The increased trafficking of the calcium channel subunit alpha2delta-1 to presynaptic terminals in neuropathic pain is inhibited by the alpha2delta ligand pregabalin

Abstract

Neuropathic pain results from damage to the peripheral sensory nervous system, which may have a number of causes. The calcium channel subunit alpha(2)delta-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of neuropathic pain, and this is causally related to the onset of allodynia, in which a non-noxious stimulus becomes painful. The therapeutic drugs gabapentin and pregabalin (PGB), which are both alpha(2)delta ligands, have antiallodynic effects, but their mechanism of action has remained elusive. To investigate this, we used an in vivo rat model of neuropathy, unilateral lumbar spinal nerve ligation (SNL), to characterize the distribution of alpha(2)delta-1 in DRG neurons, both at the light- and electron-microscopic level. We found that, on the side of the ligation, alpha(2)delta-1 was increased in the endoplasmic reticulum of DRG somata, in intracellular vesicular structures within their axons, and in the plasma membrane of their presynaptic terminals in superficial layers of the dorsal horn. Chronic PGB treatment of SNL animals, at a dose that alleviated allodynia, markedly reduced the elevation of alpha(2)delta-1 in the spinal cord and ascending axon tracts. In contrast, it had no effect on the upregulation of alpha(2)delta-1 mRNA and protein in DRGs. In vitro, PGB reduced plasma membrane expression of alpha(2)delta-1 without affecting endocytosis. We conclude that the antiallodynic effect of PGB in vivo is associated with impaired anterograde trafficking of alpha(2)delta-1, resulting in its decrease in presynaptic terminals, which would reduce neurotransmitter release and spinal sensitization, an important factor in the maintenance of neuropathic pain.

Figure 1.

Ipsilateral presynaptic increase of α₂δ-1 in the superficial and deeper layers of the dorsal horn after L5/L6 SNL is reduced by chronic PGB at a dose that alleviated neuropathic pain. A, Representative α₂δ-1 immunofluorescence in a montage of 10 images of a transverse L5 spinal cord section 14 d after SNL. α₂δ-1 Immunofluorescence, Green; nuclear staining, blue; ipsi, ipsilateral side (side of ligation); asterisk (*), fold in section; rectangles, regions of interest for fluorescence intensity profiles; arrow, ipsilateral motor neurons with elevated α₂δ-1 immunofluorescence at the level of ligation. Scale bar, 250 μm. B, Normalized fluorescence intensity profiles (FI_NORM) of the superficial layers [Lissauer's tract (Lt), lamina I (I), and lamina II (lam II) and deeper layers (from lamina III onward)] of the ipsilateral (black line) and contralateral (contra) (gray line) L5 (n = 4) and L4 (B, inset; n = 11) dorsal horn 14 d after SNL. Profiles are normalized to the contralateral FI maximum. Error bars represent SEM. For clarity, only error bars at 100 μm in lamina II and at 200 μm in lamina III are shown. Statistical analysis was as follows: nonpaired Student's t test comparing ipsilateral to contralateral side, ***p < 0.001; *p < 0.05; ns (not significant), p > 0.5. C–F, Representative electron micrographs of ipsilateral (C, D) and contralateral (E, F) immunogold labeling of α₂δ-1 in the dorsal horn (lamina I–III) of L4 and L5 spinal cord sections 14 d after SNL. Scale bar, 0.5 μm. Arrowheads, Presynaptic sites at the plasma membrane of excitatory axon terminal boutons (b, characterized by their round synaptic vesicles and opposing postsynaptic density); arrows, postsynaptic sites at the extrasynaptic plasma membrane of dendritic shafts (Den); double arrows, intracellular dendritic sites. For clarity, not all immunoparticles are labeled. G, Ratio of presynaptic to postsynaptic number of particles in the ipsilateral (filled bars) and contralateral (open bars) L4 and L5 spinal cord dorsal horn. H, Paw withdrawal frequencies to an innocuous mechanical stimulus (von Frey 6 g) applied to the ipsilateral hindpaw of SNL plus saline- (blue squares; n = 12) or SNL plus PGB-treated animals (red circles; n = 11). Error bars represent SEM. Statistical analysis was as follows: nonparametric Mann–Whitney U test, *p < 0.05. Rats were injected with PGB or saline starting at day 1 after SNL (day 0, dotted line) and killed at day 9. I, Normalized fluorescence intensity profiles of ipsi L5 saline- (blue line; n = 13), contra L5 saline- (black line; n = 13), ipsi L5 PGB- (red line; n = 16), and contra L5 PGB-treated (gray line; n = 16) dorsal horn regions. I, Inset, Ipsilateral α₂δ-1 increase compared with contralateral side at 100 μm (lam II) in L4–L6 dorsal horn after saline (blue bars) or PGB (red bars) injections. Error bars represent SEM. Statistical analysis was as follows: nonpaired Student's t test comparing saline to PGB treatment, ***p < 0.001; **p < 0.01. Number of sections is given above bars.

Figure 2.

α₂δ-1 is increased in dorsal roots after SNL and in the ipsilateral ascending tracts from the level of SNL to the brainstem. A, Z-projection of 10 optical sections of the ipsilateral (left) and contralateral (right) proximal L5 dorsal root. α₂δ-1 Immunofluorescence, Green; nuclear staining, blue. Scale bar, 20 μm. B–E, Electron micrographs of ipsilateral (B, C) and contralateral (D, E) L4 and L5 DRG axons. α₂δ-1 immunoparticles at intracellular membranes (arrow) in myelinated (m) and nonmyelinated axons (ax). Scale bar, 0.5 μm. F, Quantification of immunoparticles (10² particles/100 μm²) in ipsilateral (filled bars) and contralateral (open bars) L4 and L5 DRG axons. G, H, α₂δ-1 immunoperoxidase staining in lumbar L5 (G), L4 (G, inset), and cervical C1 (H) spinal cord sections, 14 d after SNL. I, α₂δ-1 Immunostaining in the brainstem (approximately bregma −14.40), 14 d after SNL. Scale bars, 200 μm. Arrows, α₂δ-1 staining in the ipsilateral fasciculus gracilis (fg); dcs, dorsal corticospinal tract; cu, cuneate fasciculus; ng, nucleus gracilis.

Figure 3.

Upregulation of α₂δ-1 mRNA after SNL is not affected by chronic PGB. A, Q-PCR results for α₂δ-1 mRNA levels (percentage) in pooled ipsilateral L5/L6 DRGs (ipsi 5 + 6) from sham-operated animals (open bars; n = 5), from animals 7 d (n = 4; gray bars) or 14 d (n = 7; black bars) after SNL and from SNL plus saline (horizontal striped bar; n = 4) or SNL plus PGB animals (hatched bar; n = 4). Data are normalized to the respective contralateral side. Error bars represent SEM. Statistical analysis was as follows: one-way ANOVA with p = 0.0031 using SNK post test: **p < 0.01; *p < 0.05. Note: Increase in SNL plus PGB animals was not significantly different from SNL plus saline animals in SNK post test (p > 0.05). B, Q-PCR results for α₂δ-2 mRNA levels (percentage) in pooled ipsilateral L5/L6 DRGs (ipsi 5 + 6) from either sham-operated animals (open bars, n = 5) or from animals 7 d (n = 4; gray bars) or 14 d (n = 7; black bars) after SNL. Data are normalized to the respective contralateral side. Error bars represent SEM. Statistical analysis was as follows: one-way ANOVA with p < 0.0001 using SNK post test, ***p < 0.001.

Figure 4.

SNL-mediated ipsilateral increase of α₂δ-1 protein levels in DRG somata is not affected by chronic PGB. A, Single images of ipsilateral (left) and contralateral (right) L5 DRG sections 14 d after ligation. α₂δ-1 Immunofluorescence, Green; nuclear staining, blue. Scale bar, 20 μm. B, Mean intracellular FD [in arbitrary units (a.u.) per square micrometer] of ipsilateral (filled bars) and contralateral (open bars) small (<700 μm²), medium (700–1100 μm²), and large (>1100 μm²) DRG somata 14 d after SNL. Error bars represent SEM. Statistical analysis was as follows: nonpaired Student's t test comparing ipsilateral to contralateral side, ***p < 0.001. Numbers of cells are given above bars. C–J, Electron micrographs of ipsilateral L4 (C) and L5 (D) DRG somata adjacent to the nucleus (N) and PM [L4 (E); L5 (F)] 14 d after ligation. Immunoparticles for α₂δ-1 are mainly associated with the ER (arrows) and also at PM (arrowheads). Micrographs of contralateral L4 (G) and L5 (H) DRG somata and PM [L4 (I); L5 (J)]. Scale bar, 0.5 μm. For clarity, not all immunoparticles are labeled. K, L, Quantification of immunoparticle density in the ER (particles/100 μm²; left) and number of particles at the PM (particles/100 μm; right) in ipsilateral (filled bars) and contralateral (open bars) L4 and L5 DRG somata. M, Images of ipsilateral L5 DRG sections from SNL plus saline or SNL plus PGB animals. α₂δ-1 Immunofluorescence, Green; nuclear staining, blue. Scale bar, 20 μm. N, Normalized fluorescence density (FD_NORM) of ipsilateral small, medium, and large DRG somata after SNL plus saline (blue bars) or SNL plus PGB treatment (red bars). Data are normalized to mean FD of respective SNL plus saline size group. Error bars represent SEM. Statistical analysis was as follows: nonpaired Student's t test comparing saline to PGB treatment, p = 0.417 for small; p = 0.801 for medium; p = 0.223 for large cell bodies. Numbers of cells analyzed are given above bars.

Figure 5.

Increase of α₂δ-1 protein with time in dorsal roots and accumulation proximal to the ligation site. A, Representative single images of contralateral L5 dorsal roots 2 d (left), 4 d (middle), and 7 d after SNL (right). B, Representative single images of ipsilateral L5 dorsal roots 2 d (left), 4 d (middle), and 7 d after SNL. α₂δ-1 Immunofluorescence, Green. Scale bar, 200 μm. C, Single image of an ipsilateral L5 spinal nerve and DRG section 4 d after ligation. α₂δ-1 Immunofluorescence, Green; outline, white; tissue boundaries determined with nuclear staining (data not shown). Scale bar, 1 mm. D, Single image of a contralateral L5 spinal nerve and DRG section 4 d after ligation. α₂δ-1 Immunofluorescence, Green; outline, white; tissue boundaries determined with nuclear staining (data not shown). Scale bar, 1 mm. Note: Stronger α₂δ-1 staining in the ipsilateral DRG neuron somata compared with the contralateral side. E, Top, Diagram of ipsilateral L5 spinal nerve showing ligation site, DRG, and dorsal root segments used for Western blotting. Middle, Representative immunoblot of α₂δ-1 expression in spinal nerve segments (lanes 1–4 with ligation site marked with X between lanes 2 and 3), DRG (lane 5), and dorsal root segments (lane 6 and 7) pooled from four animals 4 d after ligation. Bottom, Corresponding immunoblot of GAPDH expression in ipsilateral L5 spinal nerve, DRG, and dorsal root tissue. F, Quantification of three immunoblots of α₂δ-1 expression in pooled spinal nerve and dorsal root segments 4 d after SNL corrected for gel loading (GAPDH protein level) and normalized to the α₂δ-1 level in the DRGs (number of tissue segments in parentheses). Error bars represent SEM.

Figure 6.

PGB reduces cell surface expression but does not affect endocytosis of α₂δ-1. A–C, Localization of exogenous α₂δ-1 in Cos-7 cells that were either permeabilized (A) or nonpermeabilized (B) before immunostaining with anti-α₂δ-1 antibody (green). C, Staining for exogenous α₂δ-2 in permeabilized Cos-7 cells with anti-α₂δ-2 antibody (red) in presence of the anti-α₂δ-1 antibody (green). Nuclear staining, Blue. Note: Lack of green staining indicates lack of cross-reactivity of the anti-α₂δ-1 antibody. Scale bar, 20 μm. D–F, Images of surface expression of α₂δ-1 (green) in nonpermeabilized Cos-7 cells treated with 0 (saline; D), 20 (E) or 200 (F) μm PGB for 72 h. G, No primary antibody. Nuclear staining, Blue. Scale bar, 20 μm. H, Normalized fluorescence densities (FD_NORM) after saline (blue bar) and 20 or 200 μm PGB treatment (red bars). FDs were normalized to the mean FD of saline-treated cells. Error bars represent SEM. Statistical analysis was as follows: one-way ANOVA, p < 0.0001, with Bonferroni's post test, *p < 0.05 and ***p < 0.001. Number of cells examined is given above bars. I, J, Images of α₂δ-1 endocytosis assay in Cos-7 cells performed in absence (saline; I) or presence of 200 μm PGB (J). Cell surface α₂δ-1, Red; internalized α₂δ-1, green; nuclear staining, blue. Scale bar, 20 μm.