Spatio-temporal effects of iodine/thyroid hormone deficiency on neurodevelopment and plausible amelioration approaches beyond the critical window

TH and cerebral cortex development

Cerebral cortex (neocortex) development is an immensely fine-tuned process wherein different neural cell types originate from precursors and migrate to specific brain regions to form cohesive functional circuits.¹⁹

Previous studies from hypothyroid rats showed the lack of well-defined cortical layers due to aberrant neuronal migration, resulting in altered neural circuitry and brain function.²⁰ Here we reviewed the necessity of the TH for the developing cerebral cortex with the recent updates regarding the cellular and molecular action of TH on the neuroglial cells as discovered by us and other groups, with highlights of our novel findings on maternal TH-dependent neurogenesis and neuronal migration in the embryonic brain before FTF.

Maternal TH & neurogenesis

The commencement of cortical neurogenesis involves two modes of cell division of neuronal progenitors that give rise to all glutamatergic neurons.²¹ These two modes of cell division include either direct neurogenesis, wherein neuronal progenitors at the ventricular zone, known as radial glial cells (RG), undergo asymmetric division giving rise to another RG and one neuronal progeny or indirect neurogenesis where RG asymmetric division produces an intermediate progenitors cells (IPCs), which then undergo symmetric division to generate two neurons within the intermediate zone and contribute to cortical thickness.²¹ The period for neocortical neurogenesis is between embryonic day (ED) 12 to 18, which is mostly complete before the start of FTF at E17.5 in rats; these are largely dependent on maternal TH availability. The early expression of MCT8,²² DIO2,²³ and THRα1¹⁰ all coincides with the process of neurogenesis in the developing rodent cerebral cortex. Past studies showed a strong association between maternal T₄ insufficiency and impaired neocorticogenesis.^24,25 Adding new insight into this aspect, we demonstrated that maternal TH deficiency causes disrupted cell cycle kinetics and deranged neurogenesis in rat models of maternal hypothyroidism.¹⁷ We showed that maternal hypothyroidism specifically affects indirect neurogenesis governed by IPCs.¹⁷ Our results demonstrated that both IPCs' abundance and their differentiation into neurons were significantly compromised under maternal hypothyroidism.¹⁷ One of the mechanisms as to why neuronal progenitors are reduced under maternal hypothyroidism was due to increased apoptosis. This result was also supported by our earlier finding, wherein we found increased expression of neuronal nitric oxide synthase and its association with increased neuronal cell death in the developing neocortex under maternal hypothyroidism.¹⁵ Additionally, we showed that the IPCs pool, giving rise to neurons. This is determined by the cleavage orientation (symmetrical vs. asymmetrical) of neuronal progenitors and is dependent on maternal TH availability. Our results showed that the temporal dynamics of both symmetrical and asymmetrical cell division of IPCs are altered under maternal hypothyroidism and can be corrected upon timely T₄ supplementation, resulting in normalization of IPC cell division and neurogenesis¹⁷ [Figure 1]. Therefore, in summary, our study showed that IPCs maintain cortical thickness through indirect neurogenesis and provide for fine-tuning of brain functions that may suffer from maternal TH deficiency if not corrected early during development.

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Figure 1:

Maternal TH (MTH) affects early progenitor population and their divisions. (a) Photomicrographs show nestin (marker of progenitors) immunofluorescence at E14. A marked reduction in the nestin staining in the VZ of hypothyroid fetal cortex, along with thinner and stunted filaments compared to euthyroid. Scale bars 50 μM. (b) Hoechst staining showing symmetrical (vertical) and asymmetrical (horizontal and oblique) cleavages. Counting was done at ventricular surface within the area flanked by red arrows. White square denotes the inset of the area magnified. (c) Under MTH deficiency, symmetrical division was significantly less at E14 (p<0.05) while increasing at E18 (**p<0.05). (d) Asymmetrical divisions reduced from E14 to E18 (**p<0.005), interestingly on T4 treatment from E11-E13, E13-E15 and E15-E17, both symmetrical and asymmetrical divisions restored back to normal at E14, E16 and E18 respectively. The arrow in the panel b denotes plane of cleavage. The y-axis in panel c is the number of symmetrical cleavage/division and in the panel d shows the number of asymmetrical cleavage. The x-axis in panel c and d depicts embryonic days. “*” in panel c and d denotes p<0.05. (Reproduced by permission from: https://www.sciencedirect.com/science/article/abs/pii/S0014488612003056).

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Maternal TH & neuronal migration

Post-neurogenesis during cerebral cortex development, neurons born near the ventricular and sub-ventricular regions migrate radially, forming the six-layered cortical plate. During normal brain development, the extracellular protein, Reelin, secreted by the Cajal-Retzius (CR) neurons located in the marginal zone, plays a guiding role in neuronal migration and neocortical lamination.²⁶ The Reelin signaling cascade involves the binding of Reelin protein to the lipoprotein receptors, apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR), followed by phosphorylation of the disabled 1(Dab1) adaptor protein in the receptive migrating glutamatergic neurons.²⁶ Maternal TH plays a vital role in regulating neuronal migration during neocortical development;²⁷ however, the molecular events mediating morphogenic actions and the mechanism regulating reelin down-regulation under hypothyroidism remained unclear. To understand the cellular and molecular mechanisms behind the impaired migration of neurons under maternal hypothyroidism, we investigated the morphology of migrating neurons and their glial scaffolds with emphasis on the involvement of reelin signaling in the developing rat brains during the critical window period of neuronal migration. The hypothyroid fetuses isolated from hypothyroid dams showed aberrant neuronal migration due to loss of neuronal bipolarity and decrease in the number and length of radial glia, which was correctible after T₄ replacement only in early gestational stages, from embryonic day 13-15 in rats.¹⁶ The decrease in reelin levels in hypothyroid brains led us to hypothesize that the decreased reelin levels likely result from increased apoptosis in reelin-secreting CR neurons or compromised neurotrophin signaling by brain-derived neurotrophin factor (BDNF). Interestingly, we did not observe any evidence of increased cell death of CR neurons nor any down-regulation in BDNF expression.¹⁶ Puzzled by the decrease in reelin levels in the hypothyroid brain, which can be corrected by T₄ replacement, we checked if the reln gene was a direct THR target. Following bioinformatic analysis and gel shift assay, the chromatin immunoprecipitation assays indicated a TRE and physiological binding site of THRα on the reln gene and possible transcriptional control of reelin by TH.¹⁶ TH deficiency significantly reduced the expression of reelin receptors, ApoER2 and VLDLR, along with a decrease in the activation of adaptor Dab1 that further compromised the reelin signaling in the developing brain. These results helped us to understand the mechanistic basis for the requirement of adequate maternal TH levels to ensure proper fetal neocortical cytoarchitecture via proper migration of neurons.¹⁶ These findings were further supported by recent reports from other research groups, reinforcing that TH action targets multiple components in the developing brain through both genomic and nongenomic mechanisms.²⁸

TH regulated programmed cell death in developing cerebellum at its modulation by omega-3-fatty acids

The developmental process affected by TH insufficiency in the cerebellum is delayed migration of granule cells to the inner granule layer, defective dendritic arborization of Purkinje neurons, delayed myelination, and increased apoptosis of granule cells within the inner granule layer.²⁹ Our work on apoptosis-mediated pruning of neurons during cerebellar development revealed that excessive cell death under hypothyroidism was mediated via impaired neurotrophin signaling and increased mitochondrial dysfunction.^30-33 At a cellular level, hypothyroidism during early cerebellar development leads to decreased transcription and defective post-translational processing of neurotrophins, namely BDNF and nerve growth factor. This loss of neurotrophic support not only reduces the cell survival signaling but also increases the pro-apoptotic activity of a neurotrophin receptor, P75NTR, expressed on the cell surface of cerebellar granule cells.³² This finally culminates in mitochondrial dysfunction and activation of the intrinsic apoptotic cascade, leading to enhanced granule cell death.³⁰ Although T₄ supplementation within the critical window can significantly rescue the apoptotic events associated with hypothyroidism, the recovery is not perfect. Therefore, we wanted to investigate other combinatorial agents that could help to rescue the defects of hypothyroidism. In this regard, administration of omega-3-fatty acids in hypothyroid pups significantly reduced the phenotypic alteration observed in a hypothyroid cerebellum.³⁴ Omega-3-fatty acids, which are known neuroprotective agents³⁵ showed that they, independently of circulating TH levels, reversed the effects of hypothyroidism on cerebellar genes, neurotrophin levels, and neuronal apoptosis.³⁴ It is not clear how omega-3-fatty acids crosstalk with T₃ genomic signaling, but it is most likely that omega-3-fatty acid-binding nuclear receptors, such as LXRs could interact with unliganded THRs.³⁶ We also investigated the effect of combinatorial administration of iodine and omega-3 fatty acids in a rat model of hypothyroxinaemia.³⁷ Hypothyroxinemia is characterized by normal thyroid-stimulating hormone concentration but low free thyroxine (FT₄) concentration. The results of this study showed that supplementation of omega-3 fatty acids along with iodine improves neuronal architecture, neuronal survival, motor coordination, and memory function better than with either of them alone.³⁷ Therefore, given their cooperative effect on iodine supplementation-mediated correction of neurological defects,³⁷ further studies using omega-3-fatty acid along with iodine/T₄ replacement regimen should be tested to see if this can correct neuronal defects even beyond the critical window of TH action.

Epigenetic modifiers as a therapeutic option to prevent hypothyroidism-induced neurodevelopmental defects

Given the ontogenesis of THRs in the developing brain, it was widely presumed that liganded THRs mediate the physiological effects of TH during brain development. To verify this presumption, THRα/β knock-out mouse models were generated, which were supposed to mimic the hypothyroid phenotype since they lacked THRs.³⁸ Paradoxically, these animals did not mimic hypothyroid animals phenotypically and displayed normal brain structure and function.³⁸ This discrepancy suggested that the unliganded THRs may play a central role in mediating the phenotypic changes of hypothyroidism during brain development.³⁹ Since HDACs associated with unliganded THRs can repress the transcription of T₃-regulated genes,⁴⁰ we tested if an HDAC inhibitor can induce the derepression of T₃ target genes and developmental processes under hypothyroidism. Using the rat model of perinatal hypothyroidism, we demonstrated that administration of sodium valproate, which is an HDAC inhibitor, in hypothyroid rat pups not only caused a derepression of TH target genes like Bdnf, Pcp2, and Mbp but also improved the dendritic structure of cerebellar Purkinje neurons⁴¹ [Figure 2]. Furthermore, a significant improvement in hypothyroidism-induced motor and cognitive defects was also observed.⁴¹ A recent follow-up study done by Susteyo et al.⁴²(2022), further supported our findings wherein they used a specific HDAC3 inhibitor, RGFP966, and demonstrated significant rescue of cerebellar neuro-architecture and motor coordination defects in perinatal hypothyroid mice.⁴² Therefore, collectively, these findings indicate that HDAC3 plays an important role in the cerebellar developmental defects induced by perinatal hypothyroidism. Further evidence for the role of epigenetic silencing of T₃-responsive developmental processes was observed in embryonic neocortical development, where delayed astrogliogenesis was associated with increased DNA methylation coupled with decreased histone acetylation at the Gfap promoter, leading to suppression of Gfap expression under maternal hypothyroidism.⁴³ Therefore, these studies provide an insight into the role of epigenetic silencing of neurodevelopmental genes under TH insufficiency and provide a proof-of-concept to use epigenetic modulators to rescue iodine deficiency/hypothyroidism-induced neurological deficit both within and perhaps even beyond the critical window of TH action, which remains to be explored.

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Figure 2:

HDAC inhibitor Valproate (VPA) restores neuronal architecture in the hypothyroid cerebellum. (a and b) Photomicrographs showing Golgi stained Purkinje neurons of the cerebellar region in each study group at different magnifications. Red arrows denote the dendritic tree structures of Purkinje cells. (a) Magnification level 40× and (b) magnification level 100×. (Reproduced by permission from: https://pubmed.ncbi.nlm.nih.gov/26427529/).

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