Dioxins and related environmental contaminants increase TDP-43 levels

Abstract

Background: Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative condition that is characterized by progressive loss of motor neurons and the accumulation of aggregated TAR DNA Binding Protein-43 (TDP-43, gene: TARDBP). Increasing evidence indicates that environmental factors contribute to the risk of ALS. Dioxins, related planar polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that activate the aryl hydrocarbon receptor (AHR), a ligand-activated, PAS family transcription factor. Recently, exposure to these toxicants was identified as a risk factor for ALS.

Methods: We examined levels of TDP-43 reporter activity, transcript and protein. Quantification was done using cell lines, induced pluripotent stem cells (iPSCs) and mouse brain. The target samples were treated with AHR agonists, including 6-Formylindolo[3,2-b]carbazole (FICZ, a potential endogenous ligand, 2,3,7,8-tetrachlorodibenzo(p)dioxin, and benzo(a)pyrene, an abundant carcinogen in cigarette smoke). The action of the agonists was inhibited by concomitant addition of AHR antagonists or by AHR-specific shRNA.

Results: We now report that AHR agonists induce up to a 3-fold increase in TDP-43 protein in human neuronal cell lines (BE-M17 cells), motor neuron differentiated iPSCs, and in murine brain. Chronic treatment with AHR agonists elicits over 2-fold accumulation of soluble and insoluble TDP-43, primarily because of reduced TDP-43 catabolism. AHR antagonists or AHR knockdown inhibits agonist-induced increases in TDP-43 protein and TARDBP transcription demonstrating that the ligands act through the AHR.

Conclusions: These results provide the first evidence that environmental AHR ligands increase TDP-43, which is the principle pathological protein associated with ALS. These results suggest novel molecular mechanisms through which a variety of prevalent environmental factors might directly contribute to ALS. The widespread distribution of dioxins, PCBs and PAHs is considered to be a risk factor for cancer and autoimmune diseases, but could also be a significant public health concern for ALS.

Keywords: ALS; Alpha-synuclein; Ataxin-2; Fus; Gene regulation; Neurodegeneration; Promoter; Protein aggregation; Toxicants; Transcription.

Fig. 1

FICZ treatment of M17 cells increases endogenous TDP-43 expression. a Immunoblots of total lysates of M17 neuroblastoma cells, differentiated for 7 days in 10 μM RA, then treated for 7 days with AHR agonist FICZ (0.5 μM) or with FICZ and CB7993113, the antagonist (10 μM). Densitometry of monomeric TDP-43 (*) and the C-terminal fragment TDP-35 (□) shown in panel A immunoblots were quantified in b (N = 3; mean ± SEM, ANOVA w/Tukey’s; ** P < 0.01, * P < 0.05). FICZ agonism increases these TDP-43 entities and higher molecular weight species (# in panel a)

Fig. 2

Peripheral exposure to AHR agonists elevates TDP-43 protein levels in the brain. a Immunoblot of cortical tissue from mice exposed by intraperitoneal (i.p) injection to the AHR agonist 7,12-Dimethylbenz(a)anthracene (DMBA). b Densitometric analysis of TDP-43 from immunoblots reveals that peripheral i.p. exposure to DMBA leads to a significant increase in cortical TDP-43 protein, an effect that is substantially reversed by the co-injection of the AHR antagonist CB7993113. N = 6 mice per group; mean ± SEM, ANOVA w/Tukey’s; *** P < 0.001, * P < 0.05. c Scatter of normalized Tardbp mRNA levels of individual mice detected by qPCR, with means and 95% confidence intervals. N = 5 mice per group; mean ± SEM

Fig. 3

Benzo(a)pyrene treatment increases endogenous TDP-43 levels in M17 cells and in the murine brain. a Immunoblot of total lysates of M17 neuroblastoma cells, differentiated for 7 days in 10 μM RA, then treated for 7 days with vehicle, the AHR-activating toxin Benzo(a)pyrene (B(a)P; 10 μM), or with B(a)P and CB7993113, the AHR antagonist (10 μM). b Immunoblot of cortical tissue from mice exposed by intraperitoneal (i.p) injection to the AHR agonist Benzo(a)pyrene. Densitometry of monomeric TDP-43 bands shown in the immunoblots were quantified in c for the M17 cell samples (N = 3; mean ± SEM, ANOVA w/ Tukey’s; ** P < 0.01) and in d (for the murine cortical tissue (N = 4; mean ± SEM, ANOVA w/Tukey’s; ** P < 0.01, * P < 0.05). The environmental toxin, B(a)P, increases levels of endogenous TDP-43 protein in both cultured human cells and in the brains of mice exposure by intraperitoneal injection. This increase is substantially reversed in each model system by co-treatment with the AHR antagonist CB7993113

Fig. 4

Insoluble TDP-43 is elevated in motor neuron-differentiated ALS-patient derived iPSCs upon FICZ treatment. Immunoblots of RIPA insoluble material a and total lysates d from motor neurons differentiated from an ALS-affected patient carrying a G298S mutation in TARDBP. Both a short exposure of the monomeric TDP-43 band and a longer exposure of the full blot are shown in a. Densitometric analysis of TDP-43 b and TDP-35 c from RIPA insoluble immunoblots indicates that AHR agonism by 0.1 μM FICZ treatment results in increased pathological species of TDP-43. Further, co-treatment with the AHR antagonist CB7993113 (5 μM) prevents the increase in RIPA insoluble TDP-43 and TDP-35 triggered by FICZ. N = 3; mean ± SEM, ANOVA w/Tukey’s; * P < 0.05, ** P < 0.01

Fig. 5

TARDBP promoter is activated by AHR agonism. a Schematic representation of sense AHRE consensus sites “GCGTG” (red flags) of human CYP1A1 and CYP1B1 AHR responsive genes and the ALS-relevant gene, TARDBP. Green boxes are exons. b In human H4 cells, treatment (72 h) with the AHR agonist 6-Formylindolo[3,2-b]carbazole (FICZ; 0.5 μM) increased luminescence from positive control luciferase reporter CYP1B1_-3.8 kb/luc2, and human TARDBP_-4.1 kb/luc2. c In M17 cells, treatment (72 h) with the AHR agonist 6-Formylindolo[3,2-b]carbazole (FICZ; 0.5 μM) increased luminescence from the human TARDBP_-4.1 kb/luc2 luciferase reporter. This FICZ-triggered increase of TARDBP_-4.1 kb/luc2 is blocked with the AHR antagonist CB7993113 (10 μM). N = 4. An M17 doxycycline-inducible shAHR line was generated that displayed a 73% decrease in endogenous AHR mRNA transcript assessed by qPCR d; N = 3. By expressing the luciferase reporters in these M17.shAHR cells e, the increase in luminescence from the TARDBP promoter, caused by 0.5 μM FICZ treatment, was significantly blocked by inducing shAHR (using 1 μg/ml doxycycline). N = 4. f The potent environmental toxins the dioxin-like 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; 0.01uM), the polyaromatic hydrocarbon Benzo[a]pyrene (B(a)P; 10 μM) and the bacterial toxin pyocyanin (Pyo; 5 μM) activate TARDBP expression. B(e)P, the non-toxic B(a)P congener did not activate the TARDBP promoter through the AHR. N = 4. For each, mean ± SEM, ANOVA w/Tukey’s; *** P < 0.001, ** P < 0.01, * P < 0.05

Fig. 6

AHR regulates endogenous mRNA transcript levels of amyotrophic lateral sclerosis-linked genes. qPCR analysis of H4 cells treated with AHR agonist (0.5 μM FICZ, 48 h) reveals that the expression of ALS-associated genes TARDBP, SOD1, PON2 and C9ORF72 were significantly increased. N = 3; mean ± SEM, ANOVA w/Tukey’s; ** P < 0.01, * P < 0.05

Fig. 7

FICZ treatment of M17 cells increases endogenous TDP-43 stability. a Immunoblots of Avidin-agarose affinity purified (Avidin-AP) Click-iT AHA-labelled nascent proteins from lysates treated with DMSO vehicle or 0.5 μM FICZ, with a 2 h pulse of Click-iT AHA-labeling of endogenous nascent proteins then (by row) periods of culture in the absence of the Click-iT metabolite to observe degradation of AHA labelled nascent proteins. Blots are probed with anti-TARDBP antibody (see Additional file 3: Figure S4C). As negative controls for Avidin-AP, a representative sample from each time point, processed using the Click-iT chemistry but in the absence of the Biotin-alkyne chemoselective ligation tag, was also affinity purified against avidin-agarose (lanes 7: “No Biotin”). A further representative sample ligated using the Biotin-alkyne was affinity purified against control agarose resin as an additional negative control (lanes 8: “Agarose”). Densitometry of TDP-43 signal from dot blots (Additional file 3: Figure S4D) were quantified in b (n = 3; mean ± sd, curves fit by non-linear regression; t-tests were also performed for each data time point DMSO vs FICZ, ** P < 0.01, * P < 0.05). FICZ agonism of AHR increases the stability of TDP-43