Sen-ichi Furudate

Oxidoreductase Interactions Include a Role for ERp72 Engagement with Mutant Thyroglobulin from the rdw/rdw Rat Dwarf

Newly synthesized thyroglobulin (Tg), the secretory glycoprotein that serves as precursor in thyroid hormone synthesis, normally forms transient covalent protein complexes with oxidoreductases of the endoplasmic reticulum (ER). The Tg-G2320R mutation is responsible for congenital hypothyroidism in rdw/rdw rats, in which a lack of secondary thyroid enlargement (goiter) implicates death of thyrocytes as part of disease pathogenesis. We found that mutant Tg-G2320R was retained within the ER with no detectable synthesis of thyroxine, had persistent exposure of free cysteine thiols, and was associated with activated ER stress response but incomplete ER-associated degradation (ERAD). Tg-G2320R associated with multiple ER resident proteins, most notably ERp72, including covalent Tg-ERp72 interactions. In PC Cl3 thyrocytes, inducible overexpression of ERp72 increased the ability of cells to maintain Tg cysteines in a reduced state. Noncovalent interactions of several ER chaperones with newly synthesized Tg-G2320R diminished over time in parallel with ERAD of the mutant protein, yet a small ERAD-resistant Tg fraction remained engaged in covalent association with ERp72 even 2 days post-synthesis. Such covalent protein aggregates may set the stage for apoptotic thyrocyte cell death, preventing thyroid goiter formation in rdw/rdw rats.

Missing secretory granules, dilated endoplasmic reticulum, and nuclear dislocation in the thyroid gland of rdw rats with hereditary dwarfism

Previous studies on the rdw rat have suggested that its dwarfism is caused primarily by dysfunction of the thyroid gland. In this study, rat thyroid glands were analyzed endocrinologically and morphologically to clarify the primary cause of dwarfism in the rdw rat. The rdw rat showed lowered thyroid hormone (T4 and T3) levels but elevated TSH in serum. The rdw thyroid gland was almost proportional in size and it was not goiter in gross inspection. Our histological investigation produced three results that may lend important evidence in understanding the problem in the thyroid gland of rdw rats. First of all, secretory granules could not be detected in the follicular epithelial cells of the rdw. Secondly, thyroglobulin was found at very low levels in the follicular lumen by immunohistochemical analysis. In contrast, it could be detected in a substantial quantity inside the dilated rER and in the huge vacuoles that are formed by swelling of the rough endoplasmic reticulum (rER) at the basal side of the follicular epithelial cells. Additionally, the nucleus of the follicular epithelial cells was pressed to the luminal side by the enlarged rER. These morphological changes would indicate that the transport of thyroglobulin is stopped at or before the formation of the secretory granules and thyroglobulin is not secreted into the follicular lumen. The rdw characterization strongly supports that rdw dwarfism is induced by hypothyroidism due to some defect(s) in the thyroid gland. Anat Rec 259:60–66, 2000.

Developmental delay and unstable state of the testes in the rdw rat with congenital hypothyroidism

From the present study of the rdw rat, it is clear that the thyroid hormone is essential for the development and maintenance of the testes. In previous studies, the thyroid hormone has few serious effects on the testes except during the neonatal stage when the thyroid hormone receptor is mainly present. However, there is little knowledge concerning the prolonged effect of thyroid hormone deficiency throughout the rats life span. In the present study, a morphological analysis was performed on the testes of rdw rats with congenital hypothyroidism. The rdw testes required a longer time to develop into the normal adult structure. Moreover, the developed, normal structure began to degenerate after full maturation. Specific characteristics of the rdw testes include: (i) a prolonged proliferation of Sertoli cells during postnatal development; (ii) a developmental delay in the appearance of spermatocytes and spermatid; (iii) direct contact with each other for both spermatocytes and spermatids, without Sertoli cell cytoplasm completely intervening between adjacent germ cells; (iv) subsequent apoptosis of germ cells after maturation; (v) reduction in the height of the seminiferous epithelium; and (vi) lower testosterone levels in the rdw rats, especially during old age. Thus, we conclude that the thyroid hormone plays an important role in developing and maintaining normal function of testes.

Altered Cerebellum Development and Dopamine Distribution in a Rat Genetic Model with Congenital Hypothyroidism

Thyroid hormones play crucial roles in the development and functional maintenance of the central nervous system. Despite extensive studies of the neural function of thyroid hormones, little is known about the effects of hypothyroidism on behavioural traits and the mechanisms underlying such effects. In the present study, we report an investigation of congenitally hypothyroid mutant rdw rats, revealing a novel function of thyroid hormones in the central nervous system. The rdw rats were subjected to behavioural analyses such as the rotarod test, open field test and circadian activity measurement. To determine the cause of behavioural disorders, cerebellar morphogenesis was examined by immunohistochemical analysis, and the axonal transport of dopamine in the nigrostriatal pathway was analysed by high‐performance liquid chromatography and western blotting. The effects of thyroxine administration to the rdw rats were examined by behavioural analysis. The rdw rats showed severe impairment of motor coordination and balance. This could be explained by the fact that the rats showed severe retardation of cerebellar morphogenesis, which correlates with the small somata and poor dendritic arborisation of Purkinje cells and retarded migration of granule cells particularly during the first two postnatal weeks. Moreover, the rdw rats showed hypoactivity, characterised by decreased circadian locomotor activity. After weaning, thyroxine administration improved the dwarfism in rdw rats but had no effect on cerebellar function. In addition, the rdw rats showed anxiety and depression intrinsically to novel surroundings. Interestingly, the rdw rats showed high levels of dopamine in the substantia nigra and low levels in the striatum, an important centre for the coordination of behaviour. Furthermore, low levels of tubulin in the striatum were detected, indicating the aberrant axonal transport of dopamine in the nigrostriatal pathway as a result of the reduced delivery of microtubules. These findings indicate an important function of thyroid hormones in cerebellar formation and in the regulation of axonal transport of dopamine. Moreover, rdw rats will be useful for studies of brain function and behavioural disorders in congenital hypothyroidism.

Dopamine D2-Like Receptor Function is Converted from Excitatory to Inhibitory by Thyroxine in the Developmental Hippocampus

The mechanism by which a lack of thyroid hormone in the early development of the brain causes permanent mental retardation in cretins is currently unknown. On the other hand, an abnormality in dopamine‐related brain function is believed to underlie some forms of mental illness. In this study, we demonstrate that although the activation of a dopaminergic D2‐like receptor inhibited glutamatergic transmission in the hippocampal slices of normal adult rats, indicating the inhibitory action of the D2‐like receptor on glutamatergic transmission, it markedly enhanced glutamatergic transmission both in a mutant hypothyroid rat with a missense mutation in thyroglobulin and in hypothyroid rats treated with methylmercaptoimidazole (MMI), indicating the excitatory action of the D2‐like receptor on glutamatergic transmission. Paired pulse facilitation of field excitatory postsynaptic potentials was reduced by the activation of the D2‐like receptors from MMI‐induced hypothyroid rats, suggesting a presynaptic locus of the excitatory action of the D2‐like receptors. In normal rats, the excitatory D2‐like dopamine receptors were observed in the developing stages and were completely replaced by normal inhibitory responses up to adulthood. Furthermore, the continuous supplement of thyroxine from birth exerted a normalising effect on the abnormal excitatory property of D2‐like dopamine receptors in the hippocampal slices of MMI‐treated hypothyroid rats. From these results, it is suggested that thyroxine may play a crucial role in reversing the excitatory property of D2‐like dopaminergic receptors in the immature brain to an inhibitory one in the mature brain. Moreover, we suggest that the abnormal excitatory property of D2‐like dopaminergic receptors may develop in response to a lack of thyroxine and may contribute to some central nervous system deficits, including cognitive dysfunctions accompanied by hypothyroidism.

Disease proteomics of endocrine disorders revealed by two-dimensional gel electrophoresis and mass spectrometry.

Endocrine disorders such as dwarfism and diabetes show abnormalities in many different organs even if a certain hormone is the primary cause of the disease. One of the aims of proteomics is to elucidate an abnormal hormone network underlying dysfunction in the disease through quantitative and qualitative proteome analyses of various organs. In a comprehensive study of the rdw rat with hereditary dwarfism, we found the accumulation of ER proteins in the rdw thyroid. Contrary to the initial notion that the dwarfism of the rat was caused by genetic mutations related to pituitary hormones, the primary cause is a missense mutation in the thyroglobulin gene. To understand at the protein level cellular damage caused by oxidative stress, we developed a proteomic method and applied to detecting protein carbonyls in various organs of a diabetes model OLETF rat. The method would provide a means toward clarifying a comprehensive view of oxidative modifications of proteins in diabetes. We review 2‐DE‐based disease proteomics of endocrine disorders in general, with particular attention paid to our proteome projects by a 2‐DE method with an agarose IEF gel in the first dimension (agarose 2‐DE) and LC‐MS/MS.

A simplified procedure for purification of cytochrome P-450 by preparative ampholine gel for isoelectric focusing.

ABSTRACT The purification process for cytochrome P-450 is very complicated, involving five or more column chromatography steps for the final preparation. This paper describes a reduction in the number of the steps; it can be easily purified from pig testis microsomes with improved the yield. As the first step, DEAE-Toyopearl column chromatography is performed only once and then, as the second step, the partially purified cytochrome P-450 is completely purified by a preparative Ampholine PAG-plate Gel for Isoelectric Focusing. The combination reduced the purification to a two-step procedure.