Periodic Reporting for period 2 - MiCaBra (Mitochondrial Cannabinoid Receptors in the Brain) Reporting period: 2020-05-01 to 2021-10-31 Summary of the context and overall objectives of the project MiCaBra adresses the physiological and pharmacological roles of mitochondria-associated type-1 cannabinoid receptors in the brain (mtCB1). As a G Protein-Coupled Receptor, CB1 is mainly located on the plasma membrane of cells (pmCB1, neurons, astrocytes and others), where it mediates communication between the exterior and the interior of the cell itself. However, some years ago, we discovered that CB1 receptors are also functionally present inside cells, particularly in close association with important iintracellular organs like mitochondria. Here, previous studies from my lab showed that mtCB1 receptors control mitochondrial oxygen consumption and mediate cannabinoid-induced amnesic effects in a novel object recogtnition task. Thus, MiCaBra aims at detailing and deeply investigationg the potential roles of mtCB1 receptors, starting from a biochemical/cell biology point of view, up to addressing their behavioral implications. Considering the importance of CB1 receptors as the main targets of the plant cannabis sativa and as the main effector of the phyisiological endocannabinoid system, understanding their different modes of action is very important not only to better understand the mechanisms of brain functions, but also towards the development of novel therapeutic approaches for different conditions. Indeed, cannabis and cannabinoids are endowed with very interesting therapeutic potentials (analgesia, antispasm, anxiolytic, etc), but a certain number important possible side effects (amnesia, psychotic-like reponses, addiction potential, etc) limit their use in clinical settings and constitute the danger associated with their recreational use. As you can see below, our studies started addressing these issues, by demonstratin, for instance, that antisocial cannabinoid-induced behavior is specifically due to mtCB1 in astrocytes of specific brain regions. Moreover, we were recently able to differentiate the impact of pmCB1 and mtCB1 in the same neuronal circuit. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The work has been performed in a very smooth and productive way. Several papers have been published already where the contribution of MiCaBra is clearly acknowledged and represents the main source of support. In particular, however, I consider two studies as the most representative of the work performed. Please find below a short description of these studies, published in Nature (2020) and in Neuron (2021), respectively.Jimenez-Blasco et al. 2020 (Nature) Glucose metabolism links astroglial mitochondria to cannabinoid effects.Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors. 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 mtCB1 receptors 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 (Image 1)Soria-Gomez et al. 2021 (Neuron) Subcellular specificity of cannabinoid effects in striatonigral circuits.Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associatedCB1 receptors (mtCB1) located at thesame terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior (Image 2). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) These data are a clear indication that specific effects of cannabinoids are due to specific subcellular localization of the receptor. This is a novel concept in the field of GPCRs. Moreoever, these data underline the importance of bioenergetic mechanisms as proper signaling cascaddes to regulate brain functions and behavior. Energy metabolisms, therefore, is not just a mere "homekeeping" mechanisms to provide the cells of the usable energy to survive, but they represent a true signaling pathway that can orient the action of brain circuits toward one function or another. In the specific case, it is important to note that, whereas antisocial behavior and inhibition of voluntary movements (catalepsy) are amongst the most serious side effects of cannabinoid drugs (leading to potential psychotic states the first and causing vehicle accidents the second), antinociception and analgesia are "good" and therapeutically exploitable effects of these drugs. Having understood that intracellular (mitochondrial) CB1 receptors mediate important "bad" effects of cannabinoids and pmCB1 are responsible of at least one "good" effect paves the way to the development of safer drugs. For instance, the modification of cannabinoid agonists to impede their penetration into the plasma membrane might result in safer drugs with therapeutic potential and fewer side effects. Among other aspects of mtCB1 physiology and pharmacology, this possibility will be investigated in the following of MiCaBra. Summary scheme of paper Soria-Gomez et al. 2021 (Neuron) Summary scheme of paper Jimenez et al. 2020 (Nature)
