Alzheimer’s disease is a leading cause of death, but current treatments are limited in their use and primarily offer symptomatic relief. Recent developments targeting amyloid plaques have given some hope, but their true value remains equivocal. Hence, alternative therapeutic targets are required with neuroinflammation, particularly glial cells, been a recent area of interest. Indeed, there has been intense research into the role of reactive astrocytes in neurodegenerative diseases with several studies indicating that reducing their reactivity is beneficial in animal models of disease. Building on our previous work using the blood brain barrier permeable protease-activated receptor 2 (PAR2) activator, AC264613 (AC), we investigate the consequence of AC administration on mouse behaviour, astrocytic reactivity and amyloid plaque load in the 5xFAD mouse model of amyloid pathology. Our data reveal similar behavioural changes in both 5xFAD- and 5xFAD+ mice that are sex-independent. However, AC reduces both GFAP and C3 expression levels, markers for astrocyte reactivity, in female but not male 5xFAD+ mice. In contrast, AC did not affect Iba1 and CD68 expression levels, markers for microglial activation and function, in mice of either sex. Significantly, AC reduced amyloid plaque load only in female 5xFAD+ mice similar to that seen with astrocyte reactivity. These data reveal that PAR2 activation can reduce astrocyte reactivity and amyloid plaque load in 5xFAD+ mice. Our findings add further weight to the proposal that targeting astrocyte reactivity to reduce neuroinflammation in neurodegenerative disorders, in this case using PAR2 activation, may be a viable therapeutic strategy.

Protease-activated receptor 2 is proposed to be a novel target for several inflammation-related diseases but its role in the central nervous system remains unclear. Protease-activated receptor 2 activation is protective in cell death and excitotoxicity assays whereas examination into the role of protease-activated receptor 2 in vivo has been hindered due to the lack of suitable pharmacological tools. Recently, a small molecule protease-activated receptor 2 activator, AC264613 (AC), was reported to be a potent and selective protease-activated receptor 2 activator that crosses the blood-brain barrier. Furthermore, peptide mimetic molecules, for example GB88, were developed that were reported to act as protease-activated receptor 2 biased antagonists. Here, we examine their signalling pathways and neuroprotective properties in central nervous system preparations. AC induced significant increases in intracellular Ca 2+ in both neurons and astrocytes of primary hippocampal cultures, whereas in contrast, GB88 induced a small but significant reduction in intracellular Ca 2+ in both cell types. However, both AC and GB88 induced receptor internalisation when examined using fluorescently tagged protease-activated receptor 2. Both AC and GB88 did not induce neurotoxicity in organotypic hippocampal slice cultures when applied alone but reduced neurotoxicity when co-applied with kainate in excitotoxicity assays. Furthermore, both AC and GB88 reduced neurotoxicity when applied post kainate insult indicating they exhibit neuroprotective properties even after excitotoxicity is induced. These data indicate that protease-activated receptor 2 activation is neuroprotective but this is independent of Gq-induced Ca 2+ activation. Given that AC crosses the blood–brain barrier, this highlights its use as a novel tool to examine the protective properties of protease-activated receptor 2 in in vivo models of central nervous system disorders.

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 x 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.

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.

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 x 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.

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.

Disorders of the central nervous system (CNS) represent a global health challenge and an increased understanding of the CNS in both physiological and pathophysiological states is essential to tackle the problem. Modelling CNS conditions is difficult, as traditional in vitro models fail to recapitulate precise microenvironments and animal models of complex disease often have limited translational validity. Microfluidic and organ-on-chip technologies offer an opportunity to develop more physiologically relevant and complex in vitro models of the CNS. They can be developed to allow precise cellular patterning and enhanced experimental capabilities to study neuronal function and dysfunction. To improve ease-of-use of the technology and create new opportunities for novel in vitro studies, we introduce a modular platform consisting of multiple, individual microfluidic units that can be combined in several configurations to create bespoke culture environments. Here, we report proof-of-concept experiments creating complex in vitro models and performing functional analysis of neuronal activity across modular interfaces. This platform technology presents an opportunity to increase our understanding of CNS disease mechanisms and ultimately aid the development of novel therapies.

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.

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.

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.

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.

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.

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.

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.

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 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 Sacylation 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 noncanonical 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.

Louise Ritchie, Rothwell Tate, Luke H. Chamberlain, Graham Robertson (orcid.org/00000002-3841-874X), Michele Zagnoni (orcid.org/0000-0003-3198-9491), Teresa Sposito, Selina Wray (orcid.org/0000-0003-3062-7050) , John A. Wright, Clare E. Bryant (orcid.org/0000-0002-2924-0038), Nicholas J. Gay (orcid.org/0000-0002-2782-7169) and Trevor J. Bushell (orcid.org/0000-0003-4145-9670) Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, UK. Centre for Microsystems and Photonics, Electronic and Electrical Engineering, University of Strathclyde, Glasgow, G1 1XW, UK. Department of Molecular Neuroscience, UCL Institute of Neurology, 1 Wakefield Street, London WC1N 1PJ, UK. Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK. e Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK

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.