the boundaries of laminae I, II_o, and II_i are ambiguous in live spinal cord slices so it was difficult to classify lamina-specific neuron populations, a limitation of our sampling method. Thus, we pooled all recording data together for a general view of TSP4 effects in modulating synaptic transmission and regulation in superficial dorsal horn neurons. All recordings were performed at 32 ± 0.5 °C with MultiClamp 700B amplifiers (Axon Instruments, Molecular Devices, Union City, CA), Digidata 1440 analog-to-digital converters (Axon Instruments), and pClamp 10.2 software (Axon Instruments).

mEPSCs were recorded as described previously (2, 30, 33) in the presence of tetrodotoxin (TTX, 1 μm), strychnine (1 μm), bicuculline (10 μm), and 2-amino-5-phosphonopentanoic acid (AP5, 50 μm) to block TTX-sensitive Na⁺, glycinergic, GABAergic, and N-methyl-d-aspartate (NMDA) currents, respectively. Membrane potential was held at −60 mV so that N-methyl-d-aspartate receptor-mediated currents were blocked (58), which only represented <10% mEPSCs in dorsal horn neurons, and the remaining 90% mEPSCs could be blocked by AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (59). Series resistance was monitored without compensation throughout the experiment (Multiclamp 700B). Cells were excluded from analysis if the series resistance changed by >20% during the whole-cell recording. Signals were analyzed using clampfit 10.3 (Molecular Devices) after the traces were low-pass-filtered at 2 kHz. Cumulative distribution of mEPSC frequency or amplitude of individual neurons from each experimental group was analyzed with Kolmogorov-Smirnov test.

eEPSCs were similarly recorded from superficial dorsal horn neurons of L4 lumbar spinal cord slices upon stimulating (0.1 ms, 0.05 Hz) the attached dorsal roots or dorsal root entry zone with 0–500-μA stimulus intensity. At least six eEPSC events were recorded at each stimulus intensity. QX314 (5 mm) was added in intrapipette solution to prevent sodium channel activation.

Immunohistochemistry (2, 28)

Lumbar spinal cord and DRG samples were fixed in 4%paraformaldehyde overnight, cryoprotected in 30% sucrose, mounted in Optimum Cutting Temperature (O.C.T., Sakura Finetek, Torrance, CA), and sectioned with a cryostat (CM1900, Leica Microsystems, Wetzlar, Germany) into 10-μm slices. Slices of spinal cord samples were pretreated with heat-based antigen retrieval (10 nm sodium citrate, 0.05% Tween 20, pH 6.0, 5 min in a pressure cooker). Spinal cord and DRG slices were incubated with primary antibodies against Ca_vα₂δ₁ (rabbit polyclonal, custom made by Thermo Fisher Scientific Inc., Waltham, MA, for spinal cord samples after antigen retrieval; or mouse monoclonal, Sigma, for DRG samples without antigen retrieval; both antibodies were validated with Ca_vα₂δ₁ knock-out mice shown in Fig. 4, A–D), VGlut₂ (guinea pig, catalog #135404, validated previously (Refs. 28 and 60–62); Synaptic Systems, Germany), and PSD95 (rabbit, catalog #MA1-045, validated previously (Refs. 28, 63, and 64), Thermo Fisher Scientific) followed by secondary antibodies with Alexafluor 488 or 594 (Invitrogen) against IgG of corresponding species of the primary antibodies. Sample sections from control and experimental groups (sides) within the same set of experiments were stained at the same time. Samples were mounted with Vectashield containing DAPI for cell nuclei staining (Vector Laboratories, Burlingame, CA). Two images were taken from each superficial dorsal horn section randomly using a Zeiss LSM780 confocal microscope (UC Irvine Optical Biology Core) in 0.3-μm Z-stacks, and three consecutive Z stacks with the best signal were merged and used for data analysis with Volocity 6.0 (PerkinElmer Life Sciences). Briefly, images from control and experimental groups within the same set of experiment were captured with the same setting. Volocity Find Object Using Percentage Intensity function was used to define background threshold, which was used for both contralateral and injury sides within each set of experiment. Fluorescent immunoreactivities above the background level were selected for analysis. From TSP4 or saline-injected mouse samples, VGlut₂/PSD95 co-stained samples (n = 36 over 3 animals, 100 μm apart) were analyzed to determine the numbers of total VGlut₂⁺ (green), PSD95⁺ (red), and VGlut₂⁺/PSD95⁺ (yellow) puncta. Because the effect of intrathecal injection was bilateral, the ratio of VGlut₂⁺/PSD95⁺ over VGlut₂⁺/PSD95⁻ puncta from both sides was used to compare the differences between the TSP4- and saline-treated groups. From 2-week SNL samples, VGlut₂/Ca_vα₂δ₁ (n = 60 over 3 animals, 100 μm apart) co-stained samples were analyzed to determine the numbers of total Ca_vα₂δ₁ (red), VGlut₂⁺ (green), and VGlut₂⁺/Ca_vα₂δ₁⁺ (yellow) puncta. Data from the injury (ipsilateral) side were compared with that from the non-injury (contralateral) side.

Statistics

One-way or two-way ANOVA with post-tests were performed for multigroup comparisons, and unpaired Student's t tests were performed for pair-wise comparisons as indicated in figure legends. Significance was determined by a two-tailed p value < 0.05.

Results

TSP4 Interacts Directly with Ca_vα₂δ₁ in Rodent Spinal Cord in Vivo

Data from in vitro immunoprecipitation and functional assays suggest that TSP4/Ca_vα₂δ₁ forms a complex in mediating abnormal excitatory synapse formation in rat cerebral cortex (8). However, a direct binding or functional interaction between these proteins in spinal cord has not been shown. If interactions of these proteins lead to abnormal synaptogenesis and neuropathic pain states post injury, there should be direct molecular interactions between astrocyte-secreted TSP4 and Ca_vα₂δ₁ in the adult spinal cord in vivo. To test this, we first examined if TSP4 and Ca_vα₂δ₁ proteins were detectable in immunoprecipitation (IP) complexes from rodent spinal cord samples. Our results confirmed previous findings in rat cerebral cortex (8) and showed that Ca_vα₂δ₁ was detectable in TSP4 immunoprecipitates from rat and mouse spinal cord (Fig. 1A), suggesting that these proteins may interact directly or indirectly in rodent spinal cord. The differences in the patterns of bands between the spinal cord lysates and IP samples are likely due to these factors. First, IP samples were more concentrated compared with the “Lys” control samples as we loaded an equal volume of each sample onto the same blot. Under non-reducing conditions, the IP complexes might contain more target proteins and other associated proteins. Second, it is possible, but needs to be confirmed, that TSP4 antibodies might have pulled down the extracellular Ca_vα₂ domain (150 kDa) of the Ca_vα₂δ₁ subunit (175 kDa) without the Ca_vδ₁ peptide (25 kDa).

FIGURE 1.

TSP4 and Ca_vα₂δ₁ interaction. Immunoprecipitation, solid-phase binding, and surface plasmon resonance binding were performed as described under “Materials and Methods” to detect TSP4/Ca_vα₂δ₁ interaction in rodent spinal cord and in vitro. A, typical Western blots showing Ca_vα₂δ₁ co-immunoprecipitation with TSP4 proteins (IP) by anti-TSP4 antibodies from rat or mouse spinal cord (SC) samples (from n ≥ 3 each). Lys, spinal cord lysate positive control. −, no anti-TSP4 IP antibody. +, with anti-TSP4 IP antibody. Approximate positions of prestained molecular weight markers are shown on the left of each gel. B, solid phase binding showing dose-dependent FLAG-Ca_vα₂δ₁ binding to immobilized TSP4. **, p < 0.01; ***, p < 0.001 compared with no Ca_vα₂δ₁; #, p < 0.05; ###, p < 0.001 between adjacent doses by one-way ANOVA with Bonferroni post-tests. C, surface plasmon resonance binding sensogram of dose-dependent TSP4 binding to captured Ca_vα₂δ₁ (typical of three independent experiments).

To further assess whether TSP4 interacts with Ca_vα₂δ₁ directly, we examined interactions with solid phase binding and surface plasmon resonance spectroscopy assays. The solid phase binding assay confirmed that recombinant Ca_vα₂δ₁ proteins bind directly to immobilized TSP4 in a dose-dependent fashion (Fig. 1B). Conversely, surface plasmon resonance spectroscopy demonstrated dose-dependent binding of TSP4 to immobilized Ca_vα₂δ₁ on a BIAcore CM5 sensor chip with fast association and slow dissociation (Fig. 1C).

Interdependent Interactions between TSP4 and Ca_vα₂δ₁ Proteins Contribute to Pain States and Dorsal Horn Neuron Sensitization

If TSP4 induces neuropathic pain by interacting with Ca_vα₂δ₁, then blocking this interaction pharmacologically should abrogate TSP4-induced pain. It is known that gabapentin specifically binds to Ca_vα₂δ₁ (17, 18) and blocks neuropathic pain states induced by nerve injury-induced Ca_vα₂δ₁ (23–25). Thus, we tested whether gabapentin treatment could reverse TSP4-induced pain states. As reported previously (2), TSP4 intrathecal (i.t.) injection (45 μg/rat) induced tactile allodynia as evidenced by reduced PWT to von Frey filament (mechanical) stimulation 3 days post-TSP4 injection (open bars, Fig. 2A). One hour after gabapentin injection (300 μg/rat, i.t.), PWT increased to near the control levels. The effect of gabapentin was reversible because PWT were again reduced to the pretreatment level 24 h post-gabapentin treatment (filled bars, Fig. 2A). Gabapentin treatment had a similar effect on TSP4-induced thermal hyperalgesia and mechanical hyperalgesia (data not shown).

FIGURE 2.

Blocking or depleting Ca_vα₂δ₁ reverses pain states and exaggerated presynaptic excitatory input induced by elevated TSP4 or SNL. A, Gabapentin (i.t. injected 3 days post-TSP4 injection) reversed tactile allodynia induced by TSP4 (45 μg/rat, i.t.) in 1 h. Data are the means ± S.E. from n = 6 (saline) to 8 (gabapentin (GBP)) rats. ***, p < 0.001 versus pre-TSP4 by one-way ANOVA with Bonferroni post-tests. B1, mEPSCs from rat L5 superficial dorsal horn neurons 3 days after the same TSP4 injection. Top, mEPSC frequency, but not amplitude, was increased in TSP4 injected rats versus control rats injected with an equal molar dose of His-tag peptides. Bottom, gabapentin (50 μm) blocked the TSP4 effects. B2–B4, top, summarized data. Date are the means ± S.E., n ≥ 7 neurons from 4 rats in each group. **, p < 0.01 compared with control (B2) or pre-treatment (B4) level by Student's t test. B2–B4 bottom, cumulative distribution of mEPSC frequency (B2 bottom) and amplitude (B3 bottom) in neurons from control and TSP4 injected rats (p < 0.01 for comparison of mEPSC frequency; p > 0.05 for comparison of mEPSC amplitude; Kolmogorov-Smirnov test). B4 bottom, application of gabapentin (50 μm) significantly reduced the mEPSCs frequency in neurons from TSP4-injected rats to a level similar to that in control neurons (p < 0.001 for comparison of mEPSC frequency before and during gabapentin treatment in TSP4 injected group; Kolmogorov-Smirnov test). C, preemptive knockdown of Ca_vα₂δ₁ in rat dorsal spinal cord (DSC, inset) with bolus intrathecal injection (L5/6 region) of anti-Ca_vα₂δ₁ small hairpin RNA (Ca_vα₂δ₁-AAV), but not control (Control-AAV), AAV vectors (10⁶ units in 2 μl) 10 days before TSP4 injection (45 μg/10 μl/rat, i.t. at day 0) blocked TSP4-induced allodynia. Data are the means ± S.E. from seven rats each. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 versus pre-TSP4 by repeated measures two-way ANOVA analysis with Bonferroni post-tests; ##, p < 0.01 versus the control AAV group by Student's t test (inset). D1, similar intrathecal injection of Ca_vα₂δ₁, but not control, AAV into L5/6 regions of 2-week SNL rats led to a gradual reversal of established allodynia in the hind paws of the injury (Ipsi.) side without affecting the behavioral sensitivity in that of the non-injury (Cont.) side. Data are the means ± S.E. from six rats each. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001 compared with the pretreatment level by repeated measures two-way ANOVA analysis with Bonferroni post-tests. D2, dorsal spinal cord Ca_vα₂δ₁ levels were down-regulated significantly from both sides of rats similarly injected with Ca_vα₂δ₁-AAV 10 days ago compared with that in rats injected with the control AAV. Top, representative images from the same Western blot showing Ca_vα₂δ₁ levels in both sides of dorsal spinal cord samples. β-Actin bands were used for normalization of total protein loading (see “Materials and Methods”). Approximate positions of prestained molecular weight (MW) markers are shown on the left. Bottom, summarized Western blot data. Data are the means ± S.E. from six rats each. **, p < 0.01; ***, p < 0.001 compared with non-injury side. #, p < 0.05; ##, p < 0.01 compared with the same side in control-AAV treated rats by Student's t test. C, contralateral (non-injury) side. Ip, ipsilateral (injury) side.

We previously reported that TSP4-induced pain states correlated with increased mEPSC frequency, but not amplitude, in superficial dorsal horn neurons (2). If TSP4 mediates this effect via ongoing interaction with Ca_vα₂δ₁, then blocking Ca_vα₂δ₁ with gabapentin should reduce mEPSC frequency. Consistent with our previous study (2), recordings from superficial dorsal horn neurons 3 days after TSP4 injection revealed a significant >100% increase in average mEPSC frequency without significant changes in its amplitude (Fig. 2, B1, B2, and B3). After treatment with gabapentin (50 μm), elevated mEPSC frequency was dramatically decreased to the control level, but its basal level in the control group was not affected significantly (Fig. 2, B1 and B4). These changes were confirmed by Kolmogorov-Smirnov tests using cumulative distribution of mEPSC frequency or amplitude of individual neurons from each experimental group (bottom panels of Fig. 2, B2–B4). This gabapentin concentration is close to that in patient cerebrospinal fluid after chronic gabapentin treatments. It has been reported that oral 900 mg/day gabapentin treatment for 3 months can reach peak plasmid concentration ∼10 mg/liter (58 μm) 3 h after the last dose (65), and cerebrospinal fluid gabapentin concentration range was ∼20–35% that in the plasma after multiple dosing (65, 66). Collectively, these results support the idea that TSP4/Ca_vα₂δ₁-dependent processes mediate both TSP4-induced pain states and the putative pathological underpinning (increased mEPSC frequency).

To further test whether blocking TSP4/Ca_vα₂δ₁-dependent processes by biochemical knocking down Ca_vα₂δ₁ could prevent TSP4-induced pain states, we investigated if TSP4-induced behavioral hypersensitivity could be prevented by pre-emptive knockdown of Ca_vα₂δ₁ with intrathecal treatment of anti-Ca_vα₂δ₁ small hairpin RNA in AAV vectors (Ca_vα₂δ₁-AAV). We previously showed that bolus intrathecal TSP4 injection induced tactile allodynia as evidenced by progressive reduction in PWT to a tactile stimulus (2). This effect was confirmed in the present study in rats that were pretreated with intrathecal control AAV (10⁶ units in 2 μl) 10 days before bolus TSP4 injection (45 μg/rat, i.t.) (Fig. 2C). In contrast, rats pretreated with Ca_vα₂δ₁-AAV (10⁶ units in 2 μl), which diminished dorsal spinal cord Ca_vα₂δ₁ levels (Fig. 2C, inset), did not exhibit the progressive decreases in PWT that reflect tactile allodynia (Fig. 2C). These results support the conclusion that TSP4-induced allodynia does depend on Ca_vα₂δ₁.

To further test this idea in the clinically relevant neuropathic model of SNL, we investigated whether knock down of Ca_vα₂δ₁ by intrathecal Ca_vα₂δ₁-AAV could reverse SNL-induced behavioral hypersensitivity as our previous studies have implicated either Ca_vα₂δ₁ or TSP4 in neuropathic pain states (2, 24, 25, 28, 29). Bolus i.t Ca_vα₂δ₁-AAV, but not the control vector, treatments (10⁶ units in 2 μl) caused a time-dependent reversal of 2-week SNL-induced tactile allodynia without affecting the behavioral sensitivity in the contralateral (non-injury) side. The anti-allodynic effects of Ca_vα₂δ₁-AAV peaked ∼10 days after the i.t. injection (Fig. 2D1) and lasted for >2 weeks (data not shown). Western blot analysis of spinal cord samples from control and Ca_vα₂δ₁-AAV-treated rats at the peak allodynia reversal time point (10 days post-Ca_vα₂δ₁-AAV treatment) confirmed Ca_vα₂δ₁ knockdown on both sides of dorsal spinal cord. Ca_vα₂δ₁ levels on the injury side were not significantly different from levels in the non-injury side of control vector-treated rats (Fig. 2D2). The fact that Ca_vα₂δ₁ knockdown prevented allodynia development (Fig. 2C) and reversed established allodynia (Fig. 2D1) indicates that TSP-induced behavioral hypersensitivity requires ongoing interactions between TSP4 and Ca_vα₂δ₁ at the spinal cord level.

Next, we examined if blocking TSP4 could diminish pain states and putative pathological underpinning due to elevation of Ca_vα₂δ₁. We previously showed that TG mice with neuronal Ca_vα₂δ₁ overexpression have increased mEPSC frequency and pain states similar to mice with SNL injury (30, 33, 34, 59, 67). We first tested whether blocking TSP4-dependent processes with TSP4 antibodies would reverse Ca_vα₂δ₁ overexpression-induced allodynia. Consistent with previous findings (34), TG mice with neuronal Ca_vα₂δ₁ overexpression have greatly reduced PWT to mechanical stimuli (the behavioral reflection of allodynia). After treatment with the TSP4 antibody (10 μg/mouse, i.t., chicken polyclonal, validated previously; Ref. 68), PWT gradually increased to near the control level by 8 h post-treatment and then returned to the pretreatment level by 24 h (Fig. 3A1). Antibody treatment did not affect baseline thresholds in age- and sex-matched wild type (WT) mice. Heat-denatured antibody was without effect (Fig. 3A1). Similar treatment with this antibody also prevented the development and reversed established neuropathic pain states in the more clinically relevant SNL model (2).

FIGURE 3.

Depleting or blocking TSP4 reverses Ca_vα₂δ₁-induced pain states and dorsal horn neuron sensitization. A1, TSP4 antibody (Ab, chicken, 10 μg/5 μl/mouse, i.t.), but not heated TSP4 antibody, reversed allodynia induced by neuronal Ca_vα₂δ₁ overexpression in the Ca_vα₂δ₁-TG mice without affecting baseline sensitivity in WT mice. Data are the means ± S.E. from n = 5 (WT) or 6 (TG). ***, p < 0.001; ****, p < 0.0001 versus pretreatment level by repeated measures two-way ANOVA with Bonferroni post-tests. A2, TSP4 ablation did not affect baseline sensitivity but reversed tactile allodynia (top) and thermal hyperalgesia (middle) in the Ca_vα₂δ₁ TG mice. Locomotor functions were not affected in these genetically modified mice as analyzed with the Basso Mouse Scale for locomotion (BMS), bottom). Data are the means ± S.E. from n = 8–10 each group. **, p < 0.01; ***, p < 0.001 by Student's t test. B1, mEPSCs from L4 superficial dorsal horn neurons of the same mouse groups. Ca_vα₂δ₁-induced increase of mEPSC frequency in the Ca_vα₂δ₁ TG mice was blocked by TSP4 ablation. B2 top, summarized data. Data are the means ± S.E. from the number of neurons shown in parentheses that were obtained from ≥ 5 mice in each group. *, p < 0.05 versus WT by one-way ANOVA with Bonferroni post-tests. B2 bottom, cumulative distribution of mEPSC frequency and amplitude in each group (p < 0.01 for frequency comparisons between the Ca_vα₂δ₁ TG group and each of the other three groups; Kolmogorov-Smirnov test). C1, TSP4 ablation also blocked Ca_vα₂δ₁-induced increase of eEPSC amplitude in response to dorsal root entry zone stimulation without affecting baseline eEPSCs significantly. C2, summarized data, means ± S.E. from n ≥ 7 for each stimulus intensity (≥5 mice). **, p < 0.01; ***, p < 0.001 versus WT by repeated measures two-way ANOVA with Bonferroni post-tests.

As an alternative approach, we tested if genetic ablation of TSP4 from the Ca_vα₂δ₁ TG mice would eliminate behavioral hypersensitivities previously reported in Ca_vα₂δ₁ TG mice (30, 33, 34, 59). To test this, we crossed Ca_vα₂δ₁ TG mice that develop hypersensitivity to stimuli with TSP4 knock-out mice to generate Ca_vα₂δ₁ TG/TSP4 KO mice with elevated neuronal Ca_vα₂δ₁ and TSP4 ablation. Similar to our previous findings (34), Ca_vα₂δ₁ overexpression in the TG mice resulted in behavioral hypersensitivities to mechanical (allodynia; Fig. 3A2, top) and thermal (thermal hyperalgesia; Fig. 3A2, middle) stimuli. Assessment of pain sensitivity in Ca_vα₂δ₁ TG/TSP4 KO mice revealed no increased sensitivity to either of these stimuli. Behavioral thresholds in mice with TSP4 knock-out alone (TSP4 KO) were comparable with that in WT control mice (Fig. 3A2, top and middle). The locomotor function test scores of Basso Mouse Scale (BMS) in mice with these genetic modifications were similar to that in the control mice (Fig. 3A2, bottom). Thus, TSP4 basal level is not critical in maintaining basal sensory/motor functions, and differences in behavioral sensitivities to stimuli among these mouse groups are not due to changes in motor functions. Together, these findings support that TSP4/Ca_vα₂δ₁-dependent processes are also required for pain state processing induced by elevated Ca_vα₂δ₁.

We also assessed whether the increase in mEPSC frequency reported previously in Ca_vα₂δ₁ TG mice (30, 33, 59) could be normalized by deleting TSP4 from the Ca_vα₂δ₁ TG/TSP4 KO mice. Similar to our previous findings (30, 33, 59), recordings from superficial dorsal horn neurons of Ca_vα₂δ₁ TG mice revealed significantly elevated frequency, but not amplitude, of mEPSC compared with that from control WT mice (Fig. 3B). As another control, recordings from superficial dorsal horn neurons of TSP4 KO mice revealed mEPSC frequency/amplitude comparable with that seen in WT neurons (Fig. 3B). However, recordings from superficial dorsal horn neurons of Ca_vα₂δ₁ TG/TSP4 KO mice revealed that mEPSC frequency and amplitude were within the range of that in control mice (Fig. 3B). These findings were confirmed by Kolmogorov-Smirnov tests using cumulative distribution of mEPSC frequency or amplitude of individual neurons from each experimental group (bottom panels of Fig. 3B2). Collectively, these findings support that although basal level TSP4 is not required for maintaining a normal level of mEPSC, TSP4/Ca_vα₂δ₁-dependent processes are required for Ca_vα₂δ₁-induced increase of mEPSC frequency, an indication of enhanced presynaptic excitatory input.

We hypothesized that increased TSP4/Ca_vα₂δ₁ interactions in the spinal cord of the TG mice could lead to hyperexcitability of dorsal horn neurons to peripheral stimulation. To test this, we examined the amplitude of eEPSCs in L4 superficial dorsal horn neurons from the Ca_vα₂δ₁ TG mice in response to escalating intensities of stimulation to dorsal root entry zone. Compared with that from WT littermates, eEPSC amplitudes were increased at all levels of stimulus intensity tested (Fig. 3C), indicating hyperresponsiveness of the dorsal horn neurons to afferent activation as a result of Ca_vα₂δ₁ overexpression. Similar recordings in Ca_vα₂δ₁ TG/TSP4 KO mice revealed that eEPSC frequency was within the range of WT mice, as was also the case with TSP4 KO alone (Fig. 3C). These findings suggest that although basal level TSP4 is not required for maintaining normal dorsal horn neuron excitatory tone, TSP4/Ca_vα₂δ₁-dependent processes are required for Ca_vα₂δ₁-mediated dorsal horn neuron sensitization.

Interdependent Interactions between TSP4 and Ca_vα₂δ₁ Proteins Promote Aberrant Excitatory Synaptogenesis in Animal Models with Pain States

Previous studies have shown that Ca_vα₂δ₁ proteins in sensory neurons are transported to central afferent terminals in the spinal dorsal horn under normal and post-injury conditions (27, 29). Other studies have shown that TSP interactions with Ca_vα₂δ₁ promote excitatory synaptogenesis in vitro (8) and increased excitatory synapses are associated with neuropathic pain states (28, 69). Together, these findings raise the possibility that injury-induced TSP4 in spinal cord might interact with Ca_vα₂δ₁ at the pre-synaptic terminals of sensory afferents to promote aberrant excitatory synaptogenesis, which in turn would contribute to enhanced transmission along pain pathways.

To test this hypothesis, we took the first step in determining whether there was evidence for aberrant excitatory synaptogenesis with TSP4-induced pain states; when so, we tried to determine if genetic ablation of Ca_vα₂δ₁ could block TSP4-induced synaptogenesis. We generated Ca_vα₂δ₁ conditional knock-out (WT CKO) mice in which exon 6 of the Ca_vα₂δ₁ gene was floxed with loxP sites (Fig. 4, A and B). Crossing these mice with Cre-recombinase expressing mice resulted in deletion of Ca_vα₂δ₁ from Cre-expressing cells (Fig. 4C). The WT CKO mice were crossed with Advillin-Cre mice with Cre recombinase expression only in Advillin-positive sensory neurons that cover ∼94% of all DRG neurons (35). Cre-driven Ca_vα₂δ₁ deletion from DRG neurons in homozygous CKO^Adv-Cre+/− mice was confirmed by immunostaining data showing that 2-week SNL could induce DRG Ca_vα₂δ₁ up-regulation indicated as increased Ca_vα₂δ₁ antibody immunoreactivity in DRG neurons of control mice (WT CKO), as we have reported previously (25), but failed to do so in homozygous CKO^Adv-Cre+/− mice (Fig. 4D). We then immunostained sections of L4 lumbar spinal cord from TSP4 (5 μg/mouse, i.t.)-injected mice 4 days after the injection (peak pain states in control mice) with markers for excitatory synapses (VGlut₂ and post-synaptic density marker PSD95). Measurements of total number of synaptic puncta immunoreactive to both synaptic marker antibodies in spinal cord superficial dorsal horn (Fig. 4E) revealed substantial increases in excitatory synapses in TSP4 injected control WT CKO mice compared with vehicle-injected WT CKO mice. These TSP4-induced changes were not seen with Ca_vα₂δ₁ ablation in the CKO^Adv-Cre+/− mice (Fig. 4, F–H). Thus, TSP4-induced aberrant excitatory synaptogenesis in dorsal spinal cord requires TSP4/Ca_vα₂δ₁-dependent processes.

If TSP4-induced aberrant excitatory synaptogenesis in dorsal spinal cord contributes to pain processing, one would expect to see that the absence of TSP4-induced excitatory synaptogenesis in the CKO^Adv-Cre+/− mice correlates with the absence of TSP4-induced pain states. We tested this by examining behavioral sensitivity of control and CKO^Adv-Cre+/− mice after TSP4 injections (5 μg/mouse, i.t.). Similar to previous findings in rats (2), TSP4 injection into control WT CKO mice led to tactile allodynia (Fig. 5, A and B) and thermal hyperalgesia (Fig. 5C), which peaked ∼4 days after TSP4 injections that correlated temporally with a significant increase of excitatory synapses in dorsal spinal cord of these mice (Fig. 4, F–H). In contrast, similar TSP4 injections into CKO^Adv-Cre+/− mice failed to induce similar behavioral hypersensitivities (Fig. 5, A–C). This supports that TSP4-induced aberrant excitatory synaptogenesis through TSP4/Ca_vα₂δ₁-dependent processes plays a role in transmitting nociceptive signals.

FIGURE 5.

Ca_vα₂δ₁ ablation from DRG neurons blocked TSP4-induced pain states. Hind paw sensitivities to mechanical and thermal stimuli were measured in control WT CKO or CKO^Adv-Cre mice post-bolus TSP4 (5 μg/5 μl/mouse, i.t.) injection as described under “Materials and Methods.” A, time course of TSP4-induced tactile allodynia in WT CKO mice that was diminished in CKO^Adv-Cre mice with Ca_vα₂δ₁ ablation from Advillin⁺ DRG neurons. Data are the means ± S.E. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001 versus pretreatment level by repeated measures two-way ANOVA with Bonferroni post-tests. Peak allodynia (B) and thermal hyperalgesia (C) seen in WT CKO mice were blocked in CKO^Adv-Cre mice. Data are the means ± S.E. from (n) indicated. ****, p < 0.0001 versus saline group by Student's t test.

We next tested whether TSP4-induced synaptogenesis also plays a role in neuropathic pain development in the more clinically relevant SNL model. Our previous study has shown that SNL-induced allodynia is diminished in SNL TSP4 KO mice (2), suggesting a role of TSP4 in mediating neuropathic pain. Immunostaining for Ca_vα₂δ₁ revealed increases in Ca_vα₂δ₁ puncta in the superficial dorsal horn of the injury side in both WT and TSP4 KO mice 2 weeks post-SNL (Fig. 6, A and B), which correlated with severe allodynia in SNL WT, but not TSP4 KO, mice (2). Thus, elevated presynaptic Ca_vα₂δ₁ expression is not regulated by TSP4. Nerve injury also increased VGlut₂ puncta that mainly co-localized with Ca_vα₂δ₁ puncta in the WT mice (Fig. 6, A, C, and D), supporting that injury-induced Ca_vα₂δ₁ up-regulation at the presynaptic terminals of injured afferents is associated with increased numbers of excitatory synapses in dorsal spinal card. Importantly, these changes were not seen in TSP4 KO mice with SNL (Fig. 6, A, C, and D). These data support that SNL does induce aberrant excitatory synaptogenesis that also requires TSP4/Ca_vα₂δ₁-dependent processes.

FIGURE 6.

TSP4 ablation blocked SNL-induced excitatory synaptogenesis. Co-immunostaining of Ca_vα₂δ₁ with synaptic markers was performed in thin sections of dorsal spinal cord samples from two-week SNL WT or TSP4 KO mice when behavioral hypersensitivity occurred in the injury (Ipsi.) side of SNL WT mice but not TSP4 KO mice (2). A, representative images showing Ca_vα₂δ₁ (red) and VGlut₂ (green) immunoreactivity and their colocalization (yellow) in superficial dorsal horn. Scale bar = 5 μm for all image panels. Summarized data of total Ca_vα₂δ₁ immunoreactivity intensity (B), VGlut₂ immunoreactivity surface area (C), and VGlut₂⁺ puncta with (yellow) or without (green) co-localization with Ca_vα₂δ₁ immunoreactivity (D) are presented as the means ± S.E. collected from 60 images over three mice in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with non-injury (contra.) side by paired Student's t test.

Discussion

Peripheral nerve injury induces up-regulation of TSP4 and Ca_vα₂δ₁ in DRG/spinal cord that contributes to neuropathic pain states through mechanisms that were previously undefined (2, 24, 25, 27, 29, 31). Here, we provided a large body of evidence to support that TSP4/Ca_vα₂δ₁-dependent processes are required in promoting central sensitization and pain states.

Our data confirm that there is a direct interaction between TSP4 and Ca_vα₂δ₁ in rodent spinal cord and in vitro. In addition, blocking or down-regulating Ca_vα₂δ₁ can block TSP4-induced pain states and increased mEPSC frequency in spinal cord neurons. Conversely, blocking or genetically deleting TSP4 can block Ca_vα₂δ₁ overexpression-induced behavioral hypersensitivity, increased mEPSC frequency, and exaggerated eEPSC in spinal cord neurons. Furthermore, elevated TSP4 induces an increase of spinal excitatory synapses that correlates with heightened pain states, both of which can be normalized by Ca_vα₂δ₁ ablation from sensory neurons. Equally, TSP4 ablation blocks injury-induced excitatory synaptogenesis associated with elevated Ca_vα₂δ₁ without affecting nerve injury-induced Ca_vα₂δ₁ up-regulation in the dorsal horn. Together, these findings support that elevated TSP4 in dorsal spinal cord can induce central sensitization by promoting exaggerated presynaptic excitatory input, excitatory synaptogenesis, and evoked excitability of dorsal horn neurons through activation of TSP4/Ca_vα₂δ₁-dependent processes.

In combination with previous findings from peripheral nerve injury models, our data reveal the importance of TSP4/Ca_vα₂δ₁-dependent processes in mediating central sensitization and chronic pain states post injury. Although TSP4 is up-regulated within days after peripheral nerve injury in both DRG and dorsal spinal cord (1, 2), there is a significant delay in peak Ca_vα₂δ₁ up-regulation in the dorsal horn (weeks) (29), primarily due to time required for initial translocation of elevated Ca_vα₂δ₁ from DRG to pre-synaptic central terminals of sensory afferents in the dorsal horn (27, 29). The subsequent interaction of elevated TSP4 with excess pre-synaptic Ca_vα₂δ₁ in the dorsal horn then promotes aberrant excitatory synaptogenesis and dorsal horn neuron sensitization to maintain chronic pain states (Fig. 7). Although dorsal horn neurons are heterogeneous and play distinct roles in transmitting modality-specific information (56, 57), our sampling method would not allow us to perform a sensory neuron-type-specific dissection of these changes. Further studies, for example, using Cre-directed ablation of genes of interest from subpopulations of sensory neurons is necessary to provide more deep mechanistic insights about the TSP4/Ca_vα₂δ₁-dependent processes in mediating modality-specific nociception.

FIGURE 7.

Proposed mechanisms of the TSP4/Ca_vα₂δ₁ pathway in central sensitization and neuropathic pain.

Interestingly, although TSP4-induced dorsal horn neuron sensitization and behavioral hypersensitivity is a slow process that requires 4 days to reach the peak effects, gabapentin at a clinically relevant concentration can block these effects within 1 h. Even through we cannot rule out the possibility that a mechanism independent of its binging to Ca_vα₂δ₁ mediates the actions of gabapentin in reversing TSP4-induced dorsal spinal cord neuron sensitization and behavioral hyperalgesia, our data support that keeping the TSP4/Ca_vα₂δ₁-dependent processes in an active state is probably critical for the maintenance of TSP4-induced central sensitization and behavioral hypersensitivity. The fast anti-hyperalgesia actions of gabapentin may derive from interfering with the TSP4/Ca_vα₂δ₁-dependent processes that are likely key elements of pain genesis even after long term pathological changes, such as aberrant excitatory presynaptic input and synaptogenesis, are established post-TSP4 injection or peripheral nerve injury. This may explain why gabapentinoids exhibit inhibitory effects on sensitized spinal neurons (33, 70, 71) and neuropathic pain states in animal models (24, 25, 72–74) and patients (19–21, 75–82) but do not affect baseline sensory neuron excitability and sensory thresholds in control animals (33, 70, 71, 73, 83) and healthy volunteers (84, 85), who should not have increased expression of Ca_vα₂δ₁ and/or TSP4 in the sensory pathway. This may also explain why gabapentin is only effective in some, but not all, neuropathic pain patients with various etiologies (32) as it is less likely that all pain-inducing conditions are associated with increased expression of Ca_vα₂δ₁ and/or TSP4.

In summary, even though some details remained elusive, our findings provide a large body of multidimensional evidence to support that activation of the TSP4/Ca_vα₂δ₁-dependent processes is required for TSP4-induced central sensitization that leads to pain state development. Blocking this pathway may be a novel strategy for development of target-specific analgesics for chronic pain management.

Author Contributions

J. P. designed, performed, and analyzed experiments of in vitro binding, immunoprecipitation, protein expression, and purification. Y. P. Y. designed, performed, and analyzed experiments of confocal imaging and synaptogenesis. C.-Y. Z. designed, performed, and analyzed experiments of electrophysiology. K.-W. L., E. C., D.-S. K., B. V., X. Z., and N. G. designed, performed, and analyzed experiments of transgenic animal models and behavioral pharmacology. I. V. and E. P.-R. designed and constructed the shRNA-AAV vectors for the behavioral pharmacology experiments. K. S. and O. S. designed, performed, and analyzed experiments of motor functions. D. W. and G. F. designed, generated, and analyzed the conditional knockout mice. C. E. and B. B. provided the TSP4 KO mice and contributed to conception of the study. F. Z. designed and constructed the TSP4 expression vectors, assisted in protein purification, and contributed to conception of the study. Z. D. L. conceived and coordinated the study. Z. D. L., J. P., Y. P. Y., C.-Y. Z., and E. P.-R. contributed to preparation of the figures and writing the paper. Z. D. L., J. P., Y. P. Y., C.-Y. Z., O. S., C. E., and B. B. contributed to editing of the paper. All authors reviewed the results and approved the final version of the manuscript.