The small molecule CLP257 does not modify activity of the K+–Cl− co-transporter KCC2 but does potentiate GABAA receptor activity

Cardarelli, Ross A.; Jones, Karen L.; Pisella, Lucie I.; Wobst, Heike J.; McWilliams, Lisa; Sharpe, Paul T.; Burnham, Matthew P.; Baker, David; Chudotvorova, Ilona; Guyot, Justine; Silayeva, Liliya; Morrow, Danielle; Dekker, Niek; Zicha, Stephen; Davies, Paul; Holenz, Jörg; Duggan, Mark E.; Dunlop, John; Mather, Robert J.; Wang, Qi; Medina, Igor; Brandon, Nicholas J.; Deeb, Tarek Z.; Moss, Stephen J.

doi:10.1038/nm.4442

Cited by 46 publications

(36 citation statements)

References 20 publications

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“…In addition, its effect on locomotion has been replicated by virally overexpressing KCC2 in spinal inhibitory neurons (Chen et al, 2018). While the effectiveness of CLP257 has been challenged (Cardarelli et al, 2017), our results further confirm that CLP257 increases cell surface expression of KCC2 in spinal neurons (Gagnon et al, 2017;Chen et al, 2018).…”

Section: Clp257 Increases Kcc2 Membrane Expression On Lumbar Motoneuronssupporting

confidence: 69%

Enhancing KCC2 activity decreases hyperreflexia and spasticity after chronic SCI

Bilchak

Yeakle

Caron

et al. 2020

Preprint

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After spinal cord injury (SCI), the majority of individuals develop spasticity, a debilitating condition involving involuntary movements, co-contraction of antagonistic muscles, and hyperreflexia. By acting on GABAergic and Ca2+-dependent signaling, current anti-spastic medications lead to serious side effects, including a drastic decrease in motoneuronal excitability which impairs motor function and rehabilitation efforts. Exercise, in contrast, decreases spastic symptoms without decreasing motoneuron excitability. These functional improvements coincide with an increase in expression of the chloride co-transporter KCC2 in lumbar motoneurons. Thus, we hypothesized that spastic symptoms can be alleviated directly through restoration of chloride homeostasis and endogenous inhibition by increasing KCC2 activity. Here, we used the recently developed KCC2 enhancer, CLP257, to evaluate the effects of acutely increasing KCC2 extrusion capability on spastic symptoms after chronic SCI. Sprague Dawley rats received a spinal cord transection at T12 and were either bike-trained or remained sedentary for 5 weeks. Increasing KCC2 activity in the lumbar enlargement improved the rate-dependent depression of the H-reflex and reduced both phasic and tonic EMG responses to muscle stretch in sedentary animals after chronic SCI. Furthermore, the improvements due to this pharmacological treatment mirror those of exercise. Together, our results suggest that pharmacologically increasing KCC2 activity is a promising approach to decrease spastic symptoms in individuals with SCI. By acting to directly to restore endogenous inhibition, this strategy has potential to avoid severe side effects and improve the quality of life of affected individuals.Significance StatementSpasticity is a condition that develops after spinal cord injury (SCI) and causes major complications for individuals. We have previously reported that exercise attenuates spastic symptoms after SCI through an increase in expression of the chloride co-transporter KCC2, suggesting that restoring chloride homeostasis contributes to alleviating spasticity. However, the early implementation of rehabilitation programs in the clinic is often problematic due to co-morbidities. Here, we demonstrate that pharmacologically enhancing KCC2 activity after chronic SCI reduces multiple signs of spasticity, without the need for rehabilitation.

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Section: Clp257 Increases Kcc2 Membrane Expression On Lumbar Motoneuronssupporting

confidence: 69%

Enhancing KCC2 activity decreases hyperreflexia and spasticity after chronic SCI

Bilchak

Yeakle

Caron

et al. 2020

Preprint

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“…The mechanisms by which CLP290 enhances KCC2 function in neurons are not well understood (20,60,61). We investigated the role of the known phosphorylation sites on KCC2 those that either positively or negatively regulate KCC2 membrane stability and Clextrusion capacity, in the two mutant mice.…”

Section: Statisticsmentioning

confidence: 99%

“…The prodrug CLP290 was shown to have improved pharmacokinetics over CLP257 and rescued neuropathic pain in a rat model. In vitro studies proposed that the active drug CLP257 does not directly modulate KCC2 activity but potentiates GABAAR activity(60,61). Regardless, CLP290has been shown to improve outcomes in models of neuropathic pain and spinal cord injury associated with KCC2 hypofunction…”

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confidence: 99%

Targeting ischemia-induced KCC2 hypofunction rescues refractory neonatal seizures and mitigates epileptogenesis in a mouse model

Sullivan

Kipnis

Carter

et al. 2020

Preprint

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Neonatal seizures pose a clinical challenge for their early detection, acute management, and mitigation of long-term comorbidities. A major cause of neonatal seizures is hypoxic-ischemic encephalopathy that results in seizures that are frequently refractory to the first-line anti-seizure medication phenobarbital (PB). One proposed mechanism for PB-inefficacy during neonatal seizures is the reduced expression and function of the neuron-specific K+/Cl− cotransporter 2 (KCC2), the main neuronal Cl− extruder that maintains chloride homeostasis and influences the efficacy of GABAergic inhibition. To determine if PB-refractoriness after ischemic neonatal seizures is dependent upon KCC2 hypofunction and can be rescued by KCC2 functional enhancement, we investigated the recently developed KCC2 functional enhancer CLP290 in a CD-1 mouse model of refractory ischemic neonatal seizures quantified with vEEG. We report that acute CLP290 intervention can rescue PB-resistance, KCC2 expression, and the development of epileptogenesis after ischemic neonatal seizures. KCC2 phosphorylation sites have a strong influence over KCC2 activity and seizure susceptibility in adult experimental epilepsy models. Therefore, we investigated seizure susceptibility in two different knock-in mice in which either phosphorylation of S940 or T906/T1007 was prevented. We report that KCC2 phosphorylation regulates both neonatal seizure susceptibility and CLP290-mediated KCC2 functional enhancement. Our results validate KCC2 as a clinically relevant target for refractory neonatal seizures and provide insights for future KCC2 drug development.

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“…Mutation of serine 940 to alanine (S940A) in vivo reduces KCC2 function and exacerbates kainate-induced status epilepticus (30), while transient transfection of a T906/T1007 double-point alanine substitution mutant (T906A/T1007A) enhances KCC2 function in cell culture (29,(31)(32)(33)(34)(35). Unfortunately, no selective pharmacological activators of KCC2 exist (36), and so testing whether elevated KCC2 function can impact epileptiform activity in more complex systems remains hypothetical. We therefore generated a KCC2-T906A/T1007A knock-in mouse model to examine this theory.…”

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confidence: 99%

Potentiating KCC2 activity is sufficient to limit the onset and severity of seizures

Moore

Deeb

Chadchankar

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

120

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The type 2 K/Cl cotransporter (KCC2) allows neurons to maintain low intracellular levels of Cl, a prerequisite for efficient synaptic inhibition. Reductions in KCC2 activity are evident in epilepsy; however, whether these deficits directly contribute to the underlying pathophysiology remains controversial. To address this issue, we created knock-in mice in which threonines 906 and 1007 within KCC2 have been mutated to alanines (KCC2-T906A/T1007A), which prevents its phospho-dependent inactivation. The respective mice appeared normal and did not show any overt phenotypes, and basal neuronal excitability was unaffected. KCC2-T906A/T1007A mice exhibited increased basal neuronal Cl extrusion, without altering total or plasma membrane accumulation of KCC2. Critically, activity-induced deficits in synaptic inhibition were reduced in the mutant mice. Consistent with this, enhanced KCC2 was sufficient to limit chemoconvulsant-induced epileptiform activity. Furthermore, this increase in KCC2 function mitigated induction of aberrant high-frequency activity during seizures, highlighting depolarizing GABA as a key contributor to the pathological neuronal synchronization seen in epilepsy. Thus, our results demonstrate that potentiating KCC2 represents a therapeutic strategy to alleviate seizures.

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The small molecule CLP257 does not modify activity of the K+–Cl− co-transporter KCC2 but does potentiate GABAA receptor activity

Cited by 46 publications

References 20 publications

Enhancing KCC2 activity decreases hyperreflexia and spasticity after chronic SCI

Enhancing KCC2 activity decreases hyperreflexia and spasticity after chronic SCI

Targeting ischemia-induced KCC2 hypofunction rescues refractory neonatal seizures and mitigates epileptogenesis in a mouse model

Potentiating KCC2 activity is sufficient to limit the onset and severity of seizures

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