Origin and function of the pituitary generator of GH pulses – GH-Gen

Recent developments allowing cellular in vivo imaging and manipulation in freely-moving mice, and organ-scale 3D imaging of human pituitary places us in a unique position to address this long-standing question of how the temporal dynamics of hypothalamic stimulation and pituitary response are related in both mice and humans. Surprisingly, our preliminary data suggest a “Russian doll” organization of calcium signals generated by the network of pituitary (growth hormone (GH)-secreting) somatotrophs with fast calcium transients superimposed on slower, recurrent calcium waves driving coordinated GH release which is integrated to generate circhoral circulating hormone pulses. Based on these preliminary data and the complementary expertise of our proposed ANR consortium, this application will define the in vivo function of both pituitary somatotrophs and hypothalamic growth hormone releasing hormone (GHRH) neurons (the main GH secretagogue), as well as the signalling intermediates generating the highly-ordered GH pulses which regulate body growth and metabolism.

Our overarching hypothesis is that the pituitary somatotroph network functions in vivo as a pulse generator, translating GHRH input into slowly-evolving GH outputs. We are currently addressing the following specific aims: 1/ Determine GHRH neuron and GH cell activities and how this relates to hormonal output in both male and female awake mice and how these activities decline with age. We monitor in vivo (longitudinal studies where the animal is its own control) calcium activity in freely-moving mice using fluorescent reporters (GCAMP6) and implanted GRIN (gradient-index) lenses coupled to a light (2g) endomicroscope. This will aid design of novel challenge tests for GH pulsatilility in patients. 2/ Identify the mechanisms of coordinated and sustained calcium waves mediating GH pulses using a complementary set of techniques and tools: in vivo imaging and optogenetic manipulation, as well as ‘dynamic’ clamp and pharmacological agents, to determine the contribution of ion channels and other cellular/intercellular signalling molecules (identified from transcriptomic data from purified somatotrophs). 3/ Determine whether these mouse studies are translatable to the clinic for improved clinical diagnosis and treatment of pituitary GH diseases. To do so, we are establishing common expression of signalling intermediates using 3D human pituitary cell imaging and determine the result of manipulating these candidate genes using a CRISPR-Cas9 approach with injected virus.

In sum, our project will define a new level of understanding of how GH pulses are generated. Since GH is central in body growth and metabolism and the causes of = 15% of pituitary defects have been identified to date, it is of physiological importance and certainly of clinical relevance to identify the cellular mechanisms that underlie these series of calcium waves. Finally we expect that our proposed approach would be instrumental to reveal other facets of the brain-endocrine system dialogue.

1- Diving into the brain: deep-brain imaging techniques in conscious animals. Campos P, Walker JJ, Mollard P. J Endocrinol. 2020 Aug;246(2):R33-R50. doi: 10.1530/JOE-20-0028.
2- Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells.
Russell JP, Lim X, Santambrogio A, Yianni V, Kemkem Y, Wang B, Fish M, Haston S, Grabek A, Hallang S, Lodge EJ, Patist AL, Schedl A, Mollard P, Nusse R, Andoniadou CL. Elife. 2021 Jan 5;10:e59142. doi: 10.7554/eLife.59142.
3- Synaptic communication mediates the assembly of a self-organizing circuit that controls reproduction. Golan M, Boulanger-Weill J, Pinot A, Fontanaud P, Faucherre A, Gajbhiye DS, Hollander-Cohen L, Fiordelisio-Coll T, Martin AO, Mollard P. Sci Adv. 2021 Feb 19;7,eabc8475. doi: 10.1126/sciadv.abc8475.
4- Loss-of-function variantS IN SEMA3F AND PLXNA3 ENCODING semaphorin-3F and its receptor plexin-A3 respectively cause idiopathic hypogonadotropic hypogonadism. Kotan LD, Ternier G, Cakir AD, Emeksiz HC, Turan I, Delpouve G, Kardelen AD, Ozcabi B, Isik E, Mengen E, Cakir EDP, Yuksel A, Agladioglu SY, Dilek SO, Evliyaoglu O, Darendeliler F, Gurbuz F, Akkus G, Yuksel B, Giacobini P*, Kemal Topaloglu A*. Genet Med. 2021 Jan 25. doi: 10.1038/s41436-020-01087-5.
5- Neuron-Derived Neurotrophic Factor is Mutated in Congenital Hypogonadotropic Hypogonadism. Messina A, Pulli K, Santini S, Acierno J, Känsäkoski J, Cassatella D, Xu C, Casoni F, Malone SA, Ternier G, Conte D, Sidis Y, Tommiska J, Vaaralahti K, Dwyer A, Gothilf Y, Merlo GR, Santoni F, Niederländer NJ, Giacobini P*, Raivio T*, Pitteloud N*. Am J Hum Genet. 2020 jan 2;106(1):58-70. doi: 10.1016/j.ajhg.2019.12.003.

A current challenge in physiology/pathology is translating cell-transduction processes identified in vitro into the living organism, especially where cell-cell interaction and dynamics have key functional roles. The pituitary gland, regulating a range of essential physiological functions, exemplifies this challenge: stimulation from a few thousand hypothalamic neurons in the brain is relayed to hundreds of thousands of pituitary cells generating variable hormone pulses (the hypothalamic-pituitary (HP) system), which are decoded by peripheral organs into differential effects. We (Partners 1&3, Mollard & Le Tissier) have identified the organisation of pituitary cells as intermingled 3D cell networks as a key component of this system but the mechanisms by which these pituitary cells transform hypothalamic inputs into hormone pulses in normal physiology are currently unknown.

Recent developments allowing cellular in vivo imaging and manipulation in freely-moving mice, and organ-scale 3D imaging of human pituitary (Partner 2, Giacobini) places us in a unique position to address this long-standing question of how the temporal dynamics of hypothalamic stimulation and pituitary response are related in both mice and humans. Surprisingly, our preliminary data suggest a “Russian doll” organization of calcium signals generated by the network of pituitary (growth hormone (GH)-secreting) somatotrophs with fast calcium transients superimposed on slower, recurrent calcium waves driving coordinated GH release which is integrated to generate circhoral circulating hormone pulses. Based on these preliminary data and the complementary expertise of our proposed ANR consortium (Partners 1-3), this application will define the in vivo function of both pituitary somatotrophs and hypothalamic growth hormone releasing hormone (GHRH) neurons (the main GH secretagogue), as well as the signalling intermediates generating the highly-ordered GH pulses which regulate body growth and metabolism.

Our overarching hypothesis is that the pituitary somatotroph network functions in vivo as a pulse generator, translating GHRH input into slowly-evolving GH outputs. We will address this with the following specific aims: 1/ Determine GHRH neuron and GH cell activities and how this relates to hormonal output in both male and female awake mice and how these activities decline with age. We will monitor in vivo (longitudinal studies where the animal is its own control) calcium activity in freely-moving mice using fluorescent reporters (GCAMP6) and implanted GRIN (gradient-index) lenses coupled to a light (2g) endomicroscope. This will aid design of novel challenge tests for GH pulsatilility in patients. 2/ Identify the mechanisms of coordinated and sustained calcium waves mediating GH pulses using a complementary set of techniques and tools: in vivo imaging and optogenetic manipulation, as well as ‘dynamic’ clamp and pharmacological agents, to determine the contribution of ion channels and other cellular/intercellular signalling molecules (identified from transcriptomic data from purified somatotrophs). 3/ Determine whether these mouse studies are translatable to the clinic for improved clinical diagnosis and treatment of pituitary GH diseases. We will establish common expression of signalling intermediates using 3D human pituitary cell imaging and determine the result of manipulating these candidate genes using a CRISPR-Cas9 approach with injected virus.

In sum, our project will define a new level of understanding of how GH pulses are generated. Since GH is central in body growth and metabolism and the causes of = 15% of pituitary defects have been identified to date, it is of physiological importance and certainly of clinical relevance to identify the cellular mechanisms that underlie these series of calcium waves. Finally we expect that our proposed approach would be instrumental to reveal other facets of the brain-endocrine system dialogue.