Specific Detection of Type III Iodothyronine Deiodinase Protein in Chicken Cerebellar Purkinje Cells

Abstract

Because iodothyronine deiodinases play a crucial role in the regulation of the available intracellular T(3) concentration, it is important to determine their cellular localization. In brain, the presence of type III iodothyronine deiodinase (D3) seems to be important to maintain homeostasis of T(3) levels. Until now, no cellular localization pattern of the D3 protein was reported in chicken brain. In this study polyclonal antisera were produced against specific peptides corresponding to the D3 amino acid sequence. Their use in immunocytochemistry led to the localization of D3 in the Purkinje cells of the chicken cerebellum. Both preimmune serum as well as the primary antiserum exhausted with the peptide itself were used as negative controls. Extracts of chick cerebellum and liver were made in the presence of Triton X-100 to solubilize the membrane-bound deiodinases. Using these extracts in Western blot analysis, a band of the expected molecular weight ( approximately 30 kDa) could be detected in both tissues. Using a full-length (32)P-labeled type III deiodinase cRNA probe, we identified a single mRNA species in the cerebellum that was of the exact same size as the hepatic control mRNA (+/-2.4 kb). RT-PCR, followed by subcloning and sequence analysis, confirmed the expression of D3 mRNA in the chicken cerebellum. In this study we provide the first evidence of the presence of the D3 protein in a neuronal cell type, namely Purkinje cells, by means of immunocytochemical staining. We were able to detect a protein fragment corresponding to the expected molecular mass (30 kDa) for type III deiodinase by means of Western blot analysis. RT-PCR as well as Northern blot analysis confirmed the presence of D3 mRNA in the cerebellum.

Type II iodothyronine deiodinase protein in chicken choroid plexus: additional perspectives on T3 supply in the avian brain

“…Furthermore, D2 mRNA was demonstrated in elongated cell clusters throughout the brain, comparable with the results recently described by Gereben et al (2004). Comparison of the D2 protein distribution with that of D1 and D3 protein within the choroid plexus using earlier developed antisera (Verhoelst et al 2002(Verhoelst et al , 2003, led to the conclusion that neither of the two other deiodinase proteins are present in the choroid plexus. The staining in the cuboidal cells therefore solely originates from the D2 protein.…”

“…Triiodothyronine is then supplied to the neurons, which mainly express D3, for further deiodination and inactivation of the thyroid hormones (Guadaño-Ferraz et al 1997). In chicken, it has been shown that D3 protein is expressed in the Purkinje cells of the cerebellum, again a proof of a neuronal D3 expression (Verhoelst et al 2002). However, the present study reports the presence of D2 in a nonneuronal cell type, namely the epithelial cells of the choroid plexus.…”

“…New Zealand White rabbits were injected every 3 weeks and blood collection occurred within 7-10 days after immunization (final bleeding after 8 injections). Serum was separated, snap frozen using a mixture of dry ice and ethanol and stored at 80 C. The same approach was used for the production of the antisera against chicken D1 and D3, as was described in detail by Verhoelst et al (2002Verhoelst et al ( , 2003. Possible cross-reactivity with the other deiodinases was checked by means of an immunospotting test, in which the three different peptides used for the generation of the respective antisera, namely peptide D1, D3a and the D2 peptide, were spotted on a nitrocellulose membrane.…”

“…Thereafter, the tissue slices were incubated with a 1:150 dilution of the primary D2 antiserum in TBS-Triton X-100 for 1 h at room temperature and overnight at 4 C. For the D2 antiserum, both preimmune and exhausted primary antiserum (exhaustion overnight, 4 C, 200 µg D2 peptide) were tested as negative controls. For the D1 (1:200) and D3 (1:200) antisera, preimmune serum served as a negative control, since exhaustion experiments were already described before (Verhoelst et al 2002(Verhoelst et al , 2003. The slices were washed twice for 10 min with TBS-Triton X-100.…”

Environmentally Induced Oxidative Stress and Disruption of Brain Thyroid Hormone Homeostasis in Autism Spectrum Disorders

“…D2 is localized mainly in the glial cells (Guadano-Ferraz et al, 1997), but the Purkinje cell localization has been observed during specific developmental periods (Verhoelst et al, 2005). D3 is localized mainly in neurons including the Purkinje cells (Verhoelst et al, 2002). D3 activity increases in hyperthyroidism, and decreases in hypothyroidism (Dratman et al, 1983) and is thought to protect neurons from excessive T3 levels.…”