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DJ-1 deficiency in astrocytes selectively enhances mitochondrial Complex I inhibitor-induced neurotoxicity
Journal of Neurochemistry
Abstract
J. Neurochem. (2011) 117, 375–387.
Parkinson’s disease (PD) brains show evidence of mitochondrial respiratory Complex I deficiency, oxidative stress, and neuronal death. Complex I-inhibiting neurotoxins, such as the pesticide rotenone, cause neuronal death and parkinsonism in animal models. We have previously shown that DJ-1 over-expression in astrocytes augments their capacity to protect neurons against rotenone, that DJ-1 knock-down impairs astrocyte-mediated neuroprotection against rotenone, and that each process involves astrocyte-released factors. To further investigate the mechanism behind these findings, we developed a high-throughput, plate-based bioassay that can be used to assess how genetic manipulations in astrocytes affect their ability to protect co-cultured neurons. We used this bioassay to show that DJ-1 deficiency-induced impairments in astrocyte-mediated neuroprotection occur solely in the presence of pesticides that inhibit Complex I (rotenone, pyridaben, fenazaquin, and fenpyroximate); not with agents that inhibit Complexes II–V, that primarily induce oxidative stress, or that inhibit the proteasome. This is a potentially PD-relevant finding because pesticide exposure is epidemiologically-linked with an increased risk for PD. Further investigations into our model suggested that astrocytic GSH and heme oxygenase-1 antioxidant systems are not central to the neuroprotective mechanism.
... Glutamate uptake, inflammatory response, mitochondrial function Autosomal recessive Early [11][12][13] PARK8 LRRK2 Autophagy, Lysosome function Autosomal dominant Late [14,15] PARK9 ATP13A2 Inflammatory response, Lysosome function Autosomal recessive Early [16] GBA GCase (Glucocerebrosidase) Autophagy, Lysosome function Autosomal recessive Late [17] * Early onset, ages 21-50; late-onset, ages older than 50. ...
... ROS produced by astrocytes are implicated in oxidative stress in various PD models, including primary neurons and astrocytes [103][104][105][106], leading to neuronal injury and PD-like neurological deficits in animals. Most PD-like neurotoxicants, such as rotenone, MPTP/MPP+, and 6-OHDA, trigger ROS production in astrocytes [13,38,[107][108][109][110][111], and the ROS production is likely derived from the impaired mitochondrial function and dysregulated astrocytic antioxidant defense mechanisms. ...
... Moreover, the mutation of DJ-1, which causes an autosomal recessive PD, is closely associated with Nrf2 dysfunction. This is supported by the findings that DJ-1-deficient or -mutated astrocytes caused Nrf2 instability, reducing its transcriptional activities [115], and abolished astrocyte-mediated neuroprotection against PD [13,38]. However, DJ-1 overexpression in astrocytes protected dopaminergic neurons against rotenone-induced neurotoxicity in the substantia nigra of rat brain [107], suggesting that astrocytic oxidative stress plays a critical role in PD, at least in part by dysregulating Nrf2 and DJ-1. ...
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons and the aggregation of Lewy bodies in the basal ganglia, resulting in movement impairment referred to as parkinsonism. However, the etiology of PD is not well known, with genetic factors accounting only for 10–15% of all PD cases. The pathogenetic mechanism of PD is not completely understood, although several mechanisms, such as oxidative stress and inflammation, have been suggested. Understanding the mechanisms of PD pathogenesis is critical for developing highly efficacious therapeutics. In the PD brain, dopaminergic neurons degenerate mainly in the basal ganglia, but recently emerging evidence has shown that astrocytes also significantly contribute to dopaminergic neuronal death. In this review, we discuss the role of astrocytes in PD pathogenesis due to mutations in α-synuclein (PARK1), DJ-1 (PARK7), parkin (PARK2), leucine-rich repeat kinase 2 (LRRK2, PARK8), and PTEN-induced kinase 1 (PINK1, PARK6). We also discuss PD experimental models using neurotoxins, such as paraquat, rotenone, 6-hydroxydopamine, and MPTP/MPP+. A more precise and comprehensive understanding of astrocytes’ modulatory roles in dopaminergic neurodegeneration in PD will help develop novel strategies for effective PD therapeutics.
... In humans, the deglycase DJ-1 protein is encoded by the PARK7 gene, whose mutation causes one of the hereditary forms of PD [75]. Interestingly, its overexpression in reactive astrocytes has been reported in sporadic cases of PD [75], suggesting a pathophysiological role in PD. ...
... In humans, the deglycase DJ-1 protein is encoded by the PARK7 gene, whose mutation causes one of the hereditary forms of PD [75]. Interestingly, its overexpression in reactive astrocytes has been reported in sporadic cases of PD [75], suggesting a pathophysiological role in PD. Indeed, DJ-1 overexpression reduced rotenone neurotoxicity in neuron-astrocyte cocultures, whereas the opposite was found after DJ-1 deletion [75,76]. ...
... Interestingly, its overexpression in reactive astrocytes has been reported in sporadic cases of PD [75], suggesting a pathophysiological role in PD. Indeed, DJ-1 overexpression reduced rotenone neurotoxicity in neuron-astrocyte cocultures, whereas the opposite was found after DJ-1 deletion [75,76]. Its exact mechanism of action in astrocytes remains elusive, some data suggesting an effect on mitochondrial function that in turn might favor the release of paracrine-acting molecules [76,77]. ...
Despite the fact that astrocytes are the most abundant glial cells, critical for brain function, few studies have dealt with their possible role in neurodegenerative diseases like Parkinson’s disease (PD). This article explores relevant evidence on the involvement of astrocytes in experimental PD neurodegeneration from a molecular signaling perspective. For a long time, astrocytic proliferation was merely considered a byproduct of neuroinflammation, but by the time being, it is clear that astrocytic dysfunction plays a far more important role in PD pathophysiology. Indeed, ongoing experimental evidence suggests the importance of astrocytes and dopaminergic neurons’ cross-linking signaling pathways. The Wnt-1 (wingless-type MMTV integration site family, member 1) pathway regulates several processes including neuron survival, synapse plasticity, and neurogenesis. In PD animal models, Frizzled (Fzd) neuronal receptors’ activation by the Wnt-1 normally released by astrocytes following injuries leads to β-catenin-dependent gene expression, favoring neuron survival and viability. The transient receptor potential vanilloid 1 (TRPV1) capsaicin receptor also participates in experimental PD genesis. Activation of astrocyte TRPV1 receptors by noxious stimuli results in reduced inflammatory response and increased ciliary neurotrophic factor (CNTF) synthesis, which enhances neuronal survival and differentiation. Another major pathway involves IκB kinase (IKK) downregulation by ARL6ip5 (ADP-ribosylation-like factor 6 interacting protein 5, encoded by the cell differentiation-associated, JWA, gene). Typically, IKK releases the proinflammatory NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) molecule from its inhibitor. Therefore, by downregulating NF-κB inhibitor, ARL6ip5 promotes an anti-inflammatory response. The evidence provided by neurotoxin-induced PD animal models guarantees further research on the neuroprotective potential of normalizing astrocyte function in PD.
1. Introduction
Parkinson’s disease (PD) is the second most common neurodegenerative disease following Alzheimer’s disease. It is characterized by loss of dopaminergic neurons in the midbrain [1, 2] and bradykinesia, rigidity, and tremor as main clinical symptoms. Regularly, patients also display nonmotor symptoms like cognitive impairment, mood disorders, sleep alterations, dysautonomia, and hallucinations [1].
Typical, though not only, histopathological changes are the progressive loss of the nigrostriatal dopaminergic pathway and hence of the striatal dopaminergic tone [2]. Over the last 40 years, administration of the amino acid precursor of dopamine L-DOPA (L-3,4-dihydroxy-L-phenylalanine) to parkinsonian patients has been considered the most effective symptomatic treatment [3].
Abnormal accumulation of misfolded protein aggregates [4] as the Lewy bodies, made of α-synuclein [5], appears to be one of the physiopathological hallmarks of the disease. One major target of α-synuclein is Rab-1 (a member of the Ras superfamily of monomeric G proteins, Rab GTPase family), a key molecular switch of the endoplasmic reticulum-Golgi traffic pathway [6]. The α-synuclein accumulation-induced endoplasmic reticulum stress is likely a leading disruptive mechanism, responsible for the so-called “unfolded protein response” adaptive reaction [7], cytoprotective when moderate but deleterious when highly activated [8, 9]. Accumulation of α-synuclein may also originate abnormal synaptic connectivity or synaptopathy at nigrostriatal pathways and intrastriatal interneuronal connections, presumably most apparent at the initial stages of the disease.
Notwithstanding the fact that astrocytes are the most abundant glial subtype and are critical for brain function, only a few studies have historically focused on their putative role in neurodegenerative diseases like PD. Recently, however, several studies have reported that genes known to have a causative role in PD are expressed in astrocytes and have important roles in their function [10], suggesting that astrocyte dysfunction may be relevant for PD development. Furthermore, α-synuclein aggregates in astrocytes contributing to such dysfunction [11].
This review aims at summarizing the evidence for astrocyte participation in experimental PD genesis, the probable neuroprotective effect of molecules like GDNF (glial-derived neurotrophic factor), MANF (mesencephalic astrocyte-derived neurotrophic factor), and CNTF (ciliary neurotrophic factor), and the involved pathological cascades described so far, illustrating the potential use of these findings in developing new-generation neuroprotective agents. Following PubMED searches performed using “Parkinson’s Disease, astrocytes, molecular signaling” strings, relevant papers published in English or Spanish before January 1, 2018, were included, while reference sections were also scrutinized out of these publications for new studies.
2. Role of Astrocyte Dysfunction in the Genesis of Experimental Parkinson’s Disease
The glia accounts for over 50% of brain cells, comprising various cell subtypes, of which astrocytes are the most abundant [12, 13]. Although astrocytes were documented 100 years ago, relatively few studies have been designed to dig into their role in neurological disorders and diseases over time. Astrocytes can be both helpful and harmful in PD [14, 15], and a key aspect of PD pathophysiology is neuroinflammation in the central nervous system (CNS), for long considered a downstream response to dopaminergic neuronal death, definitely including the concurrence of reactive astrocytes [16, 17]. However, ongoing evidence suggests that astrocytes have a role in setting up PD pathophysiology. Astrocytes may have neuroprotective effects by producing factors like the glial cell line-derived neurotrophic factor (GDNF) [18], the mesencephalic astrocyte-derived neurotrophic factor (MANF) [19], and the ciliary neurotrophic factor (CNTF) [20]. Recently, a relative increase in the astrocytic level of senescence markers, inflammatory cytokines, and metalloproteinases was observed on postmortem substantia nigra specimens of five PD patients compared with five controls, illustrating astrocytes’ relevance in PD [21]. Furthermore, astrocytes and fibroblasts developed senescent phenotypes when exposed to the neurotoxin paraquat in human cell cultures, and conversely, neurodegeneration was attenuated in response to paraquat in a senescent astrocyte-selectively depleted mouse model [21].
This section reviews evidence from a molecular signaling perspective about the participation of astrocytes in the genesis of experimental PD and the involved molecular cascades.
2.1. Wnt/β-Catenin Signaling Cascade
The Wnt1 (wingless-type MMTV (mouse mammary tumor virus) integration site family, member 1) pathway has emerged as an essential signaling cascade regulating differentiation, neuron survival, axonal extension, synapse formation, neurogenesis, and many other processes in developing and adult tissues [22]. Little is known on the role of Wnt agonists in the midbrain [23]. In healthy human progenitor-derived astrocytes (PDAs), β-catenin leads to modulation of genes relevant to regulating aspects of glutamate neurotransmission [24]. However, the expression of Wnt components in adult astrocytes [25, 26] and the identification of activated midbrain astrocytes as candidate components of Wnt1 signaling suggest that astrocytes may be relevant sources of Wnt1 [27]. Using the proneurotoxin MPTP- (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-) lesioned mouse model, 92 mRNA species molecular profiling in the midbrain revealed a specific, robust, and persistent increase in the expression of the canonical Wnt1 agonist, but not of Wnt3a or Wnt5a, during MPTP-induced dopaminergic degeneration [28]. The activated astrocytes rescued mesencephalic dopaminergic neurons from MPP+-induced tyrosine hydroxylase-positive (TH⁺) neuron toxicity promoting dopaminergic neurogenesis through Wnt1/β-catenin signaling activation [28]. Further evidence supports that the Wnt signaling system may be reinforced following injury in the adult CNS [29]. Likewise, some studies suggest that Wnt/β-catenin activation reduces neurodegeneration in mouse models of Alzheimer’s disease [30, 31].
Growing evidence endorses the critical participation of Wnt1 in PD genesis. The neuroprotective effects of the Wnt pathway could be blocked by a Wnt1 antibody [28], and also, the Wnt1-targeted interfering RNA-induced Wnt1 depletion in midbrain astrocytes resulted in a substantial decrease in TH⁺ neuron survival upon serum deprivation and 6-OHDA or MPP+ treatment in neuron-astrocyte cocultures [32].
Furthermore, the Fzd-1 immunofluorescent signal largely increased in the rescued TH⁺ neurons in dopaminergic DA neurons cocultured with midbrain astrocytes, oppositely to the dramatic Fzd-1 receptor downregulation observed in purified neurons, either in vitro or in vivo, following the neurotoxic insult [32].
Interestingly, exogenous activation of Wnt signaling with a specific GSK-3β (glycogen synthase kinase 3) antagonist sharply amplified astrocyte-induced DA neuroprotection in MPP+-treated astroglia-neuron cocultures. Glial inserts or Wnt1 direct addition to purified DA neurons just before MPP+ insult largely conferred neuroprotection, which was blocked by a Wnt1 antibody or the Wnt antagonist Fzd-1-cysteine-rich domain, supporting the critical role of Wnt1 in dopaminergic neuron survival [28]. Over and above, pharmacological inhibition of GSK-3β activity increased neuroblasts’ population and promoted their migration towards the rostral migratory stream and the lesioned striatum in PD animal models [33]. Inhibiting GSK-3β enhanced dendritic arborization and survival of the granular neurons and stimulated neural stem cell-to-neuronal phenotype differentiation in the hippocampus of PD animal models. Figure 1 summarily illustrates the Wnt/β-catenin/Fdz-1 pathway.
... They are most commonly found in the plasma membrane, although they have also been reportedin the membranes of cellular organelles [17]. Mutations in Park7 were shown to result in the increased degradation of the lipid raft proteins flotillin-1 and caveolin-1, and to lead to Inflammatory response [14,[20][21][22] Mitochondrial function [25][26][27] Neurotrophic capacity [23][24][25][26] Oxidative stress [25,26] SNCA a-synuclein Endocytosis [35][36][37][38] Fatty acid metabolism [31] Glutamate uptake [40] Inflammatory response [37][38][39] Neurotrophic capacity [40] Water transport [40] PLA2G6 Group VI Ca 2+ -independent phospholipase A 2 (iPLA 2 ) Calcium signalling [43,44] Fatty acid metabolism [42] Inflammatory response [44] ATP13A2 Lysosomal type 5 ATPase (ATP13A2) Inflammatory response [49] Lysosome function [49] Neurotrophic capacity [49] LRRK2 Leucine-rich repeat kinase 2 (LRRK2) Autophagy [55] Lysosome function [56] GBA b-Glucocerebrosidase (GCase) Autophagy [61] Lysosome function [62] Mitochondrial function [62] PINK1 PTEN-induced putative kinase 1 (PINK1) Embryonic development [64] Mitochondrial function [65] Proliferation [64,65] PARK2 Parkin Inflammatory response [73] Mitochondrial function [71] Neuroprotection [69] Proliferation [69,70] Unfolded protein response [72] disrupted lipid raft assembly [15]. As a result of this disruption, Park7 knockout (KO) and mutant astrocyteswere found to exhibit impairedglutamate uptake [15]. ...
... They are most commonly found in the plasma membrane, although they have also been reportedin the membranes of cellular organelles [17]. Mutations in Park7 were shown to result in the increased degradation of the lipid raft proteins flotillin-1 and caveolin-1, and to lead to Inflammatory response [14,[20][21][22] Mitochondrial function [25][26][27] Neurotrophic capacity [23][24][25][26] Oxidative stress [25,26] SNCA a-synuclein Endocytosis [35][36][37][38] Fatty acid metabolism [31] Glutamate uptake [40] Inflammatory response [37][38][39] Neurotrophic capacity [40] Water transport [40] PLA2G6 Group VI Ca 2+ -independent phospholipase A 2 (iPLA 2 ) Calcium signalling [43,44] Fatty acid metabolism [42] Inflammatory response [44] ATP13A2 Lysosomal type 5 ATPase (ATP13A2) Inflammatory response [49] Lysosome function [49] Neurotrophic capacity [49] LRRK2 Leucine-rich repeat kinase 2 (LRRK2) Autophagy [55] Lysosome function [56] GBA b-Glucocerebrosidase (GCase) Autophagy [61] Lysosome function [62] Mitochondrial function [62] PINK1 PTEN-induced putative kinase 1 (PINK1) Embryonic development [64] Mitochondrial function [65] Proliferation [64,65] PARK2 Parkin Inflammatory response [73] Mitochondrial function [71] Neuroprotection [69] Proliferation [69,70] Unfolded protein response [72] disrupted lipid raft assembly [15]. As a result of this disruption, Park7 knockout (KO) and mutant astrocyteswere found to exhibit impairedglutamate uptake [15]. ...
... They are most commonly found in the plasma membrane, although they have also been reportedin the membranes of cellular organelles [17]. Mutations in Park7 were shown to result in the increased degradation of the lipid raft proteins flotillin-1 and caveolin-1, and to lead to Inflammatory response [14,[20][21][22] Mitochondrial function [25][26][27] Neurotrophic capacity [23][24][25][26] Oxidative stress [25,26] SNCA a-synuclein Endocytosis [35][36][37][38] Fatty acid metabolism [31] Glutamate uptake [40] Inflammatory response [37][38][39] Neurotrophic capacity [40] Water transport [40] PLA2G6 Group VI Ca 2+ -independent phospholipase A 2 (iPLA 2 ) Calcium signalling [43,44] Fatty acid metabolism [42] Inflammatory response [44] ATP13A2 Lysosomal type 5 ATPase (ATP13A2) Inflammatory response [49] Lysosome function [49] Neurotrophic capacity [49] LRRK2 Leucine-rich repeat kinase 2 (LRRK2) Autophagy [55] Lysosome function [56] GBA b-Glucocerebrosidase (GCase) Autophagy [61] Lysosome function [62] Mitochondrial function [62] PINK1 PTEN-induced putative kinase 1 (PINK1) Embryonic development [64] Mitochondrial function [65] Proliferation [64,65] PARK2 Parkin Inflammatory response [73] Mitochondrial function [71] Neuroprotection [69] Proliferation [69,70] Unfolded protein response [72] disrupted lipid raft assembly [15]. As a result of this disruption, Park7 knockout (KO) and mutant astrocyteswere found to exhibit impairedglutamate uptake [15]. ...
Astrocytes are the most populous glial subtype and are critical for brain function. Despite this, historically there have been few studies into the role that they may have in neurodegenerative diseases, such as Parkinson's disease (PD). Recently, however, several studies have determined that genes known to have a causative role in the development of PD are expressed in astrocytes and have important roles in astrocyte function. Here, we review these recent developments and discuss their impact on our understanding of the pathophysiology of PD, and the implications that this might have for its treatment.
... Besides MPTP, several other environmental toxins like rotenone, fenazaquin, trichloroethylene, paraquat, tebunfenpyrad, and fenpyroximate were identified to induce lossing of the nigral dopaminergic neurons in vivo models, and implicates in mitochondrial dysfunction in PD pathogenesis [50][51][52]. Like MPTP, these neurotoxins also inhibit mitochondrial complex I [53], triggering mitochondrial dysfunction by inducing ROS production, impairing ATP synthesis, increasing membrane permeability, and decreasing mitochondrial motility, which ultimately leads to neuronal damage in SN [54,55]. Inhibition of complex I to a small degree has been shown to significantly increase ROS production [56], which in turn inhibits complex I [57], creating a vicious cycle leading to mitochondrial damage and neuronal death [16]. ...
... With increased oxidative stress, DJ-1 expression is enhanced in reactive astrocytes [127], and excessive expression of DJ-1 is detected in reactive astrocytes in sporadic PD [128]. DJ-1 knockdown or KO in astrocytes impairs astrocyte-mediated neuroprotection against oxidative stress by deregulation of inflammatory responses and mitochondrial complex I damage (Fig. 2) [53,129]. ...
Parkinson's disease (PD) is a common age-related neurodegenerative disorder whose pathogenesis is not completely understood. Mitochondrial dysfunction and increased oxidative stress have been considered as major cause and central event responsible for the progressive degeneration of dopaminergic (DA) neurons in PD. Therefore, investigating mitochondrial disorders plays a role in understanding the pathogenesis of PD and can be an important therapeutic target for this disease. This study discusses the effect of environmental, genetic and biological factors on mitochondrial dysfunction and also focuseson the mitochondrial molecular mechanisms underlying neurodegeneration, and its possible therapeutic targets in PD, including reactive oxygen species generation, calcium overload, inflammasome activation, apoptosis, mitophagy, mitochondrial biogenesis, and mitochondrial dynamics. Other potential therapeutic strategies such as mitochondrial transfer/transplantation, targeting microRNAs, using stem cells, photobiomodulation, diet, and exercise were also discussed in this review, which may provide valuable insights into clinical aspects. A better understanding of the roles of mitochondria in the pathophysiology of PD may provide a rationale design of novel therapeutic interventions in our fight against PD.
... DJ-1 was originally identified as an oncogene and later associated with PD and diabetes mellitus [94]. The DJ-1 protein is expressed in reactive astrocytes and, to a lower extent, in neurons [95,96]. It is involved in mitochondrial function, apoptosis regulation, pro-survival signaling, autophagy, inflammatory responses, protection against oxidative stress, and chaperone activity [97]. ...
... DJ-1 loss of function is also associated with increased inflammatory responses. siRNA DJ-1-knockdown mouse astrocytes are less able to protect against neurotoxins such as rotenone when compared to wild-type controls [96]. Furthermore, there is a reduced expression of prostaglandin D2 synthase, which regulates anti-inflammatory responses. ...
Parkinson’s disease (PD) is a multifarious neurodegenerative disease. Its pathology is characterized by a prominent early death of dopaminergic neurons in the pars compacta of the substantia nigra and the presence of Lewy bodies with aggregated α-synuclein. Although the α-synuclein pathological aggregation and propagation, induced by several factors, is considered one of the most relevant hypotheses, PD pathogenesis is still a matter of debate. Indeed, environmental factors and genetic predisposition play an important role in PD. Mutations associated with a high risk for PD, usually called monogenic PD, underlie 5% to 10% of all PD cases. However, this percentage tends to increase over time because of the continuous identification of new genes associated with PD. The identification of genetic variants that can cause or increase the risk of PD has also given researchers the possibility to explore new personalized therapies. In this narrative review, we discuss the recent advances in the treatment of genetic forms of PD, focusing on different pathophysiologic aspects and ongoing clinical trials.
... LD50 for this product is 245 mg/kg for mammalians [3]. Laboratory studies have classified fenpyroximate (along with rotenone) as a Complex I inhibiting pesticide that is neurotoxic to astrocytes when cultured by in vitro bioassays [4]. Despite its worldwide usage as an insecticide, there are only few reported cases of its human toxicity. ...
... According to the Threshold of Toxicological Concern (TTC) fenpyroximate is classified as a Class III substance by the Cramer scheme suggesting significant toxicity or to have reactive functional groups (3). Complex I-inhibiting neurotoxins, such as the pesticide rotenone is known to cause neuronal death and parkinsonism in animal models [4]. Furthermore, pesticide exposure is epidemiologically-linked with an increased risk for Parkinson's disease [8]. ...
... Increased astrocytic DJ-1 is also found adjacent to infarcted brain regions after stroke [13]. Moreover, in astrocyte primary cultures DJ-1 regulates inflammatory responses [14], and in neuron-astrocyte cocultures astrocytic DJ-1 expression protects neurons from mitochondrial complex I-induced oxidative stress [15]. Astrocytic DJ-1 expression therefore seems to have a major role in protecting neurons from oxidative damage and may produce neuron-protective factors, although these agents have not yet been identified [16]. ...
... This may be a way not only to protect themselves, but also their neighboring neurons. Thus, studies in primary co-cultures of astrocytesneurons show that knock-down of astrocytic DJ-1 renders the neurons more susceptible to neurotoxin-induced oxidative stress [15,50]. Additionally, it has been shown that conditioned media from astrocytic cultures of wild type mice can protect neuronal cells from oxidative insult, whereas media derived from DJ-1 knock-out mice does not [51]. ...
DJ-1, a Parkinson´s disease-associated protein, is strongly up-regulated in reactive astrocytes in Parkinson´s disease. This is proposed to represent a neuronal protective response, although the mechanism has not yet been identified. We have generated a transgenic zebrafish line with increased astroglial DJ-1 expression driven by regulatory elements from the zebrafish GFAP gene. Larvae from this transgenic line are protected from oxidative stress-induced injuries as caused by MPP⁺, a mitochondrial complex I inhibitor shown to induce dopaminergic cells death. In a global label-free proteomics analysis of wild type and transgenic larvae exposed to MPP⁺, 3418 proteins were identified, in which 366 proteins were differentially regulated. In particular, we identified enzymes belonging to primary metabolism to be among proteins affected by MPP⁺ in wild type animals, but not affected in the transgenic line. Moreover, by performing protein profiling on isolated astrocytes we showed that an increase in astrocytic DJ-1 expression up-regulated a large group of proteins associated with redox regulation, inflammation and mitochondrial respiration. The majority of these proteins have also been shown to be regulated by Nrf2. These findings provide a mechanistic insight into the protective role of astroglial up-regulation of DJ-1 and show that our transgenic zebrafish line with astrocytic DJ-1 over-expression can serve as a useful animal model to understand astrocyte-regulated neuroprotection associated with oxidative stress-related neurodegenerative disease.
... Recently, it is known as a multifunctional protein that plays a significant role in regulation of transcriptional activity (Blackinton et al., 2009), has anti-apoptotic properties (Haigang et al., 2012), is a chaperone molecule folding proteins into a 3-dimensional shape and protects against oxidative stress (Taira et al., 2004) through regulation of mitochondrial homeostasis (Takahashi-Niki et al., 2012). DJ-1 is highly expressed in the astrocytes, which are specialized glial cells that defend surrounding neurons against oxidative stress induced-neuronal death by secreting antioxidants (Bandopadhyay et al., 2004;Ashley et al., 2009;Mullett and Hinkle, 2011). In cells, DJ-1 plays a neuroprotective role by translocating to the mitochondria and directly binding to NDUFA4 and ND1 subunits of mitochondrial complex I to maintain its activity and integrity (Takahashi-Niki et al., 2012;Hayashi et al., 2009). ...
... Loss of DJ-1 was found to directly inhibit complex I by blocking electron transport (Kwon et al., 2011). For example, DJ-1 knockdown in astrocytes was found significantly less neuroprotective compared to functional DJ-1 astrocytes because DJ-1 deficiency impaired the selective capacity of astrocytes to protect neurons against complex I inhibiting pesticides (Mullett and Hinkle, 2011). This is consistent with previous findings in which DJ-1 overexpression in astrocytes was able to rescue the effects of rotenone induced-complex I inhibition (Mullett and Hinkle, 2009). ...
... Similar to heavy metals, this exposure leads to disruption of the BBB and activation of astrocytes, based on numerous studies conducted in vivo and in vitro [68][69][70][71][72][73][74]. Furthermore, research has indicated that Parkinson's disease protein 7 (PARK7/DJ-1), found in astrocytes, plays a crucial role in regulating the neurotoxic consequences caused by the pesticide rotenone [75][76][77]. Additionally, maintaining astrocyte homeostasis is closely associated with other environmental factors such as gut microbiota, composition intake of dietary components, and air pollution [78][79][80][81]. ...
Astrocytes displaying reactive phenotypes are characterized by their ability to remodel morphologically, molecularly, and functionally in response to pathological stimuli. This process results in the loss of their typical astrocyte functions and the acquisition of neurotoxic or neuroprotective roles. A growing body of research indicates that these reactive astrocytes play a pivotal role in the pathogenesis of amyotrophic lateral sclerosis (ALS), involving calcium homeostasis imbalance, mitochondrial dysfunction, abnormal lipid and lactate metabolism, glutamate excitotoxicity, etc. This review summarizes the characteristics of reactive astrocytes, their role in the pathogenesis of ALS, and recent advancements in astrocyte-targeting strategies.
... Human DJ-1 is predominantly localized to the cytoplasm, but it has been reported to be translocated to the mitochondria and nucleus under oxidative stress and to protect cells from oxidative stress-induced cell death (Irrcher et al., 2010;Kim et al., 2012). On the other hand, some studies also show that DJ-1 may be localized to mitochondria even in the absence of oxidative stress, where it directly binds to a subunit of complex I and somehow maintains its activity, since knockdown of DJ-1 in cells decreased complex I activity (Hayashi et al., 2009;Mullett and Hinkle, 2011). A recent study also showed its connection to ATP synthase, where DJ-1 is required for the normal stoichiometry of ATP synthase and to facilitate positioning of the β subunit of ATP synthase to fully close the mitochondrial inner membrane leak (Chen et al., 2019). ...
Although copper is an essential nutrient crucial for many biological processes, an excessive concentration can be toxic and lead to cell death. The metabolism of this two-faced metal must be strictly regulated at the cell level. In this study, we investigated copper homeostasis in two related unicellular organisms: nonpathogenic Naegleria gruberi and the “brain-eating amoeba” Naegleria fowleri. We identified and confirmed the function of their specific copper transporters securing the main pathway of copper acquisition. Adjusting to different environments with varying copper levels during the life cycle of these organisms requires various metabolic adaptations. Using comparative proteomic analyses, measuring oxygen consumption, and enzymatic determination of NADH dehydrogenase, we showed that both amoebas respond to copper deprivation by upregulating the components of the branched electron transport chain: the alternative oxidase and alternative NADH dehydrogenase. Interestingly, analysis of iron acquisition indicated that this system is copper-dependent in N. gruberi but not in its pathogenic relative. Importantly, we identified a potential key protein of copper metabolism of N. gruberi, the homolog of human DJ-1 protein, which is known to be linked to Parkinson’s disease. Altogether, our study reveals the mechanisms underlying copper metabolism in the model amoeba N. gruberi and the fatal pathogen N. fowleri and highlights the differences between the two amoebas.
... The beginning of PD may be facilitated by an age-related malfunction of astrocytes that reduces their number, increases their cellular volume, facilitates the overlap of their processes, and increases their GFAP content [102,[133][134][135][136]. PD may also be facilitated by changes in the activity of different genes which increase the incidence of PD and are directly involved in the astrocyte biology [137][138][139][140][141][142][143][144][145][146][147][148][149]. This is the case of PARK7 (DJ-1 protein), which is involved in the glutamate uptake, mitochondrial function, oxidative stress, and inflammatory response of astrocytes [150][151][152][153][154]; PARK2 (Parkin), which is involved in the inflammatory response, neuroprotection, proliferation, and mitochondrial functions of astrocytes [149,[155][156][157]; SNCA (α-synuclein), which is involved in glutamate uptake, neurotrophic activity, water transport, and endocytosis functions of astrocytes [158][159][160][161][162]; PINK1 (PTEN-induced putative kinase 1), which is involved in proliferation and mitochondrial function of astrocytes [139,163]; GBA (β-glucorecebrosidase), which is involved in autophagy, lysosome functions, and mitochondrial functions of astrocytes [164,165]; LRRK2 (leucine-rich repeat kinase 2), which is involved in autophagy and lysosome functions of astrocytes [166][167][168]; ATP13A2 (lysosomal type 5 ATPase), which is involved in the neurotrophic activity, inflammatory response, and lysosome functions of astrocytes [169]; and PLA2G6 (group VI Ca 2+ -independent phospholipase A 2 ), which is involved in inflammatory response and calcium signaling functions of astrocytes [170,171]. Thus, the significance that the effects of these mutations have on the onset and progression of PD are probably linked to alterations in the physiological activity of astrocytes. ...
At present, there is no efficient treatment to prevent the evolution of Parkinson’s disease (PD). PD is generated by the concurrent activity of multiple factors, which is a serious obstacle for the development of etio-pathogenic treatments. Astrocytes may act on most factors involved in PD and the promotion of their neuroprotection activity may be particularly suitable to prevent the onset and progression of this basal ganglia (BG) disorder. The main causes proposed for PD, the ability of astrocytes to control these causes, and the procedures that can be used to promote the neuroprotective action of astrocytes will be commented upon, here.
... DJ-1, encoded by the PARK7 gene, causes early onset autosomal recessive PD (54) and is probably the most extensively studied. Mullett and Hinkle utilised neuron-astrocyte co-cultures to demonstrate that siRNA knockdown of DJ-1 in mouse astrocytes impairs their ability to protect against neurotoxins such as rotenone relative to wild type control astrocytes (55). In addition, studies using DJ-1 knockout mice astrocytes from postnatal day 1 cerebral cortices have also shown that loss of this gene can cause alterations in cholesterol levels and glutamate uptake via regulation of the expression of flotillin-1 and caveolin-1 (40). ...
Parkinson's disease (PD), the second most common neurodegenerative disease, is characterised by the motor symptoms of bradykinesia, rigidity and resting tremor and non-motor symptoms of sleep disturbances, constipation, and depression. Pathological hallmarks include neuroinflammation, degeneration of dopaminergic neurons in the substantia nigra pars compacta, and accumulation of misfolded α-synuclein proteins as intra-cytoplasmic Lewy bodies and neurites. Microglia and astrocytes are essential to maintaining homeostasis within the central nervous system (CNS), including providing protection through the process of gliosis. However, dysregulation of glial cells results in disruption of homeostasis leading to a chronic pro-inflammatory, deleterious environment, implicated in numerous CNS diseases. Recent evidence has demonstrated a role for peripheral immune cells, in particular T lymphocytes in the pathogenesis of PD. These cells infiltrate the CNS, and accumulate in the substantia nigra, where they secrete pro-inflammatory cytokines, stimulate surrounding immune cells, and induce dopaminergic neuronal cell death. Indeed, a greater understanding of the integrated network of communication that exists between glial cells and peripheral immune cells may increase our understanding of disease pathogenesis and hence provide novel therapeutic approaches.
... DJ-1 overexpression was observed in the astrocytes in sporadic PD (Bandopadhyay et al. 2004;Neumann et al. 2004;Mullett, Hamilton, and Hinkle 2009) and excessively oxidised form of DJ-1 was found in the brains of PD patients (Bandopadhyay et al. 2004;Choi et al. 2006). Knockdown/knockout of DJ-1 expression in astrocytes resulted in disrupted neuroprotection against oxidative stress and inflammatory responses (Peng et al. 2019;Mullett and Hinkle 2011;Larsen et al. 2011). Furthermore, both DJ-1 and PINK1 mutations were found in early onset PD cases (Tang et al. 2006) which is suggestive of a possible functional interaction between the two genes in causing PD. ...
Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial and sporadic Parkinson’s disease. LRRK2 protein is expressed prominently in immune cells, cell types whose contribution to LRRK2-associated Parkinson’s disease is increasingly being recognised. For my PhD, I investigated the effect of inflammatory stimulation using murine RAW264.7 wild-type, T1348N-LRRK2 (artificial mutation which abrogates GTP binding) and LRRK2 knockout macrophage cell lines and wild-type and G2019S-LRRK2 (natural most common PD mutation) primary microglia, as model systems. Immunoblotting and ELISA-based assays were used to investigate a detailed time course of TLR2/TLR4 stimulation with LPS and zymosan by studying LRRK2 phosphorylation at its specific phosphorylation sites, Rab and MAPK proteins and by measuring cytokine release in RAW264.7 macrophages and primary microglia. Four selective LRRK2 kinase inhibitors were also employed to understand LRRK2’s role in immune cell function and finally some bioinformatics work was performed to identify differentially expressed genes using RNA Sequencing. The latter part of my work was conducted at National Institutes of Health, USA. Data presented in this thesis is over three experimental chapters with first two chapters investigating LRRK2 functionality in terms of phosphorylation and cytokine release in RAW264.7 macrophages and the final chapter investigating it in terms of phosphorylation, cytokine release and transcriptomics work in primary microglia. RAW264.7 work confirmed Rab8 and Rab10 as substrates of LRRK2 activity with a differential response of dephosphorylated Rab10 seen with LPS stimulation. Loss of GTP binding due to T1348N and LRRK2 knockout coupled with LRRK2 kinase inhibitors and different inflammatory stimuli exhibited distinct responses in cytokine secretion, particularly with IL-10. Primary microglia work with zymosan and LRRK2 kinase inhibition also revealed varying patterns in cytokine secretion and transcriptomics work identified a wide range of differentially expressed genes with various enriched biological processes. Overall, my data demonstrates for the first time the dynamics observed in LRRK2 and Rab phosphorylation and cytokine release under different experimental conditions in these cellular models. This provides further insight into the function of LRRK2 in immune response and has relevance to understanding cellular dysfunctions when developing LRRK2 based inhibitors for clinical treatment of Parkinson’s disease.
... Reactive astrocytes induce DJ-1 expression in response to oxidative stress and release the protein extracellularly to protect neurons [81,165]. Several studies have demonstrated that DJ-1 deficiency or mutation in astrocytes impairs astrocyte-mediated neuroprotection in parkinsonian models [166][167][168]. In addition, DJ-1 deficiency impairs glutamate uptake into astrocytes by altering EAAT2 expression [169], and reduces mitochondrial motility in astrocytes [166]. ...
Parkinson’s disease (PD) is the second most common neurodegenerative disease. PD patients exhibit motor symptoms such as akinesia/bradykinesia, tremor, rigidity, and postural instability due to a loss of nigrostriatal dopaminergic neurons. Although the pathogenesis in sporadic PD remains unknown, there is a consensus on the involvement of non-neuronal cells in the progression of PD pathology. Astrocytes are the most numerous glial cells in the central nervous system. Normally, astrocytes protect neurons by releasing neurotrophic factors, producing antioxidants, and disposing of neuronal waste products. However, in pathological situations, astrocytes are known to produce inflammatory cytokines. In addition, various studies have reported that astrocyte dysfunction also leads to neurodegeneration in PD. In this article, we summarize the interaction of astrocytes and dopaminergic neurons, review the pathogenic role of astrocytes in PD, and discuss therapeutic strategies for the prevention of dopaminergic neurodegeneration. This review highlights neuron-astrocyte interaction as a target for the development of disease-modifying drugs for PD in the future.
... DJ-1 has a recognized role for the maintainance of astrocytic mitochondrial functions and the regulation of oxidative stress and inflammatory pathways (110,111,151,152 ]. Hence, DJ-1 deficiency impairs astrocyte ability to protect DAergic neurons against rotenone [153] and 6-OHDA [154], and selectively enhances mitochondrial Complex I inhibitor-induced neurotoxicity [155]. Opposedly, astrocytic over-expression of DJ-1, in vitro, prevented oxidative stress and mitochondrial dysfunction in primary neurons [156]. ...
Oxidative stress and inflammation have long been recognized to contribute to Parkinson’s disease (PD), a common movement disorder characterized by the selective loss of midbrain dopaminergic neurons (mDAn) of the substantia nigra pars compacta (SNpc). The causes and mechanisms still remain elusive, but a complex interplay between several genes and a number of interconnected environmental factors, are chiefly involved in mDAn demise, as they intersect the key cellular functions affected in PD, such as the inflammatory response, mitochondrial, lysosomal, proteosomal and autophagic functions. Nuclear factor erythroid 2 -like 2 (NFE2L2/Nrf2), the master regulator of cellular defense against oxidative stress and inflammation, and Wingless (Wnt)/β-catenin signaling cascade, a vital pathway for mDAn neurogenesis and neuroprotection, emerge as critical intertwinned actors in mDAn physiopathology, as a decline of an Nrf2/Wnt/β-catenin prosurvival axis with age underlying PD mutations and a variety of noxious environmental exposures drive PD neurodegeneration. Unexpectedly, astrocytes, the so-called “star-shaped” cells, harbouring an arsenal of “beneficial” and “harmful” molecules represent the turning point in the physiopathological and therapeutical scenario of PD. Fascinatingly, “astrocyte’s fil rouge” brings back to Nrf2/Wnt resilience, as boosting the Nrf2/Wnt resilience program rejuvenates astrocytes, in turn (i) mitigating nigrostriatal degeneration of aged mice, (ii) reactivating neural stem progenitor cell proliferation and neuron differentiation in the brain and (iii) promoting a beneficial immunomodulation via bidirectional communication with mDAns. Then, through resilience of Nrf2/Wnt/β-catenin anti-ageing, prosurvival and proregenerative molecular programs, it seems possible to boost the inherent endogenous self-repair mechanisms. Here, the cellular and molecular aspects as well as the therapeutical options for rejuvenating glia-neuron dialogue will be discussed together with major glial-derived mechanisms and therapies that will be fundamental to the identification of novel diagnostic tools and treatments for neurodegenerative diseases (NDs), to fight ageing and nigrostriatal DAergic degeneration and promote functional recovery.
... The role of DJ-1 in astrocyte-mediated neuroprotection is dependent on mitochondrial complex I (Mullett and Hinkle, 2011). Since DJ-1 serves as a redox sensor that recognize oxidative stress (Cao et al., 2014), DJ-1 mutations may contribute to PD pathogenesis by dysregulating nitrosative stress in astrocytes. ...
While glia are essential for regulating the homeostasis in the normal brain, their dysfunction contributes to neurodegeneration in many brain diseases, including Parkinson's disease (PD). Recent studies have identified that PD-associated genes are expressed in glial cells as well as neurons and have crucial roles in microglia and astrocytes. Here, we discuss the role of microglia and astrocytes dysfunction in relation to PD-linked mutations and their implications in PD pathogenesis. A better understanding of microglia and astrocyte functions in PD may provide insights into neurodegeneration and novel therapeutic approaches for PD.
... Several studies have determined that genes known to have a causative role in the development of PD are expressed in glial cells and have important roles in glial function. For e.g., DJ-1 (encoded by Park7), which is a chaperone that suppresses α-syn fibrillation [185], mediates neuroprotection through an astrocyte-dependent mechanism involving extracellular-secreted soluble factors (anti-oxidant molecules, bioenergetic molecules, cytokines, and peptide neurotrophic factors) [186][187][188]. To prove the importance of this protein in neuroprotection, Lev et al. did a KO of Park7 in astrocytes, which resulted in a decrease in astrocytes' capacity to protect DA neurons both in rotenone and 6-OHDA neurotoxin PD models (Fig. 3) [189,190]. ...
Glial cells have been identified more than 100 years ago, and are known to play a key role in the central nervous system (CNS) function. A recent piece of evidence is emerging showing that in addition to the capacity of CNS modulation and homeostasis, glial cells are also being looked like as a promising cell source not only to study CNS pathologies initiation and progression but also to the establishment and development of new therapeutic strategies. Thus, in the present review, we will discuss the current evidence regarding glial cells’ contribution to neurodegenerative diseases as Parkinson’s disease, providing cellular, molecular, functional, and behavioral data supporting its active role in disease initiation, progression, and treatment. As so, considering their functional relevance, glial cells may be important to the understanding of the underlying mechanisms regarding neuronal-glial networks in neurodegeneration/regeneration processes, which may open new research opportunities for their future use as a target or treatment in human clinical trials.
... Moreover, in PD pathophysiology, activated glia produce neurotoxic factors such as glutamate, S100B, TNF-α, prostaglandins, and reactive oxygen and nitrogen species, resulting in neuronal injury and leading to cell death [221]. The disruption of inflammatory signaling pathways has been shown to result in changes in essential astrocyte functions, including glutamate transport [210,214], water transport [214], and neurotrophic capacity [220,[222][223][224]. All of these functions are important for neuronal health, and it has been demonstrated that changes of this type in astrocytes result in the degeneration of neighboring neurons. ...
Astrocytes are key cells for adequate brain formation and regulation of cerebral blood flow as well as for the maintenance of neuronal metabolism, neurotransmitter synthesis and exocytosis, and synaptic transmission. Many of these functions are intrinsically related to neurodegeneration, allowing refocusing on the role of astrocytes in physiological and neurodegenerative states. Indeed, emerging evi- dence in the field indicates that abnormalities in the astrocytic function are involved in the pathogenesis of multiple neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS). In the present review, we highlight the physiological role of astrocytes in the CNS, including their communication with other cells in the brain. Furthermore, we discuss exciting findings and novel experimental approaches that elucidate the role of astrocytes in multiple neurological disorders.
... Mitochondrial oxidative stress also plays an important role in autophagy impairment in synucleinopathies. The antioxidant properties of the DJ-1 protein have been previously reported, as it could decrease oxidative stress and restore autophagy [169][170][171]. Increasing DJ-1 expression in astrocytes co-cultured with neurons proved to have beneficial effects against rotenone-induced cell oxidation [169]. ...
Alpha-synuclein positive-intracytoplasmic inclusions are the common denominators of the synucleinopathies present as Lewy bodies in Parkinson’s disease, dementia with Lewy bodies, or glial cytoplasmic inclusions in multiple system atrophy. These neurodegenerative diseases also exhibit cellular dyshomeostasis, such as autophagy impairment. Several decades of research have questioned the potential link between the autophagy machinery and alpha-synuclein protein toxicity in synucleinopathy and neurodegenerative processes. Here, we aimed to discuss the active participation of autophagy impairment in alpha-synuclein accumulation and propagation, as well as alpha-synuclein-independent neurodegenerative processes in the field of synucleinopathy. Therapeutic approaches targeting the restoration of autophagy have started to emerge as relevant strategies to reverse pathological features in synucleinopathies.
... Knock-out of parkin is a mutation which causes familial PD produced astroglial dysfunction and age-dependent vulnerability of neurons against oxidative stress that was corrected by supplementation with GSH [39]. DJ-1, a causative gene of a familial PD, is dominantly expressed in astrocytes to stabilize Nrf2 [40], and its knock-down in astrocytes impaired mitochondrial function and neuroprotective effects of astrocytes against dopaminergic neurotoxin [41,42]. NQO-1 expression increased in both astrocytes and neurons in the substantia nigra pars compacta of PD patients [43]. ...
In previous studies, we found regional differences in the induction of antioxidative molecules in astrocytes against oxidative stress, postulating that region-specific features of astrocytes lead region-specific vulnerability of neurons. We examined region-specific astrocytic features against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) as an oxidative stress using co-culture of mesencephalic neurons and mesencephalic or striatal astrocytes in the present study. The 6-OHDA-induced reduction of mesencephalic dopamine neurons was inhibited by co-culturing with astrocytes. The co-culture of midbrain neurons with striatal astrocytes was more resistant to 6-OHDA than that with mesencephalic astrocytes. Furthermore, glia conditioned medium from 6-OHDA-treated striatal astrocytes showed a greater protective effect on the 6-OHDA-induced neurotoxicity and oxidative stress than that from mesencephalic astrocytes. The cDNA microarray analysis showed that the number of altered genes in both mesencephalic and striatal astrocytes was fewer than that changed in either astrocyte. The 6-OHDA treatment, apparently up-regulated expressions of Nrf2 and some anti-oxidative or Nrf2-regulating phase II, III detoxifying molecules related to glutathione synthesis and export in the striatal astrocytes but not mesencephalic astrocytes. There is a profound regional difference of gene expression in astrocytes induced by 6-OHDA. These results suggest that protective features of astrocytes against oxidative stress are more prominent in striatal astrocytes, possibly by secreting humoral factors in striatal astrocytes.
... DJ-1 increases GSH and reduces neuronal damage induced by oxidative stress [24]. In contrast, Mullett and colleagues showed that DJ-1 did not affect GSH after cell treatment with rotenone [35]. We noted that DJ-1 was neuroprotective through GSH release by astrocytes after OGD/R. ...
Astrocytes are involved in neuroprotection, and DJ-1 is an important antioxidant protein that is abundantly expressed in reactive astrocytes. However, the role of DJ-1 in astrocytes’ neuroprotection in cerebral ischemia/reperfusion injury and its potential mechanism is unclear. Thus, to explore effects and mechanisms of DJ-1 on the neuroprotection of astrocytes, we used primary co-cultures of neurons and astrocytes under oxygen and glucose deprivation/reoxygenation in vitro and transient middle cerebral artery occlusion/reperfusion in vivo to mimic ischemic reperfusion insult. Lentiviral was used to inhibit and upregulate DJ-1 expression in astrocytes, and DJ-1 siRNA blocked DJ-1 expression in rats. Inhibiting DJ-1 expression led to decreases in neuronal viability. DJ-1 knockdown also attenuated total and nuclear Nrf2 and glutathione (GSH) levels in vitro and vivo. Similarly, loss of DJ-1 decreased Nrf2/ARE-binding activity and expression of Nrf2/ARE pathway-driven genes. Overexpression of DJ-1 yielded opposite results. This suggests that the mechanism of action of DJ-1 in astrocyte-mediated neuroprotection may involve regulation of the Nrf2/ARE pathway to increase GSH after cerebral ischemia/reperfusion injury. Thus, DJ-1 may be a new therapeutic target for treating ischemia/reperfusion injury.
Key Messages
• Astrocytes protect neurons in co-culture after OGD/R
• DJ-1 is upregulated in astrocytes and plays an important physiological roles in neuronal protection under ischemic conditions
• DJ-1 protects neuron by the Nrf2/ARE pathway which upregulates GSH
... DJ-1 overexpression in astrocytes protects neurons from pesticide-induced oxidative stress [123]. It appears that this is likely due to interference with complex 1 of the mitochondria [124]. When astrocytes were co-cultured with neurons, impaired mitochondria were located in the soma and proper fusion dynamics was observed in the processes. ...
α-Synuclein is an abundantly expressed protein located in the neuronal synapse which accumulates in Lewy body inclusions and synaptic aggregates in neurodegenerative diseases. Although evidence indicates that oligomeric α-synuclein can stimulate neurodegeneration, and α-synuclein amyloid fibrils can spread in a prion-like manner, the initial cause of α-synuclein dysregulation is uncertain. α-Synuclein released from the neuronal presynaptic terminal can accumulate in astrocytes early in disease progression and astrocyte dysfunction has been observed in the same disease phase. Astrocytes provide homeostatic balance to the brain parenchyma through release of neuroprotective factors and clearing of waste products to the vasculature, and astrocytic dysfunction has been shown to cause neuronal cell death. Like protein aggregation in other proteinopathies, such as neurofibrillary tangles and β-amyloid plaques, evidence is emerging that accumulation of abundantly expressed neuronal proteins could be a by-product of initial non-neuronal cellular degeneration. This review examines interactions between α-synuclein and astrocytes with a consideration of an astrocytic cause of synucleinopathy
... DJ-1 participates in mitochondrial homeostasis through regulation of mitochondrial complex I (Hayashi et al. 2009;Mullett and Hinkle 2011). It can be found mostly diffused in the cytoplasm (Nagakubo et al. 1997;Bonifati et al. 2003) and to a lesser extent in the mitochondria (Zhang et al. 2005) and nucleus (Nagakubo et al. 1997;Hod et al. 1999) Oxidation of DJ-1 at one of the three cysteines (C) at amino acid numbers 46, 53, and 106 regulates its function (Mitsumoto and Nakagawa 2001;Kinumi et al. 2004;Choi et al. 2006). ...
In the retina, oxidative stress can initiate a cascade of events that ultimately leads to a focal loss of RPE cells and photoreceptors, a major contributing factor in geographic atrophy. Despite these implications, the molecular regulation of RPE oxidative metabolism under physiological and pathological conditions remains largely unknown. DJ-1 functions as an antioxidant, redox-sensitive molecular chaperone, and transcription regulator, which protected cells from oxidative stress. Here we discuss our progress toward characterization of the DJ-1 function in the protection of RPE to oxidative stress.
... In endothelial cells, DJ-1 directly functions as an antioxidant via the oxidation of its cysteine residue [110,111]. In astrocytes, DJ-1 deficiency reduces their ability to protect neurons against the mitochondrial toxin, rotenone [112][113][114][115]. In addition, DJ-1 increases mitochondrial antioxidant H2S production in astrocytes through expression of cystathionine β-synthase (CBS), the major enzyme that catalyzes H2S production [116]. ...
Astrocytes and microglia support well-being and well-function of the brain through diverse functions in both intact and injured brain. For example, astrocytes maintain homeostasis of microenvironment of the brain through up-taking ions and neurotransmitters, and provide growth factors and metabolites for neurons, etc. Microglia keep surveying surroundings, and remove abnormal synapses or respond to injury by isolating injury sites and expressing inflammatory cytokines. Therefore, their loss and/or functional alteration may be directly linked to brain diseases. Since Parkinson's disease (PD)-related genes are expressed in astrocytes and microglia, mutations of these genes may alter the functions of these cells, thereby contributing to disease onset and progression. Here, we review the roles of astrocytes and microglia in intact and injured brain, and discuss how PD genes regulate their functions.
... Interestingly, unilateral injection of LV-hDJ-1/MuLV protected both the ipsilateral, and to a lesser extent, the contralateral SN and striatum from rotenone-induced neurodegeneration (Fig. 2). These observations support previous hypotheses that soluble factors released from astrocytes are dynamically regulated by DJ-1 expression, and may be manipulated to provide neuroprotection (Mullett and Hinkle, 2009;Hauser and Cookson, 2011;Mullett and Hinkle, 2011). To date, it remains unclear which signaling molecules are involved in this process. ...
DJ-1 is a redox-sensitive protein with several putative functions important in mitochondrial physiology, protein transcription, proteasome regulation, and chaperone activity. High levels of DJ-1 immunoreactivity are reported in astrocytes surrounding pathology associated with idiopathic Parkinson's disease, possibly reflecting the glial response to oxidative damage. Previous studies showed that astrocytic over-expression of DJ-1 in vitro prevented oxidative stress and mitochondrial dysfunction in primary neurons. Based on these observations, we developed a pseudotyped lentiviral gene transfer vector with specific tropism for CNS astrocytes in vivo to overexpress human DJ-1 protein in astroglial cells. Following vector delivery to the substantia nigra and striatum of adult Lewis rats, the DJ-1 transgene was expressed robustly and specifically within astrocytes. There was no observable transgene expression in neurons or other glial cell types. Three weeks after vector infusion, animals were exposed to rotenone to induce Parkinson's disease-like pathology, including loss of dopaminergic neurons, accumulation of endogenous α-synuclein, and neuroinflammation. Animals over-expressing hDJ-1 in astrocytes were protected from rotenone-induced neurodegeneration, and displayed a marked reduction in neuronal oxidative stress and microglial activation. In addition, α-synuclein accumulation and phosphorylation were decreased within substantia nigra dopaminergic neurons in DJ-1-transduced animals, and expression of LAMP-2A, a marker of chaperone mediated autophagy, was increased. Together, these data indicate that astrocyte-specific overexpression of hDJ-1 protects neighboring neurons against multiple pathologic features of Parkinson's disease and provides the first direct evidence in vivo of a cell non-autonomous neuroprotective function of astroglial DJ-1.
... The specific roles of astrocytes linked to other proteins whose mutations are causative of PD are poorly understood. Astrocytes lacking DJ-1 have reduced capacities to protect neurons from mitochondrial and oxidative stress insults (300,301). Nurr1/coREST in cultured astrocytes and microglia protects dopaminergic neurons from inflammation-induced cell death following lipopolysaccharide injection in mice (384). Although Nurr1 is an important regulatory factor in the generation of dopaminergic neurons, it is still too early to implicate alterations of this pathway in the pathogenesis of PD. ...
Astrogliopathy refers to alterations of astrocytes occurring in diseases of the nervous system, and it implies the involvement of astrocytes as key elements in the pathogenesis and pathology of diseases and injuries of the central nervous system. Reactive astrocytosis refers to the response of astrocytes to different insults to the nervous system, whereas astrocytopathy indicates hypertrophy, atrophy/degeneration and loss of function and pathological remodeling occurring as a primary cause of a disease or as a factor contributing to the development and progression of a particular disease. Reactive astrocytosis secondary to neuron loss and astrocytopathy due to intrinsic alterations of astrocytes occur in neurodegenerative diseases, overlap each other, and, together with astrocyte senescence, contribute to disease-specific astrogliopathy in aging and neurodegenerative diseases with abnormal protein aggregates in old age. In addition to the well-known increase in glial fibrillary acidic protein and other proteins in reactive astrocytes, astrocytopathy is evidenced by deposition of abnormal proteins such as β-amyloid, hyper-phosphorylated tau, abnormal α-synuclein, mutated huntingtin, phosphorylated TDP-43 and mutated SOD1, and PrPres, in Alzheimer's disease, tauopathies, Lewy body diseases, Huntington's disease, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease, respectively. Astrocytopathy in these diseases can also be manifested by impaired glutamate transport; abnormal metabolism and release of neurotransmitters; altered potassium, calcium and water channels resulting in abnormal ion and water homeostasis; abnormal glucose metabolism; abnormal lipid and, particularly, cholesterol metabolism; increased oxidative damage and altered oxidative stress responses; increased production of cytokines and mediators of the inflammatory response; altered expression of connexins with deterioration of cell-to-cell networks and transfer of gliotransmitters; and worsening function of the blood brain barrier, among others. Increased knowledge of these aspects will permit a better understanding of brain aging and neurodegenerative diseases in old age as complex disorders in which neurons are not the only players.
... (Kim et al., 2013a;Long-Smith et al., 2009;Reynolds et al., 2008;Zhang et al., 2005). PD-related genes PARK7, PINK1 and LRRK2 have also been reported to modulate mitochondrial dysfunction, oxidative stress and the inflammatory response of glial cells Moehle et al., 2012;Mullett and Hinkle, 2009;Mullett and Hinkle, 2011;Russo et al., 2015). Besides microglia, infiltrating cells such as T-lymphocytes, and neutrophiles have been reported to contribute to inflammation in neurodegenerative disorders including PD (Gonzalez and Pacheco, 2014;Ji et al., 2008;Rezai-Zadeh et al., 2009). ...
The loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the accumulation of protein inclusions (Lewy bodies) are the pathological hallmarks of Parkinson’s disease (PD). PD is triggered by genetic alterations, environmental/occupational exposures and aging. However, the exact molecular mechanisms linking these PD risk factors to neuronal dysfunction are still unclear. Alterations in redox homeostasis and bioenergetics (energy failure) are thought to be central components of neurodegeneration that contribute to the impairment of important homeostatic process in dopaminergic cells such as protein quality control mechanisms, neurotransmitter release/metabolism, axonal transport of vesicles and cell survival. Importantly, both bioenergetics and redox homeostasis are coupled to neuro-glial central carbon metabolism. We and others have recently established a link between the alterations in central carbon metabolism induced by PD risk factors, redox homeostasis and bioenergetics and their contribution to the survival/death of dopaminergic cells. In this review, we focus on the link between metabolic dysfunction, energy failure and redox imbalance in PD, making an emphasis in the contribution of central carbon (glucose) metabolism. The evidence summarized here strongly supports the consideration of PD as a disorder of cell metabolism.
... Whether this transcriptional and cellular response is related to the marked clinical efficacy of sulforaphane at treating ASD symptoms 27 remains to be determined. Numerous studies investigated a link between the inhibition of mitochondrial complex I, neurotoxicity and neurodegeneration in animal models 24,34,35 . Rotenone (in cluster 2) has been shown to increase Parkinson's disease risk in humans 25 . ...
Environmental factors, including pesticides, have been linked to autism and neurodegeneration risk using retrospective epidemiological studies. Here we sought to prospectively identify chemicals that share transcriptomic signatures with neurological disorders, by exposing mouse cortical neuron-enriched cultures to hundreds of chemicals commonly found in the environment and on food. We find that rotenone, a pesticide associated with Parkinson's disease risk, and certain fungicides, including pyraclostrobin, trifloxystrobin, famoxadone and fenamidone, produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism, advanced age and neurodegeneration (Alzheimer's disease and Huntington's disease). These chemicals stimulate free radical production and disrupt microtubules in neurons, effects that can be reduced by pretreating with a microtubule stabilizer, an antioxidant, or with sulforaphane. Our study provides an approach to prospectively identify environmental chemicals that transcriptionally mimic autism and other brain disorders.
... Reactive astrocytes enhance DJ-1 expression in the case of oxidative stress and release DJ-1 protein extracellularly to protect neurons [89]. Several studies demonstrated that DJ-1 deficiency or mutation in astrocytes impairs astrocyte-mediated neuroprotection in parkinsonian models [90][91][92]. Clements et al. [93] reported that Nrf2 protein without intact DJ-1 is unstable and its basal or inducible levels of transcriptional responses are decreased. These observations suggest that DJ-1 stabilizes antioxidant transcriptional regulation of Nrf2. ...
Astrocytes are the most abundant neuron-supporting glial cells in the central nervous system. The neuroprotective role of astrocytes has been demonstrated in various neurological disorders such as amyotrophic lateral sclerosis, spinal cord injury, stroke and Parkinson's disease (PD). Astrocyte dysfunction or loss-of-astrocytes increases the susceptibility of neurons to cell death, while astrocyte transplantation in animal studies has therapeutic advantage. We reported recently that stimulation of serotonin 1A (5-HT1A) receptors on astrocytes promoted astrocyte proliferation and upregulated antioxidative molecules to act as a neuroprotectant in parkinsonian mice. PD is a progressive neurodegenerative disease with motor symptoms such as tremor, bradykinesia, rigidity and postural instability, that are based on selective loss of nigrostriatal dopaminergic neurons, and with non-motor symptoms such as orthostatic hypotension and constipation based on peripheral neurodegeneration. Although dopaminergic therapy for managing the motor disability associated with PD is being assessed at present, the main challenge remains the development of neuroprotective or disease-modifying treatments. Therefore, it is desirable to find treatments that can reduce the progression of dopaminergic cell death. In this article, we summarize first the neuroprotective properties of astrocytes targeting certain molecules related to PD. Next, we review neuroprotective effects induced by stimulation of 5-HT1A receptors on astrocytes. The review discusses new promising therapeutic strategies based on neuroprotection against oxidative stress and prevention of dopaminergic neurodegeneration.
... A recent study demonstrates that selective loss of GFAP + astrocytes initiates neuronal loss [129], consistent with many previous studies showing that astrocytes can protect neurons, including in coculture models [130][131][132][133][134][135][136][137][138]. Furthermore, astrocytes may protect neurons with the help of glutathione and Hsp70, the two molecules examined closely here [131,133,[139][140][141][142][143][144][145][146]. ...
Astrocytes are one of the major cell types to combat cellular stress and protect neighboring neurons from injury. In order to fulfill this important role, astrocytes must sense and respond to toxic stimuli, perhaps including stimuli that are severely stressful and kill some of the astrocytes. The present study demonstrates that primary astrocytes that managed to survive severe proteotoxic stress were protected against subsequent challenges. These findings suggest that the phenomenon of preconditioning or tolerance can be extended from mild to severe stress for this cell type. Astrocytic stress adaptation lasted at least 96 h, the longest interval tested. Heat shock protein 70 (Hsp70) was raised in stressed astrocytes, but inhibition of neither Hsp70 nor Hsp32 activity abolished their resistance against a second proteotoxic challenge. Only inhibition of glutathione synthesis abolished astrocytic stress adaptation, consistent with our previous report. Primary neurons were plated upon previously stressed astrocytes, and the cocultures were then exposed to another proteotoxic challenge. Severely stressed astrocytes were still able to protect neighboring neurons against this injury, and the protection was unexpectedly independent of glutathione synthesis. Stressed astrocytes were even able to protect neurons after simultaneous application of proteasome and Hsp70 inhibitors, which otherwise elicited synergistic, severe loss of neurons when applied together. Astrocyte-induced neuroprotection against proteotoxicity was not elicited with astrocyte-conditioned media, suggesting that physical cell-to-cell contacts may be essential. These findings suggest that astrocytes may adapt to severe stress so that they can continue to protect neighboring cell types from profound injury.
... These astrocytic changes were shown to induce oxidative stress in neurons, and inhibition of astrocytic mPT with cyclosporine A significantly attenuated neuronal death, 87 suggesting that abnormal astrocytic function contributes to neuronal loss in AD. Similarly, astrocytes derived from DJ-1 knockout mice (a model of familial PD) showed severe mitochondrial abnormalities, including reduced mitochondrial motility, mPT induction and failure to protect neurons from oxidative injury and death, [239][240][241] showing that astrocytic mitochondrial abnormalities impair neuroprotective responses in PD. Likewise, astrocytes derived from mutant SOD1 mice showed mitochondrial dysfunction, which translated to less neuroprotective activity. ...
Selective neuron loss in discrete brain regions is a hallmark of various neurodegenerative disorders, although the mechanisms responsible for this regional vulnerability of neurons remain largely unknown. Earlier studies attributed neuron dysfunction and eventual loss during neurodegenerative diseases as exclusively cell autonomous. Although cell-intrinsic factors are one critical aspect in dictating neuron death, recent evidence also supports the involvement of other central nervous system cell types in propagating non-cell autonomous neuronal injury during neurodegenerative diseases. One such example is astrocytes, which support neuronal and synaptic function, but can also contribute to neuroinflammatory processes. Indeed, aberrations in astrocyte function have been shown to negatively impact neuronal integrity in several neurological diseases. The present review focuses on neuroinflammatory paradigms influenced by neuron–astrocyte cross-talk in the context of select neurodegenerative diseases.
Parkinson’s disease (PD) is an incurable neurodegenerative disease of high prevalence, characterized by the prominent death of dopaminergic neurons in the substantia nigra pars compacta, which produces dopamine deficiency, leading to classic motor symptoms. Although PD has traditionally been considered as a neuronal cell autonomous pathology, in which the damage of vulnerable neurons is responsible for the disease, growing evidence strongly suggests that astrocytes might have an active role in the neurodegeneration observed. In the present review, we discuss several studies evidencing astrocyte implications in PD, highlighting the consequences of both the loss of normal homeostatic functions and the gain in toxic functions for the wellbeing of dopaminergic neurons. The revised information provides significant evidence that allows astrocytes to be positioned as crucial players in PD etiology, a factor that needs to be taken into account when considering therapeutic targets for the treatment of the disease.
Fenpyroximate (FEN) is an acaricide used in agriculture / horticulture to control spider mites and leafhoppers. It inhibits the transport of mitochondrial electrons at the level of NADH-coenzyme Q oxidoreductase (complex I). Despite the implication of inhibition of mitochondrial complex I in neurotoxicity, especially in neurodegenerative diseases, data concerning FEN neurotoxicity remain limited. Thus, the present study was designed to investigate the toxic effect of FEN on rat brain tissue and on human neuroblastoma cells (SH-SY5Y).
Rat exposure to FEN at three different doses (4.8, 9.6 and 48 mg / Kg bw) for 28 consecutive days resulted in histopathological modifications in brain tissue and a significant decrease in acetylcholinesterase activity. Further, FEN significantly enhanced lipid peroxidation and protein oxidation in rat brain and disturbed activities of antioxidant enzymes (SOD, CAT, GPx, and GST). Besides, FEN was found to induce DNA damage in a significant and dose-dependent manner in rat brain as assessed by the comet assay. To better understand FEN neurotoxic effect, we monitored our study on SH-SY5Y cells. We confirm our data found in rat brain tissue. In fact, FEN induced cell mortality in a concentration dependent manner. It over-produced intracellular ROS and lipid peroxidation and enhanced SOD and CAT activities. FEN was also found to induce DNA damage in SH-SY5Y cells. Moreover, FEN induced a loss of mitochondrial membrane potential, which confirms FEN mitochondrial impairing activity. Acridine Orange-Bromure Etidium (AO-BE) cell staining indicated that FEN enhanced the percentage of apoptotic cells in a concentration dependent manner. Further, pretreatment with a general caspases inhibitor (ZVAD-FMK), reduced significantly the FEN induced cell mortality. We also shown that FEN increased caspase 3 activity. These findings suggested, for the first time, the possibility of the involvement of mitochondrial pathway in FEN-induced cell apoptosis.
Mon projet de thèse s’inscrit dans l’étude des synucléinopathies, une famille de maladies neurodégénératives. Les trois principales synucléinopathies sont la maladie de Parkinson, l’atrophie multisystématisée et la démence à corps de Lewy. Ces maladies sont caractérisées par une perte de neurones dans des régions cérébrales spécifiques et la présence d’inclusions intra-cytoplasmiques positives pour l’α-synucléine dans les neurones (Corps de Lewy) ou dans les oligodendrocytes (Inclusions gliales cytoplasmiques). Les causes d’induction de ces maladies restent encore inconnues et les traitements curatifs sont inexistants. L’objectif de mon travail de thèse visait à étudier les mécanismes neurodégénératifs et de potentielles cibles thérapeutiques dans le contexte des synucléinopathies. Je me suis tout d’abord intéressée aux mécanismes impliqués dans la transmission de l’α-synucléine issue de patients atteints de l’atrophie multisystématisée. Ce travail nous a permis de développer un potentiel nouveau modèle de l’atrophie multisystématisée chez la souris et le primate non-humain, par la transmission de l’α-synucléine dans le cerveau. Dans un deuxième temps, nous nous sommes intéressés à des cibles thérapeutiques éventuelles pour la maladie de Parkinson dans un même modèle animal de la pathologie. Nous avons pu vérifier l’efficacité et la pertinence de trois différentes stratégies ciblant plusieurs mécanismes affectés dans la maladie de Parkinson dans le but d’induire une protection des neurones dopaminergiques de la substance noire des souris. Nous avons pu démontrer une dérégulation des niveaux de zinc au cours de la pathologie qui a suscité l’intérêt de cibler son homéostasie dans le cerveau à travers une molécule chélatrice du zinc. Ensuite, la surexpression d’un facteur de transcription impliqué dans la survie des neurones dopaminergiques ainsi que dans le stress oxydatif et le protéasome a montré son intérêt comme cible thérapeutique de la maladie de Parkinson. Enfin, une molécule anti-agrégative a aussi démontré sa capacité à induire une neuroprotection. En résumé, ces travaux montrent d’abord l’importance de l’α-synucléine dans la mise en place et la progression des synucléinopathies, mais aussi la nécessité de cibler d’autres mécanismes dérégulés dans ces pathologies pour proposer des nouvelles stratégies thérapeutiques.
Neurodegenerative diseases (NDs) are one of the major health threats to human characterized by selective and progressive neuronal loss. The mechanisms of NDs are still not fully understood. The study of genetic defects and disease-related proteins offers us a window into the mystery of it, and the extension of knowledge indicates that different NDs share similar features, mechanisms, and even genetic or protein abnormalities. Among these findings, PARK7 and its production DJ-1 protein, which was initially found implicated in PD, have also been found altered in other NDs. PARK7 mutations, altered expression and posttranslational modification (PTM) cause DJ-1 abnormalities, which in turn lead to downstream mechanisms shared by most NDs, such as mitochondrial dysfunction, oxidative stress, protein aggregation, autophagy defects, and so on. The knowledge of DJ-1 derived from PD researches might apply to other NDs in both basic research and clinical application, and might yield novel insights into and alternative approaches for dealing with NDs.
Classically, the loss of vulnerable neuronal populations in neurodegenerative diseases was considered to be the consequence of cell-autonomous degeneration of neurons. However, progress in the understanding of glial function, the availability of improved animal models recapitulating the features of the human diseases, and the development of new approaches to derive glia and neurons from induced pluripotent stem cells obtained from patients, provided novel information that altered this view. Current evidence strongly supports the notion that non-cell autonomous mechanisms contribute to the demise of neurons in neurodegenerative disorders, and glia causally participate in the pathogenesis and progression of these diseases. In addition to microglia, astrocytes have emerged as key players in neurodegenerative diseases and will be the focus of the present review. Under the influence of pathological stimuli present in the microenvironment of the diseased CNS, astrocytes undergo morphological, transcriptional, and functional changes and become reactive. Reactive astrocytes are heterogeneous and exhibit neurotoxic (A1) or neuroprotective (A2) phenotypes. In recent years, single-cell or single-nuclei transcriptome analyses unraveled new, disease-specific phenotypes beyond A1/A2. These investigations highlighted the complexity of the astrocytic responses to CNS pathology.
The present review will discuss the contribution of astrocytes to neurodegenerative diseases with particular emphasis on Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and frontotemporal dementia. Some of the commonalties and differences in astrocyte-mediated mechanisms that possibly drive the pathogenesis or progression of the diseases will be summarized. The emerging view is that astrocytes are potential new targets for therapeutic interventions. A comprehensive understanding of astrocyte heterogeneity and disease-specific phenotypic complexity could facilitate the design of novel strategies to treat neurodegenerative disorders.
Recently, it has been reported that dysfunction of astrocytes is involved vulnerability of neuronal cells in several neurological disorders. Glutathione (GSH) is the most abundant intrinsic antioxidant in the central nervous system, and its substrate cysteine is readily becomes the oxidized dimeric cystine. Since neurons lack a cystine transport system, neuronal GSH synthesis depends on cystine uptake via the cystine/glutamate exchange transporter (xCT), GSH synthesis and release in/from surrounding astrocytes. The expression and release of the zinc-binding protein metallothionein (MT) in astrocytes, which is a strong antioxidant, is induced and exerts neuroprotective in the case of dopaminergic neuronal damage. In addition, the transcription factor Nrf2 induces expression of MT-1 and GSH related molecules. We previously revealed that several antiepileptic drugs, serotonin 5-HT1A receptor agonists, plant-derived chemicals (phytochemicals) increased xCT expression, Nrf2 activation, GSH or MT expression and release in/from astrocytes, and exerted a neuroprotective effect against dopaminergic neurodegeneration in Parkinson’s disease model. Our serial studies on neuroprotection via antioxidant defense mechanism of astrocytes have found three target molecular systems of astrocytes for neuroprotection: (1) xCT-GSH synthetic system, (2) Nrf2 system and (3) 5-HT1A receptor-Nrf2-MT system, 5-HT1A-S100β system. In this article, possible neuroprotective strategy for Parkinson’s disease has been reviewed targeting antioxidative molecules in astrocytes.
Cysteine is a rare but functionally important amino acid that is often subject to covalent modification. Cysteine oxidation plays an important role in many human disease processes, and basal levels of cysteine oxidation are required for proper cellular function. Because reactive cysteine residues are typically ionized to the thiolate anion (Cys-S ⁻ ), their formation of a covalent bond alters the electrostatic and steric environment of the active site. X-ray-induced photo-oxidation to sulfenic acids (Cys-SOH) can recapitulate some aspects of the changes that occur under physiological conditions. Here we propose how site-specific cysteine photo-oxidation can be used to interrogate ensuing changes in protein structure and dynamics at atomic resolution. Although this powerful approach can connect cysteine covalent modification to global protein conformational changes and function, careful biochemical validation must accompany all such studies to exclude misleading artifacts. New types of X-ray crystallography experiments and powerful computational methods are creating new opportunities to connect conformational dynamics to catalysis for the large class of systems that use covalently modified cysteine residues for catalysis or regulation.
In order to fulfill their evolutionary role as support cells, astrocytes have to tolerate intense oxidative stress under conditions of brain injury and disease. It is well known that astrocytes exposed to mild oxidative stress are preconditioned against subsequent stress exposure in dual hit models. However, it is unclear whether severe oxidative stress leads to stress tolerance, stress exacerbation, or no change in stress resistance in astrocytes. Furthermore, it is not known whether reactive astrocytes surviving intense oxidative stress can still support nearby neurons. The data in this Brief Report suggest that primary cortical astrocytes surviving high concentrations of the oxidative toxicant paraquat are completely resistant against subsequent oxidative challenges of the same intensity. Inhibitors of multiple endogenous defenses (e.g., glutathione, heme oxygenase 1, ERK1/2, Akt) failed to abolish or even reduce their stress resistance. Stress-reactive cortical astrocytes surviving intense oxidative stress still managed to protect primary cortical neurons against subsequent oxidative injuries in neuron/astrocyte co-cultures, even at concentrations of paraquat that otherwise led to more than 80% neuron loss. Although our previous work demonstrated a lack of stress tolerance in primary neurons exposed to dual paraquat hits, here we show that intensely stressed primary neurons can resist a second hit of hydrogen peroxide. These collective findings suggest that stress-reactive astroglia are not necessarily neurotoxic, and that severe oxidative stress does not invariably lead to stress exacerbation in either glia or neurons. Therefore, interference with the natural functions of stress-reactive astrocytes might have the unintended consequence of accelerating neurodegeneration. © 2019 Bhatia, Pant, Eckhoff, Gongaware, Do, Hutchison, Gleixner and Leak.
La maladie de Parkinson est une maladie neurodégénérative dont la physiopathologie fait intervenir des causes génétiques qui contribuent non seulement aux formes familiales mais aussi au développement des formes sporadiques, les plus fréquentes, en interagissant sans doute alors avec des facteurs environnementaux. La découverte des déterminants génétiques a permis d’identifier plusieurs mécanismes moléculaires contribuant au développement de la maladie. Ils en ont démontré le caractère complexe. Pour mieux comprendre l’étendue des perturbations moléculaires liées à la maladie, nous avons entrepris des analyses globales du transcriptome par le biais de puces ou de séquençage des ARNs (RNAseq) à partir de cellules mononuclées périphériques sanguines de patients présentant des formes génétiques et sporadiques de la maladie ainsi que de sujets sains.Nous avons identifiés la dérégulation de nombreux gènes dans les cellules mononuclées périphériques sanguines de sujets porteurs de la mutation G2019S de LRRK2 et de sujets sporadiques par rapport à des sujets sains appariés. L’analyse des voies et des processus cellulaires associés à ces gènes met en exergue une altération de la voie de signalisation EIF2 commune aux sujets porteurs de la mutation G2019S de LRRK2 et aux sujets sporadiques. Cette voie fait écho à la régulation de la traduction et de l’épissage, deux processus faisant partie du métabolisme des ARNs. Ces altérations sont retrouvées dans les cellules mononuclées périphériques sanguines de sujets parkinsoniens porteurs de mutations du gène ATXN2, essentiellement connu pour son rôle dans la stabilité, l’épissage et la traduction des ARNm. Cette perturbation des ARNs semble être une altération commune à l’ensemble des formes de maladie de Parkinson étudiées et pourrait donc être un mécanisme sous-tendant la maladie.Les résultats du séquençage des ARNs obtenus chez des parkinsoniens présentant une forme sporadique ou porteurs de mutations délétères ainsi que chez des sujets sains corroborent cette hypothèse en montrant d’une part des différences quantitatives et qualitatives de variants d’épissage au sein d’ARNm eux-mêmes impliqués dans le métabolisme des ARNs et d’autres part des variations de l’épissage de gènes impliqués dans des mécanismes moléculaires connus de la maladie.Ainsi, nos données s’inscrivent dans la dynamique physiopathologique actuelle qui fait état de nombreuses perturbations du métabolisme des ARNs dans les maladies neurodégénératives. Dans la maladie de Parkinson, ces défauts impliqueraient des variations quantitatives et qualitatives de variants d’épissage au sein de gènes liés à l’épissage mais aussi dans des mécanismes connus pour contribuer à la maladie. Une analyse approfondie de ces dérèglements devraient permettre de déterminer leur spécificité et d’évaluer leur potentiel en tant que marqueurs de la maladie et cibles thérapeutiques.
With the increased incidence of neurodegenerative diseases worldwide, Parkinson's disease (PD) represents the second-most common neurodegenerative disease. PD is a progressive multisystem neurodegenerative disorder characterized by a marked loss of nigrostriatal dopaminergic neurons and the formation of Lewy pathology in diverse brain regions. Although the mechanisms underlying dopaminergic neurodegeneration remain poorly characterized, data from animal models and postmortem studies have revealed that heightened inflammatory responses mediated via microglial and astroglial activation and the resultant release of proinflammatory factors may act as silent drivers of neurodegeneration. In recent years, numerous studies have demonstrated a positive association between the exposure to environmental neurotoxicants and the etiology of PD. Although it is unclear whether neuroinflammation drives pesticide-induced neurodegeneration, emerging evidence suggests that the failure to dampen neuroinflammatory mechanisms may account for the increased vulnerability to pesticide neurotoxicity. Furthermore, recent studies provide additional evidence that shifts the focus from a neuron-centric view to glial-associated neurodegeneration following pesticide exposure. In this review, we propose to summarize briefly the possible factors that regulate neuroinflammatory processes during environmental neurotoxicant exposure with a focus on the potential roles of mitochondria-driven redox mechanisms. In this context, a critical discussion of the data obtained from experimental research and possible epidemiological studies is included. Finally, we hope to provide insights on the pivotal role of exosome-mediated intercellular transmission of aggregated proteins in microglial activation response and the resultant dopaminergic neurodegeneration after exposure to pesticides. Collectively, an improved understanding of glia-mediated neuroinflammatory signaling might provide novel insights into the mechanisms that contribute to neurodegeneration induced by environmental neurotoxicant exposure.
DJ-1 was recently reported to mediate the cardioprotection of delayed hypoxic preconditioning (DHP) by suppressing hypoxia/reoxygenation (H/R)-induced oxidative stress, but its mechanism against H/R-induced oxidative stress during DHP is not fully elucidated. Here, using the well-established cellular model of DHP, we again found that DHP significantly improved cell viability and reduced lactate dehydrogenase release with concurrently up-regulated DJ-1 protein expression in H9c2 cells subjected to H/R. Importantly, DHP efficiently improved mitochondrial complex I activity following H/R and attenuated H/R-induced mitochondrial reactive oxygen species (ROS) generation and subsequent oxidative stress, as demonstrated by a much smaller decrease in reduced glutathione/oxidized glutathione ratio and a much smaller increase in intracellular ROS and malondialdehyde contents than that observed for the H/R group. However, the aforementioned effects of DHP were antagonized by DJ-1 knockdown with short hairpin RNA but mimicked by DJ-1 overexpression. Intriguingly, pharmacological inhibition of mitochondria complex I with Rotenone attenuated all the protective effects caused by DHP and DJ-1 overexpression, including maintenance of mitochondria complex I and suppression of mitochondrial ROS generation and subsequent oxidative stress. Taken together, this work revealed that preserving mitochondrial complex I activity and subsequently inhibiting mitochondrial ROS generation could be a novel mechanism by which DJ-1 mediates the cardioprotection of DHP against H/R-induced oxidative stress damage.
Resveratrol has been demonstrated to have cardioprotective effects by attenuating ischemia/reperfusion (I/R)-induced oxidative stress injury, but its in-depth molecular mechanisms against I/R-induced oxidative stress is not fully elaborated. DJ-1 plays a role in maintenance of mitochondrial complex I activity and is closely associated with oxidative stress. Therefore, this study sought to determine the contribution of DJ-1-mediated maintenance of mitochondrial complex I activity to the anti-oxidative stress effect of Resveratrol in the H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R). The results showed that Resveratrol significantly attenuated the H/R-induced viability loss and lactate dehydrogenase leakage, accompanied by decreases in intracellular reactive oxygen species (ROS) and malondialdehyde contents and increases in the reduced glutathione/oxidized glutathione ratio. Furthermore, Resveratrol increased the expression and mitochondrial translocation of DJ-1 and promoted the direct binding of DJ-1 with complex I subunits ND1 and NDUFS4, which in turn improved mitochondrial complex I activity and inhibited mitochondria-derived ROS production after H/R. Intriguingly, the anti-oxidative stress effect of Resveratrol could be partially blocked by DJ-1 siRNA and Complex I inhibitor Rotenone, respectively. Conclusively, these results indicated that DJ-1 is necessary for Resveratrol-mediated cardioprotective effects against H/R-induced oxidative stress damage, at least in part, through preserving mitochondrial complex I activity, and subsequently decreasing mitochondrial ROS generation.
Mitochondrial complex I (NADH: ubiquinone oxidoreductase; CI) is central to the electron transfer chain (ETC), oxidative phosphorylation, and ATP production in eukaryotes. CI is a multi-subunit complex with a complicated yet organized structure that optimally connects electron transfer with proton translocation and forms higher-order supercomplexes with other ETC complexes. Efforts to understand the molecular genetics, expression profile of subunits, and structure-function relationship of CI have increased over the years due to the direct role of the complex in human diseases. Although mutations in the nuclear and mitochondrial genes of CI and altered expression of subunits could potentially lower CI activity leading to mitochondrial dysfunction in many diseases, oxidative post-translational modifications (PTMs) have emerged as an important mechanism contributing to altered CI activity. These mainly include reversible and irreversible cysteine modifications, tyrosine nitration, carbonylation, and tryptophan oxidation that are generated following exposure to reactive oxygen species/reactive nitrogen species. Interestingly, oxidative PTMs could contribute either to CI damage, mitochondrial dysfunction, and ensuing cell death or a response mechanism with potential cytoprotective effects. This has also emerged as a promising field for structural biologists since analysis of PTMs could assist in understanding the structure-function relationship of the complex and correlate electron transfer mechanism with energy production. However, analysis of PTMs of CI and their contribution to CI function are incomplete in many physiological and pathological conditions. This review aims to highlight the role of oxidative PTMs in modulating CI activity with implications toward pathobiology of CNS diseases and novel therapeutics.
Oxidative stress reflects an imbalance between the overproduction and incorporation of free radicals and the dynamic ability of a biosystem to detoxify reactive intermediates. Free radicals produced by oxidative stress are one of the common features in several experimental models of diseases. Free radicals affect both the structure and function of neural cells, and contribute to a wide range of neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease. Although the precise mechanisms that result in the degeneration of neurons and the relevant pathological changes remain unclear, the crucial role of oxidative stress in the pathogenesis of neurodegenerative diseases is associated with several proteins (such as α-synuclein, DJ-1, Amyloid β and tau protein) and some signaling pathways (such as extracellular regulated protein kinases, phosphoinositide 3-kinase/Protein Kinase B pathway and extracellular signal-regulated kinases 1/2) that are tightly associated with the neural damage. In this review, we present evidence, gathered over the last decade, concerning a variety of pathogenic proteins, their important signaling pathways and pathogenic mechanisms associated with oxidative stress in Parkinson’s disease and Alzheimer’s disease. Proper control and regulation of these proteins’ functions and the related signaling pathways may be a promising therapeutic approach to the patients. We also emphasizes antioxidative options, including some new neuroprotective agents that eliminate excess reactive oxygen species efficiently and have a certain therapeutic effect; however, controversy surrounds some of them in terms of the dose and length of therapy. These agents require further investigation by clinical application in patients suffering Parkinson’s disease and Alzheimer’s disease.
DJ-1 mutations are associated to early-onset Parkinson's disease (PD) and accounts for about 1-2% of the genetic forms. The protein is involved in many biological processes and its role in mitochondrial regulation is gaining great interest, even if its function in mitochondria is still unclear. We describe a 47 year-old woman affected by a multisystem disorder characterized by progressive, early-onset Parkinsonism plus distal spinal amyotrophy, cataracts and sensory-neural deafness associated with a novel homozygous c.461C>A [p.T154K] mutation in DJ-1. Patient's cultured fibroblasts showed low ATP synthesis, high ROS levels and reduced amount of some subunits of mitochondrial complex I; biomarkers of oxidative stress also resulted abnormal in patient's blood. The clinical pattern of multisystem involvement and the biochemical findings in our patient highlights the role for DJ-1 in modulating mitochondrial response against oxidative stress.
Parkinson’s disease (PD) is a prevalent neurodegenerative movement disorder affecting millions of predominantly elderly people worldwide and remains essentially untreatable. The underlying mechanisms of selective dopaminergic neurodegeneration in the substantia nigra pars compacta in PD are still poorly understood and the sufferings of the victims of the disease are unimaginable. Intense research endeavors have been directed in delineating the molecular events in the etiopathogenesis of PD. Oxidative stress stands at the forefront amongst several plausible hypotheses of PD pathogenesis in terms of the volume and substantiality of evidence acquired through controlled technologically advanced studies of the human disease and the experimental models of PD. Despite ample evidence in support of the involvement of oxidative stress in PD pathogenesis, traditional antioxidant-based therapeutic strategies have failed in the clinic. Here we discuss lessons learnt from these failed clinical trials and new promising antioxidant-based neuroprotective strategies for therapeutic approaches that may usher hope to win the battle against this debilitating disease.
Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are two novel cell sources for studying neurodegenerative diseases. Dopaminergic neurons derived from hiPSCs/hESCs have been implicated to be very useful in Parkinson's disease (PD) research, including cell replacement therapy, disease modeling and drug screening. Recently, great efforts have been made to improve the application of hiPSCs/hESCs in PD research. Considerable advances have been made in recent years, including advanced reprogramming strategies without the use of viruses or using fewer transcriptional factors, optimized methods for generating highly homogeneous neural progenitors with a larger proportion of mature dopaminergic neurons and better survival and integration after transplantation. Here we outline the progress that has been made in these aspects in recent years, particularly during the last year, and also discuss existing issues that need to be addressed.
Mutations in the DJ-1 gene cause autosomal recessive parkinsonism in humans. Several mouse models of DJ-1 deficiency have been developed, but they do not have dopaminergic neuron cell death in the substantia nigra pars compacta (SNpc). Mitochondrial DNA (mtDNA) damage occurs frequently in the aged human SNpc but not in the mouse SNpc. We hypothesized that the reason DJ-1-deficient mice do not have dopaminergic cell death is due to an absence of mtDNA damage. We tested this hypothesis by crossing DJ-1-deficient mice with mice that have similar amounts of mtDNA damage in their SNpc as aged humans (Polg mutator mice). At 1 year of age, we counted the amount of SNpc dopaminergic neurons in the mouse brains using both colorimetric and fluorescent staining followed by unbiased stereology. No evidence of dopaminergic cell death was observed in DJ-1-deficient mice with the Polg mutator mutation. Furthermore, we did not observe any difference in dopaminergic terminal immunostaining in the striatum of these mice. Finally, we did not observe any changes in the amount of GFAP-positive astrocytes in the SNpc of these mice, indicative of a lack of astrogliosis. Altogether, our findings demonstrate the DJ-1-deficient mice, Polg mutator mice, and DJ-1-deficient Polg mutator mice have intact nigrastriatal pathways. Thus, the lack of mtDNA damage in the mouse SNpc does not underlie the absence of dopaminergic cell death in DJ-1-deficient mice.
How genetic and environmental factors interact in Parkinson disease is poorly understood. We have now compared the patterns
of vulnerability and rescue of Caenorhabditis elegans with genetic modifications of three different genetic factors implicated in Parkinson disease (PD). We observed that expressing
α-synuclein, deleting parkin (K08E3.7), or knocking down DJ-1 (B0432.2) or parkin produces similar patterns of pharmacological
vulnerability and rescue. C. elegans lines with these genetic changes were more vulnerable than nontransgenic nematodes to mitochondrial complex I inhibitors,
including rotenone, fenperoximate, pyridaben, or stigmatellin. In contrast, the genetic manipulations did not increase sensitivity
to paraquat, sodium azide, divalent metal ions (Fe(II) or Cu(II)), or etoposide compared with the nontransgenic nematodes.
Each of the PD-related lines was also partially rescued by the antioxidant probucol, the mitochondrial complex II activator,
d-β-hydroxybutyrate, or the anti-apoptotic bile acid tauroursodeoxycholic acid. Complete protection in all lines was achieved
by combining d-β-hydroxybutyrate with tauroursodeoxycholic acid but not with probucol. These results show that diverse PD-related genetic
modifications disrupt the mitochondrial function in C. elegans, and they raise the possibility that mitochondrial disruption is a pathway shared in common by many types of familial PD.
Investigators have hypothesized that consuming pesticide-contaminated well water plays a role in Parkinson's disease (PD), and several previous epidemiologic studies support this hypothesis.
We investigated whether consuming water from private wells located in areas with documented historical pesticide use was associated with an increased risk of PD.
We employed a geographic information system (GIS)-based model to estimate potential well-water contamination from agricultural pesticides among 368 cases and 341 population controls enrolled in the Parkinson's Environment and Genes Study (PEG). We separately examined 6 pesticides (diazinon, chlorpyrifos, propargite, paraquat, dimethoate, and methomyl) from among 26 chemicals selected for their potential to pollute groundwater or for their interest in PD, and because at least 10% of our population was exposed to them.
Cases were more likely to have consumed private well water and to have consumed it on average 4.3 years longer than controls (p = 0.02). High levels of possible well-water contamination with methomyl [odds ratio (OR) = 1.67; 95% confidence interval (CI), 1.00-2.78]), chlorpyrifos (OR = 1.87; 95% CI, 1.05-3.31), and propargite (OR = 1.92; 95% CI, 1.15-3.20) resulted in approximately 70-90% increases in relative risk of PD. Adjusting for ambient pesticide exposures only slightly attenuated these increases. Exposure to a higher number of water-soluble pesticides and organophosphate pesticides also increased the relative risk of PD.
Our study, the first to use agricultural pesticide application records, adds evidence that consuming well water presumably contaminated with pesticides may play a role in the etiology of PD.
Oxidative stress has been implicated in the etiology of Parkinson's disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. It is known that under conditions of oxidative stress, the transcription factor NF-E2-related factor (Nrf2) binds to antioxidant response element (ARE) to induce antioxidant and phase II detoxification enzymes. To investigate the role of Nrf2 in the process of MPTP-induced toxicity, mice expressing the human placental alkaline phosphatase (hPAP) gene driven by a promoter containing a core ARE sequence (ARE-hPAP) were used. ARE-hPAP mice were injected (30 mg/kg) once per day for 5 days and killed 7 days after the last MPTP injection. In response to this design, ARE-dependent gene expression was decreased in striatum whereas it was increased in substantia nigra. The same MPTP protocol was applied in Nrf2(+/+) and Nrf2(-/-) mice; Nrf2 deficiency increases MPTP sensitivity. Furthermore, we evaluated the potential for astrocytic Nrf2 overexpression to protect from MPTP toxicity. Transgenic mice with Nrf2 under control of the astrocyte-specific promoter for the glial fribillary acidic protein (GFAP-Nrf2) on both a Nrf2(+/+) and Nrf2(-/-) background were administered MPTP. In the latter case, only the astrocytes expressed Nrf2. Independent of background, MPTP-mediated toxicity was abolished in GFAP-Nrf2 mice. These striking results indicate that Nrf2 expression restricted to astrocytes is sufficient to protect against MPTP and astrocytic modulation of the Nrf2-ARE pathway is a promising target for therapeutics aimed at reducing or preventing neuronal death in PD.
There is growing evidence that oxidative stress and mitochondrial respiratory failure with attendant decrease in energy output are implicated in nigral neuronal death in Parkinson disease (PD). It is not known, however, which cellular elements (neurons or glial cells) are major targets of oxygen-mediated damage. 4-Hydroxy-2-nonenal (HNE) was shown earlier to react with proteins to form stable adducts that can be used as markers of oxidative stress-induced cellular damage. We report here results of immunochemical studies using polyclonal antibodies directed against HNE-protein conjugates to label the site of oxidative damage in control subjects (ages 18-99 years) and seven patients that died of PD (ages 57-78 years). All the nigral melanized neurons in one of the midbrain sections were counted and classified into three groups according to the intensity of immunostaining for HNE-modified proteins--i.e., no staining, weak staining, and intensely positive staining. On average, 58% of nigral neurons were positively stained for HNE-modified proteins in PD; in contrast only 9% of nigral neurons were positive in the control subjects; the difference was statistically significant (Mann-Whitney U test; P < 0.01). In contrast to the substantia nigra, the oculomotor neurons in the same midbrain sections showed no or only weak staining for HNE-modified proteins in both PD and control subjects; young control subjects did not show any immunostaining; however, aged control subjects showed weak staining in the oculomotor nucleus, suggesting age-related accumulation of HNE-modified proteins in the neuron. Our results indicate the presence of oxidative stress within nigral neurons in PD, and this oxidative stress may contribute to nigral cell death.
Deficiency of the antioxidant glutathione in brain appears to be connected with several diseases characterized by neuronal loss. To study neuronal glutathione metabolism and metabolic interactions between neurons and astrocytes in this respect, neuron-rich primary cultures and transient cocultures of neurons and astroglial cells were used. Coincubation of neurons with astroglial cells resulted within 24 hr of incubation in a neuronal glutathione content twice that of neurons incubated in the absence of astroglial cells. In cultured neurons, the availability of cysteine limited the cellular level of glutathione. During a 4 hr incubation in a minimal medium lacking all amino acids except cysteine, the amount of neuronal glutathione was doubled. Besides cysteine, also the dipeptides CysGly and gammaGluCys were able to serve as glutathione precursors and caused a concentration-dependent increase in glutathione content. Concentrations giving half-maximal effects were 5, 5, and 200 microM for cysteine, CysGly, and gammaGluCys, respectively. In the transient cocultures, the astroglia-mediated increase in neuronal glutathione was suppressed by acivicin, an inhibitor of the astroglial ectoenzyme gamma-glutamyl transpeptidase, which generates CysGly from glutathione. These data suggest the following metabolic interaction in glutathione metabolism of brain cells: the ectoenzyme gamma-glutamyl transpeptidase uses as substrate the glutathione released by astrocytes to generate the dipeptide CysGly that is subsequently used by neurons as precursor for glutathione synthesis.
The cause of Parkinson's disease (PD) is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate a systemic defect in mitochondrial complex I. We report that chronic, systemic inhibition of complex I by the lipophilic pesticide, rotenone, causes highly selective nigrostriatal dopaminergic degeneration that is associated behaviorally with hypokinesia and rigidity. Nigral neurons in rotenone-treated rats accumulate fibrillar cytoplasmic inclusions that contain ubiquitin and alpha-synuclein. These results indicate that chronic exposure to a common pesticide can reproduce the anatomical, neurochemical, behavioral and neuropathological features of PD.
Increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in the pathogenesis of Parkinson's disease. In experimental animals the exposure to a common herbicide, rotenone, induces features of parkinsonism; mechanistically, rotenone-induced destruction of dopaminergic neurons has been attributed to its inhibition of the activity of neuronal mitochondrial complex I. However, the role of microglia, the resident brain immune cells in rotenone-induced neurodegeneration, has not been reported. Using primary neuron-enriched and neuron/glia cultures from the rat mesencephalon, we discovered an extraordinary feature for rotenone-induced degeneration of cultured dopaminergic neurons. Although little neurotoxicity was detected in neuron-enriched cultures after treatment for 8 d with up to 20 nm rotenone, significant and selective dopaminergic neurodegeneration was observed in neuron/glia cultures 2 d after treatment with 20 nm rotenone or 8 d after treatment with 1 nm rotenone. The greatly enhanced neurodegenerative ability of rotenone was attributed to the presence of glia, especially microglia, because the addition of microglia to neuron-enriched cultures markedly increased their susceptibility to rotenone. Mechanistically, rotenone stimulated the release of superoxide from microglia that was attenuated by inhibitors of NADPH oxidase. Furthermore, inhibition of NADPH oxidase or scavenging of superoxide significantly reduced the rotenone-induced neurotoxicity. This is the first report demonstrating that microglia play a pivotal role in rotenone-induced degeneration of dopaminergic neurons. The results of this study should advance our understanding of the mechanism of action for pesticides in the pathogenesis of Parkinson's disease.
Sporadic Parkinson's disease involves multiple neuronal systems and results from changes developing in a few susceptible types of nerve cells. Essential for neuropathological diagnosis are alpha-synuclein-immunopositive Lewy neurites and Lewy bodies. The pathological process targets specific induction sites: lesions initially occur in the dorsal motor nucleus of the glossopharyngeal and vagal nerves and anterior olfactory nucleus. Thereafter, less vulnerable nuclear grays and cortical areas gradually become affected. The disease process in the brain stem pursues an ascending course with little interindividual variation. The pathology in the anterior olfactory nucleus makes fewer incursions into related areas than that developing in the brain stem. Cortical involvement ensues, beginning with the anteromedial temporal mesocortex. From there, the neocortex succumbs, commencing with high order sensory association and prefrontal areas. First order sensory association/premotor areas and primary sensory/motor fields then follow suit. This study traces the course of the pathology in incidental and symptomatic Parkinson cases proposing a staging procedure based upon the readily recognizable topographical extent of the lesions.
Increasing evidence has suggested an important role for environmental toxins such as pesticides in the pathogenesis of Parkinson's disease (PD). Chronic exposure to rotenone, a common herbicide, reproduces features of Parkinsonism in rats. Mechanistically, rotenone-induced dopaminergic neurodegeneration has been associated with both its inhibition of neuronal mitochondrial complex I and the enhancement of activated microglia. Our previous studies with NADPH oxidase inhibitors, diphenylene iodonium and apocynin, suggested that NADPH oxidase-derived superoxide might be a major factor in mediating the microglia-enhanced rotenone neurotoxicity. However, because of the relatively low specificity of these inhibitors, the exact source of superoxide induced by rotenone remains to be further determined. In this study, using primary mesencephalic cultures from NADPH oxidase--null (gp91phox-/-) or wild-type (gp91phox+/+) mice, we demonstrated a critical role for microglial NADPH oxidase in mediating microglia-enhanced rotenone neurotoxicity. In neuron--glia cultures, dopaminergic neurons from gp91phox-/- mice were more resistant to rotenone neurotoxicity than those from gp91phox+/+ mice. However, in neuron-enriched cultures, the neurotoxicity of rotenone was not different between the two types of mice. More importantly, the addition of microglia prepared from gp91phox+/+ mice but not from gp91phox-/- mice to neuron-enriched cultures markedly increased rotenone-induced degeneration of dopaminergic neurons. Furthermore, apocynin attenuated rotenone neurotoxicity only in the presence of microglia from gp91phox+/+ mice. These results indicated that the greatly enhanced neurotoxicity of rotenone was attributed to the release of NADPH oxidase-derived superoxide from activated microglia. This study also suggested that microglial NADPH oxidase may be a promising target for PD treatment.
Deletion and point (L166P) mutations of DJ-1 have recently been shown to be responsible for the onset of familial Parkinson's disease (PD, PARK7). The aim of this study was to determine the role of DJ-1 in PD. We first found that DJ-1 eliminated hydrogen peroxide in vitro by oxidizing itself. We then found that DJ-1 knockdown by short interfering RNA rendered SH-SY5Y neuroblastoma cells susceptible to hydrogen peroxide-, MPP+- or 6-hydroxydopamine-induced cell death and that cells harbouring mutant forms of DJ-1, including L166P, became susceptible to death in parallel with the loss of oxidized forms of DJ-1. These results clearly showed that DJ-1 has a role in the antioxidative stress reaction and that mutations of DJ-1 lead to cell death, which is observed in PD.
Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1. WT or C53A DJ-1 was readily oxidized in cultured cells, generating a pI 5.8 isoform, but an artificial C106A mutant was not. We observed a cysteine-sulfinic acid at C106 in crystalline DJ-1 but no modification of C53 or C46. Oxidation of DJ-1 was promoted by the crystallization procedure. In addition, oxidation-induced mitochondrial relocalization of DJ-1 and protection against cell death were abrogated in C106A but not C53A or C46A. We suggest that DJ-1 protects against neuronal death, and that this is signaled by acidification of the key cysteine residue, C106.
Mutations of the DJ-1 (PARK7) gene are linked to familial Parkinson's disease. We used gene targeting to generate DJ-1-deficient mice that were viable, fertile, and showed no gross anatomical or neuronal abnormalities. Dopaminergic neuron numbers in the substantia nigra and fiber densities and dopamine levels in the striatum were normal. However, DJ-1-/- mice showed hypolocomotion when subjected to amphetamine challenge and increased striatal denervation and dopaminergic neuron loss induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine. DJ-1-/-embryonic cortical neurons showed increased sensitivity to oxidative, but not nonoxidative, insults. Restoration of DJ-1 expression to DJ-1-/- mice or cells via adenoviral vector delivery mitigated all phenotypes. WT mice that received adenoviral delivery of DJ-1 resisted 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine-induced striatal damage, and neurons overexpressing DJ-1 were protected from oxidative stress in vitro. Thus, DJ-1 protects against neuronal oxidative stress, and loss of DJ-1 may lead to Parkinson's disease by conferring hypersensitivity to dopaminergic insults.
Investigations into the cellular and molecular biology of genes that cause inherited forms of Parkinson's disease, as well as the downstream pathways that they trigger, shed considerable light on our understanding the fundamental determinants of life and death in dopaminergic neurons. Homozygous deletion or missense mutation in DJ-1 results in autosomal recessively inherited Parkinson's disease, suggesting that wild-type DJ-1 has a favorable role in maintaining these neurons. Here, we show that DJ-1 protects against oxidative stress-induced cell death, but that its relatively modest ability to quench reactive oxygen species is insufficient to account for its more robust cytoprotective effect. To elucidate the mechanism of this cell-preserving function, we have screened out the death protein Daxx as a DJ-1-interacting partner. We demonstrate that wild-type DJ-1 sequesters Daxx in the nucleus, prevents it from gaining access to the cytoplasm, from binding to and activating its effector kinase apoptosis signal-regulating kinase 1, and therefore, from triggering the ensuing death pathway. All these steps are impaired by the disease-causing L166P mutant isoform of DJ-1. These findings suggest that the regulated sequestration of Daxx in the nucleus and keeping apoptosis signal-regulating kinase 1 activation in check is a critical mechanism by which DJ-1 exerts its cytoprotective function.
• apoptosis
• Parkinson's disease
• neuroprotection
• neurodegeneration
• oxidative stress
DJ-1 is the third gene that has been linked to Parkinson disease. Mutations in the DJ-1 gene cause early onset PD with autosomal recessive inheritance. To clarify the mechanism of DJ-1 protection, we have overexpressed the gene in cultured dopaminergic cells that were then subjected to chemical stress. In the rat dopaminergic cell line, N27, and in primary dopamine neurons, overexpression of wild type DJ-1 protected cells from death induced by hydrogen peroxide and 6-hydroxydopamine. Overexpressing the L166P mutant DJ-1 had no protective effect. By contrast, knocking down endogenous DJ-1 with antisense DJ-1 rendered cells more susceptible to oxidative damage. We have found that DJ-1 improves survival by increasing cellular glutathione levels through an increase in the rate-limiting enzyme glutamate cysteine ligase. Blocking glutathione synthesis eliminated the beneficial effect of DJ-1. Protection could be restored by adding exogenous glutathione. Wild type DJ-1 reduced cellular reactive oxygen species and reduced the levels of protein oxidation caused by oxidative stress. By a separate mechanism, overexpressing wild type DJ-1 inhibited the protein aggregation and cytotoxicity usually caused by A53T human alpha-synuclein. Under these circumstances, DJ-1 increased the level of heat shock protein 70 but did not change the glutathione level. Our data indicate that DJ-1 protects dopaminergic neurons from oxidative stress through up-regulation of glutathione synthesis and from the toxic consequences of mutant humanalpha-synuclein through increased expression of heat shock protein 70. We conclude that DJ-1 has multiple specific mechanisms for protecting dopamine neurons from cell death.
Chronic infusion of rotenone (Rot) to Lewis rats reproduces many features of Parkinson disease. Rot (3 mg/kg/day) was infused subcutaneously to male Lewis rats for 6 days using Alzet minipumps. Control rats received the vehicle only. Presence of 0.1% bovine serum albumin during the isolation procedure completely removed rotenone bound to the mitochondria. Therefore all functional changes observed were aftereffects of rotenone toxicity in vivo. In Rot rat brain mitochondria (Rot-RBM) there was a 30-40% inhibition of respiration in State 3 and State 3U with Complex I (Co-I) substrates and succinate. Rot did not affect the State 4Deltapsi of RBM and rat liver mitochondria (RLM). However, Rot-RBM required two times less Ca2+ to initiate permeability transition (mPT). There was a 2-fold increase in O*2- or H2O2 generation in Rot-RBM oxidizing glutamate. Rot infusion affected RLM little. Our results show that in RBM, the major site of reactive oxygen species generation with glutamate or succinate is Co-I. We also found that Co-II generates substantial amounts of reactive oxygen species that increased 2-fold in the Rot-RBM. Our data suggest that the primary mechanism of the Rot toxic effect on RBM consists in a significant increase of O*2- generation that causes damage to Co-I and Co-II, presumably at the level of 4Fe-4S clusters. Decreased respiratory activity diminishes resistance of RBM to Ca2+ and thus increases probability of mPT and apoptotic cell death. We suggest that the damage to Co-I and Co-II shifts O*2- generation from the CoQ10 sites to more proximal sites, such as flavines, and makes it independent of the RBM functional state.
Parkinson's disease (PD) is an idiopathic disease of the nervous system characterized by progressive tremor, bradykinesia, rigidity, and postural instability. It has been postulated that exogenous toxicants, including pesticides, might be involved in the etiology of PD. In this article we present a comprehensive review of the published epidemiologic and toxicologic literature and critically evaluate whether a relationship exists between pesticide exposure and PD. From the epidemiologic literature, there does appear to be a relatively consistent relationship between pesticide exposure and PD. This relationship appears strongest for exposure to herbicides and insecticides, and after long durations of exposure. Toxicologic data suggest that paraquat and rotenone may have neurotoxic actions that potentially play a role in the development of PD, with limited data for other pesticides. However, both the epidemiology and toxicology studies were limited by methodologic weaknesses. Particular issues of current and future interest include multiple exposures (both pesticides and other exogenous toxicants), developmental exposures, and gene-environment interactions. At present, the weight of evidence is sufficient to conclude that a generic association between pesticide exposure and PD exists but is insufficient for concluding that this is a causal relationship or that such a relationship exists for any particular pesticide compound or combined pesticide and other exogenous toxicant exposure.
: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is thought to produce parkinsonism in humans and other primates through its inhibition of complex I. The recent discovery of mitochondrial complex I deficiency in the substantia nigra of patients with Parkinson's disease has provided a remarkable link between the idiopathic disease and the action of the neurotoxin MPTP. This article shows that complex I deficiency in Parkinson's disease is anatomically specific for the substantia nigra, and is not present in another neurodegenerative disorder involving the substantia nigra. Evidence is also provided to show that there is no correlation between l-3,4-dihydroxyphenylalanine therapy and complex I deficiency. These results suggest that complex I deficiency may be the underlying cause of dopaminergic cell death in Parkinson's disease.
Two novel mutations recently have been identified in the DJ-1 gene that cause a new form of autosomal recessive, early-onset parkinsonism. Because the pathological role of this protein is unknown, we examined the issue here and report the colocalization of DJ-1 protein within a subset of pathological tau inclusions in a diverse group of neurodegenerative disorders known as tauopathies. Our study extends the view that different neurodegenerative diseases may have similar pathological mechanisms, and that these processes likely include DJ-1.
Mutations that eliminate DJ-1 expression cause a familial form of Parkinson's disease (PD). In sporadic PD, and many other neurodegenerative diseases, reactive astrocytes over-express DJ-1 whereas neurons maintain its expression at non-disease levels. Since DJ-1 has neuroprotective properties, and since astrocytes are known to support and protect neurons, DJ-1 over-expression in reactive astrocytes may reflect an attempt by these cells to protect themselves and surrounding neurons against disease progression. We used neuron–astrocyte contact and non-contact co-cultures to show that DJ-1 knock-down in astrocytes impaired their neuroprotective capacity, relative to wild-type astrocytes, against the neurotoxin rotenone. Conversely, DJ-1 over-expression in astrocytes augmented their neuroprotective capacity. Experiments using astrocyte conditioned media on neuron-only cultures suggested that astrocyte-released, soluble factors were involved in the DJ-1-dependent, astrocyte-mediated neuroprotective mechanism. Our findings support the developing view that astrocytic dysfunction, in addition to neuronal dysfunction, may contribute to the progression of a variety of neurodegenerative disorders.
1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) was administered via the intraperitoneal route to squirrel monkeys. Akinesia, rigidity and hypophonia were seen after repeated doses of 2 mg/kg. Postural tremor was present in one animal. Neuropathologic examination revealed cell loss restricted to the zona compacta of the substantia nigra. MPTP appears effective in producing an animal model for Parkinson's disease in the squirell monkey, and may be one of themore selective neurotoxins described to date.
Exposure to pesticides has been reported to increase the risk of Parkinson disease (PD), but identification of the specific pesticides is lacking. Three studies have found elevated levels of organochlorine pesticides in postmortem PD brains.
To determine whether elevated levels of organochlorine pesticides are present in the serum of patients with PD.
Case-control study.
An academic medical center.
Fifty patients with PD, 43 controls, and 20 patients with Alzheimer disease.
Levels of 16 organochlorine pesticides in serum samples.
beta-Hexachlorocyclohexane (beta-HCH) was more often detectable in patients with PD (76%) compared with controls (40%) and patients with Alzheimer disease (30%). The median level of beta-HCH was higher in patients with PD compared with controls and patients with Alzheimer disease. There were no marked differences in detection between controls and patients with PD concerning any of the other 15 organochlorine pesticides. Finally, we observed a significant odds ratio for the presence of beta-HCH in serum to predict a diagnosis of PD vs control (odds ratio, 4.39; 95% confidence interval, 1.67-11.6) and PD vs Alzheimer disease (odds ratio, 5.20), which provides further evidence for the apparent association between serum beta-HCH and PD.
These data suggest that beta-HCH is associated with a diagnosis of PD. Further research is warranted regarding the potential role of beta-HCH as a etiologic agent for some cases of PD.
The systemic rotenone model of Parkinson's disease (PD) accurately replicates many aspects of the pathology of human PD and has provided insights into the pathogenesis of PD. The major limitation of the rotenone model has been its variability, both in terms of the percentage of animals that develop a clear-cut nigrostriatal lesion and the extent of that lesion. The goal here was to develop an improved and highly reproducible rotenone model of PD. In these studies, male Lewis rats in three age groups (3, 7 or 12-14 months) were administered rotenone (2.75 or 3.0 mg/kg/day) in a specialized vehicle by daily intraperitoneal injection. All rotenone-treated animals developed bradykinesia, postural instability, and/or rigidity, which were reversed by apomorphine, consistent with a lesion of the nigrostriatal dopamine system. Animals were sacrificed when the PD phenotype became debilitating. Rotenone treatment caused a 45% loss of tyrosine hydroxylase-positive substantia nigra neurons and a commensurate loss of striatal dopamine. Additionally, in rotenone-treated animals, alpha-synuclein and poly-ubiquitin positive aggregates were observed in dopamine neurons of the substantia nigra. In summary, this version of the rotenone model is highly reproducible and may provide an excellent tool to test new neuroprotective strategies.
The Parkinson's disease (PD)-associated gene DJ-1 mediates direct neuroprotection. The up-regulation of DJ-1 in reactive astrocytes also suggests a role in glia. Here we show that DJ-1 regulates proinflammatory responses in mouse astrocyte-rich primary cultures. When treated with a Toll-like receptor 4 agonist, the bacterial endotoxin lipopolysaccharide (LPS), Dj-1-knockout astrocytes generated >10 times more nitric oxide (NO) than littermate controls. Lentiviral reintroduction of DJ-1 restored the NO response to LPS. The enhanced NO production in Dj-1(-/-) astrocytes was mediated by a signaling pathway involving reactive oxygen species leading to specific hyperinduction of type II NO synthase [inducible NO synthase (iNOS)]. These effects coincided with significantly increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and p38(MAPK) inhibition suppressed NO production and iNOS mRNA and protein induction. Dj-1(-/-) astrocytes also induced the proinflammatory mediators cyclooxygenase-2 and interleukin-6 significantly more strongly, but not nerve growth factor. Finally, primary neuron cultures grown on Dj-1(-/-) astrocytes became apoptotic in response to LPS in an iNOS-dependent manner, directly demonstrating the neurotoxic potential of astrocytic DJ-1 deficiency. These findings identify DJ-1 as a regulator of proinflammatory responses and suggest that loss of DJ-1 contributes to PD pathogenesis by deregulation of astrocytic neuroinflammatory damage.
The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys106 in human DJ-1). Mutation of Cys106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu18) in human DJ-1 that alter the oxidative propensity of Cys106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys106 to be oxidized to Cys106-sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys106-sulfinic acid is a key modification that regulates the protective function of DJ-1.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is thought to produce parkinsonism in humans and other primates through its inhibition of complex I. The recent discovery of mitochondrial complex I deficiency in the substantia nigra of patients with Parkinson's disease has provided a remarkable link between the idiopathic disease and the action of the neurotoxin MPTP. This article shows that complex I deficiency in Parkinson's disease is anatomically specific for the substantia nigra, and is not present in another neurodegenerative disorder involving the substantia nigra. Evidence is also provided to show that there is no correlation between L-3,4-dihydroxyphenylalanine therapy and complex I deficiency. These results suggest that complex I deficiency may be the underlying cause of dopaminergic cell death in Parkinson's disease.
The structure and function of mitochondrial respiratory-chain enzyme proteins were studied postmortem in the substantia nigra of nine patients with Parkinson's disease and nine matched controls. Total protein and mitochondrial mass were similar in the two groups. NADH-ubiquinone reductase (Complex I) and NADH cytochrome c reductase activities were significantly reduced, whereas succinate cytochrome c reductase activity was normal. These results indicated a specific defect of Complex I activity in the substantia nigra of patients with Parkinson's disease. This biochemical defect is the same as that produced in animal models of parkinsonism by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and adds further support to the proposition that Parkinson's disease may be due to an environmental toxin with action(s) similar to those of MPTP.
: The structure and function of mitochondrial respiratory-chain enzyme proteins were studied postmortem in the substantia nigra of nine patients with Parkinson's disease and nine matched controls. Total protein and mitochondrial mass were similar in the two groups. NADH-ubiquinone reductase (Complex I) and NADH cytochrome c reductase activities were significantly reduced, whereas succinate cytochrome c reductase activity was normal. These results indicated a specific defect of Complex I activity in the substantia nigra of patients with Parkinson's disease. This biochemical defect is the same as that produced in animal models of parkinsonism by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and adds further support to the proposition that Parkinson's disease may be due to an environmental toxin with action(s) similar to those of MPTP.
1-Methyl-4-phenylpyridinium (MPP+), the putative toxic metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inhibited NAD(H)-linked mitochondrial oxidation at the level of Complex I of the electron transport system. MPTP and MPP+ inhibited aerobic glycolysis in mouse striatal slices, as measured by increased lactate production; MPTP-induced effects were prevented by inhibition of monoamine oxidase B activity. Several neurotoxic analogs of MPTP also form pyridinium metabolites via MAO; these MPP+ analogs were all inhibitors of NAD(H)-linked oxidation by isolated mitochondria. 2'-Methyl-MPTP, a more potent neurotoxin in mice than MPTP, was also more potent than MPTP in inducing lactate accumulation in mouse brain striatal slices. Overall, the studies support the hypothesis that compromise of mitochondrial oxidative capacity is an important factor in the mechanisms underlying the toxicity of MPTP and similar compounds.
Seven patients developed chronic and severe parkinsonism after repeatedly injecting 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intravenously. Levodopa and bromocriptine controlled the symptoms; however, within months, five of the seven patients experienced dyskinesias or on-off fluctuations. Therefore, neither prolonged levodopa treatment nor progressive disease was necessary for on-off phenomena. Because the neurotoxic effects of MPTP seem limited to the substantia nigra, damage to this system alone may produce all the motor features of Parkinson's disease. MPTP differs from other neurotoxins in that it consistently produces a pure parkinsonian state.
GSH, GSSG, vitamin E, and ascorbate were measured in 14-day cultures of chick astrocytes and neurons and compared with levels in the forebrains of chick embryos of comparable age. Activities of enzymes involved in GSH metabolism were also measured. These included gamma-glutamylcysteine synthetase, GSH synthetase, gamma-glutamyl cyclotransferase, gamma-glutamyltranspeptidase, glutathione transferase (GST), GSH peroxidase, and GSSG reductase. The concentration of lipid-soluble vitamin E in the cultured neurons was found to be comparable with that in the forebrain. On the other hand, the concentration of vitamin E in the astrocytes was significantly greater in the cultured astrocytes than in the neurons, suggesting that the astrocytes are able to accumulate exogenous vitamin E more extensively than neurons. The concentrations of major fatty acids were higher in the cell membranes of cultured neurons than those in the astrocytes. Ascorbate was not detected in cultured cells although the chick forebrains contained appreciable levels of this antioxidant. GSH, total glutathione (i.e., GSH and GSSG), and GST activity were much higher in cultured astrocytes than in neurons. gamma-Glutamylcysteine synthetase activity was higher in the cultured astrocytes than in the cultured neurons. GSH reductase and GSH peroxidase activities were roughly comparable in cultured astrocytes and neurons. The high levels of GSH and GST in cultured astrocytes appears to reflect the situation in vivo. The data suggest that astrocytes are resistant to reactive oxygen species (and potentially toxic xenobiotics) and may play a protective role in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)
Dopamine (DA) deficiency in Parkinson's disease is commonly treated with L-dihydroxyphenylalanine (L-dopa), the amino acid precursor to DA. L-dopa is neurotoxic in vitro and impairs survival of metabolically stressed neurons in vivo. We examined with microdialysis of substantia nigra in awake rats the local production of hydroxyl (OH) radicals before and after systemic L-dopa. We found a dose-dependent increase in OH radical output which paralleled the rate of dopa catabolism, was not blocked by deprenyl, and was increased further by acute inhibition of mitochondrial complex I activity. Following high L-dopa doses, catabolism of dopa-derived DA can exceed capacity of nigral mechanisms to reduce formation of or detoxify free radicals.
Glutathione levels in neurons and glial cells were investigated in a neuronal-glial coculture and in separate cultures. Brain cell suspensions obtained from cerebral hemispheres of fetal rats were cultured, and after 5 days the glutathione content of this cell population, consisting mainly of neurons and astroglial cells, was 23.0 nmol/mg of cell protein, with a significantly high content in glial cells (28.0 nmol/mg of protein) in comparison with neurons (18.8 nmol/mg of protein). When the neurons and glial cells were separated and recultured in fresh medium, neuronal glutathione rapidly decreased, whereas glial glutathione remained unchanged. Cysteine is a rate-limiting precursor for glutathione synthesis, and its level was also decreased in neurons, but not in glial cells. Cysteine was taken up rapidly by both neurons and glial cells, but cystine was taken up only by glial cells. This accounts for the rapid decrease of glutathione in the cultured neurons, because the culture medium contains cystine, but not cysteine. It was also found that the cultured glial cells released cysteine into the medium. These results suggest that neurons maintain their glutathione level by taking up cysteine provided by glial cells.
Hyperoxidation phenomena are suspected to be involved in dopaminergic cell death in Parkinson's disease, which affects preferentially the neuromelanin-containing dopaminergic neurons of the substantia nigra. Glutathione peroxidase is the major protective enzyme against hydrogen peroxide toxicity. The distribution of glutathione peroxidase-containing cells was investigated by immunohistochemistry in the midbrain of four control subjects and four patients with Parkinson's disease. (1) Glutathione peroxidase-like immunoreactivity was detected exclusively in glial cells. (2) In control brains, the density of glutathione peroxidase-positive cells was higher in the vicinity of the dopaminergic cell groups known to be resistant to the pathological process of Parkinson's disease. (3) In Parkinson's disease, an increased density of glutathione peroxidase-immunostained cells was observed, surrounding the surviving dopaminergic neurons. The increase in glutathione peroxidase-containing cells was correlated with the severity in dopaminergic cell loss in the respective cell groups. The data suggest that in control brains, a low density of glutathione peroxidase-positive cells surround the dopaminergic neurons the most vulnerable to Parkinson's disease, and that in parkinsonian brains, the increased number of glutathione peroxidase-positive cells may contribute to protect neurons against pathological death. Thus, the amount of glutathione peroxidase protein-containing cells may be critical for a protective effect against oxidative stress, although it cannot be excluded that the level of the enzyme activity remains the crucial factor.
The heme released following subarachnoid hemorrhage is metabolized by heme-oxygenase (HO) to biliverdin and carbon monoxide (CO) with the release of iron. The HO reaction is important since heme may contribute to vasospasm and increase oxidative stress in cells. HO is comprised of at least two isozymes, HO-2 and HO-1. HO-1, also known as heat shock protein HSP32, is inducible by many factors including heme and heat shock. HO-2 does not respond to these stresses. To begin to examine HO activity following subarachnoid hemorrhage (SAH), the expression of HO-1 and HO-2 was investigated after experimental SAH in adult rats. Immunocytochemistry for HO-1, HO-2 and HSP70 proteins was performed at 1, 2, 3 and 4 days after injections of lysed blood, whole blood, oxyhemoglobin and saline into the cisterna magna. A large increase in HO-1 immunoreactivity was seen in cells throughout brain following injections of lysed blood, whole blood, and oxyhemoglobin but not saline. Lysed blood, whole blood and oxyhemoglobin induced HO-1 in all of the cortex, hippocampus, striatum, thalamus, forebrain white matter and in cerebellar cortex. HO-1 immunoreactivity was greatest in those regions adjacent to the basal subarachnoid cisterns where blood and oxyhemoglobin concentrations were likely highest. Double immunofluorescence studies showed the HO-1 positive cells to be predominately microglia, though HO-1 was induced in some astrocytes. HO-1 expression resolved by 48 h. HO-2 immunoreactivity was abundant but did not change following injections of blood. A generalized induction of HSP70 heat shock protein was not observed following injections of lysed blood, whole blood, oxyhemoglobin, or saline. These results suggest that HO-1 is induced in microglia throughout rat brain as a general, parenchymal response to the presence of oxyhemoglobin in the subarachnoid space and not as a stress response. This microglial HO-1 response could be protective against the lipid peroxidation and vasospasm induced by hemoglobin, by increasing heme clearance and iron sequestration, and enhancing the production of the antioxidant bilirubin.
The characteristics and kinetics of GSH efflux from the monolayer culture of rat astrocytes were investigated. GSH efflux was dependent on temperature, with a Q10 value of 2.0 between 37 and 25 degrees C. The GSH efflux rate showed a hyperbolic dependency on the intracellular GSH concentration. The data were fitted well to the Michaelis-Menten model, giving the following kinetic parameter values: Km = 127 nmol/mg of protein; Vmax = 0.39 nmol/min/mg of protein. p-Chloromercuribenzenesulfonic acid, a thiol-reactive agent impermeable to the cell membrane, lowered the GSH efflux rate by 25% without affecting the intracellular GSH content. These results suggest that a carrier is involved in the efflux of GSH. The GSH content of cultured astrocytes showed a marked increase when the cells were exposed to insults, such as sodium arsenite, cadmium chloride, and glucose/glucose oxidase that lead to the generation of hydrogen peroxide. The increase in GSH content was attributed to the induction of the cystine transport activity by the agents. Although the intracellular GSH concentration and GSH efflux were increased, the kinetics of GSH efflux were not affected by those agents that imposed the oxidative stress. Because the Km value is very large, it is suggested that astrocytes release GSH depending on their GSH concentration in a wide range.
Reticulocalbin (RCN) is a member of the EF-hand Ca(2+)-binding protein family and is a luminal protein of the endoplasmic reticulum (ER) with a molecular weight of 44,000 [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Although RCN has six repeats of a domain containing an EF-hand motif, the varying degrees of divergence of the amino acid sequences of these domains from the EF-hand consensus sequences suggested that some domains might have lost their Ca(2+)-binding capability and adopted new functions. To identify the domains involved in Ca(2+)-binding, discrete domains of RCN were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system. 45Ca2+ blot analysis of the resultant fusion proteins revealed that the first, fourth, fifth, and sixth domains bind Ca2+, however, the second and third ones do not. The fusion proteins containing all six domains, and the first and second domains, respectively, showed Ca(2+)-dependent increases in their electrophoretic mobilities, suggesting that Ca2+ induces a conformational change in reticulocalbin.
A variety of phenolic compounds are utilized for industrial production of phenol-formaldehyde resins, paints, lacquers, cosmetics, and pharmaceuticals. Skin exposure to industrial phenolics is known to cause skin rash, dermal inflammation, contact dermatitis, leucoderma, and cancer promotion. The biochemical mechanisms of cytotoxicity of phenolic compounds are not well understood. We hypothesized that enzymatic one-electron oxidation of phenolic compounds resulting in the generation of phenoxyl radicals may be an important contributor to the cytotoxic effects. Phenoxyl radicals are readily reduced by thiols, ascorbate, and other intracellular reductants (e.g., NADH, NADPH) regenerating the parent phenolic compound. Hence, phenolic compounds may undergo enzymatically driven redox-cycling thus causing oxidative stress. To test the hypothesis, we analyzed endogenous thiols, lipid peroxidation, and total antioxidant reserves in normal human keratinocytes exposed to phenol. Using a newly developed cis-parinaric acid-based procedure to assay site-specific oxidative stress in membrane phospholipids, we found that phenol at subtoxic concentrations (50 microM) caused oxidation of phosphatidylcholine and phosphatidylethanolamine (but not of phosphatidylserine) in keratinocytes. Phenol did not induce peroxidation of phospholipids in liposomes prepared from keratinocyte lipids labeled by cis-parinaric acid. Measurements with ThioGlo-1 showed that phenol depleted glutathione but did not produce thiyl radicals as evidenced by our high-performance liquid chromatography measurements of GS.-5, 5-dimethyl1pyrroline N-oxide nitrone. Additionally, phenol caused a significant decrease of protein SH groups. Luminol-enhanced chemiluminescence assay demonstrated a significant decrease in total antioxidant reserves of keratinocytes exposed to phenol. Incubation of ascorbate-preloaded keratinocytes with phenol produced an electron paramagnetic resonance-detectable signal of ascorbate radicals, suggesting that redox-cycling of one-electron oxidation products of phenol, its phenoxyl radicals, is involved in the oxidative effects. As no cytotoxicity was observed in keratinocytes exposed to 50 microM or 500 microM phenol, we conclude that phenol at subtoxic concentrations causes significant oxidative stress.
Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine<-->cysteine + cysteine-glutathione disulfide. Conditioned medium from neuron-enriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.
Heme[none1] oxygenase-1 (HO-1) and peroxiredoxin I (PrxI) are known to be oxidative stress- and heme-related proteins. The antioxidant activity of PrxI is inhibited by heme, therefore co-expression of HO-1 and PrxI is considered to be a reasonable mechanism to maintain its antioxidative function. Immunoblotting demonstrated that HO-1 and PrxI were induced around the hemorrhagic region. Immunohistochemical studies revealed that, in acute phase, HO-1 and PrxI were induced primarily in microglia. In the subacute and chronic phase, the immunoreactivity of HO-1 and PrxI in astrocytes was the most intense. These data are the first to demonstrate co-induction of HO-1 and PrxI in the brain. Our results suggest that HO-1 and PrxI are localized in a similar manner to assure the antioxidant activity of PrxI under stress conditions associated with intracerebral hemorrhage.
Reactive oxygen species not only modulate important signal transduction pathways, but also induce DNA damage and cytotoxicity in keratinocytes. Hydrogen peroxide and organic peroxides are particularly important as these chemicals are widely used in dermally applied cosmetics and pharmaceuticals, and also represent endogenous metabolic intermediates. Lipid peroxidation is of fundamental interest in the cellular response to peroxides, as lipids are extremely sensitive to oxidation and lipid-based signaling systems have been implicated in a number of cellular processes, including apoptosis. Oxidation of specific phospholipid classes was measured in normal human epidermal keratinocytes exposed to cumene hydroperoxide after metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid, using a fluorescence high-performance liquid chromatography assay. In addition, lipid oxidation was correlated with changes in membrane phospholipid asymmetry and other markers of apoptosis. Although cumene hydroperoxide produced significant oxidation of cis-parinaric acid in all phospholipid classes, one phospholipid, phosphatidylserine, appeared to be preferentially oxidized above all other species. Using fluorescamine derivatization and annexin V binding it was observed that specific oxidation of phosphatidylserine was accompanied by phosphatidylserine translocation from the inner to the outer plasma membrane surface where it may serve as a recognition signal for interaction with phagocytic macrophages. These effects occurred much earlier than any detectable changes in other apoptotic markers such as caspase-3 activation, DNA fragmentation, or changes in nuclear morphology. Thus, normal human epidermal keratinocytes undergo profound lipid oxidation with preference for phosphatidylserine followed by phosphatidylserine externalization upon exposure to cumene hydroperoxide. It is therefore likely that normal human epidermal keratinocytes exposed to similar oxidative stress in vivo would under go phosphatidylserine oxidation/translocation. This would make them targets for macrophage recognition and phagocytosis, and thus limit their potential to invoke inflammation or give rise to neoplastic transformations.
The DJ-1 gene encodes a ubiquitous, highly conserved protein. Here, we show that DJ-1 mutations are associated with PARK7, a monogenic form of human parkinsonism. The function of the DJ-1 protein remains unknown, but evidence suggests its involvement in the oxidative stress response. Our findings indicate that loss of DJ-1 function leads to neurodegeneration. Elucidating the physiological role of DJ-1 protein may promote understanding of the mechanisms of brain neuronal maintenance and pathogenesis of Parkinson's disease.
In Parkinson's disease, characteristic pathological features are the cell death of nigrostriatal dopamine neurons and the formation of Lewy bodies composed of oxidized proteins. Mitochondrial dysfunction and aggregation of abnormal proteins have been proposed to cause the pathological changes. However, the relation between these two factors remains to be clarified. In this study, the effects of mitochondrial dysfunction on the oxidative modification and accumulation of proteins were analyzed using an inhibitor of mitochondrial complex I, rotenone, and antibodies against acrolein- and dityrosine-modified proteins. Under conditions inducing mainly apoptosis in neuroblastoma SH-SY5Y cells, rotenone markedly increased oxidized proteins, especially those modified with acrolein, even though the increase in intracellular reactive oxygen and nitrogen species was only transient and was not so marked. In addition, the activity of the proteasome system degrading oxidized proteins was reduced profoundly after treatment with rotenone. The 20S beta subunit of proteasome was modified with acrolein, to which other acrolein-modified proteins were found to bind, as shown by coprecipitation with the antibody against 20S beta subunit. These results suggest that mitochondrial dysfunction, especially decreased activity of complex I, may reduce proteasome activity through oxidative modification of proteasome itself and aggregation with other oxidized proteins. This mechanism might account for the accumulation of modified protein and, at least partially, for cell death of the dopamine neurons in Parkinson's disease.
Exposure of rats to the pesticide and complex I inhibitor rotenone reproduces features of Parkinson's disease, including selective nigrostriatal dopaminergic degeneration and alpha-synuclein-positive cytoplasmic inclusions (Betarbet et al., 2000; Sherer et al., 2003). Here, we examined mechanisms of rotenone toxicity using three model systems. In SK-N-MC human neuroblastoma cells, rotenone (10 nm to 1 microm) caused dose-dependent ATP depletion, oxidative damage, and death. To determine the molecular site of action of rotenone, cells were transfected with the rotenone-insensitive single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (NDI1), which incorporates into the mammalian ETC and acts as a "replacement" for endogenous complex I. In response to rotenone, NDI1-transfected cells did not show mitochondrial impairment, oxidative damage, or death, demonstrating that these effects of rotenone were caused by specific interactions at complex I. Although rotenone caused modest ATP depletion, equivalent ATP loss induced by 2-deoxyglucose was without toxicity, arguing that bioenergetic defects were not responsible for cell death. In contrast, reducing oxidative damage with antioxidants, or by NDI1 transfection, blocked cell death. To determine the relevance of rotenone-induced oxidative damage to dopaminergic neuronal death, we used a chronic midbrain slice culture model. In this system, rotenone caused oxidative damage and dopaminergic neuronal loss, effects blocked by alpha-tocopherol. Finally, brains from rotenone-treated animals demonstrated oxidative damage, most notably in midbrain and olfactory bulb, dopaminergic regions affected by Parkinson's disease. These results, using three models of increasing complexity, demonstrate the involvement of oxidative damage in rotenone toxicity and support the evaluation of antioxidant therapies for Parkinson's disease.
Efficient functioning of maintenance and repair processes seem to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, under control of several genes termed vitagenes. These include members of the heat shock protein system, and there is now evidence that the heat shock response contributes to establishing a cytoprotective state in a wide variety of human conditions, including inflammation, neurodegenerative disorders, and aging. Among the various heat shock proteins, heme oxygenase-1 has received considerable attention; it has been recently demonstrated that heme oxygenase-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Acetyl-L-carnitine is proposed as a therapeutic agent for several neurodegenerative disorders. Accordingly, we report here that treatment of astrocytes with acetyl-L-carnitine induces heme oxygenase-1 in a dose- and time-dependent manner and that this effect was associated with up-regulation of heat shock protein 60 as well as high expression of the redox-sensitive transcription factor Nrf2 in the nuclear fraction of treated cells. In addition, we show that addition of acetyl-L-carnitine to astrocytes, prior to proinflammatory lipopolysaccharide- and interferon-gamma-induced nitrosative stress, prevents changes in mitochondrial respiratory chain complex activity, protein nitrosation and antioxidant status induced by inflammatory cytokine insult. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Particularly, manipulation of endogenous cellular defense mechanisms via acetyl-L-carnitine may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. We hypothesize that maintenance or recovery of the activity of vitagenes may delay the aging process and decrease the risk of age-related diseases.
Mutations in the DJ-1 gene cause early-onset autosomal recessive Parkinson's disease (PD), although the role of DJ-1 in the degeneration of dopaminergic neurons is unresolved. Here we show that the major interacting-proteins with DJ-1 in dopaminergic neuronal cells are the nuclear proteins p54nrb and pyrimidine tract-binding protein-associated splicing factor (PSF), two multifunctional regulators of transcription and RNA metabolism. PD-associated DJ-1 mutants exhibit decreased nuclear distribution and increased mitochondrial localization, resulting in diminished co-localization with co-activator p54nrb and repressor PSF. Unlike pathogenic DJ-1 mutants, wild-type DJ-1 acts to inhibit the transcriptional silencing activity of the PSF. In addition, the transcriptional silencer PSF induces neuronal apoptosis, which can be reversed by wild-type DJ-1 but to a lesser extent by PD-associated DJ-1 mutants. DJ-1-specific small interfering RNA sensitizes cells to PSF-induced apoptosis. Both DJ-1 and p54nrb block oxidative stress and mutant alpha-synuclein-induced cell death. Thus, DJ-1 is a neuroprotective transcriptional co-activator that may act in concert with p54nrb and PSF to regulate the expression of a neuroprotective genetic program. Mutations that impair the transcriptional co-activator function of DJ-1 render dopaminergic neurons vulnerable to apoptosis and may contribute to the pathogenesis of PD.
Parkinson's disease (PD) is a common neurodegenerative disorder that displays both sporadic and inherited forms. Exposure to several common environmental toxins acting through oxidative stress has been shown to be associated with PD. One recently identified inherited PD gene, DJ-1, may have a role in protection from oxidative stress, thus potentially linking a genetic cause with critical environmental risk factors. To develop an animal model that would allow integrative study of genetic and environmental influences, we have generated Drosophila lacking DJ-1 function. Fly DJ-1 homologs exhibit differential expression: DJ-1beta is ubiquitous, while DJ-1alpha is predominantly expressed in the male germline. DJ-1alpha and DJ-1beta double knockout flies are viable, fertile, and have a normal lifespan; however, they display a striking selective sensitivity to those environmental agents, including paraquat and rotenone, linked to PD in humans. This sensitivity results primarily from loss of DJ-1beta protein, which also becomes modified upon oxidative stress. These studies demonstrate that fly DJ-1 activity is selectively involved in protection from environmental oxidative insult in vivo and that the DJ-1beta protein is biochemically responsive to oxidative stress. Study of these flies will provide insight into the critical interplay of genetics and environment in PD.
Paraquat, MPTP, and rotenone reproduce features of Parkinson's disease (PD) in experimental animals. The exact mechanisms by which these compounds damage the dopamine system are not firmly established, but selective damage to dopamine neurons and inhibition of complex I are thought to be involved. We and others have previously documented that the toxic metabolite of MPTP, MPP+, is transported into dopamine neurons through the dopamine transporter (DAT), while rotenone is not transported by DAT. We have also demonstrated the requirement for complex I inhibition and oxidative damage in the dopaminergic neurodegeneration produced by rotenone. Based on structural similarity to MPP+, it has been proposed that paraquat exerts selective dopaminergic toxicity through transport by the DAT and subsequent inhibition of mitochondrial complex I. In this study we report that paraquat is neither a substrate nor inhibitor of DAT. We also demonstrate that in vivo exposure to MPTP and rotenone, but not paraquat, inhibits binding of 3H-dihydrorotenone to complex I in brain mitochondria. Rotenone and MPP+ were both effective inhibitors of complex I activity in isolated brain mitochondria, while paraquat exhibited weak inhibitory effects only at millimolar concentrations. These data indicate that, despite the apparent structural similarity to MPP+, paraquat exerts its deleterious effects on dopamine neurons in a manner that is unique from rotenone and MPTP.
Sporadic Parkinson's disease (PD) is most likely caused by a combination of environmental exposures and genetic susceptibilities, although there are rare monogenic forms of the disease. Mitochondrial impairment at complex I, oxidative stress, alpha-synuclein aggregation, and dysfunctional protein degradation, have been implicated in PD pathogenesis, but how they are related to each other is unclear. To further evaluated PD pathogenesis here, we used in vivo and in vitro models of chronic low-grade complex I inhibition with the pesticide rotenone. Chronic rotenone exposure in vivo caused oxidative modification of DJ-1, accumulation of alpha-synuclein, and proteasomal impairment. Interestingly, the effects become more regionally restricted such that systemic complex I inhibition eventually results in highly selective degeneration of the nigrostriatal pathway. DJ-1 modifications, alpha-synuclein accumulation, and proteasomal dysfunction were also seen in vitro and these effects could be prevented with alpha-tocopherol. Thus, chronic exposure to a pesticide and mitochondrial toxin brings into play three systems, DJ-1, alpha-synuclein, and the ubiquitin-proteasome system, and implies that mitochondrial dysfunction and oxidative stress link environmental and genetic forms of the disease.