Relationship: 309: Thyroperoxidase, Inhibition leads to TH synthesis, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic: This KER is plausibly applicable across vertebrates. Inhibition of TPO activity is widely accepted to directly impact TH synthesis. This is true for both rats and humans, as well as some fishes, frogs and birds. Most of the data supporting a causative relationship between TPO inhibition and altered TH synthesis is derived from animal studies, in vitro thyroid microsomes from rats or pigs, and a limited number of human ex vivo (Nagasaka and Hidaka, 1976; Vickers et al., 2012) and clinical studies. There are data to support that gene mutations in TPO result in congenital hypothyroidism, underscoring the essential role of TPO in human TH synthesis.

Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred THs during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnow, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH system disruptors.

Sex: The KE is plausibly applicable to both sexes. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.

Key Event Relationship Description

Thyroperoxidase (TPO) is a heme-containing apical membrane protein within the follicular lumen of thyrocytes that acts as the enzymatic catalyst for thyroid hormone (TH) synthesis (Taurog, 2005) across vertebrates. Two commonly used reference chemicals, propylthiouracil (PTU) and methimazole (MMI), are drugs that inhibit the ability of TPO to: a) activate iodine and transfer it to thyroglobulin (Tg) (Davidson et al., 1978); and, b) couple thyroglobulin (Tg)-bound iodotyrosyls to produce Tg-bound thyroxine (T4) and triiodothyronine (T3) (Taurog, 2005).

Evidence Supporting this KER

The weight of evidence supporting a direct linkage between the MIE, TPO inhibition, and the KE of decreased TH synthesis, is strong and supported by more than three decades of research in animals, including humans (Cooper et al., 1982; Cooper et al.,1983; Divi and Doerge, 1994).

The biological plausibility for this KER is rated Strong. TPO is the only enzyme capable of de novo synthesis of TH. TPO catalyzes several reactions, including the oxidation of iodide, nonspecific iodination of tyrosyl residues of thyroglobulin (Tg) to form monoiodotyrosyl (MIT) or diiodotyrosyl (DIT) residues, and the coupling of these Tg-bound iodotyrosyls to produce Tg-bound T3 and T4 (Divi and Doerge, 1994; Kessler et al., 2008; Ruf et al., 2006; Taurog et al., 1996, 2005). Therefore, inhibition of TPO activity is widely accepted to directly impact TH synthesis.

Empirical support for this KER is strong. There are several papers that have measured alterations in TPO and subsequent effects on TH synthesis across vertebrates. Taurog et al. (1996) showed decreased guicaol activity, decreased bound I¹²⁵, and subsequent decreases in newly formed T3 and T4 per molecule of Tg, following exposure to PTU, MMI and some antibiotics.  There is important evidence in mammals. Following in vivo exposure to PTU in rats (Cooper et al., 1982; 1983), there are concentration and time-dependent decreases in thyroid protein bound iodine and serum T4 and T3 that recovered one month after cessation of PTU exposure.  In addition, measures of thyroidal iodine content were highly correlated with intra-thyroidal PTU concentration. Vickers et al. (2012) demonstrated dose- and time- dependent inhibition of TPO activity in both human and rat thyroid homogenates exposed to MMI. Hassan et al. (2017, 2020) and Handa et al. (2021) predicted the level of THs in serum after treatment with PTU and MMI in rats. They developed a quantitative model by comparing dose- response data. 

Tietge et al (2010) showed decreases in thyroidal T4 following MMI exposure in Xenopus.  Also in Xenopus, Haselman et al (2020) showed decreases in thyroidal iodotyrosines (MIT/DIT) and iodothyronines (T4/T3) following exposure to MMI. Doerge et al (1998) showed that a tryphenylmethane dye, malachite green, inhibited TPO and lowered thyroxine production. A recent paper used a series of benzothiazoles and showed TPO inhibition (guicaol assay) and inhibition of TSH stimulated thyroxine release from Xenopus thyroid gland explant cultures (Hornung et al., 2015). 

Additionally, evidence is available from studies investigating responses to TPO inhibitors in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. Several other studies have also shown that chemically induced Inhibition of TPO results in reduced TH synthesis in zebrafish (Van der Ven et al., 2006; Raldua and Babin, 2009; Liu et al., 2011; Thienpont et al., 2011; Rehberger et al., 2018). A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.

Temporal Evidence: In mammals, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005). The impact of decreased TPO activity on TH synthesis is similar across all ages in mammals. Good evidence for the temporal relationship of the KER comes from thyroid system modeling (e.g., Degon et al., 2008; Fisher et al., 2013) using data from studies of iodine deficiency and chemicals that inhibit NIS. In addition, there is ample evidence of the temporal impacts of TPO inhibition on TH synthesis, using ex vivo and in vitro measures that demonstrate the time course of inhibition following chemical exposures, including some data from human thyroid microsomes and ex vivo thyroid slices (Vickers et al., 2012). Future work is needed that measures both TPO inhibition and TH production during development. 

In oviparous fish such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal THs transferred to the eggs. Embryonic TH synthesis is activated later during embryo-larval development. (See Domain of applicability)

Dose-Response Evidence: Dose-response data is available from a number of studies in mammals that correlate TPO inhibition with decreased TH production measured using a variety of endpoints including iodine organification (e.g., Taurog et al., 1996), inhibition of guicaol oxidation in thyroid microsomes (e.g., Doerge and Chang, 2002), and direct measure of thyroid gland T4 concentrations (e.g., Hornung et al., 2015). However, there is a lack of dose-response data from developmental studies showing direct linkages from TPO inhibition to thyroidal TH synthesis.

While it is clear that TPO inhibition will lead to altered TH synthesis, there is a need for data that will inform quantitative modeling of the relationship between TPO inhibition and the magnitude of effects on TH synthesis.

Data from studies on genistein highlight this uncertainty. Doerge and colleagues have demonstrated that for this compound up to 80% TPO inhibition did not result in decreased serum T4 in rats (Doerge and Chang, 2002). This is not consistent with other prototypical TPO inhibitors (e.g., PTU, MMI). Genistein is however a well-known phytoestrogen and the observed inconsistency may be the result of feedback mechanisms resulting from its estrogenic effect.

Quantitative Understanding of the Linkage

In Xenopus laevis, Haselman et al.(2020) demonstrated temporal profiles of thyroidal iodotyrosines (MIT/DIT) and iodothyronines (T4/T3), the products of TPO activity, following exposure to three different model TPO inhibitors (MMI, PTU, MBT) at multiple concentrations. This study established that, in Xenopus, measurable decreases in the products of TPO activity can occur as early as 2 days of exposure during pro-metamorphosis. However, despite consistent profiles of some iodo-species across chemicals, other iodo-species showed inconsistent profiles across chemicals. This highlights the multiple mechanisms of TPO (iodination and coupling) and differential susceptibility to inhibition of those mechanisms depending on the chemical's type of interaction with TPO. The most consistent concentration-response relationship across chemicals and over time was demonstrated by thyroidal T4, which is the most relevant product to subsequent key events. At the highest concentrations tested for each chemical, thyroidal T4 was below detection by 7 days of exposure across all three TPO inhibitors. Keeping in mind that the thyroid gland has follicular lumen space where thyroglobulin/T4 is stored until proteolysis and release to the blood, full inhibition of TPO would result in a delayed measurable response due to the time it takes to deplete stored hormones. Regardless of the delay, the results from this study imply full inhibition of TPO by each of these three chemicals at the highest test concentrations, but would require chemical residue analysis and/or toxicokinetic modeling to relate cellular/tissue concentrations at the site of TPO catalysis to levels of inhibition via Michaelis-Menten kinetic descriptions.

Profiles of thyroidal iodinated species demonstrated by Haselman et al. (2020) across three different TPO inhibitors suggests that a high level of TPO inhibition must occur in order to elicit responses in subsequent key events. Although the level of TPO inhibition is not directly quantifiable from this study, these data suggest that at least 90-100% inhibition was occurring since circulating T4 was not detectable at 10 days of exposure to the highest concentrations of MMI and MBT. However, additional efforts would be necessary to determine the minimum level of TPO inhibition that leads to a measurable decrease in thyroidal T4 and subsequently circulating T4.  Furthermore, Hassan et al. (2017, 2020) and Handa et al. (2021) predicted the level of THs in serum after treatment with PTU and MMI in rats. They developed a quantitative model by comparing dose- response data. 

There are only a limited number of studies where both TPO inhibition and iodine organification have been measured in vivo, and there is not enough data available to make any definitive quantitative correlations. One in vivo study in rats exposed to the TPO inhibitor genistein found no in vivo impact on serum TH concentrations, even when TPO was inhibited up to 80% (Chang and Doerge, 2000). Genistein is however a well-known phytoestrogen and the observed inconsistency may be the result of feedback mechanisms resulting from its estrogenic effect.

Given that this is an MIE to KE relationship, there is only one response to evaluate in the relationship. Decreased TH synthesis, as measured by responses of iodinated species in the thyroid gland, is the result of TPO inhibition, which cannot be measured directly in vivo.

In vivo, evaluations of TPO inhibition are limited to evaluation of the iodinated species, or products of TPO activity, present in the thyroid gland at a particular time. However, as stated previously, any measurable response in these iodinated species is not a discreet assessment of TPO activity given that the gland maintains storage of hormone in the follicular lumen space and any alteration of TPO activity would be detected once the stores begin to be depleted. In Xenopus laevis, Haselman et al. (2020) showed a decrease in thyroidal iodinated species after only 2 days of exposure to potent TPO inhibitor MMI during thyroid-mediated metamorphosis and within 4 days for PTU and MBT, both model TPO inhibitors. In zebrafish, Walter et al. (2019) reported a similar time frame, namely a decrease in T4 levels at 72 hpf after starting the exposure to PTU at 0-2 hpf. It should be noted that the time-scale is probably depending on the developmental stage and whether the embryo is capable of thyroid hormone synthesis, rather than on the exposure duration.

Iodine availability will impact the ability of TPO to iodinate tyrosine residues on thyroglobulin. Iodine availability to TPO can be impacted in a number of ways. First, environmental availability of iodine can vary greatly depending on whether and how much iodine exists in surface waters for aquatic organisms (gill respirators) and in the diets of both terrestrial and aquatic organisms. Second, somewhat regardless of iodine availability through environmental uptake (i.e., barring extremely high iodine exposure), iodine is actively transported into the thyroid follicular cell from the blood via sodium-iodide symporter (NIS), which has been shown to be susceptible to inhibition by, for example, perchlorate. As such, iodine availability to TPO is mediated by functional NIS. Finally, iodine is not fully available to TPO on the apical surface of the thyroid follicular cell until it is transported through the apical membrane by pendrin, an anion exchange protein - mutations or inhibition of pendrin could affect iodine availability to TPO.

Hydrogen peroxide is also needed by TPO to mediate the oxidation of iodide, which is produced locally by dual oxidase (DUOX). A mutation or inhibition of DUOX will impact local production of H₂O₂ leading to lower oxidizing potential of TPO and less organification of iodide.  

Thyroid stimulating hormone (TSH) released from the pituitary positively regulates the synthesis and release of thyroid hormones from the thyroid gland. As such, when TPO is inhibited and thyroid hormone synthesis is decreased, lower systemic levels of hormone cause feedback from the pituitary via TSH to upregulate a number of processes in the thyroid gland as a means of compensation, including (but not limited to) enhanced gene expression of NIS and thyrocyte cell proliferation (Tietge et al., 2010; Haselman et al., 2020).  

References

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Relationship: 305: TH synthesis, Decreased leads to T4 in serum, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic: This KER is plausibly applicable across vertebrates. While a majority of the empirical evidence comes from work with laboratory rodents, there is a large amount of supporting data from humans (with anti-hyperthyroidism drugs including propylthiouracil and methimazole), some amphibian species (e.g., frog), fish species (e.g., zebrafish and fathead minnow), and some avian species (e.g, chicken).  The following are samples from a large literature that supports this concept: Cooper et al. (1982; 1983); Hornung et al. (2010); Van Herck et al. (2013); Paul et al. (2013); Nelson et al. (2016); Alexander et al. (2017); Stinckens et al. (2020).

Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred thyroid hormones (TH) during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body TH      levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH system disruptors.

Sex: The KE is plausibly applicable to both sexes. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.

Key Event Relationship Description

Thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) are synthesized by NIS and TPO in the thyroid gland as iodinated thyroglobulin (Tg) and stored in the colloid of thyroid follicles across vertebrates. Secretion from the follicle into serum is a multi-step process. The first involves thyroid stimulating hormone (TSH) stimulation of the separation of the peptide linkage between Tg and TH. The next steps involve endocytosis of colloid, fusion of the endosome with the basolateral membrane of the thyrocyte, and finally release of TH into blood. More detailed descriptions of this process can be found in reviews by Braverman and Utiger (2012) and Zoeller et al. (2007).

Evidence Supporting this KER

The weight of evidence linking these two KEs of decreased TH synthesis and decreased T4 in serum is strong. It is commonly accepted dogma that decreased synthesis in the thyroid gland will result in decreased circulating TH (serum T4).

The biological relationship between two KEs in this KER is well understood and documented fact within the scientific community.

It is widely accepted that TPO inhibition leads to declines in serum T4 levels in adult mammals. This is due to the fact that the sole source for circulating T4 derives from hormone synthesis in the thyroid gland. Indeed, it has been known for decades that insufficient dietary iodine will lead to decreased serum TH concentrations due to inadequate synthesis. Strong qualitative and quantitative relationships exist between reduced TH synthesis and reduced serum T4 (Ekerot et al., 2013; Degon et al., 2008; Cooper et al., 1982; 1983; Leonard et al., 2016; Zoeller and Tan, 2007).  There is more limited evidence supporting the relationship between decreased TH synthesis and lowered circulating hormone levels during development.  Lu and Anderson (1994) followed the time course of TH synthesis, measured as thyroxine secretion rate, in non-treated pregnant rats and correlated it with serum T4 levels. Modeling of TH in the rat fetus demonstrates the quantitative relationship between TH synthesis and serum T4 concentrations (Hassan et al., 2017, 2020; Handa et al., 2021). Furthermore, a wide variety of drugs and chemicals that inhibit TPO are known to result in decreased release of TH from the thyroid gland, as well as decreased circulating TH concentrations. This is evidenced by a very large number of studies that employed a wide variety of techniques, including thyroid gland explant cultures, tracing organification of 131-I and in vivo treatment of a variety of animal species with known TPO inhibitors (King and May,1984; Atterwill et al., 1990; Brown et al., 1986; Brucker-Davis, 1998; Haselman et al., 2020; Hornung et al., 2010; Hurley et al., 1998; Kohrle, 2008; Tietge et al., 2010).

Additionally, evidence is available from studies investigating responses to TPO inhibitors in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. Several other studies have also shown that chemically induced Inhibition of TPO results in reduced TH synthesis in zebrafish (Van der Ven et al., 2006; Raldua and Babin, 2009; Liu et al., 2011; Thienpont et al., 2011; Rehberger et al., 2018). A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.

Temporal Evidence: In mammals, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005).  There are currently no studies that measured both TPO synthesis and TH production during development. However, the impact of decreased TH synthesis on serum hormones is similar across all ages in mammals. Good evidence for the temporal relationship comes from thyroid system modeling of the impacts of iodine deficiency and NIS inhibition (e.g., Degon et al., 2008; Fisher et al., 2013). In addition, recovery experiments have demonstrated that serum thyroid hormones recovered in athyroid mice following grafting of in-vitro derived follicles (Antonica et al., 2012). In Xenopus, it has been shown that depression of TH synthesis in the thyroid gland precedes depression of circulating TH within 7 days of exposure during pro-metamorphosis (Haselman et al., 2020).   

In oviparous fish such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal THs transferred to the eggs. Embryonic TH synthesis is activated later during embryo-larval development. (See Domain of applicability)

Dose-response Evidence: Dose-response data is lacking from studies that include concurrent measures of both TH synthesis and serum TH concentrations. However, data is available demonstrating correlations between thyroidal TH and serum TH concentrations during gestation and lactation during development (Gilbert et al., 2013). This data was used to develop a rat quantitative biologically-based dose-response model for iodine deficiency (Fisher et al., 2013). In Xenopus, dose-responses were demonstrated in both thyroidal T4 and circulating T4 following exposure to three TPO inhibitors (Haselman et al., 2020).

There are no inconsistencies in this KER, but there are some uncertainties. The first uncertainty stems from the paucity of data for quantitative modeling of the relationship between the degree of synthesis decrease and resulting changes in circulating T4 concentrations. In addition, most of the data supporting this KER comes from inhibition of TPO, and there are a number of other processes (e.g., endocytosis, lysosomal fusion, basolateral fusion and release) that are not as well studied.

For example, Kim et al. (2015) investigated the adverse effects of Triphenyl phosphate (TPP), a substance that disrupts the thyroid system. Therefore, Rat pituitary (GH3) and thyroid follicular cell lines (FRTL-5) were studied. In the GH3 cells, TPP led to an upregulation of the expression of important thyroid genes (tsh, tr alpha and tr beta) while T3, a positive control, downregulated the expression of these genes. In FRTL-5 cells, the expression of nis and tpo genes was significantly upregulated, suggesting that TPP stimulates TH synthesis in the thyroid gland.

In zebrafish larvae at the age of 7 days post-fertilisation (dpf), TPP exposure resulted in a significant increase in T3 and T4 concentrations and the expression of genes involved in thyroid hormone synthesis. Exposure to TPP also significantly regulated the expression of genes involved in the metabolism (dio1), transport (ttr) and excretion (ugt1ab) of THs. The down-regulation of the crh and tsh genes in the zebrafish larvae suggests the activation of a central regulatory feedback mechanism that is triggered by the increased T3 levels in vivo. Taken together, these observations indicate that TPP increases TH concentrations in early life stages of zebrafish by disrupting central regulatory and hormone synthesis pathways.

Quantitative Understanding of the Linkage

In rats, Hassan et al. (2020) demonstrated in vitro: ex vivo correlations of TPO inhibition using PTU and MMI and constructed a quantitative model relating level of TPO inhibition with changes in circulating T4 levels. They determined that 30% inhibition of TPO was sufficient to decrease circulating T4 levels by 20%. This is further supported by studies of Hassan et al. (2017) and Handa et al. (2021)

In Xenopus, Haselman et al. (2020) collected temporal and dose-response data for both thyroidal and circulating T4 which showed strong qualitative concordance of the response-response relationship. A quantitative relationship exists there in, but is yet to be demonstrated mathematically in this species. 

Fisher et al. (2013) published a quantitative biologically-based dose-response model for iodine deficiency in the rat. This model provides quantitative relationships for thyroidal T4 synthesis (iodine organification) and predictions of serum T4 concentrations in developing rats. There are other computational models that include thyroid hormone synthesis. Ekerot et al. (2012) modeled TPO, T3, T4 and TSH in dogs and humans based on exposure to myeloperoxidase inhibitors that also inhibit TPO.  This model was recently adapted for rats(Leonard et al., 2016) and Hassan et al (2017) have extended it to include the pregnant rat dam in response to TPO inhibition induced by PTU. While the original model predicted serum TH and TSH levels as a function of oral dose, it was not used to explicitly predict the relationship between serum hormones and TPO inhibition, or TH synthesis. Leonard et al. (2016) recently incorporated TPO inhibition into the model. Degon et al (2008) developed a human thyroid model that includes TPO, but does not make quantitative prediction of organification changes due to inhibition of the TPO enzyme. Further empirical support for the response-response relationship has been demonstrated in the amphibian model, Xenopus laevis, exposed to TPO inhibitors during pro-metamorphosis (Haselman et al., 2020) wherein temporal profiles were measured for both thyroidal and circulating T4.

Given that the thyroid gland contains follicular lumen space filled with stored thyroglobulin/T4, complete inhibition of thyroid hormone synthesis at a given point in time will not result in an instantaneous decrease in circulating T4. The system will be capable of maintaining sufficient circulating T4 levels until the gland stores are depleted. The time it takes to deplete stored hormone will greatly depend on species, developmental status and numerous other factors.

In Xenopus, Haselman et al. (2020) demonstrated an approximately 5 day difference between a significant decrease in thyroidal T4 preceding a significant decrease in circulating T4 while exposed to a potent TPO inhibitor (MMI) continuously during pro-metamorphosis.   

During Xenopus metamorphosis, circulating T4 steadily increases to peak levels at metamorphic climax. Therefore, during Xenopus metamorphosis, this KER is operable at an increased rate as compared to a system that is maintaining steady circulating T4 levels through homeostatic control. In this case, developmental status is a modulating factor for the rates and trajectories of these KEs. 

This KER is entirely influenced by the feedback loop between circulating T4 originating from the thyroid gland and circulating TSH originating from the pituitary. Intermediate biochemical processes exist within the hypothalamus to affirm feedback and coordinately release TSH from the pituitary. However, quantitative representations of these feedback processes are limited to models discussed previously.

In Xenopus, circulating levels of T4 increase through pro-metamorphosis indicating a "release" of feedback to allow circulating levels of T4 to increase and drive metamorphic changes (Sternberg et al., 2011). This provides evidence that homeostatic control of feedback can be developmentally dependent, and likely species dependent.  

References

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Gilbert ME, Hedge JM, Valentín-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW.  An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome.  Toxicol Sci. 2013 132(1):177-95.

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Relationship: 312: T4 in serum, Decreased leads to T4 in neuronal tissue, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The majority of the information on this KER comes from in vivo studies with rodents (mainly MCT8 knock-out mice and thyroidectomized rats) and histopathological analyses of human brain tissues derived from patients affected by AHDS (Allan-Herndon-Dudley syndrome). The evoluationary conservation of the transport of TH from circulation to the developing brain suggests, with some uncertainty, that this KER is also applicable to other mammalian species.

Key Event Relationship Description

In mammals, thyroxine (T4) in brain tissue is derived almost entirely from the circulating pool of T4 in blood. Transfer of free T4 (and to a lesser extent, T3) from serum binding proteins (thyroid binding globulin (TBG), transthyretin (TTR) and albumin; see McLean et al., 2017, for a recent review) into the brain requires transport across the blood brain barrier (BBB) and /or indirect transport from the cerebral spinal fluid (CSF) into the brain through the blood-CSF-barrier.  The blood vessels in rodents and humans expresses the main T4 transporter, MCT8, (Roberts et al. 2008), as does the choroid plexus which also expresses TTR and secretes the protein into the CSF (Alshehri et al. 2015).

T4 entering the brain through the BBB is taken up into astrocytes via cell membrane iodothyronine transporters (e.g., organic anion-transporting polypeptides OATP), monocarboxylate transporter 8 (MCT8) (Visser et al., 2011).  In astrocytes, T4 is then deiodinated by Type II deiodinase to triiodothyronine (T3) (St Germain and Galton, 1997), which is then transported via other iodothyronine transporters (MCT8) into neurons (Visser et al., 2011). While some circulating T3 may be taken up into brain tissue directly from blood (Dratman et al., 1991), the majority of neuronal T3 comes from deiodination of T4 in astrocytes. Decreases in circulating T4 will eventually result in decreased brain T3 tissue concentrations. It is also known that Type II deiodinase can be up-regulated in response to decreased T4 concentrations to maintain tissue concentrations of T3 (Pedraza et al., 2007; Lavado-Autric et al., 2013; Morse et al., 1986), except in tanycytes of the paraventricular nucleus (Fekete and Lechan, 2014).

Evidence Supporting this KER

The weight of evidence linking reductions in circulating serum TH and reduced brain concentrations of TH is moderate. Many studies support this basic linkage. However, there are compensatory mechanisms (e.g., upregulation of deiodinases, transporters) that may alter the relationship between hormones in the periphery and hormone concentrations in the brain. There is limited information available on the quantitative relationship between circulating levels of TH, these compensatory processes, and neuronal T4 concentrations, especially during development. Furthermore, in certain conditions, such as iodine deficiency, the decreases in circulating hormone might have greater impacts on tissue levels of TH (see for instance, Escobar del Rey, et al., 1989).

The biological relationship between these two KEs is strong as it is well accepted dogma within the scientific community. There is no doubt that decreased circulating T4 leads to declines in tissue concentrations of T4 and T3 in a variety of tissues, including brain. However, compensatory mechanisms (e.g., increased expression of Type 2 deiodinase) may differ during different lifestages and across different tissues, especially in different brain regions.  Similarly, the degree to which serum TH must drop to overwhelm these compensatory responses has not been established.

Several studies have shown that tissue levels, including brain, of TH are proportional to serum hormone level (Oppenheimer, 1983; Morreale de Escobar et al., 1987; 1990; Calvo et al., 1992; Porterfield and Hendrich, 1992, 1993; Broedel et al., 2003). In thyroidectomized rats, brain concentrations of T4 were decreased and Type II deiodinase (DII) activity was increased. Both brain T3 and T4 as well as DII activity returned to normal following infusion of T4 (Escobar-Morreale et al., 1995; 1997). Animals treated with PTU, MMI, or iodine deficiency during development demonstrate both lower serum and lower brain TH concentrations (Escobar-Morreale et al 1995; 1997; Taylor et al., 2008; Bastian et al., 2012; 2014; Gilbert et al., 2013).  Compared to the wildtype, a mouse MCT8 knockout model has was shown to have decreased plasma T4, decreased uptake of T4 into the brain, and decreased brian T3 concentrations, as well as increased cortical diodinase Type 2 activity and increased plasma T3 concentrations (Mayerl et al., 2014; Barez-Lopez et al., 2016). 

Temporal Evidence: The temporal relationship between serum T4 and T4 in growing neuronal tissue described in this KER is dependent on the developmental stage (Seed et al., 2005).  While all brain regions will be impacted by changes in serum hormones, brain concentrations will be a function of development stage and brain region. Data are available from thyroid hormone replacement studies that demonstrate recovery of fetal brain T3 and T4 levels (following low iodine diets or MMI exposure) to control levels after maternal thyroid hormone replacement or iodine supplementation (e.g., Calvo et al.,1990; Obregon et al., 1991). For example, Calvo et al. (1990) carried out a detailed study of the effects of TPO inhibition on serum and tissues levels of TH in gestating rats. Clear dose-dependent effects of T4 replacement, but not T3 replacement were seen in all maternal tissues. However, for fetal tissues, neither T4 nor T3, at any dose, could completely restore tissue TH levels to control levels.

Dose-Response Evidence:  There is good evidence, albeit from a limited number of studies of the correlative relationship between circulating thyroid hormone concentrations and brain tissue concentrations during fetal and early postnatal development following maternal iodine deficient diets or chemical treatments that depress serum THs (c.f., Calvo et al., 1990; Obregon et al., 1991; Morse et al.,1996).

The fact that decreased serum TH results in lower brain TH concentrations is well accepted.  However, the ability of the developing brain to compensate for insuffiencies in serum TH has not been well studied.  Limited data is available that demonstrates that changes in local deiodination in the developing brain can compensate for chemical-induced alterations in TH concentrations (e.g., Calvo et al., 1990; Morse et al., 1996; Sharlin et al., 2010). And, there are likely different quantitative relationships between these two KEs depending on the compensatory ability based on both developmental stage and specific brain region (Sharlin et al., 2010). For these reasons, the empirical support for this linkage is rated as moderate

The role of cellular transporters represents an additional uncertainly. In addition, future work on cellular transport mechanisms and deiodinase activity is likley to inform addition of new KEs and KERs between serum and brain T4.

Quantitative Understanding of the Linkage

While it is well established that decreased in serum TH levels result in decreased brain TH concentrations, particularly fetal brain concentrations, a major gap is the lack of empirical data that allow direct quantification of this relationship (Hassan et al., 2018). Recently, serum TH and brain TH were measured in fetal cortex and postnatal day 14 offspring following graded degrees of hypothyroidism induced by PTU (O’Shaughnessy et al., 2018). Results showed that brain levels TH levels at both ages were quantitatively related to serum T4 levels. Additional dose-response information is necessary to confirm these findings, and standardization of analysis for the measurements in these distinct matrices is crucial to allow comparisons to be made between independent experiments.

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Lavado-Autric R, Calvo RM, de Mena RM, de Escobar GM, Obregon MJ. Deiodinase activities in thyroids and tissues of iodine-deficient female rats. Endocrinology. 2013 Jan;154(1):529-36.

Mayerl S, Müller J, Bauer R, Richert S, Kassmann CM, Darras VM, Buder K, Boelen A, Visser TJ, Heuer H. Transporters MCT8 and OATP1C1 maintain murine brain thyroid hormone homeostasis.  J Clin Invest. 2014 May;124(5):1987-99.

McLean TR, Rank MM, Smooker PM, Richardson SJ.  Evolution of thyroid hormone distributor proteins. Mol Cell Endocrinol. 2017 Feb 27. pii: S0303-7207(17)30151-X. doi: 10.1016/j.mce.2017.02.038. [Epub ahead of print]

Morreale de Escobar, G., Obregon, M.J., and Escobar del Ray, F. (1987). Fetal and maternal thyroid hormones. Hormone Res. 26:12–27.

Morreale de Escobar, G., Calvo, R., Obregon, M.J., and Escobar del Rey, F. (1990). Contribution of maternal thyroxine to fetal thyroxine pools in normal rats near term. Endocrinology 126:2765–2767.

Morse DC, Wehler EK, Wesseling W, Koeman JH, Brouwer A. Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254). Toxicol Appl Pharmacol. 1996 Feb;136(2):269-79

Obregon MJ, Ruiz de Ona C, Calvo R, Escobar del Rey F, Morreale de Escobar G 1991 Outer ring iodothyronine deiodinases and thyroid hormone economy: Responses to iodine deficiency in the rat fetus and neonate. Endocrinology 129:2663–2673.

Oppenheimer, J.H. (1983). The nuclear Receptor-triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism, and biological action. In: Molecular Basis of Thyroid Hormone Action, eds. J.H. Oppenheimer and H.H. Samuels, pp. 1–35. New York: Academic Press.

O'Shaughnessy KL, Wood, C, Ford RL, Kosian, PA, Hotchkiss, MG, Degitz SJ, Gilbert ME. Thyroid hormone disruption in the fetal and neonatal rat: Predictive hormone measures and bioindicators of hormone action in the developing cortex. Toxicol Sci. 2018 Aug 6. doi: 10.1093/toxsci/kfy190.  [Epub ahead of print]

Pedraza PE, Obregon MJ, Escobar-Morreale HF, del Rey FE, de Escobar GM (2006) Mechanisms of adaptation to iodine deficiency in rats: thyroid status is tissue specific. Its relevance for man. Endocrinology 147:2098-2108.

Porterfield, S.P. and Hendrich, C.E. (1992). Tissue iodothyronine levels in fetuses of control and hypothyroid rats at 13 and 16 days gestation. Endocrinology 131:195–200.

Porterfield, S.P. and Hendrich, C.E. (1993). The role of thyroid hormones in prenatal neonatal neurological development-current perspectives. Endocrine Rev. 14:94–106.

Power DM, Llewellyn L, Faustino M, Nowell MA, Björnsson BT, Einarsdottir IE, Canario AV, Sweeney GE. (2001). Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. Dec;130(4):447-59.

Roberts LM, Woodford K, Zhou M, Black DS, Haggerty JE, Tate EH, Grindstaff KK, Mengesha W, Raman C, Zerangue N.  Expression of the thyroid hormone transporters monocarboxylate transporter-8 (SLC16A2) and organic ion transporter-14 (SLCO1C1) at the blood-brain barrier. Endocrinology. 2008 Dec;149(12):6251-61.

Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72.

Sharlin DS, Gilbert ME, Taylor MA, Ferguson DC, Zoeller RT. (2010).The nature of the compensatory response to low thyroid hormone in the developing brain. J Neuroendocrinol.  Mar;22(3):153-65.

St. Germain, D.L. and Galton, V.A. (1997). The deiodinase family of selenoproteins. Thyroid 7:655–668.

Taylor MA, Swant J, Wagner JJ, Fisher JW, Ferguson DC (2008) Lower thyroid compensatory reserve of rat pups after maternal hypothyroidism: correlation of thyroid, hepatic, and cerebrocortical biomarkers with hippocampal neurophysiology. Endocrinology 149:3521-3530.

Van Herck SL, Geysens S, Delbaere J, Darras VM. (2013). Regulators of thyroid hormone availability and action in embryonic chicken brain development. Gen Comp Endocrinol.190:96-104.

Visser WE, Friesema EC, Visser TJ.  Minireview: thyroid hormone transporters: the knowns and the unknowns.  Mol Endocrinol. 2011 Jan;25(1):1-14.

Relationship: 746: T4 in neuronal tissue, Decreased leads to Hippocampal gene expression, Altered

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Most of the data available has come from rodent models. The evolutionary conservation of thyroid receptors (Holzer et al., 2017) coupled with their role in TR regulated gene transcription in neurodevelopment, suggests that this KER may also be applicable to other species (see text above).

Key Event Relationship Description

Many cellular and biochemical effects of thyroid hormones (TH) are mediated through regulation of gene expression (Oppenheimer, 1983; Bernal, 2007).  Thyroxine (T4) is transferred from the serum to the brain (see KER: Thyroxine (T4) in Serum, Decreased leads to Thyroxine (T4) in Neuronal Tissue, Decreased), where it converted to triiodothyronine (T3), the level of which is highly controlled by deiodinases. T3 binds to thyroid receptors (TR) in the nucleus of neuronal and glial cells to control gene expression. It is generally accepted that the modulation of TR gene expression in the hippocampus, or any other brain region, must therefore depend on the presence of hormone in these tissues. 

Evidence Supporting this KER

The weight of evidence is moderate for TH concentrations affecting gene expression in the developing brain is (Oppenheimer and Schwartz, 1997; Oppenheimer, 1983; Bernal, 2007; Morte et al., 2010a; 2010b; Williams, 2008). Direct measurement of TH in brain tissue, and in hippocampus in particular, has shown correlations with gene expression. Therefore, it is assumed that reductions in TH-responsive genes in the hippocampus stem from reduced availability of hormone in the brain from the serum. However, studies in which there are simultaneous assessments of hippocampal concentrations of thyroid hormone and hippocampal gene expression is limited. 

The biological relationship between these two KEs is strong. It is a generally accepted fact that TH produce their actions on brain development by binding to nuclear receptors to affect gene transcription. See KER (1387): "T4 in serum, Decreased leads (non-adjacently) to Hippocampal gene expression, Altered" for more information on TR regulated genes. As the primary means whereby TH promotes its action is by binding to TR in brain, TH must be present in brain to affect this action. Circulating levels of T4 represent the primary source of T4 in the brain, which is then converted to the active hormone T3 by deiodinases within neuronal tissue. 

The empirical support for this KER is moderate. Many in vitro studies have demonstrated a relationship between hormone concentraionsTH and the induction of gene expression in brain cells, including hippocampal neurons in culture (Gil-Ibanez et al., 2015; Morte et al., 2010b). However, there are a limited number of studies investigating TH concentrations in the hippocampus and hippocampal gene expression. This is the case because thyroid hormone is difficult to measure in hippocampus and TH-induced gene expression changes can be subtle. We are aware of only four in vivo studies in which both thyroid hormones in the brain and gene expression in brain were simultaneously measured (Bastian et al., 2012; 2014; Hernandez et al., 2010; Sharlin et al., 2008). Only two of these reports, stemming from the same laboratory, specifically assessed thyroid hormone and gene expression in hippocampus. In these studies, Bastian et al., (2012; 2014) measured decrements in hippocampal T3 using RIA and correlated these reductions with alterations in the expression of myelin associated genes (Mbp, Plp), the neurotrophin, Ngf, the calcium binding protein Parv, a TH-dependent transcription factor, Hr, and Agt.

Temporal Evidence: The temporal nature of this KER on TH dependent gene regulation is developmental (Seed et al., 2005). The impact of brain TH concentrations on regulation of TR regulated genes is age-dependent for a number of genes critical for normal hippocampal development. It is widely accepted that different genes are altered dependent upon the window of exposure in the fetal, neonatal or adult brain (c.f., Pathak et al, 2011; Mohan et al., 2012; Quignodon et al., 2004; Williams, 2008). Thyroid hormone supplementation has been shown to reverse some of the effects on gene expression (Mohan et al., 2012; Liu et al., 2010; Pathak et al., 2011).

Dose-Response Evidence:  Dose-response data exists but is limited to a small number of studies and a small number of genes (Bastian et al., 2012; 2014; Sharlin et al., 2008).

There are no inconsistencies in this KER, but there are uncertainties. Uncertainties remain in the relationship of neuronal TH concentrations and gene expression in the brain because of the lack of studies simultaneously examining brain hormone and gene expression in the same study. This stems from the technological challenges associated with measuring brain hormone and the sometimes-subtle changes in brain gene expression induced by manipulations of the thyroid system. In addition, there are also some physiological actions of T4 that are mediated non-genomically at the cell membrane (Davis et al., 2016).  However, the exact role for the non-genomic effects is not well accepted or understood (Galton, 2017).

Quantitative Understanding of the Linkage

There is only one study available to date that provides empirical data on both TH concentrations and measures of gene expression changes in brain.  O'Shaughnessy et al (2018) demostrates dose-response relationships between brain T4 and T3 concentrations and changes in a variety of genes (e.g., Parv, Col11a2, Hr, Ngf) that were "statistically significant at doses that decreased brain t4 and/or T3".  There was no quantitation of this relationship reported.

References

Bastian TW, Anderson JA, Fretham SJ, Prohaska JR, Georgieff MK, Anderson GW (2012), Fetal and neonatal iron deficiency reduces thyroid hormone-responsive gene mRNA levels in the neonatal rat hippocampus and cerebral cortex. Endocrinology 153:5668-5680.

Bastian TW, Prohaska JR, Georgieff MK, Anderson GW (2014) Fetal and neonatal iron deficiency exacerbates mild thyroid hormone insufficiency effects on male thyroid hormone levels and brain thyroid hormone-responsive gene expression. Endocrinology 155:1157-1167.

Bernal J (2007) Thyroid hormone receptors in brain development and function. Nat Clin Pract Endocrinol Metab 3:249-259.

Davis, P.J., Goglia, F., Leonard, J.L., 2016. Nongenomic actions of thyroid hormone. Nat. Rev. Endocrinol. 12, 111-121.

Galton VA. The ups and downs of the thyroxine pro-hormone hypothesis. Mol Cell Endocrinol. 2017 Jan 24. pii: S0303-7207(17)30042-4. doi: 10.1016/j.mce.2017.01.029.

Gil-Ibañez P, García-García F, Dopazo J, Bernal J, Morte B.  Global Transcriptome Analysis of Primary Cerebrocortical Cells: Identification of Genes Regulated by Triiodothyronine in Specific Cell Types.  Cereb Cortex. 2017 Jan 1;27(1):706-717.

Hernandez A, Quignodon L, Martinez ME, Flamant F, St Germain DL (2010), Type 3 deiodinase deficiency causes spatial and temporal alterations in brain T3 signaling that are dissociated from serum thyroid hormone levels. Endocrinology 151:5550-5558.

Holzer G, Roux N, Laudet V. Evolution of ligands, receptors and metabolizing enzymes of thyroid signaling. Mol Cell Endocrinol. 2017 Mar 22. pii: S0303-7207(17)30191-0. doi: 10.1016/j.mce.2017.03.021.

Liu D, Teng W, Shan Z, Yu X, Gao Y, Wang S, Fan C, Wang H, Zhang H. The effect of maternal subclinical hypothyroidism during pregnancy on brain development in rat offspring.  Thyroid. 2010 Aug;20(8):909-15.

Mohan V, Sinha RA, Pathak A, Rastogi L, Kumar P, Pal A, Godbole MM (2012) Maternal thyroid hormone deficiency affects the fetal neocorticogenesis by reducing the proliferating pool, rate of neurogenesis and indirect neurogenesis. Exp Neurol 237:477-488.

Morte B, Diez D, Auso E, Belinchon MM, Gil-Ibanez P, Grijota-Martinez C, Navarro D, de Escobar GM, Berbel P, Bernal J (2010a) Thyroid hormone regulation of gene expression in the developing rat fetal cerebral cortex: prominent role of the Ca2+/calmodulin-dependent protein kinase IV pathway. Endocrinology 151:810-820.

Morte B, Ceballos A, Diez D, Grijota-Martínez C, Dumitrescu AM, Di Cosmo C, Galton VA, Refetoff S, Bernal J. (2010b) Thyroid hormone-regulated mouse cerebral cortex genes are differentially dependent on the source of the hormone: a study in monocarboxylate transporter-8- and deiodinase-2-deficient mice.  Endocrinology. 2010 May;151(5):2381-7

Oppenheimer, J. (1983). The nuclear-receptor-triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism, and biological action. Molecular Basis of Thyroid Hormone Action. J. O. a. H. Samuels. New York, Academic Press: 1-34.

Oppenheimer, J. H. and H. L. Schwartz (1997). "Molecular basis of thyroid hormone-dependent brain development." Endocr Rev 18(4): 462-75.

O'Shaughnessy KL, Wood, C, Ford RL, Kosian, PA, Hotchkiss, MG, Degitz SJ, Gilbert ME. Thyroid hormone disruption in the fetal and neonatal rat: Predictive hormone measures and bioindicators of hormone action in the developing cortex. Toxicol Sci. 2018 Aug 6. doi: 10.1093/toxsci/kfy190.  [Epub ahead of print]

Pathak A, Sinha RA, Mohan V, Mitra K, Godbole MM. 2011. Maternal thyroid hormone before the onset of fetal thyroid function regulates reelin and downstream signaling cascade affecting neocortical neuronal migration. Cerebral cortex. 21:11-21.

Quignodon L, Legrand C, Allioli N, Guadano-Ferraz A, Bernal J, Samarut J, Flamant F (2004) Thyroid hormone signaling is highly heterogeneous during pre- and postnatal brain development. J Mol Endocrinol 33:467-476.

Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72.

Sharlin DS, Tighe D, Gilbert ME, Zoeller RT (2008) The balance between oligodendrocyte and astrocyte production in major white matter tracts is linearly related to serum total thyroxine. Endocrinology 149:2527-2536.

Williams GR (2008), Neurodevelopmental and neurophysiological actions of thyroid hormone. J Neuroendocrinol. 2008 Jun;20(6):784-94

Relationship: 747: Hippocampal gene expression, Altered leads to Hippocampal anatomy, Altered

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The majority of data in support of this KER is from rodent models.  The evolutionary conservation of thyroid receptors (Holzer et al., 2017) coupled with their role in TR regulated gene transcription in neurodevelopment, suggests that this KER may also be applicable to other species. 

Key Event Relationship Description

The basic biological processes that link gene regulation in the structural formation and function of all organs of the body are similar throughout the developing organism.  In the developing brain, genes encode proteins critical for developmental events intrinsic to structural development (e.g., neurogenesis, neuronal migration, synaptogenesis, myelination). The development of the hippocampus is no exception to this general rule of biology.

Evidence Supporting this KER

The overall weight of evidence is moderate for a direct linkage between perturbation of the expression of genes in brain (and in hippocampus specifically) and neuroanatomical abnormalities.  It is widely acknowledged that the development of the structure of the hippocampus is under the control of hippocampal gene expression. However, while an extensive body of literature exists linking some genes to hippocampal structure, there is no complete compendium on the total number of genes involved, nor direct causative links between the myriad of genes and the intricate development (both timing and location) of the majority of hippocampal structure. 

The biological plausibility of this KER is rated as strong. It is well established that gene regulation controls brain development.  This also applies to the development of the hippocampus, where nuclear thyroid receptors that regulate gene transcription, directly or indirectly via transcription factor regulation, to control translation.

Empirical support for this KER is rated as strong. The number of publications in this area is extensive. A few examples are: Strange et al. (2014); Takei et al. (2016); and Shin et al. (2015). Work supporting the relationship includes use of a variety of animal models (i.e., nutritional deficiencies, chromosome abnormalities, gene deletions, knock out animals, toxicant exposures and developmental hormonal imbalance) (e.g., Frotscher, 2010; Castren and Castren, 2014; Spilker et al., 2016; Skucas et al., 2011; Lessman et al., 2011). Mutant mouse lines generated for genes involved in human cortical malformations such as doublecortin, reelin, Lis1 and Tuba1a also show gross disorganization within the hippocampus (Khalaf-Nazzal et al., 2013). Collectively, data from these studies clearly support the link between alterations in hippocampal gene expression and structural changes in hippocampal volume, cell number, and/or cytoarchitecture. A direct linkage between some specific gene targets and structural change in the hippocampus has been demonstrated using knock out and mutant mouse models (e.g., Grant et al., 1992; Lee et al., 2000; Frotscher, 2010; Castren and Castren, 2014; Spilker et al., 2016; Skucas et al., 2011; Lessman et al., 2011; Khalaf-Nazzal et al., 2013).

Temporal Evidence: The temporal nature of this KER is developmental (Seed et al., 2005). It is a well-recognized fact that there are critical developmental windows for disruption of TR-regulated genes and subsequent formation of the anatomy of the hippocampus. This has been demonstrated in multiple studies. Many of the gene-anatomy relationships critical to brain development only exist during development, or exist only to a very limited extent in the adult brain.  For example, genes controlling neuronal proliferation and migration are critically essential in hippocampal development, and their disruption results in abnormal hippocampal anatomy. Whereas, in the adult brain the genes are largely without effect as these processes are completed in the early neonatal period. In support of this, a limited number of studies have defined critical periods for the interaction of some genes and resulting neuroanatomical organization of the hippocampus (Lee et al., 2015; Favaro et al., 2009; Lee et al., 2000).  In addition, there are some ‘rescue’ experiments for a select number of genes (eg., Lee et al., 2015; Spilker et al., 2016). Several examples are described below:

In the Jacob/Nsfm knockout model, hippocampal dysplasia is seen in hippocampal areas CA1 and CA3, characterized by reduced complexity of the synapto-dendritic cytoarchitecture, shorter dendrites and fewer branches (Spilker et al., 2016). Simplified dendritic trees and reduced synaptogenesis were also observed in hippocampal primary neurons cultured from these knock out mice relative to cultures from wild type mice. The protein product of Jacob/Nsfm regulates activity-dependent brain-derived neurotrophic factor (Bdnf) transcription. Lower BDNF levels were seen in area CA1 of knock out mice on postnatal day 10. The dysplasia seen in hippocampal neuronal cultures from knock mice could be reversed by BDNF supplementation if administered in early (2-4 days in vitro) but not later (15 days in vitro) in development.

Neuroregluin-2 (Nrg2) contributes to synaptogenesis of the granule cell layer of the hippocampus. In hippocampal slice cultures, inducible microRNA targeting strategies have demonstrated early suppression of Nrg2 (4 days in vitro) but not late suppression (7 days in vitro) reduced synaptogenesis of inhibitory neurons. On the other hand, late treatment impaired the dendritic outgrowth of excitatory synaptic connections.  These effects could be eliminated with overexpression of Nrg2 (Lee et al., 2015).

Many of the gene-regulated processes involved in hippocampal development are also present in the developing cortex. In models of prenatal hypothyroidism, altered expression patterns of many genes involved in neuronal migration and apoptosis are associated with disruptions in hippocampal organization and cytoarchitecture of the cerebral cortex (Pathak et al, 2011; Mohan et al., 2012; Lui et al., 2010). Structural changes in hippocampus and cerebral cortex are dependent on time of exposure (Auso et al., 2003; Berbel et al., 2010; Pathak et al., 2011) and can be reversed with TH supplementation (Mohan et al., 2012; Pathak et al., 2011; Berbel et al., 2010).

Dose-Response Evidence: Dose-response data is lacking for this KER.  Papers that utilize knock-out and mutant models do not provide ‘dose-response’ information for gene-anatomy relationships. Studies in which genes and anatomy were reported following developmental hypothyroidism were single high-dose studies that focused on varying the developmental window of exposure, but not necessarily the dose.

There are no inconsistencies in this KER, but there are some uncertainties. Few studies exist that report both gene expression changes and structural changes in the hippocampus in same study to provide direct causative evidence for this KER. Lacking also is the specific suite of genes that are altered in the hippocampus at particular developmental times that are causal to the structural defects reported. For future research, it is critical to generate data in which the upstream KE is modulated in a ‘dose-response’ manner to better support the causative relationship. Significant data gaps also exist for basic fetal hippocampal development.

Quantitative Understanding of the Linkage

There are no data on the quantitative linkages between gene expression changes and altered hippocampal anatomy.

References

Auso E, Lavado-Autric R, Cuevas E, Del Rey FE, Morreale De Escobar G, Berbel P (2004) A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration. Endocrinology 145:4037-4047.

Berbel P, Navarro D, Ausó E, Varea E, Rodríguez AE, Ballesta JJ, Salinas M, Flores E, Faura CC, de Escobar GM.  Role of late maternal thyroid hormones in cerebral cortex development: an experimental model for human prematurity.  Cereb Cortex. 2010 20(6):1462-75.

Castrén ML, Castrén E. BDNF in fragile X syndrome. Neuropharmacology. 2014 76:729-36.

Favaro R, Valotta M, Ferri AL, Latorre E, Mariani J, Giachino C, Lancini C, Tosetti V, Ottolenghi S, Taylor V, Nicolis SK.  Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh. Nat Neurosci. 2009 12(10):1248-56.

Frotscher M. Role for Reelin in stabilizing cortical architecture. Trends Neurosci. 2010 Sep;33(9):407-14.

Grant SG, O'Dell TJ, Karl KA, Stein PL, Soriano P, Kandel ER. Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice. Science. 1992 Dec 18;258(5090):1903-10.

Holzer G, Roux N, Laudet V. Evolution of ligands, receptors and metabolizing enzymes of thyroid signaling. Mol Cell Endocrinol. 2017 Mar 22. pii: S0303-7207(17)30191-0. doi: 10.1016/j.mce.2017.03.021. [Epub ahead of print]

Khalaf-Nazzal R, Bruel-Jungerman E, Rio JP, Bureau J, Irinopoulou T, Sumia I, Roumegous A, Martin E, Olaso R, Parras C, Cifuentes-Diaz C, Francis F. Organelle and cellular abnormalities associated with hippocampal heterotopia in neonatal doublecortin knockout mice. PLoS One. 2013 Sep 2;8(9):e72622.

Lee KH, Lee H, Yang CH, Ko JS, Park CH, Woo RS, Kim JY, Sun W, Kim JH, Ho WK, Lee SH. Bidirectional Signaling of Neuregulin-2 Mediates Formation of GABAergicSynapses and Maturation of Glutamatergic Synapses in Newborn Granule Cells ofPostnatal Hippocampus. J Neurosci. 2015 Dec 16;35(50):16479-93.

Lee SM, Tole S, Grove E, McMahon AP. A local Wnt-3a signal is required fordevelopment of the mammalian hippocampus. Development. 2000 Feb;127(3):457-67.

Lessmann V, Stroh-Kaffei S, Steinbrecher V, Edelmann E, Brigadski T, Kilb W, Luhmann HJ. The expression mechanism of the residual LTP in the CA1 region of BDNF k.o. mice is insensitive to NO synthase inhibition. Brain Res. 2011 1391:14-23.

Liu D, Teng W, Shan Z, Yu X, Gao Y, Wang S, Fan C, Wang H, Zhang H.The effect of maternal subclinical hypothyroidism during pregnancy on brain development in rat offspring.  Thyroid. 2010 Aug;20(8):909-15.

Mohan V, Sinha RA, Pathak A, Rastogi L, Kumar P, Pal A, Godbole MM (2012) Maternal thyroid hormone deficiency affects the fetal neocorticogenesis by reducing the proliferating pool, rate of neurogenesis and indirect neurogenesis. Exp Neurol 237:477-488.

Pathak A, Sinha RA, Mohan V, Mitra K, Godbole MM. 2011. Maternal thyroid hormone before the onset of fetal thyroid function regulates reelin and downstream signaling cascade affecting neocortical neuronal migration. Cerebral Cortex.11-21.

Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35(8-9):664-72.

Shin JH, Kim YN, Kim IY, Choi DH, Yi SS, Seong JK.  Increased Cell Proliferations and Neurogenesis in the Hippocampal Dentate Gyrus of Ahnak Deficient Mice.  Neurochem Res. 2015 Jul;40(7):1457-62.

Skucas VA, Mathews IB, Yang J, Cheng Q, Treister A, Duffy AM, Verkman AS, Hempstead BL, Wood MA, Binder DK, Scharfman HE. Impairment of select forms ofspatial memory and neurotrophin-dependent synaptic plasticity by deletion ofglial aquaporin-4. J Neurosci. 2011 31(17):6392-7.

Spilker C, Nullmeier S, Grochowska KM, Schumacher A, Butnaru I, Macharadze T, Gomes GM, Yuanxiang P, Bayraktar G, Rodenstein C, Geiseler C, Kolodziej A,Lopez-Rojas J, Montag D, Angenstein F, Bär J, D'Hanis W, Roskoden T, MikhaylovaM, Budinger E, Ohl FW, Stork O, Zenclussen AC, Karpova A, Schwegler H, Kreutz MR.A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impared BDNFSignaling in Dendritogenesis. PLoS Genet. 2016 Mar 15;12(3):e1005907

Strange BA, Witter MP, Lein ES, Moser EI. Functional organization of the hippocampal longitudinal axis. Nat Rev Neurosci. 2014 Oct;15(10):655-69.

Takei Y, Kikkawa YS, Atapour N, Hensch TK, Hirokawa N. Defects in Synaptic Plasticity, Reduced NMDA-Receptor Transport, and Instability of Postsynaptic Density Proteins in Mice Lacking Microtubule-Associated Protein 1A. J Neurosci. 2015 35(47):15539-54.

Relationship: 749: Hippocampal anatomy, Altered leads to Hippocampal Physiology, Altered

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The majority of data in support of this KER is from rodent models. The evolutionary conservation of hippocampal anatomy in mammals, birds, and reptiles (see Hevner, 2016; Streidter, 2015) suggests, with some uncertainty, that this KER is also applicable to multiple species.

Key Event Relationship Description

The hippocampus is a highly integrated and organized communication and information processing network with millions of interconnections among its constitutive neurons (see Andersen et al, 2006). The neuronal spine is the primary site of action for synaptic interface between neurons. Although difficult to measure due to their small size, large number and variable shapes, changes in the frequency and structure of dendritic spines of hippocampal neurons has dramatic effects on synaptic physiology and plasticity (Harris et al., 1992). Anatomical integrity at a more macro-level is also essential for physiological function. The connectivity of axons emanating from one set of cells that synapse on the dendrites of the receiving cells must be intact for effective communication between neurons to be possible. Synaptogenesis is a critical step for neurons to be integrated into neural networks during development. Changes in the placement of cells within the network due to delays or alterations in neuronal migration, the absence of a full proliferation of dendritic arbors and spine upon which synaptic contacts are made, and the lagging of transmission of electrical impulses due to insufficient myelination will independently and cumulatively impair synaptic function.

Evidence Supporting this KER

The weight of evidence supporting the relationship between structural abnormalities in brain induced and altered synaptic function is moderate. There is no doubt that altered structure can lead to altered function. Many examples from knock out models, genetic mutations, prenatal alcohol, nutritional deficits demonstrate a correlative link between altered structure and impaired synaptic function within the hippocampus (Gil-Mohapel et al., 2010; Berman and Hannigan, 2000; Grant et al., 1992; Palop et al., 2010; Ieraci and Herrera, 2007). However, the scientific understanding of the causative and quantitative relationship between the two KEs is incomplete. 

The biological plausibility of alterations in hippocampal structure having an impact on synaptic function and plasticity in brain is strong. Because synaptic transmission in the hippocampus relies on the integrity of contacts and the reliability of electrical and chemical transmission between pre- and post-synaptic neurons, it is well accepted that interference on the anatomical levels will very much impact the functional output on the neurophysiological level (Knowles, 1992; Schultz and Engelhardt, 2014). 

Empirical support for this KER is rated as moderate. Numerous examples of a direct linkage between hippocampal anatomy and hippocampal physiology are evident in knock out or transgenic mouse models (eg., Lessman et al., 2011). Other data is derived from nutritional deficiencies, alcohol exposure, and hippocampal slice culture models (Berman and Hannigan, 2000; Ieraci and Herrera, 2007; Gilbert et al., 2016). Although several examples are evident to demonstrate direct linkages between alterations in hippocampal anatomy and disruptions in hippocampal physiology, there is not a mechanism, anatomical insult, or signature pattern of synaptic impairment that accompanies each of these treatments.

Below are a few examples where direct linkages have been reported and they serve to bear witness to a direct relationship between altered hippocampal anatomy and altered hippocampal physiology.

Fyn is a tyrosine kinase gene involved in synaptic plasticity. Mutations of this gene lead to a lack of expression during development and result in an increase in the number of neurons in the dentate gyrus and CA subfields of the hippocampus. Fyn mutant mice also exhibited impairments in long term potentiation in hippocampal CA1 whereas two other forms of short-term plasticity remained intact (Grant et al., 1992).

Neuroregluin-2 (NRG2) is a growth factor and is highly expressed in the hippocampal dentate where it contributes to synaptogenesis of newborn granule cells. In hippocampal slice cultures, inducible microRNA targeting strategies have demonstrated suppression of NRG2 reduced synaptogenesis of inhibitory neurons and impaired dendritic outgrowth and maturation of glutamatergic synapses. These anatomical alterations were accompanied by reductions in the amplitude of excitatory synaptic currents. The magnitude of the impairment was dependent on the timing of the infection and could be eliminated with overexpression of NRG2 in this in vitro model (Lee et al., 2015).

Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal synapogenesis, dendrite formation, and synaptic plasticity in the developing and adult nervous system (Lessmann et al., 2011; Panja and Bramham, 2014). Jacob is a protein that translocates to the nucleus upon activation of BDNF-dependent pathways and is involved in both neuronal plasticity and neurodegeneration. Hippocampal neurons in culture derived from Jacob/Nsmf knockout mice exhibit shorter neurite length, reduced branching, and a few synaptic contacts. This effect was specific to hippocampal neurons, as cortical cells derived from the same animals did not display these abnormalities. In vivo, these animals exhibited a reduction of dendritic complexity of CA1 neurons, lower number of branches, decreased spine density.  Deficits in synaptic plasticity in the form of LTP accompanied these structural impairments (Spilker et al., 2016).

In Alzheimer’s Disease, amyloid-b protein accumulates in the hippocampus and leads to the formation of amyloid plaques, neuritic dystrophy and aberrant sprouting of axon terminals of the hippocampus. In a developmental germ-line knockout mouse model, high levels of amyloid-b induced aberrant neuronal network excitability and altered innervation of inhibitory interneurons.  Deficits in hippocampal plasticity were seen in the dentate gyrus without change in basal levels of synaptic transmission. In contrast, in area CA1, synaptic transmission was impaired while measures of synaptic plasticity remained intact (Palop et al., 2007).

Other evidence for a direct linkage between hippocampal anatomy and hippocampal physiology comes from the area of adult neurogenesis. The neurogenesis process refers to the acquisition of new neurons on the hippocampus of the adult brain and is associated with enhanced hippocampal synaptic function and learning ability (Deng et al., 2010). Manipulations such as caloric restriction, exercise and hormones can enhance neurogenesis and increase synaptic transmission and plasticity (Kapoor et al., 2015; Trivino-Paredes et al., 2016; Deng et al., 2010). A reciprocal relationship also exists whereby increases in hippocampal neural activity serves to increase neurogenesis (Bruel-Jungerman et al., 2007, Bruel-Jungerman et al., 2009, Kameda et al., 2012). Manipulations that decrease hippocampal neurogenesis including exposure to antidepressants, hormone disruption, stress, and alcohol are associated with impaired synaptic function (Herrera et al., 2003; Saxe et al., 2006; Gilbert et al., 2016; Montero-Pedrazuela et al., 2006; Gil-Mohapel et al., 2006; Sofroniew et al., 2006).

Temporal Evidence: The temporal nature of this KER is developmental (Seed et al., 2005). This has been demonstrated in multiple studies. A few examples detailed above defined critical periods for the manipulation that alters the structural development of the hippocampus that persists to adulthood to disrupt the synaptic physiology measured in the hippocampus in adulthood (Lee et al., 2015; Grant et al., 1992).  A more limited number of ‘rescue’ experiments have been reported.  Lee et al (2015), using an in vitro model, demonstrated impaired synaptogenesis that was dependent on the timing of the infection and could be eliminated with overexpression of NRG2. In Spliker et al (2016), BDNF application rescued the morphological deficits in hippocampal pyramidal neurons from Jacob/Nsmf mice.

Dose-Response Evidence: Dose-response data is lacking for this KER. For future research, it is critical to generate data in which the upstream KE is modulated in a ‘dose-response’ manner to better support the causative relationship.

There are no inconsistencies in this KER, but there are uncertainties. Although several examples are evident to demonstrate direct linkages between alterations in hippocampal anatomy and disruptions in hippocampal physiology, there is not a common cellular mechanism, anatomical insult, or signature pattern of synaptic impairment that defines a common anatomically driven physiological phenotype.  In addition, it is also known that there is an interaction between physiological and anatomical development, where anatomy develops first, and can be ‘reshaped’ by the ongoing maturation of physiological function (e.g., Kutsarova et al., 2017)

Quantitative Understanding of the Linkage

Information does not exist to develop quantitative relationships between the KEs in this KER. Papers that utilize knock-out and mutant models have not provided ‘dose-response’ information for anatomy-physiology relationships.

References

Andersen, P., Morris,R., Amaral,D., Bliss,T., O'Keefe, J. (Editors). The Hippocampus Book. Oxford University Press, 2006.  ISBN: 9780195100273

Berman RF, Hannigan JH. Effects of prenatal alcohol exposure on the hippocampus: spatial behavior, electrophysiology, and neuroanatomy. Hippocampus. 2000;10(1):94-110.

Bruel-Jungerman E, Davis S, Laroche S (2007) Brain plasticity mechanisms and memory: a party of four. Neuroscientist 13:492-505.

Bruel-Jungerman E, Veyrac A, Dufour F, Horwood J, Laroche S, Davis S (2009) Inhibition of PI3K-Akt signaling blocks exercise-mediated enhancement of adult neurogenesis and synaptic plasticity in the dentate gyrus. PLoS One 4:e7901.

Deng W, Aimone JB, Gage FH (2010) New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory Nat Rev Neurosci 11:339-350.

Gil-Mohapel J, Boehme F, Kainer L, Christie BR. Hippocampal cell loss and neurogenesis after fetal alcohol exposure: insights from different rodent models.Brain Res Rev. 2010 Sep 24;64(2):283-303.

Gilbert ME, Goodman JH, Gomez J, Johnstone AF, Ramos RL. Adult hippocampal neurogenesis is impaired by transient and moderate developmental thyroid hormone disruption. Neurotoxicology. 2016 Dec 31;59:9-21.

Grant SG, O'Dell TJ, Karl KA, Stein PL, Soriano P, Kandel ER. Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice. Science. 1992 Dec 18;258(5090):1903-10.

Harris KM, Teyler TJ. Developmental onset of long-term potentiation in area CA1 of the rat hippocampus. J Physiol. 1984 Jan;346:27-48.

Herrera DG, Yague AG, Johnsen-Soriano S, Bosch-Morell F, Collado-Morente L, Muriach M, Romero FJ, Garcia-Verdugo JM (2003) Selective impairment of hippocampal neurogenesis by chronic alcoholism: protective effects of an antioxidant. Proc Natl Acad Sci U S A 100:7919-7924.

Hevner RF.  Evolution of the mammalian dentate gyrus. J Comp Neurol. 2016 524(3):578-94.

Ieraci A, Herrera DG. Single alcohol exposure in early life damages hippocampal stem/progenitor cells and reduces adult neurogenesis. Neurobiol Dis. 2007 Jun;26(3):597-605.

Kameda M, Taylor CJ, Walker TL, Black DM, Abraham WC, Bartlett PF (2012) Activation of latent precursors in the hippocampus is dependent on long-term potentiation. Transl Psychiatry 2:e72.

Kapoor R, Fanibunda SE, Desouza LA, Guha SK, Vaidya VA (2015) Perspectives on thyroid hormone action in adult neurogenesis. J Neurochem 133:599-616.

Knowles WD, Normal anatomy and neurophysiology of the hippocampal formation. J Clin Neurophysiol. 1992 Apr;9(2):252-63.

Kutsarova E, Munz M, Ruthazer ES.  Rules for Shaping Neural Connections in the Developing Brain.  Front Neural Circuits. 2017. 10:111. doi: 10.3389/fncir.2016.00111.

Lee KH, Lee H, Yang CH, Ko JS, Park CH, Woo RS, Kim JY, Sun W, Kim JH, Ho WK, Lee SH. Bidirectional Signaling of Neuregulin-2 Mediates Formation of GABAergicSynapses and Maturation of Glutamatergic Synapses in Newborn Granule Cells ofPostnatal Hippocampus. J Neurosci. 2015 Dec 16;35(50):16479-93.

Lessmann V, Stroh-Kaffei S, Steinbrecher V, Edelmann E, Brigadski T, Kilb W, Luhmann HJ. The expression mechanism of the residual LTP in the CA1 region ofBDNF k.o. mice is insensitive to NO synthase inhibition. Brain Res. 2011. 1391:14-23.

Montero-Pedrazuela A, Venero C, Lavado-Autric R, Fernandez-Lamo I, Garcia-Verdugo JM, Bernal J, Guadano-Ferraz A (2006) Modulation of adult hippocampal neurogenesis by thyroid hormones: implications in depressive-like behavior. Mol Psychiatry 11:361-371.

Palop JJ, Chin J, Roberson ED, Wang J, Thwin MT, Bien-Ly N, Yoo J, Ho KO, Yu GQ, Kreitzer A, Finkbeiner S, Noebels JL, Mucke L. Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease. Neuron. 2007 Sep 6;55(5):697-711.

Panja, D. and C. R. Bramham (2014). "BDNF mechanisms in late LTP formation: A synthesis and breakdown." Neuropharmacology 76 Pt C: 664-676.Schultz C, Engelhardt M. Anatomy of the hippocampal formation. Front Neurol Neurosci. 2014. 34:6-17

Saxe MD, Battaglia F, Wang JW, Malleret G, David DJ, Monckton JE, Garcia AD,

Sofroniew MV, Kandel ER, Santarelli L, Hen R, Drew MR. Ablation of hippocampal neurogenesis impairs contextual fear conditioning and synaptic plasticity in the dentate gyrus. Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17501-6.

Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72.

Schultz C, Engelhardt M.  Anatomy of the hippocampal formation.  Front Neurol Neurosci. 2014. 4:6-17.

Sofroniew et al., 2006

Spilker C, Nullmeier S, Grochowska KM, Schumacher A, Butnaru I, Macharadze T, Gomes GM, Yuanxiang P, Bayraktar G, Rodenstein C, Geiseler C, Kolodziej A, Lopez-Rojas J, Montag D, Angenstein F, Bär J, D'Hanis W, Roskoden T, MikhaylovaM, Budinger E, Ohl FW, Stork O, Zenclussen AC, Karpova A, Schwegler H, Kreutz MR.  A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNFSignaling in Dendritogenesis. PLoS Genet. 2016 Mar 15;12(3):e1005907

Striedter GF.  Evolution of the hippocampus in reptiles and birds.  J Comp Neurol. 2016 Feb 15;524(3):496-517

Triviño-Paredes J, Patten AR, Gil-Mohapel J, Christie BR. The effects of hormones and physical exercise on hippocampal structural plasticity. Front Neuroendocrinol. 2016. 41:23-43.

Relationship: 748: Hippocampal Physiology, Altered leads to Cognitive Function, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The majority of data in support of this KER is from rodent models. The evolutionary conservation of the role of the hippocampus in spatial cognitive functions suggests, with some uncertainty, that this KER is also applicable to other mammalian species.

Key Event Relationship Description

It is a well-accepted assertion that hippocampal synaptic integrity and plasticity are essential for spatial information processing in animals and spatial and episodic memory in humans (Burgess, 2002; Martin et al., 2000; Sweatt, 2016). A large number of studies with a variety of techniques and approaches have linked hippocampal functional deficits to decreased spatial ability, context learning, and fear learning. Study of human disease states and conditions where hippocampal function is impaired (i.e., brain trauma, Alzheimer’s disease, temporal lobe epilepsy, Down’s Syndrome), and imaging studies of hippocampal activation during memory challenge, makes itirrefutable that the hippocampus is essential for specific types of cognition abilities. Decades of animal research has reinforced this assertion.

There are many forms of synaptic plasticity and numerous ways in which physiological function of neural circuits can be assessed. Similarly, there are many forms of learning and memory and multiple tasks and specifics associated with these tasks that vary from laboratory to laboratory. An emerging field of computational cognitive neuroscience lies at the intersection of   computational neuroscience, machine learning and neural network theory. These computational and theoretical frameworks support the participation of the hippocampal synaptic transmission and plasticity in learning and memory in animals and humans (for review see: Ashby and Helie, 2012).

Evidence Supporting this KER

The weight of evidence for proper hippocampal function and episodic memory in humans and the animal analogue, spatial and fear-based context learning, is strong. Seminal studies over the past 60 years firmly established the cellular basis of behavior with synaptic plasticity (LTP and LTD). And recent work has provided details on the local hippocampal circuitry needed for memory formation and behavioral change (Sweatt, 2016). In humans, virtual reality experiments in large-scale spatial contexts demonstrate the convergence of spatial memory performance in normal patients with fMRI of the hippocampus clearly demonstrating the essentiality of hippocampal function to spatial learning (Burgess, 2002). This assertion is consistent with a wealth of animal data on hippocampal learning and memory. In rodent models, functional impairment of the hippocampus assessed using electrophysiological techniques is correlated with deficits in spatial memory typically assessed using mazes, and memory for context often assessed in fear-based learning paradigms (O’Keefe and Nadel, 1978; Clark et al., 2000; Squire, 2004; Eichenbaum, 2000; Panjo and Bramham, 2014).

The biological plausibility of the KER is rated as strong. It is well accepted that the normal hippocampal function is critical for the acquisition and memory of context and spatially mediated tasks in rodents and humans (Sweatt, 2016).

Empirical support for this KER is strong. The requisite of hippocampal integrity to optimal visuo-spatial context learning (i.e., episodic memory) in humans and spatial learning in rodents is well documented. In vivo recording in conscious behaving animals has demonstrated activity-dependent neural changes taking place in the hippocampus during spatial learning (Gruart and Delgado-Garcia, 2007). Impairments in hippocampal function induced by drugs, chemicals, lesions, mutant or knock out models that cause changes in synaptic transmission, plasticity, and hippocampal network activity, are coincident with deficits in spatial and context-based fear learning (O’Keefe and Nadel, 1978; Bannerman et al., 2014; Lynch, 2004; Verret et al., 2012). Similarly, treatments found to enhance or facilitate hippocampal synaptic transmission and plasticity are associated with improved learning and memory (Deng et al., 2010; Novkovic et al., 2015; Andrade et al., 2015; Trivino-Paredes et al., 2016).  For example, n-methyl-d-aspartate (NMDA)-mediated glutamatergic synaptic transmission is essential for the induction of hippocampal synaptic plasticity in the form of LTP. Blockade of this form of plasticity by selective NMDA-receptors blockers impairs LTP and hippocampal tests of learning and memory (reviewed in Sweatt, 2016). Perturbation of hippocampal plasticity and impaired spatial learning have been reported in adult offspring following prenatal ethanol exposure (An and Zhang, 2015).  The fyn mutant mouse (fyn is a tyrosine kinase pathway) displays impairments in hippocampal synaptic transmission and plasticity, as well asspatial learning deficits (Grant et al., 1992). Brain-derived neurotrophic factor (BDNF) knock out animals exhibit synaptic plasticity deficits and learning impairments (Aarse et al., 2016; Panja and Bramham, 2014). In the Jacob/Nfsm model which also exhibits pronounced alterations in BDNF-mediated signaling, hippocampal synaptic transmission and plasticity impairments were accompanied by deficits in contextual fear conditioning and novel location recognition tasks (Spilker et al., 2016).  Finally, in rodent models of developmental TH insufficiency, impairments in hippocampal synaptic transmission and plasticity are coincident with deficits in learning tasks that require the hippocampus (Opazo et al., 2008; Gilbert and Sui, 2006, Gilbert, 2011, Gilbert et al., 2016).

In humans, hippocampal physiology assessed using neuroimaging reveals activation of hippocampus upon engagement in spatial learning and episodic memory providing a direct linkage of these two specific KEs (Burgess, 2002). In fMRI studies of congenitally hypothyroid children, or children born to women with altered thyroid function during pregnancy, changes in hippocampal activity patterns during memory encoding and retention were observed and associated with memory impairments (Wheeler et al., 2012; 2015; Willoughby et al., 2013; 2014).

Temporal Evidence:  The temporal nature of this KER is developmental (Seed et al., 2005). This has been demonstrated in multiple studies.  It is well-recognized that there are critical developmental windows for disruption of the functional development of the hippocampus and the integrity of this structure is essential for later development of spatial ability, context learning, and fear learning. A wealth of studies have shown correlation between hippocampal LTP and spatial learning performance, as well as the role of glutamatergic synaptic transmission and BDNF-mediated signaling pathways in these processes (Bramham, 2007; Andero et al., 2014; Morris et al., 1986; Sweatt, 2016; Migaud et al., 1998). Although studies on reversibility are rare, deficits in hippocampal synaptic transmission and plasticity in slices from BDNF knockout animals can be rescued with recombinant BDNF (Patterson et al., 1996).

Dose-Response Evidence: Limited dose-response information is available.  Studies have investigated dose-dependency of both electrophysiological and behavioral impairments in animals suffering from developmental TH insufficiency (e.g., Gilbert and Sui, 2006; Gilbert, 2011; Gilbert et al., 2016).

There are no inconsistencies in this KER, but there are some uncertainties. It is a widely-held assertion that synaptic transmission and plasticity in the hippocampus underlie spatial learning (Martin et al., 2000; Gruart and Delgado-Garcia, 2007; Bramham, 2007). However, the causative relationship of which specific alterations in synaptic function are associated with specific cognitive deficits is difficult to ascertain given the many forms of learning and memory, and the complexity of synaptic interactions in even the simplest brain circuit.

Quantitative Understanding of the Linkage

Information does not exist to develop quantitative relationships between the KEs in this KER.

References

Aarse J, Herlitze S, Manahan-Vaughan D. The requirement of BDNF for hippocampal synaptic plasticity is experience-dependent. Hippocampus. 2016 Jun;26(6):739-51.

An L, Zhang T. Prenatal ethanol exposure impairs spatial cognition and synaptic plasticity in female rats. Alcohol. 2015 Sep;49(6):581-8.

Andero R, Choi DC, Ressler KJ. BDNF-TrkB receptor regulation of distributed adult neural plasticity, memory formation, and psychiatric disorders. Prog Mol Biol Transl Sci. 2014. 122:169-92.

Andrade-Talavera Y, Benito I, Casañas JJ, Rodríguez-Moreno A, Montesinos ML.  Rapamycin restores BDNF-LTP and the persistence of long-term memory in a model of Down's syndrome. Neurobiol Dis. 2015. 82:516-25

Ashby FG, Helie S. The Neurodynamics of Cognition: A Tutorial on Computational Cognitive Neuroscience. J Math Psychol. 2011 Aug 1;55(4):273-289.

Bannerman DM, Sprengel R, Sanderson DJ, McHugh SB, Rawlins JNP, Monyer H, Seeburg PH (2014) Hippocampal synaptic plasticity, spatial memory and anxiety. Nat Rev Neurosci 15:181-192.

Bramham CR. Control of synaptic consolidation in the dentate gyrus: mechanisms, functions, and therapeutic implications. Prog Brain Res. 2007. 163:453-71.

Burgess N (2002) The hippocampus, space, and viewpoints in episodic memory. Q J Exp Psychol A 55:1057-1080. Clark RE, Zola SM, Squire LR. Impaired recognition memory in rats after damage to the hippocampus. J Neurosci. 2000 Dec 1;20(23):8853-60.

Deng W, Aimone JB, Gage FH (2010) New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory Nat Rev Neurosci 11:339-350.

Gilbert ME (2011) Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicol Sci 124:432-445.

Gilbert ME, Sanchez-Huerta K, Wood C (2016) Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787.

Gilbert ME, Sui L (2006) Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22.

Grant SG, O'Dell TJ, Karl KA, Stein PL, Soriano P, Kandel ER. Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice. Science. 1992 Dec 18;258(5090):1903-10.

Gruart A, Delgado-García JM. Activity-dependent changes of the hippocampal CA3-CA1 synapse during the acquisition of associative learning in conscious mice. Genes Brain Behav. 2007 Jun;6 Suppl 1:24-31.

Lynch, M.A. (2004). Long-Term Potentiation and Memory. Physiological Reviews. 84:87-136.

Martin SJ, Grimwood PD, Morris RG. Synaptic plasticity and memory: an evaluation of the hypothesis. Annu Rev Neurosci. 2000. 23:649-711.

Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O'Dell TJ, Grant SG. Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature. 1998 Dec 3;396(6710):433-9.

Morris RG, Frey U. Hippocampal synaptic plasticity: role in spatial learning or the automaticrecording of attended experience? Philos Trans R Soc Lond B Biol Sci. 1997 Oct 29;352(1360):1489-503. Review

Novkovic T, Mittmann T, Manahan-Vaughan D. BDNF contributes to the facilitation of hippocampal synaptic plasticity and learning enabled by environmental enrichment. Hippocampus. 2015 Jan;25(1):1-15.

O’Keefe, J. and Nadel, L. (1978). The Hippocampus as a Cognitive Map. Oxford: Oxford University Press.

Opazo MC, Gianini A, Pancetti F, Azkcona G, Alarcón L, Lizana R, Noches V, Gonzalez PA, Marassi MP, Mora S, Rosenthal D, Eugenin E, Naranjo D, Bueno SM, Kalergis AM, Riedel CA (2008), Maternal hypothyroxinemia impairs spatial learning and synaptic nature and function in the offspring. Endocrinology 149:5097-5106

Panja, D. and C. R. Bramham (2014). "BDNF mechanisms in late LTP formation: A synthesis and breakdown." Neuropharmacology 76 Pt C: 664-676.

Schultz C, Engelhardt M, Anatomy of the hippocampal formation. Front Neurol Neurosci. 2014. 34:6-17

Patterson SL, Abel T, Deuel TA, Martin KC, Rose JC, Kandel ER. Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron. 1996 Jun;16(6):1137-45.

Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72.

Spilker C, Nullmeier S, Grochowska KM, Schumacher A, Butnaru I, Macharadze T, Gomes GM, Yuanxiang P, Bayraktar G, Rodenstein C, Geiseler C, Kolodziej A, Lopez-Rojas J, Montag D, Angenstein F, Bär J, D'Hanis W, Roskoden T, Mikhaylova M, Budinger E, Ohl FW, Stork O, Zenclussen AC, Karpova A, Schwegler H, Kreutz MR. A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis. PLoS Genet. 2016 Mar 15;12(3):e1005907Squire LR 2004. Memory systems of the brain: A brief history and current perspective. Neurobiology of Learning and Memory, 82: 171-177

Sweatt JD. Neural plasticity and behavior - sixty years of conceptual advances. J Neurochem. 2016 Oct;139 Suppl 2:179-199. doi: 10.1111/jnc.13580. Review. PubMed PMID: 26875778.

Triviño-Paredes J, Patten AR, Gil-Mohapel J, Christie BR. The effects of hormones and physical exercise on hippocampal structural plasticity. Front Neuroendocrinol. 2016. 41:23-43.

Verret L, Mann EO, Hang GB, Barth AM, Cobos I, Ho K, Devidze N, Masliah E, Kreitzer AC, Mody I, Mucke L, Palop JJ. Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell. 2012Apr 27;149(3):708-21.

Wheeler SM, McAndrews MP, Sheard ED, Rovet J (2012) Visuospatial associative memory and hippocampal functioning in congenital hypothyroidism. J Int Neuropsychol Soc 18:49-56.

Wheeler SM, McLelland VC, Sheard E, McAndrews MP, Rovet JF (2015) Hippocampal Functioning and Verbal Associative Memory in Adolescents with Congenital Hypothyroidism. Front Endocrinol (Lausanne) 6:163.

Willoughby KA, McAndrews MP, Rovet JF (2014) Effects of maternal hypothyroidism on offspring hippocampus and memory. Thyroid 24:576-584.

Willoughby KA, McAndrews MP, Rovet J (2013) Effects of early thyroid hormone deficiency on children's autobiographical memory performance. J Int Neuropsychol Soc 19:419-429.

Relationship: 366: Thyroperoxidase, Inhibition leads to T4 in serum, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic: Use of TPO inhibitors as anti-hyperthyroidism drugs in humans and pets (Emiliano et al., 2010; Trepanier, 2006) and effects of these drugs on serum TH concentrations in rats (US EPA, 2005), amphibian, fish and avian species (Coady et al., 2010; Grommen et al., 2011; Nelson et al., 2016; Rosebrough et al., 2006; Stinckens et al.; 2020; Tietge et al., 2012), strongly supports a causative linkage between inhibition of TPO and decreased serum T4 across species. Therefore, this KER is plausibly applicable across vertebrate species. Therefore, this KER is plausibly applicable across vertebrates.

Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred thyroid hormones during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). Therefore, iIt is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH system disruptors.

Sex: The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.

Key Event Relationship Description

Thyroperoxidase (TPO) is the enzyme that catalyzes iodine organification of thyroglobulin to produce thyroglobulin (Tg)-bound T3 and T4 in the lumen of thyroid follicles. Tg-bound THs are endocytosed across the apical lumen-follicular cell membrane, undergo thyroglobulin proteolysis, followed by hormone section into the blood stream (see Taurog, 2005 for review). This indirect KER describes the relationship of TPO inhibition to reduced circulating levels of thyroid hormone (TH) in the serum.

Evidence Supporting this KER

The weight of evidence linking thyroperoxidase inhibition to reductions in circulating serum TH is strong. Many studies support this basic linkage. There is no inconsistent data.

It is a well-accepted fact that inhibition of the only enzyme capable of synthesizing THs, TPO, results in subsequent decrease in serum TH concentrations.  A large amount of evidence from clinical and animal studies clearly support the commonly accepted dogma that inhibition of TPO leads to decreased serum THs. 

The majority of research in support of this KER involves exposure to known TPO inhibitors and measurement of serum hormones. There are many in vivo studies that link decreases in serum TH concentrations with exposure to xenobiotics that inhibit thyroperoxidase (TPO) in mammals (Brucker-Davis, 1998; Hurley, 1998; Boas et al., 2006; Crofton, 2008; Kohrle, 2008; Pearce and Braverman, 2009; Murk et al., 2013). 

While these studies support the connection between exposure to a known TPO inhibitor and decreased TH, many of these studies do not empirically measure TPO inhibition or decreased TH synthesis. Thus, many studies support the indirect linkage between TPO inhibition (for chemicals identified as TPO inhibitors in in vivo or ex vivo studies) and decreased TH, with the well accepted theory that these proceed via decreased TH synthesis. That exposure to TPO inhibitors leads to decreased serum TH concentrations, via decreased TH synthesis is strongly supported by decades of mechanistic research in a variety of species.

This indirect relationship is also evidenced by the use of clinically-relevant anti-hyperthyroidism drugs, MMI and PTU (Laurberg & Anderson, 2014; Sundaresh et al., 2013). These drugs are both recognized TPO inhibitors and are part of a standard drug-based regimen of care for clinically hyperthyroid patients including those with Grave's disease. Serum THs are measured as the bioindicator of successful treatment with anti-hyperthyroidism drugs; the actual decrease in TH synthesis in the thyroid gland is implied in the efficacious use of these drugs (Trepanier, 2006).

In rats, MMI and PTU are often used as control chemicals to decrease serum THs to study biological phenomena related to disruption of TH homeostasis (many examples, including Zoeller and Crofton, 2005; Morreale de Escobar et al, 2004; Schwartz et al., 1997; Herwig et al., 2014; Wu et al., 2013; Pathak et al., 2011). Further, MMI is recommended as a positive control for use in the Amphibian Metamorphosis (Frog) Assay within Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program (US EPA, 2009; Coady et al., 2010), an assay used to evaluate the potential for chemicals to disrupt TH homeostasis. PTU has been suggested as positive control chemical in the guidance for the Comparative Developmental Thyroid Assay (US EPA, 2005), a non-guideline assay used to evaluate the potential for chemicals to disrupt TH homeostasis during gestation and early neonatal development.

Additionally, evidence is available from studies investigating responses to TPO inhibitors in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.

Thus, an indirect key event relationship between TPO inhibition and decreased serum THs is strongly supported by a large database of clinical medicine and investigative research with whole animals (with a great deal of supporting evidence in rats and frogs).

Temporal Evidence: In mammals, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005). The qualitative impact of TPO inhibition on serum hormones is similar across all ages in mammals. The temporal nature of the impact on serum THs by TPO inhibitors in developmental exposure studies is evidenced by the duration of exposure and developmental age (Goldey et al., 1995; Ahmed et al., 2010; Tietge et al., 2010), as well as recovery after cessation of exposure (Cooke et al., 1993; Goldey et al., 1995; Sawin et al., 1998; Axelstad et al., 2008; Shibutani et al., 2009; Lasley and Gilbert, 2011). The temporal relationship between TPO inhibitor exposure duration and serum hormone decreases in adult organisms has been widely demonstrated (e.g., Hood et al., 1999; Mannisto et al., 1979). In addition, MMI and PTU induced decreases in serum T4 are alleviated by TH      replacement in both fetal and postnatal age rats (Calvo et al., 1990; Sack et al., 1995; Goldey and Crofton, 1998). Computational modeling of the thyroid also provides evidence for the indirect temporal relationship between these two KEs (e.g., Degon et al., 2008; Fisher et a., 2013).

In oviparous fish such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal thyroid hormones transferred to the eggs. Embryonic thyroid hormone synthesis is activated later during embryo-larval development. (See Domain of applicability)

Dose-Response Evidence: Empirical data is available from enough studies in animals treated with TPO inhibitors during development to make it readily accepted dogma that a dose-response relationship exists between TPO inhibition and serum TH concentrations.  Again, these studies do not empirically measure TPO inhibition or decreased TH synthesis, but rely on the strong support of decades of mechanistic research in a variety of species of the causative relationship between these KEs.  Examples of dose-responsive changes in TH concentrations following developmental exposure to TPO inhibitors include studies a variety of species, including: rodents (Blake and Henning, 1985; Goldey et al., 1995; Sawin et al., 1998); frogs (Tietge et al., 2013); fish tissue levels (Elsalini and Rohr, 2003.); and, chickens (Wishe et al., 1979). Computational modeling of the thyroid also provides evidence for the indirect dose-response relationship between these two KEs (e.g., Leonard et al., 2016; Fisher et a., 2013).

There are no inconsistencies in this KER, but there are some uncertainties. The predominant uncertainty regarding the indirect key event relationship between inhibition of TPO activity and decreased serum T4 is the quantitative nature of this relationship, i.e., to what degree must TPO be inhibited in order to decrease serum T4 by a certain magnitude. Many animal (rat) studies typically employ relatively high exposures of TPO-inhibiting chemicals that result in hypothyroidism (severe decrements in T4 and T3). Thus, a dose-response relationship between TPO inhibition and decreased serum T4 is not typically defined. However, there are numerous publications demonstrating clear dose- and duration- dependent relationships between TPO inhibitors dose and reduced serum T3 and T4 in rodent models (see for example: Cooper et al., 1983; Hood et al., 1999; Goldey et al., 2005; Gilbert, 2011). The relationship between maternal and fetal levels of hormone following chemically-induced TPO inhibiton has not been well characterized and may differ based on kinetics. Reductions in serum TH in the fetus, in rats and humans is derived from a chemical's effect on the maternal thyroid gland as well as the fetal thyroid gland.

Quantitative Understanding of the Linkage

Hassan et al. (2017, 2020) and Handa et al. (2021) worked out a quantitative model to predict serum T4 levels based on TPO inhibition in rats.

The indirect linkage between exposure to known TPO inhibitors and decreased serum TH has not been defined quantitatively. The two key event relationships that mediate this relationship (TPO inhibition leading to decreased TH synthesis, and decreased TH synthesis leading to decreased serum TH) have been incorporated into some quantitative models. A quantitative biologically-based dose-response model for iodine deficiency in the rat includes relationships between thyroidal T4 synthesis and serum T4 concentrations in developing rats Fisher et al. (2013). Ekerot et al. (2012) modeled TPO, T3, T4 and TSH in dogs and humans based on exposure to myeloperoxidase inhibitors that also inhibit TPO and was recently adapted for rat (Leonard et al., 2016). While the original model predicted serum TH and TSH levels as a function of oral dose, it was not used to explicitly predict the relationship between serum hormones and TPO inhibition, or TH synthesis. Leonard et al. (2016) recently incorporated TPO inhibition into the model. Degon et al (2008) developed a human thyroid model that includes TPO but does not make quantitative prediction of organification changes due to inhibition of the TPO enzyme.

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Relationship: 1387: T4 in serum, Decreased leads to Hippocampal gene expression, Altered

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Most of the data available has come from rodent models.

Key Event Relationship Description

Many of the physiological effects of thyroid hormones (THs) are mediated through regulation of gene expression by zinc finger nuclear receptor proteins that are encoded by thyroid hormone genes alpha (Thra) and beta (Thrb). It is widely accepted that TH regulates gene transcription during brain development (Bernal, 2007; Anderson et al., 2003). The sole source of TH to the brain is from the circulating levels of the prohormone, thyroxine (T4). Once taken up from the serum to reach the brain, T4 is converted to triiodothyronine (T3) which binds to TH nuclear receptors (TRα and TRβ). On binding, and in the presence of regulatory cofactors, transcription of certain genes is either up- or down-regulated (Oppenheimer, 1983). However, only a small number of genes have been shown to be directly influenced by TH receptor binding, and of these, most are transcription factors (Quignodon et al., 2008; Thompson and Potter, 2000; Horn and Heuer, 2010). In this manner, THs do influence a wide variety of genes.

Evidence Supporting this KER

The weight of evidence for this indirect relationship is strong. It is well established that serum TH is the primary source of brain T4 from which neuronal T3, the active hormone, is locally generated and presented to the receptors in the nucleus of neurons to control gene transcription.

The biological plausibility of this KER is rated as strong. This is consistent with the known biology of the relationship between serum TH concentrations and brain TH concentrations, and the known action of TH to mediate gene transcription in brain and many other tissues.

The empirical support for this KER is strong. A global transcriptome analysis of primary cerebrocoritical cells was recently published in which a number of genes regulated by T3 were identified (Gil-Ibanez et al., 2015). Although the bulk of literature in which serum TH reductions have been associated with gene expression changes in the brain have been focused on the cortex, several reports in hippocampus are available. Genes directly regulated by TH include the transcription factors Hr and Klf9 (Bteb) (Thompson and Potter, 2000; Cayrou et al., 2002; Denver and Williamson, 2009). The expression of a number of genes modulated by TH are expressed in the hippocampus. Many of genes that regulate processes involved in hippocampal development are also present in the developing cortex. Thus, Table 1 lists TH responsive genes whose expression in either area are altered by TH reduction. This list is not meant to be exhaustive, just exemplary.

Temporal Evidence:  The temporal nature of this KER is developmental (Seed et al., 2005). It is a well-recognized fact that there are critical developmental windows for disruption of the serum THs that result in altered gene expression in the developing brain, including the hippocampus. Rescue experiments for this endpoint of gene expression in hypothyroid models are limited. In one, a combination of T3 and T4 treatment delivered on the last day of a 3-day gestational MMI hypothyroxinemia mouse model altered the pattern of gene expression observed in the cortex of offspring relative to euthyroid controls and MMI alone (Dong et al., 2015).

Dose-Response Evidence: There are a limited number of studies that have reported on the dose-dependent nature of the correlation between serum THs and hippocampal gene expression (Bastian et al., 2012; 2014; Royland et al., 2008).

There are no inconsistencies in this KER, but there are some uncertainties. It is widely accepted that changes in serum THs will result in alterations in hippocampal gene expression. Several different animal models have been used to manipulate serum TH concentrations that also measure gene expression changes. Varying windows of exposure to TH disruption and developmental sample time and region examined have also varied across studies. However, dose-response data is lacking. Most investigations of hippocampal gene expression have employed treatments that induce severe hormone reductions induced by PTU or MMI, or by thyroidectomy. In addition, few reports have studied the genes in the hippocampus, the cortex being more accessible in young animals. Finally, when the hippocampus is the target, different genes at different ages are reported, making it difficult to compare findings.

Quantitative Understanding of the Linkage

There are no quantitative models that predict the degree of serum TH reduction that is required to alter hippocampal gene transcription. Most investigations for hippocampus have been conducted in the neonate after severe hormone reductions. Only four publications have reported dose-dependent effects on gene expression in at less than maximal hormone depletion (Bastian et al., 2012; 2014; O'Shaughnessy et al., 2018; Royland et al., 2008). O'Shaughnessy et al (2018) demostrates dose-response relationships between cortical T4 and T3 concentrations and changes in a variety of neocortical genes (e.g., Parv, Col11a2, Hr, Ngf) that were "statistically significant at doses that decreased brain t4 and/or T3". There was no quantitation of this relationship reported.

In addition, there is very little known about whether compensatory processes are available in the developing hippocampus that may modulate the impact of serum levels on hippocampal gene transcription. These available data suggest that a 40-50% decrement in serum T4 in the pup, is sufficient to observe changes in hippocampal gene expression.  This is similar to finding for loss of hearing function in rats following postnatal chemical-induced hypothyroxinemia (Crofton, 2004).

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Dowling AL, Zoeller RT. Thyroid hormone of maternal origin regulates the expression of RC3/neurogranin mRNA in the fetal rat brain. Brain research Molecular brain research. 2000. 82:126-132.

Gil-Ibanez P, Garcia-Garcia F, Dopazo J, Bernal J, Morte B. 2015. Global Transcriptome Analysis of Primary Cerebrocortical Cells: Identification of Genes Regulated by Triiodothyronine in Specific Cell Types. Cerebral cortex. Nov 2.

Horn S. and Heuer H. Thyroid hormone action during brain development: more questions than answers. Mol Cell Endocrinol. 2010 Feb 5;315(1-2):19-26.

Ibarrola N, Rodriguez-Pena A (1997) Hypothyroidism coordinately and transiently affects myelin protein gene expression in most rat brain regions during postnatal development. Brain Res 752:285-293.

Iñiguez MA, Rodriguez-Peña A, Ibarrola N, Aguilera M, Muñoz A, Bernal J. Thyroid hormone regulation of RC3, a brain-specific gene encoding a protein kinase-C substrate. Endocrinology. 1993 Aug;133(2):467-73.

Mohan V, Sinha RA, Pathak A, Rastogi L, Kumar P, Pal A, Godbole MM (2012) Maternal thyroid hormone deficiency affects the fetal neocorticogenesis by reducing the proliferating pool, rate of neurogenesis and indirect neurogenesis. Exp Neurol 237:477-488.

Morte B, Diez D, Auso E, Belinchon MM, Gil-Ibanez P, Grijota-Martinez C, Navarro D, de Escobar GM, Berbel P, Bernal J) Thyroid hormone regulation of gene expression in the developing rat fetal cerebral cortex: prominent role of the Ca2+/calmodulin-dependent protein kinase IV pathway. Endocrinology 2010a. 151:810-820.

Morte B, Ceballos A, Diez D, Grijota-Martinez C, Dumitrescu AM, Di Cosmo C, Galton VA, Refetoff S, Bernal J. Thyroid hormone-regulated mouse cerebral cortex genes are differentially dependent on the source of the hormone: a study in monocarboxylate transporter-8- and deiodinase-2-deficient mice. Endocrinology. 2010b. 151:2381-2387.

Navarro D, Alvarado M, Morte B, Berbel D, Sesma J, Pacheco P, Morreale de Escobar G, Bernal J, Berbel P.  Late Maternal Hypothyroidism Alters the Expression of Camk4 in Neocortical Subplate Neurons: A Comparison with Nurr1 Labeling. Cereb Cortex 2014. 10:2694-2706.

Oppenheimer J. The nuclear-receptor-triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism, and biological action, In: Samuels HH, eds: Molecular Basis of Thyroid Hormone Action. Academic Press: New York. 1983: 1-34.

Pathak A, Sinha RA, Mohan V, Mitra K, Godbole MM (2011). Maternal thyroid hormone before the onset of fetal thyroid function regulates reelin and downstream signaling cascade affecting neocortical neuronal migration. Cereb Cortex 21:11-21.

Quignodon L, et al. Thyroid hormone signaling is highly heterogeneous during pre- and postnatal brain development. J Mol Endocrinol 2004, 33(2), 467-476.

Royland JE, Parker JS, Gilbert ME. A genomic analysis of subclinical hypothyroidism in hippocampus and neocortex of the developing rat brain. J Neuroendocrinol. 2008 Dec;20(12):1319-38.

Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72.

Shiraki A, Saito F, Akane H, Takeyoshi M, Imatanaka N, Itahashi M, Yoshida T, Shibutani M (2014) Expression alterations of genes on both neuronal and glial development in rats after developmental exposure to 6-propyl-2-thiouracil. Toxicol Lett 228:225-234.

Thompson CC, Potter GB. Thyroid hormone action in neural development. Cereb Cortex 2000, 10(10), 939-945.

Relationship: 1388: T4 in serum, Decreased leads to Hippocampal anatomy, Altered

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Most of the available data has come from rodent models. Human clinincal studies have documented changes in hippocampal volume in children with congenital hypothyroidism (Wheeler et al., 2011).

Key Event Relationship Description

The vast majority of brain thyroxine (T4) is from the serum. Once taken up from the serum, T4 is converted to triiodothyronine (T3) which binds to the nuclear receptors (TRα and TRβ) to control thyroid-mediated gene expression (Oppenheimer, 1983). It is well established that TH regulates genes critical for brain development (Bernal, 2007; Anderson et al., 2003). As such, the structural development of the hippocampus is modulated by TR-mediated gene transcription, and alterations in serum TH can adversely impact hippocampal neuroanatomy.

Evidence Supporting this KER

The weight of evidence for this indirect relationship is strong. There is a vast amount of literature that supports this KER in multiple species.

The biological plausibility of this KER is rated as strong. The relationship is consistent with the known biology of the regulation of serum TH concentrations, brain TH concentrations, and the known action of TH to modulate genes critical for developmental processes that control structural development of the brain in general, including the hippocampus.  

The empirical support for this KER is strong. In humans, untreated congenital hypothyroidism and severe iodine deficiency are accompanied by reductions in circulating levels of TH, and result in severe structural alterations in brain size, including hippocampus (Wheeler et al., 2011). The tie to serum TH has been amply demonstrated in clinical therapy of hypothyroidism during pregnancy and in congenitaly hypothyroid children born to euthroid mothers. In addition, there is a vast amount of data from animal studies that support this relationship. Gross structural changes in the hippocampus following severe TH insufficiency are widely reported (Hasegawa et al., 2010; Powell et al.,2010; Madiera et al., 1991; 1992; Rami et al. 1986a 1986b; Madeira and Paula-Barbosa 1993; Rabie et al., 1980; Berbel et al., 1996). Other studies reveal more subtle changes in hippocampal structure such as reductions in a specific subregions of the hippocampus or of a cell type (eg. parvalbumin expressing inhibitory neurons) or synaptic component (ie synapsin, postsynaptic density proteins) or misplacement of cells within the hippocampal cell layers (Berbel et al., 1997; 2010; Gilbert et al., 2007; Auso et al., 2003; Gilbert et al., 2016; Cattani et al., 2013).  These observations at the histological level are correlated with reductions in serum T4. The most profound structural impairments are typically seen with severe reductions in both hormones.

 Additional evidence for a relationship between serum TH and hippocampal anatomy comes from the study of adult neurogenesis. The propensity of the hippocampus to generate new neurons throughout the lifetime of the organism occurs in only two brain regions, the olfactory bulb and the hippocampus. Severe reductions in circulating levels of TH in adulthood reduces both neuroprogenitor cell proliferation and survival of newly generated neurons in the neurogenic niche of the hippocampal dentate gyrus (Ambrogini et al., 2005, Montero-Pedrazuela et al., 2006, Kapoor et al., 2015).  These same effects on neurogenesis also occur during development. However, the impact of developmental TH disruption on neurogenesis in adult offspring shows that the developing brain is more sensitive to these persistent effects. For example, a reduction in the capacity for neurogenesis was recently demonstrated in adult euthyroid offspring of developmentally TH compromised dams (Gilbert et al., 2016). These data indicate a permanent deficit in the capacity for neurogenesis, a process that controls dentate gyrus volume and cell number, following moderate reductions in serum TH in the fetus/neonate.

Finally, from in vitro studies, T3 stimulation accelerates the formation of GABAergic boutons and alters the distribution of GABAergic axons among growing neurons in culture. This growth is dependent on both activity within the network and the presence of T3. It can be blocked by the T3 nuclear receptor antagonist, 1-850, or pharmacological block of synaptic activity (Westerholz et al., 2010; 2013). T3 is believed to have this effect by it action on synaptic pruning. This example reveals the dynamic interplay between synaptic activity and neuroanatomy in the developing nervous system (Kozorovitskiy 2012).

Temporal Evidence:  The temporal nature of this KER is developmental (Seed et al., 2005). It is a well-recognized fact that there are critical developmental windows for disruption of the serum THs that result in altered hippocampal anatomy. Reductions in serum TH in the neonate produced alterations in hippocampal parvalbumin-expressing neurons while the same treatment in adulthood is without effect (Gilbert et al., 2007).  In a rodent model of prenatal TH deficiency, decreased length and number of radial glial cells which are critical for neuronal migration was reversed by hormone replacement treatment to the dam (Pathak et al., 2011). Reversibility of cortical layering defects with thyroxine treatment have also been reported in models of maternal hypothyroidism (Pathak et al., 2011; Berbel et al., 2010; Mohan et al., 2012). In in vitro studies, temporal specificity of the influence of T3 on GABAergic synapses and synaptic pruning has also been demonstrated (Westerholz et al., 2013). In addition, clinical therapy of hypothyroidism during pregnancy, and in congenitally hypothyroid children born to euthroid mothers ameliorates most of the adverse impacts on the developing human brain.   

Dose-Response Evidence: There are limited data available to inform the dose-dependent nature of the correlation between serum THs and changes in hippocampal anatomy. Gilbert et al (2007) demonstrated dose-dependent declines in the expression of protein marker inhibitory neurons in both hippocampus and neocortex with graded exposures to PTU and resultant serum T4. Shiraki et al. (2014; 2016) report dose-dependent alterations in the expression patterns of several neuronal and glial protein markers in the hippocampus after developmental exposure to different doses of PTU or MMI. Gilbert et al. (2016) report dose-dependent reductions in linear morphometry and volume of hippocampal subfields following developmental exposure to the PTU.

This has been repeatedly demonstrated.  However, with some studies noted above, most investigations have been conducted in the neonate after severe hormone reductions induced by PTU, MMI or thyroidectomy. These severe changes alter a wide variety of general growth and developmental processes. In one of the few dose-response studies assessing hippocampal anatomy, alterations in simple guidenline metrics of linear morphometry and volume of hippocampal subfields following developmental exposure to the PTU were largely restricted to the high dose group, despite alterations in downstream KEs of hippocampal physiology and cognitive function. This may result from inadequacy of the assessment tools or the timing of the observations. Similarly, in chemically induced serum hormone reductions of comparable magnitude as those induced by PTU or MMI, observations of hippocampal morphology are not always seen (PTU vs ETU or mancozeb, European Commission, 2017).  Consideration of the sensitivity of neuroanatomical and neurobehavioral method used, as well as chemical kinetics that drive the reduction of maternal, fetal, or neonatal TH reduction, may be key to understanding these discrepancies. More data is needed that link more limited decrements in serum TH to specific hippocampal anatomical changes. The role of direct fetal TPO inhibition contribution to fetal TH and subsequent changes to hippocampal structure and subsequent downstream KEs in humans is a knowledge gap.   

Quantitative Understanding of the Linkage

Most investigations for hippocampal anatomy have been conducted in the neonate after severe hormone reductions. There is currently insufficient data for quantitative analysis of serum T4 and hippocampal neuroanatomy.

References

Ambrogini P, Cuppini R, Ferri P, Mancini C, Ciaroni S, Voci A, Gerdoni E, Gallo G (2005) Thyroid hormones affect neurogenesis in the dentate gyrus of adult rat. Neuroendocrinology 81:244-253.

Anderson GW, Schoonover CM, Jones SA (2003) Control of thyroid hormone action in the developing rat brain. Thyroid 13:1039-56.

Auso E, Lavado-Autric R, Cuevas E, Del Rey FE, Morreale De Escobar G, Berbel P (2004) A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration. Endocrinology 145:4037-4047.

Berbel P, Marco P, Cerezo JR, DeFelipe J (1996) Distribution of parvalbumin immunoreactivity in the neocortex of hypothyroid adult rats. Neurosci Lett 204:65-68.

Berbel P, Navarro D, Ausó E, Varea E, Rodríguez AE, Ballesta JJ, Salinas M, Flores E, Faura CC, de Escobar GM. Role of late maternal thyroid hormones in cerebral cortex development: an experimental model for human prematurity. Cereb Cortex. 2010 Jun;20(6):1462-75.

Bernal J. 2007. Thyroid hormone receptors in brain development and function. Nature clinical practice Endocrinology & metabolism. 3:249-259.

Cattani D, Goulart PB, Cavalli VL, Winkelmann-Duarte E, Dos Santos AQ,

Pierozan P, de Souza DF, Woehl VM, Fernandes MC, Silva FR, Gonçalves CA, Pessoa-Pureur R, Zamoner A. Congenital hypothyroidism alters the oxidative status, enzyme activities and morphological parameters in the hippocampus of developing rats. Mol Cell Endocrinol. 2013 Aug 15;375(1-2):14-26.

Gilbert ME, Goodman JH, Gomez J, Johnstone AF, Ramos RL. Adult hippocampal neurogenesis is impaired by transient and moderate developmental thyroid hormone disruption. Neurotoxicology. 2016. 59:9-21.

Gilbert ME, Sui L, Walker MJ, Anderson W, Thomas S, Smoller SN, Schon JP, Phani S, Goodman JH (2007) Thyroid hormone insufficiency during brain development reduces parvalbumin immunoreactivity and inhibitory function in the hippocampus. Endocrinology 148:92-102.

Hasegawa M, Kida I, Wada H (2010) A volumetric analysis of the brain and hippocampus of rats rendered perinatal hypothyroid. Neurosci Lett 479:240-244.

Kapoor R, Fanibunda SE, Desouza LA, Guha SK, Vaidya VA (2015) Perspectives on thyroid hormone action in adult neurogenesis. J Neurochem 133:599-616.

Kozorovitskiy Y, Saunders A, Johnson CA, Lowell BB, Sabatini BL. Recurrent network activity drives striatal synaptogenesis. Nature. 2012 May 13;485(7400):646-50.

Madeira MD, Cadete-Leite A, Andrade JP, Paula-Barbosa MM (1991) Effects of hypothyroidism upon the granular layer of the dentate gyrus in male and female adult rats: a morphometric study. J Comp Neurol 314:171-186.

Madeira MD, Paula-Barbosa MM (1993) Reorganization of mossy fiber synapses in male and female hypothyroid rats: a stereological study. J Comp Neurol 337:334-352.

Madeira MD, Sousa N, Lima-Andrade MT, Calheiros F, Cadete-Leite A, Paula-Barbosa MM (1992) Selective vulnerability of the hippocampal pyramidal neurons to hypothyroidism in male and female rats. J Comp Neurol 322:501-518.

Mohan V, Sinha RA, Pathak A, Rastogi L, Kumar P, Pal A, Godbole MM (2012) Maternal thyroid hormone deficiency affects the fetal neocorticogenesis by reducing the proliferating pool, rate of neurogenesis and indirect neurogenesis. Exp Neurol 237:477-488.

Montero-Pedrazuela A, Venero C, Lavado-Autric R, Fernandez-Lamo I, Garcia-Verdugo JM, Bernal J, Guadano-Ferraz A (2006) Modulation of adult hippocampal neurogenesis by thyroid hormones: implications in depressive-like behavior. Mol Psychiatry 11:361-371.

Oppenheimer J. The nuclear-receptor-triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism, and biological action, In: Samuels HH, eds: Molecular Basis of Thyroid Hormone Action. Academic Press: New York. 1983: 1-34.

Pathak A, Sinha RA, Mohan V, Mitra K, Godbole MM (2011) Maternal thyroid hormone before the onset of fetal thyroid function regulates reelin and downstream signaling cascade affecting neocortical neuronal migration. Cereb Cortex 21:11-21.

Powell MH, Nguyen HV, Gilbert M, Parekh M, Colon-Perez LM, Mareci TH, Montie E (2012) Magnetic resonance imaging and volumetric analysis: novel tools to study the effects of thyroid hormone disruption on white matter development. Neurotoxicology 33:1322-1329.

Rabie A, Clavel MC, Legrand J (1980) Analysis of the mechanisms underlying increased histogenetic cell death in developing cerebellum of the hypothyroid rat: determination of the time required for granule cell death. Brain Res 190:409-414.

Rami A, Patel AJ, Rabie A (1986a) Thyroid hormone and development of the rat hippocampus: morphological alterations in granule and pyramidal cells. Neuroscience 19:1217-1226.

Rami A, Rabie A, Patel AJ (1986b) Thyroid hormone and development of the rat hippocampus: cell acquisition in the dentate gyrus. Neuroscience 19:1207-1216.

Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72.

Shiraki A, Saito F, Akane H, Takeyoshi M, Imatanaka N, Itahashi M, Yoshida T, Shibutani M (2014) Expression alterations of genes on both neuronal and glial development in rats after developmental exposure to 6-propyl-2-thiouracil. Toxicol Lett 228:225-234.

Shiraki A, Saito F, Akane H, Akahori Y, Imatanaka N, Itahashi M, Yoshida T, Shibutani M. Gene expression profiling of the hippocampal dentate gyrus in an adult toxicity study captures a variety of neurodevelopmental dysfunctions in rat models of hypothyroidism. J Appl Toxicol. 2016 Jan;36(1):24-34.

Westerholz S, de Lima AD, Voigt T. Thyroid hormone-dependent development of early cortical networks: temporal specificity and the contribution of trkB and mTOR pathways. Front Cell Neurosci. 2013. 7:121.

Westerholz S, de Lima AD, Voigt T. Regulation of early spontaneous network activity and GABAergic neurons development by thyroid hormone. Neuroscience. 2010 Jun 30;168(2):573-89.

Wheeler SM, Willoughby KA, McAndrews MP, Rovet JF.  Hippocampal size and memory functioning in children and adolescents with congenital hypothyroidism.  J Clin Endocrinol Metab. 2011. 96(9):E1427-34

Relationship: 1389: T4 in serum, Decreased leads to Hippocampal Physiology, Altered

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Most of the data to support this KER are derived from rodent studies.

Key Event Relationship Description

Thyroid hormones are critical for normal development of the structure and function of the brain, including the hippocampus (Anderson et al., 2003; Bernal, 2007). Brain concentrations of T4 are dependent on transport of primarily T4 from serum, with subsequent conversion to T3 in the astrocytes by deiodinase and transfer to nuclear receptors within the neuron. This is followed by TH dependent gene transcription that influences hippocampal structural development and subsequent physiological function.

Evidence Supporting this KER

The weight of evidence for this indirect relationship is moderate. A wide variety studies have been performed in several labs in which thyroid hormone reductions in serum induced by chemicals/treatments, acting at a variety of target sites to disrupt hormonal status, is coincident with altered hippocampal physiology and/or plasticity. These include inhibition of TPO, NIS, dietary insufficiencies of iodine, and upregulation of liver catabolism, NIS inhibition, or dietary manipulation of iodine. Most of the data available is from the model TPO inhibitors, PTU and MMI, and this data documents enduring hippocampal physiological impairments in adult offspring following a period of transient serum TH insufficiencies in the pre- and post-natal period. Serum hormones are reported for the neonate and the dam at the termination of exposure, and recovery of hormonal status in the adult has been demonstrated in a number of studies despite the persistence of the hippocampal deficit. A few laboratories have reported dose-dependent effects at less than maximal hormone depletion.

The biological plausibility of this KER is rated as strong. The relationship is consistent with the known biology of how TH control development of hippocampal physiology.

Empirical support for this indirect KER is rated as strong. Empirical data from studies that measure serum TH concentrations and then assess alterations in synaptic function in the hippocampus have come from several laboratories. This work has employed in vivo, ex vivo and in vitro preparations from developmentally exposed animals.

Most of the in vivo neurophysiological assessments have been performed in the dentate gyrus. Excitatory and inhibitory synaptic transmission were reduced by PTU in a dose-dependent fashion (Gilbert and Sui, 2006; Gilbert et al., 2007; Gilbert, 2011). Serum T4 decrements in dams and pups were positively correlated with the synaptic impairments.  Serum T4 and hippocampal excitatory transmission were also reduced in pups from dams exposed to perchlorate (Gilbert et al., 2008) and iodine deficiency (Gilbert et al., 2013). However, serum T4 reductions induced by the complex PCB mixture, A1254, were associated with increases not decrements in excitatory response amplitudes (Gilbert et al., 2003).

Impaired synaptic transmission and plasticity in the form of long-term potentiation (LTP) and long-term depression (LTD) have been reported using in vitro and ex vivo preparations (Sui and Gilbert, 2003; Sui et al., 2005; 2007; Gilbert and Sui, 2006; Gilbert and Paczkowski, 2003; Gilbert, 2011; Taylor et al., 2008; Vara et al., 2002), Dong et al., 2005; Gilbert, 2003; 2004; 2011; Gilbert et al., 2016; ), .

In many studies these observations have been reported under conditions of severe hypothyroidism induced primarily by TPO-inhibitors MMI and PTU or severe iodine deficiency (Vara et al., 2002; Dong et al., 2005). In others, researchers produced graded degrees of TH insufficiency in dams and pups by administering varying doses of PTU, perchlorate, or dietary iodine deficiency, and reported dose-dependency of the observed effects.  This work has provided increased confidence in the relationship between TH insufficiency and functional impairment of the hippocampus, and the specificity of the observed effects to be mediated by TH insufficiency (Gilbert and Sui, 2006; Gilbert, 2011; Gilbert et al., 2013; 2016).

As described in the KER entitled “Hippocampal anatomy, altered leads to Hippocampal Physiology, Altered”, there is dynamic reciprocal interplay between neuroanatomy and physiology, particularly evident in the developing nervous sytem, making it difficult to parse the effects of one independently of the other (Kozorovitsky et al., 2012). In the in vitro studies of Westerholz et al (2010; 2013), T3-induced increase in GABAgeric synapses is activity-dependent, in that the anatomical changes described required both spontaneous electical activity in the network in addition to thyroid hormone. The electrophysiological competence of that emerging synaptic network was similarly dependent upon the hormone stimulation in addition to the growth of the GABAergic neurons. In this manner, TH can directly influence the formation of emerging cortical networks. Although demontrated using cortical neurons, it is expected that very similar processes occur in the developing neural networks of the hippocampus.

Temporal Evidence: The temporal nature of this KER is developmental (Seed et al., 2005). It is a well-recognized fact that there are critical developmental windows for disruption of the serum THs that result in altered physiological function in the dentate gyrus (Gilbert 2011; Sanchez-Huerta et al., 2015).  Rescue experiments have not been performed in developmental hypothyroid models. In in vitro studies, temporal specificity of the influence of T3 on network activity has been demonstrated (Westerholz et al., 2013).

Dose-Response Evidence:  There are several reports on the dose-dependent nature of the correlation between serum THs and changes in hippocampal physiology albeit from a limited number of laboratories (Taylor et al., 2008; Gilbert et al., 2007; 2016; Gilbert 2011; Gilbert and Sui, 2006).

There are no inconsistencies in this KER, but there are some remaining uncertainties. It is widely accepted that changes in serum THs during development will result in alterations in behavior controlled by the hippocampus. This has been repeatedly demonstrated in animal models and in humans. However, most studies have been performed under conditions of severe hypothyroidism induced primarily by TPO-inhibitors MMI and PTU, or severe iodine deficiency. In addition, it is also known that there is an interaction between physiological and anatomical development, where anatomy develops first, and can be ‘reshaped’ by the ongoing maturation of physiological function (e.g., Kutsarova et al., 2017).

Quantitative Understanding of the Linkage

Insufficient data exist to date that could be used to develop a quantitative predictive model of neurophysiological in hippocampus from serum TH concentrations. The dynamic range over which neurophysiological endpoints can vary is small complicating the development of quantitative relationships between degree of TH insufficiency and magnitude of neurophysiological impairment.

References

Anderson GW, Schoonover CM, Jones SA (2003) Control of thyroid hormone action in the developing rat brain. Thyroid 13:1039-56.

Bernal J. 2007. Thyroid hormone receptors in brain development and function. Nature clinical practice Endocrinology & metabolism. 3:249-259.

Dong J, Yin H, Liu W, Wang P, Jiang Y, Chen J. Congenital iodine deficiency and hypothyroidism impair LTP and decrease C-fos and C-jun expression in rat hippocampus. Neurotoxicol 2005; 26:417-426.

Gilbert ME. Perinatal exposure to polychlorinated biphenyls alters excitatory synaptic transmission and short-term plasticity in the hippocampus of the adult rat. Neurotoxicology. 2003 Dec;24(6):851-60.

Gilbert ME. Alterations in synaptic transmission and plasticity in area CA1 of adult hippocampus following developmental hypothyroidism. Brain Res Dev 2004 Jan 31;148(1):11-8

Gilbert ME. Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicol Sci. 2011;124(2):432-445.

Gilbert ME, Hedge JM, Valentín-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW. An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome. Toxicol Sci. 2013 Mar;132(1):177-95.

Gilbert ME, Paczkowski C. Propylthiouracil (PTU)-induced hypothyroidism in the developing rat impairs synaptic transmission and plasticity in the dentate gyrus of the adult hippocampus. Brain Res Dev Brain Res. 2003 Oct 10;145(1):19-29

Gilbert ME, Sanchez-Huerta K, Wood C. Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology. 2016 Feb;157(2):774-87.

Gilbert ME, Sui L. Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res. 2006 Jan 19;1069(1):10-2

Gilbert ME, Sui L. Developmental exposure to perchlorate alters synaptic transmission in hippocampus of the adult rat. Environ Health Perspect. 2008 Jun;116(6):752-60.

Gilbert ME, Sui L, Walker MJ, Anderson W, Thomas S, Smoller SN, Schon JP, Phani S, Goodman JH. Thyroid hormone insufficiency during brain development reduces parvalbumin immunoreactivity and inhibitory function in the hippocampus. Endocrinology. 2007 Jan;148(1):92-102. PubMed PMID: 17008398.

Kozorovitskiy Y, Saunders A, Johnson CA, Lowell BB, Sabatini BL. Recurrent network activity drives striatal synaptogenesis. Nature. 2012 May 13;485(7400):646-50.

Sánchez-Huerta K, Pacheco-Rosado J, Gilbert ME. Adult onset-hypothyroidism: alterations in hippocampal field potentials in the dentate gyrus are largely associated with anaesthesia-induced hypothermia. J Neuroendocrinol. 2015 Jan;27(1):8-19.

Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72.

Sui L, Anderson WL, Gilbert ME. Impairment in short-term but enhanced long-term synaptic potentiation and ERK activation in adult hippocampal area CA1 following developmental thyroid hormone insufficiency. Toxicol Sci. 2005 May;85(1):647-56.

Sui L, Gilbert ME. Pre- and postnatal propylthiouracil-induced hypothyroidism impairs synaptic transmission and plasticity in area CA1 of the neonatal rat hippocampus. Endocrinology. 2003 Sep;144(9):4195-203.

Taylor MA, Swant J, Wagner JJ, Fisher JW, Ferguson DC. Lower thyroid compensatory reserve of rat pups after maternal hypothyroidism: correlation of thyroid, hepatic, and cerebrocortical biomarkers with hippocampal neurophysiology. Endocrinology. 2008 Jul;149(7):3521-30. doi: 10.1210/en.2008-0020. PubMed PMID: 18372327.

Vara H, Muñoz-Cuevas J, Colino A. Age-dependent alterations of long-term synaptic plasticityin thyroid-deficient rats. Hippocampus. 2003;13(7):816-25.

Westerholz S, de Lima AD, Voigt T. Thyroid hormone-dependent development of early cortical networks: temporal specificity and the contribution of trkB and mTOR pathways. Front Cell Neurosci. 2013 Aug 6;7:121.

Westerholz S, de Lima AD, Voigt T. Regulation of early spontaneous network activity and GABAergic neurons development by thyroid hormone. Neuroscience. 2010Jun 30;168(2):573-89.

Relationship: 403: T4 in serum, Decreased leads to Cognitive Function, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

There is a plethora of data supporting this KER in rats, mice, and humans.

Key Event Relationship Description

Thyroid hormones (TH) are critical for normal development of the structure and function of the brain, including hippocampal development and cognitive function (Anderson et al., 2003; Bernal, 2007; Willoughby et al., 2014).   Brain concentrations of T4 are dependent on transfer of T4 from serum, through the vascular endothelia, into astrocytes.  In astrocytes, T4 is converted to T3 by deiodinase and subsequently transferred to neurons cellular membrane transporters. In the brain T3 controls transcription and translation of genes responsible for normal hippocampal structural and functional development. Clearly the brain circuitry controlling cognitive function is complex and is not solely accomplished by the functionality of the hippocampus. However, it is well documented that normal hippocampal structure and physiology are critical for the development of cognitive function. Thus, there is an indisputable indirect link between serum T4 and cognitive function.

Evidence Supporting this KER

The weight of evidence for this indirect relationship is strong. Alterations in serum TH concentrations are very well correlated with adverse impacts on cognitive behaviors such as learning and memory. This includes a large amount of literature, from more than four decades of research, that links hypothyroidism and/or hypothyroxinemia with alterations in spatial cognitive function, a hippocampal dependent behavior. A number of reviews are cited below that are primarily from humans and rodents, but this indirect relationship has also been shown for a number of other species.

In humans, severe serum TH reductions that accompany congenital hypothyroidism dramatically impair brain function and lead to severe mental retardation. Lower global IQ scores, language delays and weak verbal skills, motor weakness, attentional deficits and learning impairments accompany low serum TH in children (Derksen-Lubsen and Verkerk 1996). Standard tests of IQ function in children born to mothers with even marginal hypothyoidism during pregnancy or in children with a defective thyroid gland who are then treated remain approximately 6 points below expected values. Selective deficits on visual spatial, motor, language, memory and attention tests are observed, the exact phenotype largely dependent on the developmental window over which the insufficiency occurred and the severity of the hormone deficit (Mirabella et al. 2000; Rovet 2002; Zoeller and Rovet 2004; Willoughby et al 2014). Indeed, this link is recognized as being so clinically important that T4 and TSH are monitored in all newborns in the US.   

In rodent models, reductions in serum TH induced by TPO inhibitors such as MMI and PTU, when induced during development, lead to a variety of neurobehavioral impairments. These impairments can occur in the sensory, motor, and cognitive domains. The specific phenotype is dependent on both the window of exposure, the duration of exposure, and the severity of the hormone reduction (Zoeller and Rovet, 2004).  This includes more than four decades of work linking serum TH changes to alterations in hippocampal-dependent spatial behaviors (Akaike et al., 2004; Axelstand etal., 2008; Brosvic et al; Kawada et al, 1988; Friedhoff et al, 2000; Gilbert and Sui, 2006; Gilbert et al., 2016; Gilbert, 2011).

The biological plausibility of this KER is rated as strong. The relationship is consistent with the known biology of how the relationship between serum TH concentrations, brain TH concentrations, and TH control of brain development.

Empirical support for this KER is rated as strong. Empirical data from studies that measure serum TH concentrations and then assess alterations in cognitive function, including hippocampal dependent behaviors, is vast. The qualitative relationship between reduced serum hormone levels and adverse cognitive outcomes is well accepted in endocrinology, as well as developmental neuroendocrinology. Indeed, the relationship between serum T4 and T3 levels and adverse neurodevelopmental outcomes (e.g., IQ loss in children) is beyond reproach.

Temporal Evidence: The temporal nature of this KER is developmental (Seed et al., 2005). It is a well-recognized fact that there are critical developmental windows for disruption of the serum THs that result in cognitive function.  In humans, hormone insufficiency that occurs in mid-pregnancy due to maternal drops in serum hormone, and that which occurs in late pregnancy due to disruptions in the fetal thyroid gland lead to different patterns of cognitive impairment (Zoeller and Rovet, 2004). In animal models, deficits in hippocampal-dependent cognitive tasks result from developmental, but not adult hormone deprivation (Gilbert and Sui, 2006; Gilbert et al., 2016; Axelstad et al, 2009; Gilbert, 2011; Opazo et al., 2008). Replacement studies have demonstrated that varying adverse neurobehavioral outcomes, including cognitive function, can be reduced or eliminated if T4 (and/or T3) treatment is given during the critical windows (e.g., Kawada et al., 1988; Goldey and Crofton, 1998; Reid et al., 2007).

Dose-Response Evidence: An increasing amount of literature is now available that provides clear evidence of the ‘dose-response’ nature of this KER.  Most research over that last 40 years has employed high doses of chemicals, or chemicals plus thyroidectomies, that results in severe depletion of circulating thyroid hormones. More recently, researchers produced graded degrees of TH insufficiency in dams and pups by administering varying doses of chemicals and have correlated them to the dose-dependency of the observed effects.  This work has provided increased confidence in the relationship between serum TH decrements and a variety of neurodevelopmental impairments, and also to the specificity of the observed effects on brain development that is directly mediated by TH insufficiency (Goldey et al., 1995; Crofton, 2004; Gilbert and Sui, 2006; Gilbert, 2011; Bastian et al., 2014; Royland et al., 2008; Sharlin et al., 2008).

There are no inconsistencies in this KER, but there are some remaining uncertainties. It is widely accepted that changes in serum THs during development will result in alterations in behavior controlled by the hippocampus. This has been repeatedly demonstrated in animal models and in humans. A major uncertainty is the precise relationship between the degree, timing and duration of serum TH changes that leads to these behavioral deficits.

Inconsistencies may also exist for chemicals other than classical TPO inhibitors that may reduce serum TH and induce impairments in cognitive function, but through action on other endocrine systems, or via direct action on the brain in the absence of an intervening endocrine action.  

Quantitative Understanding of the Linkage

Except for a quantitative relationship between serum T4 and hearing loss in rodents (Crofton, 2004), there are no other reports of development of quantitative predictive models linking serum TH and adverse neurological outcomes. Insufficient data exist to develop a quantitative predictive model of adverse cognitive outcomes from serum TH concentrations. However, evidence from human studies suggests that decreases as low as 25% in serum T4 in pregnant women will yield small decrements in IQ in children (e.g., Haddow et al., 1995). Since publication of this seminal paper, several reports have appeared providing supportive if not direct confirmatory data on the association of reductions in maternal or early postnatal serum TH and adverse neurodevelopmental outcomes (e.g., Rovet and Willoughby, 2010, Wheeler et al., 2011, Willoughby et al., 2014, Wheeler et al., 2015; Pop et al., 1999, Pop et al., 2003, Kooistra et al., 2006, Henrichs et al., 2010, Korevaar et al., 2016). Based on these data, regulatory authorities have used 10 and/or 20% changes in serum T4 as a point of departure for hazard assessments in rodent studies (EPA, 2011).

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