Thyroid hormone analogues: Promising therapeutic avenues to improve the neurodevelopmental outcomes of intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a pregnancy complication impairing fetal growth and development. The compromised development is often attributed to disruptions of oxygen and nutrient supply from the placenta, resulting in a number of unfavourable physiological outcomes with impaired brain and organ growth. IUGR is associated with compromised development of both grey and white matter, predisposing the infant to adverse neurodevelopmental outcomes, including long-lasting cognitive and motor difficulties. Cerebral thyroid hormone (TH) signalling, which plays a crucial role in regulating white and grey matter development, is dysregulated in IUGR, potentially contributing to the neurodevelopmental delays associated with this condition. Notably, one of the major TH transporters, monocarboxylate transporter-8 (MCT8), is deficient in the fetal IUGR brain. Currently, no effective treatment to prevent or reverse IUGR exists. Management strategies involve close antenatal monitoring, management of maternal risk factors if present and early delivery if IUGR is found to be severe or worsening in utero. The overall goal is to determine the most appropriate time for delivery, balancing the risks of preterm birth with further fetal compromise due to IUGR. Drug candidates have shown either adverse effects or little to no benefits in this vulnerable population, urging further preclinical and clinical investigation to establish effective therapies. In this review, we discuss the major neuropathology of IUGR driven by uteroplacental insufficiency and the concomitant long-term neurobehavioural impairments in individuals born IUGR. Importantly, we review the existing clinical and preclinical literature on cerebral TH signalling deficits, particularly the impaired expression of MCT8 and their correlation with IUGR. Lastly, we discuss the current evidence on MCT8-independent TH analogues which mimic the brain actions of THs by being metabolised in a similar manner as promising, albeit underappreciated approaches to promote grey and white matter development and improve the neurobehavioural outcomes following IUGR.

Role of thyroid hormones in normal and abnormal central nervous system myelination in humans and rodents.

Thyroid hormones (THs) are instrumental in promoting the molecular mechanisms which underlie the complex nature of neural development and function within the central nervous system (CNS) in vertebrates. The key neurodevelopmental process of myelination is conserved between humans and rodents, of which both experience peak fetal TH concentrations concomitant with onset of myelination. The importance of supplying adequate levels of THs to the myelin producing cells, the oligodendrocytes, for promoting their maturation is crucial for proper neural function. In this review we examine the key TH distributor and transport proteins, including transthyretin (TTR) and monocarboxylate transporter 8 (MCT8), essential for supporting proper oligodendrocyte and myelin health; and discuss disorders with impaired TH signalling in relation to abnormal CNS myelination in humans and rodents. Furthermore, we explore the importance of using novel TH analogues in the treatment of myelination disorders associated with abnormal TH signalling.

Structural Analysis of the Complex of Human Transthyretin with 3',5'-Dichlorophenylanthranilic Acid at 1.5 Å Resolution.

Human transthyretin (hTTR) can form amyloid deposits that accumulate in nerves and organs, disrupting cellular function. Molecules such as tafamidis that bind to and stabilize the TTR tetramer can reduce such amyloid formation. Here, we studied the interaction of VCP-6 (2-((3,5-dichlorophenyl)amino)benzoic acid) with hTTR. VCP-6 binds to hTTR with 5 times the affinity of the cognate ligand, thyroxine (T4). The structure of the hTTR:VCP-6 complex was determined by X-ray crystallography at 1.52 Šresolution. VCP-6 binds deeper in the binding channel than T4 with the 3',5'-dichlorophenyl ring binding in the 'forward' mode towards the channel centre. The dichlorophenyl ring lies along the 2-fold axis coincident with the channel centre, while the 2-carboxylatephenylamine ring of VCP-6 is symmetrically displaced from the 2-fold axis, allowing the 2-carboxylate group to form a tight intermolecular hydrogen bond with Nζ of Lys15 and an intramolecular hydrogen bond with the amine of VCP-6, stabilizing its conformation and explaining the greater affinity of VCP-6 compared to T4. This arrangement maintains optimal halogen bonding interactions in the binding sites, via chlorine atoms rather than iodine of the thyroid hormone, thereby explaining why the dichloro substitution pattern is a stronger binder than either the diiodo or dibromo analogues.

A Comparative Review of Mixed Mammary Tumors in Mammals.

Mixed tumors are characterized by the histological identification of two or more cell types. Commonly, a mixture of epithelial and myoepithelial cells is included in abundant stroma, which can consist of myxoid, chondroid or bony matrices. Spontaneously arising mixed tumors are rare lesions in the human breast but are common in human salivary glands and canine mammary glands. Subtle histopathological characteristics and overlapping attributes of malignant lesions with other benign lesions can lead to a diagnostic challenge. Mixed tumors can present as benign or malignant. While malignant mixed tumors are quite rare in the human breast they have a poor prognosis. Benign mixed mammary tumors occur more frequently in female dogs than in humans and are usually associated with a good prognosis. This review will provide a comprehensive overview of mixed mammary tumors, across various mammalian species.

The affinity of transthyretin for T3 or T4 does not determine which form of the hormone accumulates in the choroid plexus.

Normal development of the brain is dependent on the required amounts of thyroid hormones (THs) reaching specific regions of the brain during each stage of ontogeny. Many proteins are involved with regulation of TH bioavailability in the brain: the TH distributor protein transthyretin (TTR), TH transmembrane transporters (e.g. MCT8, MCT10, LAT1, OATP1C1) and deiodinases (D1, D2 and D3) which either activate or inactivate THs. Previous studies revealed that in mammals, T4, but not T3, accumulated in the choroid plexus and then entered the cerebrospinal fluid. In all mammalian species studied so far, TTR binds T4 with higher affinity than T3, whereas TTR in non-mammalian vertebrates binds T3 with higher affinity than T4. We investigated if the form of TH preferentially bound by TTR influenced the form of the TH that accumulated in the choroid plexus and consequently other areas of the brain. We measured the mRNA levels corresponding to TTR, MCT8, MCT10, LAT1, OATP1C1, D1, D2 and D3 in the brains of chickens at 11days post-hatching. TTR, D3 and OATP1C1 expression were found to be highly concentrated in the choroid plexus. D1, MCT8 and MCT10 mRNA levels were slightly greater in the choroid plexus than in other areas of the brain while D2 mRNA levels were lower. LAT1 mRNA was evenly expressed throughout the brain. Therefore, the choroid plexus appears to be a structure which exhibits sophisticated control of TH levels within the brain. We also measured the uptake of intravenously injected 125I-T3 and 125I-T4 into brains of chickens of the same age. 125I-T4 but not 125I-T3 accumulated in the choroid plexus and optic lobes. Therefore, the form of TH preferentially bound by TTR does not determine the form of TH that accumulates in the choroid plexus and other areas of the brain. As for mammals, T3 present in the avian brain therefore seems mainly produced locally by conversion of T4 into T3 by D2.

Expression of thyroid hormone transporters and deiodinases at the brain barriers in the embryonic chicken: Insights into the regulation of thyroid hormone availability during neurodevelopment.

Thyroid hormones (THs) are key regulators in the development of the vertebrate brain. Therefore, TH access to the developing brain needs to be strictly regulated. The brain barriers separate the central nervous system from the rest of the body and impose specific transport mechanisms on the exchange of molecules between the general circulation and the nervous system. As such they form ideal structures for regulating TH exchange between the blood and the brain. To investigate the mechanism by which the developing brain regulates TH availability, we investigated the ontogenetic expression profiles of TH transporters, deiodinases and the TH distributor protein transthyretin (TTR) at the brain barriers during embryonic and early postnatal development using the chicken as a model. In situ hybridisation revealed expression of the TH transporters monocarboxylate transporter 8 (MCT8) and 10 (MCT10), organic anion transporting polypeptide 1C1 (OATP1C1) and L-type amino acid transporter 1 (LAT1) and the inactivating type 3 deiodinase (D3) in the choroid plexus which forms the blood-cerebrospinal fluid barrier. This was confirmed by quantitative PCR which additionally indicated strongly increasing expression of TTR as well as detectable expression of the activating type 2 deiodinase (D2) and the (in)activating type 1 deiodinase (D1). In the brain capillaries forming the blood-brain barrier in situ hybridisation showed exclusive expression of LAT1 and D2. The combined presence of LAT1 and D2 in brain capillaries suggests that the blood-brain barrier forms the main route for receptor-active T3 uptake into the embryonic chicken brain. Expression of multiple transporters, deiodinases and TTR in the choroid plexus indicates that the blood-cerebrospinal fluid barrier is also important in regulating early TH availability. The impact of these barrier systems can be deduced from the clear difference in T3 and T4 levels as well as the T3/T4 ratio between the developing brain and the general circulation. We conclude that the tight regulation of TH exchange at the brain barriers from early embryonic stages is one of the factors needed to allow the brain to develop within a relative microenvironment.

Delayed development of specific thyroid hormone-regulated events in transthyretin null mice.

Thyroid hormones (THs) are vital for normal postnatal development. Extracellular TH distributor proteins create an intravascular reservoir of THs. Transthyretin (TTR) is a TH distributor protein in the circulatory system and is the only TH distributor protein synthesized in the central nervous system. We investigated the phenotype of TTR null mice during development. Total and free 3',5',3,5-tetraiodo-L-thyronine (T(4)) and free 3',3,5-triiodo-L-thyronine (T(3)) in plasma were significantly reduced in 14-day-old (P14) TTR null mice. TTR null mice also displayed a delayed suckling-to-weaning transition, decreased muscle mass, delayed growth, and retarded longitudinal bone growth. In addition, ileums from postnatal day 0 (P0) TTR null mice displayed disordered architecture and contained fewer goblet cells than wild type. Protein concentrations in cerebrospinal fluid from P0 and P14 TTR null mice were higher than in age-matched wild-type mice. In contrast to the current literature based on analyses of adult TTR null mice, our results demonstrate that TTR has an important and nonredundant role in influencing the development of several organs.

Expression of Wnt signaling skeletal development genes in the cartilaginous fish, elephant shark (Callorhinchus milii).

Jawed vertebrates (Gnasthostomes) are broadly separated into cartilaginous fishes (Chondricthyes) and bony vertebrates (Osteichthyes). Cartilaginous fishes are divided into chimaeras (e.g. ratfish, rabbit fish and elephant shark) and elasmobranchs (e.g. sharks, rays and skates). Both cartilaginous fish and bony vertebrates are believed to have a common armoured bony ancestor (Class Placodermi), however cartilaginous fish are believed to have lost bone. This study has identified and investigated genes involved in skeletal development in vertebrates, in the cartilaginous fish, elephant shark (Callorhinchus milii). Ctnnb1 (ß-catenin), Sfrp (secreted frizzled protein) and a single Sost or Sostdc1 gene (sclerostin or sclerostin domain-containing protein 1) were identified in the elephant shark genome and found to be expressed in a number of tissues, including cartilage. ß-catenin was also localized in several elephant shark tissues. The expression of these genes, which belong to the Wnt/ß-catenin pathway, is required for normal bone formation in mammals. These findings in the cartilaginous skeleton of elephant shark support the hypothesis that the common ancestor of cartilaginous fishes and bony vertebrates had the potential for making bone.

Deficiency of 5-hydroxyisourate hydrolase causes hepatomegaly and hepatocellular carcinoma in mice.

With the notable exception of humans, uric acid is degraded to (S)-allantoin in a biochemical pathway catalyzed by urate oxidase, 5-hydroxyisourate (HIU) hydrolase, and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase in most vertebrate species. A point mutation in the gene encoding mouse HIU hydrolase, Urah, that perturbed uric acid metabolism within the liver was discovered during a mutagenesis screen in mice. The predicted substitution of cysteine for tyrosine in a conserved helical region of the mutant-encoded HIU hydrolase resulted in undetectable protein expression. Mice homozygous for this mutation developed elevated platelet counts secondary to excess thrombopoietin production and hepatomegaly. The majority of homozygous mutant mice also developed hepatocellular carcinoma, and tumor development was accelerated by exposure to radiation. The development of hepatomegaly and liver tumors in mice lacking Urah suggests that uric acid metabolites may be toxic and that urate oxidase activity without HIU hydrolase function may affect liver growth and transformation. The absence of HIU hydrolase in humans predicts slowed metabolism of HIU after clinical administration of exogenous urate oxidase in conditions of uric acid-related pathology. The data suggest that prolonged urate oxidase therapy should be combined with careful assessment of toxicity associated with extrahepatic production of uric acid metabolites.

Enhanced re-myelination in transthyretin null mice following cuprizone mediated demyelination.

Thyroid hormones (THs) impact nearly every tissue in the body, including the adult and developing central nervous system. The distribution of THs around the body is facilitated by specific TH distributor proteins including transthyretin (TTR). In addition to being produced in the liver, TTR is synthesized in the choroid plexus of the brain. The synthesis of TTR by choroid plexus epithelial cells allows transport of THs from the blood into the brain. Adequate supply of THs to the brain is required for developmental myelination of axons and the maintenance of mature myelin throughout adult life, essential for the proper conduction of nerve impulses. Insufficient THs in developing mice results in hypo-myelination (thinner myelin around axons). However, confounding evidence demonstrated that in developing brain of TTR null mice, hyper-myelination of axons was observed in the corpus callosum. This raised the question whether increased myelination occurs during re-myelination in the adult brain following targeted demyelination. To investigate the effect of TTR during re-myelination, cuprizone induced depletion of myelin in the corpus callosum of adult mice was initiated, followed by a period of myelin repair. Myelin thickness was measured to assess re-myelination rates for 6 weeks. TTR null mice displayed expedited rates of early re-myelination, preferentially re-myelinating smaller axons compared to those of wild type mice. Furthermore, TTR null mice produced thicker myelin than wild type mice during re-myelination. These results may have broader implications in understanding mechanisms governing re-myelination, particularly in potential therapeutic contexts for acquired demyelinating diseases such as multiple sclerosis.

Evolution of the parathyroid hormone family and skeletal formation pathways.

Bone is considered to be a feature of higher vertebrates and one of the features that was required for the movement from water onto land. But there are a number of evolutionarily important species that have cartilaginous skeletons, including sharks. Both bony and cartilaginous fish are believed to have a common ancestor who had a bony skeleton. A number of factors and pathways have been shown to be involved in the development and maintenance of bony skeleton including the Wnt pathway and the parathyroid hormone gene family. The study of these pathways and factors in cartilaginous animals may shed light on the evolution of the vertebrate skeleton.

Maturation Process, Nutritional Profile, Bioactivities and Utilisation in Food Products of Red Pitaya Fruits: A Review.

Red pitaya (Hylocereus polyrhizus, red pulp with pink peel), also known as dragon fruit, is a well-known species of pitaya fruit. Pitaya seeds and peels have been reported to exhibit higher concentrations of total polyphenols, beta-cyanins and amino acid than pulp, while anthocyanins (i.e., cyanidin 3-glucoside, delphinidin 3-glucoside and pelargonidin 3-glucoside) were only detected in the pulp extracts. Beta-cyanins, phenolics and flavonoids were found to increase gradually during fruit maturation and pigmentation appeared earlier in the pulp than peel. The phytochemicals were extracted and purified by various techniques and broadly used as natural, low-cost, and beneficial healthy compounds in foods, including bakery, wine, dairy, meat and confectionery products. These bioactive components also exhibit regulative influences on the human gut microbiota, glycaemic response, lipid accumulation, inflammation, growth of microbials and mutagenicity, but the mechanisms are yet to be understood. The objective of this study was to systematically summarise the effect of red pitaya's maturation process on the nutritional profile and techno-functionality in a variety of food products. The findings of this review provide valuable suggestions for the red pitaya fruit processing industry, leading to novel formulations supported by molecular research.

Roles of Parathyroid Hormone-Related Protein (PTHrP) and Its Receptor (PTHR1) in Normal and Tumor Tissues: Focus on Their Roles in Osteosarcoma.

Osteosarcoma (OS) is the most common primary bone tumor and originates from bone forming mesenchymal cells and primarily affects children and adolescents. The 5-year survival rate for OS is 60 to 65%, with little improvement in prognosis during the last four decades. Studies have demonstrated the evolving roles of parathyroid hormone-related protein (PTHrP) and its receptor (PTHR1) in bone formation, bone remodeling, regulation of calcium transport from blood to milk, regulation of maternal calcium transport to the fetus and reabsorption of calcium in kidneys. These two molecules also play critical roles in the development, progression and metastasis of several tumors such as breast cancer, lung carcinoma, chondrosarcoma, squamous cell carcinoma, melanoma and OS. The protein expression of both PTHrP and PTHR1 have been demonstrated in OS, and their functions and proposed signaling pathways have been investigated yet their roles in OS have not been fully elucidated. This review aims to discuss the latest research with PTHrP and PTHR1 in OS tumorigenesis and possible mechanistic pathways. This review is dedicated to Professor Michael Day who died in May 2020 and was a very generous collaborator.

The Role of Transthyretin in Oligodendrocyte Development.

Transthyretin (TTR) is a protein that binds and distributes thyroid hormones (THs) in blood and cerebrospinal fluid. Previously, two reports identified TTR null mice as hypothyroid in the central nervous system (CNS). This prompted our investigations into developmentally regulated TH-dependent processes in brains of wildtype and TTR null mice. Despite logical expectations of a hypomyelinating phenotype in the CNS of TTR null mice, we observed a hypermyelination phenotype, synchronous with an increase in the density of oligodendrocytes in the corpus callosum and anterior commissure of TTR null mice during postnatal development. Furthermore, absence of TTR enhanced proliferation and migration of OPCs with decreased apoptosis. Neural stem cells (NSCs) isolated from the subventricular zone of TTR null mice at P21 revealed that the absence of TTR promoted NSC differentiation toward a glial lineage. Importantly, we identified TTR synthesis in OPCs, suggestive of an alternate biological function in these cells that may extend beyond an extracellular TH-distributor protein. The hypermyelination mechanism may involve increased pAKT (involved in oligodendrocyte maturation) in TTR null mice. Elucidating the regulatory role of TTR in NSC and OPC biology could lead to potential therapeutic strategies for the treatment of acquired demyelinating diseases.

Parathyroid hormone receptor 1 (PTHR1) is a prognostic indicator in canine osteosarcoma.

Osteosarcoma (OS) is the most common malignant primary bone tumour in humans and dogs. Several studies have established the vital role of parathyroid hormone-related protein (PTHrP) and its receptor (PTHR1) in bone formation and remodeling. In addition, these molecules play a role in the progression and metastasis of many human tumour types. This study investigated the expression of PTHR1 and PTHrP in canine OS tissues and assessed their prognostic value. Formalin-fixed, paraffin-embedded tissue samples from 50 dogs diagnosed with primary OS were immunolabeled with antibodies specific for PTHR1 and PTHrP. The immunostaining intensity of tumours from patients with OS was correlated with survival time. Both PTHR1 and PTHrP were detected in all OS samples (n = 50). Dogs with OS tumours showing high immunostaining intensity for PTHR1 (n = 36) had significantly shorter survival times (p = 0.028, Log Rank; p = 0.04, Cox regression) when compared with OS that had low immunostaining intensity for PTHR1 (n = 14).PTHrP immunostaining intensity did not correlate with survival time (p > 0.05). The results of this study indicate that increased expression of PTHR1 antigen in canine OS is associated with poor prognosis. This suggests that PTHR1 may be useful as a prognostic indicator in canine OS.

Thyroid Hormone Distributor Proteins During Development in Vertebrates.

Thyroid hormones (THs) are ancient hormones that not only influence the growth, development and metabolism of vertebrates but also affect the metabolism of (at least some) bacteria. Synthesized in the thyroid gland (or follicular cells in fish not having a discrete thyroid gland), THs can act on target cells by genomic or non-genomic mechanisms. Either way, THs need to get from their site of synthesis to their target cells throughout the body. Despite being amphipathic in structure, THs are lipophilic and hence do not freely diffuse in the aqueous environments of blood or cerebrospinal fluid (in contrast to hydrophilic hormones). TH Distributor Proteins (THDPs) have evolved to enable the efficient distribution of THs in the blood and cerebrospinal fluid. In humans, the THDPs are albumin, transthyretin (TTR), and thyroxine-binding globulin (TBG). These three proteins have distinct patterns of regulation in both ontogeny and phylogeny. During development, an additional THDP with higher affinity than those in the adult, is present during the stage of peak TH concentrations in blood. Although TTR is the only THDP synthesized in the central nervous system (CNS), all THDPs from blood are present in the CSF (for each species). However, the ratio of albumin to TTR differs in the CSF compared to the blood. Humans lacking albumin or TBG have been reported and can be asymptomatic, however a human lacking TTR has not been documented. Conversely, there are many diseases either caused by TTR or that have altered levels of TTR in the blood or CSF associated with them. The first world-wide RNAi therapy has just been approved for TTR amyloidosis.

Gender-specific effects of transthyretin on neural stem cell fate in the subventricular zone of the adult mouse.

Choroid plexus epithelial cells produce and secrete transthyretin (TTR). TTR binds and distributes thyroid hormone (TH) to brain cells via the cerebrospinal fluid. The adult murine subventricular zone (SVZ) is in close proximity to the choroid plexus. In the SVZ, TH determines neural stem cell (NSC) fate towards a neuronal or a glial cell. We investigated whether the loss of TTR also disrupted NSC fate choice. Our results show a decreased neurogenic versus oligodendrogenic balance in the lateroventral SVZ of Ttr knockout mice. This balance was also decreased in the dorsal SVZ, but only in Ttr knockout male mice, concomitant with an increased oligodendrocyte precursor density in the corpus callosum. Quantitative RTqPCR analysis following FACS-dissected SVZs, or marked-coupled microbeads sorting of in vitro neurospheres, showed elevated Ttr mRNA levels in neuronal cells, as compared to uncommitted precursor and glial cells. However, TTR protein was undetectable in vivo using immunostaining, and this despite the presence of Ttr mRNA-expressing SVZ cells. Altogether, our data demonstrate that TTR is an important factor in SVZ neuro- and oligodendrogenesis. They also reveal important gender-specific differences and spatial heterogeneity, providing new avenues for stimulating endogenous repair in neurodegenerative diseases.

Cell and molecular biology of transthyretin and thyroid hormones.

Advances in four areas of transthyretin (TTR) research result in this being a timely review. Developmental studies have revealed that TTR is synthesized in all classes of vertebrates during development. This leads to a new hypothesis on selection pressure for hepatic TTR synthesis during development only, changing the previous hypotheses from "onset" of hepatic TTR synthesis in adulthood to "maintaining" hepatic TTR synthesis into adulthood. Evolutionary studies have revealed the existence of TTR-like proteins (TLPs) in nonvertebrate species and elucidated some of their functions. Consequently, TTR is an excellent model for the study of the evolution of protein structure, function, and localization. Studies of human diseases have demonstrated that TTR in the cerebrospinal fluid can form amyloid, but more recently there has been recognition of the roles of TTR in depression and Alzheimer's disease. Furthermore, amyloid mutations in human TTR that are the normal residues in other species result in cardiac deposition of TTR amyloid in humans. Finally, a revised model for TTR-thyroxine entry into the cerebrospinal fluid via the choroid plexus, based on data from studies in TTR null mice, is presented. This review concentrates on TTR and its thyroid hormone binding, in development and during evolution, and summarizes what is currently known about TLPs and the role of TTR in diseases affecting the brain.

The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase.

The mechanism of binding of thyroid hormones by the transport protein transthyretin (TTR) in vertebrates is structurally well characterised. However, a homologous family of transthyretin-like proteins (TLPs) present in bacteria as well as eukaryotes do not bind thyroid hormones, instead they are postulated to perform a role in the purine degradation pathway and function as 5-hydroxyisourate hydrolases. Here we describe the 2.5 Angstroms X-ray crystal structure of the TLP from the Gram-negative bacterium Salmonella dublin, and compare and contrast its structure with vertebrate TTRs. The overall architecture of the homotetramer is conserved and, despite low sequence homology with vertebrate TTRs, structural differences within the monomer are restricted to flexible loop regions. However, sequence variation at the dimer-dimer interface has profound consequences for the ligand binding site and provides a structural rationalisation for the absence of thyroid hormone binding affinity in bacterial TLPs: the deep, negatively charged thyroxine-binding pocket that characterises vertebrate TTR contrasts with a shallow and elongated, positively charged cleft in S. dublin TLP. We have demonstrated that Sdu_TLP is a 5-hydroxyisourate hydrolase. Furthermore, using site-directed mutagenesis, we have identified three conserved residues located in this cleft that are critical to the enzyme activity. Together our data reveal that the active site of Sdu_TLP corresponds to the thyroxine binding site in TTRs.