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.

Compartmentalized role of xCT in supporting pancreatic tumor growth, inflammation and mood disturbance in mice.

xCT (Slc7a11), the specific subunit of the cystine/glutamate antiporter system xc-, is present in the brain and on immune cells, where it is known to modulate behavior and inflammatory responses. In a variety of cancers -including pancreatic ductal adenocarcinoma (PDAC)-, xCT is upregulated by tumor cells to support their growth and spread. Therefore, we studied the impact of xCT deletion in pancreatic tumor cells (Panc02) and/or the host (xCT-/- mice) on tumor burden, inflammation, cachexia and mood disturbances. Deletion of xCT in the tumor strongly reduced tumor growth. Targeting xCT in the host and not the tumor resulted only in a partial reduction of tumor burden, while it did attenuate tumor-related systemic inflammation and prevented an increase in immunosuppressive regulatory T cells. The latter effect could be replicated by specific xCT deletion in immune cells. xCT deletion in the host or the tumor differentially modulated neuroinflammation. When mice were grafted with xCT-deleted tumor cells, hypothalamic inflammation was reduced and, accordingly, food intake improved. Tumor bearing xCT-/- mice showed a trend of reduced hippocampal neuroinflammation with less anxiety- and depressive-like behavior. Taken together, targeting xCT may have beneficial effects on pancreatic cancer-related comorbidities, beyond reducing tumor burden. The search for novel and specific xCT inhibitors is warranted as they may represent a holistic therapy in pancreatic cancer.

Impact of a Caffeine Restriction Policy on Inpatients With Schizophrenia: A Pre-Post Comparison Using Electronic Health Records.

BACKGROUND/PURPOSE: Caffeine is the most commonly used psychostimulant worldwide. Although its large intake is suspected to worsen psychotic symptoms because of increasing dopamine neurotransmission, schizophrenic patients are heavier caffeine consumers than the general population. This study aims to assess the impact of a caffeine restriction policy in a psychiatric hospital on patient psychopathology, hospitalization characteristics, and psychotropic prescribing patterns. METHODS: It is a retrospective cross-sectional study based on electronic health records of a psychiatric hospital in the French-speaking area of Belgium. Two different periods were compared, the first (n = 142), in 2017, when caffeine was available in the institution and the second (n = 119), between November 2018 and November 2019 after the restriction of access to caffeine was implemented. Adult inpatients with schizophrenia or schizoaffective disorder admitted for an acute hospitalization were included. Antipsychotic exposure, benzodiazepine daily dose, Global Assessment of Functioning scores, length of hospital stay, and some other factors were tested for their potential association with the decaffeinated period. RESULTS: After adjusting for potential confounders, reduced caffeine availability inside the hospital was significantly associated with higher Global Assessment of Functioning scores at discharge (adjusted odds ratio [aOR] = 2.86, 95% confidence interval [CI] = 1.77-4.62) and shorter hospital stays (aOR = 0.68, 95% CI = 0.47-0.99) but was not associated with change in antipsychotic exposure at discharge (aOR = 1.04, 95% CI = 0.64-1.7) or benzodiazepine daily dose (aOR = 0.89, 95% CI = 0.61-1.29). CONCLUSIONS: Limiting access to caffeine in psychiatric hospitals is a simple and inexpensive intervention that should be promoted, especially for patients with schizophrenia.

Lifespan extension with preservation of hippocampal function in aged system xc--deficient male mice.

The cystine/glutamate antiporter system xc- has been identified as the major source of extracellular glutamate in several brain regions as well as a modulator of neuroinflammation, and genetic deletion of its specific subunit xCT (xCT-/-) is protective in mouse models for age-related neurological disorders. However, the previously observed oxidative shift in the plasma cystine/cysteine ratio of adult xCT-/- mice led to the hypothesis that system xc- deletion would negatively affect life- and healthspan. Still, till now the role of system xc- in physiological aging remains unexplored. We therefore studied the effect of xCT deletion on the aging process of mice, with a particular focus on the immune system, hippocampal function, and cognitive aging. We observed that male xCT-/- mice have an extended lifespan, despite an even more increased plasma cystine/cysteine ratio in aged compared to adult mice. This oxidative shift does not negatively impact the general health status of the mice. On the contrary, the age-related priming of the innate immune system, that manifested as increased LPS-induced cytokine levels and hypothermia in xCT+/+ mice, was attenuated in xCT-/- mice. While this was associated with only a very moderate shift towards a more anti-inflammatory state of the aged hippocampus, we observed changes in the hippocampal metabolome that were associated with a preserved hippocampal function and the retention of hippocampus-dependent memory in male aged xCT-/- mice. Targeting system xc- is thus not only a promising strategy to prevent cognitive decline, but also to promote healthy aging.

Corticostriatal dysfunction and social interaction deficits in mice lacking the cystine/glutamate antiporter.

The astrocytic cystine/glutamate antiporter system xc- represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system xc-, xCT (xCT-/- mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT-/- mice. A proteomic and kinomic screen of the striatum of xCT-/- mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT-/- mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system xc- plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior.

Characterization of a Novel Aspect of Tissue Scarring Following Experimental Spinal Cord Injury and the Implantation of Bioengineered Type-I Collagen Scaffolds in the Adult Rat: Involvement of Perineurial-like Cells?

Numerous intervention strategies have been developed to promote functional tissue repair following experimental spinal cord injury (SCI), including the bridging of lesion-induced cystic cavities with bioengineered scaffolds. Integration between such implanted scaffolds and the lesioned host spinal cord is critical for supporting regenerative growth, but only moderate-to-low degrees of success have been reported. Light and electron microscopy were employed to better characterise the fibroadhesive scarring process taking place after implantation of a longitudinally microstructured type-I collagen scaffold into unilateral mid-cervical resection injuries of the adult rat spinal cord. At long survival times (10 weeks post-surgery), sheets of tightly packed cells (of uniform morphology) could be seen lining the inner surface of the repaired dura mater of lesion-only control animals, as well as forming a barrier along the implant-host interface of the scaffold-implanted animals. The highly uniform ultrastructural features of these scarring cells and their anatomical continuity with the local, reactive spinal nerve roots strongly suggest their identity to be perineurial-like cells. This novel aspect of the cellular composition of reactive spinal cord tissue highlights the increasingly complex nature of fibroadhesive scarring involved in traumatic injury, and particularly in response to the implantation of bioengineered collagen scaffolds.

Regulators of G protein signalling as pharmacological targets for the treatment of neuropathic pain.

Neuropathic pain, a specific type of chronic pain resulting from persistent nervous tissue lesions, is a debilitating condition that affects about 7% of the population. This condition remains particularly difficult to treat because of the poor understanding of its underlying mechanisms. Drugs currently used to alleviate this chronic pain syndrome are of limited benefit due to their lack of efficacy and the elevated risk of side effects, especially after a prolonged period of treatment. Although drugs targeting G protein-coupled receptors (GPCR) also have several limitations, such as progressive loss of efficacy due to receptor desensitization or unavoidable side effects due to wide receptor distribution, the identification of several molecular partners that contribute to the fine-tuning of receptor activity has raised new opportunities for the development of alternative therapeutic approaches. Regulators of G protein signalling (RGS) act intracellularly by influencing the coupling process and activity of G proteins, and are amongst the best-characterized physiological modulators of GPCR. Changes in RGS expression have been documented in a range of models of neuropathic pain, or after prolonged treatment with diverse analgesics, and could participate in altered pain processing as well as impaired physiological or pharmacological control of nociceptive signals. The present review summarizes the experimental data that implicates RGS in the development of pain with focus on the pathological mechanisms of neuropathic pain, including the impact of neuropathic lesions on RGS expression and, reciprocally, the influence of modifying RGS on GPCRs involved in the modulation of nociception as well as on the outcome of pain. In this context, we address the question of the relevance of RGS as promising targets in the treatment of neuropathic pain.

Constitutive downregulation protein kinase C epsilon in hSOD1G93A astrocytes influences mGluR5 signaling and the regulation of glutamate uptake.

Accumulating evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is a non-cell-autonomous process and that impaired glutamate clearance by astrocytes, leading to excitotoxicity, could participate in progression of the disease. In astrocytes derived from an animal model of ALS (hSOD1G93A rats), activation of type 5 metabotropic glutamate receptor (mGluR5) fails to increase glutamate uptake, impeding a putative dynamic neuroprotective mechanism involving astrocytes. Using astrocyte cultures from hSOD1G93A rats, we have demonstrated that the typical Ca2+ oscillations associated with mGluR5 activation were reduced, and that the majority of cells responded with a sustained elevation of intracellular Ca2+ concentration. Since the expression of protein kinase C epsilon isoform (PKCɛ) has been found to be considerably reduced in astrocytes from hSOD1G93A rats, the consequences of manipulating its activity and expression on mGluR5 signaling and on the regulation of glutamate uptake have been examined. Increasing PKCɛ expression was found to restore Ca2+ oscillations induced by mGluR5 activation in hSOD1G93A -expressing astrocytes. This was also associated with an increase in glutamate uptake capacity in response to mGluR5 activation. Conversely, reducing PKCɛ expression in astrocytes from wild-type animals with specific PKCɛ-shRNAs was found to alter the mGluR5 associated oscillatory signaling profile, and consistently reduced the regulation of the glutamate uptake-mediated by mGluR5 activation. These results suggest that PKCɛ is required to generate Ca2+ oscillations following mGluR5 activation, which support the regulation of astrocytic glutamate uptake. Reduced expression of astrocytic PKCɛ could impair this neuroprotective process and participate in the progression of ALS.

Post-operative pain in mice is prolonged by diet-induced obesity and rescued by dietary intervention.

The prevalence of obesity has increased at an alarming rate during past decades. Obesity is associated with pathophysiological disorders that can evolve and increase the risk of heart disease, diabetes and hypertension. While the impact of diabetes on post-operative recovery is now known, the consequences of obesity on post-operative pain remain much less explored. Here, we show that obesity affects post-operative pain resolution and leads to a chronic pain state in mice. Several mechanisms were identified as implicated in the prolonged post-operative pain. Indeed, we found that following a hind paw incision, high fat diet prolonged glial cell activation in the spinal cord. It also altered the expression of neurotrophins and increased inflammatory and endoplasmic reticulum stress markers in both central and peripheral nervous systems. Moreover, we show that a dietary intervention, leading to weight reduction and decreased inflammation, was able to restore normal pain sensitivity in mice suffering from chronic pain for more than 10 weeks. In conclusion, our data demonstrate that obesity is responsible for pain chronicization. This is clearly of importance in a clinical post-operative setting.

PKC epsilon-dependent calcium oscillations associated with metabotropic glutamate receptor 5 prevent agonist-mediated receptor desensitization in astrocytes.

A critical role has been assigned to protein kinase C (PKC)ε in the control of intracellular calcium oscillations triggered upon activation of type 5 metabotropic glutamate receptor (mGluR5) in cultured astrocytes. Nevertheless, the physiological significance of this particular signalling profile in the response of astrocytes to glutamate remains largely unknown. Considering that kinases are frequently involved in the regulation of G protein-coupled receptors, we have examined a putative link between the nature of the calcium signals and the response regulation upon repeated exposures of astrocytes to the agonist (S)-3,5-dihydroxyphenylglycine. We show that upon repeated mGluR5 activations, a robust desensitization was observed in astrocytes grown in culture conditions favouring the peak-plateau-type response. At variance, in cell cultures where calcium oscillations were predominating, the response was fully preserved even during repeated challenges with the agonist. Pharmacological inhibition of PKCε or genetic suppression of this isoform using shRNA was found to convert an oscillatory calcium profile to a sustained calcium mobilization and this latter profile was subject to desensitization upon repetitive mGluR5 activation. Our results suggest a yet undocumented scheme in which the activity of PKCε contributes to preserve the receptor sensitivity upon repeated or sustained activations. Cover Image for this issue: doi: 10.1111/jnc.13797.