A secretagogin locus of the mammalian hypothalamus controls stress hormone release

Abstract

A hierarchical hormonal cascade along the hypothalamic-pituitary-adrenal axis orchestrates bodily responses to stress. Although corticotropin-releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin's Ca(2+) sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogin's loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness.

Keywords: Ca2+ sensor; HPA axis; acute stress; vesicular release.

Figure 1. Secretagogin locus in the paraventricular nucleus of the hypothalamus

1. A–A₃ Secretagogin⁺ neurons populate the paraventricular, dorsolateral, ventromedial, periventricular and arcuate nuclei of the mouse hypothalamus (Paxinos & Franklin, 2001). Red circles denote the localization of neuronal perikarya and their relative densities. AHP, anterior hypothalamus; Arc, arcuate nucleus; DM, dorsomedial nucleus; ME, median eminence; Pe, periventricular nucleus; PVN, paraventricular nucleus, PVNm, magnocellular part; SON, supraoptic nucleus; VM, ventromedial nucleus.

2. B–B₂ Largely non-overlapping distribution of AVP⁺, oxytocin⁺ and secretagogin⁺ (sgcn⁺) neurons in the magnocellular PVN. The mouse supraoptic nucleus (SON) harbored vasopressin⁺ and oxytocin⁺ but not secretagogin⁺ neurons. Open arrowheads pinpoint single-labeled neurons. Solid arrowhead denotes AVP/secretagogin dual-labeling. lv, lateral ventricle; PVNm, magnocellular part of the paraventricular nucleus; scgn, secretagogin.

3. B₃, B₄ Secretagogin⁺ neurons had smaller somatic diameters than AVP⁺ or oxytocin⁺ neurons yet without a difference in their diameter quotient, a measure of ovoid profiles (*P < 0.05, Student’s t-test).

4. C–D₃ Terminal-like profiles in the posterior pituitary (arrowheads in C) suggesting that secretagogin can co-exist, even if infrequently with oxytocin. Solanum tuberosum lectin (Sol. tub) was used to identify blood vessels.

Data information: Scale bars: 100 μm (B), 10 μm (B₁, B₂), 2 μm (C–D₃)

Figure 2. Ultrastructural analysis suggests the prevalence of membrane-bound secretagogin

1. Secretagogin (scgn) distribution at the ultrastructural level as revealed by pre-embedding silver-enhanced immunogold labeling. Secretagogin (arrowheads) was localized to membranous organelles in the perikarya (A), particularly the plasmalemma (A₁) and endoplasmic reticulum (ER) in neuronal soma (s; A₂). Open rectangles in (A) denote the location of insets. Semi-transparent shading is used to visually dissociate subcellular compartments in (A–C).

2. Pre-embedding secretagogin labeling (arrowheads) was also seen in dendrite (d) segments.

3. In axo-dendritic terminals (ax), secretagogin was closely associated with synaptic vesicles along the plasmalemma (arrowheads).

4. Quantitative analysis of subcellular secretagogin distribution upon electron microscopy detection of silver-enhanced gold particles. Particles were considered as membrane bound when they were at < 50 nm of a membrane (plasma membrane or endomembrane; i.e., secretory vesicle, Golgi or endoplasmic reticulum). In the soma of PVN neurons, significantly more particles were found in the cytosol as along the plasma membrane (**P < 0.01). In contrast, membrane association predominated in axonal nerve endings in the median eminence (*P < 0.05). Note that a significant proportion of particles was found adjacent to endomembranes in all subcellular compartments studied.

Data information: Scale bars:, 1 μm (A), 500 nm (B, C), 200 nm (A₁, A₂).

Figure 3. Electrophysiological classification of secretagogin⁺ parvocellular neurons

1. A–A₃ Biocytin-filled (arrow) neuron immunonegative for secretagogin yet containing AVP/oxytocin (a mixture of magnocellular markers was used in triple-labeling experiments). Scale bars: 50 μm (A), 8 μm (A₃). lv, lateral ventricle; PVN, paraventricular nucleus.

2. B–B₂ Secretagogin⁺ biocytin-filled neuron (arrow) lacking AVP/oxytocin immunosignal. Scale bars: 8 μm.

3. C, C₁ Dendritic reconstruction of secretagogin⁻ and secretagogin⁺ neurons at their actual location in the paraventricular nucleus of the hypothalamus.

4. D Electrophysiological characteristics of magnocellular and parvocellular neurons. The waveform of repetitive action potential firing is shown on the left, while differential channel characteristics are depicted on the right. Examples of secretagogin⁺ neurons are shown in red.

5. E Pie diagrams showing the grouping of neurons in the PVN (upper row) and in adjacent pre-autonomic areas (lower row) cumulatively based on electrophysiological criteria listed in Supplementary Table S1. Secretagogin⁺ neurons typically belonged to Ia and IIa subtypes.

Figure 4. Molecular identity of secretagogin⁺ paraventricular neurons

Single-cell mRNA transcriptome profiling of dissociated cells from the mouse paraventricular nucleus.

1. A Differential clustering based on secretagogin (Scgn), neuropeptide, hormone and hormone receptor mRNA expression. Secretagogin-expressing(⁺) neurons typically contained corticotropin-releasing hormone (Crh) and Nr3c1 mRNA transcripts.

2. A₁ Clusters of gene transcripts from 130 cells reveal the phenotypic segregation of PVN neurons. Increasing mRNA copy numbers were depicted by a color gradient from deep blue (not detected) to red (high numbers). Secretagogin⁺ neurons are indicated by red arrows.

3. B Venn diagrams depicting the (non-)overlapping relationships of select mRNA transcripts used to molecularly define PVN neurons.

4. B₁ Cumulative ratio of phenotypic diversity among secretagogin⁺ neurons. Note that Crh and/or GABA (derived from Gad1, Gad2 and Slc32a1 mRNA expression) co-existed with secretagogin in parvocellular neurons.

5. C, C₁ Secretagogin co-localization with GFP in the PVN (solid arrowheads) of adult CRH-GFP (BAC) reporter mice (Alon et al, 2009). Open arrowhead pinpoints a single-labeled neuron.

6. D–I₃ We found secretagogin (scgn) co-expressed with CRH in both neuronal soma (solid arrowheads; D₁–D₃) and axon terminal-like specializations (arrowheads; E–E₂) in the PVN. Likewise, secretagogin⁺ neurons harbored, yet infrequently, tyrosine hydroxylase (TH; solid arrowheads; F–F₃), GABA (G–G₂), somatostatin (Sst; H–H₃) and galanin (I–I₂). Open rectangles denote the general location of insets. Open arrowheads indicate the lack of co-localization.

Data information: Scale bars: 40 μm (D, F, H), 25 μm (C₁), 10 μm (D₃, F₃, G₂, H₃), 4 μm (E₂, I₂).

Figure 5. Secretagogin is a Ca²⁺ sensor protein

1. A–A₂ Secretagogin co-existed in the majority of CRH⁺ nerve endings in the median eminence (ME). Open rectangle denotes the general location of (B, B₁).

2. B, B₁ Large axon terminals in the median eminence were immunopositive for secretagogin with silver-intensified immunogold particles (open arrowheads) associated with axonal membrane and dense-core vesicles (see Fig 1D for quantitative data). Solid arrowheads denote silver deposit particles proximal to the plasmalemma.

3. C Immunoprecipitation using an anti-secretagogin antibody in Ca²⁺-free and Ca²⁺-loaded conditions was subtractively used to decipher the Ca²⁺-dependent protein interactions. Silver-stained gel is shown.

4. C₁ Ontology classification of the 99 protein hits based on primary function assignments. Unbiased MALDI-TOF proteomics was used to identify interacting proteins. The most abundant hits (Supplementary Table S3 is referred to for details on individual proteins) are proteins implicated in vesicle fusion, trafficking, transport and formation and the regulation of vesicle exocytosis. “Other” refers to a group of proteins without known function.

5. D Single-cell transcriptomics was used to validate the above proteome data by clustering mRNA transcripts encoding proteins that underpin vesicular release processes (red color labels highest mRNA abundance, whereas dark blue color indicates the lack of mRNA expression).

6. E–E₃ Rab3, a family of vesicular fusion and transport proteins (Schluter et al, 2006), was found co-localized with secretagogin (arrowheads) in the median eminence. Neuronal soma in the PVN lacked appreciable co-localization. Note that neither our MALDI-TOF analysis nor our histochemical probing allowed the precise identification of individual Rab3C-E family members.

Data information: Scale bars: 10 μm (A₁, E₃), 7 μm (E), 500 nm (B), 150 nm (B₁).

Figure 6. Secretagogin regulates CRH release in vitro and in vivo

1. A, A₁ siRNA-mediated secretagogin (scgn) knockdown in cultured hypothalamic neurons, as indicated by reduced secretagogin immunoreactivity (A) and decreased CRH content in the culture medium (A₁).

2. B–B₂ Transient overexpression of secretagogin in immortalized CRH-expressing mHypoE-N44 hypothalamic cells significantly reduced CRH immunofluorescence intensity in secretagogin⁺ cell bodies, indirectly supporting enhanced CRH release. All experiments were performed in triplicate. Scale bar: 50 nm.

3. C, C₁ siRNA-mediated in vivo silencing of secretagogin mRNA expression in the PVN provoked somatic CRH accumulation (arrowheads). Scale bar: 150 μm.

4. C₂ Quantitative analysis demonstrating significantly increased somatic CRH contents. Note that somatic secretagogin levels remained unchanged, which we interpret as data on a neuronal contingent not affected by siRNA silencing. The lack of secretagogin/CRH co-localization suggests that secretagogin expression fell below detection threshold in many CRH⁺ neurons.

5. D, D₁ Individual data points show maximal CRH fluorescence intensity (gray scale arbitrary unit (a.u.) expression) in PVN neurons that have low or no secretagogin expression after siRNA infusion.

Data information: Data in (C₂) were normalized to those in non-targeting siRNA controls. *P < 0.05 versus control.

Figure 7. Secretagogin⁺ neurons gate the CRH–ACTH axis in response to acute stress

1. A, A₁ Paraventricular secretagogin-immunoreactive (scgn⁺) neurons weakly expressed CRH (open arrowheads) but not c-fos under control conditions. Scale bars: 300 μm (A), 25 μm (A₁).

2. B, B₁ Stress induced by subcutaneous injection of formalin-triggered co-expression of c-fos and CRH in secretagogin⁺ paraventricular neurons (arrowheads). Scale bars: 300 μm (B), 25 μm (B₁).

3. C, D In vivo siRNA-mediated silencing of secretagogin expression in the PVN occluded the stress-induced surge of serum ACTH levels (C) and significantly reduced the increase in plasma corticosterone (D). *P < 0.05 versus control.