Periodic Reporting for period 4 - THYRAGE (Resetting the THYRoid axis for prevention of AGE-related diseases and co-morbidities)
Periodo di rendicontazione: 2020-07-01 al 2021-06-30
We addressed the hypothesis that inappropriate thyroid hormone action in target cells is a common mechanism underlying age-related degenerative diseases and co-morbidities. Although regulation of systemic thyroid status is well understood and underpins treatment of common thyroid disease, less is known about the importance of local regulation of thyroid hormone action in tissue development, homeostasis and repair. During evolution, this complex temporal and cell-specific regulation has been optimized for development and reproductive fitness but NOT for ageing. Humans with their exceptional longevity are thus exposed to a prolonged period of suboptimal local thyroid hormone action. In the population, thyroid status is a continuous variable that has been related to fracture risk, muscle mass and cognitive decline. In healthy longevity, thyroid status is characterized by thyroid stimulating hormone (TSH) in the upper half of the reference range. Here, we assessed how local regulation of thyroid hormone action controls tissue homeostasis and repair, and whether its dysregulation is a common mechanism underlying age-related disease. We focussed on osteoporosis, osteoarthritis, neuro-degeneration and sarcopenia as paradigm age-related disorders whose rising prevalence impose an increasing social and economic burden on European Societies. Using cutting-edge technology, our objectives were to (i) identify thyroid hormone dependent biomarkers for disease susceptibility in bone, cartilage, central nervous system, and skeletal muscle, (ii) manipulate cell-specific thyroid hormone action in these tissues, and (iii) develop cell-type specific modulators of thyroid hormone action.
Human studies assessed the effect of manipulation of thyroid status on markers of tissue maintenance and repair. We performed challenge studies with a low doses of TSH and T3 in participants from the Leiden Longevity Study. Results from TSH challenge study indicate that members from long-lived families have lower thyroidal response to TSH. Results from T3 challenge study point to similar T3 turnover and similar negative T3 feedback control of TSH secretion in members from long-lived families and controls. After the TSH and T3 challenges, transient increases in markers of bone turnover were observed, as well as a transient increase in IGF-1 and a decrease in cortisol with similar effects in members of long-lived families + controls. Subclinical hypothyroidism (SH) is biochemically defined by elevated circulating levels of TSH and normal circulating levels of thyroid hormone. P1 is PI in 2 collaborative randomized controlled trials that test effect of levothyroxine supplementation in older adults with SH. In line with lack of an effect on the primary endpoint and other secondary endpoints we observed no effect of levothyroxine on markers of bone turnover. These studies were complemented by several large human observational studies. In an individual participant data meta-analysis of observational studies subclinical hypo- and hyperthyroidism were not associated with cognitive function, cognitive decline or incident dementia. In Mendelian Randomization studies, we found no evidence that genetically determined TSH was associated with Bone Mineral Density, Diabetes or anaemia. In a mixed-methods project utilizing both Mendelian randomization and metabolomics data we found a potentially causal association between TSH and risk of coronary disease and robust associations between thyroid status and an unfavourable lipid profile. In mouse models, we used inducible cell-specific gene targeting approaches to manipulate cell-specific thyroid hormone action in adult bone, cartilage and skeletal muscle and determine the consequences in established provocation models of osteoporosis, osteoarthritis, and sarcopenia. Similarly, non-viral transfection of short hairpin RNA was employed to manipulate cell-specific thyroid hormone action in the neural stem cell niche to determine the consequences in established provocation models of neurodegenerative disease. Extensive pilot studies confirmed major bone and joint phenotypes in mice with over-expression of Dio3 and optimised the dose, route of administration and frequency of administration of tamoxifen for intervention studies using reporter mice as well as optimising recombination frequencies following activation of Cre. Robust power calculations informed experiments with conditional expression of Cre recombinase. Preliminary data analysis from comprehensive experiments indicate that cell-specific conditional over-expression of DIO3 in bone-forming osteoblasts and/or articular chondrocytes resulting in restricted cell-specific thyroid hormone deficiency prevents (i) ovariectomy-induced trabecular bone loss and (ii) prevents DMM surgical induction of osteoarthritis. These findings support the hypothesis that cell-specific manipulation of tissue thyroid status in skeletal tissues regulates the pathogenesis and outcome of chronic bone and joint damage in experimental models of osteoporosis and osteoarthritis. Studies examining the neural stem cell niche indicate that thyroid hormone signalling is preferentially required for neural stem cell (NSC) commitment towards neuronal precursor cells (while the absence of thyroid hormone signalling favours commitment towards oligodendrocyte precursor cells). In line, the neuronal lineage was found to be more sensitive to brain hypothyroidism induced by absence of TH transporters (Mct8/Oatp1c1 DKO mice) than the oligodendroglial lineage. Moreover, the TH-distributor protein Transthyretin (TTR) was found to be a crucial effector of TH signalling-driven NSC fate choice toward a neuronal fate. In studies examining the role of TH in skeletal muscle, it was observed that Dio3 overexpression in muscle resulting in reduced TH signalling in skeletal muscle myofibers was protective for muscle loss upon hind limb muscle denervation. Preliminary data indicate that attenuation of TH signaling in muscle satellite cells increased their activation, their proliferative potential and improved their engraftment. These data suggest that during the early phase of the muscle regeneration a local hypothyroidism is required to activate MuSCs and favor their proliferation.
P6 has successfully developed the chemistry for stable and labile linkage of the TH-antagonist NH3 to various tissue-specific peptides (incl GLP-1, NPY, secretin,calcitonin),as well as PEI polymer-NH3 conjugates.These compounds were tested by P2,P4,P5 in several different in-vitro cellular assays and in 1 in-vivo animal model (TH-indicator mice). P6 also successfully synthesized several peptide-rT3 conjugates (NPY-rT3, both labile and stable linkers) which are being evaluated for the in-vitro and in-vivo efficacy.
P6 has successfully developed the chemistry for stable and labile linkage of the TH-antagonist NH3 to various tissue-specific peptides (incl GLP-1, NPY, secretin,calcitonin),as well as PEI polymer-NH3 conjugates.These compounds were tested by P2,P4,P5 in several different in-vitro cellular assays and in 1 in-vivo animal model (TH-indicator mice). P6 also successfully synthesized several peptide-rT3 conjugates (NPY-rT3, both labile and stable linkers) which are being evaluated for the in-vitro and in-vivo efficacy.
By bringing together academic and industrial experts, THYRAGE integrated clinical research, basic science and drug development. Using methods that were well established in the laboratories of THYRAGE consortium partners, proof-of-principle studies comprised: (i) Manipulation of thyroid status, (ii) Cell-specific manipulation of thyroid hormone action, and (iii) Cell-specific delivery of thyroid hormone derivatives. This work has resulted in a better understanding of the role of different parameters of thyroid status and cell-specific thyroid hormone action in the pathophysiology of osteoporosis, osteoarthritis, neurological disorders and sarcopenia.