KCC2 knockdown impairs glycinergic synapse maturation in cultured spinal cord neurons

Schwale, Chrysovalandis; Schumacher, Stefanie; Bruehl, Claus; Titz, Stefan; Schlicksupp, Andrea; Kokocinska, Mirka; Kirsch, Joachim; Draguhn, Andreas; Kuhse, Jochen

doi:10.1007/s00418-015-1397-0

Cited by 13 publications

(15 citation statements)

References 44 publications

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“…Simultaneously, the intracellular chloride concentration is reduced due to chloride extrusion by the KCC2 and KCC3 chloride-potassium co-transporters to the point where it supports efficient synaptic inhibition (47). A recent study that inhibited KCC2 function in cultured spinal neurons during this period found that the elevated chloride concentration reduced the number and size of ␣1 GlyR dendritic clusters without affecting neighboring ␣2 GlyR clusters (48). Concomitantly, glycinergic miniature IPSCs were reduced in amplitude and frequency although GABAergic IPSCs were not affected.…”

Section: Discussionmentioning

confidence: 99%

Investigating the Mechanism by Which Gain-of-function Mutations to the α1 Glycine Receptor Cause Hyperekplexia

Zhang

Bode

Nguyen

et al. 2016

Journal of Biological Chemistry

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Hyperekplexia is a rare human neuromotor disorder caused by mutations that impair the efficacy of glycinergic inhibitory neurotransmission. Loss-of-function mutations in the GLRA1 or GLRB genes, which encode the α1 and β glycine receptor (GlyR) subunits, are the major cause. Paradoxically, gain-of-function GLRA1 mutations also cause hyperekplexia, although the mechanism is unknown. Here we identify two new gain-of-function mutations (I43F and W170S) and characterize these along with known gain-of-function mutations (Q226E, V280M, and R414H) to identify how they cause hyperekplexia. Using artificial synapses, we show that all mutations prolong the decay of inhibitory postsynaptic currents (IPSCs) and induce spontaneous GlyR activation. As these effects may deplete the chloride electrochemical gradient, hyperekplexia could potentially result from reduced glycinergic inhibitory efficacy. However, we consider this unlikely as the depleted chloride gradient should also lead to pain sensitization and to a hyperekplexia phenotype that correlates with mutation severity, neither of which is observed in patients with GLRA1 hyperekplexia mutations. We also rule out small increases in IPSC decay times (as caused by W170S and R414H) as a possible mechanism given that the clinically important drug, tropisetron, significantly increases glycinergic IPSC decay times without causing motor side effects. A recent study on cultured spinal neurons concluded that an elevated intracellular chloride concentration late during development ablates α1β glycinergic synapses but spares GABAergic synapses. As this mechanism satisfies all our considerations, we propose it is primarily responsible for the hyperekplexia phenotype.

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Section: Discussionmentioning

confidence: 99%

Investigating the Mechanism by Which Gain-of-function Mutations to the α1 Glycine Receptor Cause Hyperekplexia

Zhang

Bode

Nguyen

et al. 2016

Journal of Biological Chemistry

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“…Third, we considered whether a neurodevelopmental defect may result in fewer glycinergic synapses in adult motor neurons. This idea stemmed from a recent study that concluded that an elevated intracellular chloride concentration late during development ablates α1β glycinergic synapses but spares GABAergic synapses (Schwale et al, 2016). As this mechanism satisfies all our considerations, we propose it is primarily responsible for the gain-offunction hyperekplexia phenotype.…”

supporting

confidence: 61%

“…Simultaneously, the intracellular chloride concentration is reduced due to chloride extrusion by the KCC2 and KCC3 chloride-potassium co-transporters to the point where it supports efficient synaptic inhibition (48). A recent study that inhibited KCC2 function in cultured spinal neurons during this period found that the elevated chloride concentration reduced the number and size of a1 GlyR dendritic clusters without affecting neighbouring a2 GlyR clusters (49).…”

Section: Proposed Mechanism Of Hyperekplexia By Gain-of-function Glramentioning

confidence: 99%

Physiological properties of Glycinergic synapses with defined subunit compositions

Zhang¹

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Glycine receptors (GlyRs) mediate fast inhibitory neurotransmission in the spinal cord and brainstem. Four GlyR subunits (α1 -3, β) have been identified in humans, and their differential anatomical distributions result in a diversity of synaptic isoforms with unique physiological and pharmacological properties. However, despite their importance in controlling neuronal function, little is known about these properties. To address this, we developed an 'artificial' synapse system which allows to control over the synaptic GlyR subunit composition. We induced the formation of recombinant synapses between cultured spinal neurons and HEK293 cells expressing GlyR subunits of interest plus the synapse-promoting molecule, neuroligin-2A. In the heterosynapses thus formed, recombinant α1β and α3β GlyRs mediated fast decaying inhibitory postsynaptic currents (IPSCs) whereas α2β GlyRs mediated slow decaying IPSCs. These results are consistent with the fragmentary information available from native synapses and single channel kinetic studies. As β subunit incorporation is considered essential for localizing GlyRs at the synapse, we were surprised that α1 -3 homomers supported robust IPSCs with β subunit incorporation accelerating IPSC rise and decay times in α2β and α3β heteromers only. Finally, heterosynapses incorporating α1 D80A β and α1 A52S β GlyRs exhibited accelerated IPSC decay rates closely resembling those recorded in native synapses from mutant mice homozygous for these mutations, providing an additional validation of our technique. As our model system successfully recapitulates the effects of known GlyR disease mutations, we employed it to investigate the effects of new GlyR disease mutations.Hyperekplexia is a human neuromotor disorder caused by mutations that impair glycinergic neurotransmission. We investigated the mechanism by which gain-of-function GlyR mutations cause hyperekplexia. We identified two new gain-of-function mutations (I43F, W170S) and characterised these along with the known gain-of-function mutations (Q226E, V280M, R414H) to identify how they cause hyperekplexia. Using 'artificial' synapses, we show that all mutations prolong the decay of IPSCs and induce spontaneous activation. As these effects may deplete the chloride electrochemical gradient, hyperekplexia could potentially result from reduced glycinergic inhibitory efficacy. However, we consider this unlikely as it should also lead to pain sensitization and a hyperekplexia phenotype that correlates with mutation severity, neither of which is observed in patients. We also rule out small increases in IPSC decay times (as caused by W170S and R414H)as a possible mechanism given that the clinically-important drug, tropisetron, increases glycinergic IPSC decay times without causing motor side effects. A recent study concluded that an elevated intracellular chloride concentration late during development ablates α1β glycinergic synapses but iii spares GABAergic synapses. As this mechanism satisfies all our considerations, we propose it is pr...

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“…In addition, enhancement of KCC2 restores spinal inhibition and reserves neuropathic pain (58). KCC2 knockdown impairs glycinergic synapse maturation in cultured spinal cord neurons (59). Taken together, decrease of KCC2 destroyed chloride homeostasis and affected the inhibition of inhibitory neurons.…”

Section: Volume 3 May 2016mentioning

confidence: 99%

The Inhibitory Neuronal Circuit of Spinal Cord in Neuropathic Pain

Fu¹,

Wang²

2016

J Anesth Perioper Med

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Aim of review:Inhibitory interneurons, including GABAergic neurons and glycine neurons, regulate nociceptive information and non-nociceptive information in spinal dorsal horn. Emerging evidence showed that disinhibition of inhibitory interneurons of neuronal circuit in spinal dorsal horn is a pivotal mechanism of neuropathic pain after nerve injury. Method: In this view, we summarized the recent researches of the structure of inhibitory neurons in spinal dorsal horn and disinhibition of inhibitory interneurons after nerve injury and discussed the primary mechanism. Recent findings: Much progress has been made with the construction of inhibitory neuronal network in spinal dorsal horn and the dysfunction of inhibitory interneurons in these networks since inhibitory interneurons in spinal dorsal horn firstly integrate nociceptive information and non-nociceptive information from primary afferent fiber and separate non-nociceptive stimuli from nociceptive information. Disinhibitory of inhibitory interneurons underlies hyperalgesia and allodynia after nerve injury. Summary: Loss of inhibitory function of neurons in inhibitory network in the dorsal horn contributes to hyperalgesia and allodynia. The findings of these inhibitory networks provide a new evidence for preventing and curing neuropathic pain.ABSTRACT N europathic pain is a challenge for clinicians because the treatment of neuropathic pain is still unsatisfactory. Therefore, increasing understanding of the mechanisms that underlie neuropathic pain will be beneficial for the discovery of new molecular therapy targets. The gate control theory of pain is one of important mechanisms of pain. The theory is the idea that physical pain is modulated by interaction between different neurons. According to the postulate of Melzack and Wall (1), the nerve fibers project to the substantia gelatinosa (SG) of the dorsal horn and the first central transmission cells of the spinal cord. Inhibitory interneurons in the SG act as the gate and determine which signals should reach the T cells and then go further through the spinothalamic tract to reach the brain. Thus, spinal inhibitory interneurons play an important role in maintaining normal pain. Spinal dorsal horn is the first site of integration of nociceptive and nonnociceptive information within the central nervous system (CNS). Under normal physiological conditions, spinal inhibitory interneurons, including gammaaminobutyric acid (GABA) neurons and glycine neurons, form axo-axonic contacts with the termination of primary afferent fiber (PAF), exerting presynaptic inhibition control over sensory transmission. Meanwhile, these inhibitory interneurons generate postsynaptic inhibi-

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KCC2 knockdown impairs glycinergic synapse maturation in cultured spinal cord neurons

Cited by 13 publications

References 44 publications

Investigating the Mechanism by Which Gain-of-function Mutations to the α1 Glycine Receptor Cause Hyperekplexia

Investigating the Mechanism by Which Gain-of-function Mutations to the α1 Glycine Receptor Cause Hyperekplexia

Physiological properties of Glycinergic synapses with defined subunit compositions

The Inhibitory Neuronal Circuit of Spinal Cord in Neuropathic Pain

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