Ultradian Secretion of Growth Hormone in Mice: Linking Physiology With Changes in Synapse Parameters Using Super-Resolution Microscopy.

Neuroendocrine circuits are orchestrated by the pituitary gland in response to hypothalamic hormone-releasing and inhibiting factors to generate an ultradian and/or circadian rhythm of hormone secretion. However, mechanisms that govern this rhythmicity are not fully understood. It has been shown that synaptic transmission in the rodent hypothalamus undergoes cyclical changes in parallel with rhythmic hormone secretion and a growing body of evidence suggests that rapid rewiring of hypothalamic neurons may be the source of these changes. For decades, structural synaptic studies have been utilizing electron microscopy, which provides the resolution suitable for visualizing synapses. However, the small field of view, limited specificity and manual analysis susceptible to bias fuel the search for a more quantitative approach. Here, we apply the fluorescence super-resolution microscopy approach direct Stochastic Optical Reconstruction Microscopy (dSTORM) to quantify and structurally characterize excitatory and inhibitory synapses that contact growth hormone-releasing-hormone (GHRH) neurons during peak and trough values of growth hormone (GH) concentration in mice. This approach relies on a three-color immunofluorescence staining of GHRH and pre- and post-synaptic markers, and a quantitative analysis with a Density-Based Spatial Clustering of Applications with Noise (DBSCAN) algorithm. With this method we confirm our previous findings, using electron microscopy, of increased excitatory synaptic input to GHRH neurons during peak levels of GH. Additionally, we find a shift in synapse numbers during low GH levels, where more inhibitory synaptic inputs are detected. Lastly, we utilize dSTORM to study novel aspects of synaptic structure. We show that more excitatory (but not inhibitory) pre-synaptic clusters associate with excitatory post-synaptic clusters during peaks of GH secretion and that the numbers of post-synaptic clusters increase during high hormone levels. The results presented here provide an opportunity to highlight dSTORM as a valuable quantitative approach to study synaptic structure in the neuroendocrine circuit. Importantly, our analysis of GH circuitry sheds light on the potential mechanism that drives ultradian changes in synaptic transmission and possibly aids in GH pulse generation in mice.

Regulated resurfacing of a somatostatin receptor storage compartment fine-tunes pituitary secretion.

The surfacing of the glucose transporter GLUT4 driven by insulin receptor activation provides the prototypic example of a homeostasis response dependent on mobilization of an intracellular storage compartment. Here, we generalize this concept to a G protein-coupled receptor, somatostatin receptor subtype 2 (SSTR2), in pituitary cells. Following internalization in corticotropes, SSTR2 moves to a juxtanuclear syntaxin-6-positive compartment, where it remains until the corticotropes are stimulated with corticotropin releasing factor (CRF), whereupon SSTR2 exits the compartment on syntaxin-6-positive vesicular/tubular carriers that depend on Rab10 for their fusion with the plasma membrane. As SSTR2 activation antagonizes CRF-mediated hormone release, this storage/resurfacing mechanism may allow for a physiological homeostatic feedback system. In fact, we find that SSTR2 moves from an intracellular compartment to the cell surface in pituitary gland somatotropes, concomitant with increasing levels of serum growth hormone (GH) during natural GH cycles. Our data thus provide a mechanism by which signaling-mediated plasma membrane resurfacing of SSTR2 can fine-tune pituitary hormone release.

Dissecting the molecular mechanisms that impair stress granule formation in aging cells.

Aging affects numerous aspects of cell biology, but the senescence-associated changes in the stress response are only beginning to emerge. To obtain mechanistic insights into these events, we examined the formation of canonical and non-canonical stress granules (SGs) in the cytoplasm. SG generation is a key event after exposure to physiological or environmental stressors. It requires the SG-nucleating proteins G3BP1 and TIA-1/TIAR and stress-related signaling events. To analyze SG formation, we used two independent models of somatic cell aging. In both model systems, cellular senescence impaired the assembly of two SG classes: (i) it compromised the formation of canonical SGs, and (ii) skewed the production of non-canonical SGs. We dissected the mechanisms underlying these senescence-dependent changes in granule biogenesis and identified several specific targets that were modulated by aging. Thus, we demonstrate a depletion of G3BP1 and TIA-1/TIAR in senescent cells and show that the loss of G3BP1 contributed to impaired SG formation. We further reveal that aging reduced Sp1 levels; this transcription factor regulated G3BP1 and TIA-1/TIAR abundance. The assembly of canonical SGs relies on the phosphorylation of translation initiation factor eIF2α. We show that senescence can cause eIF2α hyperphosphorylation. CReP is a subunit of protein phosphatase 1 and critical to reverse the stress-dependent phosphorylation of eIF2α. We demonstrate that the loss of CReP correlated with the aging-related hyperphosphorylation of eIF2α. Together, we have identified significant changes in the stress response of aging cells and provide mechanistic insights. Based on our work, we propose that the decline in SG formation can provide a new biomarker to evaluate cellular aging.