Immunoglobulin A Autoreactivity toward Brain Enriched and Apoptosis-Regulating Proteins in Saliva of Athletes after Acute Concussion and Subconcussive Impacts.

The diagnosis and management of concussion is hindered by its diverse clinical presentation and assessment tools reliant on subjectively experienced symptoms. The biomechanical threshold of concussion is also not well understood, and asymptomatic concussion or "subconcussive impacts" of variable magnitudes are common in contact sports. Concerns have risen because athletes returning to activity too soon have an increased risk of prolonged recovery or long-term adverse health consequences. To date, little is understood on a molecular level regarding concussion and subconcussive impacts. Recent research suggests that neuroinflammatory mechanisms may serve an important role subsequent to concussion and possibly to subconcussive impacts. These studies suggest that autoantibodies may be a valuable tool for detection of acute concussion and monitoring for changes caused by cumulative exposure to subconcussive impacts. Hence, we aimed to profile the immunoglobulin (Ig)A autoantibody repertoire in saliva by screening a unique sport-related head trauma biobank. Saliva samples (n = 167) were donated by male and female participants enrolled in either the concussion (24-48 h post-injury) or subconcussion (non-concussed participants having moderate or high cumulative subconcussive impact exposure) cohorts. Study design included discovery and verification phases. Discovery aimed to identify new candidate autoimmune targets of IgA. Verification tested whether concussion and subconcussion cohorts increased IgA reactivity and whether cohorts showed similarities. The results show a significant increase in the prevalence of IgA toward protein fragments representing 5-hydroxytryptamine receptor 1A (HTR1A), serine/arginine repetitive matrix 4 (SRRM4) and FAS (tumor necrosis factor receptor superfamily member 6) after concussion and subconcussion. These results may suggest that concussion and subconcussion induce similar physiological effects, especially in terms of immune response. Our study demonstrates that saliva is a potential biofluid for autoantibody detection in concussion and subconcussion. After rigorous confirmation in much larger independent study sets, a validated salivary autoantibody assay could provide a non-subjective quantitative means of assessing concussive and subconcussive events.

Exploring Endocytosis and Intracellular Trafficking of the Human Serotonin1A Receptor.

G protein-coupled receptors (GPCRs) represent the largest class of receptors involved in signal transduction across cell membranes and are major drug targets in all clinical areas. Endocytosis of GPCRs offers a regulatory mechanism for sustaining their signaling within a stringent spatiotemporal regime. In this work, we explored agonist-induced endocytosis of the human serotonin1A receptor stably expressed in HEK-293 cells and the cellular machinery involved in receptor internalization and intracellular trafficking. The serotonin1A receptor is a popular GPCR implicated in neuropsychiatric disorders such as anxiety and depression and serves as an important drug target. In spite of its pharmacological relevance, its mechanism of endocytosis and intracellular trafficking is less understood. In this context, we have utilized a combination of robust population-based flow cytometric analysis and confocal microscopic imaging to address the path and fate of the serotonin1A receptor during endocytosis. Our results, utilizing inhibitors of specific endocytosis pathways and intracellular markers, show that the serotonin1A receptor undergoes endocytosis predominantly via the clathrin-mediated pathway and subsequently recycles to the plasma membrane via recycling endosomes. These results would enhance our understanding of molecular mechanisms of GPCR endocytosis and could offer novel insight into the underlying mechanism of antidepressants that act via the serotonergic pathway. In addition, our results could be relevant in understanding cell (or tissue)-specific GPCR endocytosis.

Neurological impairment in experimental antiphospholipid syndrome is associated with increased ligand binding to hippocampal and cortical serotonergic 5-HT1A receptors.

The antiphospholipid syndrome (APS) is an autoimmune disease where the presence of high titers of circulating autoantibodies causes thrombosis with consecutive infarcts. In experimental APS (eAPS), a mouse model of APS, behavioral abnormalities develop in the absence of vessel occlusion or infarcts. Using brain hemispheres of control and eAPS mice with documented neurological and cognitive deficits, we checked for lymphocytic infiltration, activation of glia and macrophages, as well as alterations of ligand binding densities of various neurotransmitter receptors to unravel the molecular basis of this abnormal behavior. Lymphocytic infiltrates were immunohistochemically characterized using antibodies against CD3, CD4, CD8 and forkhead box P3 (Foxp3), respectively. GFAP, Iba1 and CD68-immunohistochemistry was performed, to check for activation of astrocytes, microglia and macrophages. Ligand binding densities of NMDA, AMPA, GABAA and 5-HT1A receptors were analyzed by in vitro receptor autoradiography. No significant inflammatory reaction occurred in eAPS mice. There was neither activation of astrocytes or microglia nor accumulation of macrophages. Binding values of excitatory and inhibitory neurotransmitter receptors were largely unchanged. However, ligand binding densities of the modulatory serotonergic 5-HT1A receptors in the hippocampus and in the primary somatosensory cortex of eAPS mice were significantly upregulated which is suggested to induce the behavioral abnormalities observed.

[Effect of activation of serotonin 5-HT1A-receptors on the immune response in mice of the ASC strain with depressive-like behavior].

Selective activation of serotonin 5-HT(1A)-receptors produced different effects on immunological reactivity in mice of ASC strain with genetic predisposition to depressive-like behavior, and parental CBA and AKR strains displaying no depressive reactions. Administration of 5-HT(1A)-receptors agonist 8-OH-DPAT at low dose (0.1 mg/kg) affecting upon presynaptic receptors resulted in immunostimulation in CBA mice and did not change the immune response level in mice of ASC strain. Activation of postsynaptic 5-HT(1A)-receptors with higher dose of 8-OH-DPAT (1.0 mg/kg) caused immunosuppression in CBA and AKR strains while under the same conditions the immune response of ASC mice was increased. Decrease the immune reactions in ASC mice was observed only after application of 8-OHDPAT at dose of 5 mg/kg. The changes of functional activity of pre- and postsynaptic 5-HT(1A)-receptors under a high predisposition to depressive-like behavior providing different effects of this receptor activation on immune function are discussed.

Serotonin and its 5-HT1 receptor in human mastocytosis.

CONTEXT: Human mastocytosis is a rare disease, in which the serotonergic system may be involved. OBJECTIVE: The objective of the present study was to examine the possible presence of serotonin (5-HT) and its 5-HT1A receptor (R) in the skin of patients with mastocytosis. In addition, the effect of the 5-HT1AR was tested on human mastocytosis cells, cultured in vitro. MATERIALS AND METHODS: The expression of 5-HT and 5-HT1AR in patients with urticaria pigmentosa and mastocytoma was studied using immunohistochemistry. The effects of 8-OH-DPAT, an agonist of 5-HT1AR, on the proliferation (cell number), viability, apoptosis, spontaneous release of histamine, as well as a possible 5-HT metabolism, in the human HMC-1 mast cell line, were investigated. RESULTS: Both 5-HT and 5-HT1AR were expressed in the mast cells in biopsies of mastocytoma and urticaria pigmentosa, as well as in HMC-1 cells. However, no metabolism of 5-HT by the cell line could be detected by the methodology used. The 5-HT1AR agonist had no significant effect on the viability and number of HMC-1 cells, and was without effect on the apoptosis. At concentrations of 10⁻6 mol/L and 10⁻8-10⁻¹° mol/L (i.e. also at physiological concentrations), the agonist inhibited histamine release by these cells by as much as 30%. CONCLUSION: These findings indicate that 5-HT and its 5-HT1AR are expressed in human mastocytosis and that an agonist of the 5-HT1AR might be of value in the treatment of these patients.

Generation and characterization of a specific polyclonal antibody against the mouse serotonin receptor 1A: a state-of-the-art recommendation on how to characterize antibody specificity.

A series of different antibodies against serotonin receptor 1A (5HT1A_R) have been reported although only limited information on the specificity of these antibodies and the antigens recognized is available. Herein, we characterized reactivity of an antibody by a gel-based proteomics method that should represent a model how antibodies may be defined in the future. An antibody against the 5HT1A_R was generated, used for immunoprecipitation and immunoblotting on blue-native gels containing a 5HT1A_R complex. The 5HT1A_R was isolated from tissue and was defined by nano-LC-ESI-MS/MS. A single band on the native gel and a single spot representing the denatured receptor in the 3rd dimensional step of gel electrophoresis was detected. Immunoprecipitation revealed a single band for the denatured 5HT1A_R. Herein, a procedure is proposed to characterize an antibody by the use of a robust method unambiguously identifying and characterizing the antigen, 5HT1A_R, from mouse whole brain.

Characterization of the 5-HT1A receptor of the honeybee (Apis mellifera) and involvement of serotonin in phototactic behavior.

Serotonin plays a key role in modulating various physiological and behavioral processes in both protostomes and deuterostomes. The vast majority of serotonin receptors belong to the superfamily of G-protein-coupled receptors. We report the cloning of a cDNA from the honeybee (Am5-ht1A) sharing high similarity with members of the 5-HT(1) receptor class. Activation of Am5-HT(1A) by serotonin inhibited the production of cAMP in a dose-dependent manner (EC(50) = 16.9 nM). Am5-HT(1A) was highly expressed in brain regions known to be involved in visual information processing. Using in vivo pharmacology, we could demonstrate that Am5-HT(1A) receptor ligands had a strong impact on the phototactic behavior of individual bees. The data presented here mark the first comprehensive study-from gene to behavior-of a 5-HT(1A) receptor in the honeybee, paving the way for the eventual elucidation of additional roles of this receptor subtype in the physiology and behavior of this social insect.

[Presynaptic 5-HT1A receptors in immunomodulation].

It is shown that a selective agonist of 5-HT1A receptors 8-OH-DPAT in a low dose (0.1 mg/kg), which is known to affect mainly the presynaptic 5-HT1A receptors increased the immune response at the peak of reactions (the forth or fifth day after immunization with sheep red blood cells - SRBC) in CBA mice and Wistar rats. Treatment of the animals with the drug 15 min prior to antigen injection raised the number of plaque-forming cells (lgM-PFC) and rosette-forming cells (RFC) in the spleen. The preliminary blockade of 5-HT1A receptor with a selective antagonist of 5-HT1A receptors WAY-100635 (0.1 mg/kg) prevented the immunostimulating effect of 5-HT 1A receptors agonist 8-OH-DPAT, whereas WAY-100635 administration alone in the same dose didn't change the immune response. Activation of 5-HT1A receptors under conditions of electrical lesion of 5-HTergic neurons of the nucleus raphe was unable to enhance the immune reactions, as it did in sham-operated rats. The data obtained indicate that the somatodendric 5-HT1A autoreceptors are involved in immunomodulation.

Expression of serotonergic receptors in psoriatic skin.

Psoriasis appears to be influenced by stress, which causes release of adrenal hormones. Serotonin, or hormonal actions on serotonin and serotonin receptors, may have a role in psoriasis. Distribution of serotonin receptors was studied in involved and noninvolved skin in patients with psoriasis and compared to normal skin, by using immunohistochemistry and antibodies to 5-HT1A, 5-HT2A and 5-HT3 receptors (R). There was a decreased (P<0.001) number of 5-HT1AR positive cells, the majority being tryptase positive, in involved and noninvolved psoriatic papillary dermis, compared to normal skin. 5-HTlAR expression was also found in the upper part of the epidermis, on vessel walls and on melanocytes. 5-HT2AR expressing papillary mononuclear cells, CD3 positive, were increased (P<0.001 and P<0.01, respectively) in involved and noninvolved psoriatic skin, compared to normal skin, an increase (P<0.01) also being found in the involved compared to noninvolved skin. Expression of 5-HT3R could be found in the basal epidermal layer of noninvolved but not in the involved skin of psoriasis, where it was only found in the acrosyringium. The present findings are compatible with the 5-HT1A and 5-HT2A receptors having antagonistic functions, and raise the possibility of using receptor specific drugs in the treatment of psoriasis.

[Activation and blocking of 5-HT1A receptors influence on the immune response in CBA mice].

5-HT1A type serotonin receptors influence the immunomodulating action of the selective preparations 8-OH-DPAT (5-HT1A receptor agonist) and WAY-100635 (5-HT1A receptor antagonist) in CBA mice. The activation of 5HT1A receptors with 8-OH-DPAT (1 mg/kg) decreased, while their blocking with WAY-100635 (1 mg/kg) increased the reaction intensity at the peak of response to immunization with ram erythrocytes. Preliminary blocking of the 5-HT1A receptors with WAY-100635 prevented the inhibiting action of 8-OH-DPAT.

Desensitization of 5-HT1A receptors by 5-HT2A receptors in neuroendocrine neurons in vivo.

An imbalance between serotonin-2A (5-HT2A) and 5-HT1A receptors may underlie several mood disorders. The present studies determined whether 5-HT2A receptors interact with 5-HT1A receptors in the rat hypothalamic paraventricular nucleus (PVN). The sensitivity of the hypothalamic 5-HT1A receptors was measured as oxytocin and adrenocorticotropic hormone (ACTH) responses to the 5-HT1A receptor agonist (+)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide [(+)8-OH-DPAT] (40 microg/kg s.c.). The 5-HT(2A/2C) receptor agonist (-)DOI [(-)-1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane HCl] (1 mg/kg s.c.) injected 2 h prior to (+)8-OH-DPAT significantly reduced the oxytocin and ACTH responses to (+)8-OH-DPAT, producing a heterologous desensitization of the 5-HT1A receptors. Microinjection of the 5-HT2A receptor antagonist MDL100,907 [(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol; 0, 10, or 20 nmol, 15 min prior to (-)DOI] into the PVN dose-dependently prevented the desensitization of 5-HT1A receptors induced by the 5-HT2A receptor agonist (-)DOI. Double-label immunocytochemistry revealed a high degree of colocalization of 5-HT1A and 5-HT2A receptors in the oxytocin and corticotropin-releasing factor neurons of the PVN. Thus, activation of 5-HT2A receptors in the PVN may directly induce a heterologous desensitization of 5-HT1A receptors within individual neuroendocrine cells. These findings may provide insight into the long-term adaptation of 5-HT1A receptor signaling after changes in function of 5-HT2A receptors; for example, during pharmacotherapy of mood disorders.

Autoantibodies against four kinds of neurotransmitter receptors in psychiatric disorders.

There is a hypothesis that autoimmune abnormalities in neurotransmitter receptors might cause some psychiatric disorders. Using a sensitive radioligand assay, we detected serum autoantibodies to recombinant human muscarinic cholinergic receptor 1 (CHRM1, 34.4%), mu-opioid receptor (OPRM1, 13.1%), 5-hydroxytryptamine receptor 1A (HTR1A, 7.4%), and dopamine receptor D2 (DRD2, 4.9%) in 122 psychiatric patients. Positive antibodies to CHRM1 were found in 34.1%, 34.9%, 33.3%, and 9.1% of patients with schizophrenic disorders (n=44), mood disorders (n=63), other psychiatric disorders (n=15) and autoimmune diseases (n=33), respectively. All three patients with neuroleptic maliganant syndrome had high activities of autoantibodies to CHRM1, OPRM1, and/or HTR1A. Our data suggest that autoimmunity to neurotransmitter receptors might be associated with the induction of psychiatric symptoms and have some relation to neuroleptic malignant syndrome.