A simple image processing pipeline to sharpen topology maps in multi-wavelength interference microscopy.

Multi-wavelength standing wave (SW) microscopy and interference reflection microscopy (IRM) are powerful techniques that use optical interference to study topographical structure. However, the use of more than two wavelengths to image the complex cell surface results in complicated topographical maps, and it can be difficult to resolve the three-dimensional contours. We present a simple image processing method to reduce the thickness and spacing of antinodal fringes in multi-wavelength interference microscopy by up to a factor of two to produce clearer and more precise topographical maps of cellular structures. We first demonstrate this improvement using model non-biological specimens, and we subsequently demonstrate the benefit of our method for reducing the ambiguity of surface topography and revealing obscured features in live and fixed-cell specimens.

Mesoscale standing wave imaging.

Standing wave (SW) microscopy is a method that uses an interference pattern to excite fluorescence from labelled cellular structures and produces high-resolution images of three-dimensional objects in a two-dimensional dataset. SW microscopy is performed with high-magnification, high-numerical aperture objective lenses, and while this results in high-resolution images, the field of view is very small. Here we report upscaling of this interference imaging method from the microscale to the mesoscale using the Mesolens, which has the unusual combination of a low-magnification and high-numerical aperture. With this method, we produce SW images within a field of view of 4.4 mm × 3.0 mm that can readily accommodate over 16,000 cells in a single dataset. We demonstrate the method using both single-wavelength excitation and the multi-wavelength SW method TartanSW. We show application of the method for imaging of fixed and living cells specimens, with the first application of SW imaging to study cells under flow conditions.

Anti-CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems.

Anti-CD52 antibody (anti-CD52-Ab) leads to a rapid depletion of T and B cells, followed by reconstitution of immune cells with tolerogenic characteristics. However, very little is known about its effect on innate immune cells. In this study, experimental autoimmune encephalomyelitis mice were administered murine anti-CD52-Ab to investigate its effect on dendritic cells and monocytes/macrophages in the periphery lymphoid organs and the central nervous system (CNS). Our data show that blood and splenic innate immune cells exhibited significantly increased expression of MHC-II and costimulatory molecules, which was associated with increased capacity of activating antigen-specific T cells, at first day but not three weeks after five daily treatment with anti-CD52-Ab in comparison with controls. In contrast to the periphery, microglia and infiltrating macrophages in the CNS exhibited reduced expression levels of MHC-II and costimulatory molecules after antibody treatment at both time-points investigated when compared to controls. Furthermore, the transit response of peripheral innate immune cells to anti-CD52-Ab treatment was also observed in the lymphocyte-deficient SCID mice, suggesting the changes are not a direct consequence of the mass depletion of lymphocytes in the periphery. Our study demonstrates a dynamic and tissue-specific modulation of the innate immune cells in their phenotype and function following the antibody treatment. The findings of differential modulation of the microglia and infiltrating macrophages in the CNS in comparison with the innate immune cells in the peripheral organs support the CNS-specific beneficial effect of alemtuzumab treatment on inhibiting neuroinflammation in multiple sclerosis patients.

Use of sedative-hypnotic medications and risk of dementia: A systematic review and meta-analysis.

AIMS: Growing evidence suggests an association between the use of sedative-hypnotic medications and risk of dementia. The aim of this study is to examine this association using a meta-analysis approach. METHODS: MEDLINE (PubMed) and Scopus were systematically searched for studies published in English only. The quality of studies was evaluated using the Newcastle-Ottawa scale, and an overall odds ratio was pooled using a random-effects model. RESULTS: A total of 35 articles were included in the analysis. Pooled odds ratios (ORs) for dementia from all records were (OR; 1.33, 95% CI 1.19-1.49) for benzodiazepine (BZD) combined use (Subgroup-1), (OR: 1.46, 95% CI 1.23-1.73) for short-acting BZD use (Subgroup-2), (OR: 1.72, 95% CI 1.48-1.99) for long-acting BZD use (Subgroup-3), (OR: 1.13, 95% CI 0.97-1.32) for BZDs without specification of duration of action (Subgroup-4), (OR: 1.64, 95% CI 1.13-2.38) for the combined BZDs and Z-drugs, (OR: 1.43, 95% CI 1.17-1.74) for Z-drugs only, (OR: 1.14, 95% CI 0.88-1.46) for antidepressant use, (OR: 0.97, 95% CI 0.68-1.39) for antipsychotic use and (OR: 0.98, 95% CI 0.85-1.13) for anticonvulsant use. When sensitivity analysis was performed, association between overall use of BZDs and short-acting BZDs with the increased risk of dementia disappeared after exclusion of studies that were not adjusted for age covariate (OR: 1.2, 95% CI 1.0-1.44) and (OR: 1.22, 95% CI 0.75-2.01), respectively. Adjustment for protopathic bias by introduction of a lag period showed no evidence of increased risk of dementia with the use of BZDs (Subgroup-1) (OR: 1.14, 95% CI 0.82-1.58), Z-drugs (OR: 1.29, 95% CI 0.78-2.13), and combined BZDs and Z-drugs (OR: 1.51, 95% CI 0.91-2.53). Combined use of BZDs and Z-drugs showed more positive association when only studies of non-user design were analysed (OR: 2.75, 95% CI 2.23-3.39). CONCLUSIONS: All the investigated sedative-hypnotics showed no association with increased risk of dementia except for BZDs. However, the observed association with BZDs did not persist after exclusion of studies with potential reverse causation and confounding by indication. Therefore, this association needs to be assessed carefully in future research.

Mitogen-activated protein kinase phosphatase-2 deletion modifies ventral tegmental area function and connectivity and alters reward processing.

Mitogen-activated protein kinases (MAPKs) regulate normal brain functioning, and their dysfunction is implicated in a number of brain disorders. Thus, there is great interest in understanding the signalling systems that control MAPK functioning. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in foetal development, the immune system, cancer and synaptic plasticity and memory. In the present study, we performed an unbiased investigation using MKP-2-/- mice to assess whether MKP-2 plays a global role in modulating brain function. Local cerebral glucose utilization is significantly increased in the ventral tegmental area (VTA) of MKP-2-/- mice, with connectivity analysis revealing alterations in VTA functional connectivity, including a significant reduction in connectivity to the nucleus accumbens and hippocampus. In addition, spontaneous excitatory postsynaptic current frequency, but not amplitude, onto putative dopamine neurons in the VTA is increased in MKP-2-/- mice, which indicates that increased excitatory drive may account for the increased VTA glucose utilization. Consistent with modified VTA function and connectivity, in behavioural tests MKP-2-/- mice exhibited increased sucrose preference and impaired amphetamine-induced hyperlocomotion. Overall, these data reveal that MKP-2 plays a role in modulating VTA function and that its dysfunction may contribute to brain disorders in which altered reward processing is present.

The development of proteinase-activated receptor-2 modulators and the challenges involved.

Protease-activated receptor-2 (PAR2) has been extensively studied since its discovery in the mid-1990. Despite the advances in understanding PAR2 pharmacology, it has taken almost 25 years for the first inhibitor to reach clinical trials, and so far, no PAR2 antagonist has been approved for human use. Research has employed classical approaches to develop a wide array of PAR2 agonists and antagonists, consisting of peptides, peptoids and antibodies to name a few, with a surge in patent applications over this period. Recent breakthroughs in PAR2 structure determination has provided a unique insight into proposed PAR2 ligand binding sites. Publication of the first crystal structures of PAR2 resolved in complex with two novel non-peptide small molecule antagonists (AZ8838 and AZ3451) revealed two distinct binding pockets, originally presumed to be allosteric sites, with a PAR2 antibody (Fab3949) used to block tethered ligand engagement with the peptide-binding domain of the receptor. Further studies have proposed orthosteric site occupancy for AZ8838 as a competitive antagonist. One company has taken the first PAR2 antibody (MEDI0618) into phase I clinical trial (NCT04198558). While this first-in-human trial is at the early stages of the assessment of safety, other research into the structural characterisation of PAR2 is still ongoing in an attempt to identify new ways to target receptor activity. This review will focus on the development of novel PAR2 modulators developed to date, with an emphasis placed upon the advances made in the pharmacological targeting of PAR2 activity as a strategy to limit chronic inflammatory disease.

Interleukin-16 inhibits sodium channel function and GluA1 phosphorylation via CD4- and CD9-independent mechanisms to reduce hippocampal neuronal excitability and synaptic activity.

Interleukin 16 (IL-16) is a cytokine that is primarily associated with CD4+ T cell function, but also exists as a multi-domain PDZ protein expressed within cerebellar and hippocampal neurons. We have previously shown that lymphocyte-derived IL-16 is neuroprotective against excitotoxicity, but evidence of how it affects neuronal function is limited. Here, we have investigated whether IL-16 modulates neuronal excitability and synaptic activity in mouse primary hippocampal cultures. Application of recombinant IL-16 impairs both glutamate-induced increases in intracellular Ca2+ and sEPSC frequency and amplitude in a CD4- and CD9-independent manner. We examined the mechanisms underlying these effects, with rIL-16 reducing GluA1 S831 phosphorylation and inhibiting Na+ channel function. Taken together, these data suggest that IL-16 reduces neuronal excitability and synaptic activity via multiple mechanisms and adds further evidence that alternative receptors may exist for IL-16.

The C-terminal domain of zDHHC2 contains distinct sorting signals that regulate intracellular localisation in neurons and neuroendocrine cells.

The S-acyltransferase zDHHC2 mediates dynamic S-acylation of PSD95 and AKAP79/150, which impacts synaptic targeting of AMPA receptors. zDHHC2 is responsive to synaptic activity and catalyses the increased S-acylation of PSD95 that occurs following action potential blockade or application of ionotropic glutamate receptor antagonists. These treatments have been proposed to increase plasma membrane delivery of zDHHC2 via an endosomal cycling pathway, enhancing substrate accessibility. To generate an improved understanding of zDHHC2 trafficking and how this might be regulated by neuronal activity, we searched for intramolecular signals that regulate enzyme localisation. Two signals were mapped to the C-terminal tail of zDHHC2: a non-canonical dileucine motif [SxxxLL] and a downstream NP motif. Mutation of these signals enhanced plasma membrane accumulation of zDHHC2 in both neuroendocrine PC12 cells and rat hippocampal neurons, consistent with reduced endocytic retrieval. Furthermore, mutation of these signals also increased accumulation of the enzyme in neurites. Interestingly, several threonine and serine residues are adjacent to these sorting motifs and analysis of phospho-mimetic mutants highlighted a potential role for phosphorylation in regulating the efficacy of these signals. This study offers new molecular insight into the signals that determine zDHHC2 localisation and highlights a potential mechanism to regulate these trafficking signals.

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling.

Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT.

The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC-neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing.

Proteinase-activated receptors (PARs) as targets for antiplatelet therapy.

Since the identification of the proteinase-activated receptor (PAR) family as mediators of serine protease activity in the 1990s, there has been tremendous progress in the elucidation of their pathophysiological roles. The development of drugs that target PARs has been the focus of many laboratories for the potential treatment of thrombosis, cancer and other inflammatory diseases. Understanding the mechanisms of PAR activation and G protein signalling pathways evoked in response to the growing list of endogenous proteases has yielded great insight into receptor regulation at the molecular level. This has led to the development of new selective modulators of PAR activity, particularly PAR1. The mixed success of targeting PARs has been best exemplified in the context of inhibiting PAR1 as a new antiplatelet therapy. The development of the competitive PAR1 antagonist, vorapaxar (Zontivity), has clearly shown the value in targeting PAR1 in acute coronary syndrome (ACS); however the severity of associated bleeding with this drug has limited its use in the clinic. Due to the efficacy of thrombin acting via PAR1, strategies to selectively inhibit specific PAR1-mediated G protein signalling pathways or to target the second thrombin platelet receptor, PAR4, are being devised. The rationale behind these alternative approaches is to bias downstream thrombin activity via PARs to allow for inhibition of pro-thrombotic pathways but maintain other pathways that may preserve haemostatic balance and improve bleeding profiles for widespread clinical use. This review summarizes the structural determinants that regulate PARs and the modulators of PAR activity developed to date.

Protease-activated receptor 2 activation induces behavioural changes associated with depression-like behaviour through microglial-independent modulation of inflammatory cytokines.

RATIONALE: Major depressive disorder (MDD) is a leading cause of disability worldwide but currently prescribed treatments do not adequately ameliorate the disorder in a significant portion of patients. Hence, a better appreciation of its aetiology may lead to the development of novel therapies. OBJECTIVES: In the present study, we have built on our previous findings indicating a role for protease-activated receptor-2 (PAR2) in sickness behaviour to determine whether the PAR2 activator, AC264613, induces behavioural changes similar to those observed in depression-like behaviour. METHODS: AC264613-induced behavioural changes were examined using the open field test (OFT), sucrose preference test (SPT), elevated plus maze (EPM), and novel object recognition test (NOR). Whole-cell patch clamping was used to investigate the effects of PAR2 activation in the lateral habenula with peripheral and central cytokine levels determined using ELISA and quantitative PCR. RESULTS: Using a blood-brain barrier (BBB) permeable PAR2 activator, we reveal that AC-264613 (AC) injection leads to reduced locomotor activity and sucrose preference in mice but is without effect in anxiety and memory-related tasks. In addition, we show that AC injection leads to elevated blood sera IL-6 levels and altered cytokine mRNA expression within the brain. However, neither microglia nor peripheral lymphocytes are the source of these altered cytokine profiles. CONCLUSIONS: These data reveal that PAR2 activation results in behavioural changes often associated with depression-like behaviour and an inflammatory profile that resembles that seen in patients with MDD and therefore PAR2 may be a target for novel antidepressant therapies.

In vivo real-time multiphoton imaging of T lymphocytes in the mouse brain after experimental stroke.

BACKGROUND AND PURPOSE: To gain a better understanding of T cell behavior after stroke, we have developed real-time in vivo brain imaging of T cells by multiphoton microscopy after middle cerebral artery occlusion. METHODS: Adult male hCD2-GFP transgenic mice that exhibit green fluorescent protein-labeled T cells underwent permanent left distal middle cerebral artery occlusion by electrocoagulation (n=6) or sham surgery (n=6) and then multiphoton laser imaging 72 hours later. RESULTS: Extravasated T cell number significantly increased after middle cerebral artery occlusion versus sham. Two T cell populations existed after middle cerebral artery occlusion, possibly driven by 2 T cell subpopulations; 1 had significantly lower and the other significantly higher track velocity and displacement rate than sham. CONCLUSIONS: The different motilities and behaviors of T cells observed using our imaging approach after stroke could reveal important mechanisms of immune surveillance for future therapeutic exploitations.

Two-pore potassium ion channels are inhibited by both G(q/11)- and G(i)-coupled P2Y receptors.

Two-pore potassium (K(2P)) ion channels and P2Y receptors modulate the activity of neurones and are targets for the treatment of neuronal disorders. Here we have characterised their interaction. In cells coexpressing the Galpha(i)-coupled hP2Y(12) receptor, ADP and ATP significantly inhibited hK(2P)2.1 currents. This was abolished by pertussis toxin (PTX), the hP2Y(12) antagonist AR-C69931MX, the hP2Y(1) antagonist MRS2179 and by mutating potential PKA/PKC phosphorylation sites in the channel C terminal. In cells coexpressing the Galpha(q/11)-coupled hP2Y(1) receptor, ADP and ATP also inhibited hK(2P)2.1 currents, which were abolished by MRS2179, but unaffected by AR-C69931MX and PTX. When both receptors were coexpressed with K(2P)2.1 channels, ADP-induced inhibition was antagonised by AR-C69913MX and MRS2179, but not PTX. Thus, both Galpha(q/11)- and Galpha(i)-coupled P2Y receptors inhibit K(2P) channels and the action of hP2Y(12) receptors appears to involve co-activation of endogenous hP2Y(1) receptors. This represents a novel mechanism by which P2Y receptors may modulate neuronal activity.

Increased Levels of IL-16 in the Central Nervous System during Neuroinflammation Are Associated with Infiltrating Immune Cells and Resident Glial Cells.

Interleukin (IL)-16, a CD4+ immune cell specific chemoattractant cytokine, has been shown to be involved in the development of multiple sclerosis, an inflammatory demyelinating disease of the central nervous system (CNS). While immune cells such as T cells and macrophages are reported to be the producers of IL-16, the cellular source of IL-16 in the CNS is less clear. This study investigates the correlation of IL-16 expression levels in the CNS with the severity of neuroinflammation and determines the phenotype of cells which produce IL-16 in the CNS of experimental autoimmune encephalomyelitis (EAE) mice. Our data show that IL-16 expression is significantly increased in the brain and spinal cord tissues of EAE mice compared to phosphate buffered saline (PBS) immunised controls. Dual immunofluorescence staining reveals that the significantly increased IL-16+ cells in the CNS lesions of EAE mice are likely to be the CD45+ infiltrating immune cells such as CD4+ or F4/80+ cells and the CNS resident CD11b+ microglia and GFAP+ astrocytes, but not NeuN+ neurons. Our data suggest cytokine IL-16 is closely involved in EAE pathology as evidenced by its increased expression in the glial and infiltrating immune cells, which impacts the recruitment and activation of CD4+ immune cells in the neuroinflammation.

An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton.

Conventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart. Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminated regions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescence microscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs with different axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased the sampling density from 50 to 98% over seven anti-nodal planes, with no notable change in axial or lateral resolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with images of the membrane and cytoskeleton of living and fixed cells.

Astrocytic activation and an inhibition of MAP kinases are required for proteinase-activated receptor-2-mediated protection from neurotoxicity.

Proteinase-activated receptor-2 (PAR-2) expression levels are altered in several CNS disorders with these changes being proposed to either exacerbate or diminish the disease state depending on the cell type in which this occurs. Here we present data investigating the consequence of PAR-2 activation on kainate (KA)-induced neurotoxicity in organotypic hippocampal slices cultures (OHSC). Exposure of OHSC to the PAR-2 activators trypsin or Ser-Leu-Ile-Gly-Arg-Leu (SLIGRL) induced no neurotoxicity when applied alone but was neuroprotective against KA-induced neurotoxicity. SLIGRL-mediated neuroprotection involved astrocytic activation as the neuroprotective effect was abolished following OHSC pre-treatment with fluoroacetate. Moreover, co-application of either reparixin or LY341495, antagonists of the CXCR2 chemokine receptor and metabotropic glutamate receptors respectively, inhibited the SLIGRL-mediated neuroprotection. SLIGRL application inhibited both p38 MAPK and ERK activity in OHSC, but not the JNK 1/2 signalling pathway. Accordingly, the co-application of the p38 MAPK and ERK inhibitors SB203580 and UO126 reduced KA-induced cell death, mimicking PAR-2-mediated neuroprotection. These data indicate that PAR-2 activation is neuroprotective and involves astrocytic activation, gliotransmitter release, and the subsequent inhibition of MAPK signalling cascades, providing further evidence for PAR-2 as an interesting therapeutic target in certain CNS disorders.

Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy.

We present here a fast optical sectioning method for mesoscopy based on HiLo microscopy, which makes possible imaging of specimens of up to 4.4 mm × 3 mm × 3 mm in volume in under 17 hours (estimated for a z-stack comprising 1000 images excluding computation time) with subcellular resolution throughout. Widefield epifluorescence imaging is performed with the Mesolens using a high pixel-number camera capable of sensor-shifting to generate a 259.5 Megapixel image, and we have developed custom software to perform HiLo processing of the very large datasets. Using this method, we obtain comparable sectioning strength to confocal laser scanning microscopy (CLSM), with sections as thin as 6.8 ± 0.2 µm and raw acquisition speed of 1 minute per slice which is up to 30 times faster than CLSM on the full field of view (FOV) of the Mesolens of 4.4 mm with lateral resolution of 0.7 µm and axial resolution of 7 µm. We have applied this HiLo mesoscopy method to image fixed and fluorescently stained hippocampal neuronal specimens and a 5-day old zebrafish larva.

The therapeutic effect of anti-CD52 treatment in murine experimental autoimmune encephalomyelitis is associated with altered IL-33 and ST2 expression levels.

Experimental autoimmune encephalomyelitis (EAE) mice were administered with murine anti-CD52 antibody to investigate its therapeutic effect and whether the treatment modulates IL-33 and ST2 expression. EAE severity and central nervous system (CNS) inflammation were reduced following the treatment, which was accompanied by peripheral T and B lymphocyte depletion and reduced production of various cytokines including IL-33, while sST2 was increased. In spinal cords of EAE mice, while the number of IL-33+ cells remained unchanged, the extracellular level of IL-33 protein was significantly reduced in anti-CD52 antibody treated mice compared with controls. Furthermore the number of ST2+ cells in the spinal cord of treated EAE mice was downregulated due to decreased inflammation and immune cell infiltration in the CNS. These results suggest that treatment with anti-CD52 antibody differentially alters expression of IL-33 and ST2, both systemically and within the CNS, which may indicate IL-33/ST2 axis is involved in the action of the antibody in inhibiting EAE.

Toll-like receptor 3 activation impairs excitability and synaptic activity via TRIF signalling in immature rat and human neurons.

Toll like receptor 3 (TLR3) belongs to a family of pattern recognition receptors that recognise molecules found on pathogens referred to as pathogen associated molecular patterns (PAMPs). Its involvement in innate immunity is well known but despite its presence in the central nervous system (CNS), our knowledge of its function is limited. Here, we have investigated whether TLR3 activation modulates synaptic activity in primary hippocampal cultures and induced pluripotent stem cell (iPSC)-derived neurons. Synaptically driven spontaneous action potential (AP) firing was significantly reduced by the TLR3 specific activator, poly I:C, in a concentration-dependent manner following both short (5 min) and long exposures (1h) in rat hippocampal cultures. Notably, the consequence of TLR3 activation on neuronal function was reproduced in iPSC-derived cortical neurons, with poly I:C (25 µg/ml, 1h) significantly inhibiting sAP firing. We examined the mechanisms underlying these effects, with poly I:C significantly reducing peak sodium current, an effect dependent on the MyD88-independent TRIF dependent pathway. Furthermore, poly I:C (25 µg/ml, 1h) resulted in a significant reduction in miniature excitatory postsynaptic potential (mEPSC) frequency and amplitude and significantly reduced surface AMPAR expression. These novel findings reveal that TLR3 activation inhibits neuronal excitability and synaptic activity through multiple mechanisms, with this being observed in both rat and human iPSC-derived neurons. These data might provide further insight into how TLR3 activation may contribute to neurodevelopmental disorders following maternal infection and in patients with increased susceptibility to herpes simplex encephalitis.