Non-Pigmented Ciliary Epithelium-Derived Extracellular Vesicles Loaded with SMAD7 siRNA Attenuate Wnt Signaling in Trabecular Meshwork Cells In Vitro.

Primary open-angle glaucoma is established by the disruption of trabecular meshwork (TM) function. The disruption leads to increased resistance to the aqueous humor (AH), generated by the non-pigmented ciliary epithelium (NPCE). Extracellular vesicles (EVs) participate in the communication between the NPCE and the TM tissue in the ocular drainage system. The potential use of NPCE-derived EVs to deliver siRNA to TM cells has scarcely been explored. NPCE-derived EVs were isolated and loaded with anti-fibrotic (SMAD7) siRNA. EV's structural integrity and siRNA loading efficiency were estimated via electron microscopy and fluorescence. Engineered EVs were added to pre-cultured TM cells and qRT-PCR was used to verify the transfer of selected siRNA to the cells. Western blot analysis was used to evaluate the qualitative effects on Wnt-TGFß2 proteins' expression. EVs loaded with exogenous siRNA achieved a 53% mRNA knockdown of SMAD7 in TM cells, resulting in a significant elevation in the levels of ß-Catenin, pGSK3ß, N-Cadherin, K-Cadherin, and TGFß2 proteins in TM cells. NPCE-derived EVs can be used for efficient siRNA molecule delivery into TM cells, which may prove to be beneficial as a therapeutic target to lower intraocular pressure (IOP).

Trabecular meshwork's collagen network formation is inhibited by non-pigmented ciliary epithelium-derived extracellular vesicles.

The present research aims to determine whether the application of non-pigmented ciliary epithelium cells derived extracellular vesicles to human trabecular meshwork cells affects the formation and secretion of collagen type I to the extracellular matrix formation. Following the extraction of non-pigmented ciliary epithelium derived extracellular vesicles by a precipitation method, their size and concentration were determined using tunable resistive pulse sensing technology. Extracellular vesicles were incubated with trabecular meshwork cells for 3 days. Morphological changes of collagen type I in the extracellular matrix of trabecular meshwork cells were visualized using confocal microscopy and scanning electron microscopy. A Sirius Red assay was used to determine the total amount of collagen. Finally, collagen type I expression levels in the extracellular matrix of trabecular meshwork cells were quantified by cell western analysis. We found that non-pigmented ciliary epithelium extracellular vesicles were very effective at preventing collagen fibres formation by the trabecular meshwork cells, and their secretion to the extracellular matrix was significantly reduced (P < .001). Morphological changes in the extracellular matrix of trabecular meshwork cells were observed. Our study indicates that non-pigmented ciliary epithelium extracellular vesicles can be used to control collagen type I fibrillogenesis in trabecular meshwork cells. These fibrils net-like structure is responsible for remodelling the extracellular matrix. Moreover, we suggest that targeting collagen type I fibril assembly may be a viable treatment for primary open-angle glaucoma abnormal matrix deposition of the extracellular matrix.

Crosstalk between MicroRNA and Oxidative Stress in Primary Open-Angle Glaucoma.

Reactive oxygen species (ROS) plays a key role in the pathogenesis of primary open-angle glaucoma (POAG), a chronic neurodegenerative disease that damages the trabecular meshwork (TM) cells, inducing apoptosis of the retinal ganglion cells (RGC), deteriorating the optic nerve head, and leading to blindness. Aqueous humor (AH) outflow resistance and intraocular pressure (IOP) elevation contribute to disease progression. Nevertheless, despite the existence of pharmacological and surgical treatments, there is room for the development of additional treatment approaches. The following review is aimed at investigating the role of different microRNAs (miRNAs) in the expression of genes and proteins involved in the regulation of inflammatory and degenerative processes, focusing on the delicate balance of synthesis and deposition of extracellular matrix (ECM) regulated by chronic oxidative stress in POAG related tissues. The neutralizing activity of a couple of miRNAs was described, suggesting effective downregulation of pro-inflammatory and pro-fibrotic signaling pathways, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), transforming growth factor-beta 2 (TGF-ß2), Wnt/ß-Catenin, and PI3K/AKT. In addition, with regards to the elevated IOP in many POAG patients due to increased outflow resistance, Collagen type I degradation was stimulated by some miRNAs and prevented ECM deposition in TM cells. Mitochondrial dysfunction as a consequence of oxidative stress was suppressed following exposure to different miRNAs. In contrast, increased oxidative damage by inhibiting the mTOR signaling pathway was described as part of the action of selected miRNAs. Summarizing, specific miRNAs may be promising therapeutic targets for lowering or preventing oxidative stress injury in POAG patients.

Influence of Anti-Glaucoma Drugs on Uptake of Extracellular Vesicles by Trabecular Meshwork Cells.

BACKGROUND: Extracellular vesicles (EVs) are capable of manipulating cellular functions for the maintenance of biological homeostasis and disease progression, such as in glaucoma disease. These nano-particles carry a net negative surface charge under physiological conditions that can contribute to EVs:EVs interaction and their uptake by target cells. PURPOSE: To investigate the effect of glaucoma drugs on EVs physicochemical characters and the implications for their uptake by trabecular meshwork (TM) cells. METHODS: TM or non-pigmented ciliary epithelium (NPCE) cells derived EVs were incubated with commercial anti-glaucoma formulation, Timolol maleate, Brinzolamide or Benzalkonium Cl and their size and zeta potential (ZP) and physical interactions of EVs derived from NPCE cells and TM cells were evaluated. The contribution of EVs interactions to up-take by TM cells was examined using fluorescence-activated cell sorting. RESULTS: EVs size and ZP were affected by the ionic strength of the buffer rather than EVs type. Commercial glaucoma eye drops, including ß-blocker, α-2-agonist and prostaglandin analogs, reduced NPCE EVs ZP, whereas exposure of EVs to carbonic anhydrase inhibitor caused an increase in the ZP. A correlation was found between increased ZP values and increased NPCE EVs uptake by TM cells. We were able to show that Benzalkonium chloride stands behind this ZP effect and not Timolol or Brinzolamide. CONCLUSION: Altogether, our findings demonstrate that EVs size, surface membrane charge, and ionic strength of the surrounding have an impact on EVs:EVs interactions, which affect the uptake of NPCE EVs by TM cells.

Non-pigmented ciliary epithelium derived extracellular vesicles uptake mechanism by the trabecular meshwork.

Consistent with increasing findings, extracellular vesicles (EVs), consider as a major constituents of the aqueous humor, have a role as signaling mediators in glaucoma. Following secretion, EVs hold immense promise for utilization as bio-therapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Yet, the specific pathway utilizing for transferring signals by EVs in the ocular drainage system is not fully understood. Hence, the objective of this study was to examine internalization mechanisms by which Non-Pigmented Ciliary Epithelium (NPCE)-derived EVs deliver their signals to the Trabecular Meshwork (TM) cells. EVs were isolated and size and concentration were determined. Internalization study of treated EVs with Proteinase-K to achieve removal of surface membrane proteins on EVs was conducted. Energy dependent uptake mechanism was examined under various temperatures. Using uptake inhibitors endocytosis, phagocytosis, and Wnt-TGFß2 signaling were investigated. TM cells exposed to NPCE EVs demonstrate a significant decrease in the levels of two proteins in two Wnt-TGFb2 signaling proteins levels: p-GSK3ß and ß-catenin. A significant decrease in the uptake by TM cells of Proteinase-K-treated EVs was found, followed by attenuation of the Wnt-TGFß2 proteins expression. Energy dependent uptake revealed a significant decrease in EVs internalization. The exposure of TM cells to endocytosis uptake inhibitors abolished the decrease of the Wnt-TGFß2 proteins levels. Exposure to phagocytosis uptake inhibitor resulted in a partial inhibition of NPCE EVs effect in TM cells. The attenuation of proteins expression levels following uptake inhibitors treatment or EVs membrane proteins removal indicates that Wnt-TGFß2 signaling in TM cells is mediated through NPCE EVs surface proteins in an active manner that involves endocytosis-dependent routes.

Extracellular Vesicles Mediate Anti-Oxidative Response-In Vitro Study in the Ocular Drainage System.

The importance of extracellular vesicles (EVs) as signaling mediators has been emphasized for several pathways with only limited data regarding their role as protective messages during oxidative stress (OS). The ocular drainage system is unique by being continuously exposed to OS and having a one-way flow of the aqueous humor carrying EVs taking role in glaucoma disease. Here, we aimed to examine the ability of EVs derived from the non-pigmented ciliary epithelium (NPCE)-the aqueous humor producing cells exposed to OS-to deliver protecting messages to the trabecular meshwork (TM)-the aqueous humor draining cells-a process with significance to the pathophysiology of glaucoma disease. EVs extracted from media of NPCE cells exposed to non-lethal OS and their unstressed control were incubated with TM cells. The effects of EVs derived from oxidative stressed cells on the activation of the nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1 (Nrf2-Keap1), a major OS pathway, and of the Wnt pathway, known for its role in primary open-angle glaucoma, were evaluated. EVs derived from oxidized NPCE cells significantly protected TM cells from direct OS. The TM cells uptake of EVs from oxidized NPCE and their cytosolic Nrf2 levels were significantly higher at 8 h post-exposure. EVs derived from oxidized NPCE cells significantly attenuated Wnt protein expression in TM cells and activated major antioxidant genes as measured by qRT-PCR. TM cells exposed to EVs derived from oxidized NPCE cells exhibited significantly lower OS and higher super oxide dismutase and catalase activity. Finally, we were able to show that carbonylated proteins and products of oxidized protein are presented in significantly higher levels in EVs derived from oxidized NPCE cells, supporting their suggested role in the signaling process. We hypothesize that these findings may have implications beyond understanding the pathophysiology of glaucoma disease and that transmitting signals that activate the antioxidant system in target cells represent a broad response common to many tissues communication.

Extracellular vesicle-mediated crosstalk between NPCE cells and TM cells result in modulation of Wnt signalling pathway and ECM remodelling.

Primary open-angle glaucoma is a leading cause of irreversible blindness, often associated with increased intraocular pressure. Extracellular vesicles (EVs) carry a specific composition of proteins, lipids and nucleotides have been considered as essential mediators of cell-cell communication. Their potential impact for crosstalk between tissues responsible for aqueous humour production and out-flow is largely unknown. The study objective was to investigate the effects of EVs derived from non-pigmented ciliary epithelium (NPCE) primary cells on the expression of Wnt proteins in a human primary trabecular meshwork (TM) cells and define the mechanism underlying exosome-mediated regulation that signalling pathway. Consistent with the results in TM cell line, EVs released by both primary NPCE cells and NPCE cell line showed diminished pGSK3ß phosphorylation and decreased cytosolic levels of ß-catenin in primary TM cells. At the molecular level, we showed that NPCE exosome treatment downregulated the expression of positive GSKß regulator-AKT protein but increased the levels of GSKß negative regulator-PP2A protein in TM cells. NPCE exosome protein analysis revealed 584 miRNAs and 182 proteins involved in the regulation of TM cellular processes, including WNT/ß-catenin signalling pathway, cell adhesion and extracellular matrix deposition. We found that negative modulator of Wnt signalling miR-29b was abundant in NPCE exosomal samples and treatment of TM cells with NPCE EVs significantly decreased COL3A1 expression. Suggesting that miR-29b can be responsible for decreased levels of WNT/ß-catenin pathway. Overall, this study highlights a potential role of EVs derived from NPCE cells in modulating ECM proteins and TM canonical Wnt signalling.

Extracellular vesicles have variable dose-dependent effects on cultured draining cells in the eye.

The role of extracellular vesicles (EVs) as signal mediators has been described in many biological fields. How many EVs are needed to deliver the desired physiological signal is yet unclear. Using a normal trabecular meshwork (NTM) cell culture exposed to non-pigmented ciliary epithelium (NPCE)-derived EVs, a relevant model for studying the human ocular drainage system, we addressed the EVs dose-response effects on the Wnt signaling. The objective of the study was to investigate the dosing effects of NPCE-derived EVs on TM Wnt signaling. EVs were isolated by PEG 8000 method from NPCE and RPE cells (used as controls) conditioned media. Concentrations were determined by Tunable Resistive Pulse Sensing method. Various exosomes concentration were incubated with TM cells, for the determination of mRNA (ß-Catenin, Axin2 and LEF1) and protein (ß-Catenin, GSK-3ß) expression using real-time quantitative PCR and Western blot, respectively. Exposure of NTM cells for 8 hrs to low EVs concentrations was associated with a significant decreased expression of ß-Catenin, GSK-3ß, as opposed to exposure to high exosomal concentrations. Pro-MMP9 and MMP9 activities were significantly enhanced in NTM cells treated with high EV concentrations of (X10) as compared to low EV concentrations of either NPCE- or RPE-derived EVs and to untreated control. Our data support the concept that EVs biological effects are concentration-dependent at their target site. Specifically in the present study, we described a general dose-response at the gene and MMPs activity and a different dose-response regarding key canonical Wnt proteins expression.

Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system.

PURPOSE: Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood. METHODS: Exosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot. RESULTS: Exosomes derived from NPCE cells were purified and detected as small rounded 50-140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118-1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3ß and reduced ß-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of ß-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of ß-catenin in the nuclear fraction was noted, relative to the control. CONCLUSIONS: The data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles affect canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy.

Severe Mental Illness and Acute Stress: A Study of Service Utilization in a Conflict Zone.

Patients suffering from severe mental illness (SMI) are considered especially vulnerable to stress. In this study, their use of acute stress services in a military context affecting civilian populations was assessed, using naturally occurring data. The proportion of patients with a previously known SMI, defined as any chronic psychotic disorder or bipolar disorder, among all civilians examined at a center for treatment of stress during a military conflict versus at the ER in usual times, was compared, using the Chi square statistical test. Among 354 subjects examined at the center for treatment of stress, 12 had a SMI diagnosis. Among 404 subjects examined at the ER in usual times, 16 had a SMI diagnosis. Patients with SMI were under-represented, but not in a statistically significant manner, at the center for treatment of stress (χ2 = 0.31, p = ns). Although these results may imply that patients with SMI are not more vulnerable to external stress than the general population, we believe that they may have difficulties in seeking immediate help in such traumatogenic contexts. In order to reduce the occurrence of PTSD and gain efficacy in the treatment of the primary disorder, psychiatric services should perhaps make a reaching out effort to identify and examine these patients in the community. .

The Patient-Psychiatrist Relationship on the Axis of the Other and the Same.

The patient-psychiatrist relationship is a cornerstone of psychiatric professionalism and ethics. We discuss this topic along the axis of the Other and the Same, concepts defined by continental philosophy. The self of Anglo-American philosophy is typically described in individualistic terms. Individualism, autonomy and ideal self are valorized within the current model of care. These characteristics belong to the Lacanian Imaginary Order, which is the core of narcissism. Patients may yearn for another model of interaction. For Levinas, ethics should not involve a search for perfectionism and accomplishment but responsibility toward others. Ethics is, according to him, rooted in the calling into question of one's Sameness by the other's Otherness. The question of hospitality and of the welcoming of Otherness is central to his thought. Derrida further asks whether hospitality is not an interruption of the self. Hospitality may thus become a fundamental way of re-thinking clinical practices. A relationship to the Other as an-other is characterized as of Euclidian-type, establishing borders between the self and the Other, whereas a relationship to the Other as same is characterized as of fractal-type, emphasizing similarities between self and other as same and obliterating boundaries. Winnicott's object-relating versus use of object and Buber's I-you and I-it relations are also examined along the axis of Sameness and Otherness. Since psychiatric clinical practice requires to our view adequate and adaptive to and fro movements along this axis, the two forms of relating to the Other are discussed both theoretically and through a clinical case presentation.

Antidepressant-induced differential ubiquitination of ß-arrestins 1 and 2 in mononuclear leucocytes of patients with depression.

ß-Arrestins 1 and 2, cytosolic proteins known to mediate receptor desensitization, endocytosis and G protein-independent signalling, are post-translationally modified by ubiquitination regulating their ability to serve as adaptors and scaffolds. ß-Arrestins were suggested to play a role in the pathophysiology of depression and in antidepressant mechanism of action. To determine whether a depressive episode or antidepressant treatment induce significant selective differences in ß-arrestin 1 and 2 levels or their ubiquitination patterns in leucocytes of patients with depression, 46 outpatients diagnosed with a depressive episode were examined before and after 4-wk antidepressant treatment compared with age- and gender-matched control subjects. ß-Arrestin levels were measured by immunoblotting using anti-arrestin antibodies. Ubiquitination of ß-arrestins was measured using anti-ubiquitin antibodies followed by an immunoprecipitation step and immunoblotting using anti-arrestin antibodies. Antidepressants induced selective alterations in leucocyte ß-arrestin 1 and 2 levels and ubiquitination. The levels of ß-arrestin 1 and 2 and their ubiquitinated forms in leucocytes of yet untreated patients with depression were significantly decreased in a symptom severity correlated manner compared to control subjects. Antidepressants normalized ß-arrestin 1 and 2 levels and uncovered novel differences between the two isoforms: (a) while antidepressants normalized ubiquitination of ß-arrestin 1, ubiquination of ß-arrestin 2 was unaffected; (b) while under antidepressants ubiquitination extent of ß-arrestin 1 positively correlated with its level, an inverse picture of negative correlation was found between ubiquitination extent of ß-arrestin 2 and its level. We conclude that antidepressants may serve as a tool to detect functional differences between the two ß-arrestin isoforms and that through these differential effects antidepressants can induce specific alterations in alternative cellular signalling.

Non-Linear Dynamic Analysis of Inter-Word Time Intervals in Psychotic Speech.

UNLABELLED: "Language is a form and not a substance" - Ferdinand de Saussure Objective: Analyses of speech processes in schizophrenia are invariably focused on words as vocal signals. The results of such analyses are, however, strongly related to content, and may be language- and culture-dependent. Little attention has been paid to a pure measure of the form of speech, unrelated to its content: inter-words time intervals. METHOD: 15 patients with schizophrenia and 15 healthy volunteers are recorded spontaneously speaking for 10-15 min. Recordings are analyzed for inter-words time intervals using the following non-linear dynamical methods: unstable periodic orbits, correlation dimension, bi-spectral analysis, and symbolic dynamics. RESULTS: The series of inter-word time intervals in normal speech have the characteristics of a low-dimensional chaotic attractor with a correlation dimension of [Formula: see text]. Deconstruction of the attractor appears in psychosis with re-establishment after anti-psychotic treatment. Shannon entropy, a measure of the complexity in the time series, calculated from symbolic dynamics, is higher for psychotic speech, which is also characterized by higher levels of phase coupling: higher bicoherence, obtained using bi-spectral analysis. CONCLUSION: Non-linear dynamical methods applied to ITIs thus enable a content-independent, pure measure of the form of normal thought, its distortion in psychosis, and its restoration under treatment.

Antidepressants elevate GDNF expression and release from C6 glioma cells in a ß-arrestin1-dependent, CREB interactive pathway.

Glial cell line-derived neurotrophic factor (GDNF), essential for neuronal survival, plasticity and development, has been implicated in the mechanism of action of antidepressant drugs (ADs). ß-arrestin1, a member of the arrestin protein family, was found to play a role in AD mechanism of action. The present study aimed at evaluating whether the effect of ADs on GDNF in C6 rat glioma cells is exerted through a ß-arrestin1-dependent, CREB-interactive pathway. For chronic treatment, C6 rat glioma cells were treated for 3 d with different classes of ADs: imipramine - a non-selective monoamine reuptake inhibitor, citalopram - a serotonin selective reuptake inhibitor (SSRI) or desipramine - a norepinephrine selective reuptake inhibitor (NSRI) and compared to mood stabilizers (lithium and valproic acid) or to the antipsychotic haloperidol. Only ADs significantly elevated ß-arrestin1 levels in the cytosol, while reducing phospho-ß-arrestin1 levels in the cell nuclear fraction. ADs significantly increased both GDNF expression and release from the cells, but were unable to induce such effects in ß-arrestin1 knock-down cells. Chronic AD treatment significantly increased CREB phosphorylation without altering the level of total CREB in the nuclear fraction of the cells. Moreover, treatment with ADs significantly increased ß-arrestin1/CREB interaction. These findings support the involvement of ß-arrestin1 in the mechanism of action of ADs. We suggest that following AD treatment, ß-arrestin1 generates a transcription complex involving CREB essential for GDNF expression and release, thus enhancing GDNF's neuroprotective action that promotes cellular survival and plasticity when the survival and function of neurons is compromised as occurs in major depression.

Antidepressants increase ß-arrestin 2 ubiquitinylation and degradation by the proteasomal pathway in C6 rat glioma cells.

beta-Arrestins, regulators of G protein-coupled receptor-G protein coupling and receptor desensitization and internalization, function also as scaffolding proteins mediating cellular signaling events. beta-Arrestin1 was previously implicated by us in the pathophysiology of depression and in the mechanism of action of antidepressants (ADs). The ubiquitously expressed beta-arrestins1 and 2 are structurally highly homologous. There has been extensive investigation of these two proteins to determine whether they serve different roles in receptor signaling. In this study, we show that treatment of C(6) rat glioma cells with ADs of various types for 3 days resulted in decreased beta-arrestin2 levels. In contrast, beta-arrestin2 mRNA expression was found to be up-regulated by ADs. To unravel the mechanism for these opposite effects several possible beta-arrestin2 post-transcriptional events and modifications were examined. C(6) rat glioma cells transfected with beta-arrestin1-targeted short hairpin RNA showed similar effects of ADs on beta-arrestin2 levels. AD-induced decreases in beta-arrestin2 protein levels were not due to cytosolic membrane translocation. Immunoprecipitation experiments showed that ADs were able to increase coimmunoprecipitation of ubiquitin with beta-arrestin2. AD-induced increases in beta-arrestin2 ubiquitinylation led to its degradation by the proteasomal pathway, as the proteasome inhibitor N-[(phenylmethoxy)carbonyl]-l-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-l-leucinamide (MG-132) prevented antidepressant-induced decreases in beta-arrestin2 protein levels.

Normalization of GRK2 protein and mRNA measures in patients with depression predict response to antidepressants.

G-protein-coupled receptor kinases (GRKs) interfere in receptor-G-protein coupling leading to desensitization of G-protein-mediated receptor signalling. G-protein-coupled receptor signalling and its desensitization were previously implicated in the pathophysiology, diagnosis and treatment of mood disorders. The present study aimed to evaluate alterations in GRK2 protein and mRNA levels in mononuclear leukocytes (MNL) of untreated patients with major depression and the effects and time-course of antidepressant treatments on these alterations. Repeated GRK2 protein and mRNA measurements were carried in MNL of 24 patients with major depression. Each patient was examined while untreated and after 1, 2, 3 and 4 wk of antidepressant treatment; 24 healthy subjects were also studied. GRK2 protein and mRNA levels were evaluated through immunoblot analyses using monoclonal antibodies against GRK2 and reverse transcriptase-polymerase chain reaction, respectively. GRK2 protein and mRNA levels in MNL of untreated patients with major depression were significantly lower than the measures characterizing healthy subjects. The decreased GRK2 protein and mRNA levels were alleviated by antidepressant treatment. Normalization of GRK2 measures preceded, and, thus, could predict clinical improvement by 1-2 wk. These findings support the implication of GRK2 in the pathophysiology of major depression and in the mechanism underlying antidepressant-induced receptor down-regulation and therapeutic effects. GRK2 measurements in patients with depression may potentially serve for biochemical diagnostic purposes and for monitoring and predicting response to antidepressants.

Beta-arrestin signaling complex as a target for antidepressants and as a depression marker.

Beta-arrestins uncouple G protein-coupled receptors (GPCRs) from G proteins and promote their internalization, leading to desensitization and downregulation and serving as negative regulators of GPCR signaling. beta-Arrestins also function as scaffold proteins, interacting with several cytoplasmic proteins and linking GPCRs to intracellular signaling pathways such as the mitogen-activated protein kinase (MAPK) cascade. Recent work has also revealed that beta-arrestins translocate from the cytoplasm to the nucleus and associate with transcription factors such as histone acetyltransferase p300 and cyclic adenosine monophosphate (cAMP)-responsive element-binding protein. These substances also interact with regulators of transcription factors. We review findings on the effects of antidepressants on beta-arrestins and the plethora of antidepressant effects on signal transduction elements in which beta-arrestins serve as signaling scaffold proteins, focusing on the three major groups of MAPKs: extracellular signal-regulated kinases, c-Jun N-terminal kinases and p38 MAPKs, and on transcription factors and cofactors of which beta-arrestins mediate transcription regulation.

Antidepressants, beta-arrestins and GRKs: from regulation of signal desensitization to intracellular multifunctional adaptor functions.

G protein-coupled receptors (GPCR) have generated considerable interest in the pharmaceutical industry as drug targets. Theories concerning antidepressant targets of action suggested pre-synaptic monoamine reuptake mechanisms regulating GPCR activities including delayed receptor desensitization and down-regulation. GRKs and beta-arrestins translocate to the cell membrane and bind to agonist-occupied receptors. This uncouples these receptors from G proteins and promotes their internalization, leading to desensitization and down-regulation. Thus, GRKs and beta-arrestins serve as negative regulators of GPCR signaling. Recently, GPCR have been demonstrated to elicit signals through interaction with beta-arrestin as scaffolding proteins, independent of heterotrimeric G-protein coupling. beta-arrestins function as scaffold proteins that interact with several cytoplasmic proteins and link GPCR to intracellular signaling pathways such as MAPK cascades. Recent work has also revealed that beta-arrestins translocate from the cytoplasm to the nucleus and associate with transcription cofactors such as p300 and CREB. They also interact with regulators of transcription factors. We review findings concerning effects of antidepressants on GRKs and beta-arrestins and the plethora of antidepressants effects on signal transduction elements in which GRKs and beta-arrestins serve as signaling scaffold proteins, and on transcription factors and cofactors in which beta-arrestins mediate regulation of transcription. The emergence of G-protein-independent signaling pathways, through beta-arrestins, changes the way in which GPCR signaling is evaluated, from a cell biological to a pharmaceutical perspective and raises the possibility for the development of pathway specific therapeutics e.g., antidepressant medications targeting GRKs and beta-arrestin regulatory and signaling proteins.

Regulators of G-protein-coupled receptor-G-protein coupling: antidepressants mechanism of action.

There is a significant gap between advances in medication for mental disorders and the present static situation of biological diagnosis and monitoring treatment. The system of neural transmission and signal transduction is a complicated, highly regulated cascade of biochemical events. Growing evidence suggests that receptor-G-protein coupling may be involved in both the pathogenesis and treatment of mood disorders. Our knowledge concerning the basic mechanisms underlying the phenomenon of desensitization, internalization, downregulation and resensitization of the G-protein-coupled receptor has been advanced during the last decade. The present review discusses the possible involvement of regulators of G-protein-coupled receptor-G-protein coupling: beta-arrestins, G-protein-coupled receptor kinases and phosducin-like proteins, as well as beta-arrestins alternative signaling events, in the pathophysiology, diagnosis and treatment monitoring of mood disorders and in the mechanism of action of antidepressant medications.

Phosducin-like protein levels in leukocytes of patients with major depression and in rat cortex: the effect of chronic treatment with antidepressants.

The importance of signal transduction processes beyond receptors involving receptor-G protein coupling, in both the pathophysiology and the treatment of mood disorders, is well documented. Thus, regulatory elements of G protein function may play a role in the molecular mechanisms underlying these alterations. Phosducin-like proteins, a family of regulators of G protein function expressed throughout brain and body, modulate G protein function by high affinity sequestration of G protein-betagamma subunits, thus impeding G protein-mediated signal transmission by both Galpha and Gbetagamma subunits. An important consequence of Gbetagamma neutralization is the prevention of G protein-coupled receptor kinase phosphorylation resulting in a temporary protection to agonist-bound receptor desensitization. Phosducin-like protein levels were measured in brain cortices of rats chronically treated with one of five classes of antidepressants: imipramine, venlafaxine, maprotiline, citalopram, and moclobemide. None of the antidepressant treatments had any significant effect on phosducin-like protein levels. Phosducin-like protein levels were evaluated in mononuclear leukocytes from a group of 15 patients diagnosed with major depressive episode, both before the initiation of antidepressant treatment and after 4 weeks of antidepressant medication. No protein changes were found in leukocytes of either untreated patients with major depressive disorder or after 4 weeks of the treatment in comparison with healthy volunteers.