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OBJECTIVE: Mutations in the spastic paraplegia gene 11 (SPG11), encoding spatacsin, cause the most frequent form of autosomal-recessive complex hereditary spastic paraplegia (HSP) and juvenile-onset amyotrophic lateral sclerosis (ALS5). When SPG11 is mutated, patients frequently present with spastic paraparesis, a thin corpus callosum, and cognitive impairment. We previously delineated a neurodegenerative phenotype in neurons of these patients. In the current study, we recapitulated early developmental phenotypes of SPG11 and outlined their cellular and molecular mechanisms in patient-specific induced pluripotent stem cell (iPSC)-derived cortical neural progenitor cells (NPCs). METHODS: We generated and characterized iPSC-derived NPCs and neurons from 3 SPG11 patients and 2 age-matched controls. RESULTS: Gene expression profiling of SPG11-NPCs revealed widespread transcriptional alterations in neurodevelopmental pathways. These include changes in cell-cycle, neurogenesis, cortical development pathways, in addition to autophagic deficits. More important, the GSK3ß-signaling pathway was found to be dysregulated in SPG11-NPCs. Impaired proliferation of SPG11-NPCs resulted in a significant diminution in the number of neural cells. The decrease in mitotically active SPG11-NPCs was rescued by GSK3 modulation. INTERPRETATION: This iPSC-derived NPC model provides the first evidence for an early neurodevelopmental phenotype in SPG11, with GSK3ß as a potential novel target to reverse the disease phenotype. Ann Neurol 2016;79:826-840.
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α-Synuclein is readily released in human and mouse brain parenchyma, even though the normal function of the secreted protein has not been yet elucidated. Under pathological conditions, such as in Parkinson's disease, pathologically relevant species of α-synuclein have been shown to propagate between neurons in a prion-like manner, although the mechanism by which α-synuclein transfer induces degeneration remains to be identified. Due to this evidence extracellular α-synuclein is now considered a critical target to hinder disease progression in Parkinson's disease. Given the importance of extracellular α-synuclein levels, we have now investigated the molecular pathway of α-synuclein secretion in mouse brain. To this end, we have identified a novel synaptic network that regulates α-synuclein release in mouse striatum. In this brain area, the majority of α-synuclein is localized in corticostriatal glutamatergic terminals. Absence of α-synuclein from the lumen of brain-isolated synaptic vesicles suggested that they are unlikely to mediate its release. To dissect the mechanism of α-synuclein release, we have used reverse microdialysis to locally administer reagents that locally target specific cellular pathways. Using this approach, we show that α-synuclein secretion in vivo is a calcium-regulated process that depends on the activation of sulfonylurea receptor 1-sensitive ATP-regulated potassium channels. Sulfonylurea receptor 1 is distributed in the cytoplasm of GABAergic neurons from where the ATP-dependent channel regulates GABA release. Using a combination of specific agonists and antagonists, we were able to show that, in the striatum, modulation of GABA release through the sulfonylurea receptor 1-regulated ATP-dependent potassium channels located on GABAergic neurons controls α-synuclein release from the glutamatergic terminals through activation of the presynaptic GABAB receptors. Considering that sulfonylurea receptors can be selectively targeted, our study highlights the potential use of the key molecules in the α-synuclein secretory pathway to aid the discovery of novel therapeutic interventions for Parkinson's disease.
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Cuerpo Estriado/metabolismo , Canales KATP/metabolismo , Transmisión Sináptica/fisiología , alfa-Sinucleína/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Moduladores del GABA/farmacología , Humanos , Canales KATP/agonistas , Canales KATP/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de GABA-B/metabolismo , Transmisión Sináptica/efectos de los fármacosAsunto(s)
Retinitis Pigmentosa , Ataxia/genética , Catepsina D , Humanos , Linaje , Retinitis Pigmentosa/genéticaRESUMEN
Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by progressive bradykinesia, rigidity, resting tremor, and gait impairment, as well as a spectrum of non-motor symptoms including autonomic and cognitive dysfunction. The cardinal motor symptoms of PD stem from the loss of substantia nigra (SN) dopaminergic (DAergic) neurons, and it remains unclear why SN DAergic neurons are preferentially lost in PD. However, recent identification of several genetic PD forms suggests that mitochondrial and lysosomal dysfunctions play important roles in the degeneration of midbrain dopamine (DA) neurons. In this review, we discuss the interplay of cell-autonomous mechanisms linked to DAergic neuron vulnerability and alpha-synuclein homeostasis. Emerging studies highlight a deleterious feedback cycle, with oxidative stress, altered DA metabolism, dysfunctional lysosomes, and pathological alpha-synuclein species representing key events in the pathogenesis of PD. We also discuss the interactions of alpha-synuclein with toxic DA metabolites, as well as the biochemical links between intracellular iron, calcium, and alpha-synuclein accumulation. We suggest that targeting multiple pathways, rather than individual processes, will be important for developing disease-modifying therapies. In this context, we focus on current translational efforts specifically targeting lysosomal function, as well as oxidative stress via calcium and iron modulation. These efforts could have therapeutic benefits for the broader population of sporadic PD and related synucleinopathies.
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Lysosomes are acidic, membrane-bound organelles that serve as the primary catabolic compartment of the cell. They are crucial to a variety of cellular processes from nutrient storage to autophagy. Given the diversity of lysosomal functions, it is unsurprising that lysosomes are also emerging as important players in aging. Lysosomal dysfunction is implicated in several aging-related neurodegenerative diseases including Alzheimer's, Parkinson's, amyotrophic lateral sclerosis/frontotemporal dementia, and Huntington's. Although the precise role of lysosomes in the aging brain is not well-elucidated, some insight into their function has been gained from our understanding of the pathophysiology of age-dependent neurodegenerative diseases. Therapeutic strategies targeting lysosomes and autophagic machinery have already been tested in several of these diseases with promising results, suggesting that improving lysosomal function could be similarly beneficial in preserving function in the aging brain.
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Envejecimiento/metabolismo , Encéfalo/metabolismo , Lisosomas/fisiología , Enfermedades Neurodegenerativas/metabolismo , Envejecimiento/fisiología , Esclerosis Amiotrófica Lateral/metabolismo , Encéfalo/fisiología , Demencia Frontotemporal/metabolismo , Humanos , Enfermedad de Huntington/metabolismo , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/prevención & control , Enfermedad de Parkinson/metabolismoRESUMEN
This article was corrected to include a revised version of figure 2 overlooked by the Publisher during the production process.
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BACKGROUND: Motor impairment appears as a characteristic symptom of several diseases and injuries. Therefore, tests for analyzing motor dysfunction are widely applied across preclinical models and disease stages. Among those, gait analysis tests are commonly used, but they generate a huge number of gait parameters. Thus, complications in data analysis and reporting raise, which often leads to premature parameter selection. NEW METHODS: In order to avoid arbitrary parameter selection, we present here a systematic initial data analysis by utilizing heat-maps for data reporting. We exemplified this approach within an intervention study, as well as applied it to two longitudinal studies in rodent models related to Parkinson's disease (PD) and Huntington disease (HD). RESULTS: The systematic initial data analysis (IDA) is feasible for exploring gait parameters, both in experimental and longitudinal studies. The resulting heat maps provided a visualization of gait parameters within a single chart, highlighting important clusters of differences. COMPARISON WITH EXISTING METHOD: Often, premature parameter selection is practiced, lacking comprehensiveness. Researchers often use multiple separated graphs on distinct gait parameters for reporting. Additionally, negative results are often not reported. CONCLUSIONS: Heat mapping utilized in initial data analysis is advantageous for reporting clustered gait parameter differences in one single chart and improves data mining.
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Análisis de Datos , Minería de Datos/métodos , Análisis de la Marcha/métodos , Trastornos Neurológicos de la Marcha/fisiopatología , Enfermedades Neurodegenerativas/fisiopatología , Neurociencias/métodos , Animales , Minería de Datos/normas , Modelos Animales de Enfermedad , Análisis de la Marcha/normas , Trastornos Neurológicos de la Marcha/etiología , Humanos , Enfermedades Neurodegenerativas/complicaciones , Neurociencias/normas , RoedoresRESUMEN
Cell-to-cell propagation of aggregated alpha synuclein (aSyn) has been suggested to play an important role in the progression of alpha synucleinopathies. A critical step for the propagation process is the accumulation of extracellular aSyn within recipient cells. Here, we investigated the trafficking of distinct exogenous aSyn forms and addressed the mechanisms influencing their accumulation in recipient cells. The aggregated aSyn species (oligomers and fibrils) exhibited more pronounced accumulation within recipient cells than aSyn monomers. In particular, internalized extracellular aSyn in the aggregated forms was able to seed the aggregation of endogenous aSyn. Following uptake, aSyn was detected along endosome-to-lysosome and autophagosome-to-lysosome routes. Intriguingly, aggregated aSyn resulted in lysosomal activity impairment, accompanied by the accumulation of dilated lysosomes. Moreover, analysis of autophagy-related protein markers suggested decreased autophagosome clearance. In contrast, the endocytic pathway, proteasome activity, and mitochondrial homeostasis were not substantially affected in recipient cells. Our data suggests that extracellularly added aggregated aSyn primarily impairs lysosomal activity, consequently leading to aSyn accumulation within recipient cells. Importantly, the autophagy inducer trehalose prevented lysosomal alterations and attenuated aSyn accumulation within aSyn-exposed cells. Our study underscores the importance of lysosomes for the propagation of aSyn pathology, thereby proposing these organelles as interventional targets.
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Lisosomas/metabolismo , Neuronas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Trehalosa/farmacología , alfa-Sinucleína/metabolismo , Animales , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Escherichia coli/genética , Glioma/patología , Humanos , Lisosomas/efectos de los fármacos , Enfermedad de Parkinson/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/metabolismo , Sirolimus/farmacología , alfa-Sinucleína/genéticaRESUMEN
Gait and postural control dysfunction are prototypical symptoms compromising quality of life for patients with Parkinson's disease (PD). Hallmarks of cellular pathology are dopaminergic degeneration and accumulation of the cytosolic protein alpha-synuclein, linked to impaired autophagy-lysosome pathway (ALP) clearance. Physical exercise improves gait in PD patients and motor function in rodent lesion models. Moreover, exercise is considered neuroprotective and ALP induction has been reported, e.g. in human skeletal muscle, rodent peripheral and cerebral tissues. A combined analysis of how distinct exercise paradigms affect motor and central biochemical aspects of PD could maximize benefits for patients. Here we examine the effect of 4 weeks treadmill exercise intervention in 7-8 month non-lesioned mice on a) distinct gait categories, b) ALP activity, c) dopaminergic and alpha-synuclein homeostasis. The study includes wild type, alpha-synuclein knockout, and mice exclusively expressing human alpha-synuclein. Parameters of gait regularity and stability, activity, and dynamic postural control during unforced walk, were assessed by an automated system (CatWalk XT). At baseline, alpha-synuclein mouse models exhibited irregular and less active gait, with impaired dynamic postural control, compared to wild type mice. Treadmill exercise particularly improved speed and stride length, while increasing dual diagonal versus three-paw body support in both the alpha-synuclein knockout and transgenic mice. Biochemical analyses showed higher striatal tyrosine hydroxylase immuno-reactivity and reduced higher-order alpha-synuclein species in the cerebral cortex. However, no significant cerebral ALP induction was measured. In summary, treadmill exercise improved gait activity and postural stability, and promoted dopaminergic and alpha-synuclein homeostasis, without robustly inducing cerebral ALP.
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Marcha/fisiología , Condicionamiento Físico Animal/fisiología , Esfuerzo Físico/fisiología , Animales , Autofagia/fisiología , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/fisiología , Terapia por Ejercicio/métodos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/fisiología , Neuroprotección , Enfermedad de Parkinson/fisiopatología , Postura/fisiología , Sustancia Negra/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , alfa-Sinucleína/fisiologíaRESUMEN
α-Synuclein (αS) regulates vesicle exocytosis but forms insoluble deposits in Parkinson's disease (PD). Developing disease-modifying therapies requires animal models that reproduce cardinal features of PD. We recently described a previously unrecognized physiological form of αS, α-helical tetramers, and showed that familial PD-causing missense mutations shift tetramers to aggregation-prone monomers. Here, we generated mice expressing the fPD E46K mutation plus 2 homologous EâK mutations in adjacent KTKEGV motifs. This tetramer-abrogating mutant causes phenotypes similar to PD. αS monomers accumulate at membranes and form vesicle-rich inclusions. αS becomes insoluble, proteinase K-resistant, Ser129-phosphorylated, and C-terminally truncated, as in PD. These changes affect regions controlling motor behavior, including a decrease in nigrostriatal dopaminergic neurons. The outcome is a progressive motor syndrome including tremor and gait and limb deficits partially responsive to L-DOPA. This fully penetrant phenotype indicates that tetramers are required for normal αS homeostasis and that chronically shifting tetramers to monomers may result in PD, with attendant therapeutic implications.
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Trastornos Parkinsonianos/genética , alfa-Sinucleína/química , alfa-Sinucleína/genética , Animales , Antiparkinsonianos/farmacología , Encéfalo/patología , Modelos Animales de Enfermedad , Levodopa/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Neuronas/patología , Trastornos Parkinsonianos/patología , Conformación ProteicaRESUMEN
BACKGROUND: Sway is a crucial gait characteristic tightly correlated with the risk of falling in patients with Parkinsons disease (PD). So far, the swaying pattern during locomotion has not been investigated in rodent models using the analysis of dynamic footprint recording obtained from the CatWalk gait recording and analysis system. NEW METHODS: We present three methods for describing locomotion sway and apply them to footprint recordings taken from C57BL6/N wild-type mice and two different α-synuclein transgenic PD-relevant mouse models (α-synm-ko, α-synm-koxα-synh-tg). Individual locomotion data were subjected to three different signal processing analytical approaches: the first two methods are based on Fast Fourier Transform (FFT), while the third method uses Low Pass Filters (LPF). These methods use the information associated with the locomotion sway and generate sway-related parameters. RESULTS: The three proposed methods were successfully applied to the footprint recordings taken from all paws as well as from front/hind-paws separately. Nine resulting sway-related parameters were generated and successfully applied to differentiate between the mouse models under study. Namely, α-synucleinopathic mice revealed higher sway and sway itself was significantly higher in the α-synm-koxα-synh-tg mice compared to their wild-type littermates in eight of the nine sway-related parameters. COMPARISON WITH EXISTING METHOD: Previous locomotion sway index computation is based on the estimated center of mass position of mice. CONCLUSIONS: The methods presented in this study provide a sway-related gait characterization. Their application is straightforward and may lead to the identification of gait pattern derived biomarkers in rodent models of PD.
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Modelos Animales de Enfermedad , Análisis de la Marcha/métodos , Trastornos Parkinsonianos/diagnóstico , Trastornos Parkinsonianos/fisiopatología , Algoritmos , Animales , Fenómenos Biomecánicos , Pie , Análisis de Fourier , Análisis de la Marcha/instrumentación , Trastornos Neurológicos de la Marcha/diagnóstico , Trastornos Neurológicos de la Marcha/fisiopatología , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Procesamiento de Señales Asistido por Computador , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Characterizing gait is important in the study of movement disorders, also in clinical mouse models. Gait data are therefore necessary for the development of gait analysis methods and the study of diseases. This article presents gait data of two α-synucleinopathic transgenic mouse models and their non-transgenic littermate, backcrossed into the C57BL/6N genetic background. The animal gait was recorded using CatWalk system, which provides the information for each run about the paw positions, paw print sizes, and paw intensities as a function of time or video frame. A total of 90 run data files are provided in this article.
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Autophagy is a conserved catabolic pathway with emerging functions in mammalian neurodevelopment and human neurodevelopmental diseases. The mechanisms controlling autophagy in neuronal development are not fully understood. Here, we found that conditional deletion of the Forkhead Box O transcription factors FoxO1, FoxO3, and FoxO4 strongly impaired autophagic flux in developing neurons of the adult mouse hippocampus. Moreover, FoxO deficiency led to altered dendritic morphology, increased spine density, and aberrant spine positioning in adult-generated neurons. Strikingly, pharmacological induction of autophagy was sufficient to correct abnormal dendrite and spine development of FoxO-deficient neurons. Collectively, these findings reveal a novel link between FoxO transcription factors, autophagic flux, and maturation of developing neurons.
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Autofagia/fisiología , Factores de Transcripción Forkhead/metabolismo , Morfogénesis/fisiología , Neurogénesis/fisiología , Animales , Separación Celular/métodos , Células Cultivadas , Ratones Transgénicos , Neuronas/metabolismoRESUMEN
The autophagy-lysosome pathway (ALP) regulates intracellular homeostasis of the cytosolic protein SNCA/alpha-synuclein and is impaired in synucleinopathies, including Parkinson disease and dementia with Lewy bodies (DLB). Emerging evidence suggests that ALP influences SNCA release, but the underlying cellular mechanisms are not well understood. Several studies identified SNCA in exosome/extracellular vesicle (EV) fractions. EVs are generated in the multivesicular body compartment and either released upon its fusion with the plasma membrane, or cleared via the ALP. We therefore hypothesized that inhibiting ALP clearance 1) enhances SNCA release via EVs by increasing extracellular shuttling of multivesicular body contents, 2) alters EV biochemical profile, and 3) promotes SNCA cell-to-cell transfer. Indeed, ALP inhibition increased the ratio of extra- to intracellular SNCA and upregulated SNCA association with EVs in neuronal cells. Ultrastructural analysis revealed a widespread, fused multivesicular body-autophagosome compartment. Biochemical characterization revealed the presence of autophagosome-related proteins, such as LC3-II and SQSTM1. This distinct "autophagosome-exosome-like" profile was also identified in human cerebrospinal fluid (CSF) EVs. After a single intracortical injection of SNCA-containing EVs derived from CSF into mice, human SNCA colocalized with endosome and neuronal markers. Prominent SNCA immunoreactivity and a higher number of neuronal SNCA inclusions were observed after DLB patient CSF EV injections. In summary, this study provides compelling evidence that a) ALP inhibition increases SNCA in neuronal EVs, b) distinct ALP components are present in EVs, and c) CSF EVs transfer SNCA from cell to cell in vivo. Thus, macroautophagy/autophagy may regulate EV protein composition and consequently progression in synucleinopathies.
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Autofagosomas/metabolismo , Autofagia/fisiología , Exosomas/metabolismo , Lisosomas/metabolismo , Neuronas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Autofagosomas/efectos de los fármacos , Autofagia/efectos de los fármacos , Biomarcadores/metabolismo , Células Cultivadas , Cloroquina/farmacología , Exosomas/efectos de los fármacos , Exosomas/ultraestructura , Humanos , Enfermedad por Cuerpos de Lewy/metabolismo , Lisosomas/efectos de los fármacos , Macrólidos/farmacología , Ratones , Neuronas/efectos de los fármacos , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Transporte de Proteínas , Ratas Sprague-DawleyRESUMEN
Oxidative stress (OS), mitochondrial dysfunction, and dysregulation of alpha-synuclein (aSyn) homeostasis are key pathogenic factors in Parkinson's disease. Nevertheless, the role of aSyn in mitochondrial physiology remains elusive. Thus, we addressed the impact of aSyn specifically on mitochondrial response to OS in neural cells. We characterize a distinct type of mitochondrial fragmentation, following H2O2 or 6-OHDA-induced OS, defined by spherically-shaped and hyperpolarized mitochondria, termed "mitospheres". Mitosphere formation mechanistically depended on the fission factor Drp1, and was paralleled by reduced mitochondrial fusion. Furthermore, mitospheres were linked to a decrease in mitochondrial activity, and preceded Caspase3 activation. Even though fragmentation of dysfunctional mitochondria is considered to be a prerequisite for mitochondrial degradation, mitospheres were not degraded via Parkin-mediated mitophagy. Importantly, we provide compelling evidence that aSyn prevents mitosphere formation and reduces apoptosis under OS. In contrast, aSyn did not protect against Rotenone, which led to a different, previously described donut-shaped mitochondrial morphology. Our findings reveal a dichotomic role of aSyn in mitochondrial biology, which is linked to distinct types of stress-induced mitochondrial fragmentation. Specifically, aSyn may be part of a cellular defense mechanism preserving neural mitochondrial homeostasis in the presence of increased OS levels, while not protecting against stressors directly affecting mitochondrial function.
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Apoptosis/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , alfa-Sinucleína/metabolismo , Animales , Caspasa 3/metabolismo , Células Cultivadas , Dinaminas , GTP Fosfohidrolasas/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/efectos de los fármacos , Dinámicas Mitocondriales/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Mitofagia/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Rotenona/farmacología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The glutamate transporter xCT (SCL7a11, system Xc-, SXC) is an emerging key player in glutamate/cysteine/glutathione homeostasis in the brain and in cancer. xCT expression correlates with the grade of malignancy. Here, we report on the use of the U.S. Food and Drug Administration and EMA-approved xCT inhibitor, sulfasalazine (SAS) in gliomas. SAS does not affect cell viability in gliomas at concentrations below 200 µM. At higher concentrations SAS becomes gliomatoxic. Mechanistically SAS inhibits xCT and induces ferroptotic cell death in glioma cells. There is no evidence for impact on autophagic flux following SAS application. However, SAS can potentiate the efficacy of the standard chemotherapeutic and autophagy-inducing agent temozolomide (Temcat, Temodal or Temodar®). We also investigated SAS in non-transformed cellular constituents of the brain. Neurons and brain tissue are almost non-responding to SAS whereas isolated astrocytes are less sensitive towards SAS toxicity compared to gliomas. In vivo SAS treatment does not affect experimental tumor growth and treated animals revealed comparable tumor volume as untreated controls. However, SAS treatment resulted in reduced glioma-derived edema and, hence, total tumor volume burden as revealed by T2-weighted magnetic resonance imaging. Altogether, we show that SAS can be utilized for targeting the glutamate antiporter xCT activity as a tumor microenvironment-normalizing drug, while crucial cytotoxic effects in brain tumors are minor.