Cannabinoids, the active components of cannabis, exert numerous acute effects in the brain by engaging cannabinoid CB 1 receptors (CB 1 Rs). However, tolerance emerges rapidly after repeated drug exposure, undermining the efficacy of cannabinoid-based therapies and contributing to cannabis-associated adverse effects. Although the processes of CB 1 R short-term desensitization (i.e., receptor uncoupling and internalization) are well characterized, the mechanisms underlying CB 1 R long-term tolerance (i.e., downregulation of receptor protein levels) remain elusive. Here, we identify a ubiquitin-dependent pathway that couples CB 1 R activation to its proteasomal degradation. We show that cannabinoids engage a G q/11 -PLC-PKC signaling cascade that phosphorylates and activates the E3 ubiquitin ligase neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L), promoting its recruitment to CB 1 R and the ubiquitination of four specific lysine residues. This modification targets the receptor for proteasomal clearance, reducing neuronal CB 1 R abundance in vitro and in the mouse brain. Using molecular, pharmacological, and circuit-specific rescue approaches, we demonstrate that preventing CB 1 R ubiquitination stabilizes receptor levels and abolishes behavioral cannabinoid tolerance in mice without impairing acute drug responses. These findings reveal a molecular mechanism that controls CB 1 R stability and identify NEDD4L-mediated ubiquitination as a central driver of cannabinoid tolerance.

Acquired or innate lack of response to standard HER2-targeted therapies remains a clinical issue in patients with HER2-positive breast cancer. Here, we investigated the role of the cannabinoid CB2 receptor (CB2R) in trastuzumab resistance. In human breast cancer samples, a decreased expression of HER2-CB2R heterodimers following neoadjuvant treatment, due to CB2R downregulation, was linked to poor long-term outcomes. Using various preclinical models, we demonstrate that CB2R drives trastuzumab resistance. Mechanistically, CB2R loss enabled cancer cells to evade antitumor IFN-γ signaling while promoting a shift from HER2-CB2R to HER2-EGFR heterodimers, thus reducing dependence on HER2 and increasing reliance on EGFR-mediated pathways. Moreover, EGFR inhibition restored trastuzumab sensitivity. In summary, we reveal an unprecedented role for CB2R as a key regulator of oncogenic and immune signaling in response to anti-HER2 therapy and its potential as a predictive biomarker of therapeutic efficacy. We also propose dual HER2/EGFR targeting and non-CB2R-selective cannabinoid therapies as potential strategies to overcome CB2R-mediated trastuzumab resistance. Together, these findings position the endocannabinoid system as a pivotal and actionable node to elucidate, anticipate, and counteract resistance to HER2-targeted therapies.

Oligodendrocytes, the myelin-producing cells in the central nervous system, derive from oligodendrocyte progenitor cells during both development and remyelination. The intricate process of oligodendrocyte differentiation, which involves several transitional stages before generating fully mature myelin-producing cells, is tightly regulated by extracellular signals. Despite advances in identifying key regulators, the comprehensive network of signaling molecules governing oligodendrocyte maturation in the healthy, and more importantly, the demyelinated brain remains incompletely understood. The endocannabinoid system has been recognized as a key regulator of oligodendroglial lineage progression, contributing to myelin formation and regeneration. In particular, CB1 cannabinoid receptors are prominent modulators of the initial steps of oligodendrocyte progenitor cell differentiation. However, the potential role of CB1 receptors in the terminal stages of oligodendrocyte differentiation remains poorly explored. Here, using a new conditional genetic mouse model in which the CB1 receptor-encoding gene was selectively ablated from differentiated oligodendrocytes, together with the cuprizone diet-induced demyelination paradigm, we demonstrate that CB1 receptors are essential for the terminal stages of oligodendroglial maturation during myelin regeneration. Specifically, we found that CB1 receptor gene inactivation in newly regenerated oligodendrocytes hinders oligodendroglial cell maturation and brain remyelination, exacerbates glial activation and axonal damage, and abolishes motor function recovery. Taken together, our findings demonstrate a crucial role for CB1 receptors in modulating oligodendrocyte maturation during myelin regeneration and supports the notion that appropriate endocannabinoid signaling in the oligodendroglial lineage is essential for achieving efficient remyelination.

Cannabinoids, the active components of cannabis, exert numerous acute effects in the brain by engaging cannabinoid CB1 receptors (CB1Rs). However, tolerance emerges rapidly after repeated drug exposure, undermining the efficacy of cannabinoid-based therapies and contributing to cannabis-associated adverse effects. Although the processes of CB1R short-term desensitization (i.e., receptor uncoupling and internalization) are well characterized, the mechanisms underlying CB1R long-term tolerance (i.e., downregulation of receptor protein levels) remain elusive. Here, we identify a ubiquitin-dependent pathway that couples CB1R activation to its proteasomal degradation. We show that cannabinoids engage a Gq/11-PLC-PKC signaling cascade that phosphorylates and activates the E3 ubiquitin ligase neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L), promoting its recruitment to CB1R and the ubiquitination of four specific lysine residues. This modification targets the receptor for proteasomal clearance, reducing neuronal CB1R abundance in vitro and in the mouse brain. Using molecular, pharmacological, and circuit-specific rescue approaches, we demonstrate that preventing CB1R ubiquitination stabilizes receptor levels and abolishes behavioral cannabinoid tolerance in mice without impairing acute drug responses. These findings reveal a molecular mechanism that controls CB1R stability and identify NEDD4L-mediated ubiquitination as a central driver of cannabinoid tolerance.

Blood-brain barrier (BBB) alterations contribute to stress vulnerability and the development of depressive behaviors. In contrast, neurovascular adaptations underlying stress resilience remain unclear. Here we report that high expression of astrocytic cannabinoid receptor 1 (CB1) in the nucleus accumbens (NAc) shell, particularly in the end-feet ensheathing blood vessels, is associated with resilience during chronic social stress in adult male mice. Viral-mediated overexpression of Cnr1 in astrocytes of the NAc shell results in baseline anxiolytic effects and dampens stress-induced anxiety- and depression-like behaviors in male mice. It promotes the expression of vascular-related genes and reduces astrocyte inflammatory response and morphological changes following an immune challenge with the cytokine interleukin-6, linked to stress susceptibility and mood disorders. Physical exercise and antidepressant treatment increase the expression of astrocytic Cnr1 in the perivascular region in male mice. In human tissue from male donors with major depressive disorder, we observe loss of CNR1 in the NAc astrocytes. Our findings suggest a role for the astrocytic endocannabinoid system in stress responses via modulation of the BBB.

Dysregulation of the endocannabinoid system (eCBS) and the loss of CB1 receptors (CB1R) in the basal ganglia are well-established hallmarks of Huntington’s disease (HD). As a result, significant research efforts have focused on targeting the eCBS to alleviate motor disturbances associated with the disease. Beyond its role in motor control, the eCBS is a complex signaling network critically involved in regulating learning and memory. Despite this, the potential involvement of eCBS dysfunction in the cognitive decline characteristic of HD, often manifested well before motor dysfunction, has remained largely unexplored. CB1R expression in the hippocampus was evaluated in both human HD samples and HD mouse models (R6/1 and HdhQ7/Q111 models, including both sexes) using Western blotting, immunohistochemistry, and radioligand binding assays. To restore CB1R function, CB1R agonist WIN-55212–2 was systemically administered, or viral vectors encoding CB1R were locally infused into the hippocampus of HD mice. A multidisciplinary approach combining behavioral, biochemical, electrophysiological, and morphological analyses, was employed to investigate the molecular mechanisms underlying the effects of CB1R activation in the context of HD-related cognitive dysfunction. In both human HD samples and HD mouse models, CB1R protein levels were reduced in the hippocampus, accompanied by structural synaptic alterations and impairment in spatial, recognition and working memory. Moreover, hippocampal depolarization-induced suppression of inhibition was significantly disrupted in R6/1 mice. Administration of WIN-55212–2 successfully restored these synaptic and cognitive deficits. Immunohistochemical analysis revealed that the CB1R decrease was specifically localized to GABAergic interneurons within the hippocampus. Notably, targeted restoration of CB1R expression in these interneurons via viral vector delivery was sufficient to rescue hippocampal-dependent memory deficits in HD mice. This study suggests that impaired CB1R function in hippocampal GABAergic interneurons contributes to memory dysfunction in HD.

Endocannabinoid signaling exerts a neurodevelopmental regulatory role via CB1 cannabinoid receptors (CB1Rs), which control pyramidal neuron differentiation, migration, and axonal guidance. Here, we investigated the long-lasting consequences of transient prenatal CB1R downregulation within the mouse prefrontal cortex by assessing its impact on gene expression, neuronal electrophysiological properties, and animal behavioral traits. Transient loss of CB1Rs as induced by in-utero small-interference RNA electroporation at Embryonic Day 14.5, when upper-layer neurons are generated, arrested cell migration leading to ectopic neurons that populated deep layers. Whole-cell current-clamp recordings showed that ectopic neurons are less excitable (increased afterhyperpolarization amplitude, decreased sag, lower firing frequency) than deep-layer-native pyramidal neurons. Differentially expressed genes (DEGs), identified by microarray characterization of FACS-sorted electroporated neurons, were significantly enriched in pathways related to cortical development, regulation of cell migration, neurotransmitter secretion, and cytoskeletal organization. Gene set enrichment analysis also supported enrichment in pathways associated with neurodegenerative disorders and synaptic function. The gene expression profile of siCB1R-derived neurons showed DEGs that had been previously associated with intellectual disability, schizophrenia, and autism. Venn diagrams unveiled one common DEG for neuropsychiatric risk databases and CB1R expression manipulation, namely, the transcription factor ZBTB20. Prenatal knockdown of CB1Rs induced long-lasting behavioral alterations in the adult offspring of either sex, with an impairment of social interaction and motor behavior in siCB1R-derived adult mice. Taken together, these findings highlight the role of CB1Rs in controlling the development of pyramidal neurons in the prefrontal cortex and support the contribution of altered endocannabinoid signaling to neuropsychiatric vulnerability.

The E3 ligase substrate adapter cereblon (CRBN), the primary target of clinical agents thalidomide and lenalidomide, recognizes endogenous substrates bearing the C-terminal cyclic imide modification. Although C-terminal cyclic imides can form spontaneously, an enzyme that regulates their formation and thereby promotes a biological pathway connecting substrates to CRBN is unknown. Here we report that protein carboxymethyltransferase (PCMT1) promotes formation of C-terminal cyclic imides on C-terminal asparagine residues of CRBN substrates. PCMT1 and CRBN coregulate the levels of metabolic enzymes including glutamine synthetase and inorganic pyrophosphatase 1 in vitro, in cells and in vivo, and this regulation is associated with the proepileptic phenotype of CRBN knockout mouse models. The discovery of an enzyme that regulates CRBN substrates through the C-terminal cyclic imide reveals a previously unknown biological pathway that is perturbed by thalidomide derivatives and provides a biochemical basis for the connection between multiple biological processes and CRBN.

Mammary gland development occurs primarily in adulthood, undergoing extensive expansion during puberty followed by cycles of functional specialization and regression with every round of pregnancy/lactation/involution. This process is ultimately driven by the coordinated proliferation and differentiation of mammary epithelial cells. However, the endogenous molecular factors regulating these developmental dynamics are still poorly defined. Endocannabinoid signaling is known to determine cell fate-related events during the development of different organs in the central nervous system and the periphery. Here, we report that the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) plays a pivotal role in adult mammary gland development. Specifically, it is required for luminal lineage specification in the mammary gland, and it promotes hormone-driven secretory differentiation of mammary epithelial cells by controlling the endogenous levels of anandamide and the subsequent activation of cannabinoid CB 1 receptors. Together, our findings shed light on the role of the endocannabinoid system in breast development and point to FAAH as a therapeutic target in milk-production deficits.

Defects in myelin homeostasis have been reported in many neuropathological conditions. Cannabinoid compounds have been shown to efficiently promote myelin regeneration in animal models of demyelination. However, it is still unknown whether this action relies mostly on a cell autonomous effect on oligodendroglial-lineage-NG2 cells. By using conditional genetic mouse models, here we found that cannabinoid CB1 receptors located on NG2 cells are required for oligodendroglial differentiation and myelin regeneration after demyelination. Selective CB1 receptor gene depletion in NG2 cells following toxin-induced demyelination disrupted oligodendrocyte regeneration and functional remyelination and exacerbated axonal damage. These deficits were rescued by pharmacological blockade of the RhoA/ROCK/Cofilin pathway. Conversely, tetrahydrocannabinol administration promoted oligodendrocyte regeneration and functional remyelination in wild-type but not Ng2-CB1-deficient mice. Overall, this study identifies CB1 receptors as essential modulators of remyelination and support the therapeutic potential of cannabinoids for promoting remyelination in neurological disorders.

Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB 1 receptor (CB 1 R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB 1 R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB 1 R and impairs the CB 1 R-G i/o -cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB 1 R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB 1 R could constitute a new therapy for this orphan disease.

Growth-associated protein of 43 kDa (GAP43) is a key cytoskeleton-associated component of the presynaptic terminal that facilitates neuroplasticity. Downregulation of GAP43 expression has been associated to various psychiatric conditions in humans and evokes hippocampus-dependent memory impairments in mice. Despite the extensive studies conducted on hippocampal GAP43 in past decades, however, very little is known about its roles in modulating the excitatory versus inhibitory balance in other brain regions. We recently generated conditional knock-out mice in which the Gap43 gene was selectively inactivated in either telencephalic glutamatergic neurons (Gap43fl/fl ;Nex1Cre mice, hereafter Glu-GAP43-/- mice) or forebrain GABAergic neurons (Gap43fl/fl ;Dlx5/6Cre mice, hereafter GABA-GAP43-/- mice). Here, we show that Glu-GAP43-/- but not GABA-GAP43-/- mice of either sex show a striking hyperactive phenotype when exposed to a novel environment. This behavioral alteration of Glu-GAP43-/- mice was linked to a selective activation of dorsal-striatum neurons, as well as to an enhanced corticostriatal glutamatergic transmission and an abrogation of corticostriatal endocannabinoid-mediated long-term depression. In line with these observations, GAP43 was abundantly expressed in corticostriatal glutamatergic terminals of wild-type mice. The novelty-induced hyperactive phenotype of Glu-GAP43-/- mice was abrogated by chemogenetically inhibiting corticostriatal afferences with a Gi-coupled "designer receptor exclusively activated by designer drugs" (DREADDs), thus further supporting that novelty-induced activity is controlled by GAP43 at corticostriatal excitatory projections. Taken together, these findings show an unprecedented regulatory role of GAP43 in the corticostriatal circuitry and provide a new mouse model with a delimited neuronal-circuit alteration for studying novelty-induced hyperactivity, a phenotypic shortfall that occurs in diverse psychiatric diseases.

En este trabajo se presentan los resultados de una investigación llevada a cabo en el Instituto Tecnológico de Santo Domingo en colaboración con la Universidad de Puerto Rico, a partir de la implementación de un curso Virtual de Física Mecánica. Se incluyen resultados de pruebas estandarizadas realizadas antes y después de que los estudiantes aborden los diferentes temas en la modalidad virtual y se comparan con resultados de la modalidad presencial. Se tienen en consideración criterios de los usuarios del curso virtual, y propuestas de cambios que deben hacerse para mejorar las versiones posteriores. Una propuesta que surgió a partir de los señalamientos realizados, fue la de incorporar objetos virtuales de aprendizaje, como detonadores de la motivación prácticamente nula, que se detectó en los estudiantes. Un equipo de investigación transdisciplinaria diseñó y puso a prueba un objeto de aprendizaje virtual transdisciplinario (OVAt) para evaluar si lograba superar algunas de las dificultades detectadas en estudiantes de ciencias físicas. Para ello se construyó un primer OVAt con el tema de movimiento de proyectiles. Se presentan los resultados de la aplicación del OVAt diseñado. Se construyeron instrumentos de evaluación auténtica para ponderar la ejecución disciplinar, así como medir aspectos cualitativos.

Melanoma is one of the deadliest forms of cancer. Most melanoma deaths are caused by distant metastases in several organs, especially the brain, the so-called melanoma brain metastases (MBMs). However, the precise mechanisms that sustain the growth of MBMs remain elusive. Recently, the excitatory neurotransmitter glutamate has been proposed as a brain-specific, pro-tumorigenic signal for various types of cancers, but how neuronal glutamate shuttling onto metastases is regulated remains unknown. Here, we show that the cannabinoid CB1 receptor (CB1R), a master regulator of glutamate output from nerve terminals, controls MBM proliferation. First, in silico transcriptomic analysis of cancer-genome atlases indicated an aberrant expression of glutamate receptors in human metastatic melanoma samples. Second, in vitro experiments conducted on three different melanoma cell lines showed that the selective blockade of glutamatergic NMDA receptors, but not AMPA or metabotropic receptors, reduces cell proliferation. Third, in vivo grafting of melanoma cells in the brain of mice selectively devoid of CB1Rs in glutamatergic neurons increased tumour cell proliferation in concert with NMDA receptor activation, whereas melanoma cell growth in other tissue locations was not affected. Taken together, our findings demonstrate an unprecedented regulatory role of neuronal CB1Rs in the MBM tumour microenvironment.

Astrocytes are usually viewed as archetypical glycolytic cells. A study now shows that fatty acid oxidation in astrocytes rearranges the respiratory chain to a configuration that safeguards neuronal functionality and proper cognitive performance in mice.

High-grade gliomas constitute the most frequent and aggressive form of primary brain cancer in adults. These tumors express cannabinoid CB 1 and CB 2 receptors, as well as other elements of the endocannabinoid system. Accruing preclinical evidence supports that pharmacological activation of cannabinoid receptors located on glioma cells exerts overt anti-tumoral effects by modulating key intracellular signaling pathways. The mechanism of this cannabinoid receptor-evoked anti-tumoral activity in experimental models of glioma is intricate and may involve an inhibition not only of cancer cell survival/proliferation, but also of invasiveness, angiogenesis, and the stem cell-like properties of cancer cells, thereby affecting the complex tumor microenvironment. However, the precise biological role of the endocannabinoid system in the generation and progression of glioma seems very context-dependent and remains largely unknown. Increasing our basic knowledge on how (endo)cannabinoids act on glioma cells could help to optimize experimental cannabinoid-based anti-tumoral therapies, as well as the preliminary clinical testing that is currently underway.

Increasing evidence indicates that a key factor in neurodegenerative diseases is the activation of the unfolded protein response (UPR) caused by an accumulation of misfolded proteins in the endoplasmic reticulum (ER stress). Particularly, in Huntington's disease (HD) mutant huntingtin (mHtt) toxicity involves disruption of the ER-associated degradation pathway and loss of the ER protein homeostasis leading to neuronal dysfunction and degeneration. Besides the role of the UPR in regulating cell survival and death, studies that demonstrate the contribution of sustained UPR activation, particularly of PERK signaling, in memory disturbances and synaptic plasticity deficiencies are emerging. Given the contribution of hippocampal dysfunction to emotional and cognitive deficits seen in HD, we have analyzed the involvement of ER stress in HD memory alterations. We have demonstrated that at early disease stages, ER stress activation manifested as an increase in GRP78 and CHOP is observed in the hippocampus of R6/1 mice. Genetic reduction of GRP78 expression resulted in preventing hippocampal-dependent memory alterations but no motor deficits. Accordingly, hippocampal neuropathology namely, dendritic spine loss and accumulation of mHtt aggregates was ameliorated by GRP78 reduction. To elucidate the signaling pathways, we found that the inactivation of PERK by GSK2606414 restored spatial and recognition memories in R6/1 mice and rescued dendritic spine density in CA1 pyramidal neurons and protein levels of some specific immediate early genes. Our study unveils the critical role of the GRP78/PERK axis in memory impairment in HD mice and suggests the modulation of PERK activation as a novel therapeutic target for HD intervention.

Clinical management of breast cancer (BC) metastasis remains an unmet need as it accounts for 90% of BC-associated mortality. Although the luminal subtype, which represents >70% of BC cases, is generally associated with a favorable outcome, it is susceptible to metastatic relapse as late as 15 years after treatment discontinuation. Seeking therapeutic approaches as well as screening tools to properly identify those patients with a higher risk of recurrence is therefore essential. Here, we report that the lipid-degrading enzyme fatty acid amide hydrolase (FAAH) is a predictor of long-term survival in patients with luminal BC, and that it blocks tumor progression and lung metastasis in cell and mouse models of BC. Together, our findings highlight the potential of FAAH as a biomarker with prognostic value in luminal BC and as a therapeutic target in metastatic disease.

Supplementary Tables 1-2, Figure 1 from Cannabinoids Inhibit Glioma Cell Invasion by Down-regulating Matrix Metalloproteinase-2 Expression