Potential Neurotoxic Effects of Glioblastoma-Derived Exosomes in Primary Cultures of Cerebellar Neurons via Oxidant Stress and Glutathione Depletion

High-grade gliomas are the most fatal brain tumors. Grade 4 gliomas are called glioblastoma multiforme (GBM), which are associated with the poorest survival and a 5-year survival rate of less than 4%. Many patients with GBM developed concomitant cognitive dysfunctions and epilepsy. Although the cognitive decline is well defined in glioblastomas, the neurotoxic factors underlying this pathology are not well understood in GBM patients. In this study, we aimed to investigate whether GBM-derived exosomes play a role in neuronal toxicity. For this purpose, exosomes obtained from T98G and U373 GBM cells were applied to primary neuron culture at different concentrations. Subsequently, MTT, LDH, GSH, TAS, and TOS tests were performed. Both GBM-derived exosomes induced a dose-dependent and statistically significant increase of LDH release in cerebellar neurons. MTT assay revealed as both T98G and U373 GBM-derived exosomes induced dose-dependent neurotoxic effects in cerebellar neurons. To the best of our knowledge, this study is the first study demonstrating the toxic potential of GBM-derived exosomes to primary neurons, which may explain the peritumoral edema and cognitive decline in GBM patients.


Introduction
Gliomas are the most lethal tumors affecting the brain. Among these, glioblastoma multiforme (GBM) is the most advanced brain tumor and it is associated with the poorest survival rate, with a 5-year survival rate of less than <4% [1]. GBM accounts for about 80% of primary brain malignancies and influences more than 17,000 patients every year in the USA [1]. For many patients with GBM, deficits in cognitive functioning and epilepsy are

Glioblastoma Cell Lines and Primary Neuron Cell Culture
The GBM cell lines T98G and U373 were kindly provided by the researchers of the department of veterinary pharmacology and toxicology of Ataturk University (Erzurum, Turkey). Briefly, the cells were resuspended in fresh medium (Dulbecco's modified eagle medium (DMEM), Fetal bovine serum (FBS; 15%), and antibiotic suspension 1% (penicillin, streptomycin, and amphotericin B). Then, the cells were plated into 150 cm 2 cell culture flasks (Corning, Corning, NY, USA) and cultured in a CO 2 incubator under appropriate conditions (5% CO 2 ; 37 • C).
Sprague Dawley newborn rats born within 24 h were used to obtain cerebellum neurons in order to test the effects of glioblastoma-derived exosomes. Briefly, after sedation, the rats were rapidly decapitated, brain tissue was collected, and cerebellar tissue was isolated with the help of a scalpel. Then, a single cell suspension was obtained with Trypsin-Ethylenediaminetetraacetic acid (EDTA) (0.25% trypsin-0.02% EDTA) dissociation. The cells were then centrifuged at 1200 rpm for 5 min. Then the cells were resuspended in an appropriate medium (88% NBM (Neuro-basal medium, Gibco, Waltham, MA, USA) with 10% FBS (Fetal bovine solution, Gibco, USA), 2% B-27 (Thermo Fisher, Bremen, Germany), 0.1% antibiotic (Penicillin-Streptomycin), and amphotericin B (Thermo Fisher, Germany). The cells were incubated at 5% CO 2 at 37 • C for 10 days by changing the medium every 3 days.

Exosome Isolation and Primary Neuron Cell Treatment
To obtain GBM-derived exosomes, T98G and U373 cell lines were plated in T75 cm 2 flasks. After 70% confluency in 150 cm 2 cell culture flasks (Corning, USA), 25 mL of complete medium was added. Subsequently, the Total Exosome Isolation Reagent (Invitrogen™ -Cat. 4478359, Waltham, MA, USA) protocol for the effective isolation of exosomes was used as previously described [22,23]. Briefly, conditioned medium and reagent (2:1) were mixed and incubated at 2 • C to 8 • C overnight. After incubation, the samples were centrifuged at 10,000× g for 1 h at 2 • C to 8 • C in order to pull down the exosomes at the bottom of the tube.
A wide range of exosome concentrations was used as follows: 1-5-10-50-100 µg/mL. Such concentrations were selected on the basis of similar studies reported in literature [24][25][26][27]. In detail, exosomes at different concentrations were added into 96 well plates, containing primary neuron cells at 85% confluence to assess the toxicity of GBM-derived exosomes. After 24 h of incubation (at 37 • C, 5% CO 2 and 95% humidity), MTT assay and immunohistochemical staining were performed and TAS, TOS, GSH and LDH levels were evaluated. Untreated cells were used as controls for biochemical and immunofluorescence evaluations.

MTT Assay
After 24 h of treatment with the GBM-derived exosomes, 10 µL of MTT solution (1:10) was added to each well plate. After 4 h of incubation, 100 µL of DMSO solution was added to all wells. The optical density of the dissolved formazan crystals was determined at a wavelength of 570 nm by using the Multiskan™ GO Microplate Spectrophotometer reader.

Total Oxidant Status (TOS) and Total Antioxidant Status (TAS)
TOS and TAS (Rel Assay Diagnostics ® Company, Gaziantep, Turkey) were determined with spectrophotometry (Multiskan™ GO Microplate Spectrophotometer reader, Waltham, MA, USA). The chromogenic xylenol orange was used to evaluate TOS levels as under acid conditions, the oxidizing potential of samples can oxidize Fe 2+ to Fe 3+ which binds xylenol orange and convert it into a blue-purple complex. To determine the TOS levels, 75 µL of cell supernatant and 500 µL of reactive 1 solution were added to each well and then read at 590 nm. For the second reading, 25 µL of reactive 2 solution was added to the wells and the absorbance was read at 590 nm (TOS = ∆example/∆ST2 × 20). To evaluate the TAS levels, ABTS (2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), a chromogenic substance that can be converted by oxidizing agent into its radical monocation form ABTS+ (colored), was used. More in detail the TAS of the samples was determined and calculated by measuring the absorbance of ABTS+ at 660 nm. Trolox is an analog of Vitamin E and has a similar antioxidant state to that of Vitamin E. Trolox is used as a reference substance for total antioxidant status. During this assay, 500 µL of reactive 1 solution was added to the wells containing 30 µL of cell supernatant. The first read was done at 660 nm. For the second read, 75 µL of reactive 2 solution was added in the wells and absorbance was read at 660 nm (TAS = (∆ST1 − ∆example)/(∆ST1 − ∆ST2)).

Lactate Dehydrogenase (LDH) Assay
LDH assay test was performed using a commercially available test kit from Cayman Chemical Co., Ltd., (Ann Arbor, MI, USA). Briefly, the cell culture medium was centrifuged at 400× g for 5 min at room temperature. A volume of 100 µL of the supernatant was added to 100 µL of the reaction solution (LDH Assay Buffer, LDH Substrate Mix) and incubated with gentle shaking on an orbital shaker for 30 min at room temperature. Finally, the absorbance was read at 490 nm wavelength.

Glutathione (GSH) Assay
The glutathione (GSH) measurement is based on the principle of glutathione reacting with dithionitrobenzoic acid (DTNB) to produce thio-nitrobenzoic acid and glutathione disulfide. Optical density (OD) is measured spectrophotometrically at 450 nm ± 2 nm wavelength. The GSH concentration in the samples tested is calculated by comparing the OD of the samples with the standard curve.
GSH + DTNB → GSSH + TNB For GSH determination, Elabscience kit (Houston, TX, USA) was employed and the intensity of the yellow color formed by nitrobenzoic acid was read in a spectrophotometer at 450 nm wavelength.

Statistical Analyses
Statistical analyses were performed using one-way analysis of variance (ANOVA) with Tukey's HSD for post-hoc comparisons using the SPSS 22.0 software and the results were presented as median ± SEM. A p-value of p < 0.05 was considered statistically significant. All the experiments were performed in triplicate. 8-OHdG fluorescent signal was observed in five different microscopic fields.

Alteration of Glutathione (GSH) Levels in Cerebellar Neurons after GBM-Derived Exosomes Treatment
The analysis of GSH revealed a dose-dependent reduction of GSH levels in cerebellar neurons after the exposure to both T98G and U373 GBM-derived exosomes. In particular, statistical differences in GSH levels were observed in cerebellar neurons treated with 50 and 100 µg/mL of exosomes. Of note, GSH depletion was more prominent in cells exposed to U373 GBM-derived exosomes compared to T98G-derived exosomes showing almost 50% reduction of GSH compared to non-treated cells (Figure 1).

Lactate Dehydrogenase (LDH) Levels in Cerebellar Neurons after GBM-Derived Exosomes Treatment
Both T98G-and U373-derived exosomes induced a dose-dependent and statistically significant increase of LDH release in the culture medium of cerebellar neurons. LDH levels increased in a dose-dependent manner mainly in cerebellar neurons exposed to T98G-derived exosomes compared to those treated with U373 exosomes (p < 0.01) at low concentrations (≥5 µg/mL). In cerebellar neurons exposed to U373-derived exosomes, the statistical significance (p < 0.05) was reached at a concentration of 50 µg/mL and

Lactate Dehydrogenase (LDH) Levels in Cerebellar Neurons after GBM-Derived Exoso Treatment
Both T98G-and U373-derived exosomes induced a dose-dependent and statis significant increase of LDH release in the culture medium of cerebellar neurons. LD els increased in a dose-dependent manner mainly in cerebellar neurons exposed to derived exosomes compared to those treated with U373 exosomes (p < 0.01) at low centrations (≥5 µg/mL). In cerebellar neurons exposed to U373-derived exosomes, th tistical significance (p < 0.05) was reached at a concentration of 50 µg/mL and occ more evidently at 100 µg/mL (p < 0.01) ( Figure 2). The increased levels of LDH de strated the harmful potential of GBM-derived exosomes able to induce damages in bellar neurons and, consequently, neuron cell death.

Treatment with GBM-Derived Exosomes Reduces Cell Viability of Cerebellar Neurons Vitro
The treatment with increasing doses of T98G-and U373-derived exosomes in a dose-dependent and statistically significant depletion of MTT activity in cerebella rons. As observed for the LDH levels, T98G-derived exosomes also induced a mor nounced depletion of MTT activity. An amount of 1 µg/mL of T98G-derived exo depleted MTT with a statistically significant difference of p < 0.05. Higher dosag µg/mL depleted MTT in a more significant manner (p < 0.01). In cerebellar neuro posed to U373-derived exosomes, a significant reduction of cell viability (p < 0.05 obtained at 5 and 10 µg/mL while a more consistent reduction of the MTT activit observed at 50 and 100 µg/mL of exosomes (p < 0.01) ( Figure 3).

Lactate Dehydrogenase (LDH) Levels in Cerebellar Neurons after GBM-Derived Exosom Treatment
Both T98G-and U373-derived exosomes induced a dose-dependent and statisti significant increase of LDH release in the culture medium of cerebellar neurons. LDH els increased in a dose-dependent manner mainly in cerebellar neurons exposed to T derived exosomes compared to those treated with U373 exosomes (p < 0.01) at low centrations (≥5 µg/mL). In cerebellar neurons exposed to U373-derived exosomes, th tistical significance (p < 0.05) was reached at a concentration of 50 µg/mL and occu more evidently at 100 µg/mL (p < 0.01) (Figure 2). The increased levels of LDH dem strated the harmful potential of GBM-derived exosomes able to induce damages in bellar neurons and, consequently, neuron cell death.

Treatment with GBM-Derived Exosomes Reduces Cell Viability of Cerebellar Neurons I Vitro
The treatment with increasing doses of T98G-and U373-derived exosomes ind a dose-dependent and statistically significant depletion of MTT activity in cerebellar rons. As observed for the LDH levels, T98G-derived exosomes also induced a more nounced depletion of MTT activity. An amount of 1 µg/mL of T98G-derived exoso depleted MTT with a statistically significant difference of p < 0.05. Higher dosage µg/mL depleted MTT in a more significant manner (p < 0.01). In cerebellar neuron posed to U373-derived exosomes, a significant reduction of cell viability (p < 0.05) obtained at 5 and 10 µg/mL while a more consistent reduction of the MTT activity observed at 50 and 100 µg/mL of exosomes (p < 0.01) (Figure 3).

Treatment with GBM-Derived Exosomes Reduces Cell Viability of Cerebellar Neurons In Vitro
The treatment with increasing doses of T98G-and U373-derived exosomes induced a dose-dependent and statistically significant depletion of MTT activity in cerebellar neurons. As observed for the LDH levels, T98G-derived exosomes also induced a more pronounced depletion of MTT activity. An amount of 1 µg/mL of T98G-derived exosomes depleted MTT with a statistically significant difference of p < 0.05. Higher dosages ≥ 5 µg/mL depleted MTT in a more significant manner (p < 0.01). In cerebellar neurons exposed to U373-derived exosomes, a significant reduction of cell viability (p < 0.05) was obtained at 5 and 10 µg/mL while a more consistent reduction of the MTT activity was observed at 50 and 100 µg/mL of exosomes (p < 0.01) (Figure 3).

Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) in Cerebellar Neurons Treated with T98G and U373 GBM-Derived Exosomes
Both T98G-and U373-derived exosomes induced dose-dependent and statisti significant reduction of TAS. The extent of TAS depletion occurred with higher sig cance when cerebellar neurons were exposed to T98G-derived exosomes. At concen tions ≥5 µg/mL of T98G-derived exosomes, TAS depletion occurred with a statistical nificance of p < 0.01. In cerebellar neurons exposed to U373-derived exosomes, the st tical significances of TAS depletions were p < 0.05 and p < 0.01 for exosome concentrat  when cerebellar neurons were exposed to T98G-derived exosomes. At concentrations ≥5 µg/mL of T98G-derived exosomes, TAS depletion occurred with a statistical significance of p < 0.01. In cerebellar neurons exposed to U373-derived exosomes, the statistical significances of TAS depletions were p < 0.05 and p < 0.01 for exosome concentrations of 50 and 100 µg/mL, respectively (Figure 4).

Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) in Cerebellar Neurons Treated with T98G and U373 GBM-Derived Exosomes
Both T98G-and U373-derived exosomes induced dose-dependent and statisti significant reduction of TAS. The extent of TAS depletion occurred with higher sig cance when cerebellar neurons were exposed to T98G-derived exosomes. At conce tions ≥5 µg/mL of T98G-derived exosomes, TAS depletion occurred with a statistica nificance of p < 0.01. In cerebellar neurons exposed to U373-derived exosomes, the s tical significances of TAS depletions were p < 0.05 and p < 0.01 for exosome concentra of 50 and 100 µg/mL, respectively (Figure 4). Conversely to what was observed for TAS levels, the analysis of TOS revealed cerebellar neurons treated with T98G-and U373-derived exosomes showed a statis increase of TOS levels with a dose-dependent trend. The extent of TOS increase was m marked in cerebellar neurons exposed to T98G-derived exosomes where a statistica crement was observed at 1 µg/mL (p < 0.05) and ≥5 µg/mL (p < 0.01 for all dosages cerebellar neurons, exposed to U373 GBM-derived exosomes, TOS levels statisticall crease at dose of 5 µg/mL (p < 0.05). Dosages equal to or higher than 10 µg/mL ind more significant levels of TOS (p < 0.01 for all dosages) ( Figure 5).

Control
Exo U373   Conversely to what was observed for TAS levels, the analysis of TOS revealed that cerebellar neurons treated with T98G-and U373-derived exosomes showed a statistical increase of TOS levels with a dose-dependent trend. The extent of TOS increase was more marked in cerebellar neurons exposed to T98G-derived exosomes where a statistical increment was observed at 1 µg/mL (p < 0.05) and ≥5 µg/mL (p < 0.01 for all dosages). In cerebellar neurons, exposed to U373 GBM-derived exosomes, TOS levels statistically increase at dose of 5 µg/mL (p < 0.05). Dosages equal to or higher than 10 µg/mL induced more significant levels of TOS (p < 0.01 for all dosages) ( Figure 5).

Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) in Cerebellar Neurons Treated with T98G and U373 GBM-Derived Exosomes
Both T98G-and U373-derived exosomes induced dose-dependent and statisti significant reduction of TAS. The extent of TAS depletion occurred with higher sig cance when cerebellar neurons were exposed to T98G-derived exosomes. At conce tions ≥5 µg/mL of T98G-derived exosomes, TAS depletion occurred with a statistica nificance of p < 0.01. In cerebellar neurons exposed to U373-derived exosomes, the st tical significances of TAS depletions were p < 0.05 and p < 0.01 for exosome concentra of 50 and 100 µg/mL, respectively (Figure 4). Conversely to what was observed for TAS levels, the analysis of TOS revealed cerebellar neurons treated with T98G-and U373-derived exosomes showed a statis increase of TOS levels with a dose-dependent trend. The extent of TOS increase was m marked in cerebellar neurons exposed to T98G-derived exosomes where a statistica crement was observed at 1 µg/mL (p < 0.05) and ≥5 µg/mL (p < 0.01 for all dosages cerebellar neurons, exposed to U373 GBM-derived exosomes, TOS levels statisticall crease at dose of 5 µg/mL (p < 0.05). Dosages equal to or higher than 10 µg/mL ind more significant levels of TOS (p < 0.01 for all dosages) ( Figure 5).

Discussion
Despite the numerous advancements regarding the pharmacological treatments of tumors [28,29] and the novel high-sensitive techniques developed for the early diagnosis of pathologies [30][31][32][33][34], glioblastoma still represents a major issue in neuro-oncology. Although some genetic and epigenetic biomarkers for the early diagnosis of GBM are under investigation [35][36][37], different clinical signs can predict the onset of advanced tumors. Indeed, many patients with GBM present deficits in cognitive functioning and epilepsy due to brain compression that could be the first indicator of disease recurrence [4,5]. Besides mechanical dysfunction due to tumor growth and brain compression, other cellular and molecular mechanisms are involved in the development of GBM-associated brain disorders. Among these, glioblastoma cells could release factors responsible for neurotoxic effects as well as immunomodulatory factors with paracrine effects towards the surrounding neurological structures directly or through cancer-derived exosomes [38,39]. Katrib and colleagues (2019) have established a strict relationship among inflammation, GBM oxidative stress, and neurological deficit by identifying a panel of genes involved in these processes [40]. Similarly, Lange and colleagues (2021) have recently highlighted a link between glioblastoma and tumor-associated epilepsy describing the processes leading to the dysregulation of the glutamatergic signaling, including imbalance of the redox system [41]. In addition, the imbalance of the brain redox status due to the presence of GBM has been also associated with the presence of parenchymal and peritumoral edema [42]. However, the identification of the specific determinants responsible for such detrimental associations between GBM and neuronal structure has not been elucidated yet.
In order to evaluate the detrimental effects of GBM-derived exosomes, this study wanted to assess the potential neurotoxic effects mediated by GBM-derived exosomes on primary neurons.
For this purpose, cerebellar neurons were treated with increasing doses of GBMderived exosomes obtained from two different GBM cell lines and subsequently several parameters were evaluated to assess the neurotoxic potential of such exosomes. Our results showed that neuronal cell viability decreased significantly after treatment with ≥1 µg/mL or ≥5 µg/mL of T98G-and U373-derived exosomes, respectively. MTT assay thus supports the hypothesis of the neuronal toxicity mediated by GBM-derived exosomes demonstrating a dose-dependent decrease of cell viability. This detrimental effect was also observed by analyzing the levels of GSH and LDH, two of the most recognized markers of oxidative stress and cell damage. In particular, both GSH and LDH has been widely associated with cognitive impairment and neurodegeneration [43][44][45]. In this context, Backos and coworkers showed that the accumulation of 2-deoxy-D-ribose (2dDR) due to the intratumoral necrotic processes occurring in GBM can modify the function of enzymes involved in GSH production, thus leading to oxidative stress-related alterations in the brain [46]. Similarly, several studies showed that LDH levels are increased in cancer patients, including GBM where LDH seems to be involved in cancer progression and metastases [47,48]. In addition, it was demonstrated that high levels of LDH induce longevity and neurodegeneration in animal models [49].
In our study, GSH levels were significantly reduced after treatment with exosome concentrations of 50 µg/mL and 100 µg/mL, while LDH levels increased significantly with ≥5 µg/mL or ≥50 µg/mL of T98G-or U373-derived exosomes, respectively. These data suggest how the redox potential of neuronal cells is significantly affected by the neurotoxic cargo of GBM-derived exosomes. As a consequence, the impaired antioxidant abilities of neurons lead to the accumulation of cellular damages and cell death as demonstrated by the increasing levels of LDH.
To further support these preliminary findings, the analysis of Total Antioxidant Status showed a significant reduction of the antioxidant potential of cerebellar neurons after the treatment with ≥5 µg/mL of T98G-derived exosomes or ≥50 µg/mL of U373-derived exosomes. Conversely, the analysis of the Total Oxidant Status showed an oppositive trend; TOS increased significantly when cerebellar neurons were exposed with a concentration of ≥1 µg/mL or ≥5 µg/mL of T98G-or U373-derived exosomes, respectively. Noteworthy, different results were obtained by using T98G-and U373-derived exosomes at the same concentrations. A possible explanation may be related to the different molecular cargo of T98G exosomes compared to that of U373 exosomes. In this context, Spinelli and colleagues (2018) have demonstrated that different GBM cells and molecular subtypes have totally different extracellular vesicle profiles [50]. Therefore, in the near future, the molecular profiling of both T98G and U373 exosomes will be mandatory to clearly identify proteins or other molecules (ncRNA, DNA fragments, etc.) responsible for the neurotoxic effects observed in primary cerebellar neurons.
The results here obtained are in line with other studies demonstrating the effects of GBM exosomes on tumor and brain microenvironment. In particular, it was demonstrated that GBM exosomes are able to affect brain homeostasis inducing a pro-inflammatory microenvironment due to the activation of monocytes, macrophages microglia, astrocytes and endothelial cells as well as vascular alterations mediated by the growth factors proangiogenic cargo consisting of EGFR, VEGF, angiogenin, PDGF, coagulation factor VIIa, etc. [51][52][53].
As regards the oxidant potential of GBM-derived exosomes here demonstrated, a possible explanation may be related to the transfer of mitochondrial damages which could increase neuron damages. In particular, Guescini and colleagues first discovered that glioblastoma cells and astrocyte release exosomes carrying mitochondrial proteins and mtDNA which transfers in neuron cells have been associated with the development of Alzheimer's disease [54,55]. Other studies have demonstrated that mitochondrial genome (mtDNA) and even whole mitochondria are mobile and can be transferred to surrounding cells through exosomes and mitochondrial-derived vesicles inducing the activation of oxidative processes [56]. These findings may explain the high levels of 8-OHdG and the alterations of TOS and TAS observed in our experiments.
Overall, these results demonstrated that GBM-derived exosomes are able to increase the oxidative stress in cerebellar neurons through the reduction of the cellular antioxidant defenses and the increase of oxidative damages as demonstrated in our experiments. Therefore, our study further enriched the knowledge on exosome functions in brain diseases. Indeed, a plethora of studies highlighted the pivotal role of exosomes in GBM, Alzheimer's disease, Parkinson's disease, epilepsy, and other brain disorders, independently [38,[57][58][59][60]. However, here, for the first time, an interconnection between GBM exosomes and neuronal damages responsible for neuronal disorders has been observed.

Conclusions
To the best of our knowledge, this is the first study that demonstrates the toxic effects of glioblastoma-derived exosomes in primary neurons. The results here obtained may explain the peritumoral edema and cognitive decline often observed in glioblastoma patients. Of note, the results here obtained are merely exploratory as the precise GBM exosome factors involved in the neurotoxic effects observed in cerebellar neurons are still unknown. However, our findings will pave the way to novel investigation studies aimed at evaluating the precise molecular cargo of GBM-derived exosomes. Indeed, further in vitro and in vivo functional studies are needed to identify the precise molecules responsible for neurotoxicity and to assess the potential therapeutic effects of drugs able to reduce tumor growth, edema, and cognitive decline.
In conclusion, the identification of a neurotoxic effect of GBM exosomes suggests how exosomes can be considered as both biomarkers and targets and their characterization could significantly improve the management of GBM patients.

Conflicts of Interest:
The authors declare no conflict of interest.