Persistent 6-OH-BDE-47 exposure impairs functional neuronal maturation and alters expression of neurodevelopmentally-relevant chromatin remodelers

Persistent 6-OH-BDE-47 exposure impairs functional neuronal maturation and alters expression of... Polybrominated diphenyl ethers (PBDEs) are a pervasive class of brominated flame retardants that are present in the envi- ronment at particularly high levels, especially in the United States. Their environmental stability, propensity for bioaccu- mulation, and known potential for neurotoxicity has evoked interest regarding their effects on the developing nervous system. Exposure to PBDEs has been strongly associated with neurodevelopmental disorders. However, the details of their mechanistic roles in such disorders are incompletely understood. Here, we report the effects of one of the most prevalent congeners, BDE-47, and its hydroxylated metabolites on the maturation and function of embryonic rat cortical neurons. Prolonged exposure to 6OH-BDE-47 produces the strongest effects amongst the parent BDE-47 congener and its tested hydroxylated metabolites. These effects include: i) disruption of transcriptional responses to neuronal activity, ii) dysregula- tion of multiple genes associated with neurodevelopmental disorders, and intriguingly, iii) altered expression of several subunits of the developmentally-relevant BAF (Brg1-associated factors) chromatin remodeling complex, including the key subunit BAF170. Taken together, our data indicate that persistent exposure to 6OH-BDE-47 may interfere with neurodeve- lopmental chromatin remodeling mechanisms and gene transcription programs, which in turn are likely to interfere with downstream processes such as synapse development and overall functional maturity of neurons. Results from this study have identified a novel aspect of 6OH-BDE-47 toxicity and open new avenues to explore the effects of a ubiquitous environ- mental toxin on epigenetic regulation of neuronal maturation and function. Key words: neurodevelopment; BDE-47 exposure; Arc; activity-induced transcription; BAF complex Introduction to their abundance in consumer products including furniture, electronics, and clothing and therefore within human house- Polybrominated diphenyl ethers (PBDEs) are a widely-used class of organohalogenated flame retardants that were popularized— holds [1]. Moreover, the environmental stability and strong lipo- and mandated for use in some cases—in the 1970 s. This has led philicity of these compounds enables their bioaccumulation, Received 30 May 2017; revised 8 November 2017; accepted 9 November 2017 V C The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2| Environmental Epigenetics, 2018, Vol. 4, No. 1 specifically in lipid rich adipose and brain tissue [2, 3]. These associated with behavioral deficits in mice [34], 3) disruption of characteristics have raised the issue of potential toxicity of endocrine signalling– primarily thought to be enabled by struc- these compounds, leading to the investigation of their effects tural similarity with triiodothyronine and thyroxine, as well as on human health [4]. Despite known dangers, the usage and with ligands of other hormone receptors [33–37], and 4) pertur- manufacturing of specific congeners has only recently become bation of calcium homeostasis which has been demonstrated in restricted and/or banned in the United States and Europe, and several cell types including human neuronal precursors [8, 38]. their presence in the environment will persist for decades to Despite much progress, a description of how PBDE exposures come [5]. In addition to parent congeners, toxicity may also are related to NDDs remains incomplete; it is not clear how and arise from metabolic products of PBDEs. In mammals, cyto- to what extent known molecular and cellular effects of PBDEs chrome p450 enzymes metabolize PBDEs to form hydroxylated- contribute to these adverse phenotypes. BDEs (OH-PBDEs) [6], some of which may be over an order of A largely unexplored point of convergence that may help to magnitude more toxic than the parent compound (e.g., 6OH- provide an integrative explanation of the relationship between BDE-47) [7–9]. Buildup of such toxic metabolites and their parent the various effects of PBDE exposures and adverse behavioral PBDEs from both natural and anthropogenic sources has led to outcomes is epigenetic regulation, specifically of gene transcrip- significant bioaccumulation in humans over the past few deca- tion during neurodevelopment. Epigenetic mechanisms are a des [10–12]. Human exposure studies suggest that several OH- major driving force of normal neurodevelopment [39], and PBDEs are present in serum at concentrations similar to, and many neurodevelopmental complications involve dysregulation sometimes higher than, those of parent PBDEs [13, 14]. Of con- of gene transcription [40, 41]. Although several epigenetic com- cern for early neurodevelopment, PBDE and OH-PBDE levels ponents of neurodevelopment are now well known, there have been shown to be especially high in young children com- remains much more to be characterized [42], especially in pared to adults, likely due to mobilization of the compounds regards to how they interact with environmental challenges through breastmilk and high exposure rates from household like PBDE exposure. Considering this, we sought to explore the products and dust [15, 16]. Concerns of such bioaccumulation ways in which exposure to BDE-47, or its hydroxylated metabo- are exacerbated by the ability of PBDEs to cross over the pla- lites, influences neuronal maturation and function, as well as centa, exposing developing fetuses to these toxins [17, 18]. the expression of NDD candidate genes, including many epige- Ominously, several lines of evidence now point towards a netic regulators. potential role of PBDEs in contributing to various neurodevelop- mental disorders (NDDs), whose rates of occurrence have grown Methods to alarming levels in the US [19]. The association between developmental exposures to PBDEs Plasmids and Sub-Cloning and NDDs has been extensively studied using mammalian A commercial shRNA construct for BAF170 (CCCAAACTGC models. Animals exposed to various PBDEs during prenatal and/or postnatal periods exhibit long-lasting behavioral abnor- TAGGGAAATTA) was obtained from Sigma. This shRNA sequence was inserted into pLKO.1-puro (designed by RNAi con- malities, including deficiencies in motor activity and cognitive functions [20–23]. Along similar lines, epidemiological studies sortium or TRC; obtained from Addgene) and then packaged in human populations have reported significant associations into lentiviruses. Self-inactivating HIV lentivirus particles were of maternal neonatal PBDE exposure with deficits in motor produced by transfecting 293 T cells with the shRNA vector, behavior, Intelligence Quotient (IQ) ratings (showing intellectual envelope (pMD2.G; Addgene), and packaging plasmids (psPAX2; disability), and attention and cognitive functions in children Addgene) using a previously described protocol [43]. The BAF170 [24–29]. Recently, an expert panel identified 70-100% probability expression construct in a lentiviral backbone was a kind gift that exposure to PBDEs contributes to IQ loss and intellectual from Dr. Trevor Archer (NIEHS, NIH) [44]. BAF170 expression disability that cost the EU public an estimated e9.59 billion from this construct was validated by Western blotting. [30]. Among other relevant findings, significant positive correla- tion has been established between concentration of PBDEs Dissociated Neuronal Culture, RNAi and Cell Treatment in postpartum breast milk and increased externalizing behavio- Cultures of cortical neurons were prepared from embryonic day ral problems [31] (reminiscent of Attention Deficiency 18 Sprague Dawley rats (UC Merced IACUC approval: AUP#13- Hyperactivity Disorder (ADHD)-like behavior in older children), 0007 and AUP#16-0004). Dissociated cortical neurons were as well as between PBDE concentration in peripheral or cord plated in Neurobasal medium (Invitrogen) supplemented with blood and increased risk of attention symptoms and poorer 25 lM glutamate (Sigma-Aldrich) and 0.5 mM L-glutamine social competency scores [32]. (Sigma-Aldrich) and either B27 (Invitrogen) or NS21 and main- While there is a large amount of correlative evidence for tained in a similar medium without glutamate. NS21 was pre- these effects on humans, disentangling the complex mecha- pared in the laboratory as previously described [45]. Neurons nisms of PBDE toxicity has been a difficult task. Of the 209 PBDE were routinely used for induction assays between 10–16 days congeners, a few have been the focus of research due to their in vitro. For infection with recombinant lentiviruses, the viral prevalence. 2, 2’, 4, 4’-tetrabromodiphenyl ether (BDE-47) is supernatant was diluted in neuronal media and cells were among the most prevalent PBDE congeners in the environment, infected at a multiplicity of infection ranging from 2 to 5. To with a correspondingly high body burden in humans [16]. induce gene transcription under basal conditions using synaptic Appropriately, much research has been focused on identifying circuits, we co-treated neurons with 50 lM Bicuculline (Sigma- the ways in which BDE-47, and to a lesser extent, its hydroxy- lated metabolites, affect neural cells. Several potential media- Aldrich) and 75 lM 4-Aminopyridine (Acros Organics) [46]. To induce gene transcription extra-synaptically, we blocked activ- tors of the toxic effects of BDE-47 have been identified, including: 1) oxidative stress following the uncoupling of mito- ity with 1 lM TTX (Calbiochem) and induced the MAP-kinase chondrial function by disruption of oxidative phosphorylation pathways with 1 lM phorbol 12-myristate 13-acetate (PMA; [33], 2) alteration of DNA methylation patterns which were Sigma-Aldrich) [47]. BDE-47 and metabolites used to treat Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 3 cultures were obtained from AccuStandard (BDE-047 N, HBDE- Images were captured with a Keyence BZ9000-E microscope at 4003 N, HBDE-4004 N, HBDE-4005 N). 40X magnification. RNA Extraction and Gene Transcription Quantitation Cell Viability Assay Total RNA was isolated from dissociated neurons using the Cell viability was assessed by an MTT assay (Biotium) [48] GeneJET RNA Purification Kit (Thermo) with an off-column wherein mitochondrial activity is detected colorimetrically fol- DNase (Promega) digestion. cDNA was synthesized using MuLV lowing incubation of cells with a tetrazolium salt. The assay reverse transcriptase (Promega), random primers (Promega), was conducted according to manufacturer’s instruction, except oligo dTs (Promega), and RNase inhibitors (Thermo Scientific). that MTT incubation time was shortened to 30 min to avoid Quantitative real-time PCR (qRT-PCR) was performed to quan- reaching a plateau where differences in product formation tify mRNA levels of specific transcripts using iTaq Universal would be indistinguishable and reagent volumes were propor- Sybr Green Supermix (BioRad) and the BIO-RAD CFX Connect tionally scaled up to appropriate amounts for 24-well plates. realtime PCR Detection System. Pre-mRNA levels were esti- mated as previously described [43]. Statistics Error bars represent standard error of mean throughout this Sample Preparation for Electrophoresis article. Statistical comparison of datasets was performed by one Neurons were lysed in ice-cold 1X RIPA buffer (25 mM Tris, pH way ANOVA with Fisher[CVOAPS]s LSD (Figs 2C and 6A)or by 7.5, 150 mM NaCl, 1% Na- deoxycholate, 0.1% SDS, 0.1% NP-40) two way ANOVA with Tukey[CVOAPS]s post hoc test (all other supplemented with 1: 100 protease inhibitor cocktail (Sigma- figures). Biological replicates are indicated throughout as N in Aldrich-Aldrich). Lysed neurons were sheared by sonication corresponding figure legends. Biological replicates constitute V R (low setting; three cycles on Bioruptor ), cell debris pelleted at cell culture preparations from independent dams. 15 000 rpm for 5 min at 4 C, and clarified supernatant trans- ferred to pre-chilled 1.5 mL microcentrifuge tube. Total cell Results extracts were denatured at 95 C, for 5 min, using either home- made 5X Laemmli buffer, 2X-, or 4X-Laemmli sample buffer Characterization of E18 Primary Rat Neuronal Cultures (both from BIO-RAD). We dissected brain tissue from the pups of timed-pregnant Sprague-Dawley rats (Rattus norvegicus) on embryonic day 18 Western Blotting and Imaging (E18) to obtain dissociated cortical neurons. Cells were then plated as monolayers in supplemented Neurobasal growth Denatured protein samples were resolved on 4-20%- (BIO-RAD medium. The identity of these cultured cells was characterized cat. no. 4568095) or 4-15%- (BIO-RAD cat. no. 456-1083) Mini V R over the first week of growth by immunocytochemistry (ICC) PROTEAN gels in Tris/Glycine/SDS (BIO-RAD cat. no. 1610772). Resolved proteins were transferred onto LF PVDF membrane, using antibodies raised against Nestin, DCX, and NeuN (neural progenitor, differentiating neuron, and differentiated neuronal using the BIO-RAD TBT RTA kit and protocol (cat. no. 1704272). markers, respectively) (Fig. 1)[49–51]. The cultures were com- PVDF membranes were incubated at 4 C overnight with appro- posed of cells exhibiting expression of: Nestin and DCX on day 0 priate primary antibodies in 1X TBS-T with 0.5% BSA at 1: 1000 in vitro (DIV 0), predominantly DCX expression by DIV 4, and dilution. Primary antibodies included the following antibodies: bActin (ThermoFisher Scientific AM4302), BAF170 (CST 12760), largely NeuN expression by DIV 7. Based on the expression of these markers, we identified our DIV 0 cells to be a mixture of BAF155 (CST 11956), Brg1 (CST 49360), BAF47 (CST 91735). Next both neuronal progenitors and differentiating neurons, whereas day, membranes were washed three times in 1X-TBST, probed V R DIV 4 and DIV 7 cells were predominantly differentiating and with appropriate Alexa Fluor secondary antibodies (Life differentiated neurons respectively. Utilizing cultures of this Technologies) for 40 min at room temperature, washed three times with 1X TBS-T, and imaged using BIO-RAD Multiplex nature allowed us to investigate the effects of exposure to BDE- TM 47 and its hydroxylated metabolites across various early stages ChemiDoc Imaging System. of neuronal maturation in vitro. Immunocytochemistry and Microscopy 6OH-BDE-47 Impairs Neuronal Maturation and Activity- Antibodies for immunocytochemistry were used at dilutions Induced Gene Transcription between 1: 100–1: 500 and include the following: NeuN Mature neurons sense environmental cues through neuronal (Millipore ABN78), Doublecortin (CST 4604 S), Nestin (Invitrogen MA1-91657). Neurons were washed twice with 1X ice-cold PBS activity and can respond via activity-induced gene transcription (Fisher Sci). The cells were then incubated with 4% paraformal- [52]. Therefore, we sought to test the effects of PBDE exposure dehyde (Sigma-Aldrich) in 1X PBS for 15 min at room tempera- on functional maturation by conducting neuronal activity- ture and then washed twice with 1X PBS, permeabilized at room induced gene transcription assays using a widely studied, temperature for 20 min with 0.5% Triton X-100 (Fisher Sci), neuron-specific immediate early gene (IEG), Arc, as a readout washed twice and blocked for 30 min with 10% goat serum (referred to from here on as ‘activity-induced gene transcription (Gibco) in 1X PBS. Cells were incubated at 4 C overnight in 3% assay’). Assays were conducted in cultures that were chronically goat serum in 1X PBS with primary antibodies. Next day, pri- exposed to 1 lM of BDE-47 or one of its hydroxylated metabo- mary antibody solution was removed and cells were washed lites– 3OH-BDE-47 (3OH), 5OH-BDE-47 (5OH), and 6OH-BDE-47 thrice with 0.05% Tween (Fisher) in 1X PBS (0.05% PBS-T), and (6OH) and neuronal activity was induced using Bicuculline and V R incubated with appropriate Alexa Fluor secondary antibody 4AP (Bic þ 4AP). Arc pre-mRNA, the direct output of transcrip- (Lifetech) for 45 min, washed thrice with 0.05% PBS-T, cured tion, was detected at 15 min after induction and quantified overnight with Prolong Anti-Fade Gold with DAPI and imaged. by quantitative real-time PCR (qRT-PCR) using intron-exon Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4| Environmental Epigenetics, 2018, Vol. 4, No. 1 positive neurons between untreated and 6OH treated cultures (Fig. 2D and E). Similar results were obtained with Tuj1, another marker for differentiated neurons (Fig. 2F), indicating that neurons can attain their fate and differentiate irrespec- tive of exposure to 6OH in our culture conditions. Therefore, reasons for 6OH exposure-related lack of functional matura- tion (Fig. 2B) likely lie elsewhere. Next, to explore any differences in 6OH effects between dif- ferentiating and differentiated neurons, we conducted addi- tional activity-induced gene transcription assays, exposing cells to a range of 6OH concentrations from both DIV 0 and DIV 7. From these assays, a dose-dependent activity-induced Arc transcriptional response was observed in cultures exposed at both DIV 0 and DIV 7 (Fig. 2G and H). Interestingly 0.5 lM 6OH exposure, which does not significantly affect cell viability, pro- duced effects that were not statistically different from 1 lM exposure. Taken together, these results indicate that chronic 6OH exposure impairs functional neuronal maturation while differentially affecting cell viability depending on the exposure dose. 6OH-BDE-47 Exposure Impairs Synaptic and Extra- Synaptic Modules of Activity-Induced Transcription Transcription of IEGs in response to neuronal activity relies on a cascade of intracellular components starting with func- tional synapses, then transduction of signals from the syn- apse to the nucleus, and finally chromatin accessibility in the nucleus itself [55, 56]. As Bic þ 4AP treatment induces neuro- nal activity by GABAergic disinhibition, any of these factors affected by 6OH could be contributing to the decreased pro- duction of nascent Arc pre-mRNA in Figure 2.As such, results Figure 1: Characterization of neuronal cultures across the first week of growth from Bic þ 4AP induction assays potentially reflect an accu- by immunocytochemistry. Rat cortical neurons were obtained by dissecting and mulation of effects arising from dysregulation at any point dissociating cortices from E18 pup brains. Cultures derived from these cells from thesynapse to thenucleus. Therefore, to morespecifi- were stained using antibodies against several markers of neuronal maturation: cally test if6OH wasacting at synapses or further down- Nestin (neuronal precursor), DCX (differentiating neurons), and NeuN (differen- stream, we induced Arc transcription intracellularly at the tiated neurons). Staining was conducted at indicated time points. Scale bar ¼ 25lm. N¼3 signal transduction level by stimulating the Mitogen Activated Protein Kinase (MAPK) pathway, which is known to transduce signals for rapid neuronal gene transcription [57, spanning primers. We found that transcription of Arc was sig- 58]. Our laboratory has established that neuronal rapid IEGs, nificantly attenuated in cultures exposed to 6OH from DIV 0-10, including Arc [43], may be induced extrasynaptically by acti- but not in cultures exposed to other compounds (Fig. 2B) sug- vating the MAPK pathway with 1 lMofthe proteinkinase C gesting that chronic exposure to 6OH may interfere with func- (PKC) activator phorbol 12-myristate 13-acetate (PMA) while tional maturation of neurons. blocking neuronal activity with 1 lMtetrodotoxin (TTX), a Lack of functional maturation could be an inadvertent fall- potent sodium channel blocker (Fig. 3A)[59]. Using this extra- out of 6OH-induced neurotoxicity or impaired neuronal differ- synaptic induction protocol, we found that Arc is induced entiation. Any cytotoxicity in cells exposed to different doses within 15 min. This Arc pre-mRNA induction was inhibited of 6OH was measured by MTT assay. This range of doses was significantly by 1 lM, but not 0.5 lMor 0.1 lM, 6OH exposure selected based on an approximation of environmentally rele- (Fig. 3C). Although 1 lM 6OH exposure caused a certain vant exposures. The reasoning and justification for this amount of cell death (Fig. 2C), theeffect of thesamedoseseen approximation is detailed further in the discussion. While in this assay may not be entirely due to neurotoxicity because 1 lM 6OH was found to induce a significant reduction in cell the Arc induction values are normalized by an internal control viability after chronic exposure for ten days, 0.5 and 0.1 lM (Gapdh, representing live cells). Additionally, comparing 6OH doses did not (Fig. 2C). Next, we studied the effect of 6OH Figs 2G and 3C, our data indicate that prolonged exposure to on neuronal differentiation. 6OH has been previously shown the non-lethal dose of 6OH (0.5 lM) may impair synaptic for- to inhibit differentiation at lower concentrations than its mation and/or functions (Bic þ 4AP assays; Fig. 2G), but spares parent compound in adult neural progenitor cells and human extra-synaptic cascades of events that lead up to Arc tran- neuronal precursors [53, 54]. To verify any effect of 6OH on scription (TTX þ PMA assays; Fig. 3C). neuronal differentiation in our system, we stained for neuro- One major suspected mode of action for PBDE toxicity is dys- nal fate markers (NeuN and Tuj1) in DIV 7 neurons after pro- regulation of thyroid hormone homeostasis, including disrup- longed exposure to the non-cytotoxic 0.5 lM6OH dose tion of thyroid hormone levels, transport, and receptor activity (Fig. 2D–F). On DIV 7, when neurons are differentiated in our [4]. These effects are thought to be mediated by structural simi- system (Fig. 1), there was no difference in fraction of NeuN larity between PBDEs and thyroid signaling molecules [7]. To Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 5 Figure 2: Chronic 6OH exposure is detrimental to neuronal maturation and function. Activity induced Arc expression assays were conducted by treating cultures with Bic and 4AP for 15 min. (A) Timeline depicting PBDE exposure durations leading up to activity induction assays. Ten day exposures were started either con- currently with plating (DIV 0) or after a week of growth (DIV 7) to assess differences in effect on differentiating versus differentiated neurons. (B)Induction of Arc after a 10-day exposure to 1lM BDE-47 or one of its hydroxylated metabolites, N¼3. (C) Assessment of cell viability following 10 days of 1lM 6OH-BDE-47 exposure. Viability was assessed colorimetrically by MTT assay. N¼3. (D) Representative images of NeuN and DAPI stained cells on DIV 7 following treatment with 0.5lM 6OH starting at DIV 0. Scale bar ¼ 50lm(E) Quantification of NeuN and DAPI staining represented in D, (2, 562 untreated and 2, 634 6OH treated cells counted). (F) Representative western blot for Tuj1 (a marker of differentiated neurons) in cells exposed to 0.5lM 6OH-BDE-47 for seven days, N¼3. (G)Induction of Arc after a ten-day exposure to various concentrations of 6OH, N¼3. (H) Similar assay as shown in (G) except 6OH exposure was started on DIV 7, N¼4. * P < 0.05, ** P < 0.01. ns ¼ non-significant test if the observed effects of 6OH in our assays are mediated already. Elevated levels of T were shown to rescue 10 lMBDE- primarily by disruption of intracellular thyroid signaling, cul- 47-induced reduction of neuronal migration in another study tures were co-exposed to 1 lM 6OH and either 30 or 3 nM triiodo- [53]. In our assays, elevated levels of T did not rescue 1 lM 6OH- thyronine (T ), which is present at low levels in culture media induced attenuation of Arc induction following Bic þ 4AP Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6| Environmental Epigenetics, 2018, Vol. 4, No. 1 neurotoxic effects, opening the possibility of a thyroid-hormone pathway-independent mechanism of 6OH toxicity. BDE-47 and Its Hydroxylated Metabolites Dysregulate Expression of NDD Candidate Genes Observing that chronic exposure to 6OH disrupts neuronal activity-related function in dosage-dependent manners, and that the effects are not primarily mediated by disruption of thyroid hor- mone homeostasis, we sought to identify other potential mecha- nisms. To accomplish this, we screened 15 NDD candidate genes, coding for proteins of various functions important for neuronal development and function (Table 1), for changes in mRNA levels following exposure to 1 lM BDE-47 or its hydroxylated metabolites. Exposures were started at various time points to assess effects dur- ing neuronal differentiation. We found that exposure to BDE-47, 3OH, and 5OH altered mRNA levels of only a few genes, while 6OH exposure altered mRNA levels of 11 out of 15 genes at the earliest and middle time points (exposures at DIV 0 and DIV 4), and a smaller subset when differentiated neurons were exposed starting at DIV 7 (Fig. 4). This observation suggests that 6OH exposure may have temporally specific effects, with immature neurons being affected to a seemingly greater extent. Following up on the wide- spread effect caused by 6OH, we measured mRNA levels of the same 15 genes after exposure to a range of 6OH concentrations over the various time points (Fig. 5). Dysregulation of mRNA levels by 6OH was found to be dose-dependent, with transcripts of a few genes exhibiting significant changes at concentrations as low as 10 nM (Arid1b, Tbl1xr1,and Adnp, data not shown). Of particular interest are Arid1b and Smarcc2,both of which code for subunits of the BAF chromatin remodeling complex [60], as well as Shank3,a high confidence autism candidate gene whose protein product is involved in the organization of the postsynaptic density [61]. In our screen, Shank3 was significantly dysregulated by 6OH in an oppos- ingmannerdepending on thetimeof exposure(Figs 4 and 5). Together, these screens demonstrate dysregulation of mRNA lev- els of several NDD candidate genes by chronic 6OH exposure in a mammalian model, and further validate the dose- and time-of- exposure dependence of 6OH toxicity. They also provide many potential novel targets for further investigation. While changes in mRNA levels following 6OH exposure can be informative of altered cellular states, they do not necessarily indicate direct effects on the transcription of target genes. This is due to mRNA abundance being a function of the rate of transcrip- tion as well as of transcript decay, which can vary greatly [62, 63]. To verify if 6OH-induced changes to mRNA levels were due to a Figure 3: Effects of 6OH exposure are at the synaptic and extrasynaptic level and direct effect on the transcription of target genes, we designed pri- are not primarily mediated by disruption of thyroid hormone signaling. Activity mers to detect pre-mRNAs, the short-lived, direct product of tran- induced Arc expression assays were conducted by treating cultures with Bic and 4AP or TTX and PMA for 15 min after ten days of 6OH exposure starting at the scription by RNA polymerase II. We focused on genes strongly time of plating. (A) Depiction of different modes of gene transcription assays. affected in differentiating neurons (cultures with exposure start- Note: Bicþ4AP induction (Fig. 2) is reliant on synaptic activity whereas ing on DIV0), and measured their respective pre-mRNA levels at TTXþPMA induction bypasses synapses and jumpstarts signaling cascades time points within that window of exposure, where early neuro- extra-synaptically. (B) Timeline depicting 6OH exposures with or without addi- tion of triiodothyronine (T3) followed by various activity induction assays. (C) nal differentiation is occurring (DIV0-5). Interestingly, as revealed Induction of Arc via direct activation of the MAPK pathway with PMA while by MTT assays and unlike what is seen in differentiated neurons blocking membrane activity with TTX in cultures exposed to various concentra- (Fig. 2C), 1 lM 6OH did not negatively impact cell viability by DIV3 tions of 6OH N¼5. (D) Induction of Arc with Bicþ4AP following exposure to 6OH (Fig. 6A). In these cells, as revealed by pre-mRNA assays, 6OH with and without additions of T3 to outcompete potential interactions of 6OH exposure impacted most genes only at the mRNA level, perhaps with thyroid hormone transporters and receptors N¼3. *P < 0.05, **P < 0.01 via indirect mechanisms that influence mRNA half-lives. stimulation, although the addition of 30 nM T alone did signifi- However, expression profiles of three of the tested genes were cantly enhance induced Arc transcription (Fig. 3D). Together, found to be significantly dysregulated at the pre-mRNA level – intriguingly, all of which encode BAF complex subunits or inter- these results indicate that boosted levels of thyroid hormone cannot rescue effects of 6OH exposure, perhaps including its acting proteins (Smarcc2, Arid1b, and Adnp). (Fig. 6B). Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 7 Table 1: Neurodevelopmental disorder candidate genes screened for mRNA dysregulation following chronic exposure to BDE-47 or its hydroxy- lated metabolites Gene Description/Function Associated Brain Disorders Adnp Activity-Dependent Neuroprotective Protein: zinc finger transcription factor that modulates ASD [81], ID [82] p53 activity and interacts with BAF chromatin remodeling complexes to regulate transcription Arid1b AT-Rich Interaction Domain 1B (BAF250b): DNA interacting protein, subunit of the neural pre- ASD [83], ID [82] cursor and neuron specific BAF chromatin remodeling complexes Cbx4 Chromobox 4: component of a Polycomb group (PcG) PRC1-like complex which is involved in ASD [84] transcriptional repression of many genes throughout development and mediates monoubi- quination of histone 2A lysine 119 residues Ctnnb1 Catenin Beta 1: component of the canonical Wnt signaling pathway, coactivator of TCF/LEF ASD [81], ID [82] transcription factors that activate Wnt responsive genes which play diverse neurodevelop- mental roles such as central nervous system patterning, and neural stem and precursor cell proliferation Dyrk1a Dual Specificity Tyrosine Phosphorylation-Regulated Kinase 1A: a nuclear protein kinase that ASD [81], ID [82] catalyzes autophosphorylation of serine/threonine and tyrosine residues, thought to play a role in regulating signaling pathways contributing to cell proliferation and neurodevelopment Med13l Mediator Complex Subunit 13 Like: a subunit of the mediator complex, a transcriptional coacti- ASD [84], ID [82] vator for RNA polymerase II transcribed genes that is recruited to promoters by binding with regulatory proteins thus serving as a scaffold for the preinitiation complex, involved in early neurodevelopment Ncor1 Nuclear Receptor Corepressor 1: corepressor of thyroid-hormone and retinoic-acid receptor tar- ASD [84], HD [85] get genes, known to recruit histone deacetylases that promote the formation of condensed chromatin structure that prevents access of transcription factors and thus represses transcription Npas2 Neuronal PAS Domain Protein 2: transcription factor that is a core component of the circadian ASD [86] clock, also known to regulate the transcription of metabolism, cell cycle, and DNA repair related genes Phf2 PHD Finger Protein 2: a zinc-finger-like plant homeodomain containing demethylase that acts ASD [84] on both histone and non-histone proteins, dimerizes with Arid5b at target promoters and demethylates histone 3 lysine 9 (H3K9me2) residues leading to transcriptional activation Rps6ka2 Ribosomal Protein S6 Kinase A2: a member of the RSK family of serine/threonine kinases that ASD [87] phosphorylates members of the mitogen-activated protein kinase signalling pathway, regu- lates processes such as cell growth, survival, and proliferation Shank3 SH3 and Multiple Ankyrin Repeat Domains 3: major scaffold protein of the postsynaptic density ASD [88], ID [89], SCZ [90] that organizes neurotransmitter receptors, ion channels, and other membrane proteins via interactions with the actin cytoskeleton, plays a role in dendritic spine maturation as well as synaptic formation and plasticity Smarcc2 SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin Subfamily C ASD [91], ID [92], SCZ [41] Member 2 (BAF170): subunit of the neural precursor and neuron specific BAF chromatin remodeling complexes, thought to be a scaffolding subunit required for stabilization of other BAF subunits, involved in transcriptional activation and repression, known to regulate corti- cal thickness and neural progenitor proliferation Sp1 Specificity Protein 1: a zinc finger transcription factor that binds GC-rich motifs at many pro- ASD [93] moters, involved in many cellular processes such as differentiation, growth, apoptosis, and recruitment of chromatin remodeling enzymes including Brg1 and Brg associated factors to regulate transcription Tbl1xr1 Transducin Beta Like 1 X-Linked Receptor 1: thought to be a component of the NCoR and ASD [81], ID [94] HDAC3 repressive complexes, and is required for transcriptional activation by a variety of transcription factors Ubr3 Ubiquitin-Protein Ligase E3-Alpha-3: targets proteins for degradation by ubiquinitination, may ASD [81] be involved in chromatin regulation and transcriptional silencing (by similarity) Abbreviations: ASD- autism spectrum disorder, ID- intellectual disability, SCZ- schizophrenia, HD- Huntington[CVOAPS]s disease 6OH-BDE-47 Dysregulates Expression of Smarcc2 that play important and fundamental roles in driving neurode- (BAF170) and Other BAF Complex Subunits velopmental processes [64]. BAF170 is thought to act as a scaf- One of the genes exhibiting 6OH-induced changes in pre-mRNA folding subunit that is critical for the stability of these level, Smarcc2, codes for BAF170, a core subunit of the mamma- complexes [65]. Therefore, to test the effect of 6OH on expres- lian SWI/SNF (SWItch/Sucrose Non-Fermentable)-like BAF com- sion of other BAF subunits, we screened mRNA levels of other plex. BAF complexes are ATP-dependent chromatin remodelers known subunits of the neuronal precursor and neuron specific Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 8| Environmental Epigenetics, 2018, Vol. 4, No. 1 Figure 4: Exposure to BDE-47 and its hydroxylated metabolites dysregulates mRNA levels of neurodevelopmental disorder candidate genes. 15 neurodevelopmental disorder-associated genes screened for changes in mRNA abundance by qRT-PCR following exposures to 1lM BDE-47 or one of its hydroxylated metabolites for periods indicated below each heatmap. Each row depicts changes in mRNA level for a gene while each column reflects exposure to a different compound. Results are displayed as fold change relative to control. Cells with a fold change above two have saturated coloring. * P < 0.05, ** P < 0.01. N¼3–5 Figure 5: Dysregulation of neurodevelopmental disorder candidate gene mRNA levels by 6OH is dose- and time-of-exposure-dependent. 15 neurodevelopmental disor- der-associated genes screened for changes in mRNA abundance by qRT-PCR following exposures to various concentrations of 6OH for periods indicated below each heatmap. Each row depicts changes in mRNA level for a gene while each column reflects exposure to a different concentration of 6OH. Results are displayed as fold change relative to control. Cells with a fold change above two have saturated coloring. * P < 0.05, ** P < 0.01. N¼3–4 BAF complexes (npBAF/nBAF) and found significant upregula- baffling, such vacillating outcomes may be explained if expres- tion of several additional subunits, namely, Brm, Brg1, BAF60a, sion of BAF170 oscillates during neuronal differentiation and BAF53a, and BAF47 (Fig. 7A). Focusing on BAF170, we then 6OH exposure shifts the oscillation frame (Fig. 7B). This hypoth- sought to confirm 6OH-induced dysregulation of this major BAF esis is supported by previous work that found the expression of subunit at the protein level by Western blotting. Here, to our BAF170 to be very dynamic across mouse corticogenesis [66] bewilderment, we encountered two opposing outcomes with and biphasic in H1 embryonic stem cells [44]. To test this possi- almost equal frequency across replicates. In some trials, chronic bility, we collected samples at 6 h intervals after plating for the exposure of differentiating neurons to 0.1-1 lM 6OH downregu- first 48 h and then assessed BAF170 protein levels by western lated BAF170 protein levels. In others, BAF170 protein levels blotting. As shown in Fig. 7C (quantified in Fig. 7D), BAF170 lev- were upregulated by the same treatment (Fig. 7B). Initially els were found to oscillate with time. Next, we attempted to Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 9 AB Figure 6: Exposure to 6OH dysregulates pre-mRNA levels of a subset of genes affected at the mRNA level. (A) Assessment of cell viability following 3 days of exposure to 1lM 6OH. Viability was assessed colorimetrically by an MTT assay. N¼3. (B) A subset of neurodevelopmental disorder associated genes found to have dysregulated mRNA levels following 6OH exposure were screened for changes in pre-mRNA abundance which better reflect effects on transcription of a given gene. Time points within the earliest exposure period (DIV 0-7) were chosen to more clearly observe direct effects of 6OH toxicity. *P < 0.05, **P < 0.01. N¼3–7 quantify such oscillation with and without 6OH exposure. To other subunits, namely BAF60a and BAF47 whose mRNA levels our surprise, these oscillations varied greatly between biological were significantly upregulated following 6OH exposure (Fig. 7A), Ns, occluding any statistical analysis after averaging data from exhibited reduced protein levels following loss of BAF170 by multiple trials. Despite such variations, 6OH treatment consis- RNAi (Fig. 8G). Taken together, our data suggest that BAF170 depletion, not tently altered the oscillatory pattern in every N (5 attempted; three displayed in Fig. 7E), providing support to our ‘altered its overexpression, likely impairs neuronal maturation via synapse-dependent mechanisms, but not extra-synaptic com- BAF170 oscillation’ hypothesis (Fig. 7B). These BAF170 protein data, alongside its altered pre-mRNA ponents of activity-induced gene transcription. As effects of BAF170-KD are highly evocative of the effects of 6OH exposure, and mRNA levels, and the observed alterations to mRNA levels we propose that environmentally-relevant doses of 6OH may of other BAF subunits, indicate that 6OH exposure likely influ- act via dysregulation of the BAF complex, especially BAF170, to ences BAF complex composition/stability and, therefore, poten- interfere with neuronal maturation and function. tially impacts BAF-dependent functions during neuronal differentiation, including chromatin remodeling and regulation of gene transcription. Discussion In this study, we have demonstrated several novel aspects of Role of BAF170 in Neuronal Maturation and Activity PBDE neurotoxicity. Initially, we show the effects of chronic Induced Arc Transcription nanomolar range exposures to BDE-47 and its hydroxylated Finally, to directly study effects of BAF170 dysregulation on metabolites on neuronal activity-induced gene transcription functional maturation of neurons, as would be expected due to and functional maturation of primary embryonic rat cortical 6OH-induced oscillation, we bi-directionally altered its levels in neurons. We then demonstrate how similar exposures impact developing cells (DIV 0-4) by either overexpressing BAF170, or transcription of NDD candidate genes by quantification of depleting it via RNAi (lentivirus-mediated delivery of BAF170 mRNA and pre-mRNA levels. One of the strongest candidates shRNA). Both methods were validated by western blot (Fig. 8D from our screen, Smarcc2, then prompted us to focus on the and G). We then performed synapse-dependent and synapse- effects of 6OH exposure on BAF170 expression and, preliminar- independent Arc induction assays. The phenotypic outcomes of ily, on BAF170-related regulation of neuronal maturation by these assays were unaffected by overexpression of BAF170 measuring levels of activity-induced Arc expression. Together, (Fig. 8B and C). Interestingly, depletion of BAF170 impaired Arc our results indicate that exposure to the hydroxylated BDE-47 induction in the synapse-dependent Bic þ 4AP assay (Fig. 8E), metabolite 6OH strongly influences functional maturation of but not in the synapse-independent PMA þ TTX assay (Fig. 8F). neurons, potentially independent of thyroid hormone disrup- This observation is reminiscent of the effects of lower 6OH tion mechanisms– perhaps instead exerting effects mediated in doses on Arc induction using these protocol (Figs 2 and 3D). part at the level of epigenetic regulation via disruption of BAF Additionally, we found that depletion of BAF170 reduces protein chromatin remodeling complexes. levels of other BAF subunits (Fig. 8G). These observations sup- Our utilization of the activity-induced transcription of an port the idea that BAF170 acts as a scaffolding component in immediate early gene (Arc) as a readout of functional neuronal BAF complexes and indicates that dysregulation of BAF170 may maturation is, to the best of our knowledge, a novel approach effectively alter BAF complex stability and composition (Fig. 7A) in assessing the neurotoxicity of PBDEs. Immediate early gene [65]. Interestingly, BAF155 protein levels did not decrease while inductionisa strong tool to this end asit reliesonseveral Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 10 | Environmental Epigenetics, 2018, Vol. 4, No. 1 C D Figure 7: BAF170 protein levels and mRNA levels of BAF complex subunits are dysregulated by 6OH exposure. (A) Abundance of various BAF protein mRNAs estimated by qRT-PCR following exposure to various concentrations of 6OH from DIV 0-7, N¼3–4. (B) Top: BAF170 protein levels observed by western blotting following 48 h of exposure to various concentrations of 6OH. N¼6. Bottom: opposing outcomes can be explained if BAF170 is expressed in an oscillatory manner and 6OH exposures dis- rupt regulatory mechanisms shaping the temporal expression patterns. (C) Time-course western blot for BAF170 with samples collected every 6 h after plating for the first 48 h. (D) Quantification of BAF170 band intensities from blot shown in (C) as an example and evidence of BAF170 oscillation in normal cells. (E) Quantification of similar time-course experiments as in (C), with collections every 6 h starting 24 h after plating. Note the inherent variation in oscillatory pattern. Cells were either untreated or exposed to 1 lM 6OH at the time of plating. N¼4 (3 represented) *P < 0.05, **P < 0.01. Note: Because of inherent variation in the oscillatory pattern, averag- ing these data sets ‘flatten’ out the phenotype and is therefore not attempted critical cellular mechanisms that are established across neuro- interferes with neuronal maturation likely at the synaptic nal maturation, ranging from proper synaptic function to level. As mentioned previously, the range of PBDE doses used in nuclear regulation of gene transcription (Fig. 3A), allowing us to broadly assess the extent of PBDE-induced effects on func- this study were estimated based on prior work done in mamma- tional neuronal maturation. Further, inducing Arc expression lian models to approximate relevant concentrations observed in both synaptically (Bic þ 4AP) and extra-synaptically the brains of exposed animals. To determine this range, an (TTX þ PMA) allowed us to hone in on where these neurotoxic exhaustive review was conducted of literature reporting levels effects are being exerted. These assays reveal that 6OH-BDE-47 of BDE-47 accumulation in rodent brain following either chronic Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 11 BC D EF G Figure 8: BAF170 contributes to the stability of BAF subunits and proper circuit formation, but is not required for MAPK stimulated Arc transcription. (A) Depiction of the timing of lentiviral mediated manipulation of BAF170 levels and subsequent activity induced Arc induction assays. (B, C) Arc induction following Bicþ4AP or TTXþPMA stimulation in cells overexpressing BAF170, N¼3. (D) Representative western blot validating BAF170 overexpression construct. (E,F) Arc induction following Bicþ4AP or TTXþPMA stimulation in cells depleted of BAF170, N¼3. (G) Representative western blots demonstrating the effect of BAF170 knockdown on protein levels of other BAF complex subunits, N¼3. **P < 0.01. ns ¼ non-significant or single dose administration [67–73]. After converting the com- reported accumulated concentrations in rodent brain, is low rel- monly reported unit of ng/g of toxin to nmol/L (converting from ative to previous in vitro studies, and is on the same order of lipid concentration to molarity), the average concentration of magnitude as reported human levels of PBDEs and their BDE-47 was determined to be 597 nM. Studies investigating the metabolites. in vitro effects of PBDEs have often been conducted using con- It has been previously demonstrated that micromolar BDE- centrations in the low to mid micromolar range. Summarizing 47 exposures decrease both neuronal and oligodendrocyte dif- human studies, the USEPA’s 2010 report assessing the environ- ferentiation in human neural progenitor cells [53]. Another mental prevalence of PBDEs estimated the body burden of total study found that BDE-47 and 6OH decrease neuronal and oligo- PBDEs in the general population to be 30-100 ng/g (approxi- dendrocyte differentiation of mouse adult neural stem cells mately 60 to 200 nM), with toddlers tending to have higher body (aNSC), with 6OH producing an effect following nanomolar burdens compared to older children and adults. Importantly, exposures, at concentrations approximately 20 times lower BDE-47 alone was estimated to account for 50% of total PBDE than its parent compound [54]. Using activity-induced gene concentrations [16]. It has also been reported that hydroxylated transcription assays as a readout in cultured embryonic rat cort- metabolites of PBDEs accumulate in human serum and are ical neurons, we found that chronic nanomolar exposure to nearly as abundant (approximately 45% of total detectable 6OH disrupts functional maturation. These effects were found PBDEs) as parent PBDE compounds in human fetal blood sam- to be independent of 6OH cytotoxicity or alteration of differen- ples collected in the United States [13, 14]. Considering the tiation for 0.5 lM exposures. Many of the observed effects were reported brain accumulation levels of BDE-47 in rodents, con- found to be dose-dependent whereas transcriptional profiles centrations commonly used for in vitro studies to date, and the (Figs 4 and 5) were additionally sensitive to time of exposure. prevalence of BDE-47 and its hydroxylated metabolites in Additionally, these functional effects appear to be mediated humans, we used a range of 100-1, 000 nM BDE-47 and its at both synaptic and extra-synaptic levels, as evidenced by hydroxylated metabolites for our studies. This range spans decreases in Arc induction following Bic þ 4AP and TTX þ PMA Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 12 | Environmental Epigenetics, 2018, Vol. 4, No. 1 treatments. Effects on IEG transcription by both assays indicate Accordingly, we have shown that reduction of BAF170 levels that 6OH, at higher doses, may act to compromise cell viability, by RNAi, but not its overexpression, negatively impacts neuro- disrupt mature synapse formation, as well as interfere with cel- nal maturation using Arc induction as a readout, recapitulating lular mechanisms at or downstream of the MAPK pathway that the effect of exposure to 0.5 lM 6OH following Bic þ 4AP treat- regulate activity-induced Arc transcription (Figs 2 and 3). ment. The lack of effect seen after BAF170 depletion on extra- Interestingly, addition of excess levels of the thyroid hormone synaptically induced Arc (TTX-PMA protocol), as was the case triiodothyronine (T3) did not produce any phenotypic rescue (as with lower doses of 6OH in Figure 3C, indicates that BAF170- seen previously for BDE-47 induced impairment of neuronal containing BAF complexes may not be necessary for nuclear migration) [53] of the observed attenuation of Arc induction. regulation of activity-induced Arc transcription, but most likely This advocates for additional modes of PBDE toxicity, perhaps contribute to synapse maturation and function via transcription acting alongside disruption of thyroid hormone signaling. regulation of other genes. These findings are consistent with However, we also note that 1 lM 6OH-induced cytotoxicity parti- recent evidence supporting the idea that BAF complexes are ally confounds interpretation of this attempted T3 rescue, as crucial for synapse formation and maturation during early neu- reduction of overall cell viability may obscure potential reversal ronal development [75]. The same study implicated a role of of the effects of 6OH exposure by T3. To explore alternate modes Brg1, a core BAF complex ATPase, in activity-induced gene tran- of PBDE toxicity within a context of the relation of PBDE expo- scription 6 h after onset of activity. However, as our data indi- sure to NDDs, we screened 15 NDD candidate genes, many of cate, BAF170-containing BAF complexes are not specifically which are epigenetic regulators (Table 1), for changes in mRNA required for Arc induction at early time points if activity is level. Subsequently, we found that, while exposure to the induced extra-synaptically, likely owing to it[CVOAPS]s poised parent compound and its other hydroxylated metabolites sig- transcriptional status [43]. This lack of effect following BAF170 nificantly impacted only a few of these targets, 6OH dysregu- depletion and TTX þ PMA treatment may be gene-specific as lated mRNA levels of many of the screened genes. These various IEGs are regulated in differing manners [43, 52, 76]. included Shank3, which encodes an important synaptic protein, While our current work potentially opens new avenues to supporting our inference related to detrimental synaptic effects explore the effects imposed by hydroxylated BDE-47 metabo- of persistent 6OH exposure. lites on epigenetic regulation during neuronal maturation and Of the genes dysregulated by 6OH exposure, a smaller activity-induced gene expression, there are limitations to our subset additionally exhibited altered levels of pre-mRNA, indi- study. Although we have demonstrated that exposure to 6OH cating a direct effect of 6OH on transcription of these genes. has a strong effect on BAF170 expression, from the pre-mRNA Surprisingly, each of the genes whose pre-mRNA was found to protein level, the effect is of a complex nature, whose mecha- to be significantly dysregulated encode BAF chromatin remodel- ing complex subunits or interacting proteins (Adnp, Arid1b, nistic basis we do not yet fully understand. Consistant with our Smarcc2). One of these genes, Smarcc2, encodes a critical core findings, the multiphasic expression pattern of BAF170 has subunit known to contribute to overall BAF complex stability been previously demonstrated during corticogenesis in mice and composition, BAF170. Protein levels of BAF170 were subse- [66] and in H1 embryonic stem cells [44], but further investiga- quently found to be dysregulated by 6OH exposure, though in a tion will be required to fully characterize the expression profile complex oscillatory manner that remains to be fully character- of BAF170 and regulatory mechanisms that shape it. ized. It may be the case that as lack of BAF170, but not over- Additionally, like much PBDE research, we have investigated abundance, is detrimental for neurons (as we have shown in the effects of exposure to a single toxin at a time, while the Fig. 8), the cells are responding to repeated periods of lack of developing human nervous system is realistically exposed to sufficient BAF170 by upregulating Smarcc2, while protein levels complex mixtures of pollutants. Some studies have been com- vary due to post-translational regulation. pleted demonstrating effects of coexposures to environmental BAF170 is a striking candidate whose dysregulation may pollutants both in vitro and in vivo using combinations of PBDE contribute to the observed effects of 6OH toxicity, owing to its congeners, other toxins including polychlorinated biphenyls critical contribution to BAF complex function. It has been shown (PCBs), and commercially available mixtures such as DE-71 [77– to regulate the stability of BAF chromatin remodeling com- 80]. More such studies will be needed to address the effects plexes by acting as a scaffolding subunit and preventing degra- resulting from environmentally relevant mixtures of com- dation of other BAF proteins. Our results support this notion pounds, especially with concern to their metabolites. and demonstrate a reduction of protein levels of several BAF Finally, our results show that nanomolar range exposures to subunits upon BAF170-KD (Fig. 8G), an effect previously only the BDE-47 hydroxylated metabolite 6OH strongly impact sev- seen with removal of both BAF170 and another interchangeable eral aspects of neuronal maturation and function and that these complex subunit, BAF155 [65]. This may indicate that BAF170 effects are likely, at least in part, mediated by disruption of BAF plays a larger role in stabilizing BAF complexes or specific subu- chromatin remodeling complexes via dysregulation of BAF170 nits during the periods of neuronal maturation investigated in expression. Going forward, it will be important to fully charac- this study. This stabilizing aspect of BAF170 function, making it terize the regulatory mechanisms governing the intricate critical for BAF complex function as a whole, is intriguing as expression of BAF170 to discern precisely how the observed BAF complexes have well known roles in neuronal differentia- PBDE-induced dysregulation is produced. Additional investiga- tion and maturation [60], and are thought to contribute to regu- tion of the underlying mechanisms that mediate the neurotoxic lating activity-induced gene transcription [74–76]. As such, effects of PBDE at the level of epigenetic regulation may lead to periods of 6OH induced lack of BAF170 during early stages a better understanding of how exposure to these compounds where differentiation and maturation are taking place in our leads to developmental complications and NDDs as well as cultures may compromise the functional maturation of cells, leading to the observed decrease in Arc expression when to further characterization of fundamental neuroepigenetic assayed at later time-points. processes. Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 13 with dioxins, dibenzofurans, and polychlorinated biphenyls. Acknowledgements J Occup Environ Med 2005;47:199–211. This study was supported by start-up funds from UC 16. U.S. Environmental Protection Agency (EPA). 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Persistent 6-OH-BDE-47 exposure impairs functional neuronal maturation and alters expression of neurodevelopmentally-relevant chromatin remodelers

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Abstract

Polybrominated diphenyl ethers (PBDEs) are a pervasive class of brominated flame retardants that are present in the envi- ronment at particularly high levels, especially in the United States. Their environmental stability, propensity for bioaccu- mulation, and known potential for neurotoxicity has evoked interest regarding their effects on the developing nervous system. Exposure to PBDEs has been strongly associated with neurodevelopmental disorders. However, the details of their mechanistic roles in such disorders are incompletely understood. Here, we report the effects of one of the most prevalent congeners, BDE-47, and its hydroxylated metabolites on the maturation and function of embryonic rat cortical neurons. Prolonged exposure to 6OH-BDE-47 produces the strongest effects amongst the parent BDE-47 congener and its tested hydroxylated metabolites. These effects include: i) disruption of transcriptional responses to neuronal activity, ii) dysregula- tion of multiple genes associated with neurodevelopmental disorders, and intriguingly, iii) altered expression of several subunits of the developmentally-relevant BAF (Brg1-associated factors) chromatin remodeling complex, including the key subunit BAF170. Taken together, our data indicate that persistent exposure to 6OH-BDE-47 may interfere with neurodeve- lopmental chromatin remodeling mechanisms and gene transcription programs, which in turn are likely to interfere with downstream processes such as synapse development and overall functional maturity of neurons. Results from this study have identified a novel aspect of 6OH-BDE-47 toxicity and open new avenues to explore the effects of a ubiquitous environ- mental toxin on epigenetic regulation of neuronal maturation and function. Key words: neurodevelopment; BDE-47 exposure; Arc; activity-induced transcription; BAF complex Introduction to their abundance in consumer products including furniture, electronics, and clothing and therefore within human house- Polybrominated diphenyl ethers (PBDEs) are a widely-used class of organohalogenated flame retardants that were popularized— holds [1]. Moreover, the environmental stability and strong lipo- and mandated for use in some cases—in the 1970 s. This has led philicity of these compounds enables their bioaccumulation, Received 30 May 2017; revised 8 November 2017; accepted 9 November 2017 V C The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2| Environmental Epigenetics, 2018, Vol. 4, No. 1 specifically in lipid rich adipose and brain tissue [2, 3]. These associated with behavioral deficits in mice [34], 3) disruption of characteristics have raised the issue of potential toxicity of endocrine signalling– primarily thought to be enabled by struc- these compounds, leading to the investigation of their effects tural similarity with triiodothyronine and thyroxine, as well as on human health [4]. Despite known dangers, the usage and with ligands of other hormone receptors [33–37], and 4) pertur- manufacturing of specific congeners has only recently become bation of calcium homeostasis which has been demonstrated in restricted and/or banned in the United States and Europe, and several cell types including human neuronal precursors [8, 38]. their presence in the environment will persist for decades to Despite much progress, a description of how PBDE exposures come [5]. In addition to parent congeners, toxicity may also are related to NDDs remains incomplete; it is not clear how and arise from metabolic products of PBDEs. In mammals, cyto- to what extent known molecular and cellular effects of PBDEs chrome p450 enzymes metabolize PBDEs to form hydroxylated- contribute to these adverse phenotypes. BDEs (OH-PBDEs) [6], some of which may be over an order of A largely unexplored point of convergence that may help to magnitude more toxic than the parent compound (e.g., 6OH- provide an integrative explanation of the relationship between BDE-47) [7–9]. Buildup of such toxic metabolites and their parent the various effects of PBDE exposures and adverse behavioral PBDEs from both natural and anthropogenic sources has led to outcomes is epigenetic regulation, specifically of gene transcrip- significant bioaccumulation in humans over the past few deca- tion during neurodevelopment. Epigenetic mechanisms are a des [10–12]. Human exposure studies suggest that several OH- major driving force of normal neurodevelopment [39], and PBDEs are present in serum at concentrations similar to, and many neurodevelopmental complications involve dysregulation sometimes higher than, those of parent PBDEs [13, 14]. Of con- of gene transcription [40, 41]. Although several epigenetic com- cern for early neurodevelopment, PBDE and OH-PBDE levels ponents of neurodevelopment are now well known, there have been shown to be especially high in young children com- remains much more to be characterized [42], especially in pared to adults, likely due to mobilization of the compounds regards to how they interact with environmental challenges through breastmilk and high exposure rates from household like PBDE exposure. Considering this, we sought to explore the products and dust [15, 16]. Concerns of such bioaccumulation ways in which exposure to BDE-47, or its hydroxylated metabo- are exacerbated by the ability of PBDEs to cross over the pla- lites, influences neuronal maturation and function, as well as centa, exposing developing fetuses to these toxins [17, 18]. the expression of NDD candidate genes, including many epige- Ominously, several lines of evidence now point towards a netic regulators. potential role of PBDEs in contributing to various neurodevelop- mental disorders (NDDs), whose rates of occurrence have grown Methods to alarming levels in the US [19]. The association between developmental exposures to PBDEs Plasmids and Sub-Cloning and NDDs has been extensively studied using mammalian A commercial shRNA construct for BAF170 (CCCAAACTGC models. Animals exposed to various PBDEs during prenatal and/or postnatal periods exhibit long-lasting behavioral abnor- TAGGGAAATTA) was obtained from Sigma. This shRNA sequence was inserted into pLKO.1-puro (designed by RNAi con- malities, including deficiencies in motor activity and cognitive functions [20–23]. Along similar lines, epidemiological studies sortium or TRC; obtained from Addgene) and then packaged in human populations have reported significant associations into lentiviruses. Self-inactivating HIV lentivirus particles were of maternal neonatal PBDE exposure with deficits in motor produced by transfecting 293 T cells with the shRNA vector, behavior, Intelligence Quotient (IQ) ratings (showing intellectual envelope (pMD2.G; Addgene), and packaging plasmids (psPAX2; disability), and attention and cognitive functions in children Addgene) using a previously described protocol [43]. The BAF170 [24–29]. Recently, an expert panel identified 70-100% probability expression construct in a lentiviral backbone was a kind gift that exposure to PBDEs contributes to IQ loss and intellectual from Dr. Trevor Archer (NIEHS, NIH) [44]. BAF170 expression disability that cost the EU public an estimated e9.59 billion from this construct was validated by Western blotting. [30]. Among other relevant findings, significant positive correla- tion has been established between concentration of PBDEs Dissociated Neuronal Culture, RNAi and Cell Treatment in postpartum breast milk and increased externalizing behavio- Cultures of cortical neurons were prepared from embryonic day ral problems [31] (reminiscent of Attention Deficiency 18 Sprague Dawley rats (UC Merced IACUC approval: AUP#13- Hyperactivity Disorder (ADHD)-like behavior in older children), 0007 and AUP#16-0004). Dissociated cortical neurons were as well as between PBDE concentration in peripheral or cord plated in Neurobasal medium (Invitrogen) supplemented with blood and increased risk of attention symptoms and poorer 25 lM glutamate (Sigma-Aldrich) and 0.5 mM L-glutamine social competency scores [32]. (Sigma-Aldrich) and either B27 (Invitrogen) or NS21 and main- While there is a large amount of correlative evidence for tained in a similar medium without glutamate. NS21 was pre- these effects on humans, disentangling the complex mecha- pared in the laboratory as previously described [45]. Neurons nisms of PBDE toxicity has been a difficult task. Of the 209 PBDE were routinely used for induction assays between 10–16 days congeners, a few have been the focus of research due to their in vitro. For infection with recombinant lentiviruses, the viral prevalence. 2, 2’, 4, 4’-tetrabromodiphenyl ether (BDE-47) is supernatant was diluted in neuronal media and cells were among the most prevalent PBDE congeners in the environment, infected at a multiplicity of infection ranging from 2 to 5. To with a correspondingly high body burden in humans [16]. induce gene transcription under basal conditions using synaptic Appropriately, much research has been focused on identifying circuits, we co-treated neurons with 50 lM Bicuculline (Sigma- the ways in which BDE-47, and to a lesser extent, its hydroxy- lated metabolites, affect neural cells. Several potential media- Aldrich) and 75 lM 4-Aminopyridine (Acros Organics) [46]. To induce gene transcription extra-synaptically, we blocked activ- tors of the toxic effects of BDE-47 have been identified, including: 1) oxidative stress following the uncoupling of mito- ity with 1 lM TTX (Calbiochem) and induced the MAP-kinase chondrial function by disruption of oxidative phosphorylation pathways with 1 lM phorbol 12-myristate 13-acetate (PMA; [33], 2) alteration of DNA methylation patterns which were Sigma-Aldrich) [47]. BDE-47 and metabolites used to treat Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 3 cultures were obtained from AccuStandard (BDE-047 N, HBDE- Images were captured with a Keyence BZ9000-E microscope at 4003 N, HBDE-4004 N, HBDE-4005 N). 40X magnification. RNA Extraction and Gene Transcription Quantitation Cell Viability Assay Total RNA was isolated from dissociated neurons using the Cell viability was assessed by an MTT assay (Biotium) [48] GeneJET RNA Purification Kit (Thermo) with an off-column wherein mitochondrial activity is detected colorimetrically fol- DNase (Promega) digestion. cDNA was synthesized using MuLV lowing incubation of cells with a tetrazolium salt. The assay reverse transcriptase (Promega), random primers (Promega), was conducted according to manufacturer’s instruction, except oligo dTs (Promega), and RNase inhibitors (Thermo Scientific). that MTT incubation time was shortened to 30 min to avoid Quantitative real-time PCR (qRT-PCR) was performed to quan- reaching a plateau where differences in product formation tify mRNA levels of specific transcripts using iTaq Universal would be indistinguishable and reagent volumes were propor- Sybr Green Supermix (BioRad) and the BIO-RAD CFX Connect tionally scaled up to appropriate amounts for 24-well plates. realtime PCR Detection System. Pre-mRNA levels were esti- mated as previously described [43]. Statistics Error bars represent standard error of mean throughout this Sample Preparation for Electrophoresis article. Statistical comparison of datasets was performed by one Neurons were lysed in ice-cold 1X RIPA buffer (25 mM Tris, pH way ANOVA with Fisher[CVOAPS]s LSD (Figs 2C and 6A)or by 7.5, 150 mM NaCl, 1% Na- deoxycholate, 0.1% SDS, 0.1% NP-40) two way ANOVA with Tukey[CVOAPS]s post hoc test (all other supplemented with 1: 100 protease inhibitor cocktail (Sigma- figures). Biological replicates are indicated throughout as N in Aldrich-Aldrich). Lysed neurons were sheared by sonication corresponding figure legends. Biological replicates constitute V R (low setting; three cycles on Bioruptor ), cell debris pelleted at cell culture preparations from independent dams. 15 000 rpm for 5 min at 4 C, and clarified supernatant trans- ferred to pre-chilled 1.5 mL microcentrifuge tube. Total cell Results extracts were denatured at 95 C, for 5 min, using either home- made 5X Laemmli buffer, 2X-, or 4X-Laemmli sample buffer Characterization of E18 Primary Rat Neuronal Cultures (both from BIO-RAD). We dissected brain tissue from the pups of timed-pregnant Sprague-Dawley rats (Rattus norvegicus) on embryonic day 18 Western Blotting and Imaging (E18) to obtain dissociated cortical neurons. Cells were then plated as monolayers in supplemented Neurobasal growth Denatured protein samples were resolved on 4-20%- (BIO-RAD medium. The identity of these cultured cells was characterized cat. no. 4568095) or 4-15%- (BIO-RAD cat. no. 456-1083) Mini V R over the first week of growth by immunocytochemistry (ICC) PROTEAN gels in Tris/Glycine/SDS (BIO-RAD cat. no. 1610772). Resolved proteins were transferred onto LF PVDF membrane, using antibodies raised against Nestin, DCX, and NeuN (neural progenitor, differentiating neuron, and differentiated neuronal using the BIO-RAD TBT RTA kit and protocol (cat. no. 1704272). markers, respectively) (Fig. 1)[49–51]. The cultures were com- PVDF membranes were incubated at 4 C overnight with appro- posed of cells exhibiting expression of: Nestin and DCX on day 0 priate primary antibodies in 1X TBS-T with 0.5% BSA at 1: 1000 in vitro (DIV 0), predominantly DCX expression by DIV 4, and dilution. Primary antibodies included the following antibodies: bActin (ThermoFisher Scientific AM4302), BAF170 (CST 12760), largely NeuN expression by DIV 7. Based on the expression of these markers, we identified our DIV 0 cells to be a mixture of BAF155 (CST 11956), Brg1 (CST 49360), BAF47 (CST 91735). Next both neuronal progenitors and differentiating neurons, whereas day, membranes were washed three times in 1X-TBST, probed V R DIV 4 and DIV 7 cells were predominantly differentiating and with appropriate Alexa Fluor secondary antibodies (Life differentiated neurons respectively. Utilizing cultures of this Technologies) for 40 min at room temperature, washed three times with 1X TBS-T, and imaged using BIO-RAD Multiplex nature allowed us to investigate the effects of exposure to BDE- TM 47 and its hydroxylated metabolites across various early stages ChemiDoc Imaging System. of neuronal maturation in vitro. Immunocytochemistry and Microscopy 6OH-BDE-47 Impairs Neuronal Maturation and Activity- Antibodies for immunocytochemistry were used at dilutions Induced Gene Transcription between 1: 100–1: 500 and include the following: NeuN Mature neurons sense environmental cues through neuronal (Millipore ABN78), Doublecortin (CST 4604 S), Nestin (Invitrogen MA1-91657). Neurons were washed twice with 1X ice-cold PBS activity and can respond via activity-induced gene transcription (Fisher Sci). The cells were then incubated with 4% paraformal- [52]. Therefore, we sought to test the effects of PBDE exposure dehyde (Sigma-Aldrich) in 1X PBS for 15 min at room tempera- on functional maturation by conducting neuronal activity- ture and then washed twice with 1X PBS, permeabilized at room induced gene transcription assays using a widely studied, temperature for 20 min with 0.5% Triton X-100 (Fisher Sci), neuron-specific immediate early gene (IEG), Arc, as a readout washed twice and blocked for 30 min with 10% goat serum (referred to from here on as ‘activity-induced gene transcription (Gibco) in 1X PBS. Cells were incubated at 4 C overnight in 3% assay’). Assays were conducted in cultures that were chronically goat serum in 1X PBS with primary antibodies. Next day, pri- exposed to 1 lM of BDE-47 or one of its hydroxylated metabo- mary antibody solution was removed and cells were washed lites– 3OH-BDE-47 (3OH), 5OH-BDE-47 (5OH), and 6OH-BDE-47 thrice with 0.05% Tween (Fisher) in 1X PBS (0.05% PBS-T), and (6OH) and neuronal activity was induced using Bicuculline and V R incubated with appropriate Alexa Fluor secondary antibody 4AP (Bic þ 4AP). Arc pre-mRNA, the direct output of transcrip- (Lifetech) for 45 min, washed thrice with 0.05% PBS-T, cured tion, was detected at 15 min after induction and quantified overnight with Prolong Anti-Fade Gold with DAPI and imaged. by quantitative real-time PCR (qRT-PCR) using intron-exon Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4| Environmental Epigenetics, 2018, Vol. 4, No. 1 positive neurons between untreated and 6OH treated cultures (Fig. 2D and E). Similar results were obtained with Tuj1, another marker for differentiated neurons (Fig. 2F), indicating that neurons can attain their fate and differentiate irrespec- tive of exposure to 6OH in our culture conditions. Therefore, reasons for 6OH exposure-related lack of functional matura- tion (Fig. 2B) likely lie elsewhere. Next, to explore any differences in 6OH effects between dif- ferentiating and differentiated neurons, we conducted addi- tional activity-induced gene transcription assays, exposing cells to a range of 6OH concentrations from both DIV 0 and DIV 7. From these assays, a dose-dependent activity-induced Arc transcriptional response was observed in cultures exposed at both DIV 0 and DIV 7 (Fig. 2G and H). Interestingly 0.5 lM 6OH exposure, which does not significantly affect cell viability, pro- duced effects that were not statistically different from 1 lM exposure. Taken together, these results indicate that chronic 6OH exposure impairs functional neuronal maturation while differentially affecting cell viability depending on the exposure dose. 6OH-BDE-47 Exposure Impairs Synaptic and Extra- Synaptic Modules of Activity-Induced Transcription Transcription of IEGs in response to neuronal activity relies on a cascade of intracellular components starting with func- tional synapses, then transduction of signals from the syn- apse to the nucleus, and finally chromatin accessibility in the nucleus itself [55, 56]. As Bic þ 4AP treatment induces neuro- nal activity by GABAergic disinhibition, any of these factors affected by 6OH could be contributing to the decreased pro- duction of nascent Arc pre-mRNA in Figure 2.As such, results Figure 1: Characterization of neuronal cultures across the first week of growth from Bic þ 4AP induction assays potentially reflect an accu- by immunocytochemistry. Rat cortical neurons were obtained by dissecting and mulation of effects arising from dysregulation at any point dissociating cortices from E18 pup brains. Cultures derived from these cells from thesynapse to thenucleus. Therefore, to morespecifi- were stained using antibodies against several markers of neuronal maturation: cally test if6OH wasacting at synapses or further down- Nestin (neuronal precursor), DCX (differentiating neurons), and NeuN (differen- stream, we induced Arc transcription intracellularly at the tiated neurons). Staining was conducted at indicated time points. Scale bar ¼ 25lm. N¼3 signal transduction level by stimulating the Mitogen Activated Protein Kinase (MAPK) pathway, which is known to transduce signals for rapid neuronal gene transcription [57, spanning primers. We found that transcription of Arc was sig- 58]. Our laboratory has established that neuronal rapid IEGs, nificantly attenuated in cultures exposed to 6OH from DIV 0-10, including Arc [43], may be induced extrasynaptically by acti- but not in cultures exposed to other compounds (Fig. 2B) sug- vating the MAPK pathway with 1 lMofthe proteinkinase C gesting that chronic exposure to 6OH may interfere with func- (PKC) activator phorbol 12-myristate 13-acetate (PMA) while tional maturation of neurons. blocking neuronal activity with 1 lMtetrodotoxin (TTX), a Lack of functional maturation could be an inadvertent fall- potent sodium channel blocker (Fig. 3A)[59]. Using this extra- out of 6OH-induced neurotoxicity or impaired neuronal differ- synaptic induction protocol, we found that Arc is induced entiation. Any cytotoxicity in cells exposed to different doses within 15 min. This Arc pre-mRNA induction was inhibited of 6OH was measured by MTT assay. This range of doses was significantly by 1 lM, but not 0.5 lMor 0.1 lM, 6OH exposure selected based on an approximation of environmentally rele- (Fig. 3C). Although 1 lM 6OH exposure caused a certain vant exposures. The reasoning and justification for this amount of cell death (Fig. 2C), theeffect of thesamedoseseen approximation is detailed further in the discussion. While in this assay may not be entirely due to neurotoxicity because 1 lM 6OH was found to induce a significant reduction in cell the Arc induction values are normalized by an internal control viability after chronic exposure for ten days, 0.5 and 0.1 lM (Gapdh, representing live cells). Additionally, comparing 6OH doses did not (Fig. 2C). Next, we studied the effect of 6OH Figs 2G and 3C, our data indicate that prolonged exposure to on neuronal differentiation. 6OH has been previously shown the non-lethal dose of 6OH (0.5 lM) may impair synaptic for- to inhibit differentiation at lower concentrations than its mation and/or functions (Bic þ 4AP assays; Fig. 2G), but spares parent compound in adult neural progenitor cells and human extra-synaptic cascades of events that lead up to Arc tran- neuronal precursors [53, 54]. To verify any effect of 6OH on scription (TTX þ PMA assays; Fig. 3C). neuronal differentiation in our system, we stained for neuro- One major suspected mode of action for PBDE toxicity is dys- nal fate markers (NeuN and Tuj1) in DIV 7 neurons after pro- regulation of thyroid hormone homeostasis, including disrup- longed exposure to the non-cytotoxic 0.5 lM6OH dose tion of thyroid hormone levels, transport, and receptor activity (Fig. 2D–F). On DIV 7, when neurons are differentiated in our [4]. These effects are thought to be mediated by structural simi- system (Fig. 1), there was no difference in fraction of NeuN larity between PBDEs and thyroid signaling molecules [7]. To Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 5 Figure 2: Chronic 6OH exposure is detrimental to neuronal maturation and function. Activity induced Arc expression assays were conducted by treating cultures with Bic and 4AP for 15 min. (A) Timeline depicting PBDE exposure durations leading up to activity induction assays. Ten day exposures were started either con- currently with plating (DIV 0) or after a week of growth (DIV 7) to assess differences in effect on differentiating versus differentiated neurons. (B)Induction of Arc after a 10-day exposure to 1lM BDE-47 or one of its hydroxylated metabolites, N¼3. (C) Assessment of cell viability following 10 days of 1lM 6OH-BDE-47 exposure. Viability was assessed colorimetrically by MTT assay. N¼3. (D) Representative images of NeuN and DAPI stained cells on DIV 7 following treatment with 0.5lM 6OH starting at DIV 0. Scale bar ¼ 50lm(E) Quantification of NeuN and DAPI staining represented in D, (2, 562 untreated and 2, 634 6OH treated cells counted). (F) Representative western blot for Tuj1 (a marker of differentiated neurons) in cells exposed to 0.5lM 6OH-BDE-47 for seven days, N¼3. (G)Induction of Arc after a ten-day exposure to various concentrations of 6OH, N¼3. (H) Similar assay as shown in (G) except 6OH exposure was started on DIV 7, N¼4. * P < 0.05, ** P < 0.01. ns ¼ non-significant test if the observed effects of 6OH in our assays are mediated already. Elevated levels of T were shown to rescue 10 lMBDE- primarily by disruption of intracellular thyroid signaling, cul- 47-induced reduction of neuronal migration in another study tures were co-exposed to 1 lM 6OH and either 30 or 3 nM triiodo- [53]. In our assays, elevated levels of T did not rescue 1 lM 6OH- thyronine (T ), which is present at low levels in culture media induced attenuation of Arc induction following Bic þ 4AP Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6| Environmental Epigenetics, 2018, Vol. 4, No. 1 neurotoxic effects, opening the possibility of a thyroid-hormone pathway-independent mechanism of 6OH toxicity. BDE-47 and Its Hydroxylated Metabolites Dysregulate Expression of NDD Candidate Genes Observing that chronic exposure to 6OH disrupts neuronal activity-related function in dosage-dependent manners, and that the effects are not primarily mediated by disruption of thyroid hor- mone homeostasis, we sought to identify other potential mecha- nisms. To accomplish this, we screened 15 NDD candidate genes, coding for proteins of various functions important for neuronal development and function (Table 1), for changes in mRNA levels following exposure to 1 lM BDE-47 or its hydroxylated metabolites. Exposures were started at various time points to assess effects dur- ing neuronal differentiation. We found that exposure to BDE-47, 3OH, and 5OH altered mRNA levels of only a few genes, while 6OH exposure altered mRNA levels of 11 out of 15 genes at the earliest and middle time points (exposures at DIV 0 and DIV 4), and a smaller subset when differentiated neurons were exposed starting at DIV 7 (Fig. 4). This observation suggests that 6OH exposure may have temporally specific effects, with immature neurons being affected to a seemingly greater extent. Following up on the wide- spread effect caused by 6OH, we measured mRNA levels of the same 15 genes after exposure to a range of 6OH concentrations over the various time points (Fig. 5). Dysregulation of mRNA levels by 6OH was found to be dose-dependent, with transcripts of a few genes exhibiting significant changes at concentrations as low as 10 nM (Arid1b, Tbl1xr1,and Adnp, data not shown). Of particular interest are Arid1b and Smarcc2,both of which code for subunits of the BAF chromatin remodeling complex [60], as well as Shank3,a high confidence autism candidate gene whose protein product is involved in the organization of the postsynaptic density [61]. In our screen, Shank3 was significantly dysregulated by 6OH in an oppos- ingmannerdepending on thetimeof exposure(Figs 4 and 5). Together, these screens demonstrate dysregulation of mRNA lev- els of several NDD candidate genes by chronic 6OH exposure in a mammalian model, and further validate the dose- and time-of- exposure dependence of 6OH toxicity. They also provide many potential novel targets for further investigation. While changes in mRNA levels following 6OH exposure can be informative of altered cellular states, they do not necessarily indicate direct effects on the transcription of target genes. This is due to mRNA abundance being a function of the rate of transcrip- tion as well as of transcript decay, which can vary greatly [62, 63]. To verify if 6OH-induced changes to mRNA levels were due to a Figure 3: Effects of 6OH exposure are at the synaptic and extrasynaptic level and direct effect on the transcription of target genes, we designed pri- are not primarily mediated by disruption of thyroid hormone signaling. Activity mers to detect pre-mRNAs, the short-lived, direct product of tran- induced Arc expression assays were conducted by treating cultures with Bic and 4AP or TTX and PMA for 15 min after ten days of 6OH exposure starting at the scription by RNA polymerase II. We focused on genes strongly time of plating. (A) Depiction of different modes of gene transcription assays. affected in differentiating neurons (cultures with exposure start- Note: Bicþ4AP induction (Fig. 2) is reliant on synaptic activity whereas ing on DIV0), and measured their respective pre-mRNA levels at TTXþPMA induction bypasses synapses and jumpstarts signaling cascades time points within that window of exposure, where early neuro- extra-synaptically. (B) Timeline depicting 6OH exposures with or without addi- tion of triiodothyronine (T3) followed by various activity induction assays. (C) nal differentiation is occurring (DIV0-5). Interestingly, as revealed Induction of Arc via direct activation of the MAPK pathway with PMA while by MTT assays and unlike what is seen in differentiated neurons blocking membrane activity with TTX in cultures exposed to various concentra- (Fig. 2C), 1 lM 6OH did not negatively impact cell viability by DIV3 tions of 6OH N¼5. (D) Induction of Arc with Bicþ4AP following exposure to 6OH (Fig. 6A). In these cells, as revealed by pre-mRNA assays, 6OH with and without additions of T3 to outcompete potential interactions of 6OH exposure impacted most genes only at the mRNA level, perhaps with thyroid hormone transporters and receptors N¼3. *P < 0.05, **P < 0.01 via indirect mechanisms that influence mRNA half-lives. stimulation, although the addition of 30 nM T alone did signifi- However, expression profiles of three of the tested genes were cantly enhance induced Arc transcription (Fig. 3D). Together, found to be significantly dysregulated at the pre-mRNA level – intriguingly, all of which encode BAF complex subunits or inter- these results indicate that boosted levels of thyroid hormone cannot rescue effects of 6OH exposure, perhaps including its acting proteins (Smarcc2, Arid1b, and Adnp). (Fig. 6B). Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 7 Table 1: Neurodevelopmental disorder candidate genes screened for mRNA dysregulation following chronic exposure to BDE-47 or its hydroxy- lated metabolites Gene Description/Function Associated Brain Disorders Adnp Activity-Dependent Neuroprotective Protein: zinc finger transcription factor that modulates ASD [81], ID [82] p53 activity and interacts with BAF chromatin remodeling complexes to regulate transcription Arid1b AT-Rich Interaction Domain 1B (BAF250b): DNA interacting protein, subunit of the neural pre- ASD [83], ID [82] cursor and neuron specific BAF chromatin remodeling complexes Cbx4 Chromobox 4: component of a Polycomb group (PcG) PRC1-like complex which is involved in ASD [84] transcriptional repression of many genes throughout development and mediates monoubi- quination of histone 2A lysine 119 residues Ctnnb1 Catenin Beta 1: component of the canonical Wnt signaling pathway, coactivator of TCF/LEF ASD [81], ID [82] transcription factors that activate Wnt responsive genes which play diverse neurodevelop- mental roles such as central nervous system patterning, and neural stem and precursor cell proliferation Dyrk1a Dual Specificity Tyrosine Phosphorylation-Regulated Kinase 1A: a nuclear protein kinase that ASD [81], ID [82] catalyzes autophosphorylation of serine/threonine and tyrosine residues, thought to play a role in regulating signaling pathways contributing to cell proliferation and neurodevelopment Med13l Mediator Complex Subunit 13 Like: a subunit of the mediator complex, a transcriptional coacti- ASD [84], ID [82] vator for RNA polymerase II transcribed genes that is recruited to promoters by binding with regulatory proteins thus serving as a scaffold for the preinitiation complex, involved in early neurodevelopment Ncor1 Nuclear Receptor Corepressor 1: corepressor of thyroid-hormone and retinoic-acid receptor tar- ASD [84], HD [85] get genes, known to recruit histone deacetylases that promote the formation of condensed chromatin structure that prevents access of transcription factors and thus represses transcription Npas2 Neuronal PAS Domain Protein 2: transcription factor that is a core component of the circadian ASD [86] clock, also known to regulate the transcription of metabolism, cell cycle, and DNA repair related genes Phf2 PHD Finger Protein 2: a zinc-finger-like plant homeodomain containing demethylase that acts ASD [84] on both histone and non-histone proteins, dimerizes with Arid5b at target promoters and demethylates histone 3 lysine 9 (H3K9me2) residues leading to transcriptional activation Rps6ka2 Ribosomal Protein S6 Kinase A2: a member of the RSK family of serine/threonine kinases that ASD [87] phosphorylates members of the mitogen-activated protein kinase signalling pathway, regu- lates processes such as cell growth, survival, and proliferation Shank3 SH3 and Multiple Ankyrin Repeat Domains 3: major scaffold protein of the postsynaptic density ASD [88], ID [89], SCZ [90] that organizes neurotransmitter receptors, ion channels, and other membrane proteins via interactions with the actin cytoskeleton, plays a role in dendritic spine maturation as well as synaptic formation and plasticity Smarcc2 SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin Subfamily C ASD [91], ID [92], SCZ [41] Member 2 (BAF170): subunit of the neural precursor and neuron specific BAF chromatin remodeling complexes, thought to be a scaffolding subunit required for stabilization of other BAF subunits, involved in transcriptional activation and repression, known to regulate corti- cal thickness and neural progenitor proliferation Sp1 Specificity Protein 1: a zinc finger transcription factor that binds GC-rich motifs at many pro- ASD [93] moters, involved in many cellular processes such as differentiation, growth, apoptosis, and recruitment of chromatin remodeling enzymes including Brg1 and Brg associated factors to regulate transcription Tbl1xr1 Transducin Beta Like 1 X-Linked Receptor 1: thought to be a component of the NCoR and ASD [81], ID [94] HDAC3 repressive complexes, and is required for transcriptional activation by a variety of transcription factors Ubr3 Ubiquitin-Protein Ligase E3-Alpha-3: targets proteins for degradation by ubiquinitination, may ASD [81] be involved in chromatin regulation and transcriptional silencing (by similarity) Abbreviations: ASD- autism spectrum disorder, ID- intellectual disability, SCZ- schizophrenia, HD- Huntington[CVOAPS]s disease 6OH-BDE-47 Dysregulates Expression of Smarcc2 that play important and fundamental roles in driving neurode- (BAF170) and Other BAF Complex Subunits velopmental processes [64]. BAF170 is thought to act as a scaf- One of the genes exhibiting 6OH-induced changes in pre-mRNA folding subunit that is critical for the stability of these level, Smarcc2, codes for BAF170, a core subunit of the mamma- complexes [65]. Therefore, to test the effect of 6OH on expres- lian SWI/SNF (SWItch/Sucrose Non-Fermentable)-like BAF com- sion of other BAF subunits, we screened mRNA levels of other plex. BAF complexes are ATP-dependent chromatin remodelers known subunits of the neuronal precursor and neuron specific Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 8| Environmental Epigenetics, 2018, Vol. 4, No. 1 Figure 4: Exposure to BDE-47 and its hydroxylated metabolites dysregulates mRNA levels of neurodevelopmental disorder candidate genes. 15 neurodevelopmental disorder-associated genes screened for changes in mRNA abundance by qRT-PCR following exposures to 1lM BDE-47 or one of its hydroxylated metabolites for periods indicated below each heatmap. Each row depicts changes in mRNA level for a gene while each column reflects exposure to a different compound. Results are displayed as fold change relative to control. Cells with a fold change above two have saturated coloring. * P < 0.05, ** P < 0.01. N¼3–5 Figure 5: Dysregulation of neurodevelopmental disorder candidate gene mRNA levels by 6OH is dose- and time-of-exposure-dependent. 15 neurodevelopmental disor- der-associated genes screened for changes in mRNA abundance by qRT-PCR following exposures to various concentrations of 6OH for periods indicated below each heatmap. Each row depicts changes in mRNA level for a gene while each column reflects exposure to a different concentration of 6OH. Results are displayed as fold change relative to control. Cells with a fold change above two have saturated coloring. * P < 0.05, ** P < 0.01. N¼3–4 BAF complexes (npBAF/nBAF) and found significant upregula- baffling, such vacillating outcomes may be explained if expres- tion of several additional subunits, namely, Brm, Brg1, BAF60a, sion of BAF170 oscillates during neuronal differentiation and BAF53a, and BAF47 (Fig. 7A). Focusing on BAF170, we then 6OH exposure shifts the oscillation frame (Fig. 7B). This hypoth- sought to confirm 6OH-induced dysregulation of this major BAF esis is supported by previous work that found the expression of subunit at the protein level by Western blotting. Here, to our BAF170 to be very dynamic across mouse corticogenesis [66] bewilderment, we encountered two opposing outcomes with and biphasic in H1 embryonic stem cells [44]. To test this possi- almost equal frequency across replicates. In some trials, chronic bility, we collected samples at 6 h intervals after plating for the exposure of differentiating neurons to 0.1-1 lM 6OH downregu- first 48 h and then assessed BAF170 protein levels by western lated BAF170 protein levels. In others, BAF170 protein levels blotting. As shown in Fig. 7C (quantified in Fig. 7D), BAF170 lev- were upregulated by the same treatment (Fig. 7B). Initially els were found to oscillate with time. Next, we attempted to Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 9 AB Figure 6: Exposure to 6OH dysregulates pre-mRNA levels of a subset of genes affected at the mRNA level. (A) Assessment of cell viability following 3 days of exposure to 1lM 6OH. Viability was assessed colorimetrically by an MTT assay. N¼3. (B) A subset of neurodevelopmental disorder associated genes found to have dysregulated mRNA levels following 6OH exposure were screened for changes in pre-mRNA abundance which better reflect effects on transcription of a given gene. Time points within the earliest exposure period (DIV 0-7) were chosen to more clearly observe direct effects of 6OH toxicity. *P < 0.05, **P < 0.01. N¼3–7 quantify such oscillation with and without 6OH exposure. To other subunits, namely BAF60a and BAF47 whose mRNA levels our surprise, these oscillations varied greatly between biological were significantly upregulated following 6OH exposure (Fig. 7A), Ns, occluding any statistical analysis after averaging data from exhibited reduced protein levels following loss of BAF170 by multiple trials. Despite such variations, 6OH treatment consis- RNAi (Fig. 8G). Taken together, our data suggest that BAF170 depletion, not tently altered the oscillatory pattern in every N (5 attempted; three displayed in Fig. 7E), providing support to our ‘altered its overexpression, likely impairs neuronal maturation via synapse-dependent mechanisms, but not extra-synaptic com- BAF170 oscillation’ hypothesis (Fig. 7B). These BAF170 protein data, alongside its altered pre-mRNA ponents of activity-induced gene transcription. As effects of BAF170-KD are highly evocative of the effects of 6OH exposure, and mRNA levels, and the observed alterations to mRNA levels we propose that environmentally-relevant doses of 6OH may of other BAF subunits, indicate that 6OH exposure likely influ- act via dysregulation of the BAF complex, especially BAF170, to ences BAF complex composition/stability and, therefore, poten- interfere with neuronal maturation and function. tially impacts BAF-dependent functions during neuronal differentiation, including chromatin remodeling and regulation of gene transcription. Discussion In this study, we have demonstrated several novel aspects of Role of BAF170 in Neuronal Maturation and Activity PBDE neurotoxicity. Initially, we show the effects of chronic Induced Arc Transcription nanomolar range exposures to BDE-47 and its hydroxylated Finally, to directly study effects of BAF170 dysregulation on metabolites on neuronal activity-induced gene transcription functional maturation of neurons, as would be expected due to and functional maturation of primary embryonic rat cortical 6OH-induced oscillation, we bi-directionally altered its levels in neurons. We then demonstrate how similar exposures impact developing cells (DIV 0-4) by either overexpressing BAF170, or transcription of NDD candidate genes by quantification of depleting it via RNAi (lentivirus-mediated delivery of BAF170 mRNA and pre-mRNA levels. One of the strongest candidates shRNA). Both methods were validated by western blot (Fig. 8D from our screen, Smarcc2, then prompted us to focus on the and G). We then performed synapse-dependent and synapse- effects of 6OH exposure on BAF170 expression and, preliminar- independent Arc induction assays. The phenotypic outcomes of ily, on BAF170-related regulation of neuronal maturation by these assays were unaffected by overexpression of BAF170 measuring levels of activity-induced Arc expression. Together, (Fig. 8B and C). Interestingly, depletion of BAF170 impaired Arc our results indicate that exposure to the hydroxylated BDE-47 induction in the synapse-dependent Bic þ 4AP assay (Fig. 8E), metabolite 6OH strongly influences functional maturation of but not in the synapse-independent PMA þ TTX assay (Fig. 8F). neurons, potentially independent of thyroid hormone disrup- This observation is reminiscent of the effects of lower 6OH tion mechanisms– perhaps instead exerting effects mediated in doses on Arc induction using these protocol (Figs 2 and 3D). part at the level of epigenetic regulation via disruption of BAF Additionally, we found that depletion of BAF170 reduces protein chromatin remodeling complexes. levels of other BAF subunits (Fig. 8G). These observations sup- Our utilization of the activity-induced transcription of an port the idea that BAF170 acts as a scaffolding component in immediate early gene (Arc) as a readout of functional neuronal BAF complexes and indicates that dysregulation of BAF170 may maturation is, to the best of our knowledge, a novel approach effectively alter BAF complex stability and composition (Fig. 7A) in assessing the neurotoxicity of PBDEs. Immediate early gene [65]. Interestingly, BAF155 protein levels did not decrease while inductionisa strong tool to this end asit reliesonseveral Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 10 | Environmental Epigenetics, 2018, Vol. 4, No. 1 C D Figure 7: BAF170 protein levels and mRNA levels of BAF complex subunits are dysregulated by 6OH exposure. (A) Abundance of various BAF protein mRNAs estimated by qRT-PCR following exposure to various concentrations of 6OH from DIV 0-7, N¼3–4. (B) Top: BAF170 protein levels observed by western blotting following 48 h of exposure to various concentrations of 6OH. N¼6. Bottom: opposing outcomes can be explained if BAF170 is expressed in an oscillatory manner and 6OH exposures dis- rupt regulatory mechanisms shaping the temporal expression patterns. (C) Time-course western blot for BAF170 with samples collected every 6 h after plating for the first 48 h. (D) Quantification of BAF170 band intensities from blot shown in (C) as an example and evidence of BAF170 oscillation in normal cells. (E) Quantification of similar time-course experiments as in (C), with collections every 6 h starting 24 h after plating. Note the inherent variation in oscillatory pattern. Cells were either untreated or exposed to 1 lM 6OH at the time of plating. N¼4 (3 represented) *P < 0.05, **P < 0.01. Note: Because of inherent variation in the oscillatory pattern, averag- ing these data sets ‘flatten’ out the phenotype and is therefore not attempted critical cellular mechanisms that are established across neuro- interferes with neuronal maturation likely at the synaptic nal maturation, ranging from proper synaptic function to level. As mentioned previously, the range of PBDE doses used in nuclear regulation of gene transcription (Fig. 3A), allowing us to broadly assess the extent of PBDE-induced effects on func- this study were estimated based on prior work done in mamma- tional neuronal maturation. Further, inducing Arc expression lian models to approximate relevant concentrations observed in both synaptically (Bic þ 4AP) and extra-synaptically the brains of exposed animals. To determine this range, an (TTX þ PMA) allowed us to hone in on where these neurotoxic exhaustive review was conducted of literature reporting levels effects are being exerted. These assays reveal that 6OH-BDE-47 of BDE-47 accumulation in rodent brain following either chronic Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 11 BC D EF G Figure 8: BAF170 contributes to the stability of BAF subunits and proper circuit formation, but is not required for MAPK stimulated Arc transcription. (A) Depiction of the timing of lentiviral mediated manipulation of BAF170 levels and subsequent activity induced Arc induction assays. (B, C) Arc induction following Bicþ4AP or TTXþPMA stimulation in cells overexpressing BAF170, N¼3. (D) Representative western blot validating BAF170 overexpression construct. (E,F) Arc induction following Bicþ4AP or TTXþPMA stimulation in cells depleted of BAF170, N¼3. (G) Representative western blots demonstrating the effect of BAF170 knockdown on protein levels of other BAF complex subunits, N¼3. **P < 0.01. ns ¼ non-significant or single dose administration [67–73]. After converting the com- reported accumulated concentrations in rodent brain, is low rel- monly reported unit of ng/g of toxin to nmol/L (converting from ative to previous in vitro studies, and is on the same order of lipid concentration to molarity), the average concentration of magnitude as reported human levels of PBDEs and their BDE-47 was determined to be 597 nM. Studies investigating the metabolites. in vitro effects of PBDEs have often been conducted using con- It has been previously demonstrated that micromolar BDE- centrations in the low to mid micromolar range. Summarizing 47 exposures decrease both neuronal and oligodendrocyte dif- human studies, the USEPA’s 2010 report assessing the environ- ferentiation in human neural progenitor cells [53]. Another mental prevalence of PBDEs estimated the body burden of total study found that BDE-47 and 6OH decrease neuronal and oligo- PBDEs in the general population to be 30-100 ng/g (approxi- dendrocyte differentiation of mouse adult neural stem cells mately 60 to 200 nM), with toddlers tending to have higher body (aNSC), with 6OH producing an effect following nanomolar burdens compared to older children and adults. Importantly, exposures, at concentrations approximately 20 times lower BDE-47 alone was estimated to account for 50% of total PBDE than its parent compound [54]. Using activity-induced gene concentrations [16]. It has also been reported that hydroxylated transcription assays as a readout in cultured embryonic rat cort- metabolites of PBDEs accumulate in human serum and are ical neurons, we found that chronic nanomolar exposure to nearly as abundant (approximately 45% of total detectable 6OH disrupts functional maturation. These effects were found PBDEs) as parent PBDE compounds in human fetal blood sam- to be independent of 6OH cytotoxicity or alteration of differen- ples collected in the United States [13, 14]. Considering the tiation for 0.5 lM exposures. Many of the observed effects were reported brain accumulation levels of BDE-47 in rodents, con- found to be dose-dependent whereas transcriptional profiles centrations commonly used for in vitro studies to date, and the (Figs 4 and 5) were additionally sensitive to time of exposure. prevalence of BDE-47 and its hydroxylated metabolites in Additionally, these functional effects appear to be mediated humans, we used a range of 100-1, 000 nM BDE-47 and its at both synaptic and extra-synaptic levels, as evidenced by hydroxylated metabolites for our studies. This range spans decreases in Arc induction following Bic þ 4AP and TTX þ PMA Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 12 | Environmental Epigenetics, 2018, Vol. 4, No. 1 treatments. Effects on IEG transcription by both assays indicate Accordingly, we have shown that reduction of BAF170 levels that 6OH, at higher doses, may act to compromise cell viability, by RNAi, but not its overexpression, negatively impacts neuro- disrupt mature synapse formation, as well as interfere with cel- nal maturation using Arc induction as a readout, recapitulating lular mechanisms at or downstream of the MAPK pathway that the effect of exposure to 0.5 lM 6OH following Bic þ 4AP treat- regulate activity-induced Arc transcription (Figs 2 and 3). ment. The lack of effect seen after BAF170 depletion on extra- Interestingly, addition of excess levels of the thyroid hormone synaptically induced Arc (TTX-PMA protocol), as was the case triiodothyronine (T3) did not produce any phenotypic rescue (as with lower doses of 6OH in Figure 3C, indicates that BAF170- seen previously for BDE-47 induced impairment of neuronal containing BAF complexes may not be necessary for nuclear migration) [53] of the observed attenuation of Arc induction. regulation of activity-induced Arc transcription, but most likely This advocates for additional modes of PBDE toxicity, perhaps contribute to synapse maturation and function via transcription acting alongside disruption of thyroid hormone signaling. regulation of other genes. These findings are consistent with However, we also note that 1 lM 6OH-induced cytotoxicity parti- recent evidence supporting the idea that BAF complexes are ally confounds interpretation of this attempted T3 rescue, as crucial for synapse formation and maturation during early neu- reduction of overall cell viability may obscure potential reversal ronal development [75]. The same study implicated a role of of the effects of 6OH exposure by T3. To explore alternate modes Brg1, a core BAF complex ATPase, in activity-induced gene tran- of PBDE toxicity within a context of the relation of PBDE expo- scription 6 h after onset of activity. However, as our data indi- sure to NDDs, we screened 15 NDD candidate genes, many of cate, BAF170-containing BAF complexes are not specifically which are epigenetic regulators (Table 1), for changes in mRNA required for Arc induction at early time points if activity is level. Subsequently, we found that, while exposure to the induced extra-synaptically, likely owing to it[CVOAPS]s poised parent compound and its other hydroxylated metabolites sig- transcriptional status [43]. This lack of effect following BAF170 nificantly impacted only a few of these targets, 6OH dysregu- depletion and TTX þ PMA treatment may be gene-specific as lated mRNA levels of many of the screened genes. These various IEGs are regulated in differing manners [43, 52, 76]. included Shank3, which encodes an important synaptic protein, While our current work potentially opens new avenues to supporting our inference related to detrimental synaptic effects explore the effects imposed by hydroxylated BDE-47 metabo- of persistent 6OH exposure. lites on epigenetic regulation during neuronal maturation and Of the genes dysregulated by 6OH exposure, a smaller activity-induced gene expression, there are limitations to our subset additionally exhibited altered levels of pre-mRNA, indi- study. Although we have demonstrated that exposure to 6OH cating a direct effect of 6OH on transcription of these genes. has a strong effect on BAF170 expression, from the pre-mRNA Surprisingly, each of the genes whose pre-mRNA was found to protein level, the effect is of a complex nature, whose mecha- to be significantly dysregulated encode BAF chromatin remodel- ing complex subunits or interacting proteins (Adnp, Arid1b, nistic basis we do not yet fully understand. Consistant with our Smarcc2). One of these genes, Smarcc2, encodes a critical core findings, the multiphasic expression pattern of BAF170 has subunit known to contribute to overall BAF complex stability been previously demonstrated during corticogenesis in mice and composition, BAF170. Protein levels of BAF170 were subse- [66] and in H1 embryonic stem cells [44], but further investiga- quently found to be dysregulated by 6OH exposure, though in a tion will be required to fully characterize the expression profile complex oscillatory manner that remains to be fully character- of BAF170 and regulatory mechanisms that shape it. ized. It may be the case that as lack of BAF170, but not over- Additionally, like much PBDE research, we have investigated abundance, is detrimental for neurons (as we have shown in the effects of exposure to a single toxin at a time, while the Fig. 8), the cells are responding to repeated periods of lack of developing human nervous system is realistically exposed to sufficient BAF170 by upregulating Smarcc2, while protein levels complex mixtures of pollutants. Some studies have been com- vary due to post-translational regulation. pleted demonstrating effects of coexposures to environmental BAF170 is a striking candidate whose dysregulation may pollutants both in vitro and in vivo using combinations of PBDE contribute to the observed effects of 6OH toxicity, owing to its congeners, other toxins including polychlorinated biphenyls critical contribution to BAF complex function. It has been shown (PCBs), and commercially available mixtures such as DE-71 [77– to regulate the stability of BAF chromatin remodeling com- 80]. More such studies will be needed to address the effects plexes by acting as a scaffolding subunit and preventing degra- resulting from environmentally relevant mixtures of com- dation of other BAF proteins. Our results support this notion pounds, especially with concern to their metabolites. and demonstrate a reduction of protein levels of several BAF Finally, our results show that nanomolar range exposures to subunits upon BAF170-KD (Fig. 8G), an effect previously only the BDE-47 hydroxylated metabolite 6OH strongly impact sev- seen with removal of both BAF170 and another interchangeable eral aspects of neuronal maturation and function and that these complex subunit, BAF155 [65]. This may indicate that BAF170 effects are likely, at least in part, mediated by disruption of BAF plays a larger role in stabilizing BAF complexes or specific subu- chromatin remodeling complexes via dysregulation of BAF170 nits during the periods of neuronal maturation investigated in expression. Going forward, it will be important to fully charac- this study. This stabilizing aspect of BAF170 function, making it terize the regulatory mechanisms governing the intricate critical for BAF complex function as a whole, is intriguing as expression of BAF170 to discern precisely how the observed BAF complexes have well known roles in neuronal differentia- PBDE-induced dysregulation is produced. Additional investiga- tion and maturation [60], and are thought to contribute to regu- tion of the underlying mechanisms that mediate the neurotoxic lating activity-induced gene transcription [74–76]. As such, effects of PBDE at the level of epigenetic regulation may lead to periods of 6OH induced lack of BAF170 during early stages a better understanding of how exposure to these compounds where differentiation and maturation are taking place in our leads to developmental complications and NDDs as well as cultures may compromise the functional maturation of cells, leading to the observed decrease in Arc expression when to further characterization of fundamental neuroepigenetic assayed at later time-points. processes. Downloaded from https://academic.oup.com/eep/article-abstract/4/1/dvx020/4798935 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Persistent 6-OH-BDE-47 exposure and its effect | 13 with dioxins, dibenzofurans, and polychlorinated biphenyls. Acknowledgements J Occup Environ Med 2005;47:199–211. This study was supported by start-up funds from UC 16. U.S. Environmental Protection Agency (EPA). 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Environmental EpigeneticsOxford University Press

Published: Jan 1, 2018

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