TH synthesis, Decreased leads to BDNF, Reduced

The importance of TH in brain development has been recognised and investigated for many decades (Bernal, 2011; Williams 2008). Several human studies have shown that low levels of circulating maternal TH, even in the modest degree, can lead to neurophysiological deficits in the offspring, including learning and memory deficits, or even cretinism in most severe cases (Zoeller and Rovet, 2004; Henrichs et al., 2010). The levels of serum TH at birth are not always informative, as most of the neurological deficits are present despite the normal thyroid status of the newborn. That means that the cause of these impairments is rooted in the early stages of the neuronal development during the gestational period. The nature and the temporal occurrence of these defects suggest that TH may exert their effects through the neurotrophins, as they are the main regulators of neuronal system development (Lu and Figurov, 1997). Among them, BDNF represents the prime candidate because of its critical role in CNS development and its ability to regulate synaptic transmission, dendritic structure and synaptic plasticity in adulthood (Binder and Scharfman, 2004). Additionally, hippocampus and neocortex are two of the regions characterized by the highest BDNF expression (Kawamoto et al., 1996), and are also key brain areas for learning and memory functions.

Many in vivo studies have focused on the determination of the relationship between TH-mediated effects and BDNF expression in the brain. The majority of the work has been performed by evaluating the effects of TH insufficiency on BDNF developmental expression profile. The results, despite some differences, are showing a trend toward BDNF down-regulation triggered by decrease of TH synthesis.

Reductions in BDNF mRNA and protein were observed in hypothyroid rat models exposed to the TPO inhibitors methimazole (MMI) or propylthiouracil (PTU), and perchlorate (NIS inhibitor) (Koibuchi et al., 1999; 2001; Sinha et al., 2009; Neveu and Arenas, 1996; Lasley and Gilbert, 2011). These studies supported direct associations between decreased levels of TH and reduced BDNF expression in the developmental cerebellum, hippocampus and cortex. The dose-response relationship could not be evaluated in these studies, as they were conducted in conditions of severe maternal hypothyroidism, namely after exposure to very high doses of the chemicals.

- Koibuchi et al., 2001: In this in vivo study, newborn mice were rendered hypothyroid by administering MMI (TPO inhibitor) and perchlorate (NIS inhibitor) in drinking water to their mothers. Neurotrophin-3 (NT-3) and BDNF gene expression was depressed in the perinatal hypothyroid cerebellum. Furthermore, the expression of retinoid-receptor-related orphan nuclear hormone receptor-alpha (ROR-alpha), an orphan nuclear receptor that plays critical roles in Purkinje cell development, was also decreased. Morphologically, disappearance of the external granule cell layer was retarded and arborization of Purkinje cell dendrite was decreased, events that were also observed in hypothyroid rats, suggesting impairment in neuronal differentiation.

- Chakraborty et al., 2012: In this in vivo study, PTU (TPO inhibitor) exposure in rat dams (4 ppm in drinking water) significantly decreased the levels of free T4 (~ 33% decrease vs control, at PND 7) and total T4 (~ 38% decrease vs control, at PND 7) in the offspring, and hippocampal BDNF protein levels in the offspring at 3 and 7 PNDs (~ 25% decrease of hippocampal BDNF, at PND 7, in female pups vs untreated female control). No significant BDNF reductions were observed in either the cerebellum or brain stem.

- Blanco et al., 2013: In this in vivo study, a significant dose-dependent down-regulation of hippocampal BDNF mRNA (~ 32% decrease vs control) in combination with the dose-dependent reduction of plasma TH (T4: ~ 25% decrease vs control; T3: ~ 14% decrease vs control), was also shown in Sprague Dawley rats after exposure to BDE-99 (2 mg/kg/day, through gavage, from gestation day 6 to PND 21).

- Shafiee et al., 2016: This in vivo study investigated the effects of PTU (TPO inhibitor, 100 mg/L) in pregnant rats to evaluate the effects elicited by maternal hypothyroidism in the offspring. PTU was added to the drinking water from gestation day 6 to PND 21. Analysis of hippocampal BDNF levels, and learning and memory tests were performed on PNDs 45-52 on pups. These results indicated that hypothyroidism during the fetal period and the early postnatal period was associated with: (i) ~ 70% reduction of total serum T4, (ii) ~ 5-fold increase of total serum TSH levels (on PND 21; no significant differences could be found at the end of the behavioral testing, on PND 52), (iii) reduction of hippocampal BDNF protein levels (~ 8% decrease vs control), (iv) impairment of spatial learning and memory, in both male and female rat offspring.

- Gilbert et al., 2016: In this in vivo study, developmental PTU treatment dramatically reduced the activity-dependent expression of neurotrophins and related genes in neonate hippocampus (e.g., at PND14, rats treated with 3 ppm PTU, underwent reduction of Bdnft by ~25%, Bdnfiv by ~10%, Ngf by ~25%, and Pval by ~50%) and was accompanied by deficits in hippocampal-based learning (e.g., mean latency to find a hidden platform, at 2^nd trial resulted ~60% higher in rats treated with 10 ppm PTU).

- Abedelhaffez and Hassan, 2013: This in vivo study in rats reported that methimazole (MMI, a TPO inhibitor)-induced hypothyroidism reduced plasma free T3, free T4 and significantly increased TSH in the pups, showing also reduced hippocampal and cerebellar BDNF levels.

- da Conceição et al., 2016: In this in vivo study, thyroidectomized (i.e,. hypothyroid) adult Wistar rats showed significant increase of serum TSH (~ 750% increase vs control rats), decrease of T4 (~ 80% decrease vs control) and T3 serum levels (~ 45% decrease vs control), together with a reduced hippocampal expression of MCT8 (~ 83% decrease vs control rats), TH receptor alfa (TRα1) (~ 77 % decrease vs control), deiodinase type 2 (DIO2) (~ 90% decrease vs control), and BDNF mRNA expression in hippocampus (~ 75% decrease vs control).

- Cortés et al., 2012: In this in vivo study, adult male Sprague-Dawley rats were treated with 6-propyl-2-thiouracil (PTU, a TPO inhibitor) (0.05% in drinking water) for 20 days to induce hypothyroidism. PTU-treated rats showed decrease serum fT4 (~ 70% decrease vs control) and tT3 (~ 45% decrease vs control) levels, and increased TSH levels (~ 9.5-fold increase over control). The hippocampus of hypothyroid adult rats displayed increased apoptosis levels in neurons and astrocyte and reactive gliosis compared with controls. The glutamatergic synapses from the stratum radiatum of CA3 from hypothyroid rats, contained lower postsynaptic density (PSD) than control rats (~ 25% lower PSD than control). This observation was in agreement with a reduced content of NMDAR subunits (NR1 and NR2A/B subunits, both subunits: ~ 25% decrease vs control) at the PSD in hypothyroid animals. Additionally, the hippocampal amount of BDNF mRNA (assessed by in situ hybridization) was higher (~ 4.8-fold increase over control) of hypothyroid rats, while the content of TrkB protein (BDNF receptor) was reduced (~ 30% decrease vs control) at the PSD of the CA3 region of hypothyroid rats, compared with controls. Even though BDNF levels were increased, the decrease of BDNF receptor (TrkB) compromises the signalling pathway under BDNF control.

- Jang et al., 2012: In this in vivo study, pregnant female C57BL/6 mice (F0) were exposed to BPA (0.1-10 mg/kg) from gestation day 6 to 17, and female offspring (F2) from F1 generation mice were analysed. High-dose BPA (10 mg/kg) caused neurocognitive deficit (i.e., reduced memory retention) as shown by passive avoidance testing (~ 33% decrease vs control) in F2 mice. Furthermore, 10 mg/kg BPA decreased the hippocampal levels of BDNF (~ 35% lower vs control) in F2 mice. These results suggest that BPA exposure (NIS inhibitor) in pregnant mothers could decrease hippocampal neurogenesis and cognitive function in future generations.

- Pathak et al, 2011: In this in vivo study, the effect of maternal TH deficiency on neocortical development was investigated. Rat dams were maintained on MMI (TPO inhibitor) from GD6 until sacrifice. Decreased number and length of radial glia, loss of neuronal bipolarity, and impaired neuronal migration were recovered with early TH replacement (at E13-15). BDNF mRNA resulted downregulated (80% decrease at E14) while trkB expression was increased (2-fold) in hypothyroid fetuses at E14 stage. TH levels in the brain were not measured in this study.

- Neveu and Arenas, 1996: This in vivo study found that early hypothyroidism (by PTU administration to rat dams) decreased the expression of neurotrophin 3 (NT-3) and BDNF mRNA (70% reduction in BDNF level at PND30). Grafting of PND3 hypothyroid rats with cell lines expressing high levels of NT-3 or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at PND15.

Despite the fact that many in vivo studies have shown a correlation between hypothyroidism and decreased BDNF expression in the brain, no clear consensus can be reached by the overall evaluation of the existing data. There are numerous conflicting studies showing no significant alterations in BDNF mRNA or protein levels (Alvarez-Dolado et al., 1994; Bastian et al., 2010; 2012; Royland et al., 2008; Lasley and Gilbert, 2011). However, the results of these studies cannot exclude the possibility of temporal- or region-specific BDNF effects as a consequence of foetal hypothyroidism. A transient TH-dependent BDNF reduction in early postnatal life can be followed by a period of normal BDNF levels or, on the contrary, normal BDNF expression in the early developmental stages is not predictive of equally normal BDNF expression throughout development. Moreover, significant differences in study design, the assessed brain regions, the age and the method of assessment in the existing studies, further complicate result interpretation.

While PTU (TPO inhibitor) has been shown to decrease brain BDNF levels and expression in offspring born from PTU-treated rat dams (Shafiee et al. 2016; Chakraborty et al., 2012; Gilbert et al. 2016), in Cortés et al., 2012 study (in vivo), treatment of adult male Sprague-Dawley rats with PTU induced an increase in the amount of BDNF mRNA in the hippocampus, while the content of TrkB, the receptor for BDNF, resulted reduced at the postsynaptic density (PSD) of the CA3 region compared with controls. Treated rats presented also thinner PSD than control rats, and a reduced content of NMDAr subunits (NR1 and NR2A/B subunits) at the PSD in hypothyroid animals. These indicate differential effects elicited by PTU (i.e., TPO inhibition) on BDNF expression/regulation comparing the adult vs foetal brain. However, even though BDNF levels were increased, the decrease of BDNF receptor (TrkB) compromises the signalling pathway under BDNF control.