Autophagy and neuroprotection in astrocytes exposed to 6-hydroxydopamine is negatively regulated by NQO2: a potential novel target in Parkinson’s disease.

doi:10.21203/rs.3.rs-2510273/v1

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Autophagy and neuroprotection in astrocytes exposed to 6-hydroxydopamine is negatively regulated by NQO2: a potential novel target in Parkinson’s disease.

https://doi.org/10.21203/rs.3.rs-2510273/v1

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published 07 Dec, 2023

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Dopaminergic degeneration is a central feature of Parkinson’s disease (PD), but glial dysfunction may accelerate or trigger neuronal death. In fact, astrocytes play a key role in maintenance of the blood-brain barrier and detoxification. 6-hydroxydopamine (6OHDA) is used to induce PD in rodent models due to its specific toxicity to dopaminergic neurons, but its effect on astrocytes has been poorly investigated. Here, we show that 6OHDA dose-dependently impairs autophagy in human U373 and primary murine astrocytes in the absence of cell death. LC3II downregulation was observed 6 to 48 hours after treatment. Interestingly, 6OHDA enhanced NRH:quinone oxidoreductase 2 (NQO2) expression and activity in U373 cells, even if 6OHDA is not its substrate. The autophagic flux was restored by inhibition of NQO2 with S29434, which correlated with a partial reduction of oxidative stress in response to 6OHDA in human and murine astrocytes. NQO2 inhibition also increased neuroprotective capability of U373 cells, since S29434 protected dopaminergic SHSY5Y cells from 6OHDA-induced cell death when co-cultured with astrocytes. Silencing of NQO2 attenuated toxic effects of 6OHDA on autophagy. Finally, the analysis of Gene Expression Ominibus datasets showed elevated NQO2 gene expression in the blood cells of early-stage PD patients. These data support a toxifying function of NQO2 in dopaminergic degeneration via negative regulation of autophagy and neuroprotection in astrocytes, suggesting a novel pharmacological target in PD.

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PD is a neurodegenerative disorder mainly characterized by progressive loss of dopaminergic neurons in the substantia nigra. Although the exact mechanisms of Dopaminergic neuronal loss in SN are not fully understood, mitochondrial damage and oxidative stress (OS) due to alterations in iron and redox metabolism, protein misfolding and aggregation, defective autophagy and “prion-like protein infection” are considered involved in the onset and progression of PD [1–4].

Autophagy is a catabolic process responsible for the removal of protein aggregates and damaged organelles, via lysosomal digestion [5, 6]. Autophagy machinery is also involved in the regulation of related processes with an increasingly recognized role in PD, like vesicular transport, exo- and phagocytosis [5, 7]. Numerous genetic mutations encoding components of the autophagic machinery have been identified as disease risk factors [2, 8, 9]. Notably, autophagy dysregulation, is also associated with reactive oxygen species (ROS) in both cellular signaling and damage [10]. In particular, all chemical agents inducing ROS and dopaminergic damage, known as Parkinsonian toxins, have been shown to dysregulate or impair autophagy [2, 11] both in neurons as well as in astrocytes.

Astrocytes maintain redox balance in the brain through antioxidant production and detoxification [12], contribute to the formation and maintenance of the blood–brain barrier [13] and regulate inflammatory responses in the central nervous system [14, 15]. Increasing evidence highlights the contribution of astrocyte dysfunction to the pathogenesis of several neurodegenerative conditions, including PD [16]. DA metabolism is the main source of ROS in the brain [3]. Functional glial cells protect neurons against OS by metabolizing DA via monoamine oxidase-B (MAOB) and catechol-O-methyltransferase (COMT) and by a battery of antioxidants enzymes present in astrocytes [17]. A prolonged dysfunction of astrocytes, involving autophagy and ROS detoxification processes, could increase the vulnerability of DA neurons and advance their degeneration during aging, leading to PD [16, 18].

6OHDA is widely used to investigate the pathogenesis of PD in pre-clinical studies since it induces PD-like symptoms in rodents [19]. 6OHDA is a DA analog, which can be produced by DA hydroxylation in presence of Fe^+ 2 iron and H₂O₂. 6OHDA is considered to be a purely synthetic toxin, but several observations suggest a possibility of its endogenous origin likely during DA catabolism. Indeed, it has been detected in caudatal samples as well as in the urine of PD patients [20, 21] and in mice brains following long-term L-dopa administration [22]. It shows specific PD-like effects [23, 24], via generation of toxic DA oxidation products. Upon injection in the brain, it causes selective death of dopaminergic neurons and pro-inflammatory activation of microglia, while its effects on the astrocytic compartment are a matter of debate [25, 26]. The mechanisms of 6OHDA toxicity are complex and involve rapid auto-oxidation, leading to generation of hydrogen peroxide and hydroxyl radicals [27] and impairment of mitochondrial energy production [28, 29]. It is also unclear if any endogenous toxifying enzymes such as quinone oxidoreductase 2 (NQO2/QR2), may contribute to this process[30, 31].

NQO2 is a cytosolic and ubiquitously expressed flavoprotein that catalyzes the two-electron reduction of quinone to hydroquinones [32]. NQO2, structurally related to NQO1, is considered as a phase II detoxifying enzyme, but it enhances the production of activated quinones and ROS in the presence of certain substrates [32, 33]. NQO2 emerged as a possible target in PD more than two decades ago. A case-control Japanese study identified a positive association of a non-familiar form of PD with a D (deletion) polymorphism in the NQO2 promoter region. This “gain of function” variant of the promoter lacking Sp3 transcriptional suppressor binding site was 3.46 times more frequent in PD patients than in healthy subjects [34]. Several other data support a potential role of NQO2 in PD and other neurodegenerative diseases [35]. NQO2 may play a role in dopamine metabolism as a catechol quinone reductase [36] and may negatively influence memory formation and the learning process in mice [37]. Next, a chronic OS induced by a parkinsonian toxin paraquat (PQ) is potently inhibited by a specific NQO2 inhibitor S29434/NMDPEF [38] in vitro and in vivo, in rats, following an intranigral as well a systemic injection of PQ [31]. PQ has been shown to inhibit basal autophagy, while the treatment with S29434 stimulated autophagy in astrocytes [30]. Recent data indicate that the function of this enzyme is not limited to generation of OS and inhibition of autophagy, but NQO2 plays a role in flavone-induced autophagy acting as a receptor of pro-autophagic ligands including flavonoids and S29434 [39].

Here, we investigated the toxic effects of 6OHDA and a related role of NQO2 in human and murine astrocytes. We show that 6OHDA potently and dose-dependently inhibited autophagic flux in U373 and primary mouse astrocytes. Even if the contribution of NQO2 to 6OHDA-induced OS appeared less specific compared to PQ, S29434 fully restored and enhanced autophagy in astrocytes, and reduced neuronal damage in DA neurons co-cultured with astrocytes. A possible role of NQO2 in PD pathogenesis is supported by higher expression of NQO2 in majority of PD patients compared to healthy subjects. These data indicate a toxifying function of NQO2 in dopaminergic degeneration via negative regulation of autophagy and neuroprotection in astrocytes, suggesting it as a novel pharmacological target in PD.

Dose-dependent downregulation of autophagy by 6OHDA and stimulation of autophagy by NQO2 inhibition.

Figure 1

To examine the effects of 6OHDA on autophagy in astrocytes, we assessed LC3II levels in U373 cells, cultured under optimal growth conditions (70–80% confluency, 4.5 g/L glucose). LC3 II levels were not significantly modulated at lower 6OHDA concentrations and significantly reduced by 50 and 100 µM toxin already 6 h after the treatment (Fig. 1A,B) as well as at 24 h (Fig. 1C,D). As expected, we observed an increased reduction of LC3II upon 24 h with respect to 6 h (Supplementary Fig. S1).

To assess if the reduction of LC3II in response to 6OHDA was a true and long-lasting inhibition of autophagic flux, we examined the LC3II levels in the presence and absence of the lysosomal inhibitor, chloroquine (ClQ) at 24 h after treatment. In the same experiment U373 cells were co-treated with NQO2 inhibitor S29434 to address whether LC3II modulation by 6OHDA was NQO2-dependent or not. Indeed, 6OHDA (100 mM) had a strong negative effect on the autophagic flux at 24 h (Fig. 1C,D), as well as 50 mM at 48 h (Fig. 1E,F). 25 mM 6OHDA had no significant effect on LC3II induction (Fig. 1E,F). The control samples in the presence of ClQ were several times higher but reduced by more than 50% or unchanged in 6OHDA-treated samples, depending on the dose, thus confirming a suppression of LC3II by higher concentrations of this toxin. Remarkably, the addition of S29434 potently restored LC3II levels in 6OHDA-intoxicated U373 cells compared to cells without S29434 (Fig. 1C-F). This was associated with a significant increase in p62/SQSTM1 levels after 24 h post-treatment (Fig. 1C,G), but not at 6 and 48 h post-treatment (Fig. 1A,E, G). Interestingly, a similar time-dependent pattern of induction was observed for NQO2 (Supplementary Fig. S2).

The effects of 6OHDA on ROS levels

Figure 2

Astrocytes are known for their capacity to counteract OS induced by various sources. Therefore, the observed here lack of autophagy stimulation could depend on the absence of OS in response to 6OHDA. For this reason, we measured the OS induced by 6OHDA. Interestingly, the fluorescein derivative DCF-DA detected a significant OS increase only at higher doses of 6OHDA (Fig. 2A), which effectively suppressed autophagy (Fig. 1B), suggesting that 6OHDA-induced OS inhibits autophagy, similarly to what was demonstrated for PQ-induced OS [30]. Next, we compared PQ- to 6OHDA-induced OS and the impact of S29434 on ROS formation in response to both compounds, as detected by a fluorescent probe MitoSox. This dye monitors the temporal changes of hydroxyradical and superoxide in mitochondria and cytoplasm. 6OHDA (100 µM) induced a time-dependent increase in MitoSox fluorescence, with a remarkable peak of activity 15 h after the treatment (Fig. 2B), much stronger than in case of 100 µM PQ. Co-treatment with S29434 (10 µM) attenuated ROS induction by 6OHDA at each tested time point, but the effect was only partial, ranging from 30–50% reduction, compared to the almost full attenuation of the MitoSox signal in the presence of PQ and S29434 (Fig. 2B). Thus, OS induced by 6OHDA is more potent, but less persistent and less sensitive to S29434 compared to PQ -induced OS. Nevertheless, the findings in Figs. 1 and 2 indicate that 6OHDA-induced toxicity suppresses basal autophagy, which can be equally well restored by S29434 in astrocytes as previously shown for PQ, despite important differences in OS dynamics between both Parkinsonian toxins.

6OHDA effects on cellular and in vitro NQO2 activity

Figure 3

Next, we addressed if 6OHDA can influence cellular NQO2 activity in astroglial cells when added to cell culture. To this end, U373 cells were treated for 24 h with 6OHDA (50 and 100 mM) and cell lysates were assayed for their capacity to reduce menadione (K3) and oxidase BNAH, which are commonly used substrate and co-substrate, respectively, for the detection of NQO2 activity. U373 cell lysates pre-treated with 6OHDA showed higher NQO2 activity than control cells, in the presence of K3, but also when no exogenous substrate was used (Fig. 3A,B). Next, we addressed if 6OHDA, or its autooxidation products, which are rapidly produced in solution [40], might be substrates of quinone oxidoreductase activity of NQO2 in vitro. 6OHDA was added as the only substrate in the reaction mixture containing purified recombinant human NQO2 and BNAH as a co-substrate. 6OHDA dose-dependently tended to accelerate BNAH oxidation, but this effect was not statistically significant when compared to a spontaneous BNAH decay in the presence of the enzyme (without substrate) and more than 50 times lower when compared to NQO2 activity in the presence of K3 (100 mM) used as a positive control (Fig. 3C,D). These data indicate that 6OHDA is not a substrate of NQO2, but 6OHDA dose-dependently induces NQO2 activity in astrocytes, which can be well explained by an increased expression of NQO2 in response to 6OHDA (Supplementary Fig. S2).

U373 astrocytes are resistant to 6OHDA-induced toxicity in optimal culture conditions in dopaminergic neuroblastoma cells.

The negative effect on autophagy in U373 astroglial cells might be a consequence of a general 6OHDA-induced toxicity leading to cell death. Nevertheless, we were able to detect only a background cell death (~ 2%) in optimal culture conditions, which were standard conditions applied in all autophagy experiments presented before, even 3 days after addition of 6OHDA (Fig. 4A). To detect a significant cell mortality in response to 6OHDA, U373 cells had to be exposed to additional stress factors such as low glucose conditions (Fig. 4B) or seeding at very low density (Fig. 4C). Interestingly, S29434 efficiently reduced cell death, also in sub-optimal growth conditions, partially dependent on the presence of the toxin (Fig. 4B,C), with a significant effect at lower concentrations of 6OHDA (25 and 50 µM) in sparsely seeded U373 cells (Fig. 4C).

Figure 4

Importantly, U373 cells overexpressing NQO2, were more sensitive to 6OHDA and showed a higher mortality than U373 when assessed at the low cell density (Supplementary Fig. S3), thus supporting a toxifying effect of NQO2 in cells exposed to 6OHDA. Next, we examined the potential involvement of NQO2 in 6OHDA–induced toxicity in neuroblastoma cells (SH-SY5Y), a commonly used in vitro model for dopaminergic neurons. SH-SY5Y were much more sensitive to 6OHDA compared to U373 cells and were massively dying already 24 h after the addition of 10 µM of 6OHDA. The treatment with S29434 did not prevent cell death in SH-SY5Y cells (Fig. 4D).

Thus, 6OHDA reduces autophagy flux in U373 cells, even though these cells are highly resistant to 6OHDA-induced cell death. This contrasts with SH-SY5Y cells that rapidly die in response to 6OHDA and are not protected by S29434.

Silencing of NQO2 partially restores autophagy in U373 cells exposed to 6OHDA

Next, we determined if NQO2 is a key player in 6OHDA-induced autophagy dysfunction. To answer this question, we silenced NQO2 in 6OHDA-treated U373 cells. Our results indicated a dose-dependent decrease of LC3II levels in cells 24 hours after the treatment with 25–50 µM 6OHDA (Fig. 5A,B). However, NQO2 silencing restored or enhanced autophagy in cells exposed to higher (50 µM) and lower (25 µM) doses of toxin, respectively, although the effect was statistically significant only with 50 µM 6OHDA (Fig. 5B). In fact, a stimulatory effect on LC3II levels was statistically significant in cells expressing lower levels of NQO2 (siNQO2) in response to 25 µM 6OHDA doses when analyzed by less stringent statistical tests (unpaired T test, P = 0.0418; one-way ANOVA and uncorrected Fisher’s LSD test, P = 0.0145). These data confirm the importance of NQO2 in toxin-induced autophagy defect in astrocytes.

Figure 5

Suppression of basal autophagy by 6OHDA in primary murine astrocytes.

To confirm the glioprotective effect of S29434 in astrocytes, we established the primary cultures of cortical astrocytes from C3H mice and treated them with 6OHDA (50 and 100 µM) in the absence and presence of S29434. These concentrations of the parkinsonian toxin were not able to induce any significant cell death (data not shown), but WB analysis revealed that 24 h treatment with 6OHDA inhibited autophagic flux by decreasing LC3II levels in a dose-dependent fashion. This was evident only in the presence of ClQ, since in these experimental conditions (90–100% confluent monolayers), it was more difficult to detect LC3II without pre-incubation with ClQ (Fig. 5C). Of note, S29434 increased LC3II levels in cells exposed to 6OHDA indicating an involvement of NQO2 in the negative regulation of autophagy in murine astrocytes under 6OHDA stress, although it did not induce a statistically significant increase of LC3II levels in control cells (Fig. 5D). This may suggest a lower sensitivity of murine NQO2 or murine autophagy machinery to S29434 compound with respect to human U373 astrocytes.

S29434 enhances the protective effect of U373 cells on dopaminergic SH-SY5Y cells against 6OHDA-induced cell death in co-culture experiments.

Figure 6

The primary role of astrocytes is to protect neurons from oxidative insults. The neuroprotective capacity of astrocytes can be assessed in vitro in human astrocyte–neuron co-cultures. We developed a simple co-culture assay by overlaying U373 astrocyte monolayers with SH-SY5Y cells and scoring viability of respective cell populations in the presence of toxic insults [30]. In this assay, SH-SY5Y cells were labeled with a green-fluorescent dye (DFCA) before mixing the two cell types. Subsequently, the monolayers of naïve U373 or U373 cells overexpressing NQO2 (N-over cells, Fig. 6E) were overlayed with a DFCA-labelled dopaminergic cells. After 24 h, co-cultured cells were exposed to 6OHDA for the next 48 h in presence and absence of S29434 at 10 µM (Fig. 6B). Cell mortality was assessed by flow cytometry with 7-AAD staining (Fig. 6A). We found that dopaminergic SH-SY5Y cells were potently protected by U373 monolayers from 6OHDA-induced cell death, and we could observe SH-SY5Y mortality only at very high doses of 6OHDA (50 and 100 µM), while NQO2-overexp U373 were more sensitive to the neurotoxin and less protective with respect to SH-SY5Y cells (Fig. 6A,C). Importantly, the protective effect of U373 astrocytes was strongly enhanced by S29434, both in normal and in N-over cells (Fig. 6A-C). NQO2 overexpression as well as its inhibition by S29434 in astrocytes exerted a highly significant effect especially for SH-SY5Y cells when analyzed by three-way ANOVA (Fig. 6D). This experiment demonstrates that modulating NQO2 activity and protein levels has a strong impact on dopaminergic neuron survival, when in co-culture with astrocytes, in contrast to monoculture shown in Fig. 4D.

The analysis of NQO2 gene expression in PD patients reveals differences compared to healthy individuals.

So far, our data suggest that high NQO2 levels or activity may increase sensitivity of astrocytes and nearby neurons to OS and parkinsonian toxins. Yet, it is not clear, if high NQO2 levels may predispose to PD or other OS-dependent neurodegenerative diseases in humans, because NQO2 gene expression as well as protein levels have never been analyzed in PD patients extensively. Public gene expression databases are unexplored resources in this respect. For this reason, we analyzed the Gene Expression Ominibus (GEO) datasets of Affymetrix Human Genome arrays. We identified few studies that analyzed gene expression in PD patients of thousands of genes, including NQO2. One study analyzed genome-wide gene expression in the whole blood of early-stage PD patients (n = 50) and compared them to healthy donors (n = 22) [41]. The analysis of GSE6613 datasets revealed a high variability of NQO2 expression in PD patients, but most cases [> 75%] showed higher expression then the mean expression in healthy patients (Fig. 7A). We also observed a tendency to higher NQO2 expression in white blood cells of PD patients in a smaller dataset (GSE100054) comprising 10 cases and 10 controls (Fig. 7B) [42]. Finally, we analyzed the GSE8397 datasets from post-mortem brain specimens of 24 late-stage PD patients and 16 controls [43, 44], available for two probes targeting NQO2 gene region (GSE8397-U133A and U133B). Surprisingly, lateral and medial Substantia nigra (L/M-SN) of PD patients presented a significantly lower expression of NQO2 compared to healthy donors (Fig. 7C), even after excluding very low expression outsiders (p = 0.04, n = 21 vs n = 15). In contrast, the expression levels detected by the second probe, corresponding to long-noncoding RNA (lnRNA) within NQO2 gene were found higher in majority of PD samples (75%) compared to the average lnRNA expression in healthy samples (Fig. 7D). GSE8397 dataset contains also the data from superior frontal gyrus (SFG) but owing to low number of samples (n = 5), it is difficult to draw any conclusions (Fig. 7C,D). These data strongly support the involvement of NQO2 in PD etiology.

Figure 7

Previously, we have reported that prolonged OS induced by PQ leads to an unexpected autophagy inhibition in astrocytes, while S29434, a specific NQO2 inhibitor, protects U373 astrocytes against PQ-induced autophagy defect and cell death [30]. While molecular mechanisms of NQO2 ligand/inhibitor-mediated autophagy remain to be determined, several other questions raised with respect to a toxifying role of NQO2 in glial degeneration. First, it was not clear if a role of NQO2 in OS and autophagy regulation can be generalized to other parkinsonian toxins with a more selective dopaminergic toxicity, such as 6OHDA. Second, little is known regarding the expression levels and mutations of NQO2 in PD, that would support a possible role of this oxidoreductase in PD. Here, we addressed both issues.

6OHDA is a neurotoxin widely used to investigate cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in PD. Similarly to other parkinsonian toxins, this neurotoxicant reproduces the main cellular processes of PD, such as OS, neurodegeneration, neuroinflammation, and neuronal death by apoptosis, although 6OHDA-induced animal PD model does not exhibit some human symptoms such as the presence of Lewy bodies [45]. The mechanism of 6OHDA toxicity has been linked to ROS production by extracellular auto-oxidation [46], leading to intracellular OS induction in various cell types [30, 47]. In agreement with this, we observed that mitochondrial and cytoplasmic ROS increase in a dose- and time-dependent fashion also in U373 cells, but the intensity and duration of the ROS burst was totally different than in case of PQ. In fact, OS induced by 6OHDA was more rapidly increasing and disappearing and several times stronger than in case of PQ. Nevertheless, the effects of both toxins on autophagy were similar and consistent with a concept that persistent and/or high levels of OS inhibit, rather than stimulating autophagy. Indeed, the exposure of U373 astrocytes to increasing 6OHDA concentrations had no effect or significantly downregulated autophagic machinery as evidenced by more than 50% decrease in LC3 lipidation when 100 µM 6OHDA was applied for 24 h. This correlated with a significant increase in p62 levels at 24 h after addition of 6OHDA (Fig. 1C,G), but not at 48 h (Fig. 1H) as previously found for PQ [30]. It is likely, however, that lack of p62 upregulation is due to very low solubility of p62 aggregates that accumulate at later time points and can be detected only by a specific high-detergent lysis. In addition, the exposure of astrocytes, under optimal culture conditions, to different concentrations of 6OHDA led to no or marginal lethality. In contrast, in our previous study we were not able to distinguish between PQ–induced general toxicity and PQ-dependent autophagy dysfunction in astrocytes, since a clear reduction of autophagic flux occurred at 24 and 48 h and preceded a massive cell death that took place at 72 h [30]. Thus, despite evident differences in OS dynamics and cellular toxicity, both parkinsonian toxins efficiently lead to autophagy dysfunction in astrocytes. This observation is fundamental for understanding why these toxins lead to a degenerative process in the brain. In light of our findings, it takes place because the oxidative damage induced by parkinsonian toxins is not mitigated by efficient autophagic response in astrocytes, leading to glial degeneration and poor or no neuroprotection. In fact, we would expect a strong induction of glial autophagy as it was widely reported for dopaminergic neurons in the presence of rotenone, paraquat, MPTP, 6OHDA and other parkinsonian toxins [11]. However, it does not occur in astrocytes, likely due to certain pathways activated by toxins, comprising reported here NQO2 pathway, that counterbalance any proautophagic signals triggered by OS. This concept is well supported not only by our observations in astrocytes [2, 30], but also more recently by other authors in several alternative models of PD based on neurotoxicants [48–52].

Autophagy emerged as a critical mediator of antioxidant responses in both cellular signaling and cellular damage[10]. Its deregulation or impairment by genetic or epigenetic mechanisms is strictly linked to the progression of PD [2, 53] and several reports suggest an autophagy defect in astrocytes in addition to autophagy dysregulation in dopaminergic neurons [18, 54, 55]. Accordingly, autophagy has been implicated in protective cellular responses in astrocytes [52, 56–58]. Thus, it is likely that 6OHDA-induced astrocytes dysfunction is at least partially mediated by autophagy inhibition. Future studies should shed more light on the critical role of autophagy in glial compartment. The important contribution of this work to our understanding of complex mechanisms involved in the etiology of PD, is the identification of NQO2 as a player in the autophagy impairment in astrocytes.

The role of NQO2 in neurodegenerative process is also supported by another finding reported in this work, i.e., the upregulation of protein levels and activity of NQO2 in cells exposed to 6OHDA. This observation is in line with our previous findings obtained with cells exposed to PQ and then assayed in the reactions containing BNAH and K3 [31]. In addition, we showed that cells pre-treated with 6OHDA sustain faster BNAH oxidation in the absence of exogenous substrates, such as K3 (Fig. 3A), suggesting that 6OHDA metabolism leads to the production of quinones that act as substrates for NQO2 in vivo. Such quinones do not seem to be produced in vitro and 6OHDA itself is not a substrate for NQO2, under our experimental conditions (Fig. 3C), but could be produced in vivo. In fact, our previous results obtained with PQ, support this hypothesis, since U373 lysates of cells pre-exposed to PQ in vivo and then assayed for NQO2 activity without exogenous substrates showed higher oxidative capacity for BNAH, although we were not able to detect any increase in NQO2 levels [30]. Future experiments would be required to validate these hypotheses.

Another novel finding of the present work is that NQO2 contributes to 6OHDA-induced ROS induction, but much less than to the autophagy impairment in astrocytes. In fact, S29434, a potent NQO2 inhibitor almost doubled autophagy levels in control cells and fully restored or even upregulated autophagic flux in U373 cells exposed to 6OHDA (Fig. 1D,F). In contrast, S29434 had a moderate effect on OS induced by the toxin, reducing it by around 25% at the peak oxidative burst (Fig. 2B), suggesting that the main beneficial activity of the compound is linked to autophagy stimulation. However, the murine astrocytes were less responsive to S29434, since the compound produced a significant stimulation of autophagy only in 6OHDA-treated cells, as judged by the increase of LC3II in control cells and no effect on the Beclin-1 levels (Fig. 5C,D). Overall, due to the reported specificity of S29434 towards NQO2 [38], these and our previous data strongly suggest that NQO2 is an important modulator of autophagy. The question whether it is due to the enzymatic activity of NQO2 or to the formation of NQO2 complexes with protein partners hypothetically regulated by S29434 and other inhibitors, as suggested previously [39], is not yet clear.

A possible role of NQO2 in PD progression is further supported by another important finding presented here, i.e., the difference in expression levels of NQO2 between PD patients and healthy donors. Higher expression of NQO2 in PD was first suggested by Harada et al [34]. NQO2 in 2001 who described a positive association of a common form of PD with D (deletion) polymorphism in the NQO2 promoter region. This “gain of function” genetic variant of the promoter without Sp3 transcriptional suppressor binding site [59] was 3.46 times more frequent in PD patients than in healthy subjects in Japanese population [34]. Only two attempts to reproduce the findings of Harada’s work have been done so far. Among them one study yielded discordant results [60], while another minor work confirmed them [61]. More recent and extensive studies of NQO2 gene or protein expression in PD patients are missing, but higher NQO2 protein levels were reported in postmortem brain specimens of patients with Alzheimer disease (AD) [62] and an elevated NQO2 expression was convincingly associated with learning deficit in rodent models [37]. The data retrieved from GSE data sets, suggest the opposite in postmortem SN specimens of late-stage PD patients, namely a statistically significant reduction of NQO2 mRNA levels in all analyzed samples. Such, a reduction is further substantiated by a higher expression of lnRNA NQO2, which might repress the expression of regular NQO2 mRNA as other long-noncoding transcripts in colon cancer, where lnRNA NQO2 was found to be significantly modulated [63]. These data are unexpected, but do not exclude that higher NQO2 expression, might be a trigger or risk factor of neurodegenerative changes at early stages of PD. This hypothesis is supported by the evidence coming from the analysis of NQO2 expression in whole blood of 50 early PD cases, which revealed that majority (39/50 (~ 75%) of early PD patients present significantly higher levels of NQO2 then its mean expression in healthy individuals. We do not know if the levels of blood born transcripts predict the brain expression of a given gene, but a higher NQO2 expression in the blood may indicate a more active promoter. According to our analysis, the higher NQO2 expression is 3 times more frequent in PD patients than in controls, thus well in line with the observations of the study by Harada et al [34].

In conclusion, our observations suggest that drugs targeting NQO2 may stimulate autophagy as well as protect against dopamine quinone and hydroxyquinone toxicity. Thus, S29434 by acting as a protective agent on astrocytes, might delay neuronal damages in PD and other pathological conditions concerning OS and autophagy impairment mediated by NQO2, suggesting that NQO2 inhibitors might have a therapeutic potential for the treatment of cerebral injuries involving oxidative neurodegeneration. The vast literature suggesting a protective effect of several natural polyphenols in neurodegeneration strongly supports this idea, since NQO2 is a well-documented target of these compounds [39, 64]. Future studies should validate this hypothesis.

Reagents and antibodies

Unless otherwise specified, all reagents were purchased from Sigma-Aldrich/MERCK (Darmstadt, Germany): 6OHDA (H4381, 100 mM stock in bi-distilled H2O (H2Ob)), paraquat (M2254; 100 mM stock in H2O), chloroquine (ClQ, C6628; 25 mM stock in H2Ob). Except for 6OHDA that was freshly prepared for each experiment and stored at -80°C for another use within one week, other reagents were kept in aliquots at − 80°C for longer times and thawed shortly before each treatment. S29434, previously well characterized [38], and used under the name of NMDPEF [30, 31] was stored as a 10 mM stock in DMSO (D4540) at − 80°C. The antibodies (Abs) used for Western Blotting (WB) were: polyclonal rabbit anti-LC3A/B (Abcam, ab1280025 used 1:2000), anti-p62 (MBL, PM045 used 1:1000), anti-GAPDH, clone FL335 (Santa Cruz Biotech., sc-25778 used 1:200), anti-NQO2 (ProteinTech, 15767-1-AP used 1:1000) and monoclonal mouse anti-α-Tubulin (Sigma-Aldrich, T6074 used 1:500).

Cell culture

Astrocytoma U373 cells were a kind gift of M. Pollicita e S. Aquaro, Tor Vergata University, Rome. Cells after thawing were expanded in high glucose (4,5% glucose) Dulbecco’s modified Eagle’s medium (DMEM) (Carlo Erba srl, Milan, Italy, FA30WL0101500) and then routinely cultured in the same medium or gradually switched to low-glucose (1% glucose) DMEM (FA30WL0060500) when indicated. Each type of DMEM was supplemented with 10% Foetal Bovine serum (FBS) (Life Technologies, Monza MB, Italy, 10500-064), 1:100 Penicillin-Streptomycin (Pen Strep, Life Technologies, 15070- 063), and 1:100 L-Glutamine (PAA Laboratories GmbH, Austria, M00409- 2722) to constitute a standard medium and cells were cultured under standard 5% CO2 conditions. If not otherwise specified for most experiments, U373 cells were seeded 2 days before or 3 days before the treatments at the density 32 x 10³ or 16 x 10³ x cm^− 2 (300 or 150 x 10³ per 3.5 cm plates), respectively and the last medium change was performed 24 h before the treatment with a desired compound. For longer treatments (48 h and 72 h), 6OHDA was re-added every 24 h and medium change was performed every 48 h. ClQ was added 2 or 3 h before the cell lysis as indicated. The stressful culture conditions were generated by switching the cells cultured for one week in high-glucose to low-glucose or by seeding them at low density (6,4 x 10³ x cm^− 2) for 2 days before treatments. Neuroblastoma SHSY5Y cells were cultured in F-12K medium plus high glucose DMEM, supplemented with 10% FBS, Pen Strep and L-Glutamine as standard medium.

Western Blotting (WB)

See Supplementary Methods

ROS detection in live cells

ROS were detected by two alternative ROS fluorescent indicators: a dichlorofluorescein (DCF) derivative, namely 5(6)-carboxy-2′,7′ dichlorofluorescein diacetate (CA-DCF-DA; Sigma-Aldrich, 21884) or MitoSOX (3,8-phenanthridinediamine, 5-(6′-triphenylphosphoniumhexyl)-5,6 dihydro-6- phenyl). The first method detected cytoplasmic ROS in live cells attached to cultured dish by fluorimeter, while the second method detected ROS in trypsin-detached cells by FACS analysis. Both methods were performed as previously described [30], with minor modifications. Briefly, U373 cells were plated at 1,2 × 10⁴ cells/per well on 24 well plates in low-glucose DMEM standard medium, 2 days before treatment. Mitosox was used at the concentration 5 µM in RPMI 1640 w/o Phenol Red.

NQO2 Activity Measurements

NQO2 enzymatic activity assays were performed as described previously [39] with the following modifications. The cellular NQO2 activity was measured in U373 cell lysates. For this purpose, the cells were cultured for two days and when sub-confluent treated 6OHDA for 24 h and then scraped and homogenized in ice-cold GPS buffer (50 mM Tris–HCl, 3 mM n-octyl-d-glucopyranoside pH 7.5) with 10 strokes of tight glass douncer on ice. After clearing by centrifugation, the lysates were stored at – 80°C. After thawing, the lysate (5 mL containing 8 to 15 mg protein) were aliquoted on 96-well plates placed on ice and then equilibrated for 10 min at RT. The reaction was initiated by the addition of GPS buffer supplemented with 50 mM of the co-substrate 1-benzyl-1,4- dihydro-nicotinamide (BNAH; Santa Cruz Biotech., 208609) with or without 50 mM menadione (K3) (Supelco, Merk Life Science S.r.l., Milan, Italy) as substrate in a total volume of 200 mL. Testing of 6OHDA as NQO2 substrate was performed with 50 or 100 ng/mL of human recombinant NQO2 (Sigma-Aldrich, Q0380) in GPS buffer plus 100 mM BNAH and different concentrations of 6OHDA or 100 mM K3 as a positive control in a total volume of 200 mL. BNAH supplemented with an appropriate amount of NQO2 and vehicle instead of 6OHDA was used as a negative control. The reaction was initiated by the addition of the recombinant enzyme to each column of 96-well plates. The exact time of pipetting the enzyme (time 0) was taken to calculate the delay between the fluorescence reading and the reaction start. BNAH fluorescence was monitored by 1420 Multilabel reader Victor 2 fluorimeter (Perkin Elmer) for 15 to 20 min. Data were analyzed by Microsoft Excel and GraphPad Prism 9.0 software (GraphPad, San Diego, CA, USA) for fitting the decay curves and finding unknown values by curve interpolation. The velocity (V) of the reaction was determined from the fluorescence change as the mean reduction of mmol of BNAH (in 200 mL) per minute for the first 5 min after the addition of the enzyme. The activity was calculated as V divided by the enzyme/protein amount added to the reaction.

Preparation of NQO2-overexpresing (N-over) U373 cells

See Supplementary Methods

Flow cytometry (FACS) analysis of cell viability

See Supplementary Methods

Co-culture of astroglial and dopaminergic cells

U373 cells and U373 N-over cells were seeded and cultured on 24-well plates in high-glucose DMEM. Briefly, U373 cells plated at a density of 40 X 10³ cm^− 2 while U373 Over cells were plated at the density between 30 x 10³ cm^− 2 under standard conditions to achieve 90% confluent monolayer after 2 days. Before plating SH-SY5Y cells were labelled with CellTracker Green CMFDA (chloromethylfluorescein diacetate, Molecular Probes, Life Technologies, U.S.A.) dye. Briefly, SHSY5Y cells, grown to 80–90% confluency, were labelled with 5 µM CMFDA in PBS for 15 min. Cells were washed once in PBS and fresh regular medium was added for 30 min. At the end, the cells were trypsinized (Trypsin-EDTA (0.05%), Gibco, Life Technologies, U.S.A.), and resuspended in fresh culture medium to a desired cell concentration. Next, 5 x 10⁴ cm^− 2 SH-SY5Y cells were seeded over U373 monolayers. After 24 h the co-cultures were treated for 48h with 6OHDA and/or S29434. At the end of the treatment the cells were washed once in PBS (Carlo Erba srl, FA30WL0615500), trypsinized for 3 min and collected with 0,5 ml in F12-K medium plus high - glucose supplemented with 10% FBS in flow cytometry round tubes. The cells were then pelleted by centrifugation for 5 min at 1000 x g and resuspended in 400 µL PBS containing 1% FBS and EDTA 0.5mM. Cell mortality was determined by 7-AAD (BD, 344563; used 1:10) added 2–3 min before FACS analysis.

U373 cells transfection and silencing NQO2

U373 cells cultured in high glucose standard medium were seeded on 6-well plates one day before transfection at the density 4 x 10⁴ cm^− 2 cells. Non-silencing control siRNA, [siEGFP, (EHUEGFP)] and a set of human NQO2-targeting esiRNA (EMU042521) were purchased from Merk, Sigma-Aldrich. Transfection of U373 cells was performed using Transfection Reagent (Merk, S1452) and Opti-MEM (Life Technologies, 51985026) according to the manufacturer's instructions. Briefly, siRNA (150 nM) and Transfection Reagent (1:1 ml/pMol siRNA) were diluted in Opti-MEM and incubated for 15–20 min at RT. Cells were incubated in Opti-MEM containing siRNA-Transfection Reagent complexes for 4 h and then incubated in culture medium containing high glucose w/o PenStrep. After 20 h the medium was exchanged for a regular high-glucose medium. The treatments were initiated 48 h after transfection and carried for the next 24 h. ClQ 25µM was added 3 h before the end of the experiment. After the treatments the cells were lysed and assayed by WB.

Animal procedures

Mice used for astrocyte isolation were WT pups of C3H/HeOuJ strain. One or two day-old (P1/P2) pups were euthanized by head decapitation. The heads were placed into sterile PBS at RT and meninges were carefully removed. The animal procedures were carried according to EU Directive 2010/63/EU and approved by Italian Ministry of Health by the project authorization Nr. 613-2017-PR. This study complied with ARRIVE guidelines (https://arriveguidelines.org/).

Isolation and Culture of Mouse Cortical Astrocytes

Primary astrocytes were isolated from cerebral cortices prepared as described above and according to a previously published protocol [30]. Briefly, the cortices were transferred to new dishes with sterile PBS and then dissected, minced, and then mechanically dissociated. Minced cortices were centrifuged (800 g, 8 min, 4°C) and the pellet was digested in DMEM containing 10 mg/mL type I DNase for 15 min on a benchtop orbital shaker (100 RPM) at RT. The reaction was stopped by adding ice-cold DMEM and the homogenate was pelleted by centrifugation (800 g, 8 min, 4°C). The cells were plated in 10 cm dish (1 brain/dish) in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco), 1% P/S (Gibco). The next day medium was changed to remove non-adherent cells. Thereafter, the medium was changed every 2 days. The cultures were maintained for approximately 1 week or until 90% confluent at 37°C and 5% CO₂. Cells were then removed from the dish with the addition of trypsin-EDTA 0.05% (Invitrogen) and re-plated on 6-well plates for experiments at the at the density of 500,000 cells per well. The treatments were performed one day after seeding.

Phase – contrast and fluorescence microscope

Phase – contrast and fluorescence images were acquired with LEICA DMI 4000 B fluorescent microscope, equipped with LEICA DFC 350 FX camera.

Microarray data information and Gene Expression Omnibus (GEO) datasets analysis

See Supplementary Methods

Data analysis and statistical procedures

Statistical analysis of all the experiments was expressed as means ± SEM for the number n of independent experiments or as means ± SD for the number n of independent samples, from a representative experiment, as indicated for each figure. Numerical data from all the experiments were evaluated by Analysis of Variance (ANOVA). If Brown-Forsythe’s test for equality of variances was positive Welch’s ANOVA or Kruskal-Wallis test was performed, otherwise Ordinary One-way ANOVA or occasionally three-way ANOVA were performed. Multiple comparisons between groups were performed by one of suggested post-tests according to GraphPad PRISM 9.0 software guidelines http://www.graphpad.com/ or occasionally by unpaired t test as indicated in figure legends.

Data availability statement

The original images of blots presented in this work are displayed in the Supplementary Information. All the other datasets generated and analyzed during the current study are available from the corresponding author upon request.

Acknowledgments

Special thanks to thank prof. Maria Cristina Caroleo, University Magna Graecia (UMG), Catanzaro for critical reading of the manuscript and useful suggestions. We also thank prof. Walter Agosti, for help with flow cytometry analysis and dr. Concetta Riillo, UMG, Catanzaro, Italy, for technical support with cell culture and sample preparation. Finally, we acknowledge prof. Stefano Aquaro and dr Michela Pollicita, University Tor Vergata, Rome and Rossella Russo, University of Calabria, Rende (Cs), Italy for the kind gift of U373 cells and neuroblastoma SHSK5Y cells, respectively.

Funding

This research was funded by Basic Research Activity Fund (FABR) 2017 grant and PhD program of Italian Ministry of Education, University and Research (M.I.U.R) and PON03PE 00078 grant (NUTRAMED Consortium) awarded to E.J. and V.M. respectively.

Author Contributions Statement

E.J. designed all experiments, supervised the work, performed some experiments, analyzed the data, wrote the main manuscript text and prepared all the figures, M.P. performed and analyzed the experiments and contributed to writing the manuscript, Concetta Martino performed experiments, J.N.G.W. analyzed the data and prepared figures, V.M. provided the fundings, J.B. and K.R. provided reagents, suggested and analyzed experiments and contributed to writing the manuscript. All authors reviewed the manuscript.

Ethics declaration

The authors declare no competing interests.

The authors declare that the current study complied with ARRIVE guidelines (https://arriveguidelines.org/).

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No competing interests reported.

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Autophagy and neuroprotection in astrocytes exposed to 6-hydroxydopamine is negatively regulated by NQO2: a potential novel target in Parkinson’s disease.

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Abstract

Figures

Introduction

Results

The effects of 6OHDA on ROS levels

Silencing of NQO2 partially restores autophagy in U373 cells exposed to 6OHDA

Discussion

Methods

Reagents and antibodies

Cell culture

Western Blotting (WB)

ROS detection in live cells

NQO2 Activity Measurements

Preparation of NQO2-overexpresing (N-over) U373 cells

Flow cytometry (FACS) analysis of cell viability

Co-culture of astroglial and dopaminergic cells

U373 cells transfection and silencing NQO2

Animal procedures

Isolation and Culture of Mouse Cortical Astrocytes

Phase – contrast and fluorescence microscope

Microarray data information and Gene Expression Omnibus (GEO) datasets analysis

Data analysis and statistical procedures

Declarations

References

Additional Declarations

Supplementary Files

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