ABSTRACT 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

the 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 cells and primary murine astrocytes in the absence of cell death.

LC3II downregulation was observed 6 to 48 h after treatment. Interestingly, 6OHDA enhanced NRH:quinone oxidoreductase 2 (NQO2) expression and activity in U373 cells, even if 6OHDA turned

with astrocytes. The toxic effects of 6OHDA on autophagy were attenuated by silencing NQO2 in human cells and primary astrocytes from _NQO2_−/− mice. Finally, the analysis of Gene

Expression Omnibus 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 potential pharmacological target in PD. SIMILAR CONTENT BEING VIEWED BY OTHERS MITOCHONDRIAL GLUTAMINE

TRANSPORTER SLC1A5_VAR, A POTENTIAL TARGET TO SUPPRESS ASTROCYTE REACTIVITY IN PARKINSON’S DISEASE Article Open access 09 November 2022 METABOLIC ALTERATIONS IN PARKINSON’S DISEASE

ASTROCYTES Article Open access 02 September 2020 CONNEXIN 43 IS DOWNREGULATED IN ADVANCED PARKINSON’S DISEASE IN MULTIPLE BRAIN REGIONS WHICH CORRELATES WITH SYMPTOMS Article Open access 25

March 2025 INTRODUCTION PD is a neurodegenerative disorder mainly characterized by progressive loss of dopaminergic neurons in the _substantia nigra_. The exact mechanisms of dopaminergic

neuronal loss in the SN are not fully understood, but several factors have been implicated in the onset and progression of PD. Among them are mitochondrial damage and oxidative stress (OS)

caused by alterations in iron and redox metabolism and/or excessive production of toxic dopamine metabolites, followed by protein misfolding and aggregation associated with defective

autophagy and “prion-like protein infection”1,2,3,4. Autophagy is a catabolic process responsible for the removal of protein aggregates and damaged organelles via lysosomal digestion5,6.

Autophagy machinery is also involved in the regulation of related processes with an increasingly recognized role in PD, such as vesicular transport, exocytosis and phagocytosis5,7. Numerous

genetic mutations encoding components of the autophagic machinery have been identified as disease risk factors1,8,9. Notably, autophagy dysregulation is also associated with reactive oxygen

species (ROS) in both cellular signaling and damage10. In particular, all chemical agents inducing ROS and dopaminergic damage, known as Parkinsonian toxins, have been shown to dysregulate

or impair autophagy1,11 in both neurons and astrocytes. Astrocytes maintain redox balance in the brain through antioxidant production and detoxification12, contribute to the formation and

maintenance of the blood–brain barrier13 and regulate inflammatory responses in the central nervous system14,15. Increasing evidence highlights the contribution of astrocyte dysfunction to

the pathogenesis of several neurodegenerative conditions, including PD16. DA metabolism is the main source of ROS in the brain17. Functional glial cells protect neurons against OS by

metabolizing DA via monoamine oxidase-B (MAO-B) and catechol-_O_-methyltransferase (COMT) and by a battery of antioxidant enzymes present in astrocytes18. 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 PD16,19. 6OHDA is

widely used to investigate the pathogenesis of PD in preclinical studies since it induces PD-like symptoms in rodents20. 6OHDA is a DA analog that can be produced by DA hydroxylation in the

presence of Fe2+ iron and H2O2. 6OHDA is considered to be a purely synthetic toxin, but several observations suggest the possibility of its endogenous origin, likely during DA catabolism.

Indeed, it has been detected in caudate samples as well as in the urine of PD patients21,22 and in mouse brains following long-term L-dopa administration23. It shows specific PD-like

effects24,25 due to its toxic oxidation metabolites. Upon injection in the brain, it causes selective death of dopaminergic neurons and proinflammatory activation of microglia, while its

effects on the astrocytic compartment are a matter of debate26,27. The mechanisms of 6OHDA toxicity are complex and involve rapid auto-oxidation, leading to the generation of hydrogen

peroxide and hydroxyl radicals28 and impairment of mitochondrial energy production29,30. It is also unclear whether any endogenous toxifying enzymes, such as quinone oxidoreductase 2

(NQO2/QR2), may contribute to this process31,32. NQO2 is a cytosolic and ubiquitously expressed flavoprotein that catalyzes the two-electron reduction of quinone to hydroquinone33. NQO2,

structurally related to NQO1, is considered a phase II detoxifying enzyme, but it enhances the production of activated quinones and ROS in the presence of certain substrates33,34. NQO2

emerged as a possible target in PD more than two decades ago. A case–control Japanese study identified a positive association of a nonfamiliar form of PD with a D (deletion) polymorphism in

the NQO2 promoter region. This “gain of function” variant of the promoter lacking the Sp3 transcriptional suppressor binding site was 3.46 times more frequent in PD patients than in healthy

subjects35. Several other data support a potential role of NQO2 in PD and other neurodegenerative diseases36. NQO2 may play a role in dopamine metabolism as a catechol quinone reductase37

and may negatively influence memory formation and learning processes in mice38. Next, chronic OS induced by the parkinsonian toxin paraquat (PQ) is potently inhibited by the specific NQO2

inhibitor S29434/NMDPEF 39 in vitro and in vivo in rats following intranigral and systemic injection of PQ32. PQ has been shown to inhibit basal autophagy, while treatment with S29434

stimulated autophagy in astrocytes31. Recent data indicate that the function of this enzyme is not limited to the generation of OS and inhibition of autophagy, but NQO2 plays a role in

flavone-induced autophagy by acting as a receptor of proautophagic ligands, including flavonoids and S2943440. Here, we investigated the toxic effects of 6OHDA and the 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, but S29434 restored and enhanced autophagy in

astrocytes and reduced neuronal damage in DA neurons cocultured with astrocytes. A possible role of NQO2 in PD pathogenesis is supported by the higher expression of NQO2 in the majority of

PD patients when 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 potential pharmacological target in PD. RESULTS DOSE-DEPENDENT DOWNREGULATION OF AUTOPHAGY BY 6OHDA AND STIMULATION OF AUTOPHAGY BY NQO2 INHIBITION 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 were significantly reduced by 50 and 100 µM toxin 6 h after the treatment (Fig. 1A,B) as well as at 24 h (Fig. 1C,D). As expected,

we observed an increased reduction in LC3II at 24 h with respect to 6 h (Supplementary Fig. S1). To assess whether the reduction in 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 cotreated with the NQO2 inhibitor S29434 to address whether LC3II modulation by 6OHDA was NQO2-dependent. Indeed, 6OHDA (100 mM) had a strong negative effect on autophagic flux at

24 h (Fig. 1C,D), as well as 50 mM at 48 h (Fig. 1E,F). Treatment with 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 (Fig. 1C,G) but not at 6 and 48 h posttreatment (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 Astrocytes are known for their capacity to counteract OS induced by various sources. Therefore, the lack of autophagy stimulation

observed here 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, similar to what was

demonstrated for PQ-induced OS31. Next, we compared PQ- to 6OHDA-induced OS and the impact of S29434 on ROS formation in response to both compounds, as detected by the fluorescent probe

MitoSox. This dye monitors the temporal changes in hydroxyradical and superoxide anions in the mitochondria and cytoplasm. 6OHDA (100 µM) induced a time-dependent increase in MitoSox

fluorescence, with a remarkable peak of activity 15 h posttreatment (Fig. 2B), much stronger than in the case of 100 µM PQ. Cotreatment with S29434 (10 µM) attenuated ROS induction by 6OHDA

at each tested time point, but the effect was only partial, ranging from a 30% to 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 than 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 Next, we addressed whether 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 substrates and cosubstrates, respectively, for the detection of NQO2 activity. U373 cell lysates pretreated 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 whether 6OHDA or its autooxidation products, which are rapidly produced in solution41, might be

substrates of the quinone oxidoreductase activity in vitro. 6OHDA was added as the only substrate in the reaction mixture containing purified recombinant human NQO2 and BNAH as a

cosubstrate. 6OHDA dose-dependently tended to accelerate BNAH oxidation, but this effect was not statistically significant when compared to 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), which was 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 general 6OHDA-induced toxicity leading to cell death. Nevertheless, we were able to detect only background cell death (≈2%) in optimal culture

conditions, which were standard conditions applied in all autophagy experiments (Fig. 4A). To detect significant cell mortality in response to 6OHDA, U373 cells had to be exposed to

additional stress factors, such as incubation in low glucose after high-glucose medium (Fig. 4B) or seeding at very low density (Fig. 4C). Interestingly, S29434 efficiently reduced cell

death, also in suboptimal 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). Importantly, U373 cells overexpressing NQO2, were more sensitive to 6OHDA and showed higher mortality than U373 cells when assessed at a 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 cells were much more sensitive to 6OHDA than U373 cells and were massively dying 24 h after

the addition of 10 µM 6OHDA. Treatment with S29434 did not prevent cell death in SH-SY5Y cells (Fig. 4D). Thus, 6OHDA reduces autophagic 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. SUPPRESSION OF BASAL AUTOPHAGY BY 6OHDA

IN U373 CELLS AND PRIMARY MURINE ASTROCYTES IS DEPENDENT ON NQO2 STATUS Next, we determined whether 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 in LC3II levels in cells 24 h after 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 (si_NQO2_) 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). In conclusion, the silencing of NQO2 partially

restores autophagy in cells exposed to 6OHDA. These data support the importance of NQO2 in toxin-induced autophagy defects in human astrocytes. To confirm the glioprotective effect of S29434

in astrocytes, we established 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 preincubation with ClQ (Fig. 5C). Of note, S29434 increased LC3II levels in cells exposed to 6OHDA, indicating the involvement of NQO2 in the negative regulation of

autophagy in murine astrocytes under 6OHDA stress, although it did not induce a statistically significant increase in LC3II levels in control cells (Fig. 5D). To further assess the role of

NQO2 in autophagic flux regulation upon 6OHDA intoxication, we analyzed LC3II levels in primary cortical astrocytes from mice lacking NQO2 (_NQO2-/-_) and compared them with their

_wild-type_ (_WT_) counterparts. These cells were plated and analyzed under the standard protocol, but another anti-LC3 antibody was employed, allowing LC3II detection w/o ClQ. The analysis

of LC3 expression upon 24 h of treatment with 6OHDA revealed a downregulation of LC3II only in _WT_ but not in KO astrocytes (Fig. 5E), and the difference was statistically significant in

the presence of ClQ (Fig. 5F). The absence of NQO2 also attenuated the upregulation of p62 in response to 6OHDA (Fig. 5E,G), which suggests a role of NQO2 in the regulation of autophagy in

astrocytes in response to oxidative insults. S29434 ENHANCES THE PROTECTIVE EFFECT OF U373 CELLS ON DOPAMINERGIC SH-SY5Y CELLS AGAINST 6OHDA-INDUCED CELL DEATH IN COCULTURE EXPERIMENTS The

primary role of astrocytes is to protect neurons from oxidative insults. Their neuroprotective capacity can be assessed in vitro in human astrocyte-neuron cocultures. We developed a simple

coculture assay by overlaying U373 astrocyte monolayers with SH-SY5Y cells and scoring the viability of the respective cell populations in the presence of toxic insults31. 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 overlaid with a DFCA-labeled dopaminergic cells. After 24 h, cocultured cells were exposed to 6OHDA for the next 48 h in the presence or absence of 10 µM S29434 (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 N-over U373 cells 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 cells 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). As expected, the extent of the

protection by S29434, calculated after subtracting basal mortality of SH-SY5Y cells in coculture with normal U373 cells (around 3 to 5%), was dependent on the concentration of 6OHDA and was

around 44% at 100 µM, but reached full protection at lower doses, such as 10 µM (Fig. S5A and S5B). These experiments demonstrate that modulating NQO2 activity and protein levels has a

strong impact on dopaminergic neuron survival when in coculture with astrocytes, in contrast to monoculture, as shown in Fig. 4D. THE ANALYSIS OF NQO2 GENE EXPRESSION IN PD PATIENTS REVEALS

DIFFERENCES COMPARED TO HEALTHY INDIVIDUALS Thus far, our data suggest that high NQO2 levels or activity may increase the sensitivity of astrocytes and nearby neurons to OS and parkinsonian

toxins. However, it is not clear whether high NQO2 levels may predispose patients to PD or other OS-dependent neurodegenerative diseases because _NQO2_ gene expression and protein levels

have never been analyzed in PD patients. Public gene expression databases are unexplored resources in this respect. We analyzed the Gene Expression Omnibus (GEO) datasets of Affymetrix Human

Genome arrays. We identified a 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)42. The analysis of GSE6613 datasets revealed a high variability and nonnormal distribution of _NQO2_

expression in early PD patients, but most cases [73%] showed higher expression than the central tendency in healthy patients, which was statistically significant with and without two

potential outliers (Fig. 7A). We also observed a higher median _NQO2_ expression in white blood cells of PD patients in a smaller dataset (GSE100054) comprising 10 cases and 9 controls, but

it was not significant (Fig. 7B)43. Finally, we analyzed the GSE8397 datasets from postmortem brain specimens of 24 late-stage PD patients and 16 controls44,45 available for two probes

targeting the _NQO2_ gene (GSE8397-U133A and U133B). Surprisingly, lateral and media _substantia nigra_ (L/M-SN) from PD patients presented significantly lower expression of _NQO2_ than that

from healthy donors, even after excluding three very low expression outliers (p = 0.04, n = 21 vs n = 15). In this case, 71% of PD cases showed lower NQO2 expression compared to the mean of

control cases (Fig. 7C). In contrast, the expression levels detected by the second probe, corresponding to long noncoding RNA (lncRNA) of _NQO2_ (_lncNQO2_), showed no significant increase

in PD samples compared to the mean _lncNQO2_ expression levels in healthy samples (Fig. 7D). The GSE8397 dataset also contains the data from the _superior frontal gyrus_ (SFG), but owing to

the low number of samples (n = 5), it is difficult to draw any conclusions (Fig. 7C,D). These data strongly support a possible involvement of NQO2 in PD etiology. DISCUSSION The important

finding of the present work is the identification of NQO2 as a player in autophagy and in the impairment of neuroprotective capacity in astrocytes induced by 6OHDA. This compound is a model

dopaminergic neurotoxicant, but may also represent toxic endogenous dopamine metabolites20,23. Similar to other parkinsonian toxins, 6OHDA reproduces the main cellular processes of PD , such

as OS, neurodegeneration, neuroinflammation, and neuronal death by apoptosis, although it is controversial whether 6OHDA-induced animal PD models accumulate a-synuclein aggregates similar

to Lewy bodies, typical features of human PD46. The mechanism of 6OHDA toxicity has been linked to ROS production by extracellular auto-oxidation47, leading to intracellular OS induction in

various cell types31,48. In agreement with this, we observed that mitochondrial and cytoplasmic ROS increased in a dose- and time-dependent fashion in U373 cells, but the intensity and

duration of the ROS burst were totally different than in the case of PQ. In fact, OS induced by 6OHDA was more rapidly increasing and disappearing and several times stronger than that

induced by PQ. Nevertheless, the effects of both neurotoxins on autophagy were similar and consistent with the concept that persistent and/or high levels of OS inhibit rather than stimulate

autophagy. Indeed, the exposure of U373 astrocytes to increasing 6OHDA doses had no effect or significantly downregulated autophagic machinery as evidenced by a 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 the addition of 6OHDA (Fig. 1C,G) but not at 48 h (Fig. 1H), as

previously found for PQ31. It is likely, however, that the lack of p62 upregulation is due to the very low solubility of p62 aggregates that accumulate at later time points. In addition, the

exposure of astrocytes to different concentrations of 6OHDA led to no or marginal lethality under optimal culture conditions. 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 in autophagic flux preceded massive cell death that took place

at 72 h31. Thus, despite evident differences in OS dynamics and cellular toxicity, both toxins efficiently lead to autophagy dysfunction in astrocytes. In contrast to the data presented

here, a net induction of autophagy was widely reported for dopaminergic neurons in vitro in response to 6OHDA, MPTP, and other parkinsonian toxins11. However, this apparent autophagy

stimulation in vitro by 6OHDA is not sufficient since many potent autophagy inducers protect dopaminergic neurons from cell death1,49,50. A net induction of autophagy does not occur in

astrocytes, likely because certain inhibitory pathways activated by toxins, comprising the NQO2 pathway reported here, counterbalance any proautophagic signals triggered by OS. This concept

is well supported not only by our observations in astrocytes1,31 but also by other authors in several alternative models of PD based on neurotoxicants51,52,53,54,55. However, robust evidence

of net autophagic flux modulation by 6OHDA or other dopamine metabolites in _vivo_ is missing. Only one paper that investigated 6OHDA effects on autophagy in rodents reported LC3II and p62

upregulation in lesioned SN samples, which would suggest inhibition of autophagic flux, but these findings were relative to 4 weeks upon treatment and were not confirmed by cotreatment with

lysosomotropic agents50. Importantly, all authors agree that pharmacological stimulation of autophagy by Torin1, curcumin or NRF2 inducers in 6OHDA-treated rodents correlates with

neuroprotective effects49,50,55, suggesting that 6OHDA causes defective rather than excessive autophagy. When autophagic flux is reduced by decreased LC3II production, autophagosome

transport blockade and/or defective lysosomal function, specialized forms of autophagy, such as mitophagy are also impaired, leading to the accumulation of dysfunctional mitochondria.

Autophagy and mitophagy impairment by genetic or epigenetic mechanisms is strictly linked to the progression of PD1,9, and recent reports suggest that a possible autophagy/mitophagy defect

may reside in astrocytes19,56. With emerging evidence demonstrating a broader role of glial mitochondria in detoxification processes57 and astrocyte dysfunction in PD pathogenesis16,19, it

is likely that parkinsonian toxins, such as 6OHDA and PQ, damage mitochondria and impair glial mitophagy at the same time, leading to the impairment of their neuroprotective capacity, which

heavily relies on mitochondrial function in astrocytes57,58. Therefore, it is possible that mitochondria are involved in the neuroprotective effect of the NQO2 inhibitor presented in this

work (Fig. 6), even though NQO2 is a cytoplasmic enzyme33, and preliminary experiments exclude its mitochondrial localization (E. Janda personal communication). Future studies should shed

more light on a possible role of NQO2 in mitophagy and mitochondrial function in the glial compartment. A potential role of NQO2 in the neurodegenerative process is also supported by its

upregulation of both protein levels and activity in cells exposed to 6OHDA (Fig. 3; Fig. S2). This observation is in line with our previous findings obtained with cells exposed to PQ and

then assayed in reactions containing BNAH and K332. In addition, we showed that cells pretreated with 6OHDA sustained 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 are not produced in vitro and 6OHDA itself is not a

substrate for NQO2 (Fig. 3C) but could be produced in cells_._ Our previous results obtained with PQ also support this hypothesis31, but futher experiments are required to validate it.

Another novel finding of the present work is that NQO2 contributes much less to 6OHDA-induced ROS induction than to autophagy impairment in astrocytes. In fact, S29434 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 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 (Fig. 5C,D). Nevertheless, the experiments with

_NQO2_ − / − primary astrocytes provide key evidence for an important role of NQO2 in the negative regulation of LC3II induction. In contrast to _WT_ cells, astrocytes lacking NQO2 were

resistant to 6OHDA-induced LC3II downregulation, and the upregulation of p62 was significantly reduced in these cells, indicating that autophagic flux was less impaired by 6OHDA in the

absence of NQO2. In addition, we observed a statistically significant reduction in LC3II levels and lower p62 expression in knockout cells, indicating differences in autophagic machinery

between _WT_ and _NQO2_-deficient astrocytes. To our knowledge, this is the first analysis of autophagic marker expression in _NQO2_ − / − cells. Overall, these and our previous data as well

as a recent work reporting that negative regulation of autophagy by fluoride is NQO2-dependent59 strongly suggest that NQO2 is an important modulator of autophagy. The question of whether

it is mediated by its enzymatic activity or by the formation of NQO2 complexes with its protein partners hypothetically regulated by S29434 and its other inhibitors, as suggested

previously39, needs to be further investigated. Another important issue is the relevance of our in vitro model to complex pathogenic mechanisms in PD. The causative role of toxic dopamine

metabolites in PD pathogenesis is well supported by several observations60, but robust evidence of autophagic flux inhibition by 6OHDA or other dopamine metabolites in vivo is missing in the

literature and in the present paper. In addition, the coculture of U373 cells with SH-SY5Y cells provides a proof of concept that the toxicity of 6OHDA to neurons can be attenuated by

modulating NQO2 levels and activity in astrocytes, but it is far from modelling complex interactions that take place _in_ vivo. Thus, to address the NQO2 role in PD, it would be necessary to

examine first 6OHDA effects on autophagy in vivo and then to verify the protective effects of NQO2 inhibitors. On the other hand, induced pluripotent stem cell-derived organoids of

dopaminergic neurons and astrocytes would be a better model to study the role of NQO2 in neuroprotective capacity of astrocytes61. In spite of these weak points, our study provides important

clinical evidence supporting 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.35, who described a positive association of a common form of PD with the D

(deletion) polymorphism in the _NQO2_ promoter region. This “gain of function” genetic variant of the promoter lacking the Sp3 transcriptional suppressor binding site62 was 3.46 times more

frequent in PD patients than in healthy subjects in the Japanese population35. Only two attempts to reproduce the findings of Harada’s work have been done so far, and one was discordant63,

while another minor work confirmed them64. 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’s disease (AD)65, and elevated NQO2 expression was convincingly associated with learning deficits in rodent models38. The data retrieved

from GSE data sets suggest the opposite in postmortem SN specimens of late-stage PD patients, namely, a statistically significant reduction in _NQO2_ mRNA levels. This is in line with a

higher expression of lnc_NQO2_, which might repress _NQO2_ expression66. These data are unexpected, but do not exclude the possibility that higher _NQO2_ expression might be a trigger or

risk factor for neurodegenerative changes in the early stages of PD. This hypothesis is supported by the evidence coming from the analysis of _NQO2_ expression in whole blood of 48 early PD

cases, which revealed that the majority (35/48 (≈73%) of early PD patients express higher _NQO2_ levels than its median expression in healthy individuals, which is statistically significant

in the analyzed population, characterized by nonnormal distribution. Considering a high degree of heterogeneity among PD patients4 as well as the presence of polymorphisms leading to high or

low _NQO2_ expression, such a non-Gaussian distribution is plausible for both healthy and PD populations. We do not know if the levels of blood-born transcripts predict the brain expression

of a given gene, but higher NQO2 expression in the blood may indicate a more active promoter. According to our analysis, elevated _NQO2_ expression is almost 3 times more frequent among PD

patients than in the control group, which is in line with the observations of the study by Harada et al.35. In conclusion, our observations suggest that drugs targeting NQO2 may stimulate

autophagy as well as protect against dopamine quinone and hydroxyquinone toxicity. Thus, S29434 and other NQO2 inhibitors by acting as protective agents in astrocytes, might delay neuronal

damage in PD and other pathological conditions involving oxidative neurodegeneration. The vast literature suggesting a protective effect of several natural polyphenols in neurodegeneration

models strongly supports this idea, since NQO2 is a well-documented target of these compounds40,67. Further studies in PD animal models and brain organoids will be necessary to validate this

interesting hypothesis. METHODS 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), and chloroquine (ClQ, C6628; 25 mM stock in H2Ob). Except for 6OHDA, which 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

characterized39 and used under the name of NMDPEF31,32 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 from M. Pollicita and S. Aquaro, Tor Vergata University,

Rome. After thawing, cells were expanded in high glucose (4.5% glucose) Dulbecco’s modified Eagle’s medium (DMEM) (Carlo Erba S.r.l., Milan, Italy, FA30WL0101500) and then maintained in

these conditions for routine culture or gradually switched to low-glucose (1% glucose) DMEM (FA30WL0060500) when indicated. Each type of DMEM was supplemented with 10% fetal 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 a density of 32 × 103 or 16 × 103 × cm−2 (300 or 150 × 103 per 3.5 cm plate), 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 the medium change was performed every 48 h. ClQ was added 2 or 3 h before 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 × 103 ×

 cm−2) for 2 days before treatments. Neuroblastoma SH-SY5Y cells were cultured in F-12 K medium plus high glucose DMEM supplemented with 10% FBS, Pen Strep and l-glutamine as standard

medium. 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

cell monolayers by fluorimeter, while the second method detected ROS in trypsin-detached cells by FACS analysis. The DCF-based method was performed as previously described31, with minor

modifications. U373 cells were plated at 2 × 104/cm2 in 96-well plates 2 days before treatments, but phenol-free DMEM (Gibco, Life Tech. 11054020) was used as the basal medium. 6OHDA was

added 6 h before analysis in triplicate for each concentration. Cell staining with CA-DCF-DA was performed 2 h before analysis. For this purpose, the conditioned medium +/− 6OHDA was

temporarily removed, and the cells were incubated with prewarmed basal phenol-free DMEM (Gibco, Life Tech. 11054020) containing 20 mM CA-DCF-DA or vehicle (DMSO) for 30 min. Stained cells

were rinsed in phenol-free DMEM and incubated again for 1.5 h with the conditioned medium. All medium changes and washes were performed gently to avoid any shear, osmotic or other types of

stress, drop by drop placed on the plate wall with the warm medium. Fluorescein signal detection of cell monolayers was performed on a Victor 2 multilabel fluorimeter (Perkin-Elmer,

Singapore). Background readings (cells stained with DMSO) were subtracted for the analysis of fluorescence of DCF-DA-stained cells. For detection of mitochondrial ROS, 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 a concentration of 5 µM in phenol-free DMEM. WESTERN BLOTTING

(WB) See Supplementary Methods. NQO2 ACTIVITY MEASUREMENTS NQO2 enzymatic activity assays were performed as described previously40 with the following modifications. Cellular NQO2 activity

was measured in U373 cell lysates. For this purpose, the cells were cultured for two days to reach a 70–90% density and then treated with 6OHDA for 24 h, scraped and homogenized in ice-cold

GPS buffer (50 mM Tris–HCl, 3 mM n-octyl-d-glucopyranoside pH 7.5) with 10 strokes by a tight glass douncer on ice. After clearing by centrifugation, the lysates were stored at − 80 °C.

After thawing, lysates (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 cosubstrate 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 an NQO2 substrate was performed with 50 or 100 ng/mL 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 a 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) to fit the decay curves and find 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-OVEREXPRESSING (N-OVER) U373 CELLS See Supplementary

Methods. FLOW CYTOMETRY (FACS) ANALYSIS OF CELL VIABILITY See Supplementary Methods. COCULTURE 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 were plated at a density of 40 × 103 cm−2, while U373 N-over cells were plated at a density between 30 × 103 cm−2 under standard

conditions to achieve a 90% confluent monolayer after 2 days. Before plating, SH-SY5Y cells were labeled with CellTracker Green CMFDA (chloromethyl fluorescein diacetate, Molecular Probes,

Life Tech., USA) dye. Briefly, SHSY5Y cells, grown to 80–90% confluency, were labeled with 5 µM CMFDA in PBS for 15 min. Cells were washed once in PBS, and fresh regular medium was added for

30 min. Finally, the cells were trypsinized (Trypsin–EDTA (0.05%), Gibco, Life Tech., USA), and resuspended in fresh culture medium to a desired cell concentration. Next, 5 × 104 cm−2

SH-SY5Y cells were seeded over U373 monolayers. After 24 h, the cocultures were treated for 48 h with 6OHDA and/or S29434. At the end of the treatment, the cells were washed once in PBS

(Carlo Erba S.r.l., FA30WL0615500), trypsinized for 3 min and collected in 0.5 ml of 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 × _g_ and resuspended in 400 µL of PBS containing 1% FBS and 0.5 mM EDTA. Cell viability and mortality were determined by 7-AAD dye (BD

Biosciences, Erenbodgem, Belgium, cat. nr 344563; used 1:10) added 2–3 min before FACS analysis on a FACSCanto II flow cytometer (BD Biosciences, Erenbodgem, Belgium). The data were acquired

in the FITC channel (for CMFDA dye) and PerCP-Cy5-5 channel (for 7-AAD dye) according to the parameters and gating criteria detailed in the Supplementary Methods and Fig. S4 (Supplementary

information link). U373 CELL TRANSFECTION AND SILENCING OF NQO2 U373 cells cultured in high glucose standard medium were seeded on 6-well plates one day before transfection at a density of 4

 × 104 cells cm−2. Nonsilencing control siRNA [_siEGFP_, (EHUEGFP)] and a set of human _NQO2_-targeting _esiRNAs_ (EMU042521) were purchased from Merck, Sigma-Aldrich. Transfection of U373

cells was performed using Transfection Reagent (Merck, 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 high-glucose culture medium containing w/o PenStrep. After 20 h, the medium was exchanged for regular high-glucose medium. The treatments were initiated 48 h after

transfection and carried for the next 24 h. ClQ (25 µM) was added 3 h before the end of the experiment. After the treatments, the cells were lysed and assayed by WB. ANIMAL PROCEDURES _NQO2_

knock-out mice generated in the C3H/HeOuJ background were previously characterized38,68. Since the first description in 2004, the original knockout mice were backcrossed with _WT_ C3H/HeOuJ

mice obtained from Charles River at least 20 times. Mice used for astrocyte isolation were WT and _NQO2_ − / − pups of the C3H/HeOuJ strain born on the same day from the same litter _WT_

and _NQO2_ − / − females crossed with the same litter males. One- to two-day-old (P1/P2) pups were euthanized by head decapitation. The heads were placed into sterile PBS at RT, and the

meninges were carefully removed. The animal procedures were carried out according to EU Directive 2010/63/EU and approved by the Italian Ministry of Health (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 protocol31. Briefly, the cortices were transferred to new dishes with sterile PBS and then dissected, minced, and

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 on 10 cm dishes (1

brain/dish) in DMEM supplemented with 10% FBS and 1% P/S (Gibco, Life Tech.). The next day, the 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 0.05% trypsin–EDTA

(Invitrogen) and replated on 6-well plates for experiments at a density of 500,000 cells per well. The treatments were performed one day after seeding. PHASE-CONTRAST AND FLUORESCENCE

MICROSCOPY 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) DATASET ANALYSIS See Supplementary Methods (Supplementary information link). DATA ANALYSIS AND STATISTICAL PROCEDURES Numerical data from all multigroup experiments

were evaluated by analysis of variance (ANOVA). If Brown-Forsythe’s test for equality of variances was positive, Welch’s ANOVA test was performed, otherwise ordinary one-way ANOVA or

occasionally three-way ANOVA was performed. Multiple comparisons between groups were performed by one of the suggested posttests according to GraphPad PRISM 9.0 software guidelines

http://www.graphpad.com/ or occasionally by unpaired T test as indicated in figure legends. Data are expressed as the means ± SEM for the number n of independent experiments or as the means 

± SD for the number of n independent samples from a representative experiment, as indicated for each figure. For patient and healthy subject expression data, D'Agostino-Pearson and

Shapiro–Wilk normality tests were performed. If the data did not pass the normality tests or groups were lower than 10, Mann–Whitney test was applied and the data were presented as medians 

+/− range; otherwise unpaired T test was used. ETHICS DECLARATION The authors declare that the current study complied with ARRIVE guidelines (https://arriveguidelines.org/). DATA

AVAILABILITY 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

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Google Scholar  Download references ACKNOWLEDGEMENTS Special thanks to 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 D.r Michela Pollicita, University Tor Vergata, Rome, and Prof. Rossella Russo, University of Calabria, Rende (Cs), Italy, for

the kind gift of U373 cells and neuroblastoma SH-SY5Y cells, respectively. FUNDING This research was funded by the Basic Research Activity Fund (FABR) 2017 grant and Ph.D. program of Italian

Ministry of Education, University and Research (M.I.U. R), awarded to E.J.; PON03PE 00078 grant (NUTRAMED Consortium) awarded to V.M. and ADOPTION (FOE, Project ‘Nuovi Biomarker Diagnostici

e Terapeutici delle Malattie Neurodegenerative’), CNR CUP:B83C21001810005, awarded to M.A. AUTHOR INFORMATION Author notes * Maddalena Parafati Present address: Department of

Pharmacodynamics, University of Florida, Gainesville, FL 32611, USA * These authors contributed equally: Maddalena Parafati and Concetta Martino. AUTHORS AND AFFILIATIONS * Laboratory of

Cellular and Molecular Toxicology, Department of Health Science, University “Magna Græcia” of Catanzaro, 88100, Catanzaro, Italy Elzbieta Janda, Maddalena Parafati, Concetta Martino, 

Francesco Crupi & Vincenzo Mollace * Center for Advanced Studies and Technology (CAST), “G. d’Annunzio” University of Chieti-Pescara, 66100, Chieti, Italy Jonahunnatha Nesson George

William * UMR 152 Pharma-Dev, Université de Toulouse III, IRD, UPS, 31400, Toulouse, France Karine Reybier * Institute for Biomedical Research and Innovation (IRIB), National Research

Council of Italy (CNR), 88100, Catanzaro, Italy Mariamena Arbitrio * Laboratory of Neuroendocrine Endocrine and Germinal Differentiation and Communication (NorDiC), Univ Rouen Normandie,

Inserm, NorDiC UMR 1239, 76000, Rouen, France Jean A. Boutin Authors * Elzbieta Janda View author publications You can also search for this author inPubMed Google Scholar * Maddalena

Parafati View author publications You can also search for this author inPubMed Google Scholar * Concetta Martino View author publications You can also search for this author inPubMed Google

Scholar * Francesco Crupi View author publications You can also search for this author inPubMed Google Scholar * Jonahunnatha Nesson George William View author publications You can also

search for this author inPubMed Google Scholar * Karine Reybier View author publications You can also search for this author inPubMed Google Scholar * Mariamena Arbitrio View author

publications You can also search for this author inPubMed Google Scholar * Vincenzo Mollace View author publications You can also search for this author inPubMed Google Scholar * Jean A.

Boutin View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS E.J. designed all experiments, supervised the work, performed experiments for Figs.

2, 3, 5 and 6, analyzed the data, wrote the main manuscript text and prepared all the figures. M.P. performed the experiments for Figs. 1 and 5 and analyzed the data, C.M. performed

experiments for Figs. 1, 4 and 6, F.C. performed experiments for Figs. 1 and 5, analyzed the data and helped with figure preparation, J.N.G.W. collected and analyzed the data for Fig. 7,

V.M., M.A. and E.J. provided the funding, J.B. and K.R. provided reagents, suggested and analyzed experiments and contributed to writing the manuscript. M. contributed to writing the revised

version of the manuscript. All authors reviewed the manuscript. CORRESPONDING AUTHORS Correspondence to Elzbieta Janda or Mariamena Arbitrio. ETHICS DECLARATIONS COMPETING INTERESTS The

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Janda, E., Parafati, M., Martino, C. _et al._ Autophagy and neuroprotection in

astrocytes exposed to 6-hydroxydopamine is negatively regulated by NQO2: relevance to Parkinson’s disease. _Sci Rep_ 13, 21624 (2023). https://doi.org/10.1038/s41598-023-44666-7 Download

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