Herstellung und Analyse einer Maus mit gezielter Inaktivierung des TRH-DE-Gens

Thyrotropin-releasing hormone (TRH) was the first hypothalamic-hypophysiotrophic releasing factor to be identified. However, more than two-thirds of immunoreactive TRH in the central nervous system (CNS) is detected outside the thyrotrophic zone of the hypothalamus, suggesting a biological function far beyond the regulation of the thyroid axis. The functions of TRH in the CNS can be conceptualized as having four distinct components: the hypothalamichypophysiotrophic neuroendocrine system, the brainstem/midbrain/spinal cord system, the limbic/cortical system and the chronobiological system. It has been proposed that the main neurobiological function is to promote homeostasis, accomplished through neuronal mechanisms resident in these four integrated systems. This is consistent with the traditional role of TRH as a regulator of metabolic homeostasis. In an extracellular context this highly potent substance is exclusively inactivated by the TRH degrading ectoenzyme (TRH-DE). TRH-DE is not known to accept any other physiological relevant substrate. In the work at hand a knock-out mouse deficient in TRH-DE was generated to study the destabilizing effects on the general TRH homeostatic system. In the mutant mouse two reporters, bacterial β-galactosidase (lacZ) and human placental alcaline phosphatase (PLAP), were put under the direct control of the endogenous TRH-DE promotor to allow specific identification of cells and cellular projections. Recombinant mice were born with an expected Mendelian ratio and no overt phenotype was observed. Regulation of the thyroid axis in TRH-DE-/- mice resulted in a normal output of TSH and thyroid hormones. The mRNA levels of all hypophyseal hormones and in particular of TSH and prolactin were near to normal. However, in compensation for the missing ectoenzyme the expression of TRH receptor 1 (TRH-R1) was markedly downregulated in the pituitary. By contrast, mRNA levels of TRH-R1 remained the same in the CNS of mutants and controls while levels of TRH-R2 were significantly elevated in the mutant brain. When tested for wheelrunning activity the mutant mice exhibited a higher night-time activity in normal light/dark cycles and a reduced total activity in constant light. With mutants the onset of activity in normal light/dark cycles was observed exactly when the lights were switched off, while controls had an earlier onset when lights were still on. This phototropic characteristic of TRH-DE-/- mice was reflected by a strong presence of TRH-DE in photosensitive tissues as indicated by the reporters. This corresponds well with the involvement of TRH in the chronobiological system, though no direct defect in circadian rhythmicity was detected. Overall the findings of this work stressed the regulatory importance of TRH-DE in the central nervous TRH-signalling rather than its contribution to neuroendocrine function.