Identification of BiP as a CB1 Receptor-Interacting Protein That Fine-Tunes Cannabinoid Signaling in the Mouse Brain.

Cannabinoids, the bioactive constituents of cannabis, exert a wide array of effects on the brain by engaging Type 1 cannabinoid receptor (CB1R). Accruing evidence supports that cannabinoid action relies on context-dependent factors, such as the biological characteristics of the target cell, suggesting that cell population-intrinsic molecular cues modulate CB1R-dependent signaling. Here, by using a yeast two-hybrid-based high-throughput screening, we identified BiP as a potential CB1R-interacting protein. We next found that CB1R and BiP interact specifically in vitro, and mapped the interaction site within the CB1R C-terminal (intracellular) domain and the BiP C-terminal (substrate-binding) domain-α. BiP selectively shaped agonist-evoked CB1R signaling by blocking an "alternative" Gq/11 protein-dependent signaling module while leaving the "classical" Gi/o protein-dependent inhibition of the cAMP pathway unaffected. In situ proximity ligation assays conducted on brain samples from various genetic mouse models of conditional loss or gain of CB1R expression allowed to map CB1R-BiP complexes selectively on terminals of GABAergic neurons. Behavioral studies using cannabinoid-treated male BiP+/- mice supported that CB1R-BiP complexes modulate cannabinoid-evoked anxiety, one of the most frequent undesired effects of cannabis. Together, by identifying BiP as a CB1R-interacting protein that controls receptor function in a signaling pathway- and neuron population-selective manner, our findings may help to understand the striking context-dependent actions of cannabis in the brain.SIGNIFICANCE STATEMENT Cannabis use is increasing worldwide, so innovative studies aimed to understand its complex mechanism of neurobiological action are warranted. Here, we found that cannabinoid CB1 receptor (CB1R), the primary molecular target of the bioactive constituents of cannabis, interacts specifically with an intracellular protein called BiP. The interaction between CB1R and BiP occurs selectively on terminals of GABAergic (inhibitory) neurons, and induces a remarkable shift in the CB1R-associated signaling profile. Behavioral studies conducted in mice support that CB1R-BiP complexes act as fine-tuners of anxiety, one of the most frequent undesired effects of cannabis use. Our findings open a new conceptual framework to understand the striking context-dependent pharmacological actions of cannabis in the brain.

Pathway-Specific Control of Striatal Neuron Vulnerability by Corticostriatal Cannabinoid CB1 Receptors.

The vast majority of neurons within the striatum are GABAergic medium spiny neurons (MSNs), which receive glutamatergic input from the cortex and thalamus, and form two major efferent pathways: the direct pathway, expressing dopamine D1 receptor (D1R-MSNs), and the indirect pathway, expressing dopamine D2 receptor (D2R-MSNs). While molecular mechanisms of MSN degeneration have been identified in animal models of striatal damage, the molecular factors that dictate a selective vulnerability of D1R-MSNs or D2R-MSNs remain unknown. Here, we combined genetic, chemogenetic, and pharmacological strategies with behavioral and neurochemical analyses, and show that the pool of cannabinoid CB1 receptor (CB1R) located on corticostriatal terminals efficiently safeguards D1R-MSNs, but not D2R-MSNs, from different insults. This cell-specific response relies on the regulation of glutamatergic signaling, and is independent from the CB1R-dependent control of astroglial activity in the striatum. These findings define cortical CB1R as a pivotal synaptic player in dictating a differential vulnerability of D1R-MSNs versus D2R-MSNs, and increase our understanding of the role of coordinated cannabinergic-glutamatergic signaling in establishing corticostriatal circuits and its dysregulation in neurodegenerative diseases.

Expression and potential role of apolipoprotein D on the death-survival balance of human colorectal cancer cells under oxidative stress conditions.

PURPOSE: Inverse correlations of apolipoprotein D (ApoD) expression with tumor growth have been shown, therefore proposing ApoD as a good prognostic marker for diverse cancer types, including colorectal cancer (CRC). Besides, ApoD expression is boosted upon oxidative stress (OS) in many pathological situations. This study aims at understanding the role of ApoD in the progression of human CRC. METHODS: Samples of CRC and distant normal tissue (n = 51) were assayed for levels of lipid peroxidation, expression profile of OS-dependent genes, and protein expression. Three single-nucleotide polymorphisms in the ApoD gene were analyzed (n = 139), with no significant associations found. Finally, we assayed the effect of ApoD in proliferation and apoptosis in the CRC HT-29 cell line. RESULTS: In CRC, lipid peroxides increase while ApoD messenger RNA and protein decrease through tumor progression, with a prominent decrease in stage I. In normal mucosa, ApoD protein is present in lamina propia and enteroendocrine cells. In CRC, ApoD expression is heterogeneous, with low expression in stromal cells commonly associated with high expression in the dysplastic epithelium. ApoD promoter is basally methylated in HT-29 cells but retains the ability to respond to OS. Exogenous addition of ApoD to HT-29 cells does not modify proliferation or apoptosis levels in control conditions, but it promotes apoptosis upon paraquat-induced OS. CONCLUSION: Our results show ApoD as a gene responding to OS in the tumor microenvironment. Besides using ApoD as marker of initial stages of tumor progression, it can become a therapeutic tool promoting death of proliferating tumor cells suffering OS.

Apolipoprotein D mediates autocrine protection of astrocytes and controls their reactivity level, contributing to the functional maintenance of paraquat-challenged dopaminergic systems.

The study of glial derived factors induced by injury and degeneration is important to understand the nervous system response to deteriorating conditions. We focus on Apolipoprotein D (ApoD), a Lipocalin expressed by glia and strongly induced upon aging, injury or neurodegeneration. Here we study ApoD function in the brain of wild type and ApoD-KO mice by combining in vivo experiments with astrocyte cultures. Locomotor performance, dopamine concentration, and gene expression levels in the substantia nigra were assayed in mice treated with paraquat (PQ). The regulation of ApoD transcription, a molecular screening of oxidative stress (OS)-related genes, cell viability and oxidation status, and the effects of adding human ApoD were tested in astrocyte cultures. We demonstrate that (1) ApoD is required for an adequate locomotor performance, modifies the gene expression profile of PQ-challenged nigrostriatal system, and contributes to its functional maintenance; (2) ApoD expression in astrocytes is controlled by the OS-responsive JNK pathway; (3) ApoD contributes to an autocrine protecting mechanism in astrocytes, avoiding peroxidated lipids accumulation and altering the PQ transcriptional response of genes involved in ROS managing and the inflammatory response to OS; (4) Addition of human ApoD to ApoD-KO astrocytes promotes survival through a mechanism accompanied by protein internalization and modulation of astroglial reactivity. Our data support that ApoD contributes to the endurance of astrocytes and decreases their reactivity level in vitro and in vivo. ApoD function as a maintenance factor for astrocytes would suffice to explain the observed protection by ApoD of OS-vulnerable dopaminergic circuits in vivo.

Apolipoprotein D alters the early transcriptional response to oxidative stress in the adult cerebellum.

The lipocalin Apolipoprotein D (ApoD), known to protect the nervous system against oxidative stress (OS) in model organisms, is up-regulated early in the mouse brain in response to the ROS generator paraquat. However, the processes triggered by this up-regulation have not been explored. We present here a study of the effect of ApoD on the early transcriptional changes upon OS in the mouse cerebellum using microarray profiling. ApoD-KO and transgenic mice over-expressing ApoD in neurons are compared to wild-type controls. In control conditions, ApoD affects the transcriptional profile of neuron and oligodendrocyte-specific genes involved in neuronal excitability, synaptic function, and myelin homeostasis. When challenged with paraquat, the absence of ApoD modifies the response of genes mainly related to OS management and myelination. Interestingly, the over-expression of ApoD in neurons almost completely abolishes the early transcriptional response to OS. We independently evaluate the expression of protein kinase Cδ, a gene up-regulated by OS only in the ApoD-KO cerebellum, and find it over-expressed in cultured ApoD-KO primary astrocytes, which points to a role for ApoD in astrocyte-microglia signaling. Our results support the hypothesis that ApoD is necessary for a proper response of the nervous system against physiological and pathological OS.

Inhibition of striatonigral autophagy as a link between cannabinoid intoxication and impairment of motor coordination.

The use of cannabis is rapidly expanding worldwide. Thus, innovative studies aimed to identify, understand and potentially reduce cannabis-evoked harms are warranted. Here, we found that Δ9-tetrahydrocannabinol, the psychoactive ingredient of cannabis, disrupts autophagy selectively in the striatum, a brain area that controls motor behavior, both in vitro and in vivo. Boosting autophagy, either pharmacologically (with temsirolimus) or by dietary intervention (with trehalose), rescued the Δ9-tetrahydrocannabinol-induced impairment of motor coordination in mice. The combination of conditional knockout mouse models and viral vector-mediated autophagy-modulating strategies in vivo showed that cannabinoid CB1 receptors located on neurons belonging to the direct (striatonigral) pathway are required for the motor-impairing activity of Δ9-tetrahydrocannabinol by inhibiting local autophagy. Taken together, these findings identify inhibition of autophagy as an unprecedented mechanistic link between cannabinoids and motor performance, and suggest that activators of autophagy might be considered as potential therapeutic tools to treat specific cannabinoid-evoked behavioral alterations.

Cannabinoid-induced motor dysfunction via autophagy inhibition.

The recreational and medical use of cannabis is largely increasing worldwide. Cannabis use, however, can cause adverse side effects, so conducting innovative studies aimed to understand and potentially reduce cannabis-evoked harms is important. Previous research conducted on cultured neural cells had supported that CNR1/CB1R (cannabinoid receptor 1), the main molecular target of cannabis, affects macroautophagy/autophagy. However, it was not known whether CNR1 controls autophagy in the brain in vivo, and, eventually, what the functional consequences of a potential CNR1-autophagy connection could be. We have now found that Δ9-tetrahydrocannabinol (THC), the major intoxicating constituent of cannabis, impairs autophagy in the mouse striatum. Administration of autophagy activators (specifically, the rapalog temsirolimus and the disaccharide trehalose) rescues THC-induced autophagy inhibition and motor dyscoordination. The combination of various genetic strategies in vivo supports the idea that CNR1 molecules located on neurons belonging to the direct (striatonigral) pathway are required for the autophagy- and motor-impairing activity of THC. By identifying autophagy as a mechanistic link between THC and motor performance, our findings may open a new conceptual view on how cannabis acts in the brain.

Oral administration of the cannabigerol derivative VCE-003.2 promotes subventricular zone neurogenesis and protects against mutant huntingtin-induced neurodegeneration.

BACKGROUND: The administration of certain cannabinoids provides neuroprotection in models of neurodegenerative diseases by acting through various cellular and molecular mechanisms. Many cannabinoid actions in the nervous system are mediated by CB1 receptors, which can elicit psychotropic effects, but other targets devoid of psychotropic activity, including CB2 and nuclear PPARγ receptors, can also be the target of specific cannabinoids. METHODS: We investigated the pro-neurogenic potential of the synthetic cannabigerol derivative, VCE-003.2, in striatal neurodegeneration by using adeno-associated viral expression of mutant huntingtin in vivo and mouse embryonic stem cell differentiation in vitro. RESULTS: Oral administration of VCE-003.2 protected striatal medium spiny neurons from mutant huntingtin-induced damage, attenuated neuroinflammation and improved motor performance. VCE-003.2 bioavailability was characterized and the potential undesired side effects were evaluated by analyzing hepatotoxicity after chronic treatment. VCE-003.2 promoted subventricular zone progenitor mobilization, increased doublecortin-positive migrating neuroblasts towards the injured area, and enhanced effective neurogenesis. Moreover, we demonstrated the proneurogenic activity of VCE-003.2 in embryonic stem cells. VCE-003.2 was able to increase neuroblast formation and striatal-like CTIP2-mediated neurogenesis. CONCLUSIONS: The cannabigerol derivative VCE-003.2 improves subventricular zone-derived neurogenesis in response to mutant huntingtin-induced neurodegeneration, and is neuroprotective by oral administration.

Astroglial monoacylglycerol lipase controls mutant huntingtin-induced damage of striatal neurons.

Cannabinoids exert neuroprotection in a wide array of preclinical models. A number of these studies has focused on cannabinoid CB1 receptors in striatal medium spiny neurons (MSNs) and the most characteristic MSN-degenerative disease, Huntington's disease (HD). Accruing evidence supports that astrocytes contribute to drive HD progression, and that they express CB1 receptors, degrade endocannabinoids, and modulate endocannabinergic transmission. However, the possible role of the astroglial endocannabinoid system in controlling MSN integrity remains unknown. Here, we show that JZL-184, a selective inhibitor of monoacylglycerol lipase (MGL), the key enzyme that deactivates the endocannabinoid 2-arachidonoylglycerol, prevented the mutant huntingtin-induced up-regulation of the pro-inflammatory cytokine tumor necrosis factor-α in primary mouse striatal astrocytes via CB1 receptors. To study the role of astroglial MGL in vivo, we injected stereotactically into the mouse dorsal striatum viral vectors that encode mutant or normal huntingtin under the control of the glial fibrillary acidic protein promoter. We observed that, in wild-type mice, pharmacological blockade of MGL with JZL-184 (8 mg/kg/day, i.p.) conferred neuroprotection against mutant huntingtin-induced striatal damage, as evidenced by the prevention of MSN loss, astrogliosis, and motor coordination impairment. We next found that conditional mutant mice bearing a genetic deletion of MGL selectively in astroglial cells (MGLfloxed/floxed;GFAP-Cre/+ mice) were resistant to mutant huntingtin-induced MSN loss, astrogliosis, and motor coordination impairment. Taken together, these data support that astroglial MGL controls the availability of a 2-arachidonoylglycerol pool that ensues protection of MSNs in the mouse striatum in vivo, thus providing a potential druggable target for reducing striatal neurodegeneration.

Aging without Apolipoprotein D: Molecular and cellular modifications in the hippocampus and cortex.

A detailed knowledge of the mechanisms underlying brain aging is fundamental to understand its functional decline and the baseline upon which brain pathologies superimpose. Endogenous protective mechanisms must contribute to the adaptability and plasticity still present in the healthy aged brain. Apolipoprotein D (ApoD) is one of the few genes with a consistent and evolutionarily conserved up-regulation in the aged brain. ApoD protecting roles upon stress or injury are well known, but a study of the effects of ApoD expression in the normal aging process is still missing. Using an ApoD-knockout mouse we analyze the effects of ApoD on factors contributing to the functional maintenance of the aged brain. We focused our cellular and molecular analyses in the cortex and hippocampus at an age representing the onset of senescence where mortality risks are below 25%, avoiding bias towards long-lived animals. Lack of ApoD causes a prematurely aged brain without altering lifespan. Age-dependent hyperkinesia and memory deficits are accompanied by differential molecular effects in the cortex and hippocampus. Transcriptome analyses reveal distinct effects of ApoD loss on the molecular age-dependent patterns of the cortex and hippocampus, with different cell-type contributions to age-regulated gene expression. Markers of glial reactivity, proteostasis, and oxidative and inflammatory damage reveal early signs of aging and enhanced brain deterioration in the ApoD-knockout brain. The lack of ApoD results in an age-enhanced significant reduction in neuronal calcium-dependent functionality markers and signs of early reduction of neuronal numbers in the cortex, thus impinging upon parameters clearly differentiating neurodegenerative conditions from healthy brain aging. Our data support the hypothesis that the physiological increased brain expression of ApoD represents a homeostatic anti-aging mechanism.

Early detection of high oxidative activity in patients with adenomatous intestinal polyps and colorectal adenocarcinoma: myeloperoxidase and oxidized low-density lipoprotein in serum as new markers of oxidative stress in colorectal cancer.

OBJECTIVES: To detect whether signs of oxidative stress appear at early stages of colorectal adenocarcinoma (CRC), particularly in the polyp stage. We also aimed to evaluate the specific entities myeloperoxidase (MPO) and oxidized low-density lipoprotein (oxLDL) as novel markers of oxidation in the plasma of patients with CRC and to study the relationship between oxidative status in plasma and patient survival. METHODS: We assayed serum or plasma specimens from healthy control subjects (n = 14), from patients with intestinal polyps (n = 39), and from patients with CRC (n = 128) to calculate the modified oxidative balance score (MOBS) using several serum markers (ß-carotene, lycopene, vitamin A, vitamin E, MPO, and oxLDL). We also assayed the levels of C-reactive protein (CRP) and obtained lipid profiles. Finally, we studied the survival of patients in relationship to oxidative status (antioxidants and pro-oxidants) and inflammation markers, and added theses data to the lipid profile for each patient. RESULTS: Oxidative stress levels increased as disease stage advanced. This increase was detected early in the polyp stage, before polyps progressed to cancer, and could be measured by the increase of such new markers as MPO and oxLDL, the decrease in antioxidants, and the MOBS value. Higher levels of oxidation correlated with lower survival. CONCLUSION: The oxidation process, which can cause mutations leading to CRC, begins development in the polyp stage. This process may be detected early by monitoring serum markers such as MPO and oxLDL.