Elsevier

Neurobiology of Aging

Volume 35, Issue 8, August 2014, Pages 1901-1912
Neurobiology of Aging

Regular article
Primary cultured astrocytes from old rats are capable to activate the Nrf2 response against MPP+ toxicity after tBHQ pretreatment

https://doi.org/10.1016/j.neurobiolaging.2014.01.143Get rights and content

Abstract

Astrocytes are key players for brain physiology, protecting neurons by releasing antioxidant enzymes; however, they are also susceptible to damage by neurotoxins. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a central regulator of the antioxidant response, and therefore, pharmacologic inducers are often used to activate this transcription factor to induce cellular protection. To date, it still remains unknown if cells from aged animals are capable of developing this response. Therefore, the purpose of this work was to determine if cortical astrocytes derived from old rats are able to respond to tertbuthyl-hydroquinene (tBHQ) pretreatment and stimulate the Nrf2-antioxidant response pathway to induce an antioxidant strategy against MPP+ toxicity, one of the most used molecules to model Parkinson's disease. Our results show that, although astrocytes from adult and old rats were more susceptible to MPP+ toxicity than astrocytes from newborn rats, when pretreated with tertbuthyl-hydroquinene, they were able to transactivate Nrf2, increasing antioxidant enzymes and developing cellular protection. These results are discussed in terms of the doses used to create protective responses.

Introduction

Aging is the main risk factor for numerous neurodegenerative disorders, and even though their accurate etiology is largely unknown, oxidative stress has been proposed as one of the primary causes that links the aging process with the establishment of most neuropathies (Simonian and Coyle, 1996, Reynolds et al., 2007), not only through the structural and functional alterations that reactive oxygen species (ROS) produce to cell biomolecules, but also because they are potential mediators of cell death by either necrosis or apoptosis (Friedlander, 2003).

Astrocytes are the most abundant glial cell type, representing more than 50% of the total cortical cells (Dringen, 2000). They are known to be important modulators of brain physiology, particularly during regenerative or protective processes, by producing and releasing several antioxidant enzymes like superoxide dismutase and glutathione precursors, which in turn support neuronal survival and stability (Kahlert and Reiser, 2004, Takuma et al., 2004). Additionally, astrocytes regulate the synaptic transmission as part of the tripartite synapse; maintain the blood–brain barrier integrity, brain cholesterol levels, and copper homeostasis (Scheiber and Dringen, 2013, Kim and De Vellis, 2005). Moreover, it is known that these cells decrease their neuroprotective capacity during aging, thereby playing critical roles in neurodegenerative diseases, because astrocytes are involved in responses to damage and stress in a multifactorial manner, by synthesizing and secreting cytokines and chemokines (Sofroniew and Vinters, 2010). This response is called reactive astrogliosis (Ting et al., 2009) and may be either harmful or beneficial because reactive astrocytes can exert both pro- and anti-inflammatory effects. Under pathologic conditions, the development of the pro-inflammatory phenotype might explain the relevance of astroglial cells in the genesis of degenerative processes in the brain (Zhang et al., 2010).

Modifications in redox state are known to modulate transcription factors (Forman et al., 2004, Jones, 2008), such as the nuclear factor erythroid-derived 2-like 2 (Nrf2). Nrf2 is a central regulator of antioxidant and phase II detoxifying enzymes. This transcription factor is an ubiquitous cytosolic protein that is continuously degraded during cellular homeostasis; however, in response to modifications in cellular redox state, Nrf2 is released from its repressor (Keap-1), phosphorylated and translocated into the nucleus, where it binds to the antioxidant response element (ARE) and induces the expression of enzymes such as γGCS and GST, which in turn are related to glutathione (GSH) metabolism (Kraft et al., 2004, Lee et al., 2003). GSH is one of the most intensively studied intracellular nonprotein-thiols because of the critical role it plays in cell biochemistry and physiology. Through maintenance of protein sulfhydryls in the appropriate redox state, GSH regulates important death and/or survival pathways. Redox changes, induced by an altered GSH and/or GSSG balance, also modulate Nrf2 release from Keap-1, and changes in GSH homeostasis have been implicated in the etiology and progression of a number of human diseases (Fernández-Checa and García-Ruiz, 2008, Darlington, 2005).

Phenols like curcumin, resveratrol, and tertbuthyl-hydroquinene (tBHQ) are well-known Nrf2 inducers in neurons and astrocytes, and have been widely used to activate the antioxidant response in both cell types (Erlank et al., 2011, Thimmulappa et al., 2002). However, it remains unknown if cells from aged animals are still capable of developing an antioxidant response in reply to such Nrf2 inducers, as it is expected for astrocytes from newborn and adult rats. Hence, the purpose of this study was to determine if astrocytes derived from old rats are able to recruit Nrf2-associated responses and evoke an antioxidant protection against an acute toxic insult. One of the most used molecules to model neurodegenerative diseases, in particular Parkinson's disease, is MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). MPTP is oxidized in glial cells, mainly in astrocytes, throughout the action of monoamine oxidase B into MPP+ (1-methyl-4-phenylpyridinium), which is further incorporated to the dopaminergic neurons where it impairs mitochondrial function, induces ROS generation and cell demise (Przedborski and Vila, 2003, Zhang et al., 2010).

Although its is generally accepted that neurons are vulnerable to the toxic actions of MPTP because of their ability to accumulate and retain MPP+, there are also some reports where MPP+ has been shown to directly exert damage on cultured astrocytes from rats. For example, it has been shown that MPP+ causes impaired energy in astrocytes by affecting mitochondrial function (Di Monte et al., 1992, Chen et al., 2008), MPP+ is concentrated by the mitochondria, where it inhibits complex I activity at the same site as the respiratory inhibitor rotenone (Krueger et al., 1990, Schapira and Olanow, 2008). In addition, it has been suggested that MPP+ toxicity in cultured astrocytes depends on oxidative and nitrergic stress (Schapira, 2008; Tsai and Lee, 1998). This evidence was preceded by comparative studies demonstrating a differential ability to accumulate MPP+ and express toxicity between rats and mice, suggesting that cultured astrocytes from the first species accumulate less MPP+ while express toxicity at higher concentrations (Tsai and Lee, 1994). Previous data from our group (Alarcón-Aguilar et al., 2014) showed that astrocytes isolated from 24-month-old rats were more susceptible to MPP+ toxicity that astrocytes from newborn and adult (9-month-old) rats. tBHQ is a known Nrf2 inductor, which has been proved in several cellular models and in young animals but not much is known of its effect on old animals. Hence, it was interesting to find out if cells derived from old animals, would still be competent to activate the Nrf2 pathway when pretreated with an inductor such as tBHQ. Therefore, in this study astrocytes derived from old animals were pretreated with tBHQ before the MPP+ insult to determine if old cells are capable to activate Nrf2 protective responses. Our data indicate that when astrocytes derived from old rats were pretreated with tBHQ, they were able to transactivate Nrf2, increasing the content of antioxidant enzymes, improving redox homesotasis measured by GSH/GSSG ratio, and developing protection against mild MPP+ toxicity, supporting the protective character of this pathway for cell survival.

Section snippets

Chemicals

All chemicals and reagents were purchased from Sigma Chemical Co (St Louis, MO, USA). The reagents obtained from other sources are detailed throughout the text.

Animals

Astrocytes were isolated from the frontal cortex of neonatal (1- to 3-day-old), adult (9-month-old), and old (24-month-old) albino Wistar rat brains (Rattus norvegicus), provided by the closed breeding colony at the Universidad Autónoma Metropolitana-Iztapalapa. A total of 40 neonatal, 80 adult, and 80 old rats were used throughout the

Astrocytes isolated from rats of different ages have differential susceptibility to tBHQ

First, to confirm that the primary cultures obtained were indeed astrocytes, GFAP expression was observed in astrocytes derived from newborn, adult, and old rats. The confocal photomicrographs shown in Fig. 1A confirm that more than 90% of cultured cells were certainly astrocytes, a result that has been previously demonstrated by our group (Alarcón-Aguilar et al., 2014). To simplify terminology, from this point on, astrocytes derived from old rats will be abbreviated as OA, whereas astrocytes

Discussion

The increase in glial reactivity that has been reported during aging, in combination with an increase in ROS production and a decrease in antioxidant content in the brain (Kanwar and Nehru, 2007) are primary features that trigger the development of neurodegenerative disorders associated with age. Noteworthy, astrocytes have been demonstrated to be susceptible to diverse toxins in particular to MPP+ (Tsai and Lee, 1994, Tsai and Lee, 1998). Previous data from our group (Alarcón-Aguilar et al.,

Disclosure statement

The authors have no conflicts of interest to disclose.

Acknowledgements

The authors thank Dr Rocío González-Vieira from UAM-I for animal supply and Dr A. Hernández from CINVESTAV for generously donating us the actin antibody; the authors also thank the CBS-UAMI Confocal Core for confocal images acquisition and analysis. This work was supported by CONACyT CB-2006-1-59659 and CB 2012-1-178349. As well as the “Red Temática de Investigación en Salud y Desarrollo Social” from CONACyT and INGER DI-PI004/2012. Alarcón-Aguilar is a CONACyT scholarship holder.

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