Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling.

The cannabinoid 1 receptor (CB 1 R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system, with key roles during neurotransmitter release and synaptic plasticity. Upon ligand activation, CB 1 Rs may signal in three different spatiotemporal waves. The first wave, which is transient (,10 minutes) and initiated by heterotrimeric G proteins, is followed by a second wave (.5 minutes) that is mediated by b-arrestins. The third and final wave occurs at intracellular compartments and could be elicited by G proteins or b-arrestins. This complexity presents multiple challenges, including the correct classification of receptor ligands, the identification of the signaling pathways regulated by each wave, and the underlying molecular mechanisms and physiologic impacts of these waves. Simultaneously, it provides new opportunities to harness the therapeutic potential of the cannabinoid system and other GPCRs. Over the last several years, we have significantly expanded our understanding of the mechanisms and pathways downstream from the CB 1 R. The identification of receptor mutations that can bias signaling to specific pathways and the use of siRNA technology have been key tools to identifying which signaling cascades are controlled by G proteins or b-arrestins. Here, we review our current knowledge on CB 1 R signaling, with particular emphasis on the mechanisms and cascades mediated by b-arrestins downstream from the CB 1 R.

We have previously shown that blood astrocytic-origin extracellular vesicles (AEVs) from Alzheimer’s disease (AD) patients contain high complement levels. To test the hypothesis that circulating EVs from AD patients can induce complement-mediated neurotoxicity involving Membrane Attack Complex (MAC) formation, we assessed the effects of immunocaptured AEVs (using anti-GLAST antibody), in comparison with neuronal-origin (N)EVs (using anti-L1CAM antibody), and nonspecific CD81+ EVs (using anti-CD81 antibody), from the plasma of AD, frontotemporal lobar degeneration (FTLD), and control participants. AEVs (and, less effectively, NEVs) of AD participants induced Membrane Attack Complex (MAC) expression on recipient neurons (by immunohistochemistry), membrane disruption (by EthD-1 assay), reduced neurite density (by Tuj-1 immunohistochemistry), and decreased cell viability (by MTT assay) in rat cortical neurons and human iPSC-derived neurons. Demonstration of decreased cell viability was replicated in a separate cohort of autopsy-confirmed AD patients. These effects were not produced by CD81+ EVs from AD participants or AEVs/NEVs from FTLD or control participants, and were suppressed by the MAC inhibitor CD59 and other complement inhibitors. Our results support the stated hypothesis and should motivate future studies on the roles of neuronal MAC deposition and AEV/NEV uptake, as effectors of neurodegeneration in AD.

Background: Synaptic loss is a feature of multiple sclerosis pathology that can be seen even in normal-appearing gray matter. Opsonization of synapses with complement components may underlie pathologic synapse loss. Objective: We sought to determine whether circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (NEVs and AEVs) provide biomarkers reflecting complement-mediated synaptic loss in multiple sclerosis. Methods: From plasma of 61 people with multiple sclerosis (46 relapsing–remitting multiple sclerosis (RRMS) and 15 progressive MS) and 31 healthy controls, we immunocaptured L1CAM + NEVs and GLAST + AEVs. We measured pre- and post-synaptic proteins synaptopodin and synaptophysin in NEVs and complement components (C1q, C3, C3b/iC3b, C4, C5, C5a, C9, Factor B, and Factor H) in AEVs, total circulating EVs, and neat plasma. Results: We found lower levels of NEV synaptopodin and synaptophysin in MS compared to controls ( p < 0.0001 for both). In AEVs, we found higher levels of multiple complement cascade components in people with MS compared to controls; these differences were not noted in total EVs or neat plasma. Strikingly, there were strong inverse correlations between NEV synaptic proteins and multiple AEV complement components in MS, but not in controls. Conclusion: Circulating EVs could identify synaptic loss in MS and suggest a link between astrocytic complement production and synaptic loss.

Exosomes derived from chondroitin sulfate proteoglycan (CSPG) 4 type neural precursor cells (CSPG4Es) were purified from human plasma by sequential immunoabsorption with anti-CSPG4 and anti-platelet growth factor receptor α mAb to characterize the potential in vivo roles of CSPG4 cells in neuronal repair. Hepatocyte growth factor, fibroblast growth factors (FGFs)-2 and -13, and type 1 insulin-like growth factor (IGF-1), which enhance neuronal survival and functions, were quantified in CSPG4E extracts. For CSPG4Es of 24 healthy control subjects, mean levels of hepatocyte growth factor, FGF-13, and IGF-1, but not FGF-2, were significantly higher by up to 7-fold than in their neuronal-derived exosomes, and mean levels of all 4 growth factors were significantly higher by up to 8-fold than in their astrocyte-derived exosomes. Mean CSPG4E levels of all growth factors were significantly lower in patients with mild Alzheimer disease (AD) ( n = 24) than in age- and sex-matched cognitively normal control subjects ( n = 24). Mean CSPG4E levels of all growth factors were also significantly lower in 15 patients at the stage of moderate dementia from AD (AD) and at their preclinical stage 3 to 8 yr earlier (AD), with no differences between values at stages AD and AD. Current findings suggest that CSPG4 cells export in exosomes higher levels of neurotrophic factors than neurons or astrocytes and that CSPG4E neurotrophic factors are diminished early in AD, with no significant progression of decreases later in the course.-Goetzl, E. J., Nogueras-Ortiz, C., Mustapic, M., Mullins, R. J., Abner, E. L., Schwartz, J. B., Kapogiannis, D. Deficient neurotrophic factors of CSPG4-type neural cell exosomes in Alzheimer disease.

Accumulating evidence suggests that neuroinflammation is involved in bipolar disorder (BD) pathogenesis. The tumor necrosis factor-alpha (TNF-α) antagonist infliximab was recently reported to improve depressive symptoms in a subpopulation of individuals with BD and history of childhood maltreatment. To explore the mechanistic mediators of infliximab’s effects, we investigated its engagement with biomarkers of cellular response to inflammation derived from plasma extracellular vesicles enriched for neuronal origin (NEVs). We hypothesized that infliximab, compared to placebo, would decrease TNF-α receptors (TNFRs) and nuclear factor-kappa B (NF-κB) pathway signaling biomarkers, and that history of childhood abuse would moderate infliximab’s effects. We immunocaptured NEVs from plasma samples collected at baseline and at weeks 2, 6, and 12 (endpoint) from 55 participants of this clinical trial and measured NEV biomarkers using immunoassays. A subset of participants (n = 27) also underwent whole-brain magnetic resonance imaging at baseline and endpoint. Childhood physical abuse moderated treatment by time interactions for TNFR1 (χ2 = 9.275, p = 0.026), NF-κB (χ2 = 13.825, p = 0.003), and inhibitor of NF-κB (IκBα)α (χ2 = 7.990, p = 0.046), indicating that higher levels of physical abuse were associated with larger biomarker decreases over time. Moreover, the antidepressant response to infliximab was moderated by TNFR1 (χ2 = 7.997, p = 0.046). In infliximab-treated participants, reductions in TNFR1 levels were associated with improvement of depressive symptoms, an effect not detected in the placebo group. Conversely, reductions in TNFR1 levels were associated with increased global cortical thickness in infliximab- (r = −0.581, p = 0.029), but not placebo-treated, patients (r = 0.196, p = 0.501). In conclusion, we report that NEVs revealed that infliximab engaged the TNFR/NF-κB neuro-inflammatory pathway in individuals with BD, in a childhood trauma-dependent manner, which was associated with clinical response and brain structural changes.

Circulating neuronal extracellular vesicles (NEVs) of Alzheimer’s disease (AD) patients show high Tau and β-amyloid (Aβ) levels, whereas their astrocytic EVs (AEVs) contain high complement levels. To validate EV proteins as AD biomarkers, we immunocaptured NEVs and AEVs from plasma collected from fifteen wild type (WT), four 2xTg-AD, nine 5xFAD, and fifteen 3xTg-AD mice and assessed biomarker relationships with brain tissue levels. NEVs from 3xTg-AD mice had higher total Tau (p = 0.03) and p181-Tau (p = 0.0004) compared to WT mice. There were moderately strong correlations between biomarkers in NEVs and cerebral cortex and hippocampus (total Tau: cortex, r = 0.4, p = 0.009; p181-Tau: cortex, r = 0.7, p < 0.0001; hippocampus, r = 0.6, p < 0.0001). NEVs from 5xFAD compared to other mice had higher Aβ42 (p < 0.005). NEV Aβ42 had moderately strong correlations with Aβ42 in cortex (r = 0.6, p = 0.001) and hippocampus (r = 0.7, p < 0.0001). AEV C1q was elevated in 3xTg-AD compared to WT mice (p = 0.005); AEV C1q had moderate-strong correlations with C1q in cortex (r = 0.9, p < 0.0001) and hippocampus (r = 0.7, p < 0.0001). Biomarkers in circulating NEVs and AEVs reflect their brain levels across multiple AD mouse models supporting their potential use as a “liquid biopsy” for neurological disorders.

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