Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin.

Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT4 (5-HT4R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT4R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT4R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections.

Establishment of a Highly Sensitive Assay for Detection of Hepatitis E Virus-Specific Immunoglobulins.

Hepatitis E, a liver disease caused by infection with the hepatitis E virus (HEV), is a worldwide emerging disease. The diagnosis is based on the detection of viral RNA and of HEV-specific immunoglobulins (Ig). For the latter, various assays are commercially available but still lack harmonization. In this study, a Luminex-based multiplex serological assay was established that measures the presence of total IgG, IgA, and IgM antibodies, targeting a short peptide derived from the viral E2 protein. For the validation, 160 serum samples with a known HEV serostatus were used to determine the assay cutoff and accuracy. Thereby, HEV IgG- and RNA-positive sera were identified with a sensitivity of 100% and a specificity of 98% (95% confidence interval [CI], 94% to 100%). Application of the assay by retesting 514 serum samples previously characterized with different HEV-IgG or total antibody tests revealed a high level of agreement between the assays (Cohen's kappa, 0.58 to 0.99). The established method is highly sensitive and specific and can be easily implemented in a multiplex format to facilitate rapid differential diagnostics with a few microliters of sample input.

Validation of HAV biomarker 2A for differential diagnostic of hepatitis A infected and vaccinated individuals using multiplex serology.

BACKGROUND: Worldwide about 1.5 million clinical cases of hepatitis A virus (HAV) infections occur every year and increasingly countries are introducing HAV vaccination into the childhood immunization schedule with a single dose instead of the originally licenced two dose regimen. Diagnosis of acute HAV infection is determined serologically by anti-HAV-IgM detection using ELISA. Additionally anti-HAV-IgG can become positive during the early phase of symptoms, but remains detectable after infection and also after vaccination against HAV. Currently no serological marker allows the differentiation of HAV vaccinated individuals and those with a past infection with HAV. Such differentiation would greatly improve evaluation of vaccination campaigns and risk assessment of HAV outbreaks. Here we tested the HAV non-structural protein 2A, important for the capsid assembly, as a biomarker for the differentiation of the immune status in previously infected and vaccinated individuals. METHODS: HAV antigens were recombinantly expressed as glutathione-S-transferase (GST) fusion proteins. Using glutathione tagged, magnetic fluorescent beads (Luminex®), the proteins were affinity purified and used in a multiplex serological assay. The multiplex HAV assay was validated using 381 reference sera in which the immune status HAV negative, vaccinated or infected was established using the Abbott ARCHITECT® HAVAb-IgM or IgG, the commercial HAV ELISA from Abnova and documentation in vaccination cards. RESULTS: HAV multiplex serology showed a sensitivity of 99% and specificity of 95% to detect anti-HAV IgG/IgM positive individuals. HAV biomarker 2A allowed the differentiation between previously infected and vaccinated individuals. HAV vaccinated individuals and previously infected individuals could be identified with 92% accuracy. CONCLUSION: HAV biomarker 2A can be used to differentiate between previously HAV-vaccinated and naturally infected individuals. Within a multiplex serological approach this assay can provide valuable novel information in the context of outbreak investigations, longitudinal population based studies and evaluations of immunization campaigns.

Synaptic Remodeling Depends on Signaling between Serotonin Receptors and the Extracellular Matrix.

Rewiring of synaptic circuitry pertinent to memory formation has been associated with morphological changes in dendritic spines and with extracellular matrix (ECM) remodeling. Here, we mechanistically link these processes by uncovering a signaling pathway involving the serotonin 5-HT7 receptor (5-HT7R), matrix metalloproteinase 9 (MMP-9), the hyaluronan receptor CD44, and the small GTPase Cdc42. We highlight a physical interaction between 5-HT7R and CD44 (identified as an MMP-9 substrate in neurons) and find that 5-HT7R stimulation increases local MMP-9 activity, triggering dendritic spine remodeling, synaptic pruning, and impairment of long-term potentiation (LTP). The underlying molecular machinery involves 5-HT7R-mediated activation of MMP-9, which leads to CD44 cleavage followed by Cdc42 activation. One important physiological consequence of this interaction includes an increase in neuronal outgrowth and elongation of dendritic spines, which might have a positive effect on complex neuronal processes (e.g., reversal learning and neuronal regeneration).