From the Cover: BDE-47 and BDE-49 Inhibit Axonal Growth in Primary Rat Hippocampal Neuron-Glia Co-Cultures via Ryanodine Receptor-Dependent Mechanisms

Abstract

Polybrominated diphenyl ethers (PBDEs) are widespread environmental contaminants associated with adverse neurodevelopmental outcomes in children and preclinical models; however, the mechanisms by which PBDEs cause developmental neurotoxicity remain speculative. The structural similarity between PBDEs and nondioxin-like (NDL) polychlorinated biphenyls (PCBs) suggests shared toxicological properties. Consistent with this, both NDL PCBs and PBDEs have been shown to stabilize ryanodine receptors (RyRs) in the open configuration. NDL PCB effects on RyR activity are causally linked to increased dendritic arborization, but whether PBDEs similarly enhance dendritic growth is not known. In this study, we quantified the effects of individual PBDE congeners on not only dendritic but also axonal growth since both are regulated by RyR-dependent mechanisms, and both are critical determinants of neuronal connectivity. Neuronal-glial co-cultures dissociated from the neonatal rat hippocampus were exposed to BDE-47 or BDE-49 in the culture medium. At concentrations ranging from 20 pM to 2 µM, neither PBDE congener altered dendritic arborization. In contrast, at concentrations ≥ 200 pM, both congeners delayed neuronal polarization resulting in significant inhibition of axonal outgrowth during the first few days in vitro. The axon inhibitory effects of these PBDE congeners occurred independent of cytotoxicity, and were blocked by pharmacological antagonism of RyR or siRNA knockdown of RyR2. These results demonstrate that the molecular and cellular mechanisms by which PBDEs interfere with neurodevelopment overlap with but are distinct from those of NDL PCBs, and suggest that altered patterns of neuronal connectivity may contribute to the developmental neurotoxicity of PBDEs.

Keywords: PBDE; axon; calcium signaling; developmental neurotoxicity; neuronal morphogenesis; ryanodine receptor..

FIG. 1

BDE-47 and BDE-49 do not alter dendritic growth in cultured hippocampal neurons. Cells dissociated from P1 rat hippocampi were transfected with MAP2B-eGFP at DIV 6. On DIV 7, cultures were exposed to vehicle (DMSO diluted 1:1000) or varying concentrations of BDE-47 or BDE-49 for 48 h. A, Representative photomicrographs of DIV 9 neurons expressing MAP2B-eGFP following exposure to vehicle, BDE-47 (200 nM) or BDE-49 (200 nM). PCB 95 (200 nM) was added to a subset of cultures as a positive control. B, Quantification of dendritic length in GFP+ neurons. Data from one experiment presented as the mean ± SE (n = 30–40 neurons per condition). Experiments were repeated in 3 independent dissections with comparable results from each experiment. *P < .05 relative to vehicle control. Scale bar = 25 µm.

FIG. 2

BDE-47 and BDE-49 and their hydroxylated metabolites reduce axon length in cultured hippocampal neurons. Cells dissociated from P1 rat hippocampi were exposed to vehicle or varying concentrations of BDE-47, BDE-49, 6-OH-BDE-47, or 4′-OH-BDE-49 beginning 3 h after plating. After a 48 h exposure, DIV 2 neurons were fixed and immunostained for tau-1. A, Representative photomicrographs of DIV 2 hippocampal neurons exposed to vehicle, BDE-47 (200 nM) or BDE-49 (200 nM). B, Quantification of axon length in tau-1 immunopositive cells in cultures exposed to BDE-47 or BDE-49 or (C) 6-OH-BDE-47 or 4′-OH-BDE-47. Data presented as the mean ± SE (n = 70–90 neurons from 3 independent dissections in all groups except for the 20 pM group in which n = 40 neurons from 3 independent dissections). **P < .01, ***P < .001 relative to vehicle control. Scale bar = 10 µm.

FIG. 3

BDE-47 and BDE-49 are not cytotoxic at concentrations that decrease axon length. A, LDH release into the media and B, live-dead staining using calcein AM and propidium iodide were used to assess cell viability in dissociated hippocampal cultures on DIV 2 following a 48 h exposure to vehicle or varying concentrations of BDE 47 or BDE 49. 0.1% Triton X-100 was used as a positive control. Data presented as mean ± SE (n = 3 independent dissections). *P < .05, ***P < .0001 relative to vehicle control.

FIG. 4

BDE-47 and BDE-49 do not decrease tau-1 protein in mature cultures. At DIV 7, hippocampal neurons were exposed to BDE-47 (A) or BDE-49 (B) for 48 h. At the end of the exposure, cells were lysed for western blotting and probed with antibodies specific for tau-1 (axonal cytoskeletal protein) and GAPDH (loading control) as shown in representative western blots (top panels). Bar graphs (bottom panel) represent densitometric data. Densitometric values of tau-1 immunopositive bands were normalized to densitometric values for GAPDH immunopositive bands within the same sample. Data presented as mean ± SE (n = 3 independent dissections). ***P < .001 relative to vehicle control.

FIG. 5

BDE-47 and BDE-49 delay the development of polarity in hippocampal neurons. Polarity was quantified in dissociated hippocampal cell cultures based on the subcellular distribution of GAP-43 immunoreactivity and morphometric criteria. A, Representative photomicrographs of hippocampal neurons at different stages of polarization. B, Ontogeny of polarity in hippocampal cultures grown under the culture conditions used in this study. C, Percent of polarized (Stage 3) neurons at DIV 2 following a 48 h exposure to vehicle, BDE-47 (200 nM) or BDE-49 (200 nM). Data presented as mean ± SE (n = 9 coverslips collected from 3 independent dissections, 70–150 neurons each coverslip). *P < .05 relative to vehicle control. Scale bar = 10 µm.

FIG. 6

Pharmacological antagonism of RyR Ca²⁺channels blocks the inhibitory effects of BDE-47 or BDE-49 on axon length. Dissociated hippocampal cell cultures were pre-treated with the L-type Ca²⁺channel blocker verapamil (30 µM), the IP₃ receptor blocker xestospongin C (1 µM) or the RyR blocker FLA365 (10 µM) 30 min prior to addition of vehicle, BDE-47 (200 nM) or BDE-49 (200 nM). After 48 h, cultures were fixed and immunostained for Tau-1. Axon length was quantified in cultures treated with pharmacological inhibitors in the absence (A) or presence (B) of PBDEs. Data presented as the mean ± SE (n = 30–60 neurons per condition from 3 cultures derived from a single dissection). This experiment was repeated in culture derived from 3 independent dissections with comparable results. *P < .05 relative to vehicle control, #P < .05 relative to BDE-matched cultures not treated with a pharmacological inhibitor.

FIG. 7

BDE-47 (200 nM) and BDE-49 (200 nM) reduce axon length via RyR2-dependent mechanism(s). A, Representative photomicrographs of axonal growth cones in DIV 2 hippocampal cultures co-labeled with fluorescently tagged phalloidin (green) and antibody selective for RyR1 (red, left) or RyR2 (red, right). B, Quantitative analyses of axonal length in Cy5 positive neurons. Dissociated hippocampal cells were electroporated with Cy5-labeled scrambled (control), RYR1 or RYR2 siRNA prior to plating. Data presented as the mean ± SE (n = 90 neurons collected from 3 independent dissections). *P < .05 relative to vehicle control, #P < .05 relative to cultures transfected with control siRNA and exposed to the same BDE. Scale bar = 5 µm.