Thyroid hormone (TH) is essential for the regulation of many physiological processes, especially growth, organ development, energy metabolism and cardiovascular effects. TH acts via the TH receptors (TR) α and β. By binding to thyroid hormone responsive elements (TREs) on the DNA, TRs regulate expression of TH target genes. Thus, TRs are mainly characterized as ligand dependent transcription factors and regulation of gene expression and protein synthesis is considered the canonical mode of TH/TR action. The demonstration that the ligand-bound TRs α and β also mediate activation of the phosphatidylinositol-3-kinase (PI3K) pathway established noncanonical TH/TR action as an additional mode of TH signaling. Recently, TR mutant mouse models allowed to determine the underlying mode of TH/TR action, either canonical or noncanonical TH/TR signaling, for several physiological TH effects in vivo: Regulation of the hypothalamic-pituitary-thyroid axis requires DNA-binding of TRβ, whereas hepatic triglyceride content appears to be regulated by noncanonical TRβ signaling. TRα mediated effects in bone development are dependent on DNA-binding, whereas several cardiovascular TRα effects are rapid and independent from DNA-binding. Therefore, noncanonical TH/TR action contributes to the overall effects of TH in physiology.

Background Hypothyroidism impairs cardiovascular health and contributes to endothelial dysfunction with reduced vasodilation. How triiodothyronine (T3) and its receptors are involved in the regulation of vasomotion is not yet fully understood. In general, thyroid hormone receptors (TRs) either influence gene expression (canonical action) or rapidly activate intracellular signaling pathways (noncanonical action). Here we aimed to characterize the T3 action underlying the mechanism of arterial vasodilation and blood pressure regulation. Methods Mesenteric arteries were isolated from male rats, wildtype (WT) mice, TRα knockout (TRα 0) mice and from knock-in mice with a mutation in the DNA-binding domain (TRα GS). In this mutant, DNA-binding and, thus, canonical action is abrogated while noncanonical signaling is preserved. In a wire myograph system, the isolated vessels were pre-constricted with norepinephrine. The response to T3 was measured, and the resulting vasodilation (Δ force [mN]) was normalized to maximum contraction with norepinephrine and expressed as percent vasodilation after maximal pre-constriction with norepinephrine (% NE). Isolated vessels were treated with T3 (1x10 -15 to 1x10 -5 mol/L) alone and in combination with the endothelial NO-synthase (eNOS) inhibitor L-NG-Nitroarginine methyl ester (L-NAME) or the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. The endothelium was removed to determine the contribution of T3 to endothelium-dependent vasodilation. The physiological relevance of T3-induced vasodilation was determined by in vivo arterial blood pressure measurements in male and female mice. Results T3 treatment induced vasodilation of mesenteric arteries from WT mice within 2 minutes (by 21.5±1.7% NE). This effect was absent in arteries from TRα 0 mice (by 5.3±0.6% NE, P<0.0001 vs. WT) but preserved in TRαGS arteries (by 17.2±1.1% NE, n.s. vs. WT). Inhibition of either eNOS or PI3K reduced T3-mediated vasodilation from 52.7±4.5% NE to 28.5±4.1% NE and 22.7±2.9% NE, respectively. Removal of the endothelium abolished the T3-mediated vasodilation in rat mesenteric arteries (by 36.7±5.4% NE vs. 3.5±6.2% NE). In vivo, T3 injection led to a rapid decrease of arterial blood pressure in WT (by 13.9±1.9 mmHg) and TRαGS mice (by 12.4±1.9 mmHg), but not in TRα0 mice (by 4.1±1.9 mmHg). Conclusions These results demonstrate that T3 acting through noncanonical TRa action impacts cardiovascular physiology by inducing endothelium-dependent vasodilation within minutes via PI3K and eNOS activation.

Hypothyroidism has been shown to reduce infarct size in rats, but the underlying mechanisms are unclear. We used isolated pressure-constant perfused hearts of control, hypothyroid and hyperthyroid mice and measured infarct size, functional parameters and phosphorylation of key molecules in cardioprotective signaling with matched heart rate. Compared with controls, hypothyroidism was cardioprotective, while hyperthyroidism was detrimental with enlarged infarct size. Next, we asked how thyroid hormone receptor α (TRα) affects ischemia/reperfusion (IR) injury. Thus, canonical and noncanonical TRα signaling was investigated in the hearts of (i) mice lacking TRα (TRα0), (ii) with a mutation in TRα DNA-binding domain (TRαGS) and (iii) in hyperthyroid TRα0 (TRα0hyper) and TRαGS mice (TRαGShyper). TRα0 mouse hearts were protected against IR injury. Furthermore, infarct size was reduced in the hearts of TRαGS mice that lack canonical TRα signaling but maintain noncanonical TRα action. Hyperthyroidism did not increase infarct size in TRα0 and TRαGS mouse hearts. These cardioprotective effects were not associated with increased phosphorylation of key proteins of RISK, SAFE and eNOS pathways. In summary, chronic hypothyroidism and the lack of canonical TRα signaling are cardioprotective in IR injury and protection is not due to favorable changes in hemodynamics.

Background One of the hallmarks of Alzheimer's Disease (AD), as with some other neurodegenerative diseases, is the misfolding and aggregation of proteins, such as amyloid‐beta and tau. Tau pathology is also believed to propagate trans‐synaptically from neuron to neuron. Either prevention of propagation, or the removal of aggregated tau, is a potential approach for AD modification. Method Deubiquitinating enzymes (DUBs) maintain ubiquitin homeostasis by removing ubiquitin modifications from target proteins, thereby altering protein function, stability, and signaling. We hypothesize that the modulation of the ubiquitin‐proteasome system with DUB inhibitors will lead to decreased tau aggregation either directly (by inhibiting the removal of ubiquitin from tau thereby increasing its degradation) or indirectly (via modulation of other relevant pathways, like autophagy). To select relevant DUBs, we developed a phenotypic assay to assess the effect of knocking down ∼100 DUBs on the clearance of tau aggregates in hiPSC – derived neurons, assayed using high content image analysis. Result We show that addition of recombinant tau seeds to hiPSC – derived cortical neurons expressing a fluorescent tau reporter construct, leads to formation of tau aggregates, which we can robustly and reproducibly reduce by knocking down specific DUBs. Conclusion The DUB enzymes revealed in our phenotypic screen have been validated in confirmatory studies and have the potential to become novel targets for the treatment of AD. In collaboration with Mission Therapeutics we will develop selective, potent DUB inhibitors for preclinical target validation.

Tau protein fibrillization is implicated in the pathogenesis of several neurodegenerative diseases collectively known as Tauopathies. For decades, investigating Tau fibrillization in vitro has required the addition of polyanions or other co-factors to induce its misfolding and aggregation, with heparin being the most commonly used. However, heparin-induced Tau fibrils exhibit high morphological heterogeneity and a striking structural divergence from Tau fibrils isolated from Tauopathies patients’ brains at ultra- and macro-structural levels. To address these limitations, we developed a quick, cheap, and effective method for producing completely co-factor-free fibrils from all full-length Tau isoforms and mixtures thereof. We show that Tau fibrils generated using this ClearTau method – ClearTau fibrils - exhibit amyloid-like features, possess seeding activity in biosensor cells and hiPSC-derived neurons, retain RNA-binding capacity, and have morphological properties and structures more reminiscent of the properties of the brain-derived Tau fibrils. We present the proof-of-concept implementation of the ClearTau platform for screening Tau aggregation-modifying compounds. We demonstrate that these advances open opportunities to investigate the pathophysiology of disease-relevant Tau aggregates and will facilitate the development of Tau pathology-targeting and modifying therapies and PET tracers that can distinguish between different Tauopathies.