Exploring the role of neuronal-enriched extracellular vesicle miR-93 and interoception in major depressive disorder.

Major depressive disorder (MDD) is associated with interoceptive processing dysfunctions, but the molecular mechanisms underlying this dysfunction are poorly understood. This study combined brain neuronal-enriched extracellular vesicle (NEEV) technology and serum markers of inflammation and metabolism with Functional Magnetic Resonance Imaging (fMRI) to identify the contribution of gene regulatory pathways, in particular micro-RNA (miR) 93, to interoceptive dysfunction in MDD. Individuals with MDD (n = 41) and healthy comparisons (HC; n = 35) provided blood samples and completed an interoceptive attention task during fMRI. EVs were separated from plasma using a precipitation method. NEEVs were enriched by magnetic streptavidin bead immunocapture utilizing a neural adhesion marker (L1CAM/CD171) biotinylated antibody. The origin of NEEVs was validated with two other neuronal markers - neuronal cell adhesion molecule (NCAM) and ATPase Na+/K+ transporting subunit alpha 3 (ATP1A3). NEEV specificities were confirmed by flow cytometry, western blot, particle size analyzer, and transmission electron microscopy. NEEV small RNAs were purified and sequenced. Results showed that: (1) MDD exhibited lower NEEV miR-93 expression than HC; (2) within MDD but not HC, those individuals with the lowest NEEV miR-93 expression had the highest serum concentrations of interleukin (IL)-1 receptor antagonist, IL-6, tumor necrosis factor, and leptin; and (3) within HC but not MDD, those participants with the highest miR-93 expression showed the strongest bilateral dorsal mid-insula activation during interoceptive versus exteroceptive attention. Since miR-93 is regulated by stress and affects epigenetic modulation by chromatin re-organization, these results suggest that healthy individuals but not MDD participants show an adaptive epigenetic regulation of insular function during interoceptive processing. Future investigations will need to delineate how specific internal and external environmental conditions contribute to miR-93 expression in MDD and what molecular mechanisms alter brain responsivity to body-relevant signals.

Exploring the role of neuronal-enriched extracellular vesicle miR-93 and interoception in major depressive disorder.

Major depressive disorder (MDD) is associated with interoceptive processing dysfunctions, but the molecular mechanisms underlying this dysfunction are poorly understood. This study combined brain Neuronal-Enriched Extracellular Vesicle (NEEV) technology and serum markers of inflammation and metabolism with Functional Magnetic Resonance Imaging (fMRI) to identify the contribution of gene regulatory pathways, in particular micro-RNA (miR) 93, to interoceptive dysfunction in MDD. Individuals with MDD (n = 44) and healthy comparisons (HC; n = 35) provided blood samples and completed an interoceptive attention task during fMRI. EVs were separated from plasma using a precipitation method. NEEVs were enriched by magnetic streptavidin bead immunocapture utilizing a neural adhesion marker (CD171) biotinylated antibody. NEEV specificities were confirmed by ow cytometry, western blot, particle size analyzer, and transmission electron microscopy. NEEV small RNAs were purified and sequenced. Results showed that: (1) MDD exhibited lower NEEV miR-93 expression than HC; (2) within MDD but not HC, those individuals with the lowest NEEV miR-93 expression had the highest serum concentrations of interleukin (IL)-1 receptor antagonist, IL-6, tumor necrosis factor, and leptin; and (3) within HC but not MDD, those participants with the highest miR-93 expression showed the strongest bilateral dorsal mid-insula activation. Since miR-93 is regulated by stress and affects epigenetic modulation by chromatin reorganization, these results suggest that healthy individuals but not MDD participants show an adaptive epigenetic regulation of insular function during interoceptive processing. Future investigations will need to delineate how specific internal and external environmental conditions contribute to miR-93 expression in MDD and what molecular mechanisms alter brain responsivity to body-relevant signals.

Association between inflammation, reward processing, and ibuprofen-induced increases of miR-23b in astrocyte-enriched extracellular vesicles: A randomized, placebo-controlled, double-blind, exploratory trial in healthy individuals.

Ibuprofen, a non-steroidal, anti-inflammatory drug, modulates inflammation but may also have neuroprotective effects on brain health that are poorly understood. Astrocyte-enriched extracellular vesicles (AEEVs) facilitate cell-to-cell communication and - among other functions - regulate inflammation and metabolism via microribonucleic acids (miRNAs). Dysfunctions in reward-related processing and inflammation have been proposed to be critical pathophysiological pathways in individuals with mood disorders. This investigation examined whether changes in AEEV cargo induced by an anti-inflammatory agent results in inflammatory modulation that is associated with reward-related processing. Data from a double-blind, randomized, repeated-measures study in healthy volunteers were used to examine the effects of AEEV miRNAs on brain activation during reward-related processing. In three separate visits, healthy participants (N = 20) received a single dose of either placebo, 200 mg, or 600 mg of ibuprofen, completed the monetary incentive delay task during functional magnetic resonance imaging, and provided a blood sample for cytokine and AEEV collection. AEEV miRNA content profiling showed that ibuprofen dose-dependently increased AEEV miR-23b-3p expression with greater increase following the 600 mg administration than placebo. Those individuals who received 600 mg and showed the highest miR-23b-3p expression also showed the (a) lowest serum tumor necrosis factor (TNF) and interleukin-17A (IL-17A) concentrations; and had the (b) highest striatal brain activation during reward anticipation. These results support the hypothesis that ibuprofen alters the composition of miRNAs in AEEVs. This opens the possibility that AEEV cargo could be used to modulate brain processes that are important for mental health.

Electronic Nicotine Delivery Systems and Serum Folate: A Case Study.

INTRODUCTION: In 2018, the US Surgeon General declared youth electronic nicotine delivery systems (ENDS) use as an epidemic. Combustible cigarettes have been shown to adversely affect folate status; however, no study to date has explored how ENDS impact folate status. METHODS: In this case study, a white 18-year-old woman was followed for a 1-month period as part of a larger study. During her participation in the study, a self-report of dietary folate consumption and use of alcohol, tobacco, and other drugs (ATOD) was collected. Each week, dried blood spots were collected to assess red blood cell (RBC) and serum folate. RESULTS: During the first 2 weeks of the study, she used ENDS and her serum folate values were depleted in the ranges of 1.91 to 4.39 µg/L. During the third week, when no ENDS were used, her serum folate value was measured at 29.44 µg/L. When ENDS use resumed during the fourth week, her serum folate value fell to 7.50 µg/L. CONCLUSIONS: This relationship suggests the need for additional studies on ENDS use and serum folate status, particularly among adolescent women who already have the lowest folate status nationally.

Altered populations of natural killer cells, cytotoxic T lymphocytes, and regulatory T cells in major depressive disorder: Association with sleep disturbance.

A subset of individuals with major depressive disorder (MDD) have impaired adaptive immunity characterized by a greater vulnerability to viral infection and a deficient response to vaccination along with a decrease in the number and/or activity of T cells and natural killer cells (NKC). Nevertheless, it remains unclear which specific subsets of lymphocytes are altered in MDD, a shortcoming we address here by utilizing an advanced fluorescence-activated cell sorting (FACS) method that allows for the differentiation of important functionally-distinct lymphocyte sub-populations. Furthermore, despite evidence that sleep disturbance, which is a core symptom of MDD, is itself associated with alterations in lymphocyte distributions, there is a paucity of studies examining the contribution of sleep disturbance on lymphocyte populations in MDD populations. Here, we measured differences in the percentages of 13 different lymphocytes and 6 different leukocytes in 54 unmedicated MDD patients (partially remitted to moderate) and 56 age and sex-matched healthy controls (HC). The relationship between self-reported sleep disturbance and cell counts was evaluated in the MDD group using the Pittsburgh Sleep Quality Index (PSQI). The MDD group showed a significantly increased percentage of CD127low/CCR4+ Treg cells, and memory Treg cells, as well as a reduction in CD56+CD16- (putative immunoregulatory) NKC counts, the latter, prior to correction for body mass index. There also was a trend for higher effector memory CD8+ cell counts in the MDD group versus the HC group. Further, within the MDD group, self-reported sleep disturbance was associated with an increased percentage of effector memory CD8+ cells but with a lower percentage of CD56+CD16- NKC. These results provide important new insights into the immune pathways involved in MDD, and provide novel evidence that MDD and associated sleep disturbance increase effector memory CD8+ and Treg pathways. Targeting sleep disturbance may have implications as a therapeutic strategy to normalize NKC and memory CD8+ cells in MDD.

IL-18 acts in synergy with IL-7 to promote ex vivo expansion of T lymphoid progenitor cells.

Although IL-18 has not previously been shown to promote T lymphopoiesis, results obtained via a novel data mining algorithm (global microarray meta-analysis) led us to explore a predicted role for this cytokine in T cell development. IL-18 is a member of the IL-1 cytokine family that has been extensively characterized as a mediator of inflammatory immune responses. To assess a potential role for IL-18 in T cell development, we sort-purified mouse bone marrow-derived common lymphoid progenitor cells, early thymic progenitors (ETPs), and double-negative 2 thymocytes and cultured these populations on OP9-Delta-like 4 stromal layers in the presence or absence of IL-18 and/or IL-7. After 1 wk of culture, IL-18 promoted proliferation and accelerated differentiation of ETPs to the double-negative 3 stage, similar in efficiency to IL-7. IL-18 showed synergy with IL-7 and enhanced proliferation of both the thymus-derived progenitor cells and the bone marrow-derived common lymphoid progenitor cells. The synergistic effect on the ETP population was further characterized and found to correlate with increased surface expression of c-Kit and IL-7 receptors on the IL-18-treated cells. In summary, we successfully validated the global microarray meta-analysis prediction that IL-18 affects T lymphopoiesis and demonstrated that IL-18 can positively impact bone marrow lymphopoiesis and T cell development, presumably via interaction with the c-Kit and IL-7 signaling axis.

Ten-color flow cytometry reveals distinct patterns of expression of CD124 and CD126 by developing thymocytes.

BACKGROUND: We have developed a 12-parameter/10-color flow cytometric staining method for the simultaneous detection and characterization of 21 mouse thymocyte subpopulations that represent discreet stages of T cell development. To demonstrate the utility of this method, we assessed cytokine receptor expression on mouse thymocyte subsets. These experiments revealed distinct patterns of surface expression of receptors for the cytokines IL-4 and IL-6. RESULTS: The IL-4 receptor α chain (CD124) was highly expressed on the earliest thymocyte subsets, then downregulated prior to T cell receptor ß-selection and finally upregulated in the CD4/CD8 double positive cells prior to positive selection. The IL-6 receptor α chain (CD126) showed a different pattern of expression. It was expressed on the most mature subsets within the CD4 and CD8 single positive (SP) compartments and was absent on all other thymocytes with the exception of a very small cKit-CD4-CD8- population. Intracellular staining of SP thymocytes for phosphorylated STAT-1 demonstrated that IL-6 signaling was confined to the most mature SP subsets. CONCLUSIONS: This 12-parameter staining methodology uses only commercially available fluorochrome-coupled monoclonal antibodies and therefore could be employed by any investigator with access to a 4-laser flow cytometer. This novel staining scheme allowed us to easily phenotype thymocyte subpopulations that span across development, from the early thymic progenitors (ETPs) to the most mature subsets of the CD4 and CD8 single positive populations.

CD28 expression redefines thymocyte development during the pre-T to DP transition.

CD27 and CD28 have emerged as indicators demarcating the transition of thymocytes through beta-selection. We found that CD28 exhibits a greater dynamic range of expression during this phase, thus it was employed to further parse the DN/CD44(-) compartment in order to assess IL-7 signaling during the beta-selection process. Plotting CD28 versus CD25 expression revealed six DN/CD44(-) populations. OP9-DL1 stromal cell co-culture was used to demonstrate a developmental linkage from DN3a (CD25(+)CD28(-/lo)) to DN3b (CD25(+)CD28(+)) to DN3c (CD25(int)CD28(+)) to DN4a (CD25(-)CD28(+)) to double positive (DP) and showed the DN4b (CD25(-)CD28(hi)) and DN4c (CD25(-)CD28(-/lo)) populations to be inefficient in producing DP cells. Using CD69 as an additional marker to further parse the DN4a population, we found the pre-DP cells to be the CD44(-)CD25(-)CD28(int)CD69(-)CD4(-/lo)CD8(-/lo) subset. Using this refined developmental scheme, IL-7R alpha expression was found to be transiently up-regulated post-beta-selection in the DN3b and DN3c subsets; however, this increase did not confer enhanced responsiveness over that observed in the DN3a population. CD28 messenger RNA expression was up-regulated in post-beta-selected cells, whereas transcripts for CD27, IL-7R alpha and Bcl-2 were lower than that observed in the DN3a population. This study refines the current thymocyte differentiation scheme to allow for more detailed evaluation of events controlling early T-cell development, specifically surrounding the beta-selection checkpoint.

Differential IL-7 responses in developing human thymocytes.

Interleukin (IL)-7 is a factor essential for mouse and human thymopoiesis. Mouse thymocytes have altered sensitivities to IL-7 at different developmental stages. CD4/CD8 double positive (DP) mouse thymocytes are shielded from the influence of IL-7 because of loss of CD127 (IL-7Ralpha). In this study, we assessed IL-7 receptor expression and IL-7 signaling in human thymocytes. We found human DP cells to be severely limited in their ability to phosphorylate STAT-5 in response to IL-7. The relative expression levels of the IL-7-inducible proteins Bcl-2 and Mcl-1 were also lower in human DP cells, consistent with a stage-specific decrease in IL-7 responsiveness. IL-7 responses were restored in a subset of cells that matured past the DP stage. Unlike the regulation of IL-7 signaling in mouse thymocytes, loss of IL-7 signaling in human DP cells was not due to absence of CD127, but instead correlated with downregulation of CD132 (common gamma chain).

Oligomerization of membrane-bound Bcl-2 is involved in its pore formation induced by tBid.

Both pro-apoptotic Bax and anti-apoptotic Bcl-2 are structurally homologous to the pore-forming domain of bacterial toxins. Bax proteins oligomerize in the mitochondrial outer membranes forming pores that release cytochrome c from the mitochondrial intermembrane space. Bcl-2 proteins also form pores that, however, are much smaller than the Bax pore. It is unknown whether Bcl-2 forms monomeric or oligomeric pores. Here, we characterized the Bcl-2 pore formation in liposomes using biophysical and biochemical techniques. The results show that the Bcl-2 pore enlarges as the concentration of Bcl-2 increases, suggesting that the pore is formed by Bcl-2 oligomers. As expected from oligomerization-mediated pore-formation, the small pores are formed earlier than the large ones. Bcl-2 oligomers form pores faster than the monomer, indicating that the oligomerization constitutes an intermediate step of the pore formation. A Bcl-2 mutant with higher affinity for oligomerization forms pores faster than wild type Bcl-2. Bcl-2 oligomers were detected in the liposomal membranes under conditions that Bcl-2 forms pores, and the extent of oligomerization was positively correlated with the pore-forming activity. Therefore, Bcl-2 oligomerizes in membranes forming pores, but the extent of oligomerization and the size of the resulting pores are much smaller than that of Bax, supporting the model that Bcl-2 is a defective Bax.

Disruption of thymopoiesis in ST6Gal I-deficient mice.

Thymocyte development is accompanied by sequential changes in cell surface glycosylation. For example, medullary thymocytes have increased levels of alpha2,3-linked sialic acid and a loss of asialo core 1 O-glycans as compared to cortical thymocytes. Some of these changes have been linked to fine tuning of the T cell receptor avidity. We analyzed ST6Gal I transcript abundance and levels of alpha2,6-linked sialic acid across thymocyte subsets. We found that ST6Gal I transcript levels increased following T cell receptor beta-selection suggesting that this sialyltransferase may influence the development of early thymocyte populations. Indeed, low levels of alpha2,6-linked sialic acid were found in the earliest T lineage cells, and then increased in T cell receptor beta-selected cells. To determine whether ST6Gal I influences T cell development, we analyzed ST6Gal I-deficient mice for disruptions in thymocyte populations. We found reduced thymic cellularity in the ST6Gal I-deficient mice starting in the early thymocyte compartments.

Effects of hepatic zonal oxygen levels on hepatocyte stress responses.

BACKGROUND: Hepatocytes spend their lifetimes in a gradient of oxygen, hormones, and enzymes. We used a three-dimensional Matrigel model to determine whether hepatocytes cultured at perivenous (zone 3) oxygen levels differed in susceptibility to anoxia-induced cell injury compared with hepatocytes cultured at periportal (zone 1) oxygen levels. MATERIALS AND METHODS: Hepatocytes were harvested from Sprague Dawley rats and cultured at 9% oxygen (hepatic zone 1) or 5% oxygen (hepatic zone 3) and stressed at 0% oxygen. Microscopy, real-time reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay were used to assess cell viability, mitochondrial potential, acute phase responses, and membrane blebbing. RESULTS: Hepatocytes cultured in Matrigel with HepatoZyme medium at zone 1 and zone 3 oxygen conditions were viable for 1 wk and showed acute phase responses as measured by interleukin-6-induced fibrinogen production. In response to 3 h anoxia, cells maintained at the perivenous oxygen level showed increased membrane blebbing and increased loss of mitochondrial membrane potential in comparison to the periportal oxygen cultured cells. Cells at perivenous oxygen also showed a reduced ability to recover following reoxygenation. CONCLUSIONS: Hepatocytes can remain viable and functional for extended periods in culture at low oxygen levels that mimic the hepatic perivenous environment, yet these cells are more susceptible to anoxia-induced damage than hepatocytes cultured at the periportal oxygen level. The small population of perivenous hepatocytes may be critical in determining the fate of the liver during ischemia/reperfusion since hepatocytes cultured at that concentration appear to be more labile in response to anoxia.

Impaired thymopoiesis in interleukin-7 receptor transgenic mice is not corrected by Bcl-2.

Murine thymocytes down-regulate IL-7 responsiveness following beta-selection and reacquire sensitivity after positive selection. To assess the potential consequences of IL-7 signaling during this phase of development, transgenic IL-7 receptor alpha (IL-7Ralpha) mice were evaluated for IL-7 responsiveness as gauged by STAT-5 phosphorylation. Transgenic IL-7Ralpha expression increased the percentage of thymocytes responsive to IL-7 yet resulted in a decrease in total thymic cellularity. Aberrant thymocyte development in transgenic mice was first manifested by a reduction of DN3 thymocytes that correlated with lower Bcl-2 expression. Surprisingly, transgenic restoration of Bcl-2 expression did not correct thymic hypocellularity induced by IL-7Ralpha overexpression. These findings demonstrate that failure to appropriately downregulate IL-7Ralpha expression interferes with thymocyte development past the pro-T stage resulting in significantly lower levels of mature thymocytes.

tBid elicits a conformational alteration in membrane-bound Bcl-2 such that it inhibits Bax pore formation.

During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multispanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multispanning form that is similar to the one found in the mitochondrial outer membrane of drug-treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay showed corresponding activities in inhibiting pore formation by Bax in vitro and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo.

Auto-activation of the apoptosis protein Bax increases mitochondrial membrane permeability and is inhibited by Bcl-2.

Interactions among Bcl-2 family proteins mediated by Bcl-2 homology (BH) regions transform apoptosis signals into actions. The interactions between BH3 region-only proteins and multi-BH region proteins such as Bax and Bcl-2 have been proposed to be the dominant interactions required for initiating apoptosis. Experimental evidence also suggests that both homo- and hetero-interactions are mediated primarily by the BH3 regions in all Bcl-2 family proteins and contribute to commitment to or inhibition of apoptosis. We found that a peptide containing the BH3 helix of Bax was not sufficient to activate recombinant Bax to permeabilize mitochondria. However, an extended peptide containing the BH3 helix and additional downstream sequences activated Bax to permeabilize mitochondria and liposomes. Bcl-2 inhibited the membrane-permeabilizing activity of peptide-activated Bax. This activity of Bcl-2 was inhibited by the extended but not the BH3-only peptide despite both peptides binding to Bcl-2 with similar affinity. Further, membrane-bound Bax activation intermediates directly activated soluble Bax further permeabilizing the membrane. Bcl-2 inhibited Bax auto-activation. We therefore propose that Bax auto-activation amplifies the initial death signal produced by BH3-only proteins and that Bcl-2 functions as an inhibitor of Bax auto-activation.

Bcl-2 homodimerization involves two distinct binding surfaces, a topographic arrangement that provides an effective mechanism for Bcl-2 to capture activated Bax.

The homo- and heterodimerization of Bcl-2 family proteins is important for transduction and integration of apoptotic signals and control of the permeability of mitochondria and endoplasmic reticulum membranes. Here we mapped the interface of the Bcl-2 homodimer in a cell-free system using site-specific photocross-linking. Bcl-2 homodimer-specific photoadducts were detected from 11 of 17 sites studied. When modeled into the structure of Bcl-2 core, the interface is composed of two distinct surfaces: an acceptor surface that includes the hydrophobic groove made by helices 2 and 8 and the loop connecting helices 4 and 5 and a donor surface that is made by helices 1-4 and the loop connecting helices 2 and 3. The two binding surfaces are on separate faces of the three-dimensional structure, explaining the formation of Bcl-2 homodimers, homo-oligomers, and Bcl-2/Bax hetero-oligomers. We show that in vitro the Bcl-2 dimer can still interact with activated Bax as a larger oligomer. However, formation of a Bax/Bcl-2 heterodimer is favored, since this interaction inhibits Bcl-2 homodimerization. Our data support a simple model mechanism by which Bcl-2 interacts with activated Bax during apoptosis in an effective manner to neutralize the proapoptotic activity of Bax.

Phospholipid bicelles that align with their normals parallel to the magnetic field.

We have recently reported phospholipid bicelles (bilayered micelles) that have positive anisotropy of the magnetic susceptibility and align with their normals parallel to an external magnetic field [J. Am. Chem. Soc. 2001, 123, 1537]. Improvements have been made via the synthesis of a new phospholipid, 1-dodecanoyl-2-(4-(4-biphenyl)butanoyl)-sn-glycero-3-phosphocholine (DBBPC). Bicelles can be formed by mixing DBBPC with a short-chain phospholipid, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) in a ratio between 5.1:1 and 6.5:1 in an aqueous medium. The (31)P NMR spectra clearly show that these bicelles align with their principal axes parallel to the magnetic field within a wide temperature range. The (31)P chemical shifts indicate that the conformation of the polar headgroup in these bicelles may be different from that in common bicelles. The phase behavior of a mixture of DBBPC/DHPC with 6:1 mole ratio was investigated in the temperature range of 10-75 degrees C using (31)P, (2)H, and (23)Na NMR. At lower temperatures (10-54 degrees C), the system is dominated by the bicellar phase. At higher temperatures (54-75 degrees C), isotropic micelles are formed and coexist with the bicelles. The partial alignment of maltotriose in the DBBPC/DHPC system was studied at three temperatures, and the (1)H-(13)C dipolar coupling constants are compared with those obtained for two other bicelle solutions.