ER-mitochondria contacts underline cannabinoid regulation of calcium signaling in astrocytes


 Intracellular calcium signaling underlies the astroglial control of brain functions. However, the cellular mechanisms regulating calcium handling by astrocytes are far from being understood. Mitochondria-endoplasmic reticulum contacts (MERCs) are key determinants of calcium dynamics, but their functional impact on astroglial regulation of brain information processing is currently unexplored. Here we show that the activation of astrocyte mitochondrial-associated CB1 receptors (mtCB1) regulates MERCs-dependent intracellular calcium signaling, thereby determining the synaptic functions of these cells. In vitro and in vivo stimulation of mtCB1 receptors promotes calcium transfer from the endoplasmic reticulum to mitochondria through a specific molecular cascade, involving AKT signaling, IPR3 receptors and different components of the mitochondrial calcium uniporter complex (MCU). Physiologically, mtCB1-dependent mitochondrial calcium uptake determines the dynamics of cytosolic calcium events in astrocytes upon endocannabinoid mobilization. Accordingly, electrophysiological recordings in hippocampal slices showed that astrocyte-specific mtCB1 receptors exclusion or dominant negative MCU expression blocks lateral synaptic potentiation, through which astrocytes integrate the activity of distant synapses. Altogether, these data reveal a cellular endocannabinoid link between astroglial MERCs and the regulation of brain network functions.


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Astrocytes represent a large proportion of brain cells and exert key metabolic, 2 structural, synaptic and protective functions 1!4 . In particular, accumulating evidence 3 supports a bidirectional communication between neurons and astrocytes at the so! 4 called tripartite synapse, formed by pre! and post!synaptic elements surrounded by 5 astroglial processes, thereby modulating information processing and behavior 2!4 . 6 Very little is known about the intracellular astroglial mechanisms required to exert 7 these functions, but it is now clear that calcium dynamics at different subcellular 8 astroglial microdomains are key functional elements of the tripartite synapse 5!7 . 9 Astroglial calcium handling is a highly sophisticated process, which is bidirectionally 10 linked to synaptic activity, and where intracellular organelles such as endoplasmic 11 reticulum (ER) and mitochondria play key active roles 8!11 . For instance, astroglial 12 microdomain signaling has been suggested to involve calcium efflux from 13 mitochondria 8 . Moreover, recent data indicate that the positioning of mitochondria 14 within astrocytic processes and their specific calcium handling properties participate 15 in the regulation of cellular functions 11 . Mitochondria/ER contacts (MERCs) are key 16 players of calcium signaling in many cells 12 and have been recently described in 17 astrocytes 13 . However, their functional involvement in astrocyte calcium signaling 18 and synaptic integration is currently unknown. 19 One of the most interesting functions of the tripartite synapse is lateral synaptic 20 potentiation (LSP), through which astrocytes, upon neuronal depolarization, are able 21 to enhance synaptic efficacy several tens of micrometers away from the stimulation 22 site 14,15 , thereby contributing to fine!tuned synaptic and circuit integration 16 . LSP 23 requires astroglial intracellular calcium elevations, which critically depend on the 24 endogenous activation of type!1 cannabinoid receptors 15,16 (CB 1 ). Discovered as the 25 main target of synthetic and plant!derived cannabinoid drugs, these G protein! 26 coupled receptors form, together with their endogenous ligands, the so!called 27 endocannabinoid system (ECS), which is a key physiological determinant of synaptic 28 and behavioral functions 17!19 . 29 Recent evidence indicates that, besides their canonical localization at plasma 30 membranes, functional intracellular CB 1 receptors can be observed in close 31 association with mitochondria 20!22 (mtCB 1 ). The activation of mtCB 1 receptors 32 decreases metabolic processes in brain cells, negatively affecting memory 1 performance 21 , social interactions 22 and likely regulating feeding behavior and 2 neuroprotection 21,23!25 . In particular, recent anatomical data showed that astrocytes 3 are endowed with a significant proportion of mtCB 1 receptors that are localized close 4 to synapses 22,26 . ER!dependent calcium signaling has been proposed as a 5 mechanism, through which astroglial CB 1 receptors trigger different functions of the 6 tripartite synapse 27,28 , including LSP 15 . However, the precise astroglial intracellular 7 mechanisms underlying these processes, the potential functional involvement of 8 MERCs and the particular implication of mtCB1 receptors are currently unknown. 9 Here we asked whether astroglial MERCs!dependent calcium signaling is regulated 10 by the activation of mtCB 1 receptors and contributes to ECS!dependent synaptic 11 plasticity. The results indicate that stimulation of CB 1 receptors promotes 12 mitochondrial calcium accumulation in cultured astrocytes and in living animals. In 13 particular, ER!dependent calcium entry into mitochondria is actively promoted by 14 mtCB 1 receptors and regulates cytosolic calcium dynamics. This phenomenon plays 15 a key role in LSP, thereby revealing an unforeseen link between mitochondrial 16 functions, astroglial activity and information processing in the brain. that this approach provides a reliable and quantitatively similar expression of either 30 CB 1 or DN22!CB 1 in cultured astrocytes ( ! ). We then used 31 super!resolution STED microscopy to analyze both the overlap and the minimal 32 distance between the mitochondrial marker Tom20 (Mitochondrial outer membrane 1 translocase 20 kD) and CB 1 or DN22!CB 1 proteins, respectively. The analysis of 2 these STED images revealed that a relatively high proportion of CB 1 protein either 3 overlapped with ( ! ) or was placed at low distance from 4 Tom20 ( ! ). Conversely, the overlap of DN22!CB 1 with 5 Tom20 was strongly reduced as compared to CB 1 ( ! ), and 6 the distance from the mitochondrial marker was significantly higher ( 7 ! ). Thus, the wild!type CB 1 protein significantly associates to astroglial 8 mitochondria, whereas the DN22!CB 1 mutant displays much lower mitochondrial 9 association.

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To simultaneously record mitochondrial and cytosolic calcium signals, we generated 11 a mitochondrial genetically encoded indicator (Mito!GCaMP6s) and we used it in 12 combination with the cytosolic indicator RCaMP2 31 . These constructs were co! increase. In our EM analysis of cultured astrocytes, almost 100% of mitochondria are 10 located less than 100 nm from ER, a distance that permits calcium exchanges  However, how astroglial calcium is regulated to provide specificity to these functions 28 is still largely elusive 71 . In this context, the double location of CB 1 receptors at 29 plasma and mitochondrial membranes might represent a way to provide signal 30 specificity to calcium increases through a two!step action. First, the activation of 31 plasma membrane CB 1 receptors might promote ER calcium release into the cytosol.

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Then, mtCB 1 receptor stimulation might locally determine the dynamics of calcium 33 spreading required to provide signaling specificity to astrocyte/neuron interactions.
1 Thus, these data pave the way to the idea that multiple control mechanisms of 2 intracellular calcium levels and dynamics are necessary for proper astroglial 3 regulation of synaptic functions. Moreover, they underline that, beyond ion absolute 4 levels, the dynamics of calcium diffusion within the cytoplasm (in this case controlled 5 by mtCB 1 receptors) determine astrocyte synaptic regulation. One example of this 6 phenomenon is the astroglial CB 1 receptor!induced LSP of excitatory synapses 15,16 .

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Our data show that the control of calcium dynamics by the activation of mtCB 1 8 receptors is required to express LSP. By allowing the coordinated activity of neurons 9 that are not necessarily synaptically connected, LSP likely represents a mechanism 10 to expand the computational impact of neuronal networks 16 . Therefore, astroglial 11 mtCB 1 receptor!dependent control of MERCs functions actively participates in 12 synaptic plasticity and likely contributes regulating complex information processing in 13 the brain.
14 Overall, our findings unravel the importance of astroglial mitochondria and MERCs in 15 shaping neuronal network activity and uncover a novel mechanism of action of CB 1 16 receptor signaling in the brain. This study is dedicated to the memory of our friend and colleague Federico Massa.

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The microscopy was done in the Bordeaux Imaging Center a service unit of the  The authors declare no competing interests.

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. Mice (7-10 weeks of age) were anaesthetized  Fig. 3j). The first minute previous and after injection 16 were removed from quantification to exclude injection effects and transients were 17 divided in two equal periods of near 15 min each. All data processing was performed 18 using custom MATLAB scripts.   the measurements were taken from distinct samples. All graphs and statistical 20 analyses were performed using GraphPad software (version 5.0 or 6.0). Results

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were expressed as means of independent data points ± s.e.m. Parametric tests were 22 used for the data and analysed using the appropriate statistical test (detailed 23 statistical data for each experiment are reported in Table S1,2).         Supplementary Fig. 1 Supplementary Fig. 1. a, Representative STED images of CB 1 -myc (upper panels) and DN22-CB 1 -myc (lower panels) transfected astrocytes. In green, Tom20 (mitochondrial marker) staining; in red, myc (CB 1 ) staining; in yellow, overlay images. Scale bar: 2 µm. b, Mean intensity quantification of CB 1 levels. c, Percentage of overlap area over Tom20 (total mitochondria). Ctrl: Control, empty-vector transfected cells. d, Representative distance map images obtained from the STED images used in (c). On the right color-distance legend. Scale bar: 2 µm. e, Percentage of CB 1 particles at given distances. *p-value<0.05, **p-value<0.01.