The Environmental Neurotoxicant PCB 95 Promotes Synaptogenesis via Ryanodine Receptor-Dependent miR132 Upregulation

“…hippocampal neurons (Impey et al., 2010), suggesting that NDL PCBs interfere with neurodevelopment by mimicking and/or enhancing the effects of neuronal activity, a critical cue in refining patterns of synaptic connectivity in the developing brain (Katz and Shatz, 1996; Yuste and Bonhoeffer, 2001).”

“PCBs are a class of synthetic organic compounds grouped according to their molecular structure as dioxin-like or non– dioxin-like (NDL) (Safe, 1994).”

“…hippocampal neurons (Impey et al., 2010), suggesting that NDL PCBs interfere with neurodevelopment by mimicking and/or enhancing the effects of neuronal activity, a critical cue in refining patterns of synaptic connectivity in the developing brain (Katz and Shatz, 1996; Yuste and Bonhoeffer, 2001).”

“…often positively correlated with cognitive capacity; however, histological studies of brains from patients diagnosed with autism spectrum disorders (Hutsler and Zhang, 2010) or fragile X syndrome (Irwin et al., 2001) have revealed significantly increased spine densities in neurons relative to…”

“Abnormalities in the density or shape of dendritic spines are associated with many neurodevelopmental disorders, including mental retardation, autism spectrum disorders (ASDs), and schizophrenia (Fukuda et al., 2005; Penzes et al., 2008; Bourgeron, 2009; Garey, 2010; Svitkina et al., 2010).”

“, 2010), anti-miR132 (Wayman et al., 2008), ACREB (Arthur et al.”

“Because RyRs are functionally coupled to NMDA receptors (Berridge, 2006; Pessah et al., 2010), our findings suggest that RyRs are required for not only PCB-induced synaptogenesis, but also activity-dependent synaptogenesis, expanding the role for RyRs in neuronal plasticity.”

“Organotypic hippocampal slices from P5 Sprague–Dawley rats (Charles River Laboratories) were cultured for 3 d, as described previously (Barria and Malinow, 2002).”

“Epidemiological studies have demonstrated a negative association between developmental exposure to environmental PCBs and measures of neuropsychological function in infancy or childhood (Seegal, 1996; Schantz et al., 2003; Carpenter, 2006; Korrick and Sagiv, 2008; Winneke, 2011).”

“The effects of external cues on spine morphology are mediated primarily by calcium-dependent signaling pathways (Cline, 2001; Saneyoshi et al., 2010), and we have recently demonstrated that calcium-dependent signaling pathways are vulnerable to modulation by polychlorinated biphenyls (PCBs) (Wayman…”

“…documenting that PCBs interfere with normal neurodevelopment in humans and experimental animal models (Seegal, 1996; Schantz et al., 2003; Carpenter, 2006; Korrick and Sagiv, 2008; Winneke, 2011), only recently have adverse outcome pathways of PCB developmental neurotoxicity been…”

“Dendritic spines are the primary site of excitatory synaptic input, and their formation is critical for normal neural function (Segal, 2005).”

“…RYR1 or RYR2 (Wayman et al., 2012b), mRFPtagged -actin (Impey et al., 2010), anti-miR132 (Wayman et al., 2008), ACREB (Arthur et al., 2004), shCREB (Wayman et al., 2006), constitutively active (ca) CREB (Cardinaux et al., 2000), tomato florescent protein (TFP) (Impey et al., 2010), and wild-type…”

“Interestingly, miR132 represses expression of methyl-CpGbinding protein-2 (MeCP2) (Klein et al., 2007).”

“…of both sexes (Charles River Laboratories), plated at a density of 3.4 10 8 cells per cm 2 on glass coverslips precoated with poly-L-lysine (Sigma; molecular weight 300,000) and maintained in Neurobasal A medium (Invitrogen) supplemented with B27 (Invitrogen) as described previously (Brewer, 1997).”

“Because the frequency of mEPSCs directly correlates with the density of functional dendritic spines (Murase et al., 2002), we also measured mEPSCs.”

“MeCP2 knock-out animals have significantly perturbed synthesis and release of BDNF, a neurotrophic factor that stimulates dendritic outgrowth and synaptogenesis (Jin et al., 2003; Wang et al., 2006; Rex et al., 2007).”

“More recently, expression of miR132 has been shown to be dysregulated in schizophrenia (Kim et al., 2010; Miller et al., 2012).”

“More recently, expression of miR132 has been shown to be dysregulated in schizophrenia (Kim et al., 2010; Miller et al., 2012).”

“Hippocampal slice cultures were transfected with pCAG-TFP various plasmids using a Helios Gene Gun (BioRad) as previously described (Barria and Malinow, 2002).”

“Dendritic spines are the primary site of excitatory synaptic input, and their formation is critical for normal neural function (Segal, 2005).”

“MeCP2 knock-out animals have significantly perturbed synthesis and release of BDNF, a neurotrophic factor that stimulates dendritic outgrowth and synaptogenesis (Jin et al., 2003; Wang et al., 2006; Rex et al., 2007).”

“…mechanisms, and this is critical for the effect of PCB 95 on synaptogenesis. miR132 mediates activitydependent spine formation in hippocampal neurons both in vitro (Impey et al., 2010) and in vivo (Hansen et al., 2013) and regulates cognitive behavior in rodent models (Hansen et al., 2010, 2013).”

“Abnormalities in the density or shape of dendritic spines are associated with many neurodevelopmental disorders, including mental retardation, autism spectrum disorders (ASDs), and schizophrenia (Fukuda et al., 2005; Penzes et al., 2008; Bourgeron, 2009; Garey, 2010; Svitkina et al., 2010).”

“Local release of Ca 2 from intracellular stores increases the size of dendritic spines (Korkotian and Segal, 1999), and inhibitory concentrations of ryanodine block BDNF-enhanced spine formation in primary hippocampal neurons (Adasme et al., 2011).”

“Epidemiological studies have demonstrated a negative association between developmental exposure to environmental PCBs and measures of neuropsychological function in infancy or childhood (Seegal, 1996; Schantz et al., 2003; Carpenter, 2006; Korrick and Sagiv, 2008; Winneke, 2011).”

“Consistent with this hypothesis, developmental exposure to PCB 95 or Aroclor 1254, a commercial mixture of PCBs enriched for NDL PCBs, impairs cognitive behavior in weanling rats at doses that enhance neuronal connectivity in vivo (Schantz et al., 1997; Kenet et al., 2007; Yang et al., 2009).”

“This possibility is supported by a recent report that PCB 95, but not other PCBs or PBDEs, are significantly higher in postmortem brains of children with a syndromic form of autism, compared with neurotypical controls (Mitchell et al., 2012).”

“…(Impey et al., 2010), anti-miR132 (Wayman et al., 2008), ACREB (Arthur et al., 2004), shCREB (Wayman et al., 2006), constitutively active (ca) CREB (Cardinaux et al., 2000), tomato florescent protein (TFP) (Impey et al., 2010), and wild-type (WT) and mutant (MT) p250GAP (Wayman et al., 2008).”

“Interestingly, in vivo studies indicate that, although miR132 is required for cognitive function, overexpression of miR132 to supraphysiological levels of miR132 compromises cognitive function coincident with significantly increased spine formation (Hansen et al., 2013).”

“Although commercial production of PCBs was banned in most countries, including the United States in the 1970s, NDL PCBs persist in environmental samples (Kostyniak et al., 2005; Hwang et al., 2006) and human tissues (Stewart et al., 1999; DeCaprio et al., 2005).”

“Abnormalities in the density or shape of dendritic spines are associated with many neurodevelopmental disorders, including mental retardation, autism spectrum disorders (ASDs), and schizophrenia (Fukuda et al., 2005; Penzes et al., 2008; Bourgeron, 2009; Garey, 2010; Svitkina et al., 2010).”

“We recently reported that PCB 95 stimulates CaMKICREB-Wnt2 signaling via RyR-dependent mechanisms to enhance dendritic growth in hippocampal neurons (Wayman et al., 2012a, b).”

“Local release of Ca 2 from intracellular stores increases the size of dendritic spines (Korkotian and Segal, 1999), and inhibitory concentrations of ryanodine block BDNF-enhanced spine formation in primary hippocampal neurons (Adasme et al., 2011).”

“The effects of external cues on spine morphology are mediated primarily by calcium-dependent signaling pathways (Cline, 2001; Saneyoshi et al., 2010), and we have recently demonstrated that calcium-dependent signaling pathways are vulnerable to modulation by polychlorinated biphenyls (PCBs) (Wayman…”

“4D). p250GAP is a GTPase activating protein that inhibits the downstream signaling of Rho-family GTPases, and it is a known target of miR132 translational silencing (Nakamura et al., 2002; Okabe et al., 2003; Zhao et al., 2003; Wayman et al., 2008).”

“Abnormalities in the density or shape of dendritic spines are associated with many neurodevelopmental disorders, including mental retardation, autism spectrum disorders (ASDs), and schizophrenia (Fukuda et al., 2005; Penzes et al., 2008; Bourgeron, 2009; Garey, 2010; Svitkina et al., 2010).”

“Epidemiological studies have demonstrated a negative association between developmental exposure to environmental PCBs and measures of neuropsychological function in infancy or childhood (Seegal, 1996; Schantz et al., 2003; Carpenter, 2006; Korrick and Sagiv, 2008; Winneke, 2011).”

“scribed (Nakazawa et al., 2003), using anti-Rb IR700 and anti-Ms IR800”

“…in significant cognitive impairment, and both mutations and duplications of the gene have been associated with Rett syndrome and ASDs (LaSalle and Yasui, 2009; Percy, 2011; Cukier et al., 2012). miR132 has also been shown to interact with fragile X mental retardation protein to regulate synapse…”

“4D). p250GAP is a GTPase activating protein that inhibits the downstream signaling of Rho-family GTPases, and it is a known target of miR132 translational silencing (Nakamura et al., 2002; Okabe et al., 2003; Zhao et al., 2003; Wayman et al., 2008).”

“Consistent with this hypothesis, developmental exposure to PCB 95 or Aroclor 1254, a commercial mixture of PCBs enriched for NDL PCBs, impairs cognitive behavior in weanling rats at doses that enhance neuronal connectivity in vivo (Schantz et al., 1997; Kenet et al., 2007; Yang et al., 2009).”

“…impairment, and both mutations and duplications of the gene have been associated with Rett syndrome and ASDs (LaSalle and Yasui, 2009; Percy, 2011; Cukier et al., 2012). miR132 has also been shown to interact with fragile X mental retardation protein to regulate synapse formation (Edbauer et al.,…”

“Although commercial production of PCBs was banned in most countries, including the United States in the 1970s, NDL PCBs persist in environmental samples (Kostyniak et al., 2005; Hwang et al., 2006) and human tissues (Stewart et al., 1999; DeCaprio et al., 2005).”

“Although commercial production of PCBs was banned in most countries, including the United States in the 1970s, NDL PCBs persist in environmental samples (Kostyniak et al., 2005; Hwang et al., 2006) and human tissues (Stewart et al., 1999; DeCaprio et al., 2005).”

“Patch-clamp experiments were performed on cultured hippocampal neurons transfected with mRFP- -actin and test plasmids as described previously (Benoist et al., 2011; Lesiak et al., 2013).”

“This is consistent with a report that RyR-dependent Ca 2 release from presynaptic internal stores is required for ethanol to increase spontaneous -aminobutyric acid release onto cerebellum Purkinje neurons (Kelm et al., 2007).”

“Abnormalities in the density or shape of dendritic spines are associated with many neurodevelopmental disorders, including mental retardation, autism spectrum disorders (ASDs), and schizophrenia (Fukuda et al., 2005; Penzes et al., 2008; Bourgeron, 2009; Garey, 2010; Svitkina et al., 2010).”

“The housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) cycling and primer sequences were as described previously (Santos and Duarte, 2008).”

“Because p250GAP inhibits Rac1 activity (Moon et al., 2003; Nakazawa et al., 2003; Taniguchi et al., 2003; Zhao et al., 2003; Nasu-Nishimura et al., 2006), the decreased translation of p250GAP effectively releases inhibition on Rac1, resulting in increased synaptogenesis.”

“Although commercial production of PCBs was banned in most countries, including the United States in the 1970s, NDL PCBs persist in environmental samples (Kostyniak et al., 2005; Hwang et al., 2006) and human tissues (Stewart et al., 1999; DeCaprio et al., 2005).”

“Thus, miRNAs are postulated to be functionally relevant targets in developmental neurotoxicity (Kaur et al., 2012; Tal and Tanguay, 2012).”

“Because p250GAP inhibits Rac1 activity (Moon et al., 2003; Nakazawa et al., 2003; Taniguchi et al., 2003; Zhao et al., 2003; Nasu-Nishimura et al., 2006), the decreased translation of p250GAP effectively releases inhibition on Rac1, resulting in increased synaptogenesis.”

“4D). p250GAP is a GTPase activating protein that inhibits the downstream signaling of Rho-family GTPases, and it is a known target of miR132 translational silencing (Nakamura et al., 2002; Okabe et al., 2003; Zhao et al., 2003; Wayman et al., 2008).”

“Thus, miRNAs are postulated to be functionally relevant targets in developmental neurotoxicity (Kaur et al., 2012; Tal and Tanguay, 2012).”

“This is consistent with recent proteomic studies showing that NDL PCBs increase expression of synaptic proteins in rat cerebellar neurons (Brunelli et al., 2012).”

“However, in light of recently published reports documenting widespread exposure of childbearing-aged women living in the United States to PCBs (Thompson and Boekelheide, 2013), even lower levels of exposure to NDL PCBs might adversely influence neuronal connectivity in the developing brain of…”

“FLA365 was a generous gift from Isaac Pessah (University of California, Davis, California).”

“Because p250GAP inhibits Rac1 activity (Moon et al., 2003; Nakazawa et al., 2003; Taniguchi et al., 2003; Zhao et al., 2003; Nasu-Nishimura et al., 2006), the decreased translation of p250GAP effectively releases inhibition on Rac1, resulting in increased synaptogenesis.”

“Patch-clamp experiments were performed on cultured hippocampal neurons transfected with mRFP- -actin and test plasmids as described previously (Benoist et al., 2011; Lesiak et al., 2013).”

“Consistent with this hypothesis, developmental exposure to PCB 95 or Aroclor 1254, a commercial mixture of PCBs enriched for NDL PCBs, impairs cognitive behavior in weanling rats at doses that enhance neuronal connectivity in vivo (Schantz et al., 1997; Kenet et al., 2007; Yang et al., 2009).”

“FLA365 was a generous gift from Isaac Pessah (University of California, Davis, California).”

“In rat primary hippocampal neurons, treatment with PCB 95 upregulated miR-132 and increased spine density [186].”

“PCB 95 also promotes the formation of dendritic spines in primary rat hippocampal neurons [149].”

“Lesiak et al demonstrated that the non-dioxin-like (NDL) polychlorinated biphenyls (PCB), PCB 95 elevated synapse formation through increased expression of miR132 (Lesiak et al., 2014).”

“…signaling via p250GAP, which in turn affects synaptic plasticity (Wayman et al. 2008; Impey et al. 2010; Lambert et al. 2010; Dhar et al. 2014; Lesiak et al. 2014). miR-132 has also been shown to be localized to axons and to regulate their extension via Rasa1 and p250GAP (Hancock et al. 2014;…”

“PCB 95 activation of the CaMKI-CREBWnt2 signaling pathway is blocked by pharmacological antagonism of RyRs using FLA 365 or siRNA knockdown of RyR1 or RyR2 (Wayman et al., 2012a).”

“Noncoding RNAs and PCBs There are a few studies investigating how EDCs alter miRNAs in peripheral reproductive tissues (Zhang and Pan, 2009; Choi et al., 2011; Kovanecz et al., 2014; Lesiak et al., 2014), but little on central reproductive neuroendocrine systems.”

“p250GAP Abnormal synaptogenesis and incorrect synaptic connectivity and function in PCB95 neurotoxicity [40]”

“miR-132↑ p250GAP Neuropsychological dysfunctions Hippocampal neurons [129]”

“Therefore, miR-132 is an important risk factor for PCB-mediated neurodevelopmental disorders (102).”

“As discussed above, PCB 95-induced increases in synaptogenesis in primary rat neurons is driven by upregulated miR132 expression [121].”

“…al., 2013), are observed in preclinical models of developmental PCB exposure, and mechanistic studies have linked PCB effects on dendritic arborization to activation of signaling pathways that map onto genes of susceptibility for NDD (reviewed in Stamou et al., 2013; and see Lesiak et al., 2014).”

“The effects of PCB exposure on dendritic growth involve the CamK1–CREB–Wnt2 signaling pathway as blockade of those factors prevents PCB-induced dendritic growth (Wayman et al., 2012a).”

“Earlier studies demonstrated that chemical-induced increases in RyR-dependent Ca signaling during development were associated with enhanced dendritic outgrowth, increased synaptic density and impaired cognitive behavior (Yang et al., 2009; Wayman et al., 2012b; Wayman et al., 2012a; Lesiak et al., 2014).”

“synaptic development in the central nervous system and with inner ear dysfunction, while acute exposure promotes dendritic spine formation in vitro and can alter synaptic transmission in vivo (Gilbert and Liang, 1998; Crofton et al., 2000a,b; Carpenter et al., 2002; Lein et al., 2007; Powers et al., 2009; Yang et al.,…”

“The effects of prenatal EDCs on developmental expression of microRNAs have not been studied in the hypothalamus, but their effects have been shown in other tissues including mouse Sertoli cells (22), whole brains, and livers (23); rat penile shafts (24) and hippocampal cultures (25); and human breast carcinoma (26, 27…”

“Further, studies from the same group found that PCBs promote synaptogenesis in cultured hippocampal neurons, as evidenced by increased dendritic spines and miniature excitatory postsynaptic currents (Lesiak et al., 2014).”

“mediated developmental neurotoxicity, most likely by mechanisms involving RyRs (Bal-Price et al., 2017; Lesiak et al., 2014; Mitchell et al., 2012; Pessah et al., 2006; Wayman et al., 2012a,b; Yang et al., 2014).”

“While developmental exposures can selectively alter specific spine shapes and sizes (Lesiak et al., 2014), more detailed and comprehensive studies are needed to characterize the dynamic effects of developmental exposures on the morphology of the spine neck and spine head.”

“Another congener, the tri-ortho substituted PCB95 (2,2’,3,5’,6- pentachloro biphenyl) is a potent inhibitor of the ryanodine receptor and was found to be increased in the brains of certain autism patients (Mitchell et al., 2012; Lesiak et al., 2014).”

“…and environmental samples (DeCaprio et al., 2005), and specifically multiple NDL-PCBs have been shown to be potent neurodevelopmental toxicants (Schantz et al., 1997; Howard et al., 2003; Yang et al., 2009; Yang and Lein, 2010; Wayman et al., 2012; Lesiak et al., 2014; Yang et al., 2014).”

“Acute and chronic exposure to polychlorinated biphenyls (PCBs) may induce neurotoxicity resulting in morphological changes of neurons, developmental defects of the nervous system, behavioral changes, and learning deficits (Winneke, 2011; Doi et al., 2013; Selvakumar et al., 2013; Lesiak et al., 2014).”

“(atp2a1), ryanodine receptor 3 (ryr3), ryanodine receptor 1 isoform b (ryr1b), voltage-dependent L-type calcium channel subunit α1S (cacna1sb), and voltage-dependent L-type calcium channel subunit α1C (cacna1c), which are associated with calcium ion transport and the synthesis of ATP (Lesiak et al., 2014; Schredelseke…”

“miR-132 was also involved in the promotion of synaptogenesis in rat hippocampal neurons by a non-dioxin-like PCB, PCB-95 [38].”

“Spines were counted and sorted manually using previously described criteria [Harris et al., 1992; Lesiak et al., 2014].”

“…(Giordano et al. 2008; Lyng and Seegal 2008; Dingemans et al. 2009; Costa et al. 2016; Chen et al. 2018), synaptic markers (Brunelli et al. 2012; Lesiak et al. 2014) and change in enzymatic activity (Reistad et al. 2006; Slotkin et al. 2008; Dishaw et al. 2011; W ojtowicz et al. 2019) as…”

“RyR activity is necessary for BDNF-induced remodeling of dendritic spines,(25) and RyR1 activity is required for activity-dependent dendritic growth and synaptogenesis.(26,27) These”

“…this induced receptor activation leads to altered neuronal growth and plasticity (Kenet et al., 2007; Wayman et al., 2012a; Wayman et al., 2012b; Lesiak et al., 2014) and marked deficits in cognitive function and behavior in mammals (Schantz et al., 1997; Roegge et al., 2006; Yang et al., 2009).”

“This reactive pathology indicates damage and ongoing insult to neurons, rather than simply developmental abnormalities as previously hypothesized (Lesiak et al. 2014; Modabbernia et al. 2017).”

“, 2014); it was suggested that RyR channels contribute to activity-induced synaptogenesis through miR132-dependent suppression of p250GAP (Lesiak et al., 2014).”

“9 Motif-Containing ProteinTrim26) is associated to brain vascular integrity in zebrafish [38], spine density [39] and synaptogenesis [40].”

“…[193] PCB 95—increased dendritic growth and spine density via RyRs Rat hippocampal neuron cultures (in vitro) Schizophrenia/Neurodevelopment [91] Multiple PCB congeners—no association with maternal PCB levels Finnish birth cohort (offspring) Schizophrenia [200] EPM: Elevated Plus Maze; HVA:…”

“Studies in rodents have shown that chlorpyrifos downregulates brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), impairing neuroprotective pathways and neurotrophin-dependent synaptic maintenance [124,128,129,154,155].”

“For instance, the ryanodine receptor (RyR)-mediated calcium release from the endoplasmic reticulum is required for miR132 upregulation, as pharmacological interventions or siRNA downregulation of RyR abolished miR132 upregulation and dendritic remodeling [58,77].”

“…dendritic arbori-zation, increase axonal complexity, and disrupt intracellular calcium dynamics (Feng et al. 2017 ; Griffin et al. 2024; Lein 2023; Lesiak et al. 2014; Sethi et al. 2018, 2021, p. 11; Way-man et al. 2012; Wong et al. 1997), PBDEs delay neuronal polarization and axonal outgrowth…”

“In addition, PCB-95 exposure was associated with an increase in synaptogenesis and spine formation through CREB-mediated upregulation of microRNA 132 (miR132) (Lesiak et al. 2014).”

“13,14 Importantly, exposure to PCBs varies within human populations.”

“An example of a current research area that may greatly benefit from pooling is the potential role of polychlorinated biphenyls (PCBs) in the etiology of ASD because the levels of PCBs are low, laboratory analyses are very costly, and the blood volumes needed to achieve acceptable sensitivity of analysis are large [26,…”