Sylvie Liabeuf

Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury

Hyperexcitability of spinal reflexes and reduced synaptic inhibition are commonly associated with spasticity after spinal cord injury (SCI). In adults, the activation of γ-aminobutyric acidA (GABAA) and glycine receptors inhibits neurons as a result of low intracellular chloride (Cl−) concentration, which is maintained by the potassium-chloride cotransporter KCC2 (encoded by Slc12a5). We show that KCC2 is downregulated after SCI in rats, particularly in motoneuron membranes, thereby depolarizing the Cl− equilibrium potential and reducing the strength of postsynaptic inhibition. Blocking KCC2 in intact rats reduces the rate-dependent depression (RDD) of the Hoffmann reflex, as is observed in spasticity. RDD is also decreased in KCC2-deficient mice and in intact rats after intrathecal brain-derived neurotrophic factor (BDNF) injection, which downregulates KCC2. The early decrease in KCC2 after SCI is prevented by sequestering BDNF at the time of SCI. Conversely, after SCI, BDNF upregulates KCC2 and restores RDD. Our results open new perspectives for the development of therapeutic strategies to alleviate spasticity.

Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2

In healthy adults, activation of γ-aminobutyric acid (GABA)A and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl–]i), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), EIPSP, in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT2A receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT2AR agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).

Developmental up-regulation of the potassium–chloride cotransporter type 2 in the rat lumbar spinal cord

The classical GABA/glycine hyperpolarizing inhibition is not observed in the immature spinal cord. GABA(A) and glycine receptors are anions channels and the efficacy of inhibitory transmission in the spinal cord is largely determined by the gradient between intracellular and extracellular chloride concentrations. The concentration of intracellular chloride in neurons is mainly regulated by two cation-chloride cotransporters, the potassium-chloride cotransporter 2 (KCC2) and the sodium-potassium-chloride co-transporter 1 (NKCC1). In this study, we measured the reversal potential of IPSPs (E(IPSP)) of lumbar motoneurons during the first postnatal week and we investigated the expression of KCC2 and NKCC1 in the ventral horn of the spinal cord from the embryonic day 17 to the postnatal day 20 in the rat. Our results suggest that the negative shift of E(IPSP) from above to below the resting membrane potential occurs during the first postnatal week when the expression of KCC2 increases significantly and the expression of NKCC1 decreases. KCC2 immunolabeling surrounded motoneurons, presumably in the plasma membrane and NKCC1 immunolabeling appeared outside this KCC2-labeled fine strip. Taken together, the present results indicate that maturation of chloride homeostasis is not completed at birth in the rat and that the upregulation of KCC2 plays a key role in the shift from depolarizing to hyperpolarizing IPSPs.

Contribution of the potassium–chloride co‐transporter KCC2 to the modulation of lumbar spinal networks in mice

Spontaneous activity is observed in most developing neuronal circuits, such as the retina, hippocampus, brainstem and spinal cord. In the spinal cord, spontaneous activity is important for generating embryonic movements critical for the proper development of motor axons, muscles and synaptic connections. A spontaneous bursting activity can be recorded in vitro from ventral roots during perinatal development. The depolarizing action of the inhibitory amino acids γ‐aminobutyric acid and glycine is widely proposed to contribute to spontaneous activity in several immature systems. During development, the intracellular chloride concentration decreases, leading to a shift of equilibrium potential for Cl− ions towards more negative values, and thereby to a change in glycine‐ and γ‐aminobutyric acid‐evoked potentials from depolarization/excitation to hyperpolarization/inhibition. The up‐regulation of the outward‐directed Cl− pump, the neuron‐specific potassium–chloride co‐transporter type 2 KCC2, has been shown to underlie this shift. Here, we investigated whether spontaneous and locomotor‐like activities are altered in genetically modified mice that express only 8–20% of KCC2, compared with wild‐type animals. We show that a reduced amount of KCC2 leads to a depolarized equilibrium potential for Cl− ions in lumbar motoneurons, an increased spontaneous activity and a faster locomotor‐like activity. However, the left–right and flexor–extensor alternating pattern observed during fictive locomotion was not affected. We conclude that neuronal networks within the spinal cord are more excitable in KCC2 mutant mice, which suggests that KCC2 strongly modulates the excitability of spinal cord networks.

Cleavage of Na+ channels by calpain increases persistent Na+ current and promotes spasticity after spinal cord injury

Upregulation of the persistent sodium current (INaP) in motoneurons contributes to the development of spasticity after spinal cord injury (SCI). We investigated the mechanisms that regulate INaP and observed elevated expression of voltage-gated sodium (Nav) 1.6 channels in spinal lumbar motoneurons of adult rats with SCI. Furthermore, immunoblots revealed a proteolysis of Nav channels, and biochemical assays identified calpain as the main proteolytic factor. Calpain-dependent cleavage of Nav channels after neonatal SCI was associated with an upregulation of INaP in motoneurons. Similarly, the calpain-dependent cleavage of Nav1.6 channels expressed in human embryonic kidney (HEK) 293 cells caused the upregulation of INaP. The pharmacological inhibition of calpain activity by MDL28170 reduced the cleavage of Nav channels, INaP in motoneurons and spasticity in rats with SCI. Similarly, the blockade of INaP by riluzole alleviated spasticity. This study demonstrates that Nav channel expression in lumbar motoneurons is altered after SCI, and it shows a tight relationship between the calpain-dependent proteolysis of Nav1.6 channels, the upregulation of INaP and spasticity.

Production of Sm37‐GAPDH, a major therapeutical target in human schistosomiasis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic metabolism and the production of energy. This probably explains why GAPDH was evidenced as a major therapeutical target in several parasitic diseases; either as a vaccine candidate or as a target for chemotherapeutic treatments. Schistosoma mansoni GAPDH (Sm37-GAPDH) is one of the main schistosome vaccine candidates. The production of recombinant Sm37-GAPDH is essential to evaluate the ability of this molecule to induce protective immunity in animals and possibly in humans. The cDNA encoding Sm37-GAPDH has been cloned and sequenced. In addition, five B cell (including the major B-cell epitope Sm35-5) and two T cell epitopes have been localized on the molecule. Different expression systems have been evaluated in respect with the production yield and the GAPDH enzymatic activity. Some of them have led to either a high production of insoluble material (E. coli) or to an inactive enzyme (Pischia pastoris). The present article describes the production setting of rSm37-GAPDH using the baculovirus-insect cell system. Large amounts of soluble rSm37-GAPDH with enzymatic activity were obtained. Most sera from individuals living in an area endemic for S. mansoni recognised the rSm37 molecule and inhibited its catalytic activity.

Activation of 5-HT2A Receptors Restores KCC2 Function and Reduces Neuropathic Pain after Spinal Cord Injury

Downregulation of the potassium chloride cotransporter type 2 (KCC2) after a spinal cord injury (SCI) disinhibits motoneurons and dorsal horn interneurons causing spasticity and neuropathic pain, respectively. We showed recently (Bos et al., 2013) that specific activation of 5-HT2A receptors by TCB-2 [(4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide] upregulates KCC2 function, restores motoneuronal inhibition and reduces SCI-induced spasticity. Here, we tested the potential analgesic effect of TCB-2 on central (thoracic hemisection) and peripheral [spared nerve injury (SNI)] neuropathic pain. We found mechanical and thermal hyperalgesia reduced by an acute administration of TCB-2 in rats with SCI. This analgesic effect was associated with an increase in dorsal horn membrane KCC2 expression and was prevented by pharmacological blockade of KCC2 with an intrathecal injection of DIOA [(dihydroindenyl)oxy]alkanoic acid]. In contrast, the SNI-induced neuropathic pain was not attenuated by TCB-2 although there was a slight increase of membrane KCC2 expression in the dorsal horn ipsilateral to the lesion. Up-regulation of KCC2 function by targeting 5-HT2A receptors, therefore, has therapeutic potential in the treatment of neuropathic pain induced by SCI but not by SNI.

Mild Intrauterine Hypoperfusion Leads to Lumbar and Cortical Hyperexcitability, Spasticity, and Muscle Dysfunctions in Rats: Implications for Prematurity

Intrauterine ischemia-hypoxia is detrimental to the developing brain and leads to white matter injury (WMI), encephalopathy of prematurity (EP), and often to cerebral palsy (CP), but the related pathophysiological mechanisms remain unclear. In prior studies, we used mild intrauterine hypoperfusion (MIUH) in rats to successfully reproduce the diversity of clinical signs of EP, and some CP symptoms. Briefly, MIUH led to inflammatory processes, diffuse gray and WMI, minor locomotor deficits, musculoskeletal pathologies, neuroanatomical and functional disorganization of the primary somatosensory and motor cortices, delayed sensorimotor reflexes, spontaneous hyperactivity, deficits in sensory information processing, memory and learning impairments. In the present study, we investigated the early and long-lasting mechanisms of pathophysiology that may be responsible for the various symptoms induced by MIUH. We found early hyperreflexia, spasticity and reduced expression of KCC2 (a chloride cotransporter that regulates chloride homeostasis and cell excitability). Adult MIUH rats exhibited changes in muscle contractile properties and phenotype, enduring hyperreflexia and spasticity, as well as hyperexcitability in the sensorimotor cortex. Taken together, these results show that reduced expression of KCC2, lumbar hyperreflexia, spasticity, altered properties of the soleus muscle, as well as cortical hyperexcitability may likely interplay into a self-perpetuating cycle, leading to the emergence, and persistence of neurodevelopmental disorders (NDD) in EP and CP, such as sensorimotor impairments, and probably hyperactivity, attention, and learning disorders.

Expression in Insect Cells of the Major Parasite Antigen Associated with Human Resistance to Schistosomiasis

We have produced and purified a functional and active recombinant S.mansoni G3PDH. To the best of our knowledge, this report is the first to describe the quantitative production of biologically active rS.mansoni G3PDH. Given that IgG antibodies to the S.mansoni G3PDH are associated with resistance to infection in human, the biologically active rSm37-G3PDH may prove important as a component of an anti S.mansoni vaccine. Recombinant antigens isolated under nondenaturating conditions which retain biological activity as in this case should resemble the natural parasite antigen on the opposite to inactive or denaturated forms.