Publications by authors named "Kumlesh K Dev"

70 Publications

EBI2 Is Temporarily Upregulated in MO3.13 Oligodendrocytes during Maturation and Regulates Remyelination in the Organotypic Cerebellar Slice Model.

Int J Mol Sci 2021 Apr 21;22(9). Epub 2021 Apr 21.

Department of Laboratory Medicine, Medical University of Gdańsk, 80-210 Gdańsk, Poland.

The EBI2 receptor regulates the immune system and is expressed in various immune cells including B and T lymphocytes. It is also expressed in astrocytes in the central nervous system (CNS) where it regulates pro-inflammatory cytokine release, cell migration and protects from chemically induced demyelination. Its signaling and expression are implicated in various diseases including multiple sclerosis, where its expression is increased in infiltrating immune cells in the white matter lesions. Here, for the first time, the EBI2 protein in the CNS cells in the human brain was examined. The function of the receptor in MO3.13 oligodendrocytes, as well as its role in remyelination in organotypic cerebellar slices, were investigated. Human brain sections were co-stained for EBI2 receptor and various markers of CNS-specific cells and the human oligodendrocyte cell line MO3.13 was used to investigate changes in EBI2 expression and cellular migration. Organotypic cerebellar slices prepared from wild-type and cholesterol 25-hydroxylase knock-out mice were used to study remyelination following lysophosphatidylcholine (LPC)-induced demyelination. The data showed that EBI2 receptor is present in OPCs but not in myelinating oligodendrocytes in the human brain and that EBI2 expression is temporarily upregulated in maturing MO3.13 oligodendrocytes. Moreover, we show that migration of MO3.13 cells is directly regulated by EBI2 and that its signaling is necessary for remyelination in cerebellar slices post-LPC-induced demyelination. The work reported here provides new information on the expression and role of EBI2 in oligodendrocytes and myelination and provides new tools for modulation of oligodendrocyte biology and therapeutic approaches for demyelinating diseases.
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http://dx.doi.org/10.3390/ijms22094342DOI Listing
April 2021

Spatiotemporal immunolocalisation of REST in the brain of healthy ageing and Alzheimer's disease rats.

FEBS Open Bio 2021 Jan 1;11(1):146-163. Epub 2020 Dec 1.

School of Life Sciences, Queens Medical Centre, University of Nottingham, UK.

In the brain, REST (Repressor Element-1 Silencing Transcription factor) is a key regulator of neuron cell-specific gene expression. Nuclear translocation of neuronal REST has been shown to be neuroprotective in a healthy ageing context. In contrast, inability to upregulate nuclear REST is thought to leave ageing neurons vulnerable to neurodegenerative stimuli, such as Alzheimer's disease (AD) pathology. Hippocampal and cortical neurons are known to be particularly susceptible to AD-associated neurodegeneration. However, REST expression has not been extensively characterised in the healthy ageing brain. Here, we examined the spatiotemporal immunolocalisation of REST in the brains of healthy ageing wild-type Fischer-344 and transgenic Alzheimer's disease rats (TgF344-AD). Nuclear expression of REST increased from 6 months to 18 months of age in the hippocampus, frontal cortex and subiculum of wild-type rats, but not in TgF344-AD rats. No changes in REST were measured in more posterior cortical regions or in the thalamus. Interestingly, levels of the presynaptic marker synaptophysin, a known gene target of REST, were lower in CA1 hippocampal neurons of 18-month TgF344-AD rats compared to 18-month wild-types, suggesting that elevated nuclear REST may protect against synapse loss in the CA1 of 18-month wild-type rats. High REST expression in ageing wild-type rats did not, however, protect against axonal loss nor against astroglial reactivity in the hippocampus. Taken together, our data confirm that changes in nuclear REST expression are context-, age- and brain region-specific. Moreover, key brain structures involved in learning and memory display elevated REST expression in healthy ageing wild-type rats but not TgF344-AD rats.
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http://dx.doi.org/10.1002/2211-5463.13036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780110PMC
January 2021

Fingolimod Rescues Demyelination in a Mouse Model of Krabbe's Disease.

J Neurosci 2020 04 3;40(15):3104-3118. Epub 2020 Mar 3.

Drug Development, School of Medicine, Trinity College Dublin D02 R590, Ireland

Krabbe's disease is an infantile neurodegenerative disease, which is affected by mutations in the lysosomal enzyme galactocerebrosidase, leading to the accumulation of its metabolite psychosine. We have shown previously that the S1P receptor agonist fingolimod (FTY720) attenuates psychosine-induced glial cell death and demyelination both and models. These data, together with a lack of therapies for Krabbe's disease, prompted the current preclinical study examining the effects of fingolimod in twitcher mice, a murine model of Krabbe's disease. Twitcher mice, both male and female, carrying a natural mutation in the galc gene were given fingolimod via drinking water (1 mg/kg/d). The direct impact of fingolimod administration was assessed via histochemical and biochemical analysis using markers of myelin, astrocytes, microglia, neurons, globoid cells, and immune cells. The effects of fingolimod on twitching behavior and life span were also demonstrated. Our results show that treatment of twitcher mice with fingolimod significantly rescued myelin levels compared with vehicle-treated animals and also regulated astrocyte and microglial reactivity. Furthermore, nonphosphorylated neurofilament levels were decreased, indicating neuroprotective and neurorestorative processes. These protective effects of fingolimod on twitcher mice brain pathology was reflected by an increased life span of fingolimod-treated twitcher mice. These findings corroborate initial studies and highlight the potential use of S1P receptors as drug targets for treatment of Krabbe's disease. This study demonstrates that the administration of the therapy known as fingolimod in a mouse model of Krabbe's disease (namely, the twitcher mouse model) significantly rescues myelin levels. Further, the drug fingolimod also regulates the reactivity of glial cells, astrocytes and microglia, in this mouse model. These protective effects of fingolimod result in an increased life span of twitcher mice.
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http://dx.doi.org/10.1523/JNEUROSCI.2346-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141882PMC
April 2020

Inhibition of Piezo1 attenuates demyelination in the central nervous system.

Glia 2020 02 9;68(2):356-375. Epub 2019 Oct 9.

School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK.

Piezo1 is a mechanosensitive ion channel that facilitates the translation of extracellular mechanical cues to intracellular molecular signaling cascades through a process termed, mechanotransduction. In the central nervous system (CNS), mechanically gated ion channels are important regulators of neurodevelopmental processes such as axon guidance, neural stem cell differentiation, and myelination of axons by oligodendrocytes. Here, we present evidence that pharmacologically mediated overactivation of Piezo1 channels negatively regulates CNS myelination. Moreover, we found that the peptide GsMTx4, an antagonist of mechanosensitive cation channels such as Piezo1, is neuroprotective and prevents chemically induced demyelination. In contrast, the positive modulator of Piezo1 channel opening, Yoda-1, induces demyelination and neuronal damage. Using an ex vivo murine-derived organotypic cerebellar slice culture model, we demonstrate that GsMTx4 attenuates demyelination induced by the cytotoxic lipid, psychosine. Importantly, we confirmed the potential therapeutic effects of GsMTx4 peptide in vivo by co-administering it with lysophosphatidylcholine (LPC), via stereotactic injection, into the cerebral cortex of adult mice. GsMTx4 prevented both demyelination and neuronal damage usually caused by the intracortical injection of LPC in vivo; a well-characterized model of focal demyelination. GsMTx4 also attenuated both LPC-induced astrocyte toxicity and microglial reactivity within the lesion core. Overall, our data suggest that pharmacological activation of Piezo1 channels induces demyelination and that inhibition of mechanosensitive channels, using GsMTx4, may alleviate the secondary progressive neurodegeneration often present in the latter stages of demyelinating diseases.
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http://dx.doi.org/10.1002/glia.23722DOI Listing
February 2020

Piezo1 regulates calcium oscillations and cytokine release from astrocytes.

Glia 2020 01 21;68(1):145-160. Epub 2019 Aug 21.

School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK.

Astrocytes are important for information processing in the brain and they achieve this by fine-tuning neuronal communication via continuous uptake and release of biochemical modulators of neurotransmission and synaptic plasticity. Often overlooked are their important functions in mechanosensation. Indeed, astrocytes can detect pathophysiological changes in the mechanical properties of injured, ageing, or degenerating brain tissue. We have recently shown that astrocytes surrounding mechanically-stiff amyloid plaques upregulate the mechanosensitive ion channel, Piezo1. Moreover, ageing transgenic Alzheimer's rats harboring a chronic peripheral bacterial infection displayed enhanced Piezo1 expression in amyloid plaque-reactive astrocytes of the hippocampus and cerebral cortex. Here, we have shown that the bacterial endotoxin, lipopolysaccharide (LPS), also upregulates Piezo1 in primary mouse cortical astrocyte cultures in vitro. Activation of Piezo1, via the small molecule agonist Yoda1, enhanced Ca influx in both control and LPS-stimulated astrocytes. Moreover, Yoda1 augmented intracellular Ca oscillations but decreased subsequent Ca influx in response to adenosine triphosphate (ATP) stimulation. Neither blocking nor activating Piezo1 affected cell viability. However, LPS-stimulated astrocyte cultures exposed to the Piezo1 activator, Yoda1, migrated significantly slower than reactive astrocytes treated with the mechanosensitive channel-blocking peptide, GsMTx4. Furthermore, our data show that activating Piezo1 channels inhibits the release of cytokines and chemokines, such as IL-1β, TNFα, and fractalkine (CX CL1), from LPS-stimulated astrocyte cultures. Taken together, our results suggest that astrocytic Piezo1 upregulation may act to dampen neuroinflammation and could be a useful drug target for neuroinflammatory disorders of the brain.
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http://dx.doi.org/10.1002/glia.23709DOI Listing
January 2020

Inhibition of the Interaction Between Group I Metabotropic Glutamate Receptors and PDZ-Domain Proteins Prevents Hippocampal Long-Term Depression, but Not Long-Term Potentiation.

Front Synaptic Neurosci 2019 18;11:13. Epub 2019 Apr 18.

Department of Neurophysiology, Medical Faculty, Ruhr University Bochum, Bochum, Germany.

The group I metabotropic glutamate (mGlu) receptor subtypes, mGlu1 and mGlu5, strongly regulate hippocampal synaptic plasticity. Both harbor PSD-95/discs-large/ZO-1 (PDZ) motifs at their extreme carboxyl terminals, which allow interaction with the PDZ domain of Tamalin, regulate the cell surface expression of group I mGlu receptors, and may modulate their coupling to signaling proteins. We investigated the functional role of this interaction in hippocampal long-term depression (LTD). Acute intracerebral treatment of adult rats with a cell-permeable PDZ-blocking peptide (pep-mGluR-STL), designed to competitively inhibit the interaction between Tamalin and group 1 mGlu receptors, prevented expression of LTD in the hippocampal CA1 region without affecting long-term potentiation (LTP) or basal synaptic transmission. Pep-mGluR-STL prevented facilitation by the group I mGlu receptor agonist, (S)-3,5-Dihydroxyphenylglycine (DHPG), and the mGlu5 agonist, (R,S)-2-chloro-5-Hydroxyphenylglycine (CHPG), of short-term depression (STD) into LTD, suggesting that Tamalin preferentially acts by mediating signaling through mGlu5. These data support that Tamalin is essential for the persistent expression of LTD and that it subserves the effective signaling of group 1 mGlu receptors.
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http://dx.doi.org/10.3389/fnsyn.2019.00013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482240PMC
April 2019

Infection Augments Expression of Mechanosensing Piezo1 Channels in Amyloid Plaque-Reactive Astrocytes.

Front Aging Neurosci 2018 22;10:332. Epub 2018 Oct 22.

Neuroimmulology & Neurotherapeutics Laboratory, School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom.

A defining pathophysiological hallmark of Alzheimer's disease (AD) is the amyloid plaque; an extracellular deposit of aggregated fibrillar Aβ peptides. Amyloid plaques are hard, brittle structures scattered throughout the hippocampus and cerebral cortex and are thought to cause hyperphosphorylation of tau, neurofibrillary tangles, and progressive neurodegeneration. Reactive astrocytes and microglia envelop the exterior of amyloid plaques and infiltrate their inner core. Glia are highly mechanosensitive cells and can almost certainly sense the mismatch between the normally soft mechanical environment of the brain and very stiff amyloid plaques via mechanosensing ion channels. Piezo1, a non-selective cation channel, can translate extracellular mechanical forces to intracellular molecular signaling cascades through a process known as . Here, we utilized an aging transgenic rat model of AD (TgF344-AD) to study expression of mechanosensing Piezo1 ion channels in amyloid plaque-reactive astrocytes. We found that Piezo1 is upregulated with age in the hippocampus and cortex of 18-month old wild-type rats. However, more striking increases in Piezo1 were measured in the hippocampus of TgF344-AD rats compared to age-matched wild-type controls. Interestingly, repeated urinary tract infections with bacteria, a common comorbidity in elderly people with dementia, caused further elevations in Piezo1 channel expression in the hippocampus and cortex of TgF344-AD rats. Taken together, we report that aging and peripheral infection augment amyloid plaque-induced upregulation of mechanoresponsive ion channels, such as Piezo1, in astrocytes. Further research is required to investigate the role of astrocytic Piezo1 in the Alzheimer's brain, whether modulating channel opening will protect or exacerbate the disease state, and most importantly, if Piezo1 could prove to be a novel drug target for age-related dementia.
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http://dx.doi.org/10.3389/fnagi.2018.00332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204357PMC
October 2018

A novel modelling mechanism of PAEL receptor and GABARAPL2 interaction involved in Parkinson's disease.

Neurosci Lett 2018 04 26;673:12-18. Epub 2018 Feb 26.

Drug Development, School of Medicine, Trinity College Dublin, Ireland. Electronic address:

Parkin associated endothelin like receptor (PAELR) is G-protein coupled and ubiquitinated by parkin, promoting its degradation. In autosomal recessive Parkinson's disease, mutations in parkin lead to PAELR aggregation in the endoplasmic reticulum (ER), ER stress, neurotoxicity and cell death. We have identified previously that the protein kinase C interacting protein (PICK1) interacts with and regulates the expression and cell toxicity of PAELR. Here, we experimentally identify and provide in-silico modelling of a novel interaction between PAELR and GABARAPL2 (γ-aminobutyrate type A receptor associated protein like 2), which is an autophagosome-specific Ub-like protein implicated in vesicle trafficking and autophagy. We show that the family of GABARAPs interact with the carboxy terminal (ct) of PAELR and find the cysteine rich region (-CCCCCC-EEC) of ct-PAELR interacts with the GABA binding site of GABARAPL2. This interaction is modelled by in-slico analysis and confirmed using affinity chromatography, showing Myc-tagged GABARAPL2 is retained by a GST fusion of the ct-PAELR. We also demonstrate that transient transfection of GABARAPL2 in HEK293 cells reduces PAELR expression. This study supports the idea that protein levels of PAELR are likely regulated by a multitude of proteins including parkin, PICK1 and GABARAPL2 via mechanisms that include ubiquitination, proteasomal degradagtion and autophagy.
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http://dx.doi.org/10.1016/j.neulet.2018.02.055DOI Listing
April 2018

Sphingosine 1-phosphate receptors regulate TLR4-induced CXCL5 release from astrocytes and microglia.

J Neurochem 2018 03 21;144(6):736-747. Epub 2018 Feb 21.

Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

Sphingosine 1-phosphate receptors (S1PR) are G protein-coupled and compose a family with five subtypes, S1P1R-S1P5R. The drug Gilenya (Novartis, Basel, Switzerland) (Fingolimod; FTY720) targets S1PRs and was the first oral therapy for patients with relapsing-remitting multiple sclerosis (MS). The phosphorylated form of FTY720 (pFTY720) binds S1PRs causing initial agonism, then subsequent receptor internalization and functional antagonism. Internalization of S1P1R attenuates sphingosine 1-phosphate (S1P)-mediated egress of lymphocytes from lymph nodes, limiting aberrant immune function in MS. pFTY720 also exerts direct actions on neurons and glial cells which express S1PRs. In this study, we investigated the regulation of pro-inflammatory chemokine release by S1PRs in enriched astrocytes and microglial cultures. Astrocytes and microglia were stimulated with lipopolysaccharide (LPS) and increases in C-X-C motif chemokine 5 (CXCL5), also known as LIX (lipopolysaccharide-induced CXC chemokine) expression were quantified. Results showed that pFTY720 attenuated LPS-induced CXCL5 (LIX) protein release from astrocytes, as did the S1P1R selective agonist, SEW2871. In addition, pFTY720 blocked messenger ribonucleic acid (mRNA) transcription of the chemokines, (i) CXCL5/LIX, (ii) C-X-C motif chemokine 10 (CXCL10) also known as interferon gamma-induced protein 10 (IP10) and (iii) chemokine (C-C motif) ligand 2 (CCL2) also known as monocyte chemoattractant protein 1 (MCP1). Interestingly, inhibition of sphingosine kinase attenuated LPS-induced increases in mRNA levels of all three chemokines, suggesting that LPS-TLR4 (Toll-like receptor 4) signalling may enhance chemokine expression via S1P-S1PR transactivation. Lastly, these observations were not limited to astrocytes since we also found that pFTY720 attenuated LPS-induced release of CXCL5 from microglia. These data highlight a role for S1PR signalling in regulating the levels of chemokines in glial cells and support the notion that pFTY720 efficacy in multiple sclerosis may involve the direct modulation of astrocytes and microglia.
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http://dx.doi.org/10.1111/jnc.14313DOI Listing
March 2018

EBI2 regulates pro-inflammatory signalling and cytokine release in astrocytes.

Neuropharmacology 2018 05 31;133:121-128. Epub 2018 Jan 31.

Drug Development, School of Medicine, Trinity College, Dublin, Ireland.

The endogenous oxysterol 7α, 25-dihydroxycholesterol (7α25HC) ligand activates the G protein-coupled receptor EBI2 to regulate T cell-dependant antibody response and B cell migration. We have demonstrated that EBI2 is expressed in human and mouse astrocytes, that 7α25HC induces intracellular signalling and astrocyte migration, and that EBI2 plays a role in the crosstalk between astrocytes and macrophages. Recently, we demonstrate that EBI2 regulates myelin development and inhibits LPC-induced demyelination. Here, we show that 7α25HC inhibits LPS- and IL17/TNF-induced pro-inflammatory cytokine release in astrocytes. We observe the following: 1. Human astrocytes treated with IL17/TNF increases the nuclear translocation of NFκB, which is attenuated by pre-treatment with 7α25HC; 2. IL17/TNF increases cell impedance in human astrocytes, which is also attenuated by pre-treatment with 7α25HC; 3. The EBI2 antagonist NIBR189 inhibits these effects of 7α25HC, supporting the role of EBI2; 4. in vivo data corroborate these in vitro findings, showing that EBI2 knock-out (KO) animals display enhanced pro-inflammatory cytokine in response to LPS challenge, in the brain. These results demonstrate a role for oxysterol/EBI2 signalling in attenuating the response of astrocytes to pro-inflammatory signals as well as limiting the levels of pro-inflammatory cytokines in the brain.
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http://dx.doi.org/10.1016/j.neuropharm.2018.01.029DOI Listing
May 2018

EBI2 receptor regulates myelin development and inhibits LPC-induced demyelination.

J Neuroinflammation 2017 Dec 16;14(1):250. Epub 2017 Dec 16.

Drug Development, School of Medicine, Trinity College, Dublin, Ireland.

Background: The G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2) is activated by 7α, 25-dihydroxycholesterol (7α25HC) and plays a role in T cell-dependant antibody response and B cell migration. Abnormal EBI2 signaling is implicated in a range of autoimmune disorders; however, its role in the CNS remains poorly understood.

Methods: Here we characterize the role of EBI2 in myelination under normal and pathophysiological conditions using organotypic cerebellar slice cultures and EBI2 knock-out (KO) animals.

Results: We find that MBP expression in brains taken from EBI2 KO mice is delayed compared to those taken from wild type (WT) mice. In agreement with these in vivo findings, we show that antagonism of EBI2 reduces MBP expression in vitro. Importantly, we demonstrate that EBI2 activation attenuates lysolecithin (LPC)-induced demyelination in mouse organotypic slice cultures. Moreover, EBI2 activation also inhibits LPC-mediated release of pro-inflammatory cytokines such as IL6 and IL1β in cerebellar slices.

Conclusions: These results, for the first time, display a role for EBI2 in myelin development and protection from demyelination under pathophysiological conditions and suggest that modulation of this receptor may be beneficial in neuroinflammatory and demyelinating disorders such as multiple sclerosis.
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http://dx.doi.org/10.1186/s12974-017-1025-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732472PMC
December 2017

Phospholipase A2 is involved in galactosylsphingosine-induced astrocyte toxicity, neuronal damage and demyelination.

PLoS One 2017 2;12(11):e0187217. Epub 2017 Nov 2.

Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

Krabbe disease is a fatal rare inherited lipid storage disorder affecting 1:100,000 births. This illness is caused by mutations in the galc gene encoding for the enzyme galactosylceramidase (GALC). Dysfunction of GALC has been linked to the toxic build-up of the galactolipid, galactosylsphingosine (psychosine), which induces cell death of oligodendrocytes. Previous studies show that phospholipase A2 (PLA2) may play a role in psychosine induce cell death. Here, we demonstrate that non-selective inhibition of cPLA2/sPLA2 and selective inhibition of cPLA2, but not sPLA2, also attenuates psychosine-induced cell death of human astrocytes. This study shows that extracellular calcium is required for psychosine induced cell death, but intracellular calcium release, reactive oxygen species or release of soluble factors are not involved. These findings suggest a cell autonomous effect, at least in human astrocytes. Supporting a role for PLA2 in psychosine-induced cell death of oligodendrocytes and astrocytes, the results show inhibition of PLA2 attenuates psychosine-induced decrease in the expression of astrocyte marker vimentin as well as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and the neuronal marker SMI-32 in organotypic slice cultures. These findings provide further mechanistic details of psychosine-induced death of glia and suggest a role for PLA2 in the process. This work also supports the proposal that novel drugs for Krabbe disease may require testing on astrocytes as well as oligodendrocytes for more holistic prediction of pre-clinical and clinical efficacy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187217PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667767PMC
December 2017

The chemokine fractalkine (CX3CL1) attenuates HO-induced demyelination in cerebellar slices.

J Neuroinflammation 2017 08 15;14(1):159. Epub 2017 Aug 15.

Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

Background: Fractalkine/CX3CR1 signalling has been implicated in many neurodegenerative and neurological diseases of the central nervous system (CNS). This signalling pathway plays an important role in regulating reactive oxygen species (ROS), as well as itself being altered in conditions of oxidative stress. Here, we investigated the effects of recombinant fractalkine (rCX3CL1) in models of hydrogen peroxide (HO)-induced demyelination and astrocyte toxicity, within organotypic cerebellar slice cultures.

Methods: Organotypic cerebellar slice cultures were generated from postnatal day 10 C57BL/6J mice to assess myelination. Immunohistochemistry was used to measure the degree of myelination. Fluorescent images were obtained using a leica SP8 confocal microscope and data analysed using ImageJ software.

Results: We show here, for the first time, that rCX3CL1 significantly attenuated bolus HO-induced demyelination as measured by expression of myelin basic protein (MBP) and attenuated reduced vimentin expression. Using the GOX-CAT system to continuously generate low levels of HO and induce demyelination, we observed similar protective effects of rCX3CL1 on MBP and MOG fluorescence, although in this model, the decrease in vimentin expression was not altered.

Conclusions: This data indicates possible protective effects of fractalkine signalling in oxidative stress-induced demyelination in the central nervous system. This opens up the possibility of fractalkine receptor (CX3CR1) modulation as a potential new target for protecting against oxidative stress-induced demyelination in both inflammatory and non-inflammatory nervous system disorders.
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http://dx.doi.org/10.1186/s12974-017-0932-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5558650PMC
August 2017

6th Annual Meeting Frontiers in Neurology 2016.

Authors:
Kumlesh K Dev

Ir J Med Sci 2017 Jul;186(Suppl 7):281-298

Department of Physiology Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

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http://dx.doi.org/10.1007/s11845-017-1645-5DOI Listing
July 2017

Demyelination induced by oxidative stress is regulated by sphingosine 1-phosphate receptors.

Glia 2017 07 4;65(7):1119-1136. Epub 2017 Apr 4.

Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

Oxidative stress is a pathological condition defined as an imbalance between production and removal of reactive oxygen species. This process causes structural cell damage, disrupts DNA repair and induces mitochondrial dysfunction. Many in vitro studies have used direct bolus application of H O to investigate the role of oxidative stress in cell culture. In this study, using mouse organotypic cerebellar slice cultures, the effects of H O -induced oxidative stress on myelination state were examined, using bolus concentrations of H O (0.1-1 mM) and low-continuous H O (∼20 μM) generated from glucose oxidase and catalase (GOX-CAT). Using these models, the potential therapeutic effects of pFTY720, an oral therapy used in multiple sclerosis, was also examined. We found bolus treatment of H O (0.5 mM) and, for the first time, low-continuous H O (GOX-CAT) to induce demyelination in organotypic slices. Both bolus H O and GOX-CAT treatments significantly decreased vimentin expression in these slice cultures as well as increased cell death in isolated astrocyte cultures. Importantly, pre-treatment with pFTY720 significantly attenuated both bolus H O and GOX-CAT-induced demyelination and the GOX-CAT-induced decrease in vimentin in cerebellar slices, without altering levels of the proinflammatory cytokines such as IL-6 and CX3CL1. We also observed increased SMI-32 immunoreactivity in the white matter tract induced by GOX-CAT indicating axonal damage, which was remarkably attenuated by pFTY720. Taken together, this data establishes a novel GOX-CAT model of demyelination and demonstrates that pFTY720 can act independently of inflammatory cytokines to attenuate decreases in vimentin, as well as axonal damage and demyelination induced by oxidative stress.
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http://dx.doi.org/10.1002/glia.23148DOI Listing
July 2017

Editorial (Thematic Issue: Common Receptor Signalling in Glial & Immune Cells).

Authors:
Kumlesh K Dev

Curr Drug Targets 2016;17(16):1828

Molecular Neuropharmacology Drug Development Deptartment of Physiology Neuroscience Institute Trinity College Dublin, Dublin Ireland.

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http://dx.doi.org/10.2174/138945011716161108230929DOI Listing
July 2018

Sphingosine-1-phosphate receptor therapies: Advances in clinical trials for CNS-related diseases.

Neuropharmacology 2017 Feb 4;113(Pt B):597-607. Epub 2016 Nov 4.

Drug Development, School of Medicine, Trinity College Dublin, Ireland. Electronic address:

The family of sphingosine-1-phosphate receptors (S1PRs) are G protein-coupled and comprise of five subtypes, S1P-S1P. These receptors are activated by the sphingolipid ligand, S1P, which is produced from the phosphorylation of sphingosine by sphingosine kinases. The activation of S1PRs modulates a host of cellular processes such as cell proliferation, migration and survival. These receptors are targeted by the drug fingolimod, a first in class oral therapy for multiple sclerosis. Importantly, S1PRs have also been implicated, in cellular experiments, pre-clinical studies and clinical trials in a range of other neurodegenerative diseases, neurological disorders and psychiatric illnesses, where S1PR drugs are proving beneficial. Overall, studies now highlight the importance of S1PRs as targets for modulating a variety of debilitating brain-related diseases. Here, we review the role of S1PRs in these illnesses. This article is part of the Special Issue entitled 'Lipid Sensing G Protein-Coupled Receptors in the CNS'.
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http://dx.doi.org/10.1016/j.neuropharm.2016.11.006DOI Listing
February 2017

Fractalkine shedding is mediated by p38 and the ADAM10 protease under pro-inflammatory conditions in human astrocytes.

J Neuroinflammation 2016 08 22;13(1):189. Epub 2016 Aug 22.

Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

Background: The fractalkine (CX3CR1) ligand is expressed in astrocytes and reported to be neuroprotective. When cleaved from the membrane, soluble fractalkine (sCX3CL1) activates the receptor CX3CR1. Although somewhat controversial, CX3CR1 is reported to be expressed in neurons and microglia. The membrane-bound form of CX3CL1 additionally acts as an adhesion molecule for microglia and infiltrating white blood cells. Much research has been done on the role of fractalkine in neuronal cells; however, little is known about the regulation of the CX3CL1 ligand in astrocytes.

Methods: The mechanisms involved in the up-regulation and cleavage of CX3CL1 from human astrocytes were investigated using immunocytochemistry, Q-PCR and ELISA. All statistical analysis was performed using GraphPad Prism 5.

Results: A combination of ADAM17 (TACE) and ADAM10 protease inhibitors was found to attenuate IL-1β-, TNF-α- and IFN-γ-induced sCX3CL1 levels in astrocytes. A specific ADAM10 (but not ADAM17) inhibitor also attenuated these effects, suggesting ADAM10 proteases induce release of sCX3CL1 from stimulated human astrocytes. A p38 MAPK inhibitor also attenuated the levels of sCX3CL1 upon treatment with IL-1β, TNF-α or IFN-γ. In addition, an IKKβ inhibitor significantly reduced the levels of sCX3CL1 induced by IL-1β or TNF-α in a concentration-dependent manner, suggesting a role for the NF-kB pathway.

Conclusions: In conclusion, this study shows that the release of soluble astrocytic fractalkine is regulated by ADAM10 proteases with p38 MAPK also playing a role in the fractalkine shedding event. These findings are important for understanding the role of CX3CL1 in healthy and stimulated astrocytes and may benefit our understanding of this pathway in neuro-inflammatory and neurodegenerative diseases.
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http://dx.doi.org/10.1186/s12974-016-0659-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4994207PMC
August 2016

In vitro and ex vivo models of multiple sclerosis.

Drug Discov Today 2016 09 3;21(9):1504-1511. Epub 2016 Jun 3.

Galway Neuroscience Centre, School of Natural Sciences, National University of Ireland, Galway, Ireland. Electronic address:

Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS). Current therapies suppress a misdirected myelin-destructive immune response. To combat the progressive, neurodestructive phase of MS, the therapeutic research focus is currently on compounds that might boost the endogenous potential of the brain to remyelinate axons, thereby achieving lesion repair. Here, we describe the testing of fingolimod on cultures of oligodendrocytes (OLs) and organotypic brain slices. We detail the protocols, pros, and cons of these in vitro and ex vivo approaches, along with the potential benefit of exploiting skin-punch biopsies from patients with MS, before concluding with a summary of future developments.
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http://dx.doi.org/10.1016/j.drudis.2016.05.018DOI Listing
September 2016

The EBI2 signalling pathway plays a role in cellular crosstalk between astrocytes and macrophages.

Sci Rep 2016 05 11;6:25520. Epub 2016 May 11.

Drug Development, School of Medicine, Trinity College, Dublin, Ireland.

EBI2 is a G protein-coupled receptor activated by oxysterol 7α, 25-dihydroxycholesterol (7α25HC) and regulates T cell-dependant antibody response and B cell migration. We recently found EBI2 is expressed in human astrocytes, regulates intracellular signalling and modulates astrocyte migration. Here, we report that LPS treatment of mouse astrocytes alters mRNA levels of EBI2 and oxysterols suggesting that the EBI2 signalling pathway is sensitive to LPS-mediated immune challenge. We also find that conditioned media obtained from LPS-stimulated mouse astrocytes induces macrophage migration, which is inhibited by the EBI2 antagonist NIBR189. These results demonstrate a role for the EBI2 signalling pathway in astrocytes as a sensor for immune challenge and for communication with innate immune cells such as macrophages.
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http://dx.doi.org/10.1038/srep25520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863252PMC
May 2016

"5th Annual Meeting Frontiers in Neurology 2015".

Authors:
Kumlesh K Dev

Ir J Med Sci 2016 May;185 Suppl 4:169-85

Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

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http://dx.doi.org/10.1007/s11845-016-1459-xDOI Listing
May 2016

The Role of the Oxysterol/EBI2 Pathway in the Immune and Central Nervous Systems.

Curr Drug Targets 2016 ;17(16):1851-1860

Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Forum 1, CH-4002, Switzerland.

Oxysterols are pleiotropic messengers interacting with multiple receptor systems. One of the cognate receptors for oxysterols is EBI2, a G protein-coupled receptor highly expressed in the cells of the immune system. Here we discuss the receptor's role in the adapted immunity and inflammation as well as the receptor's expression and function in the CNS with the focus on astrocytes. We also discuss expression and signalling of oxysterol-producing enzymes such as CH25H and CYP7B1 in the CNS and the immune system. These steps will help to elucidate a possible role for this pathway in the physiology of the central and peripheral nervous system and its possible link to human disease.
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http://dx.doi.org/10.2174/1389450117666160217123042DOI Listing
August 2017

The dual S1PR1/S1PR5 drug BAF312 (Siponimod) attenuates demyelination in organotypic slice cultures.

J Neuroinflammation 2016 Feb 8;13:31. Epub 2016 Feb 8.

Drug Development, School of Medicine, Trinity College, Dublin, Ireland.

Background: BAF312 (Siponimod) is a dual agonist at the sphingosine-1 phosphate receptors, S1PR1 and S1PR5. This drug is currently undergoing clinical trials for the treatment of secondary progressive multiple sclerosis (MS). Here, we investigated the effects of BAF312 on isolated astrocyte and microglia cultures as well as in slice culture models of demyelination.

Methods: Mouse and human astrocytes were treated with S1PR modulators and changes in the levels of pERK, pAkt, and calcium signalling as well as S1PR1 internalization and cytokine levels was investigated using Western blotting, immunochemistry, ELISA and confocal microscopy. Organotypic slice cultures were prepared from the cerebellum of 10-day-old mice and treated with lysophosphatidylcholine (LPC), psychosine and/or S1PR modulators, and changes in myelination states were measured by fluorescence of myelin basic protein and neurofilament H.

Results: BAF312 treatment of human and mouse astrocytes activated pERK, pAKT and Ca(2+) signalling as well as inducing S1PR1 internalization. Notably, activation of S1PR1 increased pERK and pAKT in mouse astrocytes while both S1PR1 and S1PR3 equally increased pERK and pAKT in human astrocytes, suggesting that the coupling of S1PR1 and S1PR3 to pERK and pAKT differ in mouse and human astrocytes. We also observed that BAF312 moderately attenuated lipopolysaccharide (LPS)- or TNFα/IL17-induced levels of IL6 in both astrocyte and microglia cell cultures. In organotypic slice cultures, BAF312 reduced LPC-induced levels of IL6 and attenuated LPC-mediated demyelination. We have shown previously that the toxic lipid metabolite psychosine induces demyelination in organotypic slice cultures, without altering the levels of cytokines, such as IL6. Importantly, psychosine-induced demyelination was also attenuated by BAF312.

Conclusions: Overall, this study suggests that BAF312 can modulate glial cell function and attenuate demyelination, highlighting this drug as a further potential therapy in demyelinating disorders, beyond MS.
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http://dx.doi.org/10.1186/s12974-016-0494-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746808PMC
February 2016

Galactosylsphingosine (psychosine)-induced demyelination is attenuated by sphingosine 1-phosphate signalling.

J Cell Sci 2015 Nov 10;128(21):3878-87. Epub 2015 Sep 10.

Drug Development, School of Medicine, Trinity College Dublin, Dublin D02 R590, Ireland

Globoid cell leukodystrophy (Krabbe disease) is a rare infantile neurodegenerative disorder. Krabbe disease is caused by deficiency in the lysosomal enzyme galactocerebrosidase (GALC) resulting in accumulation, in the micromolar range, of the toxic metabolite galactosylsphingosine (psychosine) in the brain. Here we find that psychosine induces human astrocyte cell death probably via an apoptotic process in a concentration- and time-dependent manner (EC50 ∼ 15 μM at 4 h). We show these effects of psychosine are attenuated by pre-treatment with the sphingosine 1-phosphate receptor agonist pFTY720 (fingolimod) (IC50 ∼ 100 nM). Psychosine (1 μM, 10 μM) also enhances LPS-induced (EC50 ∼ 100 ng/ml) production of pro-inflammatory cytokines in mouse astrocytes, which is also attenuated by pFTY720 (1 μM). Most notably, for the first time, we show that psychosine, at a concentration found in the brains of patients with Krabbe disease (EC50 ∼ 100 nM), directly induces demyelination in mouse organotypic cerebellar slices in a manner that is independent of pro-inflammatory cytokine response and that pFTY720 (0.1 nM) significantly inhibits. These results support the idea that psychosine is a pathogenic agent in Krabbe disease and suggest that sphingosine 1-phosphate signalling could be a potential drug target for this disorder.
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http://dx.doi.org/10.1242/jcs.169342DOI Listing
November 2015

Increased interleukin 23 (IL23) levels in schizophrenia patients treated with depot antipsychotic medication.

Cytokine 2015 May 10;73(1):196-8. Epub 2015 Mar 10.

Drug Development, School of Medicine, Trinity College Dublin, Ireland.

Background: Schizophrenia is a mental disorder, where genetic and environmental factors contribute to disease onset and progression. The immune system appears to play a role in schizophrenia, where altered cytokines levels and autoantibodies have been described. Notably however, to our knowledge, IL23 levels have not before been measured in schizophrenia patients treated with depot medication.

Methods: We examined IL23 levels in serum samples obtained from patients with schizophrenia, treated with depot medication (n=35) compared with healthy controls (n=38) and correlated these levels with treatment time, patient age and illness severity.

Results: IL23 levels were raised in depot treated groups compared with healthy controls. No correlation was observed, however, between IL23 levels and treatment time, patient age or illness severity.

Conclusions: IL23 levels are raised in schizophrenia patients prescribed with depot medication, supporting the role of aberrant cytokine signalling in schizophrenia.
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http://dx.doi.org/10.1016/j.cyto.2014.11.003DOI Listing
May 2015

EBI2 regulates intracellular signaling and migration in human astrocyte.

Glia 2015 Feb 9;63(2):341-51. Epub 2014 Oct 9.

Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

The G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2) is activated by 7α, 25-dihydroxycholesterol (7α25HC) and plays a role in T cell-dependant antibody response and B cell migration. Aberrant EBI2 signaling is implicated in a range of autoimmune disorders however its role in the CNS remains unknown. Here we characterize the functional role of EBI2 in GLIA cells using primary human astrocytes and EBI2 knockout animals. We find human and mouse astrocytes express EBI2 and the enzymes necessary for synthesis and degradation of 7α25HC. In astrocytes, EBI2 activation stimulates ERK phosphorylation, Ca(2+) signaling and induces cellular migration. These results, for the first time, demonstrate a role for EBI2 in astrocyte function and suggest that modulation of this receptor may be beneficial in neuroinflammatory disorders.
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http://dx.doi.org/10.1002/glia.22757DOI Listing
February 2015

The premium of a big pharma license deal.

Nat Biotechnol 2014 Jul;32(7):617-9

1] Drug Development, School of Medicine, Trinity College Dublin, Ireland [2] Venture Valuation AG, Zurich, Switzerland.

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http://dx.doi.org/10.1038/nbt.2946DOI Listing
July 2014

The use of cytokine signature patterns: separating drug naïve, interferon and natalizumab-treated multiple sclerosis patients.

Autoimmunity 2014 Dec 30;47(8):505-11. Epub 2014 Jun 30.

Drug Development, School of Medicine, Trinity College Dublin , Ireland .

Multiple sclerosis (MS) is an inflammatory illness characterized by demyelination and axonal neurodegeneration. Here, we used serum samples from MS patients to demonstrate if "cytokine signature" patterns can separate different patient groups better than using single cytokines. In this case, we used cytokine profiling to demonstrate if "cytokine signature" patterns can separate MS patients treated with interferon or natalizumab from drug naïve patients. Serum levels of eight individual cytokines (TNFα, IFNγ, S100B, IL-1β, IL-6, IL-8, IL-17 and IL-23) in MS patients treated with interferons (n = 11) and natalizumab (n = 14) were measured and, in general, showed reduced levels compared to drug naïve MS patients (n = 12). More evident changes were seen when analyzing "cytokine signatures" (i.e. summed value of all eight cytokines), which showed that patients treated with natalizumab and interferons showed significantly reduced cytokine signature levels than drug naïve MS patients. Moreover, patients treated with natalizumab were separated from drug naïve patients by almost 100% fidelity and that patients treated with natalizumab also had reduced levels of pro-inflammatory cytokines compared to patients treated with interferon. Overall, this study provides an example showing that the use of "cytokine signatures" may provide benefits over the analysis of single cytokines for the development of potential biomarkers.
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http://dx.doi.org/10.3109/08916934.2014.930734DOI Listing
December 2014

Fingolimod attenuates splenocyte-induced demyelination in cerebellar slice cultures.

PLoS One 2014 9;9(6):e99444. Epub 2014 Jun 9.

Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

The family of sphingosine-1-phosphate receptors (S1PRs) is G-protein-coupled, comprised of subtypes S1PR1-S1PR5 and activated by the endogenous ligand S1P. The phosphorylated version of Fingolimod (pFTY720), an oral therapy for multiple sclerosis (MS), induces S1PR1 internalisation in T cells, subsequent insensitivity to S1P gradients and sequestering of these cells within lymphoid organs, thus limiting immune response. S1PRs are also expressed in neuronal and glial cells where pFTY720 is suggested to directly protect against lysolecithin-induced deficits in myelination state in organotypic cerebellar slices. Of note, the effect of pFTY720 on immune cells already migrated into the CNS, prior to treatment, has not been well established. We have previously found that organotypic slice cultures do contain immune cells, which, in principle, could also be regulated by pFTY720 to maintain levels of myelin. Here, a mouse organotypic cerebellar slice and splenocyte co-culture model was thus used to investigate the effects of pFTY720 on splenocyte-induced demyelination. Spleen cells isolated from myelin oligodendrocyte glycoprotein immunised mice (MOG-splenocytes) or from 2D2 transgenic mice (2D2-splenocytes) both induced demyelination when co-cultured with mouse organotypic cerebellar slices, to a similar extent as lysolecithin. As expected, in vivo treatment of MOG-immunised mice with FTY720 inhibited demyelination induced by MOG-splenocytes. Importantly, in vitro treatment of MOG- and 2D2-splenocytes with pFTY720 also attenuated demyelination caused by these cells. In addition, while in vitro treatment of 2D2-splenocytes with pFTY720 did not alter cell phenotype, pFTY720 inhibited the release of the pro-inflammatory cytokines such as interferon gamma (IFNγ) and interleukin 6 (IL6) from these cells. This work suggests that treatment of splenocytes by pFTY720 attenuates demyelination and reduces pro-inflammatory cytokine release, which likely contributes to enhanced myelination state induced by pFTY720 in organotypic cerebellar slices.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099444PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049809PMC
October 2015

Targeting S1P receptors in experimental autoimmune encephalomyelitis in mice improves early deficits in locomotor activity and increases ultrasonic vocalisations.

Sci Rep 2014 May 23;4:5051. Epub 2014 May 23.

Drug Development, School of Medicine, Trinity College Dublin, Dublin, Ireland.

Fingolimod (FTY720) is an oral therapy for relapsing remitting multiple sclerosis (MS) and targets sphingosine 1-phosphate receptors (S1PRs). FTY720 also rescues animals from experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The protective effects of FTY720 in EAE are primarily scored manually by examining weight loss and limb paralysis that begins around 10-12 days after immunisation. To our knowledge, pre-clinical effects of FTY720 on animal behaviour early in EAE have not been explored. Here, we developed an automated behaviour monitoring system to examine the early effects of FTY720 on subtle pre-symptomatic behaviour of mice induced with EAE. Our automated home-cage monitoring system (AHC-MS) enabled non-contact detection of movement and ultrasonic vocalisations (USVs) of mice induced with EAE, thus allowing detection of subtle changes in mouse behaviour before paralysis occurs. Mice receiving FTY720 emit longer USVs and display higher levels of motor activity than vehicle-treated EAE mice before clinical symptoms become apparent. Importantly, this study promotes the 3Rs ethics (replacement, reduction and refinement) in the EAE animal model and may also improve pre-screening of potentially novel MS therapies. In addition, this is the first report showing the early effects of FTY720 in EAE which underscores its protective effects.
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http://dx.doi.org/10.1038/srep05051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031479PMC
May 2014