OR01-02 The Role of Thyroid Hormone Receptor Beta Signaling in the Hypothalamic Paraventricular Nucleus

Abstract A. Zhang: None. J. Pagan: None. V.A. Rodrigues: None. A.N. Hollenberg: None. R.H. Costa e Sousa: None. Thyroid hormone receptor beta (THRB) is thought to directly regulate TRH expression in the hypothalamic paraventricular nucleus (PVN). We hypothesized that disrupting THRB expression in the PVN would increase Trh mRNA expression and TH levels. To investigate this we generated PVN neuronal-specific THRBKO (THRBKOnPVN) mice by stereotaxic injection of an AAV-Cre (AAV.hSyn.Cre-GFP) in the PVN of Thrb floxed male mice. WT mice were subjected to the same procedure and used as controls. Blood was collected before surgery, 1 and 4 weeks after. At week 4, mice were euthanized and the PVN microdissected for RNA extraction. THRBKOnPVN Thrb mRNA expression was reduced to about 65% of control levels. Despite this reduction, no significant difference was observed in Trh mRNA, TSH or T4 levels, food intake, or body weight. To test sensitivity to T3, another group of THRBKOnPVN and control mice were fed a LID/PTU diet for 4 weeks. In the last week, daily T3 injections were performed. THRBKOnPVN Thrb mRNA expression was reduced to about 50% of control levels, yet Trh mRNA did not significantly differ between groups. Plasma TSH increased from 900 pg/mL to 60,000 pg/mL after 3 weeks on PTU diet and T3 treatment reduced plasma TSH to 100 pg/mL in both groups. Daily food intake showed a linear decrease after starting the PTU diet, from 6.5g to around 3g after 3 weeks in both groups. Despite TSH decreasing below euthyroid levels after T3 injections, food intake remained lower than euthyroid levels, at 4g/day. Body weight remained similar throughout the experimental protocol. Next, we decided to conduct an experiment using a different AAV (AAV.CMV.Cre-GFP), which targets both neuronal and glial cells, generating again PVN-specific THRBKO (THRBKOPVN) mice. Blood was collected before surgery, 1, 3, and 5 weeks after. Male and female mice were euthanized at week 5 and brains were collected for fluorescent microscopy analysis. The site of injection was confirmed through GFP fluorescence and those that missed the PVN were excluded. No significant difference was observed in plasma TSH or T4 levels between groups at any time point. Interestingly, THRBKOPVN male mice showed a significant increase in daily food intake from 5.5g pre-surgery to an average of 9.2g at week 3 compared to WT mice. They also gained more weight averaging 40g at week 5 compared to WTs averaging 30g. No difference was observed in female mice. To investigate if we were targeting hypophysiotropic neurons, we injected the same AAV in the PVN of tdTomato reporter mice. tdTomato fluorescence was detected in the PVN and the median eminence, showing that this technique induces recombination in hypophysiotropic neurons. In summary, reducing PVN neuronal Thrb mRNA by 50% did not affect the HPT axis, food intake, or body weight. In contrast, disrupting Thrb expression in PVN neurons and glial cells resulted in a remarkable increase in food intake and body weight. Sunday, June 2, 2024