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DJ-1 mutation decreases astroglial release of inflammatory mediators
NeuroToxicology 52 (2016) 198–203
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NeuroToxicology
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DJ-1 mutation decreases astroglial release of inflammatory mediators A.K. Ashley, A.I. Hinds, W.H. Hanneman, R.B. Tjalkens, M.E. Legare* The Center for Environmental Medicine, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1680, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 June 2015 Received in revised form 10 November 2015 Accepted 8 December 2015 Available online 12 December 2015
Mutations in DJ-1, reactive gliosis and concomitant inflammatory processes are implicated in the pathogenesis and progression of Parkinson’s disease (PD). To study the physiological consequences of DJ-1 mutation in the context of neuroinflammatory insult, primary cortical astrocytes were isolated from DJ-1 knockout mice. Astrocytes were exposed to 1 mg/mL lipopolysaccharide (LPS) for 24 h following 2 h pre-exposure to inhibitors of MEK (U0126), JNK (JNK inhibitor II) or p38 (SB203580). Real-time PCR was used to assess the LPS-induced expression of pro-inflammatory mediators cyclooxygenase 2 (COX2), inducible nitric oxide synthetase (NOS2), and tumor necrosis factor a (TNFa). LPS-induced expression of COX2 decreased similarly in DJ-1+/+ and DJ-1/ astrocytes in response to inhibition of p38, but was unaffected by inhibition of MEK or JNK. No significant alterations in NOS2 expression were observed in any inhibitor-treated cells. The inhibitors did not affect expression of TNFa; however, DJ-1/ astrocytes had consistently lower expression compared to DJ-1+/+ counterparts. Secretion of TNFa and prostaglandin E2 (PGE2) into the culture medium was significantly decreased in DJ-1/ astrocytes, and inhibition of p38 decreased this secretion in both genotypes. In conclusion, DJ-1/ astrocytes may provide decreased neuroprotection to surrounding neurons due to alterations in pro-inflammatory mediator expression. ã 2015 Elsevier Inc. All rights reserved.
Keywords: DJ-1 Astrocyte Neuroinflammation Lipopolysaccharide
1. Introduction Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disease resulting from loss of dopaminergic neurons in the substantia nigra pars compacta. Approximately 5–10% of diagnosed PD cases have been associated with genetic mutations (Perrett et al., 2015). Specifically, mutations in 7 genes are robustly associated with autosomal dominant (SNCA, LRRK2, EIF4G1, VPS35) or recessive (parkin/PARK2, PINK1, DJ1/PARK7) PD (Bertolin et al., 2015; Mbefo et al., 2015; Puschmann 2013; Triplett et al., 2015). DJ-1 is a recessive familial PD gene involved in anti oxidative function as well as mitochondrial maintenance (Cai et al., 2015; Saito et al., 2014). DJ-1 is also known to regulate the activity of phosphatase and tension homolog (PTEN) which plays a critical role in neuronal cell death in response to various insults. DJ1 protein is widely produced throughout mammalian tissues (Olzmann et al., 2004; Zhang et al., 2005; Larsen et al., 2007) and may function as an atypical peroxiredoxin-like peroxidase, capable of scavenging reactive oxygen species (Andres-Mateos et al., 2007) and mediate cellular protection through its antioxidant properties (Wilson, 2011). DJ-1 has been suggested to function as a redox-
* Corresponding author. E-mail address: [email protected] (M.E. Legare). http://dx.doi.org/10.1016/j.neuro.2015.12.007 0161-813X/ ã 2015 Elsevier Inc. All rights reserved.
dependent chaperone, promoting proper folding of a-synuclein (Shendelman et al., 2004; Zhou et al., 2006). Further, DJ-1 has been shown to accumulate in brain tissue and is elevated in the plasma and cerebrospinal fluid of individuals with PD (Waragai et al., 2006; Waragai et al., 2007). Given the myriad of key proteins that DJ-1 may modulate, including a-synuclein and Nrf2 (nuclear factor-like 2), DJ-1 could affect numerous physiological processes. Indeed, like PD, other neurological disorders have alterations in DJ-1 expression and/or function including Alzheimer’s disease (Choi et al., 2006), multiple system atrophy and Pick’s disease, (Neumann et al., 2004), multiple sclerosis (Hirotani et al., 2008), cells expressing huntingtin (Goswami et al., 2006), and ischemia/reperfusion injury (Aleyasin et al., 2007; Yanagisawa et al., 2008), indicating DJ-1 may play a significant role in maintaining CNS homeostasis (Batelli et al., 2015). Although in humans DJ-1 mutation induces PD, DJ-1 knockout mice fail to consistently recapitulate the key clinical and neuropathological features of PD (Rousseaux et al., 2012; Yamaguchi and Shen, 2007) suggesting the existence of compensatory mechanisms that may protect mice from the neurodegeneration and the consequent motor symptoms. However, recent reports suggest consistent alterations in behavioral and striatal dopamine abnormalities in the DJ-1 knockout mice (Hennis et al., 2013; Hennis et al., 2013). It is apparent that the DJ-1 mutant mouse model provides an excellent platform for investigating PD
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as a multi-factorial disease including the specific role of DJ-1 in neural cells. In utero exposure to bacterial lipopolysaccharide (LPS) has been shown to decrease the number of dopaminergic neurons as well as brain dopamine levels in offspring (Bakos et al., 2004; Carvey et al., 2003; Ling et al., 2002). Further, exposure to LPS as an adult can initiate neuronal cell loss within the substantia nigra in experimental models, and may be an environmental factor in the development of PD (Gayle et al., 2002; Herrera et al., 2000; Qin et al., 2007). LPS acts through toll-like receptors to increase the expression of numerous pro-inflammatory mediators in microglia and other cells, including interleukin1 (IL-1) (Sharif et al., 1993), interleukin 6 (IL-6) (Chung and Benveniste, 1990), mitogenactivated protein kinases (Tai et al., 2013), and TNFa (Liu et al., 2014). Recently, LPS-induced inflammation was demonstrated to induce mitochondrial dysfunction in the substantia nigra as well as the striatum, with increases in COX2 and NOS2 (Hunter et al., 2007). In animals, LPS also induces astrogliosis and neurodegeneration (Hoban et al., 2013), evidenced by increased glialfibrillary acidic protein (Cai et al., 2003). Several studies have shown that astroglia and microglia differentially expression IL-1b and TNFa following stimulation by LPS, with microglia having higher expression levels of IL-1b but TNFa expression being comparable between the two cell types (Lu et al., 2014). Reactive astrocytes consistently have high expression of DJ-1 (Saito et al., 2014), suggesting that DJ-1 has a significant role in gliosis or is elevated resultant to the gliosis (Bandopadhyay et al., 2004; Meulener et al., 2005). Interestingly, DJ-1 protein increases following exposure to LPS (Ejima et al., 2000; Mitsumoto and Nakagawa, 2001) which may indicate that DJ-1 might possibly play an important role in preventing or alleviating LPS-induced toxicity. Increasing evidence indicates that development of PD represents a labyrinth of interactions occurring over time, with contributing factors including genetic predisposition and exposure to environmental toxins. Given that pro-inflammatory mediators promote neuronal viability and prolonged inflammation contributes to neurodegeneration, we tested the hypothesis that LPSexposed astrocytes from DJ-1 mutant mice would exhibit alterations in expression of genes related to inflammation. DJ-1’s role in modulating the inflammatory response may help to elucidate pathways involved in PD and other neurological diseases.
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Colorado State University Institutional Animal Care and Use Committee. 2.2. Real-time PCR
2. Materials and methods
To assess which protein(s) of the mitogen-activated protein kinase (MAPK) pathways are involved in COX2, NOS2, and TNFa increases following LPS exposure in astrocytes, we utilized a MEK inhibitor (MEKi, 10 mM U0126, Cell Signaling), a p38 inhibitor (p38i, 30 mM SB203580, Calbiochem), or an inhibitor of JNK (JNKi, 10 mM JNK Inhibitor II, Calbiochem). Each inhibitor was suspended in DMSO (Sigma) at concentrations designed to deliver equivalent volumes of DMSO. DMSO alone was used in our control group at the same volume as inhibitor-exposed groups (0.1%). The concentrations of inhibitors selected have been demonstrated to inhibit phosphorylation of the respective protein in mouse primary cortical astrocytes previously demonstrated within our laboratory (Moreno et al., 2008). Cells were preincubated with inhibitors and astrocytes were subsequently exposed to PBS or 1 mg/mL LPS (Sigma) for 24 h, culture medium was collected for analysis, and RNA was isolated via the RNeasy Kit (Qiagen) including on-column DNase treatment. cDNA was synthesized with iScript (BioRad) to assess expression of COX2, NOS2, and TNFa via real-time PCR. Relative expression of COX2 (50 GGA GTC TGG AAC ATT GTG AAC - 30 , 50 - GTA GTA GGA GAG GTT GGA GAA G - 30 ), NOS2 (50 -TCA CGC TTG GGT CTT GTT- 30 , 50 - CAG GTC ACT TTG GTA GGA TTT G - 30 ) or TNF a (50 - GCA CCA CCA TCA AGG ACT C 30 , 50 - GAA AGG TCT GAA GGT AGG AAG G - 30 ) was measured using a BioRad iCycler utilizing SYBR green incorporation (BioRad). Expression of COX2, NOS2, and TNFa in each sample was normalized to expression of b-actin (50 -GAC AGG ATG CAG AAG GAG ATT ACT G-30 , 50 -GCT GAT CCA CAT CTG CTG GAA-30 ) via the delta–delta CT method (Livak and Schmittgen, 2001) and is reported as relative expression. Each primer set was examined prior to use to assure consistent PCR efficiency, production of one specific amplicon and absence of primer-dimers. As a negative control, each RNA sample without reverse transcriptase was also analyzed to assure the lack of genomic DNA contamination. Each RNA sample was assayed in duplicate for each primer set assessed. The relative mRNA expression of COX2 and TNFa in each genotype was compared to DJ-1+/+ astrocytes exposed to PBS and DMSO. In all PBS-treated samples, NOS2 expression was beneath the detection limit; therefore expression of NOS2 was analyzed relative to LPSexposed DJ-1+/+ levels.
2.1. Primary astrocyte isolation
2.3. Secreted TNFa, prostaglandin, and nitric oxide
DJ-1 mutant mice were generated, characterized, and genotyped as previously described (Ashley et al., 2009). Heterozygous mice were bred to maintain a colony containing all three genotypes. Primary mouse cortical astrocytes were isolated and maintained as previously described (Allen et al., 2000). This isolation method results in 98% pure astrocyte culture, as determined through immunofluorescent staining (Carbone et al., 2008). Cortices from day 0 to 1 old mouse pups were dissected and meninges removed. Tissue was digested with dispase (1.5 U/mL) in warm MEM with Earle’s Salt and L-glutamine (Fisher) supplemented with an antibiotic cocktail (Invitrogen) containing penicillin (0.001 mg/mL), streptomycin (0.001 mg/mL), and neomycin (0.002 mg/mL). Cells were plated on tissue culture dishes (BD Biosciences) at 300,000 cells/mL in culture medium described above (without dispase) supplemented with 10% fetal bovine serum (Atlas). Culture medium was changed after 24 h to remove the non-adherent microglial cells. Astrocytes were cultured for 3–4 weeks to achieve approximately 80% confluence. Culture medium was changed 24 h prior to application of any treatment to minimize serum shock. All animal procedures were approved by the
Astrocytes were cultured as described, and medium was collected and stored at 80 C for later analysis. TNFa levels in culture medium samples were determined via an enzyme-linked immunosorbent assay (ELISA, eBiosciences). Secreted levels of prostaglandin E2 were assessed with a monoclonal EIA kit with limit of detection of 39–25,000 pg/mL (Cayman Chemicals). Nitric oxide levels in culture medium were determined via nitrate/nitrite colorimetric assay (Cayman Chemicals). 2.4. Statistical analyses All statistical analyses were performed with GraphPad Prism software. Means were analyzed using two way ANOVA, and when they significantly differed (p < 0.05), Tukey’s post hoc test was employed. Four sets of astrocyte cell cultures were examined: DJ1+/+ treated with PBS, DJ-1+/+ treated with LPS, DJ-1/ treated with PBS, and DJ-1/ treated with LPS. Each set contained four treatment groups: cultures were treated with MEKi, JNKi, or p38i, or with DMSO as the vehicle control for the inhibitors. mRNA levels were measured by real-time PCR in each group for genes of
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Fig. 1. LPS-induced relative expression of COX2 decreases with inhibition of p38 but not MEK or JNK. Astrocytes were exposed to U0126 (MEKi, 10 mM), JNK inhibitor II (JNKi, 10 mM), or SB203580 (p38i, 30 mM) for 2 h, then PBS or 1 mg/mL LPS for 24 h, and RNA was isolated. Expression of COX2 was determined via real-time PCR, and values are expressed relative to DJ-1+/+ cells exposed to DMSO and PBS. * illustrates a significant difference (p < 0.05) from basal expression within the treatment group. D illustrates a significant difference (p < 0.05) from LPS treatment within the genotype.
interest. In utilizing the delta–delta CT method for analysis of realtime PCR, CT values were normalized to b-actin, then relative to DJ1+/+ treated with PBS and DMSO to yield relative expression of COX2 and TNFa. In NOS2 samples, mRNA was below the threshold of detection in PBS-treated cells, so following normalization to b-actin, samples are expressed relative to DJ-1+/+ treated with LPS, and only LPS-treated cells were compared. 3. Results The stimulatory effect of LPS on the expression of COX2 (Fig. 1) and NOS2 (Fig. 2) mRNA was similar in DJ-1+/+ and DJ-1/ astrocytes and therefore independent of genotype. However, the stimulatory effect of LPS on the relative expression of TNFa mRNA was less in DJ-1/ than wild type cells (Fig. 3). COX2 expression in either genotype was not affected by pre-incubation with U0126 or JNK inhibitor II, however pre-incubation with the p38 inhibitor SB203580 significantly decreased COX2 upregulation (Fig. 1). Expression of NOS2 was below the limit of detection in all PBStreated samples, which is consistent with reports that this gene is not constitutively expressed in astrocytes (Moreno et al., 2008). NOS2 expression was effected only by the use of the p38 inhibitor (Fig. 2) in the DJ/ genotype where inhibition diminished LPSinduced increases in NOS2 expression; additionally, this diminished expression caused by the treatment with the p38 inhibitor was seen in DJ+/+ genotype at a statistical p value of 0.06. Genotypic differences in LPS, MEK, and JNK treatment groups of the knockout model were significantly diminished in the expression of TNFa mRNA. This expression was additionally shown to be lower in DJ-1/ astrocytes in regards to the p38 treatment (Fig. 3). Secretion of PGE2 was analyzed via assaying the culture medium from both PBS and LPS exposed cells. Vehicle controls were run for all treatment groups as a matter of diligence with no observable effect In LPS treated samples, DJ-1+/+ astrocytes secreted more PGE2 compared to DJ-1/ cells (Fig. 4). Inhibition of MEK, JNK, or p38 phosphorylation significantly decreased PGE2 release from DJ-1+/+ astrocytes (p < 0.05), whereas only p38 inhibition decreased secretion of PGE2 from DJ-1/ astrocytes (p < 0.05). Secretion of NO2 was assessed in the culture medium using a total nitrate/nitrite kit, however all samples were below the detection limit of the assay (5 mM nitrate/nitrite). This is not surprising given that other investigators have only noted minimal increases in NO2 produced from primary astrocytes, even following LPS exposure (Esen et al., 2004), and these levels were similar to the assay’s detection limit. To verify that the observed decreased expression of TNFa mRNA in DJ-1/ astrocytes was physiologically relevant, secretion of TNFa into the culture medium was analyzed. Levels of TNFa in all
PBS-treated samples were below the assay detection limit (8 pg/mL); therefore, only LPS-treated samples are graphically represented (Fig. 5). Basal LPS-induced secretion of TNFa (i.e., DMSO-treated samples) into media samples was significantly lower in DJ-1/ cultures than in DJ-1+/+ cultures (p < 0.001). Inhibition of JNK significantly decreased levels of secreted TNFa into the medium by DJ-1+/+ but not DJ-1/ astrocytes. Inhibition of p38 decreased secretion of TNFa from both DJ-1+/+ and DJ-1/ astrocytes. 4. Discussion and conclusions These data report that a loss of function mutation in DJ-1 decreased production of TNFa and PGE2 in astrocytes. It is noteworthy that dysregulation of these factors occurs in astrocytes in response to numerous endogenous and exogenous stimuli. The net effect of TNFa in the CNS is dose and context dependent with studies in knockout mice showing controversial results therefore the different variables accounting for TNFa’s effect in PD have not been defined. A decrease in TNFa expression and secretion in DJ-1/ mice may decrease astrocytic support of neuronal viability during early stages of neurodegeneration. TNFa is known to regulate calcium homeostasis (Daschil and Humpel, 2014), the synthesis and release of neuronal monoamines and glutamate, potassium and calcium ion channel regulation, and production of growth factors and cytokines, as well as numerous second messengers (Neniskyte et al., 2014; Perry et al., 2002; Vitkovic et al., 2000). Furthermore, both TNFa and its receptors are constitutively expressed in various regions of the brain including the cortex, striatum, and thalamus, suggesting a role in normal cellular homeostasis (Vitkovic et al., 2000). Astrocyte-derived TNFa is essential for maintaining synaptic connectivity and plasticity (Stellwagen and Malenka, 2006). Therefore, release of cytokines by astrocytes is critical to maintaining neuronal viability and decreased secretion mediated by DJ-1 mutation likely increases neuronal vulnerability. Prostaglandins, via their specific G protein coupled receptors, have a variety of physiological functions in the central nervous system. Whereas high PGE2 concentrations are neurotoxic (Ahmad et al., 2006a; Takadera et al., 2004), lower levels of PGE2 mediate neuroprotective effects (Ahmad et al., 2005; Ahmad et al., 2006b; McCullough et al., 2004). PGE2 itself is released from astrocytes following glutamate activation (Zonta et al., 2003) and is likely a cytoprotective event; therefore decreased PGE2 release in astrocytes induced by DJ-1/ may decrease neuronal viability. Because expression of COX2, NOS2, and TNFa are all upregulated in response to LPS, an effect predominantly mediated by nuclear factor kappa B (NFkB), it is surprising that DJ-1/
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Fig. 2. LPS-induced relative expression of NOS2 tends to decrease with inhibition of p38 but not MEK or JNK. Astrocytes were exposed to U0126 (MEKi, 10 mM), JNK inhibitor II (JNKi, 10 mM), or SB203580 (p38i, 30 mM) for 2 h, then PBS or 1 mg/mL LPS for 24 h, and RNA was isolated. Expression of NOS2 was determined via real-time PCR, and values are expressed relative to DJ-1+/+ cells exposed to DMSO and LPS, as all expression levels in PBS-treated samples were too low to detect. Inhibition of p38 decrease expression compared to basal levels in DJ-1/ cells and showed depression in expression in DJ+/+ (p = 0.11). * illustrates a significant difference (p < 0.05) from basal expression within the treatment group. D illustrates a significant difference (p < 0.05) from LPS treatment within the genotype.
Fig. 3. LPS-induced relative expression of TNF a is lower in DJ-1/ astrocytes, and does not decrease following inhibition of MEK, JNK, or p38. Astrocytes were exposed to U0126 (MEKi, 10 mM), JNK inhibitor II (JNKi, 10 mM), or SB203580 (p38i, 30 mM) for 2 h, then PBS or 1 mg/mL LPS for 24 h, and RNA was isolated. Expression of TNFa was determined via real-time PCR, and values are expressed relative to DJ-1+/+ cells exposed to DMSO and PBS. * illustrates a significant difference (p < 0.05) from basal expression within the treatment group. # illustrates a significant difference (p < 0.05) from same treatment between genotypes.
Fig. 4. LPS-induced PGE2 production is decreased in DJ-1/ astrocytes. Astrocytes were exposed to U0126 (MEKi, 10 mM), JNK inhibitor II (JNKi,10 mM), or SB203580 (p38i, 30 mM) for 2 h, then 1 mg/mL LPS for 24 h, and culture medium was collected. Secretion of PGE2 into the culture medium was determined via ELISA (eBiosciences). Bars represent means +/ SEM (n = 5). # illustrates a significant difference (p < 0.05) from same treatment between genotypes. D illustrates a significant difference (p < 0.05) from LPS treatment within the genotype.
astrocytes have lower expression of only TNFa. By analyzing levels of TNFa secreted into the culture medium, we have confirmed that decreased mRNA expression leads to a decline in secreted levels of TNFa by DJ-1/ astrocytes. It is possible that mutation of DJ-1 prevents a key co-activator from participating in NFkB binding, thus resulting in the blunted response observed in our studies. DJ1 is known to prevent PIASxa from negatively regulating the androgen receptor (Takahashi et al., 2001), so removal of DJ1 might negatively affect genes downstream of this protein, and a yet unidentified protein could mediate similar effects in TNFa regulation. Mo and colleagues suggest that MEKK1 requires functional DJ-1 for full activation (Mo et al., 2008). As
MEKK1 phosphorylates and activates IkB kinase (Lee et al., 1998), it is possible that mutation of DJ-1 decreases overall phosphorylation of IkB. Furthermore, numerous proteins, which are not involved in COX2 or NOS2 regulation (Bcl3, Atf2, Egr1, and Stat) but are involved in regulating the TNFa promoter, have not been studied for DJ-1 interaction. DJ-1 may transiently interact with one or more of these proteins, or act further upstream to modulate regulation of TNFa. In the current study, we established that expression of representative pro-inflammatory genes did not respond identically to inhibition of MAPKs. Inhibition of p38 alone decreased expression of COX2. Inhibition of p38 significantly decreased
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Fig. 5. LPS-induced TNFa secretion is decreased in DJ-1/ astrocytes. Astrocytes were exposed to U0126 (MEKi, 10 mM), JNK inhibitor II (JNKi,10 mM), or SB203580 (p38i, 30 mM) for 2 h, then 1 mg/mL LPS for 24 h, and culture medium was collected. Amount of TNFa in the culture medium was determined EIA (Cayman Chemical). Bars represent means +/ SEM (n = 5). # illustrates a significant difference (p < 0.05) from same treatment between genotypes. D illustrates a significant difference (p < 0.05) from LPS treatment within the genotype.
expression of NOS2 in DJ-1/ and showed depression in DJ+/+ (p = 0.06) astrocytes compared to their respective controls. Neither MEK nor JNK inhibition affected expression of NOS2. No expression changes in COX2 were induced by inhibition of MEK, JNK, or p38. However, secretion of PGE2 was lower in DJ-1/ astrocytes. Interestingly, MEK inhibition did not affect secretion of TNFa in either genotype, whereas significant decreases were observed in DJ-1+/+ astrocytes treated with JNK inhibitor II, and inhibition of p38 decreased secretion in both genotypes. As neuroinflammation and reactive astrocytosis are clearly involved in the pathogenesis of PD as well as many other neurological disorders, the response of astrocytes to pro-inflammatory stimuli such as LPS warrants investigation. While we would have anticipated that mutation of DJ-1 would increase expression of pro-inflammatory proteins following LPS exposure, upregulation of TNFa was lower in DJ-1/ astrocytes compared to DJ-1+/+ cells. No other data to date has demonstrated alterations in TNFa levels due to knockout, knockdown, or even overexpression of DJ-1; therefore how this expression is decreased is unclear. Because the etiology of PD is still enigmatic, the early neuronal changes that occur with this disease are still largely unknown. However, given the cytoprotective effects of TNFa, it is likely that decreasing the secretion of this cytokine from astrocytes would be detrimental to neuronal viability. In support of this concept, data from Mullett and Hinkle (2009) suggests that DJ-1 knockdown astrocytes provide decreased neuronal protection due to an alteration in unidentified soluble factors released into the culture medium. In summary, mutation of DJ-1 decreased LPS-induced expression and secretion of TNFa, as well as secretion of PGE2 in primary astrocytes. This finding is the first to suggest TNFa or PGE2 could be influenced by DJ-1 providing insight into DJ-1’s role in cellular homeostasis. Because expression of COX2 was unaffected by mutation of DJ-1, yet changes in PGE2 secretion occurred, it is possible that DJ-1 may affect secretory pathways as well as protein production. Future studies are needed to discern the mechanisms by which DJ-1 alters LPS-induced astroglial regulation of cytokines and define physiological consequences of these alterations are in the context of DJ-1 related disorders. Conflict of interest There are no conflicts of interests for authors on this publication. Acknowledgements Funding for this research was provided by a Basil O’Connor Starter Scholar Research Award to Dr. Marie Legare and a College of
Veterinary Medicine and Biomedical Sciences College Research Council Award to Dr. William Hanneman. References Ahmad, A.S., Ahmad, M., de Brum-Fernandes, A.J., Dore, S., 2005. Prostaglandin EP4 receptor agonist protects against acute neurotoxicity. Brain Res. 1066, 71–77. Ahmad, A.S., Saleem, S., Ahmad, M., Dore, S., 2006a. Prostaglandin EP1 receptor contributes to excitotoxicity and focal ischemic brain damage. Toxicol. Sci. 89, 265–270. Ahmad, A.S., Zhuang, H., Echeverria, V., Dore, S., 2006b. Stimulation of prostaglandin EP2 receptors prevents NMDA-induced excitotoxicity. J. Neurotrauma 23, 1895–1903. Aleyasin, H., Rousseaux, M.W., Phillips, M., Kim, R.H., Bland, R.J., Callaghan, S., Slack, R.S., During, M.J., Mak, T.W., Park, D.S., 2007. The Parkinson’s disease gene DJ-1 is also a key regulator of stroke-induced damage. Proc. Natl. Acad. Sci. U. S. A. 104, 18748–18753. Allen, J.W., Mutkus, L.A., Aschner, M., 2000. Isolation of neonatal rat cortical astrocytes for primary cultures. Current Protocols in Toxicology. John Wiley & Sons, Inc. 11-12.14.15 (chapter 12: Unit 12.4). Andres-Mateos, E., Perier, C., Zhang, L., Blanchard-Fillion, B., Greco, T.M., Thomas, B., Ko, H.S., Sasaki, M., Ischiropoulos, H., Przedborski, S., Dawson, T.M., Dawson, V. L., 2007. DJ-1 gene deletion reveals that DJ-1 is an atypical peroxiredoxin-like peroxidase. Proc. Natl. Acad. Sci. U. S. A. 104, 14807–14812. Ashley, A.K., Hanneman, W.H., Katoh, T., Moreno, J.A., Pollack, A., Tjalkens, R.B., Legare, M.E., 2009. Analysis of targeted mutation in DJ-1 on cellular function in primary astrocytes. Toxicol. Lett. 184, 186–191. Bakos, J., Duncko, R., Makatsori, A., Pirnik, Z., Kiss, A., Jezova, D., 2004. Prenatal immune challenge affects growth, behavior, and brain dopamine in offspring. Ann. N. Y. Acad. Sci. 1018, 281–287. Bandopadhyay, R., Kingsbury, A.E., Cookson, M.R., Reid, A.R., Evans, I.M., Hope, A.D., Pittman, A.M., Lashley, T., Canet-Aviles, R., Miller, D.W., McLendon, C., Strand, C., Leonard, A.J., Abou-Sleiman, P.M., Healy, D.G., Ariga, H., Wood, N.W., de Silva, R., Revesz, T., Hardy, J.A., Lees, A.J., 2004. The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson’s disease. Brain 127, 420–430. Batelli, S., Invernizzi, R.W., Negro, A., Calcagno, E., Rodilossi, S., Forloni, G., Albani, D., 2015. The Parkinson's disease-related protein DJ-1 protects dopaminergic neurons in vivo and cultured cells from alpha-synuclein and 6hydroxydopamine toxicity. Neurodegenerative Dis. 15 (1), 13–23. Bertolin, G., Jacoupy, M., Traver, S., Ferrando-Miguel, R., Saint Georges, T., Grenier, K., Ardila-Osorio, H., Muriel, M.P., Takahashi, H., Lees, A.J., Gautier, C., Guedin, D., Coge, F., Fon, E.A., Brice, A., Corti, O., 2015. Parkin maintains mitochondrial levels of the protective Parkinson’s disease-related enzyme 17-b hydroxysteroid dehydrogenase type 10. Cell Death Differ. 16 (January) . Cai, Z., Zeng, W., Tao, K., Lu, F., Gao, G., Yang, Q., 2015. Myricitrin alleviates MPP (+)-induced mitochondrial dysfunction in a DJ-1-dependent manner in SN4741 cells. Biochem. Biophys. Res. Commun. 458 (2), 227–233. Cai, Z., Pang, Y., Lin, S., Rhodes, P.G., 2003. Differential roles of tumor necrosis factoralpha and interleukin-1 beta in lipopolysaccharide-induced brain injury in the neonatal rat. Brain Res. 975, 37–47. Carbone, D.L., Popichak, K.A., Moreno, J.A., Safe, S., Tjalkens, R.B., 2008. Suppression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nitric oxide synthase 2 expression in astrocytes by a novel diindolylmethane analog protects striatal neurons against apoptosis. Mol. Pharmacol. 75 (1), 35–43. Carvey, P.M., Chang, Q., Lipton, J.W., Ling, Z., 2003. Prenatal exposure to the bacteriotoxin lipopolysaccharide leads to long-term losses of dopamine neurons in offspring: a potential, new model of Parkinson’s disease. Front. Biosci. 8, s826–s837. Choi, J., Sullards, M.C., Olzmann, J.A., Rees, H.D., Weintraub, S.T., Bostwick, D.E., Gearing, M., Levey, A.I., Chin, L.S., Li, L., 2006. Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases. J. Biol. Chem. 281, 10816–10824.
A.K. Ashley et al. / NeuroToxicology 52 (2016) 198–203 Chung, I.Y., Benveniste, E.N., 1990. Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide IFN-gamma, and IL-1 beta. J. Immunol. 144, 2999–3007. Daschil, N., Humpel, C., 2014. Nifedipine and nimodipine protect dopaminergic substantia nigra neurons against axotomy-induced cell death in rat vibrosections via modulating inflammatory responses. Brain Res. 18 (September), 1581. Ejima, K., Nanri, H., Araki, M., Koji, T., Shibata, E., Kashimura, M., Ikeda, M., 2000. Expression of mitochondrial thioredoxin-dependent antioxidant protein, SP-22, in normal human and inflammatory mouse placentae. Placenta 21, 847–852. Esen, N., Tanga, F.Y., DeLeo, J.A., Kielian, T., 2004. Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus. J. Neurochem. 88, 746–758. Gayle, D.A., Ling, Z., Tong, C., Landers, T., Lipton, J.W., Carvey, P.M., 2002. Lipopolysaccharide (LPS)-induced dopamine cell loss in culture: roles of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide. Brain Res. Dev. Brain Res. 133 (1), 27–35. Goswami, A., Dikshit, P., Mishra, A., Mulherkar, S., Nukina, N., Jana, N.R., 2006. Oxidative stress promotes mutant huntingtin aggregation and mutant huntingtin-dependent cell death by mimicking proteasomal malfunction. Biochem. Biophys. Res. Commun. 342, 184–190. Hennis, M.R., Seamans, K.W., Marvin, M.A., Casey, B.H., Goldberg, M.S., 2013. Behavioral and neurotransmitter abnormalities in mice deficient for Parkin, DJ1 and superoxide dismutase. PLoS One 8 (12), e84894. Herrera, A.J., Castano, A., Venero, J.L., Cano, J., Machado, A., 2000. The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system. Neurobiol. Dis. 7, 429–447. Hirotani, M., Maita, C., Niino, M., Iguchi-Ariga, S., Hamada, S., Ariga, H., Sasaki, H., 2008. Correlation between DJ-1 levels in the cerebrospinal fluid and the progression of disabilities in multiple sclerosis patients. Mult. Scler. 14, 1056–1060. Hoban, D.B., Connaughton, E., Connaughton, C., Hogan, G., Thornton, C., Mulcahy, P., Moloney, T.C., Dowd, E., 2013. Further characterisation of the LPS model of Parkinson’s disease: a comparison of intra-nigral and intra-striatal lipopolysaccharide administration on motor function, microgliosis and nigrostriatal neurodegeneration in the rat. Brain Behav. Immun. 27 (1), 91–100. Hunter, R.L., Dragicevic, N., Seifert, K., Choi, D.Y., Liu, M., Kim, H.C., Cass, W.A., Sullivan, P.G., Bing, G., 2007. Inflammation induces mitochondrial dysfunction and dopaminergic neurodegeneration in the nigrostriatal system. J. Neurochem. 100, 1375–1386. Larsen, K., Madsen, L.B., Hoj, A., Bendixen, C., 2007. Porcine DJ-1: cloning of PARK7 cDNA, sequence comparison, expression analysis and chromosomal localization. Cytogenet. Genome. Res. 116, 93–99. Lee, F.S., Peters, R.T., Dang, L.C., Maniatis, T., 1998. MEKK1 activates both IkappaB kinase alpha and IkappaB kinase beta. Proc. Natl. Acad. Sci. U. S. A. 95, 9319– 9324. Ling, Z., Gayle, D.A., Ma, S.Y., Lipton, J.W., Tong, C.W., Hong, J.S., Carvey, P.M., 2002. In utero bacterial endotoxin exposure causes loss of tyrosine hydroxylase neurons in the postnatal rat midbrain. Mov. Disord. 17, 116–124. Liu, F., Zhou, R., Yan, H., Yin, H., Wu, X., Tan, Y., Li, L., 2014. Metabotropic glutamate receptor 5 modulates calcium oscillation and innate immune response induced by lipopolysaccharide in microglial cell. Neuroscience 281C (22), 24–34. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25 (4), 402–408. Lu, Y., He, M., Zhang, Y., Xu, S., Zhang, L., He, Y., Chen, C., Liu, C., Pi, H., Yu, Z., Zhou, Z., 2014. Differential pro-inflammatory responses of astrocytes and microglia involve STAT3 activation in response to 1800 MHz radiofrequency fields. PLos One 9 (10) . Mbefo, M., Fares, M.B., Paleologou, K., Oueslati, A., Yin, G., Tenreiro, S., Pinto, M., Outeiro, T., Zweckstetter, M., Masliah, E., Lashuel, H.A., 2015. The Parkinson's disease mutant E46K enhances alpha-synuclein phosphorylation in mammalian cell-lines, in yeast and in vivo. J. Biol. Chem. 290 (February (15)), 9412–9427. McCullough, L., Wu, L., Haughey, N., Liang, X., Hand, T., Wang, Q., Breyer, R.M., Andreasson, K., 2004. Neuroprotective function of the PGE2 EP2 receptor in cerebral ischemia. J. Neurosci. 24, 257–268. Meulener, M.C., Graves, C.L., Sampathu, D.M., Armstrong-Gold, C.E., Bonini, N.M., Giasson, B.I., 2005. DJ-1 is present in a large molecular complex in human brain tissue and interacts with alpha-synuclein. J. Neurochem. 93, 1524–1532. Mitsumoto, A., Nakagawa, Y., 2001. DJ-1 is an indicator for endogenous reactive oxygen species elicited by endotoxin. Free Radic. Res. 35, 885–893. Mo, J.S., Kim, M.Y., Ann, E.J., Hong, J.A., Park, H.S., 2008. DJ-1 modulates UV-induced oxidative stress signaling through the suppression of MEKK1 and cell death. Cell Death Differ. 15 (16), 1030–1041. Moreno, J.A., Sullivan, K.A., Carbone, D.L., Hanneman, W.H., Tjalkens, R.B., 2008. Manganese potentiates nuclear factor-kappaB-dependent expression of nitric oxide synthase 2 in astrocytes by activating soluble guanylate cyclase and extracellular responsive kinase signaling pathways. J. Neurosci. Res. 86 (9), 2028–2038.
203
Mullett, S.J., Hinkle, D.A., 2009. DJ-1 knock-down in astrocytes impairs astrocytemediated neuroprotection against rotenone. Neurobiol. Dis. 33 (1), 28–36. Neniskyte, U., Vilalta, A., Brown, G.C., 2014. Tumour necrosis factor alpha-induced neuronal loss is mediated by microglial phagocytosis. FEBS Lett. 588 (17), 2952–2956. Neumann, M., Muller, V., Gorner, K., Kretzschmar, H.A., Haass, C., Kahle, P.J., 2004. Pathological properties of the Parkinson’s disease-associated protein DJ-1 in alpha-synucleinopathies and tauopathies: relevance for multiple system atrophy and Pick’s disease. Acta Neuropathol. (Berl) 107, 489–496. Olzmann, J.A., Brown, K., Wilkinson, K.D., Rees, H.D., Huai, Q., Ke, H., Levey, A.I., Li, L., Chin, L.S., 2004. Familial Parkinson’s disease-associated L166P mutation disrupts DJ-1 protein folding and function. J. Biol. Chem. 279, 8506–8515. Perrett, R.M., Alexopoulou, Z., Tofaris, G.K., 2015. The endosomal pathway in Parkinson’s disease. Mol. Cell. Neurosci. 19 (10), 1016. Perry, S.W., Dewhurst, S., Bellizzi, M.J., Gelbard, H.A., 2002. Tumor necrosis factoralpha in normal and diseased brain: conflicting effects via intraneuronal receptor crosstalk? J. Neurovirol. 8, 611–624. Puschmann, A., 2013. Monogenic Parkinson’s disease and parkinsonism: clinical phenotypes and frequencies of known mutations. Parkinsonism Relat. Disord. 19 (4), 407–415. Qin, L., Wu, X., Block, M.L., Liu, Y., Breese, G.R., Hong, J.S., Knapp, D.J., Crews, F.T., 2007. Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration. Glia 55, 453–462. Rousseaux, M.W., Marcogliese, P.C., Qu, D., Hewitt, S.J., Seang, S., Kim, R.H., Slack, R. S., Schlossmacher, M.G., Lagace, D.C., Mak, T.W., Park, D.S., 2012. Progressive dopaminergic cell loss with unilateral-to-bilateral progression in a genetic model of Parkinson disease. Proc. Natl. Acad. Sci. U. S. A. 109 (39), 15918–15923. Saito, Y., Miyasaka, T., Hatsuta, H., Takahashi-Niki, K., Hayashi, K., Mita, Y., KusanoArai, O., Iwanari, H., Ariga, H., Hamakubo, T., Yoshida, Y., Niki, E., Murayama, S., Ihara, Y., Noguchi, N., 2014. Immunostaining of oxidized DJ-1 in human and mouse brains. J. Neuropathol. Exp. Neurol. 73 (7), 714–728. Sharif, S.F., Hariri, R.J., Chang, V.A., Barie, P.S., Wang, R.S., Ghajar, J.B., 1993. Human astrocyte production of tumour necrosis factor-alpha, interleukin-1 beta, and interleukin-6 following exposure to lipopolysaccharide endotoxin. Neurol. Res. 15, 109–112. Shendelman, S., Jonason, A., Martinat, C., Leete, T., Abeliovich, A., 2004. DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation. PLoS Biol. 2, e362. Stellwagen, D., Malenka, R.C., 2006. Synaptic scaling mediated by glial TNF-alpha. Nature 440, 1054–1059. Tai, W., Ye, X., Bao, X., Zhao, B., Wang, X., Zhang, D., 2013. Inhibition of Src tyrosine kinase activity by squamosamide derivative FLZ attenuates neuroinflammation in both in vivo and in vitro Parkinson’s disease models. Neuropharmacology 75, 201–212. Takadera, T., Shiraishi, Y., Ohyashiki, T., 2004. Prostaglandin E2 induced caspasedependent apoptosis possibly through activation of EP2 receptors in cultured hippocampal neurons. Neurochem. Int. 45, 713–719. Takahashi, K., Taira, T., Niki, T., Seino, C., Iguchi-Ariga, S.M., Ariga, H., 2001. DJ1 positively regulates the androgen receptor by impairing the binding of PIASx alpha to the receptor. J. Biol. Chem. 276, 37556–37563. Triplett, J., Zhang, Z., Sultana, R., Cai, J., Klein, J.B., Büeler, H., Butterfield, D.A., 2015. Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson disease. J. Neurochem. 133 (January (5)), 750–765. Vitkovic, L., Bockaert, J., Jacque, C., 2000. Inflammatory cytokines: neuromodulators in normal brain. J. Neurochem. 74, 457–471. Waragai, M., Wei, J., Fujita, M., Nakai, M., Ho, G.J., Masliah, E., Akatsu, H., Yamada, T., Hashimoto, M., 2006. Increased level of DJ-1 in the cerebrospinal fluids of sporadic Parkinson’s disease. Biochem. Biophys. Res. Commun. 345, 967–972. Waragai, M., Nakai, M., Wei, J., Fujita, M., Mizuno, H., Ho, G., Masliah, E., Akatsu, H., Yokochi, F., Hashimoto, M., 2007. Plasma levels of DJ-1 as a possible marker for progression of sporadic Parkinson’s disease. Neurosci. Lett. 425, 18–22. Wilson, M.A., 2011. The role of cysteine oxidation in DJ-1 function and dysfunction. Antioxid. Redox Signal. 15 (1), 111–122. Yamaguchi, H., Shen, J., 2007. Absence of dopaminergic neuronal degeneration and oxidative damage in aged DJ-1-deficient mice. Mol. Neurodegener. 2 (10) . Yanagisawa, D., Kitamura, Y., Inden, M., Takata, K., Taniguchi, T., Morikawa, S., Morita, M., Inubushi, T., Tooyama, I., Taira, T., Iguchi-Ariga, S.M., Akaike, A., Ariga, H., 2008. DJ-1 protects against neurodegeneration caused by focal cerebral ischemia and reperfusion in rats. J. Cereb. Blood Flow Metab. 28 (3), 563–578. Zhang, L., Shimoji, M., Thomas, B., Moore, D.J., Yu, S.W., Marupudi, N.I., Torp, R., Torgner, I.A., Ottersen, O.P., Dawson, T.M., Dawson, V.L., 2005. Mitochondrial localization of the Parkinson's disease related protein DJ-1: implications for pathogenesis. Hum. Mol. Genet. 14, 2063–2073. Zhou, W., Zhu, M., Wilson, M.A., Petsko, G.A., Fink, A.L., 2006. The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein. J. Mol. Biol. 356, 1036–1048. Zonta, M., Sebelin, A., Gobbo, S., Fellin, T., Pozzan, T., Carmignoto, G., 2003. Glutamate-mediated cytosolic calcium oscillations regulate a pulsatile prostaglandin release from cultured rat astrocytes. J. Physiol. 553, 407–414.
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NeuroToxicology
Full length article
DJ-1 mutation decreases astroglial release of inflammatory mediators A.K. Ashley, A.I. Hinds, W.H. Hanneman, R.B. Tjalkens, M.E. Legare* The Center for Environmental Medicine, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1680, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 June 2015 Received in revised form 10 November 2015 Accepted 8 December 2015 Available online 12 December 2015
Mutations in DJ-1, reactive gliosis and concomitant inflammatory processes are implicated in the pathogenesis and progression of Parkinson’s disease (PD). To study the physiological consequences of DJ-1 mutation in the context of neuroinflammatory insult, primary cortical astrocytes were isolated from DJ-1 knockout mice. Astrocytes were exposed to 1 mg/mL lipopolysaccharide (LPS) for 24 h following 2 h pre-exposure to inhibitors of MEK (U0126), JNK (JNK inhibitor II) or p38 (SB203580). Real-time PCR was used to assess the LPS-induced expression of pro-inflammatory mediators cyclooxygenase 2 (COX2), inducible nitric oxide synthetase (NOS2), and tumor necrosis factor a (TNFa). LPS-induced expression of COX2 decreased similarly in DJ-1+/+ and DJ-1/ astrocytes in response to inhibition of p38, but was unaffected by inhibition of MEK or JNK. No significant alterations in NOS2 expression were observed in any inhibitor-treated cells. The inhibitors did not affect expression of TNFa; however, DJ-1/ astrocytes had consistently lower expression compared to DJ-1+/+ counterparts. Secretion of TNFa and prostaglandin E2 (PGE2) into the culture medium was significantly decreased in DJ-1/ astrocytes, and inhibition of p38 decreased this secretion in both genotypes. In conclusion, DJ-1/ astrocytes may provide decreased neuroprotection to surrounding neurons due to alterations in pro-inflammatory mediator expression. ã 2015 Elsevier Inc. All rights reserved.
Keywords: DJ-1 Astrocyte Neuroinflammation Lipopolysaccharide
1. Introduction Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disease resulting from loss of dopaminergic neurons in the substantia nigra pars compacta. Approximately 5–10% of diagnosed PD cases have been associated with genetic mutations (Perrett et al., 2015). Specifically, mutations in 7 genes are robustly associated with autosomal dominant (SNCA, LRRK2, EIF4G1, VPS35) or recessive (parkin/PARK2, PINK1, DJ1/PARK7) PD (Bertolin et al., 2015; Mbefo et al., 2015; Puschmann 2013; Triplett et al., 2015). DJ-1 is a recessive familial PD gene involved in anti oxidative function as well as mitochondrial maintenance (Cai et al., 2015; Saito et al., 2014). DJ-1 is also known to regulate the activity of phosphatase and tension homolog (PTEN) which plays a critical role in neuronal cell death in response to various insults. DJ1 protein is widely produced throughout mammalian tissues (Olzmann et al., 2004; Zhang et al., 2005; Larsen et al., 2007) and may function as an atypical peroxiredoxin-like peroxidase, capable of scavenging reactive oxygen species (Andres-Mateos et al., 2007) and mediate cellular protection through its antioxidant properties (Wilson, 2011). DJ-1 has been suggested to function as a redox-
* Corresponding author. E-mail address: [email protected] (M.E. Legare). http://dx.doi.org/10.1016/j.neuro.2015.12.007 0161-813X/ ã 2015 Elsevier Inc. All rights reserved.
dependent chaperone, promoting proper folding of a-synuclein (Shendelman et al., 2004; Zhou et al., 2006). Further, DJ-1 has been shown to accumulate in brain tissue and is elevated in the plasma and cerebrospinal fluid of individuals with PD (Waragai et al., 2006; Waragai et al., 2007). Given the myriad of key proteins that DJ-1 may modulate, including a-synuclein and Nrf2 (nuclear factor-like 2), DJ-1 could affect numerous physiological processes. Indeed, like PD, other neurological disorders have alterations in DJ-1 expression and/or function including Alzheimer’s disease (Choi et al., 2006), multiple system atrophy and Pick’s disease, (Neumann et al., 2004), multiple sclerosis (Hirotani et al., 2008), cells expressing huntingtin (Goswami et al., 2006), and ischemia/reperfusion injury (Aleyasin et al., 2007; Yanagisawa et al., 2008), indicating DJ-1 may play a significant role in maintaining CNS homeostasis (Batelli et al., 2015). Although in humans DJ-1 mutation induces PD, DJ-1 knockout mice fail to consistently recapitulate the key clinical and neuropathological features of PD (Rousseaux et al., 2012; Yamaguchi and Shen, 2007) suggesting the existence of compensatory mechanisms that may protect mice from the neurodegeneration and the consequent motor symptoms. However, recent reports suggest consistent alterations in behavioral and striatal dopamine abnormalities in the DJ-1 knockout mice (Hennis et al., 2013; Hennis et al., 2013). It is apparent that the DJ-1 mutant mouse model provides an excellent platform for investigating PD
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as a multi-factorial disease including the specific role of DJ-1 in neural cells. In utero exposure to bacterial lipopolysaccharide (LPS) has been shown to decrease the number of dopaminergic neurons as well as brain dopamine levels in offspring (Bakos et al., 2004; Carvey et al., 2003; Ling et al., 2002). Further, exposure to LPS as an adult can initiate neuronal cell loss within the substantia nigra in experimental models, and may be an environmental factor in the development of PD (Gayle et al., 2002; Herrera et al., 2000; Qin et al., 2007). LPS acts through toll-like receptors to increase the expression of numerous pro-inflammatory mediators in microglia and other cells, including interleukin1 (IL-1) (Sharif et al., 1993), interleukin 6 (IL-6) (Chung and Benveniste, 1990), mitogenactivated protein kinases (Tai et al., 2013), and TNFa (Liu et al., 2014). Recently, LPS-induced inflammation was demonstrated to induce mitochondrial dysfunction in the substantia nigra as well as the striatum, with increases in COX2 and NOS2 (Hunter et al., 2007). In animals, LPS also induces astrogliosis and neurodegeneration (Hoban et al., 2013), evidenced by increased glialfibrillary acidic protein (Cai et al., 2003). Several studies have shown that astroglia and microglia differentially expression IL-1b and TNFa following stimulation by LPS, with microglia having higher expression levels of IL-1b but TNFa expression being comparable between the two cell types (Lu et al., 2014). Reactive astrocytes consistently have high expression of DJ-1 (Saito et al., 2014), suggesting that DJ-1 has a significant role in gliosis or is elevated resultant to the gliosis (Bandopadhyay et al., 2004; Meulener et al., 2005). Interestingly, DJ-1 protein increases following exposure to LPS (Ejima et al., 2000; Mitsumoto and Nakagawa, 2001) which may indicate that DJ-1 might possibly play an important role in preventing or alleviating LPS-induced toxicity. Increasing evidence indicates that development of PD represents a labyrinth of interactions occurring over time, with contributing factors including genetic predisposition and exposure to environmental toxins. Given that pro-inflammatory mediators promote neuronal viability and prolonged inflammation contributes to neurodegeneration, we tested the hypothesis that LPSexposed astrocytes from DJ-1 mutant mice would exhibit alterations in expression of genes related to inflammation. DJ-1’s role in modulating the inflammatory response may help to elucidate pathways involved in PD and other neurological diseases.
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Colorado State University Institutional Animal Care and Use Committee. 2.2. Real-time PCR
2. Materials and methods
To assess which protein(s) of the mitogen-activated protein kinase (MAPK) pathways are involved in COX2, NOS2, and TNFa increases following LPS exposure in astrocytes, we utilized a MEK inhibitor (MEKi, 10 mM U0126, Cell Signaling), a p38 inhibitor (p38i, 30 mM SB203580, Calbiochem), or an inhibitor of JNK (JNKi, 10 mM JNK Inhibitor II, Calbiochem). Each inhibitor was suspended in DMSO (Sigma) at concentrations designed to deliver equivalent volumes of DMSO. DMSO alone was used in our control group at the same volume as inhibitor-exposed groups (0.1%). The concentrations of inhibitors selected have been demonstrated to inhibit phosphorylation of the respective protein in mouse primary cortical astrocytes previously demonstrated within our laboratory (Moreno et al., 2008). Cells were preincubated with inhibitors and astrocytes were subsequently exposed to PBS or 1 mg/mL LPS (Sigma) for 24 h, culture medium was collected for analysis, and RNA was isolated via the RNeasy Kit (Qiagen) including on-column DNase treatment. cDNA was synthesized with iScript (BioRad) to assess expression of COX2, NOS2, and TNFa via real-time PCR. Relative expression of COX2 (50 GGA GTC TGG AAC ATT GTG AAC - 30 , 50 - GTA GTA GGA GAG GTT GGA GAA G - 30 ), NOS2 (50 -TCA CGC TTG GGT CTT GTT- 30 , 50 - CAG GTC ACT TTG GTA GGA TTT G - 30 ) or TNF a (50 - GCA CCA CCA TCA AGG ACT C 30 , 50 - GAA AGG TCT GAA GGT AGG AAG G - 30 ) was measured using a BioRad iCycler utilizing SYBR green incorporation (BioRad). Expression of COX2, NOS2, and TNFa in each sample was normalized to expression of b-actin (50 -GAC AGG ATG CAG AAG GAG ATT ACT G-30 , 50 -GCT GAT CCA CAT CTG CTG GAA-30 ) via the delta–delta CT method (Livak and Schmittgen, 2001) and is reported as relative expression. Each primer set was examined prior to use to assure consistent PCR efficiency, production of one specific amplicon and absence of primer-dimers. As a negative control, each RNA sample without reverse transcriptase was also analyzed to assure the lack of genomic DNA contamination. Each RNA sample was assayed in duplicate for each primer set assessed. The relative mRNA expression of COX2 and TNFa in each genotype was compared to DJ-1+/+ astrocytes exposed to PBS and DMSO. In all PBS-treated samples, NOS2 expression was beneath the detection limit; therefore expression of NOS2 was analyzed relative to LPSexposed DJ-1+/+ levels.
2.1. Primary astrocyte isolation
2.3. Secreted TNFa, prostaglandin, and nitric oxide
DJ-1 mutant mice were generated, characterized, and genotyped as previously described (Ashley et al., 2009). Heterozygous mice were bred to maintain a colony containing all three genotypes. Primary mouse cortical astrocytes were isolated and maintained as previously described (Allen et al., 2000). This isolation method results in 98% pure astrocyte culture, as determined through immunofluorescent staining (Carbone et al., 2008). Cortices from day 0 to 1 old mouse pups were dissected and meninges removed. Tissue was digested with dispase (1.5 U/mL) in warm MEM with Earle’s Salt and L-glutamine (Fisher) supplemented with an antibiotic cocktail (Invitrogen) containing penicillin (0.001 mg/mL), streptomycin (0.001 mg/mL), and neomycin (0.002 mg/mL). Cells were plated on tissue culture dishes (BD Biosciences) at 300,000 cells/mL in culture medium described above (without dispase) supplemented with 10% fetal bovine serum (Atlas). Culture medium was changed after 24 h to remove the non-adherent microglial cells. Astrocytes were cultured for 3–4 weeks to achieve approximately 80% confluence. Culture medium was changed 24 h prior to application of any treatment to minimize serum shock. All animal procedures were approved by the
Astrocytes were cultured as described, and medium was collected and stored at 80 C for later analysis. TNFa levels in culture medium samples were determined via an enzyme-linked immunosorbent assay (ELISA, eBiosciences). Secreted levels of prostaglandin E2 were assessed with a monoclonal EIA kit with limit of detection of 39–25,000 pg/mL (Cayman Chemicals). Nitric oxide levels in culture medium were determined via nitrate/nitrite colorimetric assay (Cayman Chemicals). 2.4. Statistical analyses All statistical analyses were performed with GraphPad Prism software. Means were analyzed using two way ANOVA, and when they significantly differed (p < 0.05), Tukey’s post hoc test was employed. Four sets of astrocyte cell cultures were examined: DJ1+/+ treated with PBS, DJ-1+/+ treated with LPS, DJ-1/ treated with PBS, and DJ-1/ treated with LPS. Each set contained four treatment groups: cultures were treated with MEKi, JNKi, or p38i, or with DMSO as the vehicle control for the inhibitors. mRNA levels were measured by real-time PCR in each group for genes of
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Fig. 1. LPS-induced relative expression of COX2 decreases with inhibition of p38 but not MEK or JNK. Astrocytes were exposed to U0126 (MEKi, 10 mM), JNK inhibitor II (JNKi, 10 mM), or SB203580 (p38i, 30 mM) for 2 h, then PBS or 1 mg/mL LPS for 24 h, and RNA was isolated. Expression of COX2 was determined via real-time PCR, and values are expressed relative to DJ-1+/+ cells exposed to DMSO and PBS. * illustrates a significant difference (p < 0.05) from basal expression within the treatment group. D illustrates a significant difference (p < 0.05) from LPS treatment within the genotype.
interest. In utilizing the delta–delta CT method for analysis of realtime PCR, CT values were normalized to b-actin, then relative to DJ1+/+ treated with PBS and DMSO to yield relative expression of COX2 and TNFa. In NOS2 samples, mRNA was below the threshold of detection in PBS-treated cells, so following normalization to b-actin, samples are expressed relative to DJ-1+/+ treated with LPS, and only LPS-treated cells were compared. 3. Results The stimulatory effect of LPS on the expression of COX2 (Fig. 1) and NOS2 (Fig. 2) mRNA was similar in DJ-1+/+ and DJ-1/ astrocytes and therefore independent of genotype. However, the stimulatory effect of LPS on the relative expression of TNFa mRNA was less in DJ-1/ than wild type cells (Fig. 3). COX2 expression in either genotype was not affected by pre-incubation with U0126 or JNK inhibitor II, however pre-incubation with the p38 inhibitor SB203580 significantly decreased COX2 upregulation (Fig. 1). Expression of NOS2 was below the limit of detection in all PBStreated samples, which is consistent with reports that this gene is not constitutively expressed in astrocytes (Moreno et al., 2008). NOS2 expression was effected only by the use of the p38 inhibitor (Fig. 2) in the DJ/ genotype where inhibition diminished LPSinduced increases in NOS2 expression; additionally, this diminished expression caused by the treatment with the p38 inhibitor was seen in DJ+/+ genotype at a statistical p value of 0.06. Genotypic differences in LPS, MEK, and JNK treatment groups of the knockout model were significantly diminished in the expression of TNFa mRNA. This expression was additionally shown to be lower in DJ-1/ astrocytes in regards to the p38 treatment (Fig. 3). Secretion of PGE2 was analyzed via assaying the culture medium from both PBS and LPS exposed cells. Vehicle controls were run for all treatment groups as a matter of diligence with no observable effect In LPS treated samples, DJ-1+/+ astrocytes secreted more PGE2 compared to DJ-1/ cells (Fig. 4). Inhibition of MEK, JNK, or p38 phosphorylation significantly decreased PGE2 release from DJ-1+/+ astrocytes (p < 0.05), whereas only p38 inhibition decreased secretion of PGE2 from DJ-1/ astrocytes (p < 0.05). Secretion of NO2 was assessed in the culture medium using a total nitrate/nitrite kit, however all samples were below the detection limit of the assay (5 mM nitrate/nitrite). This is not surprising given that other investigators have only noted minimal increases in NO2 produced from primary astrocytes, even following LPS exposure (Esen et al., 2004), and these levels were similar to the assay’s detection limit. To verify that the observed decreased expression of TNFa mRNA in DJ-1/ astrocytes was physiologically relevant, secretion of TNFa into the culture medium was analyzed. Levels of TNFa in all
PBS-treated samples were below the assay detection limit (8 pg/mL); therefore, only LPS-treated samples are graphically represented (Fig. 5). Basal LPS-induced secretion of TNFa (i.e., DMSO-treated samples) into media samples was significantly lower in DJ-1/ cultures than in DJ-1+/+ cultures (p < 0.001). Inhibition of JNK significantly decreased levels of secreted TNFa into the medium by DJ-1+/+ but not DJ-1/ astrocytes. Inhibition of p38 decreased secretion of TNFa from both DJ-1+/+ and DJ-1/ astrocytes. 4. Discussion and conclusions These data report that a loss of function mutation in DJ-1 decreased production of TNFa and PGE2 in astrocytes. It is noteworthy that dysregulation of these factors occurs in astrocytes in response to numerous endogenous and exogenous stimuli. The net effect of TNFa in the CNS is dose and context dependent with studies in knockout mice showing controversial results therefore the different variables accounting for TNFa’s effect in PD have not been defined. A decrease in TNFa expression and secretion in DJ-1/ mice may decrease astrocytic support of neuronal viability during early stages of neurodegeneration. TNFa is known to regulate calcium homeostasis (Daschil and Humpel, 2014), the synthesis and release of neuronal monoamines and glutamate, potassium and calcium ion channel regulation, and production of growth factors and cytokines, as well as numerous second messengers (Neniskyte et al., 2014; Perry et al., 2002; Vitkovic et al., 2000). Furthermore, both TNFa and its receptors are constitutively expressed in various regions of the brain including the cortex, striatum, and thalamus, suggesting a role in normal cellular homeostasis (Vitkovic et al., 2000). Astrocyte-derived TNFa is essential for maintaining synaptic connectivity and plasticity (Stellwagen and Malenka, 2006). Therefore, release of cytokines by astrocytes is critical to maintaining neuronal viability and decreased secretion mediated by DJ-1 mutation likely increases neuronal vulnerability. Prostaglandins, via their specific G protein coupled receptors, have a variety of physiological functions in the central nervous system. Whereas high PGE2 concentrations are neurotoxic (Ahmad et al., 2006a; Takadera et al., 2004), lower levels of PGE2 mediate neuroprotective effects (Ahmad et al., 2005; Ahmad et al., 2006b; McCullough et al., 2004). PGE2 itself is released from astrocytes following glutamate activation (Zonta et al., 2003) and is likely a cytoprotective event; therefore decreased PGE2 release in astrocytes induced by DJ-1/ may decrease neuronal viability. Because expression of COX2, NOS2, and TNFa are all upregulated in response to LPS, an effect predominantly mediated by nuclear factor kappa B (NFkB), it is surprising that DJ-1/
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Fig. 2. LPS-induced relative expression of NOS2 tends to decrease with inhibition of p38 but not MEK or JNK. Astrocytes were exposed to U0126 (MEKi, 10 mM), JNK inhibitor II (JNKi, 10 mM), or SB203580 (p38i, 30 mM) for 2 h, then PBS or 1 mg/mL LPS for 24 h, and RNA was isolated. Expression of NOS2 was determined via real-time PCR, and values are expressed relative to DJ-1+/+ cells exposed to DMSO and LPS, as all expression levels in PBS-treated samples were too low to detect. Inhibition of p38 decrease expression compared to basal levels in DJ-1/ cells and showed depression in expression in DJ+/+ (p = 0.11). * illustrates a significant difference (p < 0.05) from basal expression within the treatment group. D illustrates a significant difference (p < 0.05) from LPS treatment within the genotype.
Fig. 3. LPS-induced relative expression of TNF a is lower in DJ-1/ astrocytes, and does not decrease following inhibition of MEK, JNK, or p38. Astrocytes were exposed to U0126 (MEKi, 10 mM), JNK inhibitor II (JNKi, 10 mM), or SB203580 (p38i, 30 mM) for 2 h, then PBS or 1 mg/mL LPS for 24 h, and RNA was isolated. Expression of TNFa was determined via real-time PCR, and values are expressed relative to DJ-1+/+ cells exposed to DMSO and PBS. * illustrates a significant difference (p < 0.05) from basal expression within the treatment group. # illustrates a significant difference (p < 0.05) from same treatment between genotypes.
Fig. 4. LPS-induced PGE2 production is decreased in DJ-1/ astrocytes. Astrocytes were exposed to U0126 (MEKi, 10 mM), JNK inhibitor II (JNKi,10 mM), or SB203580 (p38i, 30 mM) for 2 h, then 1 mg/mL LPS for 24 h, and culture medium was collected. Secretion of PGE2 into the culture medium was determined via ELISA (eBiosciences). Bars represent means +/ SEM (n = 5). # illustrates a significant difference (p < 0.05) from same treatment between genotypes. D illustrates a significant difference (p < 0.05) from LPS treatment within the genotype.
astrocytes have lower expression of only TNFa. By analyzing levels of TNFa secreted into the culture medium, we have confirmed that decreased mRNA expression leads to a decline in secreted levels of TNFa by DJ-1/ astrocytes. It is possible that mutation of DJ-1 prevents a key co-activator from participating in NFkB binding, thus resulting in the blunted response observed in our studies. DJ1 is known to prevent PIASxa from negatively regulating the androgen receptor (Takahashi et al., 2001), so removal of DJ1 might negatively affect genes downstream of this protein, and a yet unidentified protein could mediate similar effects in TNFa regulation. Mo and colleagues suggest that MEKK1 requires functional DJ-1 for full activation (Mo et al., 2008). As
MEKK1 phosphorylates and activates IkB kinase (Lee et al., 1998), it is possible that mutation of DJ-1 decreases overall phosphorylation of IkB. Furthermore, numerous proteins, which are not involved in COX2 or NOS2 regulation (Bcl3, Atf2, Egr1, and Stat) but are involved in regulating the TNFa promoter, have not been studied for DJ-1 interaction. DJ-1 may transiently interact with one or more of these proteins, or act further upstream to modulate regulation of TNFa. In the current study, we established that expression of representative pro-inflammatory genes did not respond identically to inhibition of MAPKs. Inhibition of p38 alone decreased expression of COX2. Inhibition of p38 significantly decreased
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Fig. 5. LPS-induced TNFa secretion is decreased in DJ-1/ astrocytes. Astrocytes were exposed to U0126 (MEKi, 10 mM), JNK inhibitor II (JNKi,10 mM), or SB203580 (p38i, 30 mM) for 2 h, then 1 mg/mL LPS for 24 h, and culture medium was collected. Amount of TNFa in the culture medium was determined EIA (Cayman Chemical). Bars represent means +/ SEM (n = 5). # illustrates a significant difference (p < 0.05) from same treatment between genotypes. D illustrates a significant difference (p < 0.05) from LPS treatment within the genotype.
expression of NOS2 in DJ-1/ and showed depression in DJ+/+ (p = 0.06) astrocytes compared to their respective controls. Neither MEK nor JNK inhibition affected expression of NOS2. No expression changes in COX2 were induced by inhibition of MEK, JNK, or p38. However, secretion of PGE2 was lower in DJ-1/ astrocytes. Interestingly, MEK inhibition did not affect secretion of TNFa in either genotype, whereas significant decreases were observed in DJ-1+/+ astrocytes treated with JNK inhibitor II, and inhibition of p38 decreased secretion in both genotypes. As neuroinflammation and reactive astrocytosis are clearly involved in the pathogenesis of PD as well as many other neurological disorders, the response of astrocytes to pro-inflammatory stimuli such as LPS warrants investigation. While we would have anticipated that mutation of DJ-1 would increase expression of pro-inflammatory proteins following LPS exposure, upregulation of TNFa was lower in DJ-1/ astrocytes compared to DJ-1+/+ cells. No other data to date has demonstrated alterations in TNFa levels due to knockout, knockdown, or even overexpression of DJ-1; therefore how this expression is decreased is unclear. Because the etiology of PD is still enigmatic, the early neuronal changes that occur with this disease are still largely unknown. However, given the cytoprotective effects of TNFa, it is likely that decreasing the secretion of this cytokine from astrocytes would be detrimental to neuronal viability. In support of this concept, data from Mullett and Hinkle (2009) suggests that DJ-1 knockdown astrocytes provide decreased neuronal protection due to an alteration in unidentified soluble factors released into the culture medium. In summary, mutation of DJ-1 decreased LPS-induced expression and secretion of TNFa, as well as secretion of PGE2 in primary astrocytes. This finding is the first to suggest TNFa or PGE2 could be influenced by DJ-1 providing insight into DJ-1’s role in cellular homeostasis. Because expression of COX2 was unaffected by mutation of DJ-1, yet changes in PGE2 secretion occurred, it is possible that DJ-1 may affect secretory pathways as well as protein production. Future studies are needed to discern the mechanisms by which DJ-1 alters LPS-induced astroglial regulation of cytokines and define physiological consequences of these alterations are in the context of DJ-1 related disorders. Conflict of interest There are no conflicts of interests for authors on this publication. Acknowledgements Funding for this research was provided by a Basil O’Connor Starter Scholar Research Award to Dr. Marie Legare and a College of
Veterinary Medicine and Biomedical Sciences College Research Council Award to Dr. William Hanneman. References Ahmad, A.S., Ahmad, M., de Brum-Fernandes, A.J., Dore, S., 2005. Prostaglandin EP4 receptor agonist protects against acute neurotoxicity. Brain Res. 1066, 71–77. Ahmad, A.S., Saleem, S., Ahmad, M., Dore, S., 2006a. Prostaglandin EP1 receptor contributes to excitotoxicity and focal ischemic brain damage. Toxicol. Sci. 89, 265–270. Ahmad, A.S., Zhuang, H., Echeverria, V., Dore, S., 2006b. Stimulation of prostaglandin EP2 receptors prevents NMDA-induced excitotoxicity. J. Neurotrauma 23, 1895–1903. Aleyasin, H., Rousseaux, M.W., Phillips, M., Kim, R.H., Bland, R.J., Callaghan, S., Slack, R.S., During, M.J., Mak, T.W., Park, D.S., 2007. The Parkinson’s disease gene DJ-1 is also a key regulator of stroke-induced damage. Proc. Natl. Acad. Sci. U. S. A. 104, 18748–18753. Allen, J.W., Mutkus, L.A., Aschner, M., 2000. Isolation of neonatal rat cortical astrocytes for primary cultures. Current Protocols in Toxicology. John Wiley & Sons, Inc. 11-12.14.15 (chapter 12: Unit 12.4). Andres-Mateos, E., Perier, C., Zhang, L., Blanchard-Fillion, B., Greco, T.M., Thomas, B., Ko, H.S., Sasaki, M., Ischiropoulos, H., Przedborski, S., Dawson, T.M., Dawson, V. L., 2007. DJ-1 gene deletion reveals that DJ-1 is an atypical peroxiredoxin-like peroxidase. Proc. Natl. Acad. Sci. U. S. A. 104, 14807–14812. Ashley, A.K., Hanneman, W.H., Katoh, T., Moreno, J.A., Pollack, A., Tjalkens, R.B., Legare, M.E., 2009. Analysis of targeted mutation in DJ-1 on cellular function in primary astrocytes. Toxicol. Lett. 184, 186–191. Bakos, J., Duncko, R., Makatsori, A., Pirnik, Z., Kiss, A., Jezova, D., 2004. Prenatal immune challenge affects growth, behavior, and brain dopamine in offspring. Ann. N. Y. Acad. Sci. 1018, 281–287. Bandopadhyay, R., Kingsbury, A.E., Cookson, M.R., Reid, A.R., Evans, I.M., Hope, A.D., Pittman, A.M., Lashley, T., Canet-Aviles, R., Miller, D.W., McLendon, C., Strand, C., Leonard, A.J., Abou-Sleiman, P.M., Healy, D.G., Ariga, H., Wood, N.W., de Silva, R., Revesz, T., Hardy, J.A., Lees, A.J., 2004. The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson’s disease. Brain 127, 420–430. Batelli, S., Invernizzi, R.W., Negro, A., Calcagno, E., Rodilossi, S., Forloni, G., Albani, D., 2015. The Parkinson's disease-related protein DJ-1 protects dopaminergic neurons in vivo and cultured cells from alpha-synuclein and 6hydroxydopamine toxicity. Neurodegenerative Dis. 15 (1), 13–23. Bertolin, G., Jacoupy, M., Traver, S., Ferrando-Miguel, R., Saint Georges, T., Grenier, K., Ardila-Osorio, H., Muriel, M.P., Takahashi, H., Lees, A.J., Gautier, C., Guedin, D., Coge, F., Fon, E.A., Brice, A., Corti, O., 2015. Parkin maintains mitochondrial levels of the protective Parkinson’s disease-related enzyme 17-b hydroxysteroid dehydrogenase type 10. Cell Death Differ. 16 (January) . Cai, Z., Zeng, W., Tao, K., Lu, F., Gao, G., Yang, Q., 2015. Myricitrin alleviates MPP (+)-induced mitochondrial dysfunction in a DJ-1-dependent manner in SN4741 cells. Biochem. Biophys. Res. Commun. 458 (2), 227–233. Cai, Z., Pang, Y., Lin, S., Rhodes, P.G., 2003. Differential roles of tumor necrosis factoralpha and interleukin-1 beta in lipopolysaccharide-induced brain injury in the neonatal rat. Brain Res. 975, 37–47. Carbone, D.L., Popichak, K.A., Moreno, J.A., Safe, S., Tjalkens, R.B., 2008. Suppression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nitric oxide synthase 2 expression in astrocytes by a novel diindolylmethane analog protects striatal neurons against apoptosis. Mol. Pharmacol. 75 (1), 35–43. Carvey, P.M., Chang, Q., Lipton, J.W., Ling, Z., 2003. Prenatal exposure to the bacteriotoxin lipopolysaccharide leads to long-term losses of dopamine neurons in offspring: a potential, new model of Parkinson’s disease. Front. Biosci. 8, s826–s837. Choi, J., Sullards, M.C., Olzmann, J.A., Rees, H.D., Weintraub, S.T., Bostwick, D.E., Gearing, M., Levey, A.I., Chin, L.S., Li, L., 2006. Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases. J. Biol. Chem. 281, 10816–10824.
A.K. Ashley et al. / NeuroToxicology 52 (2016) 198–203 Chung, I.Y., Benveniste, E.N., 1990. Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide IFN-gamma, and IL-1 beta. J. Immunol. 144, 2999–3007. Daschil, N., Humpel, C., 2014. Nifedipine and nimodipine protect dopaminergic substantia nigra neurons against axotomy-induced cell death in rat vibrosections via modulating inflammatory responses. Brain Res. 18 (September), 1581. Ejima, K., Nanri, H., Araki, M., Koji, T., Shibata, E., Kashimura, M., Ikeda, M., 2000. Expression of mitochondrial thioredoxin-dependent antioxidant protein, SP-22, in normal human and inflammatory mouse placentae. Placenta 21, 847–852. Esen, N., Tanga, F.Y., DeLeo, J.A., Kielian, T., 2004. Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus. J. Neurochem. 88, 746–758. Gayle, D.A., Ling, Z., Tong, C., Landers, T., Lipton, J.W., Carvey, P.M., 2002. Lipopolysaccharide (LPS)-induced dopamine cell loss in culture: roles of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide. Brain Res. Dev. Brain Res. 133 (1), 27–35. Goswami, A., Dikshit, P., Mishra, A., Mulherkar, S., Nukina, N., Jana, N.R., 2006. Oxidative stress promotes mutant huntingtin aggregation and mutant huntingtin-dependent cell death by mimicking proteasomal malfunction. Biochem. Biophys. Res. Commun. 342, 184–190. Hennis, M.R., Seamans, K.W., Marvin, M.A., Casey, B.H., Goldberg, M.S., 2013. Behavioral and neurotransmitter abnormalities in mice deficient for Parkin, DJ1 and superoxide dismutase. PLoS One 8 (12), e84894. Herrera, A.J., Castano, A., Venero, J.L., Cano, J., Machado, A., 2000. The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system. Neurobiol. Dis. 7, 429–447. Hirotani, M., Maita, C., Niino, M., Iguchi-Ariga, S., Hamada, S., Ariga, H., Sasaki, H., 2008. Correlation between DJ-1 levels in the cerebrospinal fluid and the progression of disabilities in multiple sclerosis patients. Mult. Scler. 14, 1056–1060. Hoban, D.B., Connaughton, E., Connaughton, C., Hogan, G., Thornton, C., Mulcahy, P., Moloney, T.C., Dowd, E., 2013. Further characterisation of the LPS model of Parkinson’s disease: a comparison of intra-nigral and intra-striatal lipopolysaccharide administration on motor function, microgliosis and nigrostriatal neurodegeneration in the rat. Brain Behav. Immun. 27 (1), 91–100. Hunter, R.L., Dragicevic, N., Seifert, K., Choi, D.Y., Liu, M., Kim, H.C., Cass, W.A., Sullivan, P.G., Bing, G., 2007. Inflammation induces mitochondrial dysfunction and dopaminergic neurodegeneration in the nigrostriatal system. J. Neurochem. 100, 1375–1386. Larsen, K., Madsen, L.B., Hoj, A., Bendixen, C., 2007. Porcine DJ-1: cloning of PARK7 cDNA, sequence comparison, expression analysis and chromosomal localization. Cytogenet. Genome. Res. 116, 93–99. Lee, F.S., Peters, R.T., Dang, L.C., Maniatis, T., 1998. MEKK1 activates both IkappaB kinase alpha and IkappaB kinase beta. Proc. Natl. Acad. Sci. U. S. A. 95, 9319– 9324. Ling, Z., Gayle, D.A., Ma, S.Y., Lipton, J.W., Tong, C.W., Hong, J.S., Carvey, P.M., 2002. In utero bacterial endotoxin exposure causes loss of tyrosine hydroxylase neurons in the postnatal rat midbrain. Mov. Disord. 17, 116–124. Liu, F., Zhou, R., Yan, H., Yin, H., Wu, X., Tan, Y., Li, L., 2014. Metabotropic glutamate receptor 5 modulates calcium oscillation and innate immune response induced by lipopolysaccharide in microglial cell. Neuroscience 281C (22), 24–34. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25 (4), 402–408. Lu, Y., He, M., Zhang, Y., Xu, S., Zhang, L., He, Y., Chen, C., Liu, C., Pi, H., Yu, Z., Zhou, Z., 2014. Differential pro-inflammatory responses of astrocytes and microglia involve STAT3 activation in response to 1800 MHz radiofrequency fields. PLos One 9 (10) . Mbefo, M., Fares, M.B., Paleologou, K., Oueslati, A., Yin, G., Tenreiro, S., Pinto, M., Outeiro, T., Zweckstetter, M., Masliah, E., Lashuel, H.A., 2015. The Parkinson's disease mutant E46K enhances alpha-synuclein phosphorylation in mammalian cell-lines, in yeast and in vivo. J. Biol. Chem. 290 (February (15)), 9412–9427. McCullough, L., Wu, L., Haughey, N., Liang, X., Hand, T., Wang, Q., Breyer, R.M., Andreasson, K., 2004. Neuroprotective function of the PGE2 EP2 receptor in cerebral ischemia. J. Neurosci. 24, 257–268. Meulener, M.C., Graves, C.L., Sampathu, D.M., Armstrong-Gold, C.E., Bonini, N.M., Giasson, B.I., 2005. DJ-1 is present in a large molecular complex in human brain tissue and interacts with alpha-synuclein. J. Neurochem. 93, 1524–1532. Mitsumoto, A., Nakagawa, Y., 2001. DJ-1 is an indicator for endogenous reactive oxygen species elicited by endotoxin. Free Radic. Res. 35, 885–893. Mo, J.S., Kim, M.Y., Ann, E.J., Hong, J.A., Park, H.S., 2008. DJ-1 modulates UV-induced oxidative stress signaling through the suppression of MEKK1 and cell death. Cell Death Differ. 15 (16), 1030–1041. Moreno, J.A., Sullivan, K.A., Carbone, D.L., Hanneman, W.H., Tjalkens, R.B., 2008. Manganese potentiates nuclear factor-kappaB-dependent expression of nitric oxide synthase 2 in astrocytes by activating soluble guanylate cyclase and extracellular responsive kinase signaling pathways. J. Neurosci. Res. 86 (9), 2028–2038.
203
Mullett, S.J., Hinkle, D.A., 2009. DJ-1 knock-down in astrocytes impairs astrocytemediated neuroprotection against rotenone. Neurobiol. Dis. 33 (1), 28–36. Neniskyte, U., Vilalta, A., Brown, G.C., 2014. Tumour necrosis factor alpha-induced neuronal loss is mediated by microglial phagocytosis. FEBS Lett. 588 (17), 2952–2956. Neumann, M., Muller, V., Gorner, K., Kretzschmar, H.A., Haass, C., Kahle, P.J., 2004. Pathological properties of the Parkinson’s disease-associated protein DJ-1 in alpha-synucleinopathies and tauopathies: relevance for multiple system atrophy and Pick’s disease. Acta Neuropathol. (Berl) 107, 489–496. Olzmann, J.A., Brown, K., Wilkinson, K.D., Rees, H.D., Huai, Q., Ke, H., Levey, A.I., Li, L., Chin, L.S., 2004. Familial Parkinson’s disease-associated L166P mutation disrupts DJ-1 protein folding and function. J. Biol. Chem. 279, 8506–8515. Perrett, R.M., Alexopoulou, Z., Tofaris, G.K., 2015. The endosomal pathway in Parkinson’s disease. Mol. Cell. Neurosci. 19 (10), 1016. Perry, S.W., Dewhurst, S., Bellizzi, M.J., Gelbard, H.A., 2002. Tumor necrosis factoralpha in normal and diseased brain: conflicting effects via intraneuronal receptor crosstalk? J. Neurovirol. 8, 611–624. Puschmann, A., 2013. Monogenic Parkinson’s disease and parkinsonism: clinical phenotypes and frequencies of known mutations. Parkinsonism Relat. Disord. 19 (4), 407–415. Qin, L., Wu, X., Block, M.L., Liu, Y., Breese, G.R., Hong, J.S., Knapp, D.J., Crews, F.T., 2007. Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration. Glia 55, 453–462. Rousseaux, M.W., Marcogliese, P.C., Qu, D., Hewitt, S.J., Seang, S., Kim, R.H., Slack, R. S., Schlossmacher, M.G., Lagace, D.C., Mak, T.W., Park, D.S., 2012. Progressive dopaminergic cell loss with unilateral-to-bilateral progression in a genetic model of Parkinson disease. Proc. Natl. Acad. Sci. U. S. A. 109 (39), 15918–15923. Saito, Y., Miyasaka, T., Hatsuta, H., Takahashi-Niki, K., Hayashi, K., Mita, Y., KusanoArai, O., Iwanari, H., Ariga, H., Hamakubo, T., Yoshida, Y., Niki, E., Murayama, S., Ihara, Y., Noguchi, N., 2014. Immunostaining of oxidized DJ-1 in human and mouse brains. J. Neuropathol. Exp. Neurol. 73 (7), 714–728. Sharif, S.F., Hariri, R.J., Chang, V.A., Barie, P.S., Wang, R.S., Ghajar, J.B., 1993. Human astrocyte production of tumour necrosis factor-alpha, interleukin-1 beta, and interleukin-6 following exposure to lipopolysaccharide endotoxin. Neurol. Res. 15, 109–112. Shendelman, S., Jonason, A., Martinat, C., Leete, T., Abeliovich, A., 2004. DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation. PLoS Biol. 2, e362. Stellwagen, D., Malenka, R.C., 2006. Synaptic scaling mediated by glial TNF-alpha. Nature 440, 1054–1059. Tai, W., Ye, X., Bao, X., Zhao, B., Wang, X., Zhang, D., 2013. Inhibition of Src tyrosine kinase activity by squamosamide derivative FLZ attenuates neuroinflammation in both in vivo and in vitro Parkinson’s disease models. Neuropharmacology 75, 201–212. Takadera, T., Shiraishi, Y., Ohyashiki, T., 2004. Prostaglandin E2 induced caspasedependent apoptosis possibly through activation of EP2 receptors in cultured hippocampal neurons. Neurochem. Int. 45, 713–719. Takahashi, K., Taira, T., Niki, T., Seino, C., Iguchi-Ariga, S.M., Ariga, H., 2001. DJ1 positively regulates the androgen receptor by impairing the binding of PIASx alpha to the receptor. J. Biol. Chem. 276, 37556–37563. Triplett, J., Zhang, Z., Sultana, R., Cai, J., Klein, J.B., Büeler, H., Butterfield, D.A., 2015. Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson disease. J. Neurochem. 133 (January (5)), 750–765. Vitkovic, L., Bockaert, J., Jacque, C., 2000. Inflammatory cytokines: neuromodulators in normal brain. J. Neurochem. 74, 457–471. Waragai, M., Wei, J., Fujita, M., Nakai, M., Ho, G.J., Masliah, E., Akatsu, H., Yamada, T., Hashimoto, M., 2006. Increased level of DJ-1 in the cerebrospinal fluids of sporadic Parkinson’s disease. Biochem. Biophys. Res. Commun. 345, 967–972. Waragai, M., Nakai, M., Wei, J., Fujita, M., Mizuno, H., Ho, G., Masliah, E., Akatsu, H., Yokochi, F., Hashimoto, M., 2007. Plasma levels of DJ-1 as a possible marker for progression of sporadic Parkinson’s disease. Neurosci. Lett. 425, 18–22. Wilson, M.A., 2011. The role of cysteine oxidation in DJ-1 function and dysfunction. Antioxid. Redox Signal. 15 (1), 111–122. Yamaguchi, H., Shen, J., 2007. Absence of dopaminergic neuronal degeneration and oxidative damage in aged DJ-1-deficient mice. Mol. Neurodegener. 2 (10) . Yanagisawa, D., Kitamura, Y., Inden, M., Takata, K., Taniguchi, T., Morikawa, S., Morita, M., Inubushi, T., Tooyama, I., Taira, T., Iguchi-Ariga, S.M., Akaike, A., Ariga, H., 2008. DJ-1 protects against neurodegeneration caused by focal cerebral ischemia and reperfusion in rats. J. Cereb. Blood Flow Metab. 28 (3), 563–578. Zhang, L., Shimoji, M., Thomas, B., Moore, D.J., Yu, S.W., Marupudi, N.I., Torp, R., Torgner, I.A., Ottersen, O.P., Dawson, T.M., Dawson, V.L., 2005. Mitochondrial localization of the Parkinson's disease related protein DJ-1: implications for pathogenesis. Hum. Mol. Genet. 14, 2063–2073. Zhou, W., Zhu, M., Wilson, M.A., Petsko, G.A., Fink, A.L., 2006. The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein. J. Mol. Biol. 356, 1036–1048. Zonta, M., Sebelin, A., Gobbo, S., Fellin, T., Pozzan, T., Carmignoto, G., 2003. Glutamate-mediated cytosolic calcium oscillations regulate a pulsatile prostaglandin release from cultured rat astrocytes. J. Physiol. 553, 407–414.