The nAChR Chaperone TMEM35a (NACHO) Contributes to the Development of Hyperalgesia in Mice

Abstract

Pain is a major health problem, affecting over fifty million adults in the US alone, with significant economic cost in medical care and lost productivity. Despite evidence implicating nicotinic acetylcholine receptors (nAChRs) in pathological pain, their specific contribution to pain processing in the spinal cord remains unclear given their presence in both neuronal and non-neuronal cell types. Here we investigated if loss of neuronal-specific TMEM35a (NACHO), a novel chaperone for functional expression of the homomeric α7 and assembly of the heteromeric α3, α4, and α6-containing nAChRs, modulates pain in mice. Mice with tmem35a deletion exhibited thermal hyperalgesia and mechanical allodynia. Intrathecal administration of nicotine and the α7-specific agonist, PHA543613, produced analgesic responses to noxious heat and mechanical stimuli in tmem35a KO mice, respectively, suggesting residual expression of these receptors or off-target effects. Since NACHO is expressed only in neurons, these findings indicate that neuronal α7 nAChR in the spinal cord contributes to heat nociception. To further determine the molecular basis underlying the pain phenotype, we analyzed the spinal cord transcriptome. Compared to WT control, the spinal cord of tmem35a KO mice exhibited 72 differentially-expressed genes (DEGs). These DEGs were mapped onto functional gene networks using the knowledge-based database, Ingenuity Pathway Analysis, and suggests increased neuroinflammation as a potential contributing factor for the hyperalgesia in tmem35a KO mice. Collectively, these findings implicate a heightened inflammatory response in the absence of neuronal NACHO activity. Additional studies are needed to determine the precise mechanism by which NACHO in the spinal cord modulates pain.

Keywords: TMEM35a/NACHO; inflammation; nicotinic acetylcholine receptor; pain; spinal transcriptome.

Figure 1:

NACHO expression in mouse dorsal spinal horn. (A) Confocal image of WT spinal cord showing NACHO⁺ neurons (green), lamina I marked with CGRP⁺ fiber (blue), and lamina II marked with IB4⁺ fibers (red) of the dorsal horn. (B) Confocal image showing absence of NACHO⁺ neurons in the tmem35a KO spinal cord without changes in the dorsal horn architecture. (C, D) Enlarged image of panel A (white box) showing colocalization of NACHO and peptidergic fibers in lamina I (panel C, CGRP⁺, arrows), but little with non-peptidergic fibers (panel D, IB4⁺) in lamina II. In panel C, the pseudocolor blue was converted to purple for visual enhancement of overlapping areas (white). (E, F) Photomicrographs showing colocalization of NACHO (red) and α7 nAChR (α-Bgtx, green) in lamina I, II (panel E) and V, VI (panel F). Cell nuclei were labeled with DAPI (blue) in panel E and F. White boxes in insets indicate enlarged areas of the dorsal horn. Scale bars = 100 μm for panel A and B, and 25 μm for panel E and F.

Figure 2:

Pain behaviors in tmem35a knockout (KO) adult male mice. KO mice showed hypersensitivity to mechanical (A) heat (B) and cold (C) stimuli as compared to WT control mice. Values are mean ± SEM, t-test, ***p<0.001, ****p<0.0001.

Figure 3:

Intrathecal administration nicotine produced analgesia to heat in tmem35a KO mice. In WT mice, nicotine (0.5 nmol) produced analgesia to mechanical and heat stimuli (panel A and C), but not at a higher (1.5 nmol) nicotine dose. In the KO mice, a high dose (1.5 nmol) of nicotine produced an analgesic response to heat, but not mechanical stimuli (panel B and D). Values are mean ± SEM, 2-way ANOVA with post hoc Bonferroni’s multiple comparisons test, **p<0.01, ***p<0.001.

Figure 4:

High dose intrathecal PHA543613 administration reduced mechanical allodynia in tmem35a KO mice. In WT mice, PHA at doses of 10 nM or 50nM produced analgesia to mechanical and heat stimuli (A and C). In KO mice, only the 50 nM PHA dose reduced mechanical allodynia (B), whereas sensitivity to heat was not altered (D). Values are mean ± SEM, 2-way ANOVA with post hoc Bonferroni’s multiple comparisons test, *p<0.05, **p<0.01.

Figure 5:

Spinal cord transcriptomic analysis revealed increased neuro-inflammation in tmem35a KO mice. (A) A merged functional gene network showed a predictive reduction of intracellular transport of molecules (Z-score = −2.6, p = 0.0026) and neuroglia activation (Z-score = −1.0, p = 0.0005) concomitant with increased inflammation (Z-score = +1.4, p < 0.0001) and number of leukocytes (Z-score = +1.0, p = 0.0022). (B) Predictive activation of upstream regulators (e.g., TCF7L2, AIRE, and EHF, Z-score ≥ +2.0, p < 0.001) accompanied by reduced activity of inhibitors of inflammation (e.g., IKBKB, CHUK, Z-score < −2.2, p < 0.0001). Legends: Red/pink = upregulation, green = downregulation, orange line = leads to activation, blue line = leads to inhibition, yellow line = findings inconsistent with state of downstream molecule, and grey line = effect not predicted. Arrows indicate direction of change in activity. (C) Estimation of microglia (Iba1⁺) in mouse spinal cord dorsal horn. Number of Iba1⁺ cells were counted in the dorsal horn lamina I through III using only visible cell bodies (arrows). Compared to WT, tmem35a KO mice showed a higher number of microglia in the dorsal horn. Representative confocal image of spinal cord dorsal horn labeled for CGRP (blue), IB4 (red), and Iba1 (green). Arrows indicate Iba1⁺ microglia. Scale bar = 50 μm. Values are mean ± SEM, t-test, **p<0.01.