Peripheral inflammatory biomarkers define biotypes of bipolar depression.

We identified biologically relevant moderators of response to tumor necrosis factor (TNF)-α inhibitor, infliximab, among 60 individuals with bipolar depression. Data were derived from a 12-week, randomized, placebo-controlled clinical trial secondarily evaluating the efficacy of infliximab on a measure of anhedonia (i.e., Snaith-Hamilton Pleasure Scale). Three inflammatory biotypes were derived from peripheral cytokine measurements using an iterative, machine learning-based approach. Infliximab-randomized participants classified as biotype 3 exhibited lower baseline concentrations of pro- and anti-inflammatory cytokines and soluble TNF receptor-1 and reported greater pro-hedonic improvements, relative to those classified as biotype 1 or 2. Pretreatment biotypes also moderated changes in neuroinflammatory substrates relevant to infliximab's hypothesized mechanism of action. Neuronal origin-enriched extracellular vesicle (NEV) protein concentrations were reduced to two factors using principal axis factoring: phosphorylated nuclear factorκB (p-NFκB), Fas-associated death domain (p-FADD), and IκB kinase (p-IKKα/ß) and TNF receptor-1 (TNFR1) comprised factor "NEV1," whereas phosphorylated insulin receptor substrate-1 (p-IRS1), p38 mitogen-activated protein kinase (p-p38), and c-Jun N-terminal kinase (p-JNK) constituted "NEV2". Among infliximab-randomized subjects classified as biotype 3, NEV1 scores were decreased at weeks 2 and 6 and increased at week 12, relative to baseline, and NEV2 scores increased over time. Decreases in NEV1 scores and increases in NEV2 scores were associated with greater reductions in anhedonic symptoms in our classification and regression tree model (r2 = 0.22, RMSE = 0.08). Our findings provide preliminary evidence supporting the hypothesis that the pro-hedonic effects of infliximab require modulation of multiple TNF-α signaling pathways, including NF-κB, IRS1, and MAPK.

Synaptic and complement markers in extracellular vesicles in multiple sclerosis.

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.

Mechanisms of Biased ß-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor.

Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins ß-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 ß-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of ß-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 ß-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and ß-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 ß-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control ß-arrestin-mediated signaling.

Brain responses to intermittent fasting and the healthy living diet in older adults.

Diet may promote brain health in metabolically impaired older individuals. In an 8-week randomized clinical trial involving 40 cognitively intact older adults with insulin resistance, we examined the effects of 5:2 intermittent fasting and the healthy living diet on brain health. Although intermittent fasting induced greater weight loss, the two diets had comparable effects in improving insulin signaling biomarkers in neuron-derived extracellular vesicles, decreasing the brain-age-gap estimate (reflecting the pace of biological aging of the brain) on magnetic resonance imaging, reducing brain glucose on magnetic resonance spectroscopy, and improving blood biomarkers of carbohydrate and lipid metabolism, with minimal changes in cerebrospinal fluid biomarkers for Alzheimer's disease. Intermittent fasting and healthy living improved executive function and memory, with intermittent fasting benefiting more certain cognitive measures. In exploratory analyses, sex, body mass index, and apolipoprotein E and SLC16A7 genotypes modulated diet effects. The study provides a blueprint for assessing brain effects of dietary interventions and motivates further research on intermittent fasting and continuous diets for brain health optimization. For further information, please see ClinicalTrials.gov registration: NCT02460783.

Single-extracellular vesicle (EV) analyses validate the use of L1 Cell Adhesion Molecule (L1CAM) as a reliable biomarker of neuron-derived EVs.

Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins ß-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and ß-III-tubulin range from 30% to 63%, in contrast to 0.8%-3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.

Neuron-derived extracellular vesicles in blood reveal effects of exercise in Alzheimer's disease.

BACKGROUND: Neuron-derived extracellular vesicles (NDEVs) in blood may be used to derive biomarkers for the effects of exercise in Alzheimer's disease (AD). For this purpose, we studied changes in neuroprotective proteins proBDNF, BDNF, and humanin in plasma NDEVs from patients with mild to moderate AD participating in the randomized controlled trial (RCT) of exercise ADEX. METHODS: proBDNF, BDNF, and humanin were quantified in NDEVs immunocaptured from the plasma of 95 ADEX participants, randomized into exercise and control groups, and collected at baseline and 16 weeks. Exploratorily, we also quantified NDEV levels of putative exerkines known to respond to exercise in peripheral tissues. RESULTS: NDEV levels of proBDNF, BDNF, and humanin increased in the exercise group, especially in APOE ε4 carriers, but remained unchanged in the control group. Inter-correlations between NDEV biomarkers observed at baseline were maintained after exercise. NDEV levels of putative exerkines remained unchanged. CONCLUSIONS: Findings suggest that the cognitive benefits of exercise could be mediated by the upregulation of neuroprotective factors in NDEVs. Additionally, our results indicate that AD subjects carrying APOE ε4 are more responsive to the neuroprotective effects of physical activity. Unchanged NDEV levels of putative exerkines after physical activity imply that exercise engages different pathways in neurons and peripheral tissues. Future studies should aim to expand upon the effects of exercise duration, intensity, and type in NDEVs from patients with early AD and additional neurodegenerative disorders. TRIAL REGISTRATION: The Effect of Physical Exercise in Alzheimer Patients (ADEX) was registered in ClinicalTrials.gov on April 30, 2012 with the identifier NCT01681602.

Comparative assessment of Alzheimer's disease-related biomarkers in plasma and neuron-derived extracellular vesicles: a nested case-control study.

Introduction: Alzheimer's disease (AD) is currently defined according to biomarkers reflecting the core underlying neuropathological processes: Aß deposition, Tau, and neurodegeneration (ATN). The soluble phase of plasma and plasma neuron-derived extracellular vesicles (NDEVs) are increasingly being investigated as sources of biomarkers. The aim of this study was to examine the comparative biomarker potential of these two biofluids, as well as the association between respective biomarkers. Methods: We retrospectively identified three distinct diagnostic groups of 44 individuals who provided samples at baseline and at a mean of 3.1 years later; 14 were cognitively unimpaired at baseline and remained so (NRM-NRM), 13 had amnestic MCI that progressed to AD dementia (MCI-DEM) and 17 had AD dementia at both timepoints (DEM-DEM). Plasma NDEVs were isolated by immunoaffinity capture targeting the neuronal markers L1CAM, GAP43, and NLGN3. In both plasma and NDEVs, we assessed ATN biomarkers (Aß42, Aß40, total Tau, P181-Tau) alongside several other exploratory markers. Results: The Aß42/Aß40 ratio in plasma and NDEVs was lower in MCI-DEM than NRM-NRM at baseline and its levels in NDEVs decreased over time in all three groups. Similarly, plasma and NDEV-associated Aß42 was lower in MCI-DEM compared to NRM-NRM at baseline and its levels in plasma decreased over time in DEM-DEM. For NDEV-associated proBDNF, compared to NRM-NRM, its levels were lower in MCI-DEM and DEM-DEM at baseline, and they decreased over time in the latter group. No group differences were found for other exploratory markers. NDEV-associated Aß42/Aß40 ratio and proBDNF achieved the highest areas under the curve (AUCs) for discriminating between diagnostic groups, while proBDNF was positively associated with Mini-Mental State Examination (MMSE) score. No associations were found between the two biofluids for any assessed marker. Discussion: The soluble phase of plasma and plasma NDEVs demonstrate distinct biomarker profiles both at a single time point and longitudinally. The lack of association between plasma and NDEV measures indicates that the two types of biofluids demonstrate distinct biomarker signatures that may be attributable to being derived through different biological processes. NDEV-associated proBDNF may be a useful biomarker for AD diagnosis and monitoring.

Neuronal and Astrocytic Extracellular Vesicle Biomarkers in Blood Reflect Brain Pathology in Mouse Models of Alzheimer's Disease.

Circulating neuronal extracellular vesicles (NEVs) of Alzheimer's disease (AD) patients show high Tau and ß-amyloid (Aß) 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 Aß42 (p < 0.005). NEV Aß42 had moderately strong correlations with Aß42 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.

Exploring brain insulin resistance in adults with bipolar depression using extracellular vesicles of neuronal origin.

Accumulating evidence suggests that disrupted insulin signaling is involved in bipolar disorder (BD) pathogenesis. Herein, we aimed to directly explore the potential role of neuronal insulin signaling using an innovative technique based on biomarkers derived from plasma extracellular vesicles enriched for neuronal origin (NEVs). We leveraged plasma samples from a randomized, double-blind, placebo-controlled, 12-week clinical trial evaluating infliximab as a treatment of bipolar depression. We isolated NEVs using immunoprecipitation against neuronal marker L1CAM from samples collected at baseline and weeks 2, 6 and 12 (endpoint) and measured NEV biomarkers using immunoassays. We assessed neuronal insulin signaling at its first node (IRS-1) and along the canonical (Akt, GSK-3ß, p70S6K) and alternative (ERK1/2, JNK and p38-MAPK) pathways. A subset of participants (n = 27) also underwent whole-brain magnetic resonance imaging (MRI) at baseline and endpoint. Pre-treatment, NEV biomarkers of insulin signaling were independently associated with cognitive function and MRI measures (i.e. hippocampal and ventromedial prefrontal cortex [vmPFC] volumes). In fact, the association between IRS-1 phosphorylation at serine site 312 (pS312-IRS-1), an indicator of insulin resistance, and cognitive dysfunction was mediated by vmPFC volume. In the longitudinal analysis, patients treated with infliximab, a tumor necrosis factor-alpha antagonist with known insulin sensitizing properties, compared to those treated with placebo, had augmented phosphorylation of proteins from the alternative pathway. Infliximab responders had significant increases in phosphorylated JNK levels, relative to infliximab non-responders and placebo responders. In addition, treatment with infliximab resulted in increase in MRI measures of brain volume; treatment-related changes in the dorsolateral prefrontal cortex volume were mediated by changes in biomarkers from the insulin alternative pathway. In conclusion, our findings support the idea that brain insulin signaling is a target for further mechanistic and therapeutic investigations.

Astrocyte- and Neuron-Derived Extracellular Vesicles from Alzheimer's Disease Patients Effect Complement-Mediated Neurotoxicity.

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.

Extracellular Vesicle Biomarkers Reveal Inhibition of Neuroinflammation by Infliximab in Association with Antidepressant Response in Adults with Bipolar Depression.

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) (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.

Imaging GPCRs trafficking and signaling with total internal reflection fluorescence microscopy in cultured neurons.

Total internal reflection fluorescence (TIRF) microscopy allows probing the cellular events occurring close and at the plasma membrane. Over the last decade, we have seen a significant increase in the number of publications applying TIRF microscopy to unravel some of the fundamental biological questions regarding G protein-coupled receptors (GPCRs) function such as the mechanisms controlling receptor trafficking, quaternary structure, and signaling among others. Most of the published work has been performed in heterologous systems such as HEK293 and CHO cells, where the imaging surface available is higher and smoother when compared with the narrow processes or the smaller cell bodies of neurons. However, some publications have expanded our understanding of these events to primary cell cultures, mostly rat hippocampal and striatal neuronal cultures. Results from these cells provide a bona fide model of the complex events controlling GPCR function in living cells. We believe more work needs to be performed in primary cultures and eventually in intact tissue to complement the knowledge obtained from heterologous cell models. Here, we described a step-by-step protocol to investigate the surface trafficking and signaling from GPCRs in rat hippocampal and striatal primary cultures.

Imaging of kiss-and-run exocytosis of surface receptors in neuronal cultures.

Transmembrane proteins are continuously shuttled from the endosomal compartment to the neuronal plasma membrane by highly regulated and complex trafficking steps. These events are involved in many homeostatic and physiological processes such as neuronal growth, signaling, learning and memory among others. We have previously shown that endosomal exocytosis of the B2 adrenergic receptor (B2AR) and the GluR1-containing AMPA receptor to the neuronal plasma membrane is mediated by two different types of vesicular fusion. A rapid type of exocytosis in which receptors are delivered to the plasma membrane in a single kinetic step, and a persistent mode in which receptors remain clustered at the insertion site for a variable period of time before delivery to the cell surface. Here, by comparing the exocytosis of multiple receptors in dissociated hippocampal and striatal cultures, we show that persistent events are a general mechanism of vesicular delivery. Persistent events were only observed after 10 days in vitro, and their frequency increased with use of the calcium ionophore A23187 and with depolarization induced by KCl. Finally, we determined that vesicles producing persistent events remain at the plasma membrane, closing and reopening their fusion pore for a consecutive release of cargo in a mechanism reminiscent of synaptic kiss-and-run. These results indicate that the delivery of transmembrane receptors to the cell surface can be dynamically regulated by kiss-and-run exocytosis.

Ligand-specific endocytic dwell times control functional selectivity of the cannabinoid receptor 1.

G protein-coupled receptors (GPCRs) are the major transducers of external stimuli and key therapeutic targets in many pathological conditions. When activated by different ligands, one receptor can elicit multiple signalling cascades that are mediated by G proteins or ß-arrestin, a process defined as functional selectivity or ligand bias. However, the dynamic mechanisms underlying ß-arrestin signalling remain unknown. Here by studying the cannabinoid receptor 1 (CB1R), we identify ligand-specific endocytic dwell times, that is, the time during which receptors are clustered into clathrin pits together with ß-arrestins before endocytosis, as the mechanism controlling ß-arrestin signalling. Agonists inducing short endocytic dwell times produce little or no ß-arrestin signalling, whereas those eliciting prolonged dwell times induce robust signalling. Remarkably, extending CB1R dwell times by preventing endocytosis substantially increased ß-arrestin signalling. These studies reveal how receptor activation translates into ß-arrestin signalling and identify a mechanism to control this pathway.