Glucose metabolism links astroglial mitochondria to cannabinoid effects

Abstract

Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1,2,3,4,5. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors5,6,7. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB1 receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.

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Fig. 1: mtCB1 activation inhibits the activity of complex I by destabilization of the N-module.
Fig. 2: Astroglial mtCB1 activation decreases the level of phosphorylated NDUFS4 and inhibits the activity of complex I.
Fig. 3: MtCB1-dependent destabilization of complex I reduces levels of mROS and leads to downregulation of the astroglial HIF-1 pathway.
Fig. 4: Astroglial mtCB1 activation decreases the production of lactate and mediates the THC-induced impairment of social behaviour.

Data availability

All data generated in this manuscript are included within the paper (and its Supplementary Information files). For any further inquiries about our work please contact the corresponding authors. Source data are provided with this paper.

References

  1. 1.

    Magistretti, P. J. & Allaman, I. Lactate in the brain: from metabolic end-product to signalling molecule. Nat. Rev. Neurosci. 19, 235–249 (2018).

    CAS  PubMed  Google Scholar 

  2. 2.

    Barros, L. F. & Weber, B. CrossTalk proposal: an important astrocyte-to-neuron lactate shuttle couples neuronal activity to glucose utilisation in the brain. J. Physiol. 596, 347–350 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Bolaños, J. P. Bioenergetics and redox adaptations of astrocytes to neuronal activity. J. Neurochem. 139, 115–125 (2016).

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Suzuki, A. et al. Astrocyte-neuron lactate transport is required for long-term memory formation. Cell 144, 810–823 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Allen, N. J. & Barres, B. A. Glia — more than just brain glue. Nature 457, 675–677 (2009).

    ADS  CAS  PubMed  Google Scholar 

  6. 6.

    Hansson, E. & Rönnbäck, L. Astrocytes in neurotransmission. a review. Cell. Mol. Biol. 36, 487–496 (1990).

    CAS  PubMed  Google Scholar 

  7. 7.

    Araque, A. et al. Gliotransmitters travel in time and space. Neuron 81, 728–739 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Almeida, A., Almeida, J., Bolaños, J. P. & Moncada, S. Different responses of astrocytes and neurons to nitric oxide: the role of glycolytically generated ATP in astrocyte protection. Proc. Natl Acad. Sci. USA 98, 15294–15299 (2001).

    ADS  CAS  PubMed  Google Scholar 

  9. 9.

    Lopez-Fabuel, I. et al. Complex I assembly into supercomplexes determines differential mitochondrial ROS production in neurons and astrocytes. Proc. Natl Acad. Sci. USA 113, 13063–13068 (2016).

    CAS  PubMed  Google Scholar 

  10. 10.

    Semenza, G. L., Roth, P. H., Fang, H. M. & Wang, G. L. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J. Biol. Chem. 269, 23757–23763 (1994).

    CAS  PubMed  Google Scholar 

  11. 11.

    Busquets-Garcia, A., Bains, J. & Marsicano, G. CB1 receptor signaling in the brain: extracting specificity from ubiquity. Neuropsychopharmacology 43, 4–20 (2018).

    CAS  PubMed  Google Scholar 

  12. 12.

    Araque, A., Castillo, P. E., Manzoni, O. J. & Tonini, R. Synaptic functions of endocannabinoid signaling in health and disease. Neuropharmacology 124, 13–24 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Robin, L. M. et al. Astroglial CB1 receptors determine synaptic D-serine availability to enable recognition memory. Neuron 98, 935–944 (2018).

    CAS  PubMed  Google Scholar 

  14. 14.

    Bénard, G. et al. Mitochondrial CB1 receptors regulate neuronal energy metabolism. Nat. Neurosci. 15, 558–564 (2012).

    PubMed  Google Scholar 

  15. 15.

    Hebert-Chatelain, E. et al. A cannabinoid link between mitochondria and memory. Nature 539, 555–559 (2016).

    CAS  PubMed  Google Scholar 

  16. 16.

    Mendizabal-Zubiaga, J. et al. Cannabinoid CB1 receptors are localized in striated muscle mitochondria and regulate mitochondrial respiration. Front. Physiol. 7, 476 (2016).

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Aquila, S. et al. Human sperm anatomy: ultrastructural localization of the cannabinoid1 receptor and a potential role of anandamide in sperm survival and acrosome reaction. Anat. Rec. 293, 298–309 (2010).

    CAS  Google Scholar 

  18. 18.

    Koch, M. et al. Hypothalamic POMC neurons promote cannabinoid-induced feeding. Nature 519, 45–50 (2015).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Gutiérrez-Rodríguez, A. et al. Localization of the cannabinoid type-1 receptor in subcellular astrocyte compartments of mutant mouse hippocampus. Glia 66, 1417–1431 (2018).

    PubMed  Google Scholar 

  20. 20.

    Hebert-Chatelain, E. et al. Cannabinoid control of brain bioenergetics: exploring the subcellular localization of the CB1 receptor. Mol. Metab. 3, 495–504 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Han, J. et al. Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD. Cell 148, 1039–1050 (2012).

    CAS  PubMed  Google Scholar 

  22. 22.

    Guaras, A. M. & Enríquez, J. A. Building a beautiful beast: mammalian respiratory complex I. Cell Metab. 25, 4–5 (2017).

    CAS  PubMed  Google Scholar 

  23. 23.

    Mimaki, M., Wang, X., McKenzie, M., Thorburn, D. R. & Ryan, M. T. Understanding mitochondrial complex I assembly in health and disease. Biochim. Biophys. Acta 1817, 851–862 (2012).

    CAS  PubMed  Google Scholar 

  24. 24.

    De Rasmo, D., Panelli, D., Sardanelli, A. M. & Papa, S. cAMP-dependent protein kinase regulates the mitochondrial import of the nuclear encoded NDUFS4 subunit of complex I. Cell Signal. 20, 989–997 (2008).

    PubMed  Google Scholar 

  25. 25.

    Hammond, S. L., Leek, A. N., Richman, E. H. & Tjalkens, R. B. Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection. PLoS ONE 12, e0188830 (2017).

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Vicente-Gutierrez, C. et al. Astrocytic mitochondrial ROS modulate brain metabolism and mouse behavior. Nat. Metab. 1, 201–211 (2019).

    CAS  Google Scholar 

  27. 27.

    Chandel, N. S. et al. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc. Natl Acad. Sci. USA 95, 11715–11720 (1998).

    ADS  CAS  PubMed  Google Scholar 

  28. 28.

    Patten, D. A. et al. Hypoxia-inducible factor-1 activation in nonhypoxic conditions: the essential role of mitochondrial-derived reactive oxygen species. Mol. Biol. Cell 21, 3247–3257 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Shadel, G. S. & Horvath, T. L. Mitochondrial ROS signaling in organismal homeostasis. Cell 163, 560–569 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Semenza, G. L. Hypoxia-inducible factors: coupling glucose metabolism and redox regulation with induction of the breast cancer stem cell phenotype. EMBO J. 36, 252–259 (2017).

    CAS  PubMed  Google Scholar 

  31. 31.

    Semenza, G. L. Dynamic regulation of stem cell specification and maintenance by hypoxia-inducible factors. Mol. Aspects Med. 47–48, 15–23 (2016).

    PubMed  Google Scholar 

  32. 32.

    Almeida, A., Moncada, S. & Bolaños, J. P. Nitric oxide switches on glycolysis through the AMP protein kinase and 6-phosphofructo-2-kinase pathway. Nat. Cell Biol. 6, 45–51 (2004).

    CAS  PubMed  Google Scholar 

  33. 33.

    Herrero-Mendez, A. et al. The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1. Nat. Cell Biol. 11, 747–752 (2009).

    CAS  PubMed  Google Scholar 

  34. 34.

    Jimenez-Blasco, D., Santofimia-Castaño, P., Gonzalez, A., Almeida, A. & Bolaños, J. P. Astrocyte NMDA receptors’ activity sustains neuronal survival through a Cdk5–Nrf2 pathway. Cell Death Differ. 22, 1877–1889 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Morland, C. et al. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1: expression and action in brain. J. Neurosci. Res. 93, 1045–1055 (2015).

    CAS  PubMed  Google Scholar 

  36. 36.

    Liu, C. et al. 3,5-Dihydroxybenzoic acid, a specific agonist for hydroxycarboxylic acid 1, inhibits lipolysis in adipocytes. J. Pharmacol. Exp. Ther. 341, 794–801 (2012).

    CAS  PubMed  Google Scholar 

  37. 37.

    Pierre, K., Magistretti, P. J. & Pellerin, L. MCT2 is a major neuronal monocarboxylate transporter in the adult mouse brain. J. Cereb. Blood Flow Metab. 22, 586–595 (2002).

    CAS  PubMed  Google Scholar 

  38. 38.

    Mazuel, L. et al. A neuronal MCT2 knockdown in the rat somatosensory cortex reduces both the NMR lactate signal and the BOLD response during whisker stimulation. PLoS One 12, e0174990 (2017).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Hollis, F. et al. Mitochondrial function in the brain links anxiety with social subordination. Proc. Natl Acad. Sci. USA 112, 15486–15491 (2015).

    ADS  CAS  PubMed  Google Scholar 

  40. 40.

    Picard, M., McEwen, B. S., Epel, E. S. & Sandi, C. An energetic view of stress: focus on mitochondria. Front. Neuroendocrinol. 49, 72–85 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Melser, S. et al. Functional analysis of mitochondrial CB1 cannabinoid receptors (mtCB1) in the brain. Methods Enzymol. 593, 143–174 (2017).

    CAS  PubMed  Google Scholar 

  42. 42.

    Jollé, C., Déglon, N., Pythoud, C., Bouzier-Sore, A. K. & Pellerin, L. Development of AAV2/DJ-based viral vectors to selectively downregulate the expression of neuronal or astrocytic target proteins in the rat central nervous system. Front Mol. Neurosci. 12, 201 (2019).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Marsicano, G. et al. The endogenous cannabinoid system controls extinction of aversive memories. Nature 418, 530–534 (2002).

    ADS  CAS  PubMed  Google Scholar 

  44. 44.

    Puente, N., Bonilla-Del Río, I., Achicallende, S., Nahirney, P. C. & Grandes, P. High-resolution immunoelectron microscopy techniques for revealing distinct subcellular type 1 cannabinoid receptor domains in brain. Bio-protocol 9, e3145 (2019).

    Google Scholar 

  45. 45.

    De Rasmo, D. et al. Activation of the cAMP cascade in human fibroblast cultures rescues the activity of oxidatively damaged complex I. Free Radic. Biol. Med. 52, 757–764 (2012).

    PubMed  Google Scholar 

  46. 46.

    Acin-Perez, R., Fernandez-Silva, P., Peleato, M. L., Perez-Martos, A. & Enriquez, J. A. Respiratory active mitochondrial supercomplexes. Mol. Cell 32, 529–539 (2008).

    CAS  PubMed  Google Scholar 

  47. 47.

    Belousov, V. V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3, 281–286 (2006).

    CAS  PubMed  Google Scholar 

  48. 48.

    Darley-Usmar, V. M., Rickwood, D. & Wilson, M. T. (eds) Mitochondria: A Practical Approach (IRL, 1987).

  49. 49.

    King, T. E. in Methods in Enzymology Vol. 10 (eds Estabrook, R. W. & Pullman, M. E.) 216–225 (Academic, 1967).

  50. 50.

    Wharton, D. C. & Tzagoloff, A. in Methods in Enzymology Vol. 10 (eds Estabrook, R. W. & Pullman, M. E.) 245–250 (Academic, 1967).

  51. 51.

    Shepherd, D. & Garland, P. B. The kinetic properties of citrate synthase from rat liver mitochondria. Biochem. J. 114, 597–610 (1969).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Busquets-Garcia, A. et al. Pregnenolone blocks cannabinoid-induced acute psychotic-like states in mice. Mol. Psychiatry 22, 1594–1603 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Soria-Gómez, E. et al. Habenular CB1 receptors control the expression of aversive memories. Neuron 88, 306–313 (2015).

    PubMed  Google Scholar 

  54. 54.

    Paxinos, G. & Franklin, K. B. J. The Mouse Brain in Stereotaxic Coordinates (Academic, 2001).

  55. 55.

    Martin, P. M. & O’Callaghan, J. P. A direct comparison of GFAP immunocytochemistry and GFAP concentration in various regions of ethanol-fixed rat and mouse brain. J. Neurosci. Methods 58, 181–192 (1995).

    CAS  PubMed  Google Scholar 

  56. 56.

    Huang, H. et al. Chronic and acute intranasal oxytocin produce divergent social effects in mice. Neuropsychopharmacol. 39, 1102–1114 (2014).

    CAS  Google Scholar 

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Acknowledgements

We thank D. Gonzales, N. Aubailly, M. Carabias-Carrasco, L. Martin, E. Prieto-Garcia and all the personnel of the Animal Facilities of the NeuroCentre Magendie and University of Salamanca for mouse care; the Biochemistry Platform of Bordeaux NeuroCampus for help; S. Papa and D. De Rasmo for providing the NDUFS4 cDNA and anti-phospho-Ser173 NDUFS4 antibody, respectively; the viral vector facility headed by A. Bemelmans for producing AAVs at MIRCen; M.-C. Gaillard for help in the design and production of the gfaABC1D-NDUFS4-PM plasmid; P.-A. Vigneron for the acquisition of confocal images depicting cellular tropism of AAV PHP.eB; all the members of the Marsicano laboratory for useful discussions; V. Morales for invaluable assistance; and S. Pouvreau, G. Benard, D. Cota and G. Ferreira for critical reading of the manuscript and suggestions. This work was funded by: INSERM, the European Research Council (Endofood, ERC–2010–StG–260515 and CannaPreg, ERC-2014-PoC-640923, MiCaBra, ERC-2017-AdG-786467), Fondation pour la Recherche Medicale (FRM, DRM20101220445), the Human Frontiers Science Program, Region Nouvelle Aquitaine and Agence Nationale de la Recherche (ANR; NeuroNutriSens ANR-13-BSV4-0006, CaCoVi ANR 18-CE16-0001-02, MitObesity ANR 18-CE14-0029-01, ORUPS ANR-16-CE37-0010-01 and BRAIN ANR-10-LABX-0043) (to G.M.); NIH/NIDA (1R21DA037678-01), Spanish Ministry of Science, Innovation and Universities (MCINU/FEDER; grants SAF2016-78114-R and RED2018-102576-T), Instituto de Salud Carlos III (CB16/10/00282), an EU BATCure grant (666918), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA and Fundación Ramón Areces (to J.P.B.); Instituto de Salud Carlos III (PI18/00285; RD16/0019/0018), the European Regional Development Fund, the European Union’s Horizon 2020 Research and Innovation Programme (grant agreement 686009), Junta de Castilla y León (IES007P17) and Fundación Ramón Areces to (A.A.); French State/ANR/IdEx (ANR-10-IDEX-03-02), Eu-Fp7 (FP7-PEOPLE-2013-IEF-623638) and Ramon y Cajal Investigator Program (RYC-2017-21776) (to A.B.-G.); FRM (SPF20121226369) (to R.S.); FRM (ARF20140129235) (to L.B.); Spanish Ministry of Science, Innovation and Universities (MCINU/FEDER; grants SAF2015-64945-R and RTI2018-095311-B-I00) (to M.G.); Canada Research Chair, Alzheimer Society of Canada—Brain Canada (17-09), Natural Sciences and Engineering Research Council (RGPIN-2015-05880), Canadian Breast Cancer Foundation (2015-317342), Canadian Health Research Institute (CIHR, 388808), New Brunswick Innovation Foundation, New Brunswick Health Research Foundation and Université de Moncton (to E.H.-C.); Basque Government (IT1230-19), Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III), European Regional Development Funds-European Union (ERDF-EU; RD16/0017/0012) and MINECO/FEDER, UE (SAF2015-65034-R) (to P.G.); University of the Basque Country PhD contract (PIF 16/251) (to S.A.); POP contract (BES-2016-076766, BES-C-2016-0051) (to I.B.-D.R.); French State/ANR (ANR-10-IDEX and TRAIL ANR-10-LABX-57); French-Swiss ANR-FNS (ANR-15- CE37-0012) (to A.-K.B.-S.); SFB/TRR 58 ‘Fear, anxiety, anxiety disorders’ (subproject A04); and CRC 1193 ‘Neurobiology of resilience’ (subproject B04) (to B.L.).

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Authors

Contributions

J.P.B. and GM. conceptualized and supervised the study. D.J.-B., E.H.-C., C.V.-G., R.S., I.L.-F., M.R.-B., D.S. and A.A. performed and supervised in vitro experiments in cell and astrocyte cultures and ex vivo analysis of brain tissue; A.B.-G., C.I. and P.G.-S. performed behavioural experiments and surgical procedures in mice; E.R. and M.G. provided some CB1-KO mice to the group of J.P.B.; D.A. and A.P. performed electrophysiological experiments not shown in the manuscript; M.V. and F.J.-K. performed mouse perfusion and immunohistochemistry experiments; A.C. and L.B. produced some of the viral constructs used (for example, Syn-MitoCAT); I.B.-D.R., N.P., S.A. and P.G. performed and supervised electron microscopy experiments; M.-L.L.-R. provided pharmacological tools (HU210–Biotin); C. Jollé, N.D. and L.P. provided specific viral constructs to modulate the MCT2 transporter; C. Josephine and G.B. provided data and viral vectors for mouse retro-orbital injections; B.L. and P.-V.P. provided conceptual ideas; and A.-K.B.-S performed in vivo NMR experiments. D.J.-B., A.B.-G., E.H.-C., M.G., J.P.B. and G.M. wrote the paper with input from all authors.

Corresponding authors

Correspondence to Juan P. Bolaños or Giovanni Marsicano.

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Extended data figures and tables

Extended Data Fig. 1 Role of astroglial mtCB1 in the cannabinoid-induced decrease of respiration in forebrain mitochondria.

a, Detection of CB1 receptors on astroglial and neuronal mitochondrial membranes in the nucleus accumbens and piriform cortex of wild-type and CB1-KO mice (n = 4). am, astrocytic mitochondria; nm, neuronal mitochondria; sp, spine; ter, terminal. CB1-positive inhibitory terminals are marked in blue, astrocytes in brown, excitatory terminals in red, astrocytic mitochondria in yellow and neuronal mitochondria in green. Asterisks indicate astrocytic processes; coloured arrows point to CB1 particles at colour-matching subcellular compartments and mitochondria. Scale bars, 1 μm. #, P < 0.05 (vs WT). b, Effects of the CB1 receptor cell permeant and impermeant agonists HU210 (1 μM, n = 5) and HU210-Biotin (1 μM, n = 6), respectively, on cellular respiration of primary mouse cortical astrocyte cultures. *, P < 0.05 (versus HU210-Biotin). c, sAC inhibitor KH7 (5 μM, n = 3) effects on the HU210-induced reduction of cellular respiration in astrocyte cultures. *, P < 0.05 (versus control). d, e, THC (800 nM) (d) and WIN (100 nM) (e) effects on respiration of purified brain mitochondria from CB1-KO (n = 4) and GFAP-CB1-KO (n = 5) mice and their respective WT littermates (CB1-WT and GFAP-CB1-WT). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (versus WT conditions). Data are expressed as mean ± s.e.m. and are analysed by one-way ANOVA in a, d and e; two-way ANOVA in c and unpaired two-sided Student’s t-test in c. n represents number of mice in a, d and e and independent experiments in b and c. Statistical details, Supplementary Table 2. Source data

Extended Data Fig. 2 Activation of mtCB1 receptors inhibits complex I activity by destabilizing the N-module of complex I.

a, THC (1 μM) or HU210 (50 nM) effects on complex II–III (CII–III; left), complex IV (CIV; centre) and citrate synthase (right) activities in WT astrocyte cultures (n = 3). b, c, Quantification (n = 3) of gels represented in Fig. 1c, reporting the effects of THC on (b) mitochondrial complex I (CI) activity and (c) expression (normalized to β-ATPase) of the complex I subunits NDUFS1, NDUFV2, NDUFB8 and NDUFA9 in complex I and super-complexes (SC) of cultured astrocytes from WT mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (versus Vehicle). d, e, Quantification (n = 3) of gels represented in Fig. 1d, reporting THC or HU210 effects on (d) complex I activity and (e) expression (normalized to β-ATPase) of the complex I subunit NDUFS1 in complex I and SC of WT and CB1-KO mice. **, P < 0.01; ***, P < 0.001 (versus Vehicle); #, P < 0.05; ##, P < 0.01; ###, P < 0.001 (versus WT). f, Relative quantification (n = 3) of BNGE represented in Fig. 1e, reporting the in vivo THC effects (10 mg/Kg, 24h before) on complex I activity and expression (normalized to β-ATPase) of the complex I subunit NDUFS1 and NDUFA9 in complex I and SC of HC (left panels) or PFC (right panels) from GFAP-CB1-WT and GFAP-CB1-KO mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ##, P < 0.01; ###, P < 0.001 (versus WT). g, Representative Western Immunoblottings (left) and relative quantification of these gels (n = 3) (right) showing the effects of in vivo THC treatment (10 mg/Kg, 24h before) on expression of the complex I subunits NDUFS1, NDUFV2 and NDUFA9 of HC or PFC from GFAP-CB1-WT and GFAP-CB1-KO mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 (versus WT). Data are expressed as mean ± s.e.m. and are analysed by one-way ANOVA in ac and two-way ANOVA in dg. n represents number of independent experiments. Original gels, Supplementary Fig. 1; Statistical details, Supplementary Table 2. Source data

Extended Data Fig. 3 Activation of astroglial mtCB1 receptors decreases NDUFS4-Ser173 phosphorylation to inhibit complex I activity.

a, Quantification (n = 3) of Fig. 2a, reporting the THC (1 μM) effects on PKA-dependent phosphorylation of complex I proteins (left) and on NDUFS2 levels in WT astrocytes (right). ***, P < 0.001 (versus Vehicle). b, Quantification (n = 3) of Fig. 2b, reporting the THC effects on pNDUFS4 in WT astrocytes. ***, P < 0.001 (versus Vehicle). c, Quantification (n = 3) of Fig. 2c, reporting THC or HU210 effects on pNDUFS4 levels in WT and CB1-KO astrocytes. **, P < 0.01; ***, P < 0.001 (versus Vehicle). ##, P < 0.01; ###, P < 0.001 (versus WT-THC). d, Quantification (n = 3) of Fig. 2d, reporting THC or HU210 effects on pNDUFS4 levels in CB1-KO astrocytes (empty, CB1-WT, DN22-CB1). *, P < 0.05 (versus Vehicle). ##, P < 0.01 (versus WT). e, Quantification (n = 3) of Fig. 2e, reporting THC (10 mg/Kg, 24h before) effects on pNDUFS4 of HC (left) or PFC (right) from GFAP-CB1-WT and GFAP-CB1-KO mice. *, P < 0.05 (versus Vehicle). #, P < 0.05; ###, P < 0.001 (versus WT). f, Quantification (n = 3) of Fig. 2f, reporting the expression of NDUFS4-PM in WT astrocytes with or without THC treatment. *, P < 0.05 (versus Vehicle). g, Quantification (n = 3) of Fig. 2g, reporting NDUFS4-PM expression in WT astrocytes effects on complex I activity, NDUFS1 levels and β-ATPase expression after 24h incubation with vehicle, THC or HU210. **, P < 0.01; ***P < 0.001 (versus Vehicle). #, P < 0.05; ##, P < 0.01; ###, P < 0.001 (versus empty). h, THC or HU210 effects on citrate synthase activity of WT astrocytes in the presence or absence of NDUFS4-PM (n = 3). i, Quantification of Fig. 2i, reporting AAV-GFAP-NDUFS4-PM effects on complex I activity, NDUFS1 and NDUFA9 levels and -ATPase expression in HC (left) or PFC (right) 24h after THC treatment (10 mg/Kg). *, P < 0.05; ***P < 0.001 (versus Vehicle). #, P < 0.05; ##, P < 0.01; ###, P < 0.001 (vs AAV-Control). Data are expressed as mean ± s.e.m. and are analysed by one-way ANOVA in a, b and two-way ANOVA in ci. n represents number of independent experiments. Statistical details, Supplementary Table 2. Source data

Extended Data Fig. 4 Astroglial expression of NDUFS4-PM in the hippocampus and PFC.

Top, immunofluorescence micrographs showing the expression of AAV-gfa-ABC1D-NDUFS4-PM (fused to mRuby, red) in neurons (staining with NeuN marker, green) or astrocytes (staining with an anti-S-100β antibody, green) of the HC and the PFC. Note the large overlapping in the merged images when using the S-100-β antibody (HC, n = 10 mice; PFC, n = 8) but not the neuronal NeuN marker (n = 5 mice; PFC, n = 3). Bottom, graph quantification showing the percentage of mRuby positive cells (NDUFS4-PM positive) that colocalize with the astroglial marker S-100-β or the neuronal one NeuN. Data are expressed as mean ± s.e.m. Scale bars, 40 μm. Source data

Extended Data Fig. 5 MtCB1-dependent effects on mROS and mitochondrial membrane potential in astrocytes.

a, THC (1 μM) or HU210 (50 nM) effects on mROS in CB1-WT and CB1-KO astrocytes (n = 8) as revealed by Amplex Red fluorescence. *, P < 0.05 (versus Vehicle); #, P < 0.05; ###, P < 0.001 (versus WT). b, pHyPer-dMito micrographs (left) and quantification (right) 24h after THC (1 μM) or HU210 (50 nM) treatments in CB1-WT and CB1-KO astrocytes (n = 5). Scale bar: 40 uM. *, P < 0.05 (versus Vehicle); #, P < 0.05 (versus WT). c, Effects on mROS of NDUFS4-PM transfection per se in astrocytes from CB1-WT (n = 3). d, MitoSOX schematic example of the gating strategy and workflow for the determination of mROS in GFP+ cells by flow cytometry. e, Left, immunofluorescence micrographs showing expression of GFP three weeks after retro-orbital injection of the AAV-PHP.eB gfaABC1D-GFP. GFP is expressed in PHGDH-positive astrocytes but not in NeuN-positive neurons (n = 3). Right, immunofluorescence micrographs showing expression of GFP three weeks after retro-orbital injection of the AAV-PHP.eB hSYN-GFP. GFP is expressed in NeuN-positive neurons and not in PHGDH-positive astrocytes (n = 3). f, AAV-gfa-ABC1D-NDUFS4-PM infusion effects on the levels of mROS in HC and PFC (n = 5). Data are expressed as mean ± s.e.m. and are analysed by two-way ANOVA. n represents number of independent experiments. Statistical details are in Supplementary Table 2. Source data

Extended Data Fig. 6 Activation of astroglial mtCB1 receptors decreases glycolysis and lactate release by attenuating the HIF-1 pathway.

a, THC (1 μM) or HU210 (50 nM) effects on HIF-1 promoter activity in WT and CB1-KO astrocytes (n = 5). ***, P < 0.001 (versus Vehicle); ###, P < 0.001 (versus WT). b, THC or HU210 effects (n = 3) on mRNA levels of different HIF-1 targets (Glut3, HkII and Gadph) in WT and CB1-KO astrocytes. **, P < 0.01; ***, P < 0.001 (versus Vehicle); #, P < 0.05 (versus WT). c, Quantification (n = 3) of Fig. 3g, reporting THC and HU210 effects on HIF-1α nuclear expression in WT and CB1-KO astrocytes. *, P < 0.05; ***, P < 0.001 (versus Vehicle); #, P < 0.05; (versus WT). d, Quantification (n = 3) of Fig. 3h, reporting THC effects (10 mg/Kg, 24h before) on HIF-1α protein levels in HC (left) or PFC (right) from GFAP-CB1-WT and GFAP-CB1-KO mice. *, P < 0.05; ***, P < 0.001 (versus Vehicle); #, P < 0.05; ##, P < 0.01 (versus WT). e, Quantification (n = 3) of Fig. 3i, reporting NDUFS4-PM expression effects on the THC-induced HIF-1α expression decrease in WT astrocytes. *, P < 0.05 (versus Vehicle); ##, P < 0.01 (versus empty). f, Quantification (n = 3) of Fig. 3j, reporting the effect of local infusion of AAV-GFAP-NDUFS4-PM on the THC-mediated decrease of HIF-1a in HC (left) or PFC (right) from WT mice. *, P < 0.05 **, P < 0.01 (versus Vehicle); ##, P < 0.01 (versus AAV-Control). g, Western immunoblotting (left) and quantification (n = 3, right) showing the HIF-1α overexpression obtained by transfecting astrocytes with 1 or 0.25 μg plasmid DNA, respectively. *, P < 0.05; ***, P < 0.001 (versus empty). h, Effect of HIF-1α overexpression (n = 6) on lactate release from WT astrocytes. ***, P < 0.001 (versus empty). Data are expressed as mean ± s.e.m. and are analysed by one-way ANOVA in gh and two-way ANOVA in af. n represents number of independent experiments. Statistical details, Supplementary Table 2. Source data

Extended Data Fig. 7 Determination of mitochondrial membrane potential and apoptosis in neurons.

a, Schematic and representative example of the gating strategy and workflow for the determination of mitochondrial membrane potential (DiIC1(5)) in neurons co-cultured with astrocytes by flow cytometry. b, Effects on cultured neurons co-cultured with astrocytes from CB1-WT or CB1-KO mice previously treated with THC (1 μM) or HU210 (50 nM) on mitochondrial membrane potential (ΔΨm). **, P < 0.01; ***, P < 0.001 (versus Vehicle); ##, P < 0.01; ###, P < 0.001 (versus WT-cannabinoids). c, Effects of lactate supplementation (2 mM) on the decreased mitochondrial membrane potential (ΔΨm). ***, P < 0.001 (versus Saline); ###, P < 0.001; (versus cannabinoids). d, Schematic and representative example of the gating strategy and workflow for the determination of apoptosis (AnnV+/7AAD) in brain GFP+ cells by flow cytometry. e, Effects of ICV lactate supplementation (100 mM) on the THC-induced increase of apoptotic cell death, in Syn-positive cells (neurons) from PFC or HC (n = 5). **, P < 0.01; ***, P < 0.001 (versus Saline); ##, P < 0.01; ###, P < 0.001 (versus THC). f, g, Effect of the intravenous administration of AAV-gfa-ABC1D-NDUFS4-PM on the neuronal levels of mROS as revealed by MitoSOX analysis by fluorescence-activated cell sorting (n = 5) (f) and the apoptotic cell death in neurons as revealed by cell sorting flow cytometry (n = 5) (g). h, Effects of retro-orbital injection of AAV-PHP.eB-gfa-ABC1D-NDUFS4-PM on the THC-induced increase of apoptotic cell death, in Syn-positive cells (neurons) from PFC or HC (n = 5). Data are expressed as mean ± s.e.m. and are analysed by one-way ANOVA in b and two-way ANOVA in c and e. n represents number of independent experiments. Statistical details, Supplementary Table 2. Source data

Extended Data Fig. 8 Behavioural and metabolic effects of THC administration in GFAP-CB1-KO mice.

ad, The administration of THC (10 mg per kg, i.p.) did not affect the prepulse inhibition (PPI) of the startle response (a, Veh, n = 7 ; THC, n = 5), the locomotion assessed in the Open Field (b, n = 8), the anxiety-like responses assessed in the Open Field (c, n = 8) or in the elevated plus maze (d, n = 8). e, THC induced spontaneous alternation impairment in both in GFAP-CB1-WT (n = 15) and GFAP-CB1-KO mice (n = Veh, n = 3; THC, n = 5). ** P < 0.01 (versus Vehicle). f, THC effects (Fig. 4k) on the exploration times of social and non-social compartments in GFAP-CB1-KO mice (Veh, n = 11 ; THC, n = 16) and GFAP-CB1-WT (n = 13) littermates. *, P < 0.05; **, P < 0.01 ; ***, P < 0.001 (versus non-social). g, Locomotor effects of THC in the sociability test in GFAP-CB1-KO mice (Veh, n = 11 ; THC, n = 16) and GFAP-CB1-WT littermates (n = 13). h, Top, typical 13C-NMR spectra of brain perchloric acid extracts of a control mouse treated with Vehicle (green) or THC (blue). 1: Alanine C3; 2: Lactate C3; 3: Glutamine C3; 4: Glutamate C3; 5: Glutamine C4; 6: Glutamate C4; 7: GABA C2; 8: Glutamine C2; 9: Glutamate C2; 10: Ethylene glycol (external standard); 11: α Glucose C1; 12: β Glucose C1. Right, zoom showing the difference in the incorporation of carbone-13 into carbone 2 compared to carbone 4 between vehicle- and THC-treated. Black arrow represents the height of carbone-2 peak. Bottom, typical raw 1H-NMR spectra of brain perchloric acid extracts of vehicle treated mouse. Data are expressed as mean ± s.e.m. and are analysed by two-way ANOVA in e, f. n represents number of mice. Experiments shown in h, i were repeated in four independent experiments. Statistical details, Supplementary Table 2. Source data

Extended Data Fig. 9 Mechanisms that underlie the THC-induced impairment of social interaction.

a, THC effects (Fig. 4m) on exploration times of social and non-social compartments (Sal-Veh, n = 10; Sal-THC, n = 8; Lac-Veh, n = 10, Lac-THC, n = 10). **, P < 0.01 (versus non-social). b, Locomotor effects of THC in the sociability test in mice receiving saline or lactate (Sal-Veh, n = 9; Sal-THC, n = 7; Lac-Veh, n = 9, Lac-THC, n = 9). c, d, Effects of THC on the direct social interaction test on mice treated ICV with saline (c, Veh-Sal, n = 13; THC-Sal, n = 16) or lactate (d, Veh-Lac, n = 13; THC-Lac, n = 14). **, P < 0.01 (versus Vehicle). e, Overall social exploration through the 3 trials of the direct social interaction task in mice receiving Vehicle (Veh-Sal, n = 13; Veh-Lac, n = 13) or THC (THC-Sal, n = 16, HC-Lac, n = 14) under lactate or Saline (total exploration, 3 trials together). *, P < 0.05 (versus vehicle). f, THC effects (Fig. 4n) on exploration times of social and non-social compartments (Control-Veh, n = 37; Control-THC, n = 38; NDUFS4-Veh, n = 10; NDUSF4-THC, n = 11; MCT2-Veh, n = 15; MCT2-THC, n = 13 ; mitoCAT-Veh, n = 11; mitoCAT-THC, n = 14) in mice injected with the different viral vectors. *, P < 0.05 ; **, P < 0.01 (versus Non-social). g, Locomotor effects of THC administration in the sociability test in mice injected with the different viral vectors (Control-Veh, n = 37; Control-THC, n = 38; NDUFS4-Veh, n = 10; NDUSF4-THC, n = 11; MCT2-Veh, n = 15; MCT2-THC, n = 13 ; mitoCAT-Veh, n = 11; mitoCAT-THC, n = 14). In f, g, different batches of mice infused with different control viral vectors (Methods) were pooled as One-way ANOVA analysis of social indices shown in Fig. 4n indicated no statistical differences between control groups (P = 0.357). h, Representative images of HC and PFC for the different viral vectors infused in HC and PFC. Here, we are showing the expression of mCHERRY after infusion of AAV2/DJ-CBA-mCherry-miR30E-shMCT2 in these brain regions. i, Central (ICV) supplementation of GPR81 receptor agonist (100 nM and 1 mM, n = 5) did not rescue the social impairment induced by THC. **, P < 0.01 (main effect of THC). j, THC effects on the exploration times of social and non-social compartments in the sociability test shown in i (n = 5). *, P < 0.05 (versus non-social). k, Locomotor effects of THC administration in the sociability test in mice receiving saline or the GPR81 receptor agonist just before the test (n = 5). Data are expressed as mean ± s.e.m. and are analysed by two-way ANOVA in a, c, e, f, i, j. n represents number of mice. Statistical details, Supplementary Table 2. Source data

Extended Data Fig. 10 Neuronal expression of mitochondrial catalase in the hippocampus and PFC.

Top, immunofluorescence micrographs showing the expression of AAV-hSyn-mitoCAT (fused to mRuby, red) in neurons (staining with an anti-NeuN antibody) or astrocytes (staining with S100-β, green) of HC and PFC. Note the large overlapping in the merged images when using the NeuN antibody (HC, n = 8 mice; PFC, n = 8) but not the astroglial S100-β marker (HC, n = 4 mice; PFC, n = 4). Bottom, graph quantification showing the % of mRuby positive cells (neurons) that colocalize with the NeuN antibody. Data are mean ±s.e.m. Scale bars, 40 μm. Source data

Extended Data Fig. 11 Astroglial mtCB1 receptor activation impairs social interactions by hampering the metabolism of lactate in the brain and altering neuronal functions through a complex I–mROS–HIF-1 pathway.

(1), Activation of astroglial mtCB1 receptors reduces PKA-dependent phosphorylation of the mitochondrial complex I subunit NDUFS4, which disrupts the assembly and activity of complex I to attenuate the levels of mitochondrial reactive oxygen species (mROS). (2) The decrease in mROS levels leads to a HIF-1α-dependent reduction in the glycolytic production of lactate. (3) The diminished release of lactate from astrocytes to neurons through MCT2 results in neuronal bioenergetic and redox stress. All of these processes lead to deficits in social interaction. This scheme was created by the authors with the use of some free images from Servier Medical Art. Source data

Supplementary information

Supplementary Information

This file contains Supplementary Information Figure 1: Original immunoblot gels used for the representative images shown in Main and Extended Data Figures and Statistical Data Tables 1-2: Additional statistical details of Main (Table 1) and Extended Data (Table 2) Figures.

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Representative video of social interaction and spontaneous alternation experiments in mice.

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Representative video of social interaction and spontaneous alternation experiments in mice.

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Representative video of social interaction and spontaneous alternation experiments in mice.

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Representative video of social interaction and spontaneous alternation experiments in mice.

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Representative video of social interaction and spontaneous alternation experiments in mice.

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Representative video of social interaction and spontaneous alternation experiments in mice.

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Representative video of social interaction and spontaneous alternation experiments in mice.

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Representative video of social interaction and spontaneous alternation experiments in mice.

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Representative video of social interaction and spontaneous alternation experiments in mice.

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Representative video of social interaction and spontaneous alternation experiments in mice.

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Jimenez-Blasco, D., Busquets-Garcia, A., Hebert-Chatelain, E. et al. Glucose metabolism links astroglial mitochondria to cannabinoid effects. Nature 583, 603–608 (2020). https://doi.org/10.1038/s41586-020-2470-y

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