Pamela J. Roqué

Mechanisms of Neuroprotection by Quercetin: Counteracting Oxidative Stress and More.

Increasing interest has recently focused on determining whether several natural compounds, collectively referred to as nutraceuticals, may exert neuroprotective actions in the developing, adult, and aging nervous system. Quercetin, a polyphenol widely present in nature, has received the most attention in this regard. Several studies in vitro, in experimental animals and in humans, have provided supportive evidence for neuroprotective effects of quercetin, either against neurotoxic chemicals or in various models of neuronal injury and neurodegenerative diseases. The exact mechanisms of such protective effects remain elusive, though many hypotheses have been formulated. In addition to a possible direct antioxidant effect, quercetin may also act by stimulating cellular defenses against oxidative stress. Two such pathways include the induction of Nrf2-ARE and induction of the antioxidant/anti-inflammatory enzyme paraoxonase 2 (PON2). In addition, quercetin has been shown to activate sirtuins (SIRT1), to induce autophagy, and to act as a phytoestrogen, all mechanisms by which quercetin may provide its neuroprotection.

Neurotoxicants Are in the Air: Convergence of Human, Animal, and In Vitro Studies on the Effects of Air Pollution on the Brain

In addition to increased morbidity and mortality caused by respiratory and cardiovascular diseases, air pollution may also negatively affect the brain and contribute to central nervous system diseases. Air pollution is a mixture comprised of several components, of which ultrafine particulate matter (UFPM; <100 nm) is of much concern, as these particles can enter the circulation and distribute to most organs, including the brain. A major constituent of ambient UFPM is represented by traffic-related air pollution, mostly ascribed to diesel exhaust (DE). Human epidemiological studies and controlled animal studies have shown that exposure to air pollution may lead to neurotoxicity. In addition to a variety of behavioral abnormalities, two prominent effects caused by air pollution are oxidative stress and neuroinflammation, which are seen in both humans and animals and are confirmed by in vitro studies. Among factors which can affect neurotoxic outcomes, age is considered the most relevant. Human and animal studies suggest that air pollution (and DE) may cause developmental neurotoxicity and may contribute to the etiology of neurodevelopmental disorders, including autistic spectrum disorders. In addition, air pollution exposure has been associated with increased expression of markers of neurodegenerative disease pathologies.

Neurotoxicity of traffic-related air pollution.

&NA; The central nervous system is emerging as an important target for adverse health effects of air pollution, where it may contribute to neurodevelopmental and neurodegenerative disorders. Air pollution comprises several components, including particulate matter (PM) and ultrafine particulate matter (UFPM), gases, organic compounds, and metals. An important source of ambient PM and UFPM is represented by traffic‐related air pollution, primarily diesel exhaust (DE). Human epidemiological studies and controlled animal studies have shown that exposure to air pollution, and to traffic‐related air pollution or DE in particular, may lead to neurotoxicity. In particular, air pollution is emerging as a possible etiological factor in neurodevelopmental (e.g. autism spectrum disorders) and neurodegenerative (e.g. Alzheimers disease) disorders. The most prominent effects caused by air pollution in both humans and animals are oxidative stress and neuro‐inflammation. Studies in mice acutely exposed to DE (250–300 &mgr;g/m3 for 6 h) have shown microglia activation, increased lipid peroxidation, and neuro‐inflammation in various brain regions, particularly the hippocampus and the olfactory bulb. An impairment of adult neurogenesis was also found. In most cases, the effects of DE were more pronounced in male mice, possibly because of lower antioxidant abilities due to lower expression of paraoxonase 2. HighlightsTraffic‐related air pollution may cause neurotoxicity.Traffic‐related air pollution may contribute to neurodevelopmental and neurodegenerative disorders.Particulate matter (PM) from diesel exhaust can cause oxidative stress and neuroinflammation.

Microglia mediate diesel exhaust particle-induced cerebellar neuronal toxicity through neuroinflammatory mechanisms

In addition to the well-established effects of air pollution on the cardiovascular and respiratory systems, emerging evidence has implicated it in inducing negative effects on the central nervous system. Diesel exhaust particulate matter (DEP), a major component of air pollution, is a complex mixture of numerous toxicants. Limited studies have shown that DEP-induced dopaminergic neuron dysfunction is mediated by microglia, the resident immune cells of the brain. Here we show that mouse microglia similarly mediate primary cerebellar granule neuron (CGN) death in vitro. While DEP (0, 25, 50, 100μg/2cm2) had no effect on CGN viability after 24h of treatment, in the presence of primary cortical microglia neuronal cell death increased by 2-3-fold after co-treatment with DEP, suggesting that microglia are important contributors to DEP-induced CGN neurotoxicity. DEP (50μg/2cm2) treatment of primary microglia for 24h resulted in morphological changes indicative of microglia activation, suggesting that DEP may induce the release of cytotoxic factors. Microglia-conditioned medium after 24h treatment with DEP, was also toxic to CGNs. DEP caused a significant increase in reactive oxygen species in microglia, however, antioxidants failed to protect neurons from DEP/microglia-induced toxicity. DEP increased mRNA levels of the pro-inflammatory cytokines IL-6 and IL1-β, and the release of IL-6. The antibiotic minocycline (50μM) and the peroxisome proliferator-activated receptor-γ agonist pioglitazone (50μM) attenuated DEP-induced CGN death in the co-culture system. Microglia and CGNs from male mice appeared to be somewhat more susceptible to DEP neurotoxicity than cells from female mice possibly because of lower paraoxonase-2 expression. Together, these results suggest that microglia-induced neuroinflammation may play a critical role in modulating the effect of DEP on neuronal viability. .

Role of glutamate receptors in tetrabrominated diphenyl ether (BDE-47) neurotoxicity in mouse cerebellar granule neurons

The polybrominated diphenyl ether (PBDE) flame retardants are developmental neurotoxicants, as evidenced by numerous in vitro, animal and human studies. PBDEs can alter the homeostasis of thyroid hormone and directly interact with brain cells. Induction of oxidative stress, leading to DNA damage and apoptotic cell death is a prominent mechanism of PBDE neurotoxicity, though other mechanisms have also been suggested. In the present study we investigated the potential role played by glutamate receptors in the in vitro neurotoxicity of the tetrabromodiphenyl ether BDE-47, one of the most abundant PBDE congeners. Toxicity of BDE-47 in mouse cerebellar neurons was diminished by antagonists of glutamate ionotropic receptors, but not by antagonists of glutamate metabotropic receptors. Antagonists of NMDA and AMPA/Kainate receptors also inhibited BDE-47-induced oxidative stress and increases in intracellular calcium. The calcium chelator BAPTA-AM also inhibited BDE-47 cytotoxicity and oxidative stress. BDE-47 caused a rapid increase of extracellular glutamate levels, which was not antagonized by any of the compounds tested. The results suggest that BDE-47, by still unknown mechanisms, increases extracellular glutamate which in turn activates ionotropic glutamate receptors leading to increased calcium levels, oxidative stress, and ultimately cell death.

Quantification of synaptic structure formation in cocultures of astrocytes and hippocampal neurons.

The ability to quantify changes of synaptic structure, whether associated with the formation of synapse in early development or the degeneration of synapses in adult life in an in vitro culture system, is important for understanding the underlying mechanisms. Astrocytes play a vital role in neuronal development and functioning, including synapse formation and stabilization. The method described in this chapter allows for the determination of the modulation by astrocytes of synaptic structure formation in hippocampal neurons. Using a sandwich coculture system, highly pure, hippocampal neurons are grown in culture for 14 days on glass coverslips, after which they are inverted, without contact, over separately cultured astrocytes or pretreated astrocytes for 24 h. Neuronal immunocytochemical staining of the presynaptic marker, synaptophysin, and the postsynaptic marker, PSD-95, is used to assess the localization of synaptic proteins into pre and postsynaptic structures. Deconvolved, confocal images are used to determine a mean puncta intensity threshold for use in rendering the surface of the synaptic structures using three-dimensional object analysis software. Once rendered in three-dimensional space, automatic quantification of the number of pre- and postsynaptic specializations and the number of those structures that overlap is obtained, allowing the ability to compare how different treatments may modulate the formation of synapses. Because synapses not only consist of distinct pre- and postsynaptic specializations, but are also defined by their apposition, the determination and study of synapse formation can only benefit by methods that use all of the available data to assess the actual structure.

Co‐Culture of Neurons and Microglia

Microglia, the resident immune cells of the brain, have been implicated in numerous neurodegenerative and neurodevelopmental diseases. Activation of microglia by a variety of stimuli induces the release of factors, including pro‐ and anti‐inflammatory cytokines and reactive oxygen species, that contribute to modulating neuro‐inflammation and oxidative stress, two crucial processes linked to disorders of the central nervous system. The in vitro techniques described here will provide a set of protocols for the isolation and plating of primary cerebellar granule neurons, primary cortical microglia from a mixed glia culture, and methods for co‐culturing both cell types. These methods allow the study of how microglia and the factors they release in this shared environment mediate the effects of toxicants on neuronal function and survival. The protocols presented here allow for flexibility in experimental design, the study of numerous toxicological endpoints, and the opportunity to explore neuroprotective strategies.

Synaptic Structure Quantification in Cultured Neurons

Behavioral problems (e.g., learning and memory) following developmental exposure to toxicants suggests that dysregulation of the process of synapse formation and function may occur. The ability to assess these changes is thus of value. This unit describes a method to investigate toxicant‐induced changes to synaptic structure formation in primary hippocampal neurons using immunocytochemical labeling of the pre‐ and post‐synaptic markers synaptophysin and PSD‐95, confocal imaging, and three‐dimensional object analysis. Protocols for the long‐term culturing of primary hippocampal neurons and of primary cortical astrocytes, as well as their co‐culture, are included. While the described methods focus on how astrocytes influence synapse formation and how toxicants may interfere in this process, modifications to the experimental plan can easily be implemented. This would allow for the investigation of the effects of toxicants after treating neurons alone, or both astrocytes and neurons in co‐culture. With the common endpoint of synapse structure formation, differences between varying treatment paradigms can expand the understanding of the influence of particular toxicants on these diverse cell types and provide insight into potential mechanisms of effect and the contributions of each to synapse formation. Curr. Protoc. Toxicol. 60:12.22.1‐12.22.32.

Nutraceuticals in CNS Diseases: Potential Mechanisms of Neuroprotection

Abstract In recent years, there has been increasing attention devoted to the possibility that several nutraceuticals may act as neuroprotective agents. Such protective effects have often been ascribed to a direct antioxidant effect; however, the exact mechanisms of neuroprotection remain elusive, and alternate mechanisms have been proposed. This chapter focuses on some examples of potential mechanisms of neuroprotection at the cellular, biochemical, and molecular level. Polyphenols (particularly quercetin) are discussed as model nutraceuticals, although other molecules are mentioned and discussed to illustrate additional potential neuroprotective mechanisms. Quercetin neuroprotection may be ascribed to its direct antioxidant ability; however, it is more likely that it acts in a hormetic fashion, stimulating cellular defenses by activating the Nrf2-ARE pathway. Induction of the antioxidant/anti-inflammatory enzyme paraoxonase 2 may represent an additional novel mechanism of neuroprotection. Interference with multiple signal transduction pathways, activation of sirtuins (SIRT1), and modulation of autophagy are other mechanisms that nutraceuticals may use to provide beneficial effects in central nervous system diseases.In recent years, there has been increasing attention devoted to the possibility that several nutraceuticals may act as neuroprotective agents. Such protective effects have often been ascribed to a direct antioxidant effect; however, the exact mechanisms of neuroprotection remain elusive, and alternate mechanisms have been proposed. This chapter focuses on some examples of potential mechanisms of neuroprotection at the cellular, biochemical, and molecular level. Polyphenols (particularly quercetin) are discussed as model nutraceuticals, although other molecules are mentioned and discussed to illustrate additional potential neuroprotective mechanisms. Quercetin neuroprotection may be ascribed to its direct antioxidant ability; however, it is more likely that it acts in a hormetic fashion, stimulating cellular defenses by activating the Nrf2-ARE pathway. Induction of the antioxidant/anti-inflammatory enzyme paraoxonase 2 may represent an additional novel mechanism of neuroprotection. Interference with multiple signal transduction pathways, activation of sirtuins (SIRT1), and modulation of autophagy are other mechanisms that nutraceuticals may use to provide beneficial effects in central nervous system diseases.

Chapter 1 – Nutraceuticals in CNS Diseases: Potential Mechanisms of Neuroprotection

Abstract In recent years, there has been increasing attention devoted to the possibility that several nutraceuticals may act as neuroprotective agents. Such protective effects have often been ascribed to a direct antioxidant effect; however, the exact mechanisms of neuroprotection remain elusive, and alternate mechanisms have been proposed. This chapter focuses on some examples of potential mechanisms of neuroprotection at the cellular, biochemical, and molecular level. Polyphenols (particularly quercetin) are discussed as model nutraceuticals, although other molecules are mentioned and discussed to illustrate additional potential neuroprotective mechanisms. Quercetin neuroprotection may be ascribed to its direct antioxidant ability; however, it is more likely that it acts in a hormetic fashion, stimulating cellular defenses by activating the Nrf2-ARE pathway. Induction of the antioxidant/anti-inflammatory enzyme paraoxonase 2 may represent an additional novel mechanism of neuroprotection. Interference with multiple signal transduction pathways, activation of sirtuins (SIRT1), and modulation of autophagy are other mechanisms that nutraceuticals may use to provide beneficial effects in central nervous system diseases.In recent years, there has been increasing attention devoted to the possibility that several nutraceuticals may act as neuroprotective agents. Such protective effects have often been ascribed to a direct antioxidant effect; however, the exact mechanisms of neuroprotection remain elusive, and alternate mechanisms have been proposed. This chapter focuses on some examples of potential mechanisms of neuroprotection at the cellular, biochemical, and molecular level. Polyphenols (particularly quercetin) are discussed as model nutraceuticals, although other molecules are mentioned and discussed to illustrate additional potential neuroprotective mechanisms. Quercetin neuroprotection may be ascribed to its direct antioxidant ability; however, it is more likely that it acts in a hormetic fashion, stimulating cellular defenses by activating the Nrf2-ARE pathway. Induction of the antioxidant/anti-inflammatory enzyme paraoxonase 2 may represent an additional novel mechanism of neuroprotection. Interference with multiple signal transduction pathways, activation of sirtuins (SIRT1), and modulation of autophagy are other mechanisms that nutraceuticals may use to provide beneficial effects in central nervous system diseases.