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Supplementary Figure 3: Gating strategy for a representative sample

Supplementary Tables S1-S2

Purpose:Patients with cancer display reduced humoral responses after double-dose COVID-19 vaccination, whereas their cellular response is more comparable with that in healthy individuals. Recent studies demonstrated that a third vaccination dose boosts these immune responses, both in healthy people and patients with cancer. Because of the availability of many different COVID-19 vaccines, many people have been boosted with a different vaccine from the one used for double-dose vaccination. Data on such alternative vaccination schedules are scarce. This prospective study compares a third dose of BNT162b2 after double-dose BNT162b2 (homologous) versus ChAdOx1 (heterologous) vaccination in patients with cancer.Experimental Design:A total of 442 subjects (315 patients and 127 healthy) received a third dose of BNT162b2 (230 homologous vs. 212 heterologous). Vaccine-induced adverse events (AE) were captured up to 7 days after vaccination. Humoral immunity was assessed by SARS-CoV-2 anti-S1 IgG antibody levels and SARS-CoV-2 50% neutralization titers (NT50) against Wuhan and BA.1 Omicron strains. Cellular immunity was examined by analyzing CD4+ and CD8+ T-cell responses against SARS-CoV-2–specific S1 and S2 peptides.Results:Local AEs were more common after heterologous boosting. SARS-CoV-2 anti-S1 IgG antibody levels did not differ significantly between homologous and heterologous boosted subjects [GMT 1,755.90 BAU/mL (95% CI, 1,276.95–2,414.48) vs. 1,495.82 BAU/mL (95% CI, 1,131.48–1,977.46)]. However, homologous-boosted subjects show significantly higher NT50 values against BA.1 Omicron. Subjects receiving heterologous boosting demonstrated increased spike-specific CD8+ T cells, including higher IFNγ and TNFα levels.Conclusions:In patients with cancer who received double-dose ChAdOx1, a third heterologous dose of BNT162b2 was able to close the gap in antibody response.

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.

Purpose: Patients with cancer display reduced humoral responses after double-dose COVID-19 vaccination, whereas their cellular response is more comparable with that in healthy individuals. Recent studies demonstrated that a third vaccination dose boosts these immune responses, both in healthy people and patients with cancer. Because of the availability of many different COVID-19 vaccines, many people have been boosted with a different vaccine from the one used for double-dose vaccination. Data on such alternative vaccination schedules are scarce. This prospective study compares a third dose of BNT162b2 after double-dose BNT162b2 (homologous) versus ChAdOx1 (heterologous) vaccination in patients with cancer. Experimental Design: A total of 442 subjects (315 patients and 127 healthy) received a third dose of BNT162b2 (230 homologous vs. 212 heterologous). Vaccine-induced adverse events (AE) were captured up to 7 days after vaccination. Humoral immunity was assessed by SARS-CoV-2 anti-S1 IgG antibody levels and SARS-CoV-2 50% neutralization titers (NT50) against Wuhan and BA.1 Omicron strains. Cellular immunity was examined by analyzing CD4+ and CD8+ T-cell responses against SARS-CoV-2–specific S1 and S2 peptides. Results: Local AEs were more common after heterologous boosting. SARS-CoV-2 anti-S1 IgG antibody levels did not differ significantly between homologous and heterologous boosted subjects [GMT 1,755.90 BAU/mL (95% CI, 1,276.95–2,414.48) vs. 1,495.82 BAU/mL (95% CI, 1,131.48–1,977.46)]. However, homologous-boosted subjects show significantly higher NT50 values against BA.1 Omicron. Subjects receiving heterologous boosting demonstrated increased spike-specific CD8+ T cells, including higher IFNγ and TNFα levels. Conclusions: In patients with cancer who received double-dose ChAdOx1, a third heterologous dose of BNT162b2 was able to close the gap in antibody response.

BackgroundPatients with cancer, especially haematological cancer, are at increased risk for breakthrough COVID-19 infection. However, so far, a predictive biomarker that can assess compromised vaccine-induced anti-SARS-CoV-2 immunity in cancer patients has not been proposed.MethodsHere, we employed machine learning approaches to identify a biomarker signature based on blood cytokine and growth factors linked to vaccine response from 199 cancer patients receiving BNT162b2 vaccine.ResultsWe show that C-reactive protein (CRP; general marker of inflammation), interleukin (IL)-15 (a pro-inflammatory cytokine), IL-18 (interferon-gamma inducing factor), and placental growth factor (an angiogenic cytokine) can correctly classify patients with a diminished vaccine response assessed at day 49 with >80% accuracy. Amongst these, CRP showed the highest predictive value for poor response to vaccine administration. Importantly, this unique signature of vaccine response was present at different studied timepoints both before and after vaccination and was not majorly affected by different anti-cancer treatments.ConclusionWhile we propose a blood-based signature of cytokines and growth factors that can be employed in identifying cancer patients at continued risk of COVID-19, our data also importantly suggest that such a signature could reflect the inherent make-up of some cancer patients who are also refractive to immunotherapy.

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.

The cystine/glutamate antiporter system x

Background: Cancer patients are at higher risk of developing severe COVID-19. However, safety and efficacy of COVID-19 vaccination in cancer patients undergoing treatment is unclear. Methods: In this interventional prospective multi-cohort study, priming and booster doses of the BNT162b2 COVID-19 vaccine were administered 21 days apart to solid tumor patients receiving chemotherapy, immunotherapy, targeted- or hormonal therapy, and patients with a hematologic malignancy receiving rituximab or after allogeneic hematopoietic stem cell transplantation. Primary endpoint was anti-SARS-CoV-2 receptor binding domain (RBD) antibody level at 28 days post-booster. Anti-RBD IgG titers above 200 IU/ml was used to define high-responders as it leads to a neutralization response required for 50% protection against symptomatic SARS-CoV-2 infection in 268 independent subjects. Secondary endpoints included self-reported adverse events (AEs), antibody response over time and number of breakthrough infections after vaccination. Findings: 28 days post-booster vaccination of 197 cancer patients, antibody response was lower in the hematological cohort (geometric mean titer (GMT) 17·61 IU/mL [95% CI 7·17-43·24]) and the chemotherapy cohort (GMT 234·05 IU/mL [95% 95%CI 122·10-448·66]) when compared to healthy individuals (n=40; GMT 1844·93 IU/mL [95% CI 1383·57-2460·14]), irrespective of timing of vaccination during chemotherapy cycles. Only 55% and 7% of patients receiving chemotherapy or rituximab could be categorized as high-responders at 28 days post-booster. During the study period, five subjects tested positive for SARS-CoV-2 infection, including three after full vaccination. Local and systemic AEs were mostly mild to moderate, with only 1-3% of patients experiencing severe AEs. Local, but not systemic, AEs occurred more frequently after booster than after priming dose. Interpretation: The BNT162b2 vaccine is well-tolerated in cancer patients under active treatment. However, the antibody response of immunized cancer patients was delayed and diminished, mainly in patients receiving chemotherapy or rituximab, resulting in breakthrough infections. Trial Registration: EudraCT number 2021-000300-38 Funding: The regulatory sponsor was the Antwerp University Hospital (EudraCT number 2021-000300-38). The trial was funded by the Belgian Government through Sciensano (COVID-19_SC004, COVID- 19_SC059, COVID-19_SC061). Declaration of Interest: MP declares to have an advisory role within Remedus, all other authors declare no competing interests. Ethical Approval: The study was approved by the local ethics committee and was executed in accordance with Good Clinical Practice and the Declaration of Helsinki (ICH GCP E6(R2)).

In Parkinson's disease, striatal dopamine depletion leads to plastic changes at excitatory corticostriatal and thalamostriatal synapses. The functional consequences of these responses on the expression of behavioral deficits are incompletely understood. In addition, most of the information on striatal synaptic plasticity has been obtained in models with severe striatal dopamine depletion, and less is known regarding changes during early stages of striatal denervation. Using a partial model of nigral cell loss based on intranigral injection of the proteasome inhibitor lactacystin, we demonstrate ultrastructural changes at corticostriatal synapses with a 15% increase in the length and 30% increase in the area of the postsynaptic densities at corticostriatal synapses 1 week following toxin administration. This increase was positively correlated with the performance of lactacystin-lesioned mice on the rotarod task, such that mice with a greater increase in the size of the postsynaptic density performed better on the rotarod task. We therefore propose that lengthening of the postsynaptic density at corticostriatal synapses acts as a compensatory mechanism to maintain motor function under conditions of partial dopamine depletion. The ultrastructure of thalamostriatal synapses remained unchanged following lactacystin administration. Our findings provide novel insights into the mechanisms of synaptic plasticity and behavioral compensation following partial loss of substantia nigra pars compacta neurons, such as those occurring during the early stages of Parkinson's disease.

Proper glutamatergic neurotransmission requires a balance between glutamate release and removal. The removal is mainly catalyzed by the glutamate transporters EAAT1-3, while the glutamate-cystine exchanger (system xc− with specific subunit xCT) represents one of the release mechanisms. Previous studies of the spinal cord have focused on the cellular distribution of EAAT1-3 with special reference to the dorsal horn, but have not provided quantitative data and have not systematically compared multiple segments. Here we have studied the distribution of EAAT1-3 and xCT in sections of multiple spinal cord segments using knockout tissue as negative controls. EAAT2 and EAAT3 were evenly expressed in all gray matter areas at all segmental levels, albeit with slightly higher levels in laminae 1–4 (dorsal horn). Somewhat higher levels of EAAT2 were also seen in lamina 9 (ventral horn), while EAAT3 was also detected in the lateral spinal nucleus. EAAT1 was concentrated in laminae 1–3, lamina 10, the intermediolateral nucleus and the sacral parasympathetic nucleus, while xCT was concentrated in laminae 1–3, lamina 10 and the leptomeninges. The levels of these four transporters were low in white matter, which represents 42% of the spinal cord volume. Quantitative immunoblotting revealed that the average level of EAAT1 in the whole spinal cord was 0.6 ± 0.1% of that in the cerebellum, while the levels of EAAT2, EAAT3 and xCT were, respectively, 41.6 ± 12%, 39.8 ± 7.6%, and 30.8 ± 4.3% of the levels in the hippocampus (mean values ± SEM). Conclusions: Because the hippocampal tissue content of EAAT2 protein is two orders of magnitude higher than the content of the EAAT3, it follows that most of the gray matter in the spinal cord depends almost exclusively on EAAT2 for glutamate removal, while the lamina involved in the processing of autonomic and nociceptive information rely on a complex system of transporters.

The cystine/glutamate antiporter system xc-, with xCT as specific subunit, exchanges intracellular glutamate for extracellular cystine. It is highly expressed in the central nervous system, mainly on astrocytes, and has been shown to be the major source of extracellular glutamate in various regions of the brain, such as the striatum and hippocampus. This extrasynaptically released glutamate can affect synaptic neurotransmission. As the physiological function of system xc- in the central nervous system remains poorly understood, we studied how system xc- regulates transmission at corticostriatal synapses, one of the two major types of striatal excitatory synapses. Electrophysiological recordings identified a significant decrease in the amplitude of striatal field excitatory postsynaptic potentials in mice genetically lacking xCT (xCT-/- mice) following stimulation of corticostriatal fibers. Further, using electron microscopy, we observed depletion of glutamate immunogold labeling from corticostriatal terminalsand their corresponding dendritic spines in xCT-/- mice. Genetic deletion of xCT did not, however, affect the morphology of corticostriatal synapses, the density of cortical innervation or the density of dendritic spines in the striatum. Proteomic analysis revealed decreased expression of a wide range of proteins involved in regulating presynaptic neurotransmitter release in the striatum of xCT-/- mice, including synaptophysin, VGLUT1, and members of the synapsin, septin, and syntaxin families. In addition, kinome profiling identified changes in striatal serine/threonine kinase activity, highlighting ERK signaling as a possible node of kinase dysregulation in xCT-/- mice. Finally, we evaluated the effect of the disturbed corticostriatal communication on the behavioral phenotype of the xCT-/- mice. In the marble burying test, a paradigm sensitive to changes in corticostriatal function, we measured a significant increase in repetitive digging behavior in xCT-/- mice. Whereas spontaneous grooming behavior was not affected by genetic deletion of xCT, we have preliminary findings from the reciprocal interaction and three-chamber test, suggesting aberrant social behavior. Together, our results identify system xc- as a modulator of corticostriatal synaptic transmission that may be relevant to neuropsychiatric disorders characterized by corticostriatal dysfunction and repetitive behavior, such as obsessive-compulsive behavior and autism.

The pathological hallmarks of Parkinson’s disease are the progressive loss of nigral dopaminergic neurons and the formation of intracellular inclusion bodies, termed Lewy bodies, in surviving neurons. Accumulation of proteins in large insoluble cytoplasmic aggregates has been proposed to result, partly, from a failure in the function of intracellular protein degradation pathways. Evidence in support for such a hypothesis emerged in the beginning of the years 2000 with studies demonstrating structural and functional deficits in the ubiquitin-proteasome pathway in post-mortem nigral tissue of patients with Parkinson’s disease. These fundamental findings have inspired the development of a new generation of animal models based on the use of proteasome inhibitors to disturb protein homeostasis and trigger nigral dopaminergic neurodegeneration. In this review, we provide an updated overview of the current approaches in employing proteasome inhibitors to model Parkinson’s disease, with particular emphasis on rodent studies. In addition, the mechanisms underlying proteasome inhibition-induced cell death and the validity criteria (construct, face and predictive validity) of the model will be critically discussed. Due to its distinct, but highly relevant mechanism of inducing neuronal death, the proteasome inhibition model represents a useful addition to the repertoire of toxin-based models of Parkinson’s disease that might provide novel clues to unravel the complex pathogenesis of this disorder.

System xc-, with xCT as specific subunit, is an astrocytic antiporter that imports cystine in exchange for glutamate. System xc- is enhanced following oxidative stress and inflammation1, both present in the diseased or aged brain2. This upregulation might induce or further drive neurological dysfunction, as it could decrease the threshold for glutamate toxicity (by releasing glutamate)3 and modulate neuroinflammation (by driving the pro-inflammatory microglial phenotype)4, 5. Although inhibition of system xc- has been proposed as a therapeutic strategy for diverse neurological disorders (some age-related), reports on its function in physiological aging are sparse. We first studied the effect of genetic xCT deletion (xCT-/- mice) on life- and health-span. Next, we investigated the effect of aging on hippocampal xCT expression as well as the effect of xCT deletion on hippocampal aging (3-4 month versus 18-19-month old mice). As glutamate released via system xc- into the extrasynaptic space, could modulate synaptic transmission, we hypothesized that absence of system xc- might affect age-induced hippocampal deficits. We therefore compared age-related changes in hippocampal neurotransmission (slice electrophysiology), long-term potentiation (LTP) as well as hippocampus-dependent memory (Barnes maze, novel object location recognition task (NOLR)) between xCT-/- mice and their xCT+/+ littermates. xCT-/- mice have an increased life-span without deterioration of health-span, compared to xCT+/+ mice. Next, although no age-induced changes in xCT protein expression were seen in the brain of xCT+/+ mice (C57BL/6J background; 3-4 month versus 20-24-month old mice), xCT mRNA was significantly increased in hippocampus of 13-month old compared to 9-month old SAMP8 mice (model for accelerated aging). Aging prolongs the learning phase in the Barnes maze task for both genotypes. However, contrary to aged xCT+/+ mice and comparable to adult mice, the majority of aged xCT-/- mice use the hippocampus-dependent direct search strategy in the Barnes maze set-up at the end of the 5-day training session and they preserve this memory till 5 days after the last training session. In the NOLR, loss of system xc- induces impairment of spatial memory in adult mice. However, whereas aging negatively affects performance in the NOLR task in xCT+/+ mice, this was not the case for xCT-/- littermates and aged xCT-/- mice even perform better compared to adult xCT-/- mice. These data suggest that the hippocampal aging process is fundamentally different in mice lacking system xc-. In line with our behavioral data, basal hippocampal neurotransmission is indeed reduced in adult xCT-/- mice, while the age-related decrease in basal synaptic transmission as well as the age-induced changes in LTP that are observed in xCT+/+ mice, are prevented in the absence of system xc-. To conclude, our results demonstrate that absence of system xc- increases life-span and confers protection against age-related hippocampal dysfunction. Indeed, while system xc- seems to be important for hippocampal function in adult animals, it can become harmful during the aging process, thereby contributing to age-related memory decline.

Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System xc − or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system xc −, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT−/−) mice and irradiated mice reconstituted in xCT−/− bone marrow (BM), to their proper wild type (xCT+/+) controls. xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT+/+ mice, xCT−/− mice were equally susceptible to EAE, whereas mice transplanted with xCT−/− BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system xc − on immune cells invading the CNS participates to EAE. Since a total loss of system xc − had no net beneficial effects, these results have important implications for targeting system xc − for treatment of MS.

System xc- is a plasma membrane amino acid antiporter, of mainly glial origin, that couples the import of cystine with the export of glutamate. System xc- (specific subunit xCT) contributes substantially to ambient extracellular glutamate levels in various regions of the brain, including the striatum and hippocampus. Despite the fact that system xc- is highly expressed in the brain and is a proposed therapeutic target for various neurological disorders, including Parkinson’s disease, Alzheimer’s disease, multiple sclerosis and epilepsy, its function under physiological conditions in the central nervous system remains poorly understood. By acting as a source of glial extrasynaptic glutamate, system xc- might modulate synaptic transmission as a mechanism of neuro-glial communication. Previous electrophysiological findings indicate that system xc- delivered glutamate can inhibit excitatory synaptic neurotransmission in the cortico-accumbens pathway (Moran et al. J Neurosci. 2005; 25:6389-93) and at hippocampal CA3-CA1 synapses (Williams et al. J Neurosci. 2014; 34:16093-102). In order to gain further insight into the proposed function of system xc- as modulator of synaptic transmission, we carried out single section electron microscopy analyses of cortico-striatal excitatory synapses in adult xCT knockout (xCT-/-) and xCT wildtype (xCT+/+) mice. Our preliminary findings reveal depletion of glutamate immunogold labeling from presynaptic terminals of xCT-/- mice, an increase in the head diameter and area of spines contacted by asymmetric synapses, enlargement of the postsynaptic density and an increased occurrence of spinules, post-synaptic elements of high efficacy. These synaptic changes occurred in the absence of differences in the total density of cortico-striatal synapses or dendritic spines in the dorsolateral striatum of xCT-/- vs. xCT+/+ mice. Our results suggest the involvement of both presynaptic and postsynaptic forms of synaptic strength regulation via system xc-. Whether the structural changes we observedindicate a global increase in cortico-striatal transmission in xCT-/- mice, or conversely, represent forms of synaptic compensation, is being addressed by electrophysiological measures of excitatory post-synaptic currents in xCT-/- mice after cortical stimulation. Together, our findings shed new light on the re-organization of the glutamatergic system after genetic deletion of system xc-, and confirm the involvement of this antiporter in the control of synaptic strength in vivo.

Background Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System xc − or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. Methods Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system xc −, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT−/−) mice and irradiated mice reconstituted in xCT−/− bone marrow (BM), to their proper wild type (xCT+/+) controls. Results xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT+/+ mice, xCT−/− mice were equally susceptible to EAE, whereas mice transplanted with xCT−/− BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. Conclusions Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system xc − on immune cells invading the CNS participates to EAE. Since a total loss of system xc − had no net beneficial effects, these results have important implications for targeting system xc − for treatment of MS.

Zonisamide (ZNS), an anticonvulsant drug exhibiting symptomatic effects in Parkinson's disease (PD), was recently reported to exert neuroprotection in rodent models. One of the proposed neuroprotective mechanisms involves increased protein expression of xCT, the specific subunit of the cystine/glutamate antiporter system xc−, inducing glutathione (GSH) synthesis. Here, we investigated the outcome of ZNS treatment in a mouse model of PD based on intranigral proteasome inhibition, and whether the observed effects would be mediated by system xc−. The proteasome inhibitor lactacystin (LAC) was administered intranigrally to male C57BL/6J mice receiving repeated intraperitoneal injections of either ZNS 30 mg kg− 1 or vehicle. Drug administration was initiated three days prior to stereotaxic LAC injection and was maintained until six days post-surgery. One week after lesion, mice were behaviorally assessed and investigated in terms of nigrostriatal neurodegeneration and molecular changes at the level of the basal ganglia, including expression levels of xCT. ZNS reduced the loss of nigral dopaminergic neurons following LAC injection and the degree of sensorimotor impairment. ZNS failed, however, to modulate xCT expression in basal ganglia of lesioned mice. In a separate set of experiments, the impact of ZNS treatment on system xc− was investigated in control conditions in vivo as well as in vitro. Similarly, ZNS did not influence xCT or glutathione levels in naive male C57BL/6J mice, nor did it alter system xc− activity or glutathione content in vitro. Taken together, these results demonstrate that ZNS treatment provides neuroprotection and behavioral improvement in a PD mouse model based on proteasome inhibition via system xc− independent mechanisms.

Background System xc- is a plasma membrane amino acid antiporter, of mainly glial origin, that couples the import of cystine with the export of glutamate. System xc- (specific subunit xCT) contributes substantially to ambient extracellular glutamate levels in various regions of the brain, including the striatum and hippocampus. Despite the fact that system xc- is highly expressed in the brain and is a proposed therapeutic target for various neurological disorders, its function under physiological conditions in the central nervous system remains poorly understood. By acting as a source of glial extrasynaptic glutamate, system xc- might modulate synaptic transmission as a mechanism of neuro-glial communication. Previous electrophysiological findings indicate that system xc- delivered glutamate can inhibit excitatory synaptic neurotransmission in the corticoaccumbens pathway and at hippocampal CA3-CA1 synapses. To gain further insight into the proposed function of system xc- as modulator of synaptic transmission, we here focus on corticostriatal synapses. Methods Single section electron microscopy was carried out on VGLUT1-pre-embed and glutamate immunogold post-embed labeled slices of the dorsolateral striatum of xCT+/+ and xCT-/- mice. Various parameters related to the pre- and post-synaptic compartments were integrated on the obtained electron micrographs, including glutamate immunogold density in the presynaptic terminal and spine, area of the terminal and spine, measures of the postsynaptic density (PSD) (length, area, thickness, and maximum thickness), percentage of PSDs showing perforations, and width of the synaptic cleft. Electrophysiological measures of corticostriatal transmission were obtained by recording the amplitude of field excitatory postsynaptic potentials (fEPSPs) after stimulation of corticostriatal fibers. Finally, grooming behavior was compared between xCT-/- and xCT+/+ littermates. Results Genetic deletion of xCT led to depletion of glutamate immunogold labeling from corticostriatal terminals and their corresponding dendritic spines. Absence of xCT did not, however, affect the morphology of corticostriatal synapses, as evaluated by the area of the terminals and spines, size of the PSD, and width of the synaptic cleft. Similarly, no changes could be observed in the density of VGLUT1-positive synapses, indicating normal cortical innervation and spine density. Electrophysiological recordings revealed decreased amplitude of fEPSPs in xCT-/- mice after stimulation of corticostriatal fibers. Preliminary investigations revealed that this reduced response can be rescued by restoring physiological levels of glutamate to xCT-/- slices. Changes in corticostriatal transmission were not reflected in aberrant grooming behavior in xCT-/- mice; we could not observe any difference in the total grooming duration, the number of grooming bouts, the average bout duration or the latency to onset to grooming between xCT-/- and xCT+/+ mice. Discussion Contrary to available evidence at hippocampal and corticoaccumbens pathways, our findings indicate a positive effect of system xc- on basal synaptic transmission at corticostriatal synapses. The decreased response we observed after stimulation of corticostriatal fibers in xCT-/- mice was accompanied by depletion of glutamate immunogold labeling from corticostriatal terminals, suggesting a possible defect in presynaptic glutamate handling. Given the strong decrease (70%) in extracellular glutamate levels previously reported in this strain of mice, we hypothesize that the decreased presynaptic glutamate labeling in xCT-/- mice is related to a loss of extracellular glutamate needed to supply terminals for proper excitatory transmission. This hypothesis is supported by our preliminary results showing increased responses in xCT-/- slices after restoring physiological levels of glutamate. Together, our findings shed new light on the role of system xc- in controlling synaptic transmission, and suggest that it may play an important role in supplying presynaptic terminals with glutamate as an alternative mechanism to the glutamate-glutamine cycle. As a novel modulator of corticostriatal transmission, system xc- may be of interest as a possible therapeutic target for disorders with a corticostriatal component, such as schizophrenia or obsessive-compulsive disorder.