Tetraspanin heterogeneity of small extracellular vesicles in human biofluids and brain tissue.

Extracellular vesicles (EVs) are particles released from most cell types delimited by a lipid bilayer. Small EVs (sEVs) are nanosized (<200 nm) and include exosomes. Brain-derived sEVs may provide a source for new biomarkers of brain status. CD9, CD63, and CD81 are major members of the tetraspanin family frequently used as sEV markers. However, according to a recent report, tetraspanins were not equally expressed in all sEVs, but rather show heterogeneity that reflects the expression levels in their secretory cells. We therefore investigated tetraspanin heterogeneity of sEVs in biofluids commonly used for clinical laboratory tests, and those in the brain. Expression levels and distributions of CD9, CD63 and CD81 on sEVs were determined in serum, plasma, and cerebrospinal fluid (CSF) samples collected from each healthy donor, and in post-mortem brain tissue samples. We found heterogeneous mixes of sEVs with various tetraspanin combinations among sEVs, and the predominant types and heterogeneous patterns of tetraspanins were specific to sample type. Hierarchical clustering revealed that brain sEVs were similar to those in the CSF, but different from those in peripheral blood. Our findings both provide basic information and contribute to the development of biomarkers for neurological and psychiatric disorders.

Lysophosphatidic acid induces thrombospondin-1 production in primary cultured rat cortical astrocytes.

Lysophosphatidic acid (LPA), a brain membrane-derived lipid mediator, plays important roles including neural development, function, and behavior. In the present study, the effects of LPA on astrocyte-derived synaptogenesis factor thrombospondins (TSPs) production were examined by real-time PCR and western blotting, and the mechanism underlying this event was examined by pharmacological approaches in primary cultured rat cortical astrocytes. Treatment of astrocytes with LPA increased TSP-1 mRNA, and TSP-2 mRNA, but not TSP-4 mRNA expression. TSP-1 protein expression and release were also increased by LPA. LPA-induced TSP-1 production were inhibited by AM966 a LPA1 receptor antagonist, and Ki16425, LPA1/3 receptors antagonist, but not by H2L5146303, LPA2 receptor antagonist. Pertussis toxin, Gi/o inhibitor, but not YM-254890, Gq inhibitor, and NF499, Gs inhibitor, inhibited LPA-induced TSP-1 production, indicating that LPA increases TSP-1 production through Gi/o-coupled LPA1 and LPA3 receptors. LPA treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). LPA-induced TSP-1 mRNA expression was inhibited by U0126, MAPK/ERK kinase (MEK) inhibitor, but not SB202190, p38 MAPK inhibitor, or SP600125, JNK inhibitor. However, LPA-induced TSP-1 protein expression was diminished with inhibition of all three MAPKs, indicating that these signaling molecules are involved in TSP-1 protein production. Treatment with antidepressants, which bind to astrocytic LPA1 receptors, increased TSP-1 mRNA and protein production. The current findings show that LPA/LPA1/3 receptors signaling increases TSP-1 production in astrocytes, which could be important in the pathogenesis of affective disorders and could potentially be a target for the treatment of affective disorders.

Reduced Cerebrospinal Fluid Levels of Lysophosphatidic Acid Docosahexaenoic Acid in Patients With Major Depressive Disorder and Schizophrenia.

BACKGROUND: Lysophosphatidic acid (LPA) is involved in numerous biological processes, including neurodevelopment, chronic inflammation, and immunologic response in the central nervous system. Autotaxin (ATX) is a secreted enzyme that produces LPA from lysophosphatidylcholine (LPC). Previous studies have demonstrated decreased protein levels of ATX in cerebrospinal fluid (CSF) of patients with major depressive disorder (MDD). Based on those studies, the current study investigated the levels of lysophospholipids species including LPA and related metabolic enzymes, in CSF of patients with MDD and schizophrenia (SCZ). METHODS: The levels of lysophospholipids species and related metabolic enzymes were measured with either liquid chromatography-tandem mass spectrometry or enzyme-linked immunosorbent assay. Japanese patients were diagnosed with DSM-IV-TR. CSF was obtained from age- and sex-matched healthy controls (n = 27) and patients with MDD (n = 26) and SCZ (n = 27). RESULTS: Of all lysophospholipids species, the levels of LPA 22:6 (LPA - docosahexaenoic acid) were significantly lower in patients with MDD and SCZ than in healthy controls. These levels were negatively correlated with several clinical symptomatic scores of MDD, but not those of SCZ. In addition, the levels of LPA 22:6 were significantly correlated with the levels of LPC 22:6 among all 3 groups. On the other hand, the levels of LPA 22:6 were not correlated with ATX activity in patients with MDD and SCZ. CONCLUSION: The lower levels of LPA 22:6 in patients with MDD and SCZ suggest an abnormality of LPA 22:6 metabolism. In addition, several depressive symptoms in patients with MDD were significantly associated with the lower levels of LPA 22:6, suggesting an involvement of LPA 22:6 in the pathophysiology of MDD.

Differential anatomical and cellular expression of lysophosphatidic acid receptor 1 in adult mouse brain.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that acts as an extracellular signaling molecule through six G-protein-coupled receptors: LPA1-LPA6. Recent studies have demonstrated that LPA signaling via LPA1 receptor plays a crucial role in cognition and emotion. However, because of limited availability of reliable antibodies, it is currently difficult to identify the cell types expressing LPA1 receptor in the brain. The current study explored the cellular distribution pattern of LPA1 receptor in the brain using the LPA1 lacZ-knock-in reporter mice. In situ hybridization and immunohistochemistry revealed that LacZ gene expression in these mice reflected the expression of endogenous LPA1 receptor in the brain. Overall, some brain nuclei contained higher levels of LPA1 receptor than others. The majority of LPA1 receptor-expressing cells were Olig2+ oligodendrocytes. In addition, ALDH1l1+ astrocytes and CD31+ vascular endothelial cells also expressed LPA1 receptor. By contrast, NeuN+ neuron and Iba1+ microglia expressed little or no LPA1 receptor. The current neuroanatomical findings will aid in elucidating a role of brain LPA1 receptor, especially those involved in cognition and emotion.

Increased matrix metalloproteinases in cerebrospinal fluids of patients with major depressive disorder and schizophrenia.

BACKGROUND: Chronic inflammation of the brain has a pivotal role in the pathophysiology of major depressive disorder (MDD) and schizophrenia (SCZ). Matrix metalloproteinases (MMPs) are extracellular proteases involved in pro-inflammatory processes and interact with IL-6, which is increased in the cerebrospinal fluid (CSF) of patients with MDD and SCZ. However, MMPs in the CSF in patients with MDD and SCZ remains unclear. Therefore, we compared MMPs in the CSF of patients with MDD and SCZ to those of healthy controls (HC). METHODS: Japanese patients were diagnosed with DSM-IV-TR and clinical symptoms were assessed with the Hamilton Rating Scale for Depression for MDD and the Positive and Negative Syndrome Scale for SCZ. CSF was obtained from MDD (n=90), SCZ (n=86) and from age- and sex-matched HC (n=106). The levels of MMPs in CSF were measured with multiplex bead-based immunoassay. RESULTS: The levels of MMP-2 in CSF were higher in both MDD and SCZ than HC and were positively correlated with clinical symptomatic scores in MDD, but not in SCZ. Regardless of diagnosis, the levels of MMP-2, -7 and -10 were positively correlated with each other, and the levels of MMP-7 and -10 were higher in MDD, but not in SCZ, compared to HC. CONCLUSION: Increased CSF levels of MMP-2 in MDD and SCZ may be associated with brain inflammation. State-dependent alteration of MMP-2 and activation of cascades involving MMP-2, -7, and -10 appeared to have a role in the pathophysiology of MDD.

Reduced Serum and Cerebrospinal Fluid Levels of Autotaxin in Major Depressive Disorder.

BACKGROUND: The autotaxin/lysophosphatidic acid axis is involved in diverse biological processes including neurodevelopment, inflammation, and immunological functioning. The lysophosphatidic acid 1 receptor has been implicated in the pathophysiology of major depressive disorder and in the mechanism of action of antidepressants. However, it is unclear whether central or peripheral autotaxin levels are altered in patients with major depressive disorder. METHODS: Serum autotaxin levels were measured by an enzyme-linked immunosorbent assay in 37 patients with major depressive disorder diagnosed using DSM-IV-TR who underwent electroconvulsive therapy and were compared with those of 47 nondepressed controls matched for age and sex between January 2011 and December 2015. Patient serum levels of autotaxin before and after electroconvulsive therapy were also compared. In a separate sample set, cerebrospinal fluid autotaxin levels were compared between 26 patients with major depressive disorder and 27 nondepressed controls between December 2010 and December 2015. A potential association was examined between autotaxin levels and clinical symptoms assessed with the Hamilton Depression Rating Scale. RESULTS: Before electroconvulsive therapy, both serum and cerebrospinal fluidautotaxin levels were significantly lower in major depressive disorder patients than in controls (serum: P = .001, cerebrospinal fluid: P = .038). A significantly negative correlation between serum, but not cerebrospinal fluid, autotaxin levels and depressive symptoms was observed (P = .032). After electroconvulsive therapy, a parallel increase in serum autotaxin levels and depressive symptoms improvement was observed (P = .005). CONCLUSION: The current results suggest that serum autotaxin levels are reduced in a state-dependent manner. The reduction of cerebrospinal fluidautotaxin levels suggests a dysfunction in the autotaxin/lysophosphatidic acid axis in the brains of patients with major depressive disorder.

Shared preventive factors associated with relapse after a response to electroconvulsive therapy in four major psychiatric disorders.

AIM: The efficacy of electroconvulsive therapy (ECT) has been established in psychiatric disorders but the high rate of relapse is a critical problem. The current study sought preventative factors associated with relapse after a response to ECT in a continuum of four major psychiatric disorders. METHODS: The records of 255 patients with four psychiatric disorders (83 unipolar depression, 60 bipolar depression, 91 schizophrenia, 21 schizoaffective disorder) were retrospectively reviewed. RESULTS: The relapse-free rate of all patients at 1 year was 56.3% in the four psychiatric disorders without a difference. As a result of univariate analysis, three items could be considered as preventative factors associated with relapse: a small number of psychiatric symptom episodes before an acute course of ECT, the use of mood stabilizers, and the use of maintenance ECT. Multivariate analysis was performed, keeping age, sex, and diagnosis constant in addition to the three items, and small number of psychiatric symptom episodes before an acute course of ECT (P = 0.003), the use of lithium (P = 0.025), the use of valproate (P = 0.027), and the use of maintenance ECT (P = 0.001) were found to be significant preventative measures against relapse. CONCLUSION: The use of mood stabilizers, such as lithium and valproate, and maintenance ECT could be shared preventive factors associated with relapse after a response to ECT in four major psychiatric disorders.

Possible Association between Serum Matrix Metalloproteinase-9 (MMP-9) Levels and Relapse in Depressed Patients following Electroconvulsive Therapy (ECT).

Background: Matrix metalloproteinases are involved in neuroinflammatory processes, which could underlie depression. Serum levels of MMP-9 and MMP-2 in depressed patients are significantly altered following electroconvulsive therapy, but an association between altered matrix metalloproteinases after successful ECT and possible relapse has yet to be investigated. Methods: Serum was obtained twice, before and immediately after a course of electroconvulsive therapy, from 38 depressed patients. Serum was also collected, once, from two groups of age- and gender-matched healthy controls, 40 volunteers in each group. Possible associations between levels of matrix metalloproteinases and relapse during a 1-year follow-up period were analyzed. Results: Excluding patients who did not respond to electroconvulsive therapy and patients lost to follow-up, data from 28 patients were evaluated. Eighteen of the patients (64.3%) relapsed within 1 year. In the group that did not relapse, serum levels of MMP-9 were significantly decreased after a course of electroconvulsive therapy, but not in the group that relapsed. No association between MMP-2 and relapse was observed. Conclusion: The degree of change in serum MMP-9 change could be associated with relapse following electroconvulsive therapy in depressed patients.

Identification of Lysophosphatidic Acid Receptor 1 in Astroglial Cells as a Target for Glial Cell Line-derived Neurotrophic Factor Expression Induced by Antidepressants.

Preclinical and clinical evidence suggests that glial cell line-derived neurotrophic factor (GDNF) is important in the therapeutic effect of antidepressants. A previous study demonstrated that the tricyclic antidepressant amitriptyline induces Gαi/o activation, which leads to GDNF expression in astrocytes. However, the specific target expressed in astrocytes that mediates antidepressant-evoked Gαi/o activation has yet to be identified. Thus, the current study explored the possibility that antidepressant-induced Gαi/o activation depends on lysophosphatidic acid receptor 1 (LPAR1), a Gαi/o-coupled receptor. GDNF mRNA expression was examined using real-time PCR and Gαi/o activation was examined using the cell-based receptor assay system CellKeyTM in rat C6 astroglial cells and rat primary cultured astrocytes. LPAR1 antagonists blocked GDNF mRNA expression and Gαi/o activation evoked by various classes of antidepressants (amitriptyline, nortriptyline, mianserin, and fluoxetine). In addition, deletion of LPAR1 by RNAi suppressed amitriptyline-evoked GDNF mRNA expression. Treatment of astroglial cells with the endogenous LPAR agonist LPA increased GDNF mRNA expression through LPAR1, whereas treatment of primary cultured neurons with LPA failed to affect GDNF mRNA expression. Astrocytic GDNF expression evoked by either amitriptyline or LPA utilized, in part, transactivation of fibroblast growth factor receptor and a subsequent ERK cascade. The current results suggest that LPAR1 is a novel, specific target of antidepressants that leads to GDNF expression in astrocytes.

Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells.

Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα(i/o) inhibitor, but not by NF449, a Gα(s) inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα(ο1) and Gα(i3) by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey(TM) assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα(i/o) activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα(i/o) upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants.

The expression of glial cell line-derived neurotrophic factor mRNA by antidepressants involves matrix metalloproteinase-9 activation in rat astroglial cells.

Neurotrophic/growth factors derived from glial cells, especially astrocytes, have been implicated in mood disorders and the pharmacological effects of antidepressant drugs. Previous studies demonstrated that the release of glial cell line-derived neurotrophic factor (GDNF) induced by the tricyclic antidepressant amitriptyline was significantly inhibited by a broad-spectrum matrix metalloproteinase (MMP) inhibitor in rat C6 astroglial cells (C6 cells). However, it is unknown whether amitriptyline affects MMP enzymatic activity or expression, and the MMP subtype has yet to be identified. The current study measured the effect of antidepressants on MMP activity with gelatin zymography, an in vitro assay for enzymatic activity, in C6 cells and primary cultured rat astrocytes (primary astrocytes). Treatment with amitriptyline increased zymographic MMP-9 activity without changing MMP-9 mRNA expression in C6 cells. Several different classes of antidepressants significantly increased zymographic MMP-9 activity in C6 cells and primary astrocytes, whereas antipsychotic drugs without antidepressant pharmacological activity did not. The amitriptyline-induced expression of GDNF mRNA was completely blocked by selective inhibition of MMP-9 in C6 cells. Treatment of C6 cells and primary astrocytes with exogenous recombinant MMP-9 increased GDNF mRNA expression, similar to that observed with amitriptyline. Inhibiting MMP-3 blocked amitriptyline-induced zymographic MMP-9 activation in C6 cells and primary astrocytes, indicating that MMP-3 is necessary for MMP-9 activity. The current study suggests that MMP-9 activation is indispensable in the amitriptyline-induced expression of GDNF mRNA in astrocytes and further supports a role of astrocytic neurotrophic/growth factors in the pharmacological effect of antidepressants.

Altered Serum Levels of Matrix Metalloproteinase-2, -9 in Response to Electroconvulsive Therapy for Mood Disorders.

BACKGROUND: Inflammatory processes could underlie mood disorders. Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMP) are inflammation-related molecules. The current study sought an association between mood disorders and systemic levels of MMPs and TIMPs. METHODS: Serum was obtained from patients with mood disorders (n=21) and patients with schizophrenia (n=13) scheduled to undergo electroconvulsive therapy. Serum was also obtained from healthy controls (n=40). Clinical symptoms were assessed by the Hamilton Rating Score for Depression and the Brief Psychiatric Rating Scale. Serum levels of MMPs and TIMPs were quantified by ELISA. RESULTS: The serum levels of MMP-2 in mood disorder patients, but not in schizophrenia patients, prior to the first electroconvulsive therapy session (baseline) was significantly lower than that of healthy controls. At baseline, levels of MMP-9 and TIMP-2, -1 were not different between patients with mood disorder and schizophrenia and healthy controls. After a course of electroconvulsive therapy, MMP-2 levels were significantly increased in mood disorder patients, but MMP-9 levels were significantly decreased in both mood disorder and schizophrenia patients. In mood disorder patients, there was a significant negative correlation between depressive symptoms and serum levels of MMP-2 and a positive correlation between depressive symptoms and MMP-9. In addition, alterations of serum levels of MMP-2 and MMP-9 were significantly correlated each other and were associated with certain depressive symptoms. CONCLUSION: A change in inflammatory homeostasis, as indicated by MMP-2 and MMP-9, could be related to mood disorders, and these markers appear to be sensitive to electroconvulsive therapy.

Fibroblast growth factor 2 mRNA expression evoked by amitriptyline involves extracellular signal-regulated kinase-dependent early growth response 1 production in rat primary cultured astrocytes.

Recently, we demonstrated that several antidepressants including amitriptyline increased fibroblast growth factor 2 (FGF2) mRNA expression slowly over 24 h through de novo protein synthesis in rat primary cultured astrocytes. This study defined the signaling cascade that mediates amitriptyline-induced FGF2 production in rat primary cultured astrocytes. Amitriptyline treatment significantly increased early growth response 1 (EGR1), a transcription factor known to regulate FGF2 expression. Knockdown of EGR1 using siRNA blocked amitriptyline-evoked FGF2 mRNA expression. Treatment with several different classes of antidepressants leads to expression of EGR1 mRNA as well as FGF2 mRNA. These results confirm that EGR1 production is likely to be indispensable for the amitriptyline-evoked FGF2 mRNA expression. Signal transduction inhibitors were used to elaborate the cellular signaling cascade that leads to EGR1-mediated FGF2 expression following amitriptyline treatment. Amitriptyline-evoked EGR1-mediated FGF2 mRNA expression was blocked by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1/2 inhibitor. Furthermore, extracellular signal-regulated kinase/EGR1-mediated FGF2 mRNA expression evoked by amitriptyline was blocked by inhibitors of the FGF receptor, epidermal growth factor receptor (EGFR), and matrix metalloproteinase. Taken together, these results demonstrate that amitriptyline increases FGF2 mRNA expression through a matrix metalloproteinase/receptor tyrosine kinases (RTK) (FGF receptor and EGFR)/extracellular signal-regulated kinase/EGR1 signaling pathway in rat primary cultured astrocytes. Recent studies suggest that fibroblast growth factor 2 (FGF2) is involved in the antidepressant effect in the model of depression. The production of ERK-dependent early growth response 1 (EGR1) is a crucial part of the amitriptyline-induced FGF2 expression signaling cascade in rat primary cultured astrocytes. The findings elaborate an astrocytic mechanism that could be used to develop antidepressants.

Identification of the antidepressant effect of electroconvulsive stimulation-related genes in hippocampal astrocyte.

Major depressive disorder (MDD) remains a significant global health concern, with limited and slow efficacy of existing antidepressants. Electroconvulsive therapy (ECT) has superior and immediate efficacy for MDD, but its action mechanism remains elusive. Therefore, the elucidation of the action mechanism of ECT is expected to lead to the development of novel antidepressants with superior and immediate efficacy. Recent studies suggest a potential role of hippocampal astrocyte in MDD and ECT. Hence, we investigated antidepressant effect of electroconvulsive stimulation (ECS), an animal model of ECT, -related genes in hippocampal astrocyte with a mouse model of MDD, in which corticosterone (CORT)-induced depression-like behaviors were recovered by ECS. In this model, both of CORT-induced depression-like behaviors and the reduction of hippocampal astrocyte were recovered by ECS. Following it, astrocytes were isolated from the hippocampus of this model and RNA-seq was performed with these isolated astrocytes. Interestingly, gene expression patterns altered by CORT were reversed by ECS. Additionally, cell proliferation-related signaling pathways were inhibited by CORT and recovered by ECS. Finally, serum and glucocorticoid kinase-1 (SGK1), a multi-functional protein kinase, was identified as a candidate gene reciprocally regulated by CORT and ECS in hippocampal astrocyte. Our findings suggest a potential role of SGK1 in the antidepressant effect of ECS via the regulation of the proliferation of astrocyte and provide new insights into the involvement of hippocampal astrocyte in MDD and ECT. Targeting SGK1 may offer a novel approach to the development of new antidepressants which can replicate superior and immediate efficacy of ECT.

TCF7L2: A potential key regulator of antidepressant effects on hippocampal astrocytes in depression model mice.

Clinical and preclinical studies suggest that hippocampal astrocyte dysfunction is involved in the pathophysiology of depression; however, the underlying molecular mechanisms remain unclear. Here, we attempted to identify the hippocampal astrocytic transcripts associated with antidepressant effects in a mouse model of depression. We used a chronic corticosterone-induced mouse model of depression to assess the behavioral effects of amitriptyline, a tricyclic antidepressant. Hippocampal astrocytes were isolated using fluorescence-activated cell sorting, and RNA sequencing was performed to evaluate the transcriptional profiles associated with depressive effects and antidepressant responses. Depression model mice exhibited typical depression-like behaviors that improved after amitriptyline treatment; the depression group mice also had significantly reduced GFAP-positive astrocyte numbers in hippocampal subfields. Comprehensive transcriptome analysis of hippocampal astrocytes showed opposing responses to amitriptyline in depression group and control mice, suggesting the importance of using the depression model. Transcription factor 7 like 2 (TCF7L2) was the only upstream regulator gene altered in depression model mice and restored in amitriptyline-treated depression model mice. In fact, TCF7L2 expression was significantly decreased in the depression group. The level of TCF7L2 long non-coding RNA, which controls mRNA expression of the TCF7L2 gene, was also significantly decreased in this group and recovered after amitriptyline treatment. The Gene Ontology biological processes associated with astrocytic TCF7L2 included proliferation, differentiation, and cytokine production. We identified TCF7L2 as a gene associated with depression- and antidepressant-like behaviors in response to amitriptyline in hippocampal astrocytes. Our findings could provide valuable insights into the mechanism of astrocyte-mediated antidepressant effects.

G protein-biased LPAR1 agonism of prototypic antidepressants: Implication in the identification of novel therapeutic target for depression.

Prototypic antidepressants, such as tricyclic/tetracyclic antidepressants (TCAs), have multiple pharmacological properties and have been considered to be more effective than newer antidepressants, such as selective serotonin reuptake inhibitors, in treating severe depression. However, the clinical contribution of non-monoaminergic effects of TCAs remains elusive. In this study, we discovered that amitriptyline, a typical TCA, directly binds to the lysophosphatidic acid receptor 1 (LPAR1), a G protein-coupled receptor, and activates downstream G protein signaling, while exerting a little effect on ß-arrestin recruitment. This suggests that amitriptyline acts as a G protein-biased agonist of LPAR1. This biased agonism was specific to TCAs and was not observed with other antidepressants. LPAR1 was found to be involved in the behavioral effects of amitriptyline. Notably, long-term infusion of mouse hippocampus with the potent G protein-biased LPAR agonist OMPT, but not the non-biased agonist LPA, induced antidepressant-like behavior, indicating that G protein-biased agonism might be necessary for the antidepressant-like effects. Furthermore, RNA-seq analysis revealed that LPA and OMPT have opposite patterns of gene expression changes in the hippocampus. Pathway analysis indicated that long-term treatment with OMPT activated LPAR1 downstream signaling (Rho and MAPK), whereas LPA suppressed LPAR1 signaling. Our findings provide insights into the mechanisms underlying the non-monoaminergic antidepressant effects of TCAs and identify the G protein-biased agonism of LPAR1 as a promising target for the development of novel antidepressants.

Serum levels of high mobility group box-1 protein (HMGB1) and soluble receptors of advanced glycation end-products (RAGE) in depressed patients treated with electroconvulsive therapy.

AIMS: High mobility group box-1 (HMGB1) is one of the damage-associated molecular patterns produced by stress and induces inflammatory responses mediated by receptors of advanced glycation end-products (RAGE) on the cell surface. Meanwhile, soluble RAGE (sRAGE) exhibits an anti-inflammatory effect by capturing HMGB1. Animal models have shown upregulation of HMGB1 and RAGE in the brain or blood, suggesting the involvement of these proteins in depression pathophysiology. However, there have been no reports using blood from depressed patients, nor ones focusing on HMGB1 and sRAGE changes associated with treatment and their relationship to depressive symptoms. METHODS: Serum HMGB1 and sRAGE concentrations were measured by enzyme-linked immunosorbent assay in a group of patients with severe major depressive disorder (MDD) (11 males and 14 females) who required treatment with electroconvulsive therapy (ECT), and also in a group of 25 age- and gender-matched healthy subjects. HMGB1 and sRAGE concentrations were also measured before and after a course of ECT. Depressive symptoms were assessed using the Hamilton Rating Scale for Depression (HAMD). RESULTS: There was no significant difference in HMGB1 and sRAGE concentrations in the MDD group compared to healthy subjects. Although ECT significantly improved depressive symptoms, there was no significant change in HMGB1 and sRAGE concentrations before and after treatment. There was also no significant correlation between HMGB1 and sRAGE concentrations and the HAMD total score or subitem scores. CONCLUSION: There were no changes in HMGB1 and sRAGE in the peripheral blood of severely depressed patients, and concentrations had no relationship with symptoms or ECT.

A machine learning-based scoring system and ten factors associated with hip fracture occurrence in the elderly.

Hip fractures are fragility fractures frequently seen in persons over 80-years-old. Although various factors, including decreased bone mineral density and a history of falls, are reported as hip fracture risks, few large-scale studies have confirmed their relevance to individuals older than 80, and tools to assess contributions of various risks to fracture development and the degree of risk are lacking. We recruited 1395 fresh hip fracture patients and 1075 controls without hip fractures and comprehensively evaluated various reported risk factors and their association with hip fracture development. We initially constructed a predictive model using Extreme Gradient Boosting (XGBoost), a machine learning algorithm, incorporating all 40 variables and evaluated the model's performance using the area under the receiver operating characteristic curve (AUC), yielding a value of 0.87. We also employed SHapley Additive exPlanation (SHAP) values to evaluate each feature importance and ranked the top 20. We then used a stepwise selection method to determine key factors sequentially until the AUC reached a plateau nearly equal to that of all variables and identified the top 10 sufficient to evaluate hip fracture risk. For each, we determined the cutoff value for hip fracture occurrence and calculated scores of each variable based on the respective feature importance. Individual scores were: serum 25(OH)D levels (<10 ng/ml, score 7), femoral neck T-score (<-3, score 5), Barthel index score (<100, score 3), maximal handgrip strength (<18 kg, score 3), GLFS-25 score (≥24, score 2), number of falls in previous 12 months (≥3, score 2), serum IGF-1 levels (<50 ng/ml, score 2), cups of tea/day (≥5, score -2), use of anti-osteoporosis drugs (yes, score -2), and BMI (<18.5 kg/m2, score 1). Using these scores, we performed receiver operating characteristic (ROC) analysis and the resultant optimal cutoff value was 7, with a specificity of 0.78, sensitivity of 0.75, and AUC of 0.85. These ten factors and the scoring system may represent tools useful to predict hip fracture.

Tricyclic antidepressant amitriptyline activates fibroblast growth factor receptor signaling in glial cells: involvement in glial cell line-derived neurotrophic factor production.

Recently, both clinical and animal studies demonstrated neuronal and glial plasticity to be important for the therapeutic action of antidepressants. Antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production through monoamine-independent protein-tyrosine kinase, extracellular signal-regulated kinase (ERK), and cAMP responsive element-binding protein (CREB) activation in glial cells (Hisaoka, K., Takebayashi, M., Tsuchioka, M., Maeda, N., Nakata, Y., and Yamawaki, S. (2007) J. Pharmacol. Exp. Ther. 321, 148-157; Hisaoka, K., Maeda, N., Tsuchioka, M., and Takebayashi, M. (2008) Brain Res. 1196, 53-58). This study clarifies the type of tyrosine kinase and mechanism of antidepressant-induced GDNF production in C6 glioma cells and normal human astrocytes. The amitriptyline (a tricyclic antidepressant)-induced ERK activation was specifically and completely inhibited by fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors and siRNA for FGFR1 and -2. Treatment with amitriptyline or several different classes of antidepressants, but not non-antidepressants, acutely increased the phosphorylation of FGFRs and FGFR substrate 2α (FRS2α). Amitriptyline-induced CREB phosphorylation and GDNF production were blocked by FGFR-tyrosine kinase inhibitors. Therefore, antidepressants activate the FGFR/FRS2α/ERK/CREB signaling cascade, thus resulting in GDNF production. Furthermore, we attempted to elucidate how antidepressants activate FGFR signaling. The effect of amitriptyline was inhibited by heparin, non-permeant FGF-2 neutralizing antibodies, and matrix metalloproteinase (MMP) inhibitors. Serotonin (5-HT) also increased GDNF production through FGFR2 (Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., and Nakata, Y. (2008) J. Neurochem. 106, 244-257); however, the effect of 5-HT was not inhibited by heparin and MMP inhibitors. These results suggest that amitriptyline-induced FGFR activation might occur through an extracellular pathway, in contrast to that of 5-HT. The current data show that amitriptyline-induced FGFR activation might occur by the MMP-dependent shedding of FGFR ligands, such as FGF-2, thus resulting in GDNF production.

Decreased serum levels of thrombospondin-1 in female depressed patients.

AIM: Thrombospondin-1 (TSP-1) is an astrocyte-derived synaptogenesis-related factor. It was previously reported to be increased by chronic treatment of electroconvulsive seizure, a model of electroconvulsive therapy (ECT), in rat hippocampus. The aim of this study was to examine whether serum levels of TSP-1 are associated with depression and ECT. METHODS: Serum TSP-1 levels of major depressive disorder (MDD) patients (n = 36) and age- and gender-matched healthy controls (n = 36) were measured by TSP-1 ELISA. MDD patients were diagnosed according to the Diagnostics and Statistical Manual of Mental Disorders-IV-TR and underwent ECT. MDD patients were also analyzed for serum TSP-1 levels pre- and post-ECT. Evaluation of symptoms was obtained using the Hamilton Rating Scale for Depression. RESULTS: Serum TSP-1 levels showed significant decreases specific to female MDD patients. However, TSP-1 did not change pre- and post-ECT, did not correlate with symptoms, nor was not affected by the dose of antidepressants. CONCLUSION: Serum TSP-1 is a possible female-specific factor that reflects depressive trait, but not state.