Proteinase-activated receptor 2 is involved in the behavioural changes associated with sickness behaviour

Proteinase-activated receptor-2 (PAR2) is widely expressed in the CNS but whether it plays a key role in inflammation-related behavioural changes remains unknown. Hence, in the present study we have examined whether PAR2 contributes to behaviour associated with systemic inflammation using PAR2 transgenic mice. The onset of sickness behaviour was delayed and the recovery accelerated in PAR2-/- mice in the LPS-induced model of sickness behaviour. In contrast, PAR2 does not contribute to behaviour under normal conditions. In conclusion, these data suggest that PAR2 does not contribute to behaviour in the normal healthy brain but it plays a role in inflammation-related behavioural changes

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

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

A 340/380 nm light emitting diode illuminator for Fura-2 AM ratiometric Ca2+ imaging of live cells with better than 5 nM precision

Cytosolic Ca2+ plays an integral role in cells and the study of its dynamics can reveal much about biological processes [1]. Fura-2 can provide quantitative data on cytosolic Ca2+ changes by exciting at 340 nm and 380 nm and taking the ratio of the emission at both wavelengths [2]. Traditionally for this type of imaging an arc lamp had to be used for illumination as LEDs of the appropriate wavelengths were not available [3]. LEDs hold advantages over arc lamps by exhibiting high amplitude stability and the ability to rapidly switch between wavelengths. We aimed to test a new 340/380 nm LED system for use in ratiometric Fura-2 AM Ca2+ imaging and present results using tsA-201 cells and hippocampal neurons

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

Increased expression of IL-16 in the brain of experimental autoimmune encephalomyelitis

Multiple Sclerosis (MS) is a demyelinating disease of the CNS, whose pathophysiology involves both inflammatory and neurodegenerative components. CD4+ T cells are one of the key mediators of disease initiation and progression; however CD4 i s also the receptor for the pro-inflammatory cytokine, interleukin - 16 (IL - 16). IL - 16 has been proposed to play a role in several autoimmune diseases, but the exact role of IL - 16 in the CNS during MS initiation and progression remains unclear. Therefore, the aim of this study was to examine the expression and distribution of IL - 16 in CNS tissue and investigate whether expression levels correlate with neuro-inflammation in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. EAE was induced in 6 week old C 57BL/6J female mice by immunisation with MOG35 - 55 peptide and adjuvants. Tissue was harvested at onset (day 11), peak (day 16) and resolution (day 26), and immunofluorescence staining carried out to determine CD45, CD4 and IL - 16 expression and localisation in the brain of both control and EAE mice. In addition, co-localisation of IL - 16 with CNS and immune cell subtypes was performed using a Mesolens microscope (McConnell et al., 2016), which allows subcellular detail to be obtained from wide - field epifluorescence images. Expression of IL - 16 and CD4 was observed primarily within the lesions of cerebellum and hippocampus of the EAE brain, whereas little expression was observed in control brains. IL - 16 expression was highest at onset with 76 ±2.8% of cells ( n=3) within these lesions expressing IL - 16. This was reduced to 48±2.4% (n=3) at peak and 16 ±1.3% at resolution (n=3). Co-localization studies revealed that IL - 16 was expressed primarily by infiltrating immune cells but not by neurons or astrocytes. Co-localization of IL - 16 with immune cells in brain lesions of EAE mice suggests that infiltrating immune cells are the primary source of IL - 16. Further investigation is required if IL - 16 is pro-inflammatory or anti-inflammatory in the CNS during EAE

Protease-activated receptor 2 : are common functions in glial and immune cells linked to inflammation-related CNS disorders?

Protease-activated receptors (PARs) are a novel family of G-protein coupled receptors (GPCRs) whose activation requires the cleavage of the N-terminus by a serine protease. However recent evidence reveals that alternative routes of activation also occur and that PARs signal via multiple pathways and that pathway activation is activator-dependent. Given our increased understanding of PAR function both under physiological and pathophysiological conditions; one aspect that has remained a constant is the link between PAR2 and inflammation. PAR2 is expressed in immune cells of both the innate and adaptive immune system and has been shown to play a role in several peripheral inflammatory conditions. PAR2 is similarly expressed on astrocytes and microglia within the CNS and its activation is either protective or detrimental to CNS function depending on the conditions or disease state investigated. With a clear similarity between the function of PAR2 on both immune cells and CNS glial cells, here we have reviewed their roles in both these systems. We suggest that the recent development of novel PAR2 modulators, including those that show biased signalling, will further increase our understanding of PAR2 function and the development of potential therapeutics for CNS disorders in which inflammation is proposed to play a role

A novel microfluidic drug testing platform for studying communication between independent neural networks

Many in-vitro systems used during pre-clinical trials fail to recreate the biological complexity of the in-vivo neural microenvironment.Taking advantage of recent advances in microfluidic technology, we seek to develop a perfusion based drug discovery platform that is capable of high-throughput pharmacological profiling. This in turn will allow us to better understand how drugs influence the communication between functionally connected neural networks

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 signalling 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 (LTP) is impaired in MKP-2-/- mice compared to MKP-2+/+ controls whereas neuronal excitability, evoked synaptic transmission and paired-pulse facilitation remain unaltered. Furthermore, spontaneous excitatory postsynaptic currents (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 spontaneous EPSC 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 signalling