Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion

doi:10.1073/pnas.2213682120

. 2023 Feb 14;120(7):e2213682120.

doi: 10.1073/pnas.2213682120. Epub 2023 Feb 6.

Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion

Lora L Martucci^{1

2

3}, Jean-Marie Launay⁴, Natsuko Kawakami⁵, Cécile Sicard¹, Nathalie Desvignes¹, Mbarka Dakouane-Giudicelli², Barbara Spix⁶, Maude Têtu¹, Franck-Olivier Gilmaire¹, Sloane Paulcan¹, Jacques Callebert^{7

8}, Cyrille Vaillend¹, Franz Bracher⁹, Christian Grimm⁶, Philippe Fossier¹, Sabine de la Porte², Hirotaka Sakamoto⁵, John Morris¹⁰, Antony Galione³, Sylvie Granon¹, José-Manuel Cancela¹

Affiliations

¹ Neuroscience Paris-Saclay Institute, CNRS UMR 9197, Paris-Sud University, Paris-Saclay University, Saclay 91400, France.
² Université Paris-Saclay, Université de Versailles Saint-Quentin-en-Yvelines, Inserm, Evolution of Neuromuscular Diseases: Innovative Concepts and Practices, Versailles 78000, France.
³ Department of Pharmacology, University of Oxford, Oxford OX1 3QT, UK.
⁴ INSERM UMR-S 942, Hôpital Lariboisière, Paris Cedex 10 75475, France.
⁵ Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan.
⁶ Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine Ludwig-Maximilians-University, Munich 80336, Germany.
⁷ Laboratoire de Biochimie et Biologie Moléculaire, Hôpital Lariboisière, Paris 75010, France.
⁸ Inserm UMR-S 1144 Universités Paris Descartes-Paris Diderot, Optimisation Thérapeutique en Neuropsychopharmacologie - Faculté des Sciences Pharmaceutiques et Biologiques, Paris Descartes, Paris Paris 75006, France.
⁹ Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians University, Munich 81377, Germany.
¹⁰ Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford OX1 3QX, UK.

PMID: 36745816
PMCID: PMC9963339
DOI: 10.1073/pnas.2213682120

Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion

Lora L Martucci et al. Proc Natl Acad Sci U S A. 2023.

. 2023 Feb 14;120(7):e2213682120.

doi: 10.1073/pnas.2213682120. Epub 2023 Feb 6.

Authors

Affiliations

¹ Neuroscience Paris-Saclay Institute, CNRS UMR 9197, Paris-Sud University, Paris-Saclay University, Saclay 91400, France.
² Université Paris-Saclay, Université de Versailles Saint-Quentin-en-Yvelines, Inserm, Evolution of Neuromuscular Diseases: Innovative Concepts and Practices, Versailles 78000, France.
³ Department of Pharmacology, University of Oxford, Oxford OX1 3QT, UK.
⁴ INSERM UMR-S 942, Hôpital Lariboisière, Paris Cedex 10 75475, France.
⁵ Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan.
⁶ Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine Ludwig-Maximilians-University, Munich 80336, Germany.
⁷ Laboratoire de Biochimie et Biologie Moléculaire, Hôpital Lariboisière, Paris 75010, France.
⁸ Inserm UMR-S 1144 Universités Paris Descartes-Paris Diderot, Optimisation Thérapeutique en Neuropsychopharmacologie - Faculté des Sciences Pharmaceutiques et Biologiques, Paris Descartes, Paris Paris 75006, France.
⁹ Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians University, Munich 81377, Germany.
¹⁰ Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford OX1 3QX, UK.

PMID: 36745816
PMCID: PMC9963339
DOI: 10.1073/pnas.2213682120

Abstract

Oxytocin (OT) is a prominent regulator of many aspects of mammalian social behavior and stored in large dense-cored vesicles (LDCVs) in hypothalamic neurons. It is released in response to activity-dependent Ca²⁺ influx, but is also dependent on Ca²⁺ release from intracellular stores, which primes LDCVs for exocytosis. Despite its importance, critical aspects of the Ca²⁺-dependent mechanisms of its secretion remain to be identified. Here we show that lysosomes surround dendritic LDCVs, and that the direct activation of endolysosomal two-pore channels (TPCs) provides the critical Ca²⁺ signals to prime OT release by increasing the releasable LDCV pool without directly stimulating exocytosis. We observed a dramatic reduction in plasma OT levels in TPC knockout mice, and impaired secretion of OT from the hypothalamus demonstrating the importance of priming of neuropeptide vesicles for activity-dependent release. Furthermore, we show that activation of type 1 metabotropic glutamate receptors sustains somatodendritic OT release by recruiting TPCs. The priming effect could be mimicked by a direct application of nicotinic acid adenine dinucleotide phosphate, the endogenous messenger regulating TPCs, or a selective TPC2 agonist, TPC2-A1-N, or blocked by the antagonist Ned-19. Mice lacking TPCs exhibit impaired maternal and social behavior, which is restored by direct OT administration. This study demonstrates an unexpected role for lysosomes and TPCs in controlling neuropeptide secretion, and in regulating social behavior.

Keywords: NAADP; calcium; channel; hypothalamus; neuropeptide.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interest.

Figures

**Fig. 1.**
Plasma OT levels are altered in TPC knockout mice. (A) qPCR analysis in duplicate of TPC1 and TPC2 mRNA in the hypophysis of WT mice (each group n = 9 mice). (B) qPCR analysis in duplicate of TPC1 and TPC2 mRNA in the hypothalamus of WT mice (each group n = 9 mice). (C) Plasma OT levels (male WT n = 10, male DKO n = 11, female WT n = 4, female DKO n = 5 mice). *Refers to differences between TPC DKO mice and their respective WT. ^#Refers to differences between females and their respective strain males. (D) Plasma levels of OT in *Tpcn1^−/− (*TPC1^−/−), *Tpcn2^−/−* (TPC2^−/−) and mucolipin1 (*Trpml1^−/−*) KO mice (WT n = 4; TPC1^−/− n = 8; TPC2^−/− n = 8; TRPML1^−/− n = 4 mice). (E) Immunostaining of TPC2-tGFP reporter (in green) revealed with the anti-GFP antibody and nuclei stained with bisbenzimide (in blue) in the hypophysis containing the neurohypophysis (neurohyp) and adenohypophysis (adenohyp) (n = 2 mice). (*Top* scale bar, 200 µm.) *Lower* panel represents a magnification of the *Top* panel images (white square), (scale bar, 50 µm.) (F) Immunostaining of TPC2-tGFP reporter (in green) and nuclei stained with bisbenzimide (in blue) in the hypothalamus. Top raw shows the PVN, SON, and third ventricle (3V). The middle row shows a crop of the PVN, and the lower row shows a crop of the SON (n = 2 mice). (*Top Row* scale bar, 200 µm.) (*Middle row* scale bar, 20 µm.) (*Bottom Row* scale bar, 50 µm.) (G) Endogenous OT content in isolated hypothalami (male WT n = 20, male DKO n = 20, female WT n = 7, female DKO n = 10 mice). (H) Average number of OT neurons per slice in the PVN and SON in male mice (each group n = 7 mice) and illustrated in I. (I) Immunostaining of OT neurons from the PVN of SON of WT and TPC DKO mice. Statistical analysis were performed with unpaired t test (A–C, G and H) and one-way ANOVA followed by the post hoc Dunnett’s multiple comparison test (D). Values are expressed as mean ± SEM. *P < 0,05; **P < 0.01; ***P < 0.001 and ns, nonsignificant.

**Fig. 2.**
Pharmacological inhibition (green) or TPC deletion (red) reduced OT release. (A) OT released from isolated hypothalami after 50 mM KCl stimulation (each bar group n = 5 mice). *Refers to differences between TPC DKO mice and their respective WT. ^#Refers to differences between males and females of the same mouse strain. (B) Electron microscopy images labeled with neurophysin-I (15-nm gold particles) to distinguish OT-positive endings depicting autophagic grades: grade 0 corresponds to an absence of autophagy vacuoles within the ending, whereas grades 1, 2, and 3 correspond to a progressive increase of autophagic vacuoles. (C) Histograms of the electron microscopy showing the ratio of the area occupied by different grades of autophagic bodies in the neurohypophysial nerve endings divided by the total area of the ending (each group n = 3). (D) OT released from isolated hypothalami after 1 mM glutamate stimulation (male WT n = 20, male DKO n = 20) and after mGluR1 stimulation with 100 µM DHPG (each bar group n = 5 mice). (E) OT released from isolated hypothalami after 50 mM KCl stimulation in presence of 100 µM Ned-19, 4 µM bafilomycin A1, or 10 µM tetrandrine (each group n = 5 mice). (F) OT released from isolated hypothalami after 1mM glutamate stimulation in presence of 100 µM Ned-19 or 50 µM GPN (each bar group n = 5 mice). (G) OT released from isolated hypothalami after 100 µM DHPG (mGluR1) stimulation in presence of 100 µM Ned-19 or 10 µM tetrandrine (each bar group n = 5 mice). Statistical analyses were performed with an unpaired t test (A and D); ANOVA 2-way (C) or ANOVA 1-way (E–G) followed by post hoc Dunnett’s test. Values are expressed as mean ± SEM ns (no significant); *P < 0.05; **P < 0.01; ***P < 0.001 or ^###P < 0.001 (difference between female and male).

**Fig. 3.**
Pharmacological inhibition (green) or deletion of TPCs (red) reduced agonist-evoked Ca²⁺ response in the PVN of the hypothalamus. (A) Example of confocal Ca²⁺ imaging of WT neurons before and after glutamate stimulation. Acute slices were incubated with the Ca²⁺ sensitive probe Calbryte 520-AM. Arrows show some examples of neurons responding to 1 mM glutamate stimulation. (Scale bar, 50 µm.) (B) Shows the peak amplitude and (C and D) shows the time course graphs of glutamate-induced Ca²⁺ response in presence of 100 µM Ned-19 (green line, Ctrl n = 20, Ned-19 n = 20 cells, both n = 3 mice) or 50 µM GPN (green line, Ctrl n = 123, GPN n = 123 cells, both n = 4 mice) in hypothalamic neurons of WT mice compared to untreated control WT hypothalamic neurons (Ctrl, black line), area under the curve (AUC). (E) Glutamate-induced Ca²⁺ response in hypothalamic neurons of TPC DKO mice (red line, n = 47 cells, n = 5 mice) compared to control WT hypothalamic neurons (Ctrl, black line, n = 67 cells, n = 5 mice), area under the curve (AUC). (F) Shows the peak amplitude and (G and H) show the time course graphs of DHPG-induced Ca²⁺ response in the presence of 100 µM Ned-19 (green line, Ctrl n = 19, Ned-19 n = 19 cells, both n = 7 mice) or 50 µM GPN (green line, Ctrl n = 8, GPN n = 8, both n = 5 mice) in hypothalamic neurons of WT mice compared with untreated control WT hypothalamic neurons (Ctrl, black line), area under the curve (AUC). (I) DHPG-induced Ca²⁺ response in hypothalamic neurons of TPC DKO mice (red line, n = 57 cells, n = 14 mice) compared to control WT hypothalamic neurons (Ctrl, black line, n = 86 cells, n = 15 mice), area under the curve (AUC). Statistical analysis were performed with two-way ANOVA followed by Sidak’s test. Values are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

**Fig. 4.**
Lysosomes and TPCs control the priming of OT vesicles. (A) Confocal images in the PVN coimmunostained with OT (in green) and the lysosomal marker LAMP2 (in red). (Scale bars, 5 µm and 1 µm in the enlarged image.) (B) Electron micrograph of a dendrite (Den) from a SON neuron containing numerous neurosecretory OT vesicles (white arrows) identified by OT-associated neurophysin 1 immunoreactivity (15-nm gold particles). Lysosome distribution reveals by immunogold staining (6-nm gold particles) of LAMP1. Lysosomes appear surrounding the dense-cored vesicles and the membrane of electron lucent structures. (Scale bars, 200 nm, and 100 nm in the enlarged image.) (C) Scheme showing the priming protocol. (D) Basal levels of OT release measured in the supernatant after preincubation with 10 µM thapsigargin (Thapsi), 5 µM NAADP, or 10 µM of the TPC2 agonist TPC2-A1-N (A1-N) (Ctrl n = 10 mice; Thapsi n = 5 mice; NAADP n = 5 mice; A1-N n = 5 mice). Bars represent OT released in percentage of WT OT release without drugs. (E) OT secretion evoked by 50 mM KCl stimulation of the hypothalamus, following preincubation with various drugs. Bars represent OT released in percentage of WT OT release without drugs. Green bars show OT release in WT hypothalamus in response to thapsigargin (Thapsi) or NAADP preincubation in the presence of 100 µM Ned-19 (Ctrl WT and DKO n = 15 mice; thapsi + Ned-19 n = 4 mice; NAADP DKO n = 6 mice; all other groups n = 5 mice). Statistical analyses were performed with 1-way ANOVA followed by Dunnett’s test. Values are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 and ns, nonsignificant.

**Fig. 5.**
TPC deletion impaired maternal and social behaviors. (A) Proportion of pups retrieved by dams (WT n = 36, DKO n = 27, DKO+OT n = 30). (B) Latencies took by the dam to retrieve its first pup (WT n = 9, DKO n = 7, DKO+OT n = 10). (C) Diagram describing the social motivation test and histograms showing the time spent in contact with the empty pot or with the mouse visitor during the social motivation test. (D) Diagram describing the social novelty test and histogram showing the time spent with the familiar or the new mouse visitor during this test (WT n = 9 mice, DKO n = 10 mice, DKO+OT n = 10 mice). (E) Typical ultrasonic vocalizations (USVs) recorded during a free interaction between two adult males. (F) Proportion of each type of USVs emitted by the WT mice and the TPC DKO after intranasal NaCl administration (left and mid pie charts) and by TPC DKO mice after OT administration (right pie chart). Statistical analysis performed with A Fisher’s exact test, (B–D) unpaired two-tailed t test, (F) two-way ANOVA followed by a Chi-square post hoc test. Values are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

**Fig. 6.**
Endolysosomal TPCs control OT vesicle priming and release. Working model showing that glutamate, through mGluR1 activation, engages the NAADP pathway to stimulate Ca²⁺ release from endolysosomal TPCs. This Ca²⁺ response in its turns, participates in OT release likely by recruiting the ER Ca²⁺ by Ca²⁺-induced Ca²⁺ release. This scheme includes the priming working model we propose showing that TPCs generate the Ca²⁺ ionic signal to prime the nonreleasable vesicles into readily releasable vesicles either alone or by triggering a larger Ca²⁺ release from the ER stores (CICR). Note that for the priming process, the ER Ca²⁺ channels supporting priming remain to be identified. Abbreviations: mGluR, metabotropic glutamate receptor; CICR, Ca²⁺ -induced Ca²⁺ release; IP₃R, IP₃ receptors; LDCV, large dense core vesicle; SERCA, sarcoendoplasmic reticulum Ca²⁺ ATPase; VGCC, voltage-gated Ca²⁺ channel.

See this image and copyright information in PMC

Cited by

MASTER-NAADP: a membrane permeable precursor of the Ca²⁺ mobilizing second messenger NAADP.
Krukenberg S, Möckl F, Weiß M, Dekiert P, Hofmann M, Gerlach F, Winterberg KJ, Kovacevic D, Khansahib I, Troost B, Hinrichs M, Granato V, Nawrocki M, Hub T, Tsvilovskyy V, Medert R, Woelk LM, Förster F, Li H, Werner R, Altfeld M, Huber S, Clarke OB, Freichel M, Diercks BP, Meier C, Guse AH. Krukenberg S, et al. Nat Commun. 2024 Sep 13;15(1):8008. doi: 10.1038/s41467-024-52024-y. Nat Commun. 2024. PMID: 39271671 Free PMC article.

References

1. Dale H. H., On some physiological actions of ergot. J. Physiol. 34, 163–206 (1906). - PMC - PubMed
1. Bell W. B., The pituitary body and the therapeutic value of the infundibular extract in shock, uterine atony, and intestinal paresis. Br. Med. J. 2, 1609–1613 (1909). - PMC - PubMed
1. Ott I., Scott J. C., The action of the glandular extracts upon the contractions of the uterus. J. Exp. Med. 11, 326–330 (1909). - PMC - PubMed
1. Pedersen C. A., Prange A. J., Induction of maternal behavior in virgin rats after intracerebroventricular administration of oxytocin. Proc. Natl. Acad. Sci. U.S.A. 76, 6661–6665 (1979). - PMC - PubMed
1. Marlin B. J., Mitre M., D’amour J. A., Chao M. V., Froemke R. C., Oxytocin enables maternal behaviour by balancing cortical inhibition. Nature 520, 499–504 (2015). - PMC - PubMed

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Miscellaneous
- NCI CPTAC Assay Portal

[1] Dale H. H., On some physiological actions of ergot. J. Physiol. 34, 163–206 (1906). - PMC - PubMed

[2] Dale H. H., On some physiological actions of ergot. J. Physiol. 34, 163–206 (1906). - PMC - PubMed

[3] Bell W. B., The pituitary body and the therapeutic value of the infundibular extract in shock, uterine atony, and intestinal paresis. Br. Med. J. 2, 1609–1613 (1909). - PMC - PubMed

[4] Bell W. B., The pituitary body and the therapeutic value of the infundibular extract in shock, uterine atony, and intestinal paresis. Br. Med. J. 2, 1609–1613 (1909). - PMC - PubMed

[5] Ott I., Scott J. C., The action of the glandular extracts upon the contractions of the uterus. J. Exp. Med. 11, 326–330 (1909). - PMC - PubMed

[6] Ott I., Scott J. C., The action of the glandular extracts upon the contractions of the uterus. J. Exp. Med. 11, 326–330 (1909). - PMC - PubMed

[7] Pedersen C. A., Prange A. J., Induction of maternal behavior in virgin rats after intracerebroventricular administration of oxytocin. Proc. Natl. Acad. Sci. U.S.A. 76, 6661–6665 (1979). - PMC - PubMed

[8] Pedersen C. A., Prange A. J., Induction of maternal behavior in virgin rats after intracerebroventricular administration of oxytocin. Proc. Natl. Acad. Sci. U.S.A. 76, 6661–6665 (1979). - PMC - PubMed

[9] Marlin B. J., Mitre M., D’amour J. A., Chao M. V., Froemke R. C., Oxytocin enables maternal behaviour by balancing cortical inhibition. Nature 520, 499–504 (2015). - PMC - PubMed

[10] Marlin B. J., Mitre M., D’amour J. A., Chao M. V., Froemke R. C., Oxytocin enables maternal behaviour by balancing cortical inhibition. Nature 520, 499–504 (2015). - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion

Affiliations

Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Miscellaneous

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Miscellaneous