Implication of system xc- in neuroinflammation during the onset and maintenance of neuropathic pain.

BACKGROUND: Despite the high prevalence of neuropathic pain, treating this neurological disease remains challenging, given the limited efficacy and numerous side effects associated with current therapies. The complexity in patient management is largely attributed to an incomplete understanding of the underlying pathological mechanisms. Central sensitization, that refers to the adaptation of the central nervous system to persistent inflammation and heightened excitatory transmission within pain pathways, stands as a significant contributor to persistent pain. Considering the role of the cystine/glutamate exchanger (also designated as system xc-) in modulating glutamate transmission and in supporting neuroinflammatory responses, we investigated the contribution of this exchanger in the development of neuropathic pain. METHODS: We examined the implication of system xc- by evaluating changes in the expression/activity of this exchanger in the dorsal spinal cord of mice after unilateral partial sciatic nerve ligation. In this surgical model of neuropathic pain, we also examined the consequence of the genetic suppression of system xc- (using mice lacking the system xc- specific subunit xCT) or its pharmacological manipulation (using the pharmacological inhibitor sulfasalazine) on the pain-associated behavioral responses. Finally, we assessed the glial activation and the inflammatory response in the spinal cord by measuring mRNA and protein levels of GFAP and selected M1 and M2 microglial markers. RESULTS: The sciatic nerve lesion was found to upregulate system xc- at the spinal level. The genetic deletion of xCT attenuated both the amplitude and the duration of the pain sensitization after nerve surgery, as evidenced by reduced responses to mechanical and thermal stimuli, and this was accompanied by reduced glial activation. Consistently, pharmacological inhibition of system xc- had an analgesic effect in lesioned mice. CONCLUSION: Together, these observations provide evidence for a role of system xc- in the biochemical processes underlying central sensitization. We propose that the reduced hypersensitivity observed in the transgenic mice lacking xCT or in sulfasalazine-treated mice is mediated by a reduced gliosis in the lumbar spinal cord and/or a shift in microglial M1/M2 polarization towards an anti-inflammatory phenotype in the absence of system xc-. These findings suggest that drugs targeting system xc- could contribute to prevent or reduce neuropathic pain.

AMPKα1 Deficiency in Astrocytes from a Rat Model of ALS Is Associated with an Altered Metabolic Resilience.

Alterations in the activity of the regulator of cell metabolism AMP-activated protein kinase (AMPK) have been reported in motor neurons from patients and animal models of amyotrophic lateral sclerosis (ALS). Considering the key role played by astrocytes in modulating energy metabolism in the nervous system and their compromised support towards neurons in ALS, we examined whether a putative alteration in AMPK expression/activity impacted astrocytic functions such as their metabolic plasticity and glutamate handling capacity. We found a reduced expression of AMPK mRNA in primary cultures of astrocytes derived from transgenic rats carrying an ALS-associated mutated superoxide dismutase (hSOD1G93A). The activation of AMPK after glucose deprivation was reduced in hSOD1G93A astrocytes compared to non-transgenic. This was accompanied by a lower increase in ATP levels and increased vulnerability to this insult, although the ATP production rate did not differ between the two cell types. Furthermore, soliciting the activity of glutamate transporters was found to induce similar AMPK activity in these cells. However, manipulation of AMPK activity did not influence glutamate transport. Together, these results suggest that the altered AMPK responsiveness in ALS might be context dependent and may compromise the metabolic adaptation of astrocytes in response to specific cellular stress.

Structural investigation of human cystine/glutamate antiporter system xc - (Sxc -) using homology modeling and molecular dynamics.

The cystine/glutamate antiporter system xc - (Sxc -) belongs to the SLC7 family of plasma membrane transporters. It exports intracellular glutamate along the latter's concentration gradient as a driving force for cellular uptake of cystine. Once imported, cystine is mainly used for the production of glutathione, a tripeptide thiol crucial in maintenance of redox homeostasis and protection of cells against oxidative stress. Overexpression of Sxc - has been found in several cancer cells, where it is thought to counteract the increased oxidative stress. In addition, Sxc - is important in the central nervous system, playing a complex role in regulating glutamatergic neurotransmission and glutamate toxicity. Accordingly, this transporter is considered a potential target for the treatment of cancer as well as neurodegenerative diseases. Till now, no specific inhibitors are available. We herein present four conformations of Sxc - along its transport pathway, obtained using multi-template homology modeling and refined by means of Molecular Dynamics. Comparison with a very recently released cryo-EM structure revealed an excellent agreement with our inward-open conformation. Intriguingly, our models contain a structured N-terminal domain that is unresolved in the experimental structures and is thought to play a gating role in the transport mechanism of other SLC7 family members. In contrast to the inward-open model, there is no direct experimental counterpart for the other three conformations we obtained, although they are in fair agreement with the other stages of the transport mechanism seen in other SLC7 transporters. Therefore, our models open the prospect for targeting alternative Sxc - conformations in structure-based drug design efforts.

AMPK Modulates the Metabolic Adaptation of C6 Glioma Cells in Glucose-Deprived Conditions without Affecting Glutamate Transport.

Energy homeostasis in the central nervous system largely depends on astrocytes, which provide metabolic support and protection to neurons. Astrocytes also ensure the clearance of extracellular glutamate through high-affinity transporters, which indirectly consume ATP. Considering the role of the AMP-activated protein kinase (AMPK) in the control of cell metabolism, we have examined its implication in the adaptation of astrocyte functions in response to a metabolic stress triggered by glucose deprivation. We genetically modified the astrocyte-like C6 cell line to silence AMPK activity by overexpressing a dominant negative mutant of its catalytic subunit. Upon glucose deprivation, we found that C6 cells maintain stable ATP levels and glutamate uptake capacity, highlighting their resilience during metabolic stress. In the same conditions, cells with silenced AMPK activity showed a reduction in motility, metabolic activity, and ATP levels, indicating that their adaptation to stress is compromised. The rate of ATP production remained, however, unchanged by AMPK silencing, suggesting that AMPK mostly influences energy consumption during stress conditions in these cells. Neither AMPK modulation nor prolonged glucose deprivation impaired glutamate uptake. Together, these results indicate that AMPK contributes to the adaptation of astrocyte metabolism triggered by metabolic stress, but not to the regulation of glutamate transport.

Pharmacological evidence for the concept of spare glutamate transporters.

Through the efficient clearance of extracellular glutamate, high affinity astrocytic glutamate transporters constantly shape excitatory neurotransmission in terms of duration and spreading. Even though the glutamate transporter GLT-1 (also known as EAAT2/SLC1A2) is amongst the most abundant proteins in the mammalian brain, its density and activity are tightly regulated. In order to study the influence of changes in the expression of GLT-1 on glutamate uptake capacity, we have developed a model in HEK cells where the density of the transporter can be manipulated thanks to a tetracycline-inducible promoter. Exposing the cells to doxycycline concentration-dependently increased GLT-1 expression and substrate uptake velocity. However, beyond a certain level of induction, increasing the density of transporters at the cell surface failed to increase the maximal uptake. This suggested the progressive generation of a pool of spare transporters, a hypothesis that was further validated using the selective GLT-1 blocker WAY-213613 of which potency was influenced by the density of the transporters. The curve showing inhibition of uptake by increasing concentrations of WAY-213613 was indeed progressively rightward shifted when tested in cells where the transporter density was robustly induced. As largely documented in the context of cell-surface receptors, the existence of 'spare' glutamate transporters in the nervous tissue and particularly in astrocytes could impact on the consequences of physiological or pathological regulation of these transporters.

Validation of a System xc - Functional Assay in Cultured Astrocytes and Nervous Tissue Samples.

Disruption of the glutamatergic homeostasis is commonly observed in neurological diseases and has been frequently correlated with the altered expression and/or function of astrocytic high-affinity glutamate transporters. There is, however, a growing interest for the role of the cystine-glutamate exchanger system xc - in controlling glutamate transmission. This exchanger is predominantly expressed in glial cells, especially in microglia and astrocytes, and its dysregulation has been documented in diverse neurological conditions. While most studies have focused on measuring the expression of its specific subunit xCT by RT-qPCR or by Western blotting, the activity of this exchanger in tissue samples remains poorly examined. Indeed, the reported use of sulfur- and carbon-radiolabeled cystine in uptake assays shows several drawbacks related to its short radioactive half-life and its relatively high cost. We here report on the elaborate validation of a method using tritiated glutamate as a substrate for the reversed transport mediated by system xc -. The uptake assay was validated in primary cultured astrocytes, in transfected cells as well as in crude synaptosomes obtained from fresh nervous tissue samples. Working in buffers containing defined concentrations of Na+, allowed us to differentiate the glutamate uptake supported by system xc - or by high-affinity glutamate transporters, as confirmed by using selective pharmacological inhibitors. The specificity was further demonstrated in primary astrocyte cultures from transgenic mice lacking xCT or in cell lines where xCT expression was genetically induced or reduced. As such, this assay appears to be a robust and cost-efficient solution to investigate the activity of this exchanger in physiological and pathological conditions. It also provides a reliable tool for the screening and characterization of new system xc - inhibitors which have been frequently cited as valuable drugs for nervous disorders and cancer.

Inflammation-associated regulation of RGS in astrocytes and putative implication in neuropathic pain.

BACKGROUND: Regulators of G-protein signaling (RGS) are major physiological modulators of G-protein-coupled receptors (GPCR) signaling. Several GPCRs expressed in both neurons and astrocytes participate in the central control of pain processing, and the reduced efficacy of analgesics in neuropathic pain conditions may rely on alterations in RGS function. The expression and the regulation of RGS in astrocytes is poorly documented, and we herein hypothesized that neuroinflammation which is commonly observed in neuropathic pain could influence RGS expression in astrocytes. METHODS: In a validated model of neuropathic pain, the spared nerve injury (SNI), the regulation of RGS2, RGS3, RGS4, and RGS7 messenger RNA (mRNA) was examined up to 3 weeks after the lesion. Changes in the expression of the same RGS were also studied in cultured astrocytes exposed to defined activation protocols or to inflammatory cytokines. RESULTS: We evidenced a differential regulation of these RGS in the lumbar spinal cord of animals undergoing SNI. In particular, RGS3 appeared upregulated at early stages after the lesion whereas expression of RGS2 and RGS4 was decreased at later stages. Decrease in RGS7 expression was already observed after 3 days and outlasted until 21 days after the lesion. In cultured astrocytes, we observed that changes in the culture conditions distinctly influenced the constitutive expression of these RGS. Also, brief exposures (4 to 8 h) to either interleukin-1ß, interleukin-6, or tumor necrosis factor α caused rapid changes in the mRNA levels of the RGS, which however did not strictly recapitulate the regulations observed in the spinal cord of lesioned animals. Longer exposure (48 h) to inflammatory cytokines barely influenced RGS expression, confirming the rapid but transient regulation of these cell signaling modulators. CONCLUSION: Changes in the environment of astrocytes mimicking the inflammation observed in the model of neuropathic pain can affect RGS expression. Considering the role of astrocytes in the onset and progression of neuropathic pain, we propose that the inflammation-mediated modulation of RGS in astrocytes constitutes an adaptive mechanism in a context of neuroinflammation and may participate in the regulation of nociception.

Inhibition of the regulator of G protein signalling RGS4 in the spinal cord decreases neuropathic hyperalgesia and restores cannabinoid CB1 receptor signalling.

BACKGROUND AND PURPOSE: Regulators of G protein signalling (RGS) are major determinants of metabotropic receptor activity, reducing the lifespan of the GTP-bound state of G proteins. Because the reduced potency of analgesic agents in neuropathic pain may reflect alterations in RGS, we assessed the effects of CCG 63802, a specific RGS4 inhibitor, on pain hypersensitivity and signalling through cannabinoid receptors, in a model of neuropathic pain. EXPERIMENTAL APPROACH: The partial sciatic nerve ligation (PSNL) model in male Sprague Dawley rats was used to measure paw withdrawal thresholds to mechanical (von Frey hairs) or thermal (Hargreaves method) stimuli, during and after intrathecal injection of CCG 63802. HEK293 cells expressing CB1 receptors and conditional expression of RGS4 were used to correlate cAMP production and ERK phosphorylation with receptor activation and RGS4 action. KEY RESULTS: Treatment of PSNL rats with CCG 63802, twice daily for 7 days after nerve injury, attenuated thermal hyperalgesia during treatment. Spinal levels of anandamide were higher in PSNL animals, irrespective of the treatment. Although expression of CB1 receptors was unaffected, HU210-induced CB1 receptor signalling was inhibited in PSNL rats and restored after intrathecal CCG 63802. In transfected HEK cells expressing CB1 receptors and RGS4, inhibition of cAMP production, a downstream effect of CB1 receptor signalling, was blunted after RGS4 overexpression. RGS4 expression also attenuated the CB1 receptor-controlled activation of ERK1/2. CONCLUSIONS AND IMPLICATIONS: Inhibition of spinal RGS4 restored endogenous analgesic signalling pathways and mitigated neuropathic pain. Signalling through CB1 receptors may be involved in this beneficial effect.

The anti-inflammatory peptide stearyl-norleucine-VIP delays disease onset and extends survival in a rat model of inherited amyotrophic lateral sclerosis.

Vasoactive intestinal peptide (VIP) has potent immune modulatory actions that may influence the course of neurodegenerative disorders associated with chronic inflammation. Here, we show the therapeutic benefits of a modified peptide agonist stearyl-norleucine-VIP (SNV) in a transgenic rat model of amyotrophic lateral sclerosis (mutated superoxide dismutase 1, hSOD1(G93A)). When administered by systemic every-other-day intraperitoneal injections during a period of 80 days before disease, SNV delayed the onset of motor dysfunction by no less than three weeks, while survival was extended by nearly two months. SNV-treated rats showed reduced astro- and microgliosis in the lumbar ventral spinal cord and a significant degree of motor neuron preservation. Throughout the treatment, SNV promoted the expression of the anti-inflammatory cytokine interleukin-10 as well as neurotrophic factors commonly considered as beneficial in amyotrophic lateral sclerosis management (glial derived neuroptrophic factor, insulin like growth factor, brain derived neurotrophic factor). The peptide nearly totally suppressed the expression of tumor necrosis factor-α and repressed the production of the pro-inflammatory mediators interleukin-1ß, nitric oxide and of the transcription factor nuclear factor kappa B. Inhibition of tumor necrosis factor-α likely accounted for the observed down-regulation of nuclear factor kappa B that modulates the transcription of genes specifically involved in amyotrophic lateral sclerosis (sod1 and the glutamate transporter slc1a2). In line with this, levels of human superoxide dismutase 1 mRNA and protein were decreased by SNV treatment, while the expression and activity of the glutamate transporter-1 was promoted. Considering the large diversity of influences of this peptide on both clinical features of the disease and associated biochemical markers, we propose that SNV or related peptides may constitute promising candidates for amyotrophic lateral sclerosis treatment.

Differential regulation of glutamate transporter subtypes by pro-inflammatory cytokine TNF-α in cortical astrocytes from a rat model of amyotrophic lateral sclerosis.

Dysregulation of the astroglial glutamate transporters GLAST and GLT-1 has been implicated in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) where a loss of GLT-1 protein expression and activity is reported. Furthermore, the two principal C-terminal splice variants of GLT-1 (namely GLT-1a and GLT-1b) show altered expression ratio in animal models of this disease. Considering the putative link between inflammation and excitotoxicity, we have here characterized the influence of TNF-α on glutamate transporters in cerebral cortical astrocyte cultures from wild-type rats and from a rat model of ALS (hSOD1G93A). Contrasting with the down-regulation of GLAST, a 72 h treatment with TNF-α substantially increased the expression of GLT-1a and GLT-1b in both astrocyte cultures. However, as the basal level of GLT-1a appeared considerably lower in hSOD1G93A astrocytes, its up-regulation by TNF-α was insufficient to recapitulate the expression observed in wild-type astrocytes. Also the glutamate uptake activity after TNF-α treatment was lower for hSOD1G93A astrocytes as compared to wild-type astrocytes. In the presence of the protein synthesis inhibitor cycloheximide, TNF-α did not influence GLT-1 isoform expression, suggesting an active role of dynamically regulated protein partners in the adaptation of astrocytes to the inflammatory environment. Confirming the influence of inflammation on the control of glutamate transmission by astrocytes, these results shed light on the regulation of glutamate transporter isoforms in neurodegenerative disorders.