After cellular internalization, quercetin causes Nrf2 nuclear translocation, increases glutathione levels, and prevents neuronal death against an oxidative insult

Abstract

In this work we describe the protective effects of quercetin against H₂O₂ in 24-h-pretreated neuronal cultures. We explored quercetin availability and subcellular fate through the use of HPLC-Diode Array Detection (DAD), epifluorescence, and confocal microscopy. We focused on quercetin modulation of thiol-redox systems by evaluating changes in mitochondrial thioredoxin Trx2, the levels of total glutathione (GSH), and the expression of the γ-glutamate–cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme of GSH synthesis, by the use of Western blot, HPLC, and real-time PCR techniques, respectively. We further explored the activation of the protective NF-E2-related factor 2 (Nrf2)-dependent signaling pathway by quercetin using immunocytochemistry techniques. Our results showed rapid quercetin internalization into neurons, reaching the nucleus after its addition to the culture. Quercetin pretreatment increased total GSH levels, but did not increase Trx2. Interestingly it caused Nrf2 nuclear translocation and significantly increased GCLC gene expression. At the moment of H₂O₂ addition, intracellular quercetin or related metabolites were undetectable in the cultures although quercetin pretreatment prevented neuronal death from the oxidant exposure. Our findings suggest alternative mechanisms of quercetin neuroprotection beyond its long-established ROS scavenging properties, involving Nrf2-dependent modulation of the GSH redox system.

Introduction

Reactive oxygen and nitrogen species (ROS/RNS) are recognized for playing a dual role as both deleterious and beneficial molecules. Under physiologic conditions, the balance between the generation and the elimination of ROS/RNS maintains a redox homeostasis to ensure the correct function of redox-sensitive signaling proteins. However, when it is disturbed, oxidative processes take place, damaging biomolecules, and aberrant redox cell signaling may lead to cell death, contributing to disease onset [1], [2], [3], [4]. This recognized "two-faced" character of ROS/RNS has contributed to the establishment of a redefinition of oxidative stress from the classical “imbalance of pro-oxidants and antioxidants” to a more contemporary concept regarding a “disruption of redox signaling and control” [5], [6].

The maintenance of cellular redox homeostasis appears as a clue to the correct functioning of redox signaling and control and the prevention of oxidative stress-related diseases. In this sense, cells have developed diverse mechanisms to maintain such homeostasis and to deal with ROS/RNS produced in excess during oxidative stress [2].

A main regulator of the intracellular redox status is glutathione, a cysteine-containing tripeptide with reducing and nucleophilic properties. It exists either as a reduced form (GSH) or as an oxidized form (GSSG). This thiol is a key modulator of cell functions including antioxidant defense, redox regulation of protein thiols, and maintenance of redox homeostasis [7], [8], [9], [10], [11].

The other major thiol-dependent system underlying redox modulation functions is the family of thioredoxins (Trx). Trx isozymes play an important role in redox regulation of protein thiols involved in signal transduction and gene regulation [12]. Particularly, the mitochondrial isoform Trx2 is abundantly and widely distributed in rat brain. It is essential for cell survival, and evidence suggests it has protective effects against oxidative stress [12], [13], [14], [15].

An important number of studies on the central nervous system (CNS) support the idea that the disturbance of redox homeostasis, often associated with deficits in mitochondrial function, leads to oxidative stress events and neuronal death, thereby contributing to the development of neurodegenerative disorders such as Alzheimer and Parkinson disease [16], [17], [18]. Interestingly, several studies associate a shift in GSH levels with such pathologies [19], [20], [21], [22], [23]. In addition, a rapidly growing number of studies suggest that Trx proteins perform important functions in the CNS, including neuroprotective actions [12].

In this context, pharmacological interventions that regulate redox homeostasis could be an important strategy to promote cell survival and treat or prevent neurodegeneration [11], [19], [20], [21], [22], [23].

There is a growing interest in the neuroprotective potential of flavonoids although the exact mechanisms by which these compounds exert their benefits are not fully understood [24], [25], [26], [27]. Though these favorable effects have been largely related to their classical hydrogen-donating antioxidant activity [28], in the past 5–10 years, evidence from cell studies suggests that flavonoids can influence cellular fate by other mechanisms of action involving protein and lipid interactions leading to enzymatic modulation, interaction with several receptors, modulation of intracellular signaling cascades, and modulation of gene expression [24], [25], [27], [29], [30], [31]. In particular, flavonoids have been proposed to induce the expression of protective genes, including those encoding enzymes involved in GSH synthesis, through the activation of the NF-E2-related factor-2 (Nrf2) transcription factor pathway [32]. In addition, many of these multitarget mechanisms are redox sensitive, suggesting that the basis of the potential therapeutic capacity of flavonoids may be related to properties beyond scavenging and metal-chelating antioxidant activities.

Among the high number of naturally occurring flavonoids, quercetin is the most frequent flavonoid in the Western diet [33], [34], [35]. Studies of quercetin's neuroprotective mechanisms have been mainly focused either on ROS scavenging and metal-chelating properties or on interactions with kinase signaling cascades [29], [30], [36], [37], [38]. Less work has been done on the modulation of thiol-redox systems by quercetin and these studies have been mainly performed in nonneuronal cells [39]. Accordingly, in the present work we focused on the modulation of thiol-redox systems as a complementary antioxidant mechanism of quercetin neuroprotection. To this aim we developed a model of oxidative stress-induced neuronal death in primary cerebellar granule neurons, in which we studied neuroprotective and neurotoxic profiles of quercetin in 24-h pretreated cultures. We evaluated quercetin bioavailability in cultures during the 24 h of incubation previous to oxidative injury, and we investigated its subcellular fate in neurons by the use of its fluorogenic properties. Furthermore we studied changes in mitochondrial thioredoxin Trx2 and in cellular levels of GSH and changes in the expression of γ-glutamate–cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme of GSH synthesis, and we explored the activation of the protective Nrf2-dependent signaling pathway by quercetin.