Male rat hypothalamic extraretinal photoreceptor Opsin3 is sensitive to osmotic stimuli and light.

The light-sensitive protein Opsin 3 (Opn3) is present throughout the mammalian brain; however, the role of Opn3 in this organ remains unknown. Since Opn3 encoded mRNA is modulated in the supraoptic and paraventricular nucleus of the hypothalamus in response to osmotic stimuli, we have explored by in situ hybridization the expression of Opn3 in these nuclei. We have demonstrated that Opn3 is present in the male rat magnocellular neurones expressing either the arginine vasopressin or oxytocin neuropeptides and that Opn3 increases in both neuronal types in response to osmotic stimuli, suggesting that Opn3 functions in both cell types and that it might be involved in regulating water balance. Using rat hypothalamic organotypic cultures, we have demonstrated that the hypothalamus is sensitive to light and that the observed light sensitivity is mediated, at least in part, by Opn3. The data suggests that hypothalamic Opn3 can mediate a light-sensitive role to regulate circadian homeostatic processes.

Isoflurane Rapidly Modifies Synaptic and Cytoskeletal Phosphoproteomes of the Supraoptic Nucleus of the Hypothalamus and the Cortex.

INTRODUCTION: Despite the widespread use of general anaesthetics, the mechanisms mediating their effects are still not understood. Although suppressed in most parts of the brain, neuronal activity, as measured by FOS activation, is increased in the hypothalamic supraoptic nucleus (SON) by numerous general anaesthetics, and evidence points to this brain region being involved in the induction of general anaesthesia (GA) and natural sleep. Posttranslational modifications of proteins, including changes in phosphorylation, enable fast modulation of protein function which could be underlying the rapid effects of GA. In order to identify potential phosphorylation events in the brain-mediating GA effects, we have explored the phosphoproteome responses in the rat SON and compared these to cingulate cortex (CC) which displays no FOS activation in response to general anaesthetics. METHODS: Adult Sprague-Dawley rats were treated with isoflurane for 15 min. Proteins from the CC and SON were extracted and processed for nano-LC mass spectrometry (LC-MS/MS). Phosphoproteomic determinations were performed by LC-MS/MS. RESULTS: We found many changes in the phosphoproteomes of both the CC and SON in response to 15 min of isoflurane exposure. Pathway analysis indicated that proteins undergoing phosphorylation adaptations are involved in cytoskeleton remodelling and synaptic signalling events. Importantly, changes in protein phosphorylation appeared to be brain region specific suggesting that differential phosphorylation adaptations might underlie the different neuronal activity responses to GA between the CC and SON. CONCLUSION: In summary, these data suggest that rapid posttranslational modifications in proteins involved in cytoskeleton remodelling and synaptic signalling events might mediate the central mechanisms mediating GA.

Effects of Triiodothyronine Treatment in an Animal Model of Heart Failure with Preserved Ejection Fraction.

Background: Low levels of triiodothyronine (T3) are common in patients with heart failure (HF). Our aim was to evaluate the effects of supplementation with low and replacement doses of T3 in an animal model of HF with preserved ejection fraction (HFpEF). Methods: We evaluated four groups: ZSF1 Lean (n = 8, Lean-Ctrl), ZSF1 Obese (rat model of metabolic-induced HFpEF, n = 13, HFpEF), ZSF1 Obese treated with a replacement dose of T3 (n = 8, HFpEF-T3high), and ZSF1 Obese treated with a low-dose of T3 (n = 8, HFpEF-T3low). T3 was administered in drinking water from weeks 13 to 24. The animals underwent anthropometric and metabolic assessments, echocardiography, and peak effort testing with maximum O2 consumption (VO2max) determination at 22 weeks, and a terminal hemodynamic evaluation at 24 weeks. Afterwhile myocardial samples were collected for single cardiomyocyte evaluation and molecular studies. Results: HFpEF animals showed lower serum and myocardial thyroid hormone levels than Lean-Ctrl. Treatment with T3 did not normalize serum T3 levels, but increased myocardial T3 levels to normal levels in the HFpEF-T3high group. Body weight was significantly decreased in both the T3-treated groups, comparing with HFpEF. An improvement in glucose metabolism was observed only in HFpEF-T3high. Both the treated groups had improved diastolic and systolic function in vivo, as well as improved Ca2+ transients and sarcomere shortening and relaxation in vitro. Comparing with HFpEF animals, HFpEF-T3high had increased heart rate and a higher rate of premature ventricular contractions. Animals treated with T3 had higher myocardial expression of calcium transporter ryanodine receptor 2 (RYR2) and α-myosin heavy chain (MHC), with a lower expression of ß-MHC. VO2max was not influenced by treatment with T3. Myocardial fibrosis was reduced in both the treated groups. Three animals died in the HFpEF-T3high group. Conclusions: Treatment with T3 was shown to improve metabolic profile, myocardial calcium handling, and cardiac function. While the low dose was well-tolerated and safe, the replacement dose was associated with increased heart rate, and increased risk of arrhythmias and sudden death. Modulation of thyroid hormones may be a potential therapeutic target in HFpEF; however, it is important to take into account the narrow therapeutic window of T3 in this condition.

Translational and Posttranslational Dynamics in a Model Peptidergic System.

The cell bodies of hypothalamic magnocellular neurones are densely packed in the hypothalamic supraoptic nucleus, whereas their axons project to the anatomically discrete posterior pituitary gland. We have taken advantage of this unique anatomical structure to establish proteome and phosphoproteome dynamics in neuronal cell bodies and axonal terminals in response to physiological stimulation. We have found that proteome and phosphoproteome responses to neuronal stimulation are very different between somatic and axonal neuronal compartments, indicating the need of each cell domain to differentially adapt. In particular, changes in the phosphoproteome in the cell body are involved in the reorganization of the cytoskeleton and in axonal terminals the regulation of synaptic and secretory processes. We have identified that prohormone precursors including vasopressin and oxytocin are phosphorylated in axonal terminals and are hyperphosphorylated following stimulation. By multiomic integration of transcriptome and proteomic data, we identify changes to proteins present in afferent inputs to this nucleus.

Osmoadaptive GLP-1R signalling in hypothalamic neurones inhibits antidiuretic hormone synthesis and release.

OBJECTIVES: The excessive release of the antidiuretic hormone vasopressin is implicated in many diseases including cardiovascular disease, diabetes, obesity, and metabolic syndrome. Once thought to be elevated as a consequence of diseases, data now supports a more causative role. We have previously identified CREB3L1 as a transcription factor that co-ordinates vasopressin synthesis and release in the hypothalamus. The objective here was to identify mechanisms orchestrated by CREB3L1 that co-ordinate vasopressin release. METHODS: We mined Creb3l1 knockdown SON RNA-seq data to identify downstream target genes. We proceeded to investigate the expression of these genes and associated pathways in the supraoptic nucleus of the hypothalamus in response to physiological and pharmacological stimulation. We used viruses to selectively knockdown gene expression in the supraoptic nucleus and assessed physiological and metabolic parameters. We adopted a phosphoproteomics strategy to investigate mechanisms that facilitate hormone release by the pituitary gland. RESULTS: We discovered glucagon like peptide 1 receptor (Glp1r) as a downstream target gene and found increased expression in stimulated vasopressin neurones. Selective knockdown of supraoptic nucleus Glp1rs resulted in decreased food intake and body weight. Treatment with GLP-1R agonist liraglutide decreased vasopressin synthesis and release. Quantitative phosphoproteomics of the pituitary neurointermediate lobe revealed that liraglutide initiates hyperphosphorylation of presynapse active zone proteins that control vasopressin exocytosis. CONCLUSION: In summary, we show that GLP-1R signalling inhibits the vasopressin system. Our data advises that hydration status may influence the pharmacodynamics of GLP-1R agonists so should be considered in current therapeutic strategies.