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. 2024 Nov 20;44(47):e1442242024.
doi: 10.1523/JNEUROSCI.1442-24.2024.

Glutamine Oxidation in Mouse Dorsal Root Ganglia Regulates Pain Resolution and Chronification

Md Mamunul Haque  1 , Panjamurthy Kuppusamy  1 , Ohannes K Melemedjian  2   3   4
Affiliations

Glutamine Oxidation in Mouse Dorsal Root Ganglia Regulates Pain Resolution and Chronification

Md Mamunul Haque et al. J Neurosci. .

Abstract

Chronic pain remains a significant health challenge with limited effective treatments. This study investigates the metabolic changes underlying pain progression and resolution, uncovering a novel compensatory mechanism in sensory neurons. Using the hyperalgesic priming model in male mice, we demonstrate that nerve growth factor (NGF) initially disrupted mitochondrial pyruvate oxidation, leading to acute allodynia. Surprisingly, this metabolic disruption persisted even after the apparent resolution of allodynia. We discovered that during the resolution phase, sensory neurons exhibit increased glutamine oxidation and upregulation of the major glutamine transporter ASCT2 in dorsal root ganglia. This compensatory response plays a crucial role in pain resolution, as demonstrated by our experiments. Knockdown of ASCT2 prevents the resolution of NGF-induced allodynia and precipitates the transition to a chronic state. Furthermore, we show that the glutamine catabolite α-ketoglutarate attenuated glycolytic flux and alleviated allodynia in both acute and chronic phases of the hyperalgesic priming model. The importance of ASCT2 is further confirmed in a translational model, where its knockdown prevented the resolution of allodynia following plantar incision. These findings highlight the pivotal role of metabolic changes in pain resolution and identify ASCT2-mediated glutamine metabolism as a potential therapeutic target for chronic pain. Understanding these endogenous mechanisms that promote pain resolution can guide the development of novel interventions to prevent the transition pain from acute to chronic.

Keywords: ASCT2; chronic pain; dorsal root ganglia; hyperalgesic priming; metabolism; nerve growth factor.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
NGF-induced hyperalgesic priming model and its impact on mitochondrial function in lumbar DRGs. A, Tactile thresholds measured by von Frey filaments in mice injected with IPL vehicle or NGF. NGF induces acute tactile allodynia (acute phase; vehicle vs NGF; ****p < 0.0001) that resolves within 72 h. B, On Day 7, mice primed with NGF do not exhibit tactile allodynia (primed phase) but develop tactile allodynia after IPL PGE2 injection (chronic phase). Vehicle-treated mice do not develop allodynia post-PGE2 (vehicle vs NGF; ****p < 0.0001). C, D, Mitochondrial stress test measured by Seahorse analyzer in lumbar DRGs from NGF- or vehicle-treated mice dissected on Day 7 (C) or 3 d post-PGE2 injection (D). NGF injection alters mitochondrial respiration, reducing maximal respiration and spared respiratory capacity in chronic (post-PGE2) but not in primed state (**p < 0.01; ***p < 0.001). E, F, Western blot analysis of PDHK1, phospho-PDH S293, phospho-PDH S300, and total PDH in lumbar DRGs from NGF- or vehicle-treated mice on Day 7 (E) or 3 d post-PGE2 injection (F). NGF increases PDHK1 expression and PDH phosphorylation in both primed and chronic states and reduces PDH expression in chronic state (*p < 0.05). G, Pyruvate oxidation in lumbar DRGs from NGF- or vehicle-treated mice on Day 7. NGF priming reduces basal pyruvate oxidation, and this effect is reversed by the PDHK inhibitor DCA (20 mM). The mitochondrial pyruvate carrier inhibitor UK5099 (UK, 10 µM) was added at the end of the assay to measure pyruvate-dependent OCR (*p < 0.05; ***p < 0.001).
Figure 2.
Figure 2.
Pharmacological inhibition or siRNA-mediated knockdown of PDHK1 reverses allodynia during the chronic phase of the hyperalgesic priming model. A, Tactile thresholds measured by von Frey filaments in mice treated with IPL vehicle or NGF. Seven days posttreatment (BL, baseline), when allodynia had subsided, IPL PGE2 injection precipitated the chronic phase of hyperalgesic priming. Forty-eight hours post-PGE2, intraperitoneal administration of the PDHK inhibitor DCA (100 mg/kg) transiently normalized tactile thresholds in NGF-primed mice, while vehicle injection had no effect (IPL NGF→IPL PGE2→IP vehicle vs IPL NGF→IPL PGE2→IP DCA; *p < 0.05; **p < 0.01; ****p < 0.0001; IPL NGF→IPL PGE2→IP vehicle vs IPL vehicle groups; ####p < 0.0001). B, Tactile thresholds in mice that received IT control or PDHK1-targeting siRNA (1 μg in 5 μl) 24 and 48 h after PGE2 treatment. Knockdown of PDHK1 reversed the allodynia in NGF-primed mice (IPL NGF→IPL PGE2→IT control siRNA vs IPL NGF→IPL PGE2→IT PDHK1 siRNA; ****p < 0.0001; IPL NGF→IPL PGE2→IT control siRNA vs IPL vehicle groups; ####p < 0.0001). C, Confirmation of PDHK1 knockdown using Western blot analysis of PDHK1 expression in L4–L6 DRGs and lumbar SC following IT control or PDHK1 siRNA treatment in vehicle- and NGF-treated mice. PDHK1 siRNA treatment significantly reduced PDHK1 levels in DRGs but not in the SC (*p < 0.05; ***p < 0.001).
Figure 3.
Figure 3.
Effect of NGF on glycolysis in lumbar DRGs, and inhibition of LDHA reverses allodynia during the chronic phase of the hyperalgesic priming model. A, B, Glycolysis stress test measured by Seahorse analyzer in lumbar DRGs from NGF- or vehicle-treated mice dissected on Day 7 (A) or 3 d post-PGE2 injection (B). NGF priming increases glycolytic capacity and glycolytic reserve in DRGs in chronic phase (*p < 0.05; **p < 0.01). C, Tactile thresholds measured by von Frey filaments in NGF-primed mice treated with IP vehicle or the glycolysis inhibitor oxamate (500 mg/kg) 48 h after PGE2 injection. Oxamate administration reversed the allodynia in NGF-treated mice (IPL NGF→IPL PGE2→IP vehicle vs IPL NGF→IPL PGE2→IP oxamate; **p < 0.01; ****p < 0.0001; IPL NGF→IPL PGE2→IP vehicle vs IPL vehicle groups; ####p < 0.0001). D, Tactile thresholds in NGF-treated mice that received IT control or LDHA-targeting siRNA (1 μg in 5 μl) 24 and 48 h after PGE2 treatment. Knockdown of LDHA reversed the allodynia in NGF-primed mice (IPL NGF→IPL PGE2→IT control siRNA vs IPL NGF→IPL PGE2→IT LDHA siRNA; ****p < 0.0001; IPL NGF→IPL PGE2→IT control siRNA vs IPL vehicle groups; ####p < 0.0001). E, Confirmation of LDHA knockdown using Western blot analysis of LDHA expression in L4–L6 DRGs and lumbar SC following IT control or LDHA siRNA treatment in vehicle- and NGF-treated mice. LDHA siRNA treatment significantly reduced LDHA levels in DRGs but not in the SC (*p < 0.05; **p < 0.01).
Figure 4.
Figure 4.
Pain resolution is associated with increased glutamine utilization and ASCT2 expression in lumbar DRGs. A, B, Spare respiratory capacity measured by Seahorse analyzer in lumbar DRGs from NGF- or vehicle-treated mice dissected on Day 7 (A) or 3 d post-PGE2 injection (B). DRGs were incubated in medium with or without glutamine (Gln) during the assay. NGF injection increases spare respiratory capacity in a glutamine-dependent manner in the primed condition which is absent post-PGE2 injection (**p < 0.01; ***p < 0.001; ****p < 0.0001). C–E, Western blot analysis of ASCT2 expression in lumbar DRGs from NGF- or vehicle-treated mice on Day 1 (C), Day 7 (D), or 3 d post-PGE2 injection (E). NGF injection increases ASCT2 protein levels in DRGs on Day 7 which disappears post-PGE2 treatment (***p < 0.001).
Figure 5.
Figure 5.
siRNA-mediated knockdown of ASCT2 initiates the chronic phase in primed mice. A, Tactile thresholds measured by von Frey filaments in mice treated with IPL vehicle or NGF. Seven days posttreatment (BL, baseline), when allodynia had subsided, mice received IT control or ASCT2-targeting siRNA (1 μg in 5 μl). Knockdown of ASCT2 precipitated the chronic phase of hyperalgesic priming in NGF-primed mice, while control siRNA had no effect (IPL NGF→IT Cont siRNA vs all other groups; ****p < 0.0001). B, Confirmation of ASCT2 knockdown using Western blot analysis of ASCT2 expression in L4–L6 DRGs and lumbar SC following IT control or ASCT2 siRNA treatment in vehicle- and NGF-treated mice. ASCT2 siRNA treatment significantly reduced ASCT2 levels in DRGs but not in the SC of NGF-primed mice (*p < 0.05; **p < 0.01).
Figure 6.
Figure 6.
DKG attenuates glycolytic flux and alleviates allodynia in both acute and chronic phases of the hyperalgesic priming model. A, ECAR measured by Seahorse analyzer in lumbar DRGs from NGF- or vehicle-treated mice. NGF significantly increases glycolytic flux compared with vehicle, and this effect is profoundly attenuated by DKG treatment (****p < 0.0001). B, Tactile thresholds measured in mice treated with IPL vehicle or NGF. Intraperitoneal administration of DKG (300 mg/kg) alleviated allodynia in the acute phase post-NGF injection compared with vehicle treatment (IPL NGF→IP DKG vs IPL NGF→IP vehicle; **p < 0.01; ****p < 0.0001; IPL NGF→IP vehicle vs IPL vehicle groups; ####p < 0.0001; ###p < 0.001; ##p < 0.01). C, Tactile thresholds in NGF-primed mice that received IPL PGE2 injection to precipitate the chronic phase. IP administration of DKG 48 h post-PGE2 alleviated allodynia in the chronic phase compared with vehicle treatment (IPL NGF→IP DKG vs IPL NGF→IP vehicle; ***p < 0.001; ****p < 0.0001; IPL NGF→IP vehicle vs IPL vehicle groups; ####p < 0.0001).
Figure 7.
Figure 7.
DCA, oxamate, and DKG block glucose-induced CPA in the chronic phase of the hyperalgesic priming model. CPA was assessed using a two-chamber paradigm, where one chamber was paired with saline and the other with glucose (2 g/kg, i.p.) in vehicle or NGF-injected mice during the chronic phase. Glucose induces a significant place aversion compared with saline in NGF-primed mice (glucose paired vs saline paired; *p < 0.05). Treatment with DCA (100 mg/kg), oxamate (Oxa, 500 mg/kg), or DKG (300 mg/kg) prior to glucose pairing blocked the glucose-induced CPA in NGF-primed mice.
Figure 8.
Figure 8.
DKG attenuates UK-mediated calcium influx and alleviates PDP1 knockdown-induced allodynia. A, Cumulative average of calcium imaging traces showing the changes in Fluo4 fluorescence in dissociated DRG neurons. Cells were treated with either vehicle or DKG (1 mM), and baseline measurements were performed for 25 s. UK5099 (5 µM) was added to both groups, and the Fluo4 fluorescence was recorded for 400 s. B, AUC analysis of UK5099 revealed that DRG neurons pretreated with DKG display a significantly lower AUC of calcium responses than the vehicle (Veh) pretreated group (****p < 0.0001). C, PDP1 siRNA-induced (black arrows) allodynia was inhibited for several hours by DKG (blue arrow; 300 mg/kg, i.p.; IT PDP1 siRNA→IP DKG vs IT PDP1 siRNA→IP vehicle; ****p < 0.0001; IT PDP1siRNA→IP vehicle vs IT Cont siRNA groups; ####p < 0.0001).
Figure 9.
Figure 9.
ASCT2 siRNA treatment prevents the resolution of NGF and incision-induced allodynia. A, Intrathecal injection of ASCT2 siRNA at 3 h, Day 1, and Day 3 post-NGF administration prevents the resolution of NGF-induced allodynia for at least 84 d compared with control siRNA (IPL NGF→ASCT2 siRNA vs IPL NGF→Cont siRNA; *p < 0.05; ****p < 0.0001; IPL NGF→ASCT2 siRNA vs IPL vehicle groups; ####p < 0.0001). B, Plantar incision increases glycolytic flux in ipsilateral lumbar DRGs relative to the contralateral DRGS as measured by glycolysis stress test. Oxamate (Oxa) treatment blocks the incision-induced increase in glycolytic flux (****p < 0.0001). C, DKG treatment also blocks the incision-induced increase in glycolytic flux in lumbar DRGs (****p < 0.0001). D, Intrathecal injection of ASCT2 siRNA at 2 h, Day 1, and Day 2 postincision prevents the resolution of incisional pain for at least 93 d compared with control siRNA (incision→ASCT2 siRNA vs incision→Cont siRNA; ****p < 0.0001; incision→ASCT2 siRNA vs control groups; ####p < 0.0001).
Figure 10.
Figure 10.
Metabolic adaptations in sensory neurons during pain progression, resolution, and chronification. This schematic illustrates three phases: acute, primed, and chronic. In the acute phase, NGF or plantar incision enhance PDHK expression, limiting mitochondrial pyruvate oxidation and increasing lactate production. Extrusion of lactate and protons can activate pronociceptive channels and receptors. The primed phase shows pain resolution through increased glutamine transporter ASCT2 expression, enabling enhanced glutamine oxidation that maintains cellular homeostasis despite persistent PDHK activity. Moreover, glutamine-derived α-ketoglutarate (α-KG) interferes with lactate production. The chronic phase, triggered by IPL PGE2, depicts loss of ASCT2 upregulation, decreased glutamine utilization, and renewed lactate production. Pharmacological interventions include DCA (inhibits PDHK) and oxamate (inhibits LDHA). This model highlights the role of metabolic adaptations, particularly glutamine metabolism, in pain resolution and chronification. Created with BioRender.com.
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