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The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxin and serves multiple developmental roles. In the adult brain, while we now localize AhR mRNA to nestin-expressing neural progenitor cells in the dentate gyrus (DG) of the hippocampus, its function is unknown. This study tested the hypothesis that AhR participates in hippocampal neurogenesis and associated functions. AhR deletion and activation by the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), adversely impacted neurogenesis and cognition. Adult AhR-deficient mice exhibited impaired hippocampal-dependent contextual fear memory while hippocampal-independent memory remained intact. AhR-deficient mice displayed reduced cell birth, decreased cell survival, and diminished neuronal differentiation in the DG. Following TCDD exposure, wild-type mice exhibited impaired hippocampal-dependent contextual memory, decreased cell birth, reduced neuronal differentiation, and fewer mature neurons in the DG. Glial differentiation and apoptosis were not altered in either TCDD-exposed or AhR-deficient mice. Finally, defects observed in TCDD-exposed mice were dependent on AhR, as TCDD had no negative effects in AhR-deficient mice. Our findings suggest that AhR should be further evaluated as a potential transcriptional regulator of hippocampal neurogenesis and function, although other sites of action may also warrant consideration. Moreover, TCDD exposure should be considered as an environmental risk factor that disrupts adult neurogenesis and potentially related memory processes.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; Dioxin) is a ubiquitous environmental chemical that has been linked to immune dysfunction, reproductive defects, and cancer (White and Birnbaum 2009). Developmental TCDD exposures have also been reported to produce cognitive deficits (Haijima et al. 2010). However, whether adult TCDD exposure affects brain structure and function is unknown. TCDD exerts its toxicity by binding to the aryl hydrocarbon receptor (AhR), a ligand-activated basic Helix–Loop–Helix/Per–Arnt–Sim (bHLH/PAS) protein that regulates the expression of growth regulatory genes (Bock and Kohle 2006). While the normal physiological functions of AhR are unresolved, previous reports indicate AhR regulates neurogenesis in invertebrate systems (Qin and Powell-Coffman 2004; Kim et al. 2006). We recently identified AhR expression in neural progenitor cells (NPCs) in the mouse cerebellum and prenatal forebrain and discovered that TCDD exposure interfered with developmental neurogenesis (Williamson et al. 2005; Collins et al. 2008; Latchney et al. 2011). In addition, cerebellar neurogenesis was abnormal in AhR-deficient mice (Collins et al. 2008). Because the AhR is expressed in the adult hippocampus, including granule cells of the neurogenic dentate gyrus (Petersen et al. 2000), we hypothesize that the AhR regulates neurogenesis and associated functions, and that these processes will be disrupted by TCDD exposure.
Hippocampal NPCs proliferate in the subgranular zone (SGZ) and then differentiate into mature dentate granule neurons between 1 and 4 weeks after birth (van Praag et al. 2002). Newly generated cells subsequently integrate into hippocampal networks encoding spatial memory (Kee et al. 2007) and, after a period of maturation, become physiologically indistinguishable from existing granule neurons (van Praag et al. 2002). Although the functional role of adult neurogenesis is uncertain, it is proposed that new neurons contribute to learning and memory (Inokuchi 2011). For example, associative learning has been connected to the survival of newborn hippocampal cells (Anderson et al. 2011). Conversely, learning and memory deficits result when adult neurogenesis is ablated (Jessberger et al. 2009). While most evidence for the relationship between adult neurogenesis and cognitive processing has been correlative, the continuous incorporation of newborn neurons into the hippocampal network appears to be optimized according to cognitive demands (Zhao et al. 2008). Although the intrinsic molecular signals that orchestrate SGZ neurogenesis and function are complex, there is evidence that bHLH/PAS transcription factors participate in regulating neurogenesis (Pleasure et al. 2000).
To examine the role of the AhR in adult hippocampal neurogenesis and function, we first demonstrated the presence of AhR mRNA in nestin-expressing NPCs in the SGZ of the adult dentate gyrus. We next evaluated NPC birth, death, and differentiation in AhR-deficient and age-matched wild-type mice. We also examined the effect of TCDD on NPC maturation. Learning and memory were assessed by a contextual and auditory fear-conditioning paradigm in both AhR-deficient and TCDD-exposed mice. We demonstrate that both AhR-deficient and TCDD-exposed wild-type mice exhibited compromised hippocampal-dependent contextual memory, reduced NPC birth, and diminished neuronal differentiation. Moreover, AhR was required to mediate the adverse effects of TCDD. Although additional studies are necessary, our findings are consistent with the possibility that AhR participates in the molecular signaling cascade that controls adult hippocampal neurogenesis, revealing a novel target for TCDD neurotoxicity.
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Our observations indicate that AhR deletion or activation negatively impact hippocampal NPC birth and differentiation associated with reduced hippocampal-dependent memory function. The presence of receptor expression in SGZ precursors is consistent with the hypothesis that AhR deletion or activation directly alters signaling pathways that orchestrate hippocampal neurogenesis and related functions. However, as our studies do not modulate AhR function selectively in hippocampal NPC, alternative sites of action must also be considered. For example, there could be subtle alterations in glucose or lipid metabolism (Sato et al. 2008; Takeda et al. 2011; Kachaylo et al. 2012; Tanos et al. 2012), circadian rhythms (Pendergast and Yamazaki 2012), monoamine neurotransmitters (Byers et al. 2006; Tanida et al. 2009), thyroid hormones or adrenal cortical functions (Han et al. 2011; Spaulding 2011; Leijs et al. 2012) among others, that could contribute to the observed impairments in SGZ NPC maturation and hippocampal function (Desouza et al. 2005; Kapoor et al. 2010; Snyder et al. 2011). Therefore, it remains to be determined whether the deficits in neurogenesis and behavior result directly from AhR signaling events in NPCs.
In addition to its well-characterized role in xenobiotic detoxification, AhR also functions as a modulator of cellular signaling pathways during development in the absence of an exogenous ligand (Puga et al. 2005). However, endogenous ligands for AhR remain elusive. Apart from the observed neurogenic deficits, we determined that hippocampal memory processing is abnormal in AhR−/− mice, suggesting an associated functional link for the receptor. We also demonstrated that TCDD produces similar hippocampal neurogenic and cognitive impairments as seen in AhR−/− mice. Moreover, these adverse responses were mediated by the AhR, as AhR−/− mice were resistant to TCDD-induced neurotoxicity. It is conceivable that environmental modulation of AhR activity by TCDD diverts the receptor from performing normal physiological activity, which results in impaired hippocampal function and neurogenesis. However, it remains a formal possibility that absence of a TCDD effect in AhR−/− mice represents a “floor effect” in that a constant minimum amount of neurogenesis remains despite the presence of inhibitory signals. Together, our findings identify the AhR as a novel bHLH/PAS transcription factor that has the potential to contribute to signaling events that modulate adult hippocampal neurogenesis and related cognitive functions.
Although AhR expression in the adult dentate gyrus has previously been shown, the specific cell types expressing AhR remained unknown (Petersen et al. 2000). Using a transgenic mouse line, we built upon the previous study by demonstrating that nestin-positive NPCs express AhR in the SGZ of the adult neurogenic dentate gyrus. As nestin-positive NPCs are critical participants in the hippocampal neurogenic cascade, it is plausible that AhR expression and activity plays a role in regulating NPC proliferation and differentiation. AhR expression in NPCs also renders these cells as targets for the potent neurotoxicant, TCDD. However, additional studies are necessary to fully delineate the potential direct roles of AhR deletion or activation on hippocampal NPC maturation and function.
In both AhR−/− and TCDD-exposed wild-type mice, there was a substantial reduction of newborn cells in the adult dentate gyrus, as shown by a decrease in BrdU- and Ki67-positive cells. We speculate that the decrease in newborn cells is not because of cell death, as there was no evidence of increased activated caspase-3 staining or numbers of pyknotic nuclei in hippocampal cells compared with the respective control mice in each study. However, it is possible that our studies missed the short timeframe when apoptotic cells are detectable in the SGZ (Harburg et al. 2007; Sierra et al. 2010). A more likely possibility is that the decrease in newborn cells represents a failure of NPCs to proliferate, ultimately depleting the precursor pool and leading to fewer mature neurons. Supporting this interpretation, AhR−/− and TCDD-exposed wild-type mice both exhibited deficits in neuronal differentiation and had fewer mature granule cells. Alternatively, the cell cycle length of newborn cells could either be shortened or prolonged, also leading to reduced NPC proliferation and delayed neuronal differentiation. We cannot conclude that the reduced proliferation contributed to the diminished neuronal differentiation, because these outcomes could be independent of each other. However, the precise stage during which AhR deletion or activation impedes differentiation of NPCs into neurons remains to be determined. Our data do not reveal that modulation of AhR activity altered fate determination of newborn SGZ cells. Moreover, our studies did not detect a reduction in dentate gyrus volume, but it is still possible that granule cell density could be altered. Further investigations involving a more comprehensive time course and stereological evaluations are necessary to resolve the observed differentiation defect and potential impact on granule cell density.
Although our studies do not provide evidence that AhR is directly involved in hippocampal neurogenesis or hippocampal-dependent behavior, we propose that a functional AhR in nestin-positive NPCs contributes to proper maintenance of the NPC pool and/or NPC cell cycle kinetics, which is important for balancing NPC proliferation with neuronal differentiation. This hypothesis is supported by the similarity between our findings related to NPC maturation and the observations described in inducible Notch knockout (iKO) mice, where decreased NSCs in these mice were interpreted as a failure to maintain the precursor reservoir in the adult brain, leading to fewer mature neurons (Ables et al. 2010). We propose that abnormal AhR function, via genetic deletion or the untimely activation of this receptor by TCDD, leads to abnormal neurogenesis and cognitive processing. This hypothesis raises the important issue of indirect and/or secondary effects that may occur in AhR−/− and TCDD-exposed mice, and additional work is necessary to evaluate the numerous potential indirect mechanisms that might contribute to our observed deficits in neurogenesis and hippocampal-dependent memory processing. Our findings in AhR-deficient mice should also be interpreted with some caution because as with all lifelong global knockout mice, the possibility that our observations are indirectly because of adaptive changes during development or from signaling events unrelated to NPCs must also be considered, a AhR is widely expressed throughout the CNS and other organ systems. Future studies using inducible, cell-specific AhR knockout mice are required to draw a more direct link between AhR deletion and the associated neurological impairments in the adult hippocampus.
Whereas it was unexpected that AhR−/− and dioxin-exposed wild-type mice would exhibit phenotypic similarities, there are two reasons that might explain our experimental outcomes. One possible explanation is that TCDD has been shown to rapidly down-regulate AhR, resulting in an expression deficiency analogous to the AhR−/− mice (Pollenz 2002). However, this reason cannot fully explain our observed results because the AhR is not permanently down-regulated following ligand exposure. Instead, AhR protein is eventually recycled and returned to basal levels (Pollenz and Buggy 2006). A second possibility is that TCDD causes persistent activation of the AhR, thereby inducing a toxic response and preventing AhR from performing its functional roles. The toxic response from TCDD exposure may be because of the ligand displacing the AhR from promoter regions of gene clusters involved in cell growth to favor the regulation of xenobiotic metabolism genes. Consequently, cell growth-related genes may become transcriptionally repressed, resulting in abnormal growth regulation (Sartor et al. 2009), which could interfere with neurogenesis and associated function.
The similar deficits in hippocampal-mediated behavior and neurogenesis following AhR deletion or activation were initially surprising. However, there are other examples where either increases or decreases in transcription factors give similar functional and/or structural phenotypes in the brain, in both animal models and human disease. For instance, engrailed2-deficient and -over-expression transgenic mouse mutants exhibited a similar autistic-related cerebellar phenotype (Millen et al. 1994; Kuemerle et al. 1997; Baader et al. 1998, 1999). The loss or gain of function in MECP2, a transcriptional repressor, also generated similar neurological aberrations in humans and experimental animals (Chahrour and Zoghbi 2007). With regard to the AhR, two studies indicated that AhR-deficient or TCDD-exposed wild-type mice modulate immune system T cells in a similar manner (Quintana et al. 2008; Veldhoen et al. 2008). The similarities observed between AhR−/− and TCDD-exposed wild-type mice in our studies suggest that the AhR may serve dual roles in mediating xenobiotic metabolism and hippocampal neurogenesis. Therefore, both the expression and activity levels of the AhR are potentially important in regulating adult hippocampal structural and functional outcomes.
Our findings that aberrant neurogenesis in both AhR−/− and TCDD-exposed wild types produced parallel deficits in one type of hippocampal-based memory are consistent with the accumulating evidence that adult-born NPCs participate in hippocampal-dependent cognitive processing (Clelland et al. 2009; Stone et al. 2011). The observations that neuronal differentiation (DCX-positive cells) was persistently impaired several weeks following TCDD exposure are consistent with the idea that immature neurons undergoing maturation are important contributors to hippocampal function (Aasebo et al. 2011; Marin-Burgin et al. 2012). The absence of a sustained reduction in newborn cells, even though there was a functional deficit, further supports the idea that newborn NPCs less than 1 week old lack synaptic connections and are unable to make significant contributions to hippocampal function (Deng et al. 2009). However, contextual fear conditioning may not be a discrete measure of neurogenesis-dependent memory acquisition. There is also controversy about the role of the hippocampus in contextual fear conditioning, as other brain regions such as the amygdala are thought to participate in this learning and memory paradigm (Goosens and Maren 2001; Onishi and Xavier 2010). Moreover, the hippocampus is involved in functions beyond contextual memory such as mood regulation (Lagace et al. 2010; Sahay et al. 2011). Future studies are required not only to more thoroughly evaluate hippocampal function but also to establish a connection with neurogenesis and behavioral outcomes related to AhR signaling activity.
Our data suggest the abnormal hippocampal neurogenesis resulting from AhR deletion or inappropriate activation by TCDD could lead to a gradual depletion of NPCs and compromised neuronal maturation, thereby potentially accounting for the impaired formation of hippocampal-based memories. Our current work adds to existing research related to the impact of TCDD exposure during developmental neurogenesis by demonstrating that adult hippocampal neurogenesis may also be vulnerable to environmentally relevant doses of TCDD. It has been previously shown that in utero and lactational TCDD exposure also produces cognitive deficits in adulthood (Haijima et al. 2010). In addition, whereas the functional implications of our data are conjectural, it is noteworthy to contemplate the relationship between defective AhR signaling and age-related cognitive impairments. Deficiencies in neurogenesis have previously been observed in mouse models of neurodegenerative diseases, including Alzheimer's disease (Winner et al. 2011). Given that TCDD body burdens increase with age (Schecter et al. 2006), it is possible that developmental or adult exposure may be a risk factor for age-related cognitive dysfunction. Therefore, further research is warranted to identify the precise mechanism by which developmental or adult TCDD exposure perturbs hippocampal neurogenesis to determine the potential contribution of this ubiquitous environmental chemical in developing age-related cognitive disorders.