Lipidomic UPLC-MS/MS Profiles of Normal-Appearing White Matter Differentiate Primary and Secondary Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is a neurodegenerative inflammatory disease where an autoimmune response to components of the central nervous system leads to a loss of myelin and subsequent neurological deterioration. People with MS can develop primary or secondary progressive disease (PPMS, SPMS) and differentiation of the specific differences in the pathogenesis of these two courses, at the molecular level, is currently unclear. Recently, lipidomics studies using human biofluids, mainly plasma and cerebrospinal fluid, have highlighted a possible role for lipids in the initiation and progression of MS. However, there is a lack of lipidomics studies in MS on CNS tissues, such as normal-appearing white matter (NAWM), where local inflammation initially occurs. Herein, we developed an untargeted reverse phase ultra-performance liquid chromatography time of flight tandem mass spectrometry (RP-UPLC-TOF MSE)-based workflow, in combination with multivariate and univariate statistical analysis, to assess significant differences in lipid profiles in brain NAWM from post-mortem cases of PPMS, SPMS and controls. Groups of eight control, nine PPMS and seven SPMS NAWM samples were used. Correlation analysis of the identified lipids by RP-UPLC-TOF MSE was undertaken to remove those lipids that correlated with age, gender and post-mortem interval as confounding factors. We demonstrate that there is a significantly altered lipid profile of control cases compared with MS cases and that progressive disease, PPMS and SPMS, can be differentiated on the basis of the lipidome of NAWM with good sensitivity, specificity and prediction accuracy based on receiver operating characteristic (ROC) curve analysis. Metabolic pathway analysis revealed that the most altered lipid pathways between PPMS and SPMS were glycerophospholipid metabolism, glycerophosphatidyl inositol (GPI) anchor synthesis and linoleic acid metabolism. Further understanding of the impact of these lipid alterations described herein associated with progression will provide an increased understanding of the mechanisms underpinning progression and highlight possible new therapeutic targets.

Hypothermia increases aquaporin 4 (AQP4) plasma membrane abundance in human primary cortical astrocytes via a calcium/transient receptor potential vanilloid 4 (TRPV4)- and calmodulin-mediated mechanism.

Human aquaporin 4 (AQP4) is the primary water channel protein in brain astrocytes. Hypothermia is known to cause astrocyte swelling in culture, but the precise role of AQP4 in this process is unknown. Primary human cortical astrocytes were cultured under hypothermic (32 °C) or normothermic (37 °C) conditions. AQP4 transcript, total protein and surface-localized protein were quantified using RT-qPCR, sandwich ELISA with whole cell lysates or cell surface biotinylation, followed by ELISA analysis of the surface-localized protein, respectively. Four-hour mild hypothermic treatment increased the surface localization of AQP4 in human astrocytes to 155 ± 4% of normothermic controls, despite no change in total protein expression levels. The hypothermia-mediated increase in AQP4 surface abundance on human astrocytes was blocked using either calmodulin antagonist (trifluoperazine, TFP); TRPV4 antagonist, HC-067047 or calcium chelation using EGTA-AM. The TRPV4 agonist (GSK1016790A) mimicked the effect of hypothermia compared with untreated normothermic astrocytes. Hypothermia led to an increase in surface localization of AQP4 in human astrocytes through a mechanism likely dependent on the TRPV4 calcium channel and calmodulin activation. Understanding the effects of hypothermia on astrocytic AQP4 cell surface expression may help develop new treatments for brain swelling based on an in-depth mechanistic understanding of AQP4 translocation.

Transcriptome analysis suggests a role for the differential expression of cerebral aquaporins and the MAPK signalling pathway in human temporal lobe epilepsy.

Epilepsies are common disorders of the central nervous system (CNS), affecting up to 2% of the global population. Pharmaco-resistance is a major clinical challenge affecting about 30% of temporal lobe epilepsy (TLE) patients. Water homeostasis has been shown crucial for regulation of neuronal excitability. The control of water movement is achieved through a family of small integral membrane channel proteins called aquaporins (AQPs). Despite the fact that changes in water homeostasis occur in sclerotic hippocampi of people with TLE, the expression of AQPs in the epileptic brain is not fully characterised. This study uses microarray and ELISA methods to analyse the mRNA and protein expression of the human cerebral AQPs in sclerotic hippocampi (TLE-HS) and adjacent neocortex tissue (TLE-NC) of TLE patients. The expression of AQP1 and AQP4 transcripts was significantly increased, while that of the AQP9 transcript was significantly reduced in TLE-HS compared to TLE-NC. AQP4 protein expression was also increased while expression of AQP1 protein remained unchanged, and AQP9 was undetected. Microarray data analysis identified 3333 differentially regulated genes and suggested the involvement of the MAPK signalling pathway in TLE pathogenesis. Proteome array data validated the translational profile for 26 genes and within the MAPK pathway (e.g. p38, JNK) that were identified as differentially expressed from microarray analysis. ELISA data showed that p38 and JNK inhibitors decrease AQP4 protein levels in cultured human primary cortical astrocytes. Elucidating the mechanism of selective regulation of different AQPs and associated regulatory proteins may provide a new therapeutic approach to epilepsy treatment.

Transcriptome Analysis of Gene Expression Provides New Insights into the Effect of Mild Therapeutic Hypothermia on Primary Human Cortical Astrocytes Cultured under Hypoxia.

Hypothermia is increasingly used as a therapeutic measure to treat brain injury. However, the cellular mechanisms underpinning its actions are complex and are not yet fully elucidated. Astrocytes are the most abundant cell type in the brain and are likely to play a critical role. In this study, transcriptional changes and the protein expression profile of human primary cortical astrocytes cultured under hypoxic conditions for 6 h were investigated. Cells were treated either with or without a mild hypothermic intervention 2 h post-insult to mimic the treatment of patients following traumatic brain injury (TBI) and/or stroke. Using human gene expression microarrays, 411 differentially expressed genes were identified following hypothermic treatment of astrocytes following a 2 h hypoxic insult. KEGG pathway analysis indicated that these genes were mainly enriched in the Wnt and p53 signaling pathways, which were inhibited following hypothermic intervention. The expression levels of 168 genes involved in Wnt signaling were validated by quantitative real-time-PCR (qPCR). Among these genes, 10 were up-regulated and 32 were down-regulated with the remainder unchanged. Two of the differentially expressed genes (DEGs), p38 and JNK, were selected for validation at the protein level using cell based ELISA. Hypothermic intervention significantly down-regulated total protein levels for the gene products of p38 and JNK. Moreover, hypothermia significantly up-regulated the phosphorylated (activated) forms of JNK protein, while downregulating phosphorylation of p38 protein. Within the p53 signaling pathway, 35 human apoptosis-related proteins closely associated with Wnt signaling were investigated using a Proteome Profiling Array. Hypothermic intervention significantly down-regulated 18 proteins, while upregulating one protein, survivin. Hypothermia is a complex intervention; this study provides the first detailed longitudinal investigation at the transcript and protein expression levels of the molecular effects of therapeutic hypothermic intervention on hypoxic human primary cortical astrocytes. The identified genes and proteins are targets for detailed functional studies, which may help to develop new treatments for brain injury based on an in-depth mechanistic understanding of the astrocytic response to hypoxia and/or hypothermia.

Gene expression profiling of the astrocyte transcriptome in multiple sclerosis normal appearing white matter reveals a neuroprotective role.

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). White matter lesions in MS are surrounded by areas of non-demyelinated normal appearing white matter (NAWM) with complex pathology, including blood brain barrier dysfunction, axonal damage and glial activation. Astrocytes, the most abundant cell type within the CNS, may respond and/or contribute to lesion pathogenesis. We aimed to characterise the transcriptomic profile of astrocytes in NAWM to determine whether specific glial changes exist in the NAWM which contribute to lesion development or prevent disease progression. Astrocytes were isolated from control and NAWM by laser capture microdissection (LCM), using glial fibrillary acidic protein (GFAP) as a marker, and the astrocyte transcriptome determined using microarray analysis. 452 genes were significantly differentially expressed (208 up-regulated and 244 down-regulated, FC≥1.5 and p-value≤0.05). Within the NAWM, astrocytes were associated with significant upregulation of genes involved in the control of iron homeostasis (including metallothionein-1 and -2, ferritin light chain and transferrin), oxidative stress responses, the immune response and neurotrophic support. These findings suggest a neuroprotective role of astrocytes in the NAWM in MS.

Localisation of citrullinated proteins in normal appearing white matter and lesions in the central nervous system in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease, considered to be autoimmune in origin. Post-translational modification of central nervous system proteins, including glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP), through citrullination of arginine residues, may lead to exposure of neoepitopes, triggering autoimmunity. Here we investigated the expression of citrullinated proteins in active MS lesions, MS normal appearing white matter and control brain white matter. We demonstrate increased citrullinated GFAP and MBP by immunohistochemistry and western blotting in areas of ongoing demyelination, suggesting a pivotal role for deimination of GFAP and MBP in MS pathogenesis MS.

Innate and adaptive immune responses in neurodegeneration and repair.

Emerging evidence suggests important roles of the innate and adaptive immune responses in the central nervous system (CNS) in neurodegenerative diseases. In this special review issue, five leading researchers discuss the evidence for the beneficial as well as the detrimental impact of the immune system in the CNS in disorders including Alzheimer's disease, multiple sclerosis and CNS injury. Several common pathological mechanisms emerge indicating that these pathways could provide important targets for manipulating the immune reposes in neurodegenerative disorders. The articles highlight the role of the traditional resident immune cell of the CNS - the microglia - as well as the role of other glia astrocytes and oligodendrocytes in immune responses and their interplay with other immune cells including, mast cells, T cells and B cells. Future research should lead to new discoveries which highlight targets for therapeutic interventions which may be applicable to a range of neurodegenerative diseases.

Effect of Testosterone on Inflammatory Markers in the Development of Early Atherogenesis in the Testicular-Feminized Mouse Model.

BACKGROUND: Low levels of serum testosterone in men are associated with cardiovascular disease. Clinical studies show that testosterone replacement therapy (TRT) can improve symptoms of cardiovascular disease and reduce the inflammatory burden evident in atherosclerosis. AIM: We used an in vivo animal model to determine whether testosterone influences mediators of vascular inflammation as part of its beneficial effects on atherogenesis. METHODS: Testicular-feminized (Tfm) mice, which express low endogenous testosterone and a non-functional androgen receptor (AR), were used to assess the effect of androgen status on atheroma formation, serum lipids, and inflammatory mediators. Tfm mice were fed a high-cholesterol diet, received saline or physiological (TRT), and were compared to saline-treated XY littermates. RESULTS: A total of 28 weeks of high-cholesterol diet caused fatty streak formation in the aortic root of XY littermates and Tfm mice, an effect significantly amplified in Tfm mice. Tfm mice on normal diet showed elevated serum tumor necrosis factor-α (TFN-α) and interleukin-6 compared to XY littermates. High-cholesterol diet induced increased monocyte chemoattractant protein-1 (MCP-1) in Tfm mice, and TFN-α and MCP-1 in XY littermates. TRT reduced fatty streak formation and serum interleukin-6 in Tfm mice but had no significant effects on lipid profiles. Monocyte/macrophage staining indicated local inflammation in aortic root fatty streak areas of all mice, with TRT reducing local inflammation through plaque reduction in Tfm mice. Fractalkine (CX3CL1) and its receptor (CX3CR1) were present in fatty streaks of all mice fed a high-cholesterol diet, independent of androgen status. CONCLUSION: These results are consistent with AR-dependent and AR-independent anti-inflammatory actions of testosterone in atheroprotection, although the local anti-inflammatory mechanisms via which testosterone acts remain unknown.

siRNA knockdown of ADAM-10, but not ADAM-17, significantly reduces fractalkine shedding following pro-inflammatory cytokine treatment in a human adult brain endothelial cell line.

Fractalkine shedding is believed to occur constitutively and following induction via the activity of two membrane-bound enzymes, ADAM-10 and ADAM-17. However, our previous work suggested that ADAM-17 is not involved in the proteolytic release of fractalkine under TNF treatment of a human adult brain endothelial cell line, hCMEC/D3. The pro-inflammatory cytokine, TNF, has previously been shown to be expressed in the perivascular cuffs in multiple sclerosis. Here we sought to identify, using siRNAs to silence the expression of ADAM-10 and ADAM-17, whether ADAM-10 is responsible for TNF-induced shedding of fractalkine from the cell membrane in hCMEC/D3. Our findings suggest that ADAM-10, and not ADAM-17, is the major protease involved in fractalkine release under pro-inflammatory conditions in this human adult brain endothelial cell model.

IL-1ß down-regulates ADAMTS-13 mRNA expression in cells of the central nervous system.

ADAMTS-13 is the Von Willebrand factor (vWF) cleaving protease, responsible for the cleavage and down-regulation of the pro-thrombotic properties of ultra large VWF multimers. It is expressed predominantly by the hepatic stellate cells of the liver, but is also found to be expressed in other tissues, including brain. Reduced ADAMTS-13 is associated with a variety of thrombotic microangiopathies. Since the cellular origin and regulation of ADAMTS-13 expression in the brain is unknown, we aimed to investigate this in four different central nervous system (CNS)-derived cell lines, SHSY-5Y (human neuroblastoma), U373 (human astroglioma), CHME-3 (human foetal microglia) and hCMEC/D3 (adult human brain endothelial cells). All cell lines expressed ADAMTS-13 mRNA constitutively with neuroblastoma cells showing the highest expression. Interleukin (IL)-1ß down-regulated ADAMTS-13 mRNA expression in astroglioma cells and microglial cells whereas TNF and IL-6 treatment showed no significant differences in ADAMTS-13 mRNA expression in any cell line tested. ADAMTS-13 protein expression was reduced in a dose-dependent manner only in astroglioma cells following stimulation by IL-1ß. The ability of IL-1ß to significantly reduce ADAMTS-13 mRNA expression in human microglia and astroglioma cells suggests a role in the haemostasis of the local microenvironment under inflammatory conditions. This is the first report of ADAMTS-13 expression in cells of the CNS; however, its function remains to be determined.

MALDI-MS imaging of lipids in ex vivo human skin.

Lipidomics is a rapidly expanding area of scientific research and there are a number of analytical techniques that are employed to facilitate investigations. One such technique is matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Previous MALDI-MS studies involving lipidomic investigation have included the analysis of a number of different ex vivo tissues, most of which were obtained from animal models, with only a few being of human origin. In this study, we describe the use of MALDI-MS, MS/MS and MS imaging methods for analysing lipids within cross-sections of ex vivo human skin. It has been possible to tentatively identify lipid species via accurate mass measurement MALDI-MS and also to confirm the identity of a number of these species via MALDI-MS/MS, in experiments carried out directly on tissue. The main lipid species detected include glycerophospholipids and sphingolipids. MALDI images have been generated at a spatial resolution of 150 and 30 µm, using a MALDI quadrupole time-of-flight Q-Star Pulsar-i (TM) (Applied Biosystems/MDS Sciex, Concord, ON, Canada) and a MALDI high-definition MS (HDMS) SYNAPT G2-HDMS(TM) system (Waters, Manchester, UK), respectively. These images show the normal distribution of lipids within human skin, which will provide the basis for assessing alterations in lipid profiles linked to specific skin conditions e.g. sensitisation, in future investigations.

ADAMTS-9 expression is up-regulated following transient middle cerebral artery occlusion (tMCAo) in the rat.

The ADAMTS enzymes (a disintegrin and metalloproteinase with thrombospondin type 1-like motifs) have important roles in central nervous system (CNS) physiology and pathology. This current study aimed to analyse the expression of ADAMTS-9 following transient middle cerebral artery occlusion (tMCAo) in the rat, a model of focal cerebral ischaemia. Using real-time RT-PCR, ADAMTS-9 mRNA was demonstrated to be significantly up-regulated in tMCAo brain tissue compared to sham-operated at 24h post-ischaemia. The mature form of the ADAMTS-9 protein was only detected by Western blotting in brains subjected to tMCAo at 24h. In situ hybridisation demonstrated that ADAMTS-9 mRNA was expressed by neurones in tMCAo tissue. This study indicates that ADAMTS-9 expression is modulated in response to cerebral ischaemia in vivo and further research will resolve whether it plays a role in the subsequent degenerative or repair processes.

Expression of ADAM-17, TIMP-3 and fractalkine in the human adult brain endothelial cell line, hCMEC/D3, following pro-inflammatory cytokine treatment.

ADAM-17 expression is localised to endothelial cells in the human central nervous system (CNS) and is increased in multiple sclerosis (MS) white matter, suggesting a role in MS pathogenesis. Expression of ADAM-17, TIMP-3, and fractalkine were investigated in a human brain endothelial cell line (hCMEC/D3) after pro-inflammatory cytokine treatment. Tumour necrosis factor (TNF) significantly increased fractalkine mRNA (>100 fold) and protein expression, which was associated with increased shedding of fractalkine from the cell. Fractalkine shedding may regulate immune cell trafficking into the CNS, however, this does not appear to be directly controlled by ADAM-17 activity.

Cleavage of chemokines CCL2 and CXCL10 by matrix metalloproteinases-2 and -9: implications for chemotaxis.

Proteolytic processing of chemokines is a complex process that can result in dramatic effects on their chemotactic activity. Results from gel electrophoresis and mass spectrometry using recombinant CCL2 and CXCL10, incubated with either MMP-2 or -9, indicate that both chemokines are cleaved by the enzymes. N-terminal truncation of four amino acids from CCL2, and four or five residues from CXCL10 occurred, but removal of four residues from the C-terminus of CXCL10 was also observed with both MMPs. The speed of the reaction was chemokine-dependent, with N-terminal processing of CCL2 being complete within 3h, whereas activity of the MMPs on CXCL10 remained incomplete at 48h. The effect on the chemotactic potential of N-terminal truncation of CCL2 by MMPs-2 and -9 was investigated using in vitro migration assays. Monocytic cells exhibited a 2-fold reduction in migration to MMP-cleaved CCL2 variants, compared to intact CCL2.

ADAM-17 and TIMP3 protein and mRNA expression in spinal cord white matter of rats with acute experimental autoimmune encephalomyelitis.

Tumour necrosis factor (TNF) is a major immunomodulatory and proinflammatory cytokine implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). ADAM-17 cleaves membrane-bound TNF into its soluble form. The distribution and level of ADAM-17 expression within spinal cords of Lewis rats with EAE was investigated. ADAM-17 was associated with endothelial cells in the naïve and pre-disease spinal cords. In peak disease astrocytic and inflammatory cells expressed ADAM-17. Upregulation of ADAM-17 mRNA expression was coupled with a decrease in mRNA levels of its inhibitor TIMP3 suggesting a role for ADAM-17 in EAE pathogenesis.

Detection and localization of chemokine gene expression in autoimmune thyroid disease.

OBJECTIVE: Autoimmune thyroid disease (Hashimoto's thyroiditis and Graves' disease) is characterized by lymphocytic infiltration of the thyroid gland. Chemokines are cytokines with chemoattractant properties for a range of immune effector cells and might therefore play a significant role in the initiation and maintenance of the autoimmune process. The aim of this study was to analyse chemokine gene expression in autoimmune thyroid tissue and in cultured thyroid follicular cells (TFC). DESIGN AND PATIENTS: Immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) amplification were used to analyse the expression of monocyte chemoattractant protein (MCP)-1, RANTES (regulated upon activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, interferon (IFN)-gamma-inducible protein (IP)-10 and monokine induced by IFN-gamma (Mig) in thyroid tissue from patients with Hashimoto's thyroiditis (n = 4), Graves' disease (n = 6) and nonautoimmune multinodular goitre (n = 4). Chemokine gene expression was also examined in cultured TFC by RT-PCR. RESULTS: Expression of MCP-1, RANTES, MIP-1 alpha, MIP-1 beta, IP-10 and Mig was demonstrated in all Hashimoto's and most Graves' thyroid specimens but very little expression was detected in the nonautoimmune goitre samples. In thyroid tissue from Graves' disease patients, positive staining for chemokines was largely restricted to the lymphocytic cell infiltrate. Within thyroid tissue from Hashimoto's patients, there was evidence for the expression of all chemokines by thyroid follicular cells, suggesting a role for local chemokine synthesis by the glandular epithelial cells in the recruitment of inflammatory cells into the gland in autoimmunity. The present work also showed that expression all the chemokine genes analysed could be induced in cultured thyroid cells by IFN-gamma and interleukin (IL)-1 alpha. Expression of all the chemokines examined was not stimulated by TSH. CONCLUSION: We postulate that TFC may play a role in the pathogenesis of autoimmune thyroid disease as they are able to express the chemokines MIP-1 alpha, MIP-1 beta, MCP-1, RANTES, IP-10 and Mig that would promote the infiltration of immune cells into the thyroid gland.