Loss of STEP61 couples disinhibition to N-methyl-d-aspartate receptor potentiation in rodent and human spinal pain processing

Dedek, Annemarie; Xu, Jian; Kandegedara, Chaya M; Lorenzo, Louis‐Etienne; Godin, Antoine G.; Koninck, Yves De; Lombroso, Paul J.; Tsai, Eve C.; Hildebrand, Michael E.

doi:10.1093/brain/awz105

Cited by 57 publications

(100 citation statements)

References 48 publications

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“…The superficial layers of the spinal cord dorsal horn, laminae I and II, are a hub for processing pain-related sensory inputs [1]. Disruption in the balance of excitability within the superficial dorsal horn can drive chronic pain [2], and yet the specific molecular determinants that mediate synaptic signaling across dorsal horn neuron subpopulations remain poorly understood. The N-methyl-D-aspartate receptor (NMDAR) subtype of ionotropic glutamate receptors are critical mediators of synaptic transmission and plasticity throughout the nervous system.…”

Section: Introductionmentioning

confidence: 99%

Conserved contributions of NMDA receptor subtypes to synaptic responses in lamina II spinal neurons across early postnatal development

2020

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NMDA receptors are heteromeric complexes that contribute to excitatory synaptic transmission and plasticity. The presence of specific variants of GluN2 subunits in these complexes enables diversity in NMDA receptor function and regulation. At brain synapses, there is a switch from slow GluN2B-mediated NMDA receptors to faster GluN2Adominated NMDA receptors as well as an increase in the ratio of AMPA to NMDA receptors during early postnatal development. This glutamate receptor switch is observed across brain regions and is critical for synaptic maturation, circuit development, and associative learning. However, whether a similar receptor subunit switch occurs within pain processing neurons in the developing spinal cord remains untested. To investigate this, we performed wholecell patch clamp recordings of excitatory synaptic responses from lamina II dorsal horn neurons of one to three week-old rats. We found that GluN2B and GluN2A both prominently contribute to NMDA receptor responses at neonatal lamina II synapses, with a small contribution from GluN2D as well. Surprisingly, we found that this molecular identity of NMDA receptor responses as well as the relative contribution of AMPA receptors versus NMDA receptors did not change at lamina II synapses across early postnatal development (P7 to P21). The lack of a developmental switch and persistence of slow-decaying GluN2B-and GluN2D-mediated synaptic responses throughout neuronal maturation in the dorsal horn has implications for understanding both the regulation of synaptic glutamatergic receptors as well as spinal mechanisms of pain processing.

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Section: Introductionmentioning

confidence: 99%

Conserved contributions of NMDA receptor subtypes to synaptic responses in lamina II spinal neurons across early postnatal development

2020

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“…Together, the previous studies demonstrate causality of KCC2 downregulation in pathologic pain. Interestingly, recent results suggested that this mechanism also might apply to human spinal pathophysiology in pathologic pain (Dedek et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

“…In chronic pathologic pain, KCC2 expression is attenuated in the primary sensory gate in spinal cord dorsal horn neurons. This key pathophysiological mechanism contributes to an excitation/inhibition imbalance by corrupting inhibitory neurotransmission and causing inhibitory circuit malfunction (Braz et al, 2017;Dedek et al, 2019;Doyon et al, 2013;Ferrini et al, 2013;Kahle et al, 2014a;Li et al, 2016;Mapplebeck et al, 2019;Price et al, 2005). Notably, there is no 'back-up' protein that can rescue the KCC2 expression deficit.…”

Section: Introductionmentioning

confidence: 99%

Repurposing cell growth-regulating compounds identifies kenpaullone which ameliorates pathologic pain via normalization of inhibitory neurotransmission

Yeo

Jiang

et al. 2019

Preprint

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Inhibitory GABA-ergic neurotransmission is of fundamental relevance for the adult vertebrate central nervous system and requires low chloride ion concentration in neurons. This basic ionic-homeostatic mechanism critically relies on expression and function of KCC2, a neuroprotective ionic transporter that extrudes neuronal chloride. Attenuated expression of KCC2 causes circuit malfunction in chronic pain and other neuropsychiatric illnesses. To find new analgesics, we screened 1057 cell growth-regulating compounds in cultured mouse primary cortical neurons for enhancement of Kcc2 gene expression. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain in preclinical mouse models. In nerve-injury pain, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via Kaiso transcription factor, and transient spinal transgenesis of delta-catenin mimicked KP's analgesic effects.1D), which is accompanied by increased KCC2 protein expression and increased expression of synaptophysin ( Fig. 1E-F). This latter finding suggests increased synaptic maturation. These data indicate that our rationally designed screen identified a GSK3/CDK kinase inhibitor, KP, that enhances Kcc2/KCC2 gene expression and not KCC2-mediated chloride extrusion in CNS neurons. KP functions as Kcc2/KCC2 gene expression enhancer in mammals including humans, where we document enhanced synaptic maturation and increased KCC2 expression and function. ============================= Fig. 1========================= Fig. 1. Compound screening for enhancers of Kcc2 expression in primary cortical neurons yields KenpaulloneA, primary mouse neurons, B-C: primary rat neurons. D-F: primary human neurons. A) Top panel: Screening paradigm using three rounds of primary screen based on luciferase (LUC) activity followed by secondary screen including Kcc2 RT-qPCR and Clomeleon chloride imaging. Bottom panel: Four compound "winners" including Kenpaullone (KP). Screening conducted in primary mouse neurons. B) Primary rat cortical neurons. Bar diagram: dose-dependent increase in Kcc2 mRNA expression after KP treatment, also reflected by increased protein expression as shown by KCC2 immuno-label (micrographs). Results represent the average mRNA expression of 4 independent neuronal cultures. p<0.05, one-way ANOVA C) Primary rat cortical neurons. Neuronal [Cl-]i, measured with ratiometric chloride indicator, clomeleon, is robustly and significantly reduced after KP treatment. Note that add-on treatment with KCC2-transport blocker, VU0240551, leads to a [Cl-]i ≥120mM, for both, vehicle-treated and KP-treated, indicating that KP's chloride lowering effect relies on KCC2 chloride extruding transport function. n≥75 neurons from 3 independent cultures; * p<0.01, t-test D) Primary human cortical neurons. KCC2 mRNA increases in a KP...

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“…Inflammation-induced hyperalgesia leads to decrease in STEP61 activity (by both phosphorylation at serine 221 residue and cleavage) with concurrent increases in phosphorylated GluN2B in the spinal cord [82,83]. In 2019, we showed that STEP61 is significantly reduced at the superficial dorsal horn in a persistent inflammatory pain model and an ex vivo BDNF model of pathological pain processing [84].…”

Section: Step61 -The Linker Between Disinhibition and Increased Excitmentioning

confidence: 69%

“…The images were taken at 63x for high magnification. Quantification and changes in KCC2 intensity was detected using MATLAB routines as previously described [84]. In brief, CGRP was used as a marker of the superficial dorsal horn and only the neurons the CGRP expressing region was quantified.…”

Section: Quantification Of Kcc2 Expressionmentioning

confidence: 99%

BDNF Driven Coupling Between KCC2 Downregulation and Fyn-Dependent Phosphorylation of GluN2B NMDA Receptors in Human Spinal Pain Processing

Kandegedara¹

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Hyperexcitation of neurons in the dorsal horn of the spinal cord leads to chronic pain. In rodent pain models, the neurotrophic factor BDNF mediates loss of inhibitory signalling. This permits enhancement of excitatory GluN2B-NMDAR currents in lamina I neuronal synapses. However, whether this pathway is conserved between rodents and humans as well as between sexes is unknown. We developed a human ex vivo BDNF model of pathological pain using postmortem human spinal tissue. We found that ex vivo BDNF downregulates KCC2 and active STEP61 and upregulates active Fyn and phosphorylated GluN2B at the superficial dorsal horn in both human and rodent males. We also show that BDNF increases intracellular KCC2 of superficial dorsal horn neurons. Thus, our human model may connect the vast translational divide between rodent and human chronic pain mechanisms to identify and validate new therapeutic targets for human chronic pain.

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Loss of STEP61 couples disinhibition to N-methyl-d-aspartate receptor potentiation in rodent and human spinal pain processing

Cited by 57 publications

References 48 publications

Conserved contributions of NMDA receptor subtypes to synaptic responses in lamina II spinal neurons across early postnatal development

Conserved contributions of NMDA receptor subtypes to synaptic responses in lamina II spinal neurons across early postnatal development

Repurposing cell growth-regulating compounds identifies kenpaullone which ameliorates pathologic pain via normalization of inhibitory neurotransmission

BDNF Driven Coupling Between KCC2 Downregulation and Fyn-Dependent Phosphorylation of GluN2B NMDA Receptors in Human Spinal Pain Processing

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