Effect of the CSF1R inhibitor PLX3397 on remyelination of corpus callosum in a cuprizone-induced demyelination mouse model

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system (CNS). Despite introducing multiple immunomodulatory approaches for MS, there are still major concerns about possible ways for improving remyelination in this disease. Microglia exert essential roles in regulation of myelination processes, and interaction between colony-stimulating factor 1 (CSF1) with its receptor CSF1R is considered as a key regulator of microglial differentiation and survival. The aim of this study was to investigate possible roles for a CSF1R inhibitor PLX3397 in recovery of central myelination processes. Chronic demyelination was induced in mice by addition of 0.2% cuprizone to the chow for 12 weeks. Next, animals were undergoing a diet containing 290 mg/kg PLX3397 to induce microglial ablation. The PLX3397 treatment caused a significant decrease in the rate of expression for the CSF1/CSF1R axis, and a reduction in the protein expressions for the microglial marker Iba-1 and for the oligodendrocyte marker Olig-2. Findings from Luxol fast blue (LFB) staining and transmission electron microscopy (TEM) showed an increase in the rate of myelination for the mice receiving PLX3397. The rate of destruction in the nerve fibers and the extent of the gaps formed between layers of myelin sheaths was also reduced after the treatment with PLX3397. In addition, animals experienced an improvement in recovery of motor deficit after receiving PLX3397 (for all P < 0.05). It could be concluded that PLX3397 could retain myelination in the MS model possibly through regulation of the myelin environment.     

Soluble BAFF levels inversely correlate with peripheral B cell numbers and the expression of BAFF receptors

The TNF family member protein BAFF/BLyS is essential for B cell survival and plays an important role in regulating class switch recombination as well as in the selection of autoreactive B cells. In humans, increased concentrations of soluble BAFF are found in different pathological conditions, which may be as diverse as autoimmune diseases, B cell malignancies, and primary Ab deficiencies (PAD). Because the mechanisms that regulate BAFF levels are not well understood, we newly developed a set of mAbs against human BAFF to study the parameters that determine the concentrations of soluble BAFF in circulation. Patients with PAD, including severe functional B cell defects such as BTK, BAFF-R, or TACI deficiency, were found to have higher BAFF levels than asplenic individuals, patients after anti-CD20 B cell depletion, chronic lymphocytic leukemia patients, or healthy donors. In a comparable manner, mice constitutively expressing human BAFF were found to have higher concentrations of BAFF in the absence than in the presence of B cells. Therefore, our data strongly suggest that BAFF steady-state concentrations mainly depend on the number of B cells as well as on the expression of BAFF-binding receptors. Because most patients with PAD have high levels of circulating BAFF, the increase in BAFF concentrations cannot compensate defects in B cell development and function.     

GCH1-regulated miRNAs are potential targets for microglial activation in neuropathic pain

Neuropathic pain (NP) is a chronic pain directly caused by injury or disease of the somatosensory nervous system. Previous studies suggest that GTP cyclohydrolase I (GCH1) may play a pivotal role in microglial activation, which has been shown to be essential for NP. However, its underlying mechanisms in microglial activation remain unclear. A wide range of microRNAs (miRNAs) have been found to be involved in microglial activation-induced NP. To identify the miRNAs regulated by GCH1 and predict their functions in the progression of microglial activation, we analyzed the miRNA expression profiles of GCH1-knockdown (KD) BV2 microglial cells. Small RNA-sequencing analysis revealed 13 differentially expressed (DE) miRNAs in GCH1-KD cells. The target genes of DE miRNAs mainly participate in PI3K-Akt signaling pathway, peroxisome and ferroptosis. The miRNA–mRNA regulatory network analysis showed that GCH1, MAP4K5 and YWHAB acted as hub genes. qRT-PCR results further verified the expression levels of mmu-miR-1a-3p, mmu-miR-133a-3p, mmu-miR-7a-5p and mmu-miR-10a-5p in GCH1-KD cells, which were consistent with the sequencing data. In addition, our data indicated that overexpression of mmu-miR-133a-3p alleviated the pro-inflammatory cytokines IL-1β and IL-6 production induced by lipopolysaccharide (LPS), indicating that mmu-miR-133a-3p has a negative effect on microglial activation. Taken together, our findings suggest that many miRNAs regulated by GCH1 may be involved in microglial activation, which may provide new potential targets for GCH1 in the pathogenesis of NP.     

Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase–AIF pathway

PADs (peptidylarginine deiminases) are calcium-dependent enzymes that change protein-bound arginine to citrulline (citrullination/deimination) affecting protein conformation and function. PAD up-regulation following chick spinal cord injury has been linked to extensive tissue damage and loss of regenerative capability. Having found that human neural stem cells (hNSCs) expressed PAD2 and PAD3, we studied PAD function in these cells and investigated PAD3 as a potential target for neuroprotection by mimicking calcium-induced secondary injury responses. We show that PAD3, rather than PAD2 is a modulator of cell growth/death and that PAD activity is not associated with caspase-3-dependent cell death, but is required for AIF (apoptosis inducing factor)-mediated apoptosis. PAD inhibition prevents association of PAD3 with AIF and AIF cleavage required for its translocation to the nucleus. Finally, PAD inhibition also hinders calcium-induced cytoskeleton disassembly and association of PAD3 with vimentin, that we show to be associated also with AIF; together this suggests that PAD-dependent cytoskeleton disassembly may play a role in AIF translocation to the nucleus. This is the first study highlighting a role of PAD activity in balancing hNSC survival/death, identifying PAD3 as an important upstream regulator of calcium-induced apoptosis, which could be targeted to reduce neural loss, and shedding light on the mechanisms involved.     

Anti-inflammatory mechanism of exogenous C2 ceramide in lipopolysaccharide-stimulated microglia

Ceramide is a major molecule among the sphingolipid metabolites which are produced in the brain and other organs and act as intracellular second messengers. Although a variety of physiological roles of ceramide have been reported in the periphery and central nervous systems, the role of ceramide in microglial activation has not been clearly demonstrated. In the present study, we examined the effects of exogenous cell permeable short chain ceramides on microglial activation in vitro and in vivo. We found that C2, C6, and C8 ceramide and C8 ceramide-1-phosphate inhibited iNOS and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia. In addition, the administration of C2 ceramide suppressed microglial activation in the brains of LPS-exposed mice. By HPLC and LC/MS/MS analyses, we found that C2 ceramide on its own, rather than its modified form (i.e. ceramide-1-phosphate or long chain ceramides), mainly work by penetrating into microglial cells. Further mechanistic studies by using the most effective C2 ceramide among the short chain ceramides tested, revealed that C2 ceramide exerts anti-inflammatory effects via inhibition of the ROS, MAPKs, PI3K/Akt, and Jak/STAT pathways with upregulation of PKA and hemeoxygenase-1 expressions. Interestingly, we found that C2 ceramide inhibits TLR4 signaling by interfering with LPS and TLR4 interactions. Therefore, our data collectively suggests the therapeutic potential of short chain ceramides such as C2 for neuroinflammatory disorders such as Alzheimer's disease and Parkinson's disease.     

Modulation of the cannabinoid CB2 receptor in microglial cells in response to inflammatory stimuli

The cannabinoid system is known to be important in neuronal regulation, but is also capable of modulating immune function. Although the CNS resident microglial cells have been shown to express the CB2 subtype of cannabinoid receptor during non-immune-mediated pathological conditions, little is known about the expression of the cannabinoid system during immune-mediated CNS pathology. To examine this question, we measured CB2 receptor mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) and, by real-time PCR, found a 100-fold increase in CB2 receptor mRNA expression during EAE onset. We next determined whether microglial cells specifically express the CB2 receptor during EAE, and found that activated microglial cells expressed 10-fold more CB2 receptor than microglia in the resting state. To determine the signals required for the up-regulation of the CB2 receptor, we cultured microglial cells with combinations of gamma-interferon (IFN-gamma) and granulocyte) macrophage-colony stimulating factor (GM-CSF), which both promote microglial cell activation and are expressed in the CNS during EAE, and found that they synergized, resulting in an eight to 10-fold increase in the CB2 receptor. We found no difference in the amount of the CB2 receptor ligand, 2-arachidonylglycerol (2-AG), in the spinal cord during EAE. These data demonstrate that microglial cell activation is accompanied by CB2 receptor up-regulation, suggesting that this receptor plays an important role in microglial cell function in the CNS during autoimmune-induced inflammation.     

Morphological studies on neuroglia

The postnatal development of microglial cells was investigated in the neonatal rat brain by use of light- and electron microscopy, including enzyme-histochemical techniques. Microglial cells were selectively stained by demonstration of their nucleoside diphosphatase (NDPase) activity and classified into three types: 1) In the early postnatal period "primitive microglial cells" showing scantily ramified processes were found in the cerebral cortex, the hippocampal formation, and the hypothalamus. During the course of the first postnatal week the processes of this cell type developed gradually and the cells were transformed into typical ramified microglial cells, called "resting microglial cells". 2) "Amoeboid microglial cells "showing typical features of macrophages were characteristic of the cerebral white matter. 3) "Round microglial cells" possessing a round soma and few pseudopodia but no characteristic processes occurred in large numbers in the subventricular zone of the lateral ventricle and as single elements in the vicinity of blood vessels. Histochemically, thiamine pyrophosphatase (TPPase) was demonstrated only in the fully developed, ramified microglial cells ("resting microglial cells"), which could be readily observed in the central nervous tissue from the age of 14 day. "Round and amoeboid microglial cells" did not show TPPase activity and disappeared after 14 days of postnatal life. By use of electron microscopy, in neonatal rats NDPase activity was apparent in the plasma membrane of the three types of microglial cells ("primitive, round, and amoeboid" types). They showed basically similar submicroscopic characteristics, i.e., well-developed Golgi apparatus, long strands of rough-surfaced endoplasmic reticulum, single dense bodies and vacuoles, and numerous ribosomes. "Amoeboid microglial cells" were characterized by their well-developed cytoplasmic vacuoles and phagocytic inclusion bodies. The present study strongly suggests a mesodermal origin for these microglial elements.     

Potential Role for PAD2 in Gene Regulation in Breast Cancer Cells

The peptidylarginine deiminase (PAD) family of enzymes post-translationally convert positively charged arginine residues in substrate proteins to the neutral, non-standard residue citrulline. PAD family members 1, 2, 3, and 6 have previously been localized to the cell cytoplasm and, thus, their potential to regulate gene activity has not been described. We recently demonstrated that PAD2 is expressed in the canine mammary gland epithelium and that levels of histone citrullination in this tissue correlate with PAD2 expression. Given these observations, we decided to test whether PAD2 might localize to the nuclear compartment of the human mammary epithelium and regulate gene activity in these cells. Here we show, for the first time, that PAD2 is specifically expressed in human mammary gland epithelial cells and that a portion of PAD2 associates with chromatin in MCF-7 breast cancer cells. We investigated a potential nuclear function for PAD2 by microarray, qPCR, and chromatin immunoprecipitation analysis. Results show that the expression of a unique subset of genes is disregulated following depletion of PAD2 from MCF-7 cells. Further, ChIP analysis of two of the most highly up- and down-regulated genes (PTN and MAGEA12, respectively) found that PAD2 binds directly to these gene promoters and that the likely mechanism by which PAD2 regulates expression of these genes is via citrullination of arginine residues 2-8-17 on histone H3 tails. Thus, our findings define a novel role for PAD2 in gene expression in human mammary epithelial cells.     

Expression pattern of peptidylarginine deiminase in rat and human Schwann cells

The peptidylarginine deiminase (PAD) family of enzymes are responsible for conversion of protein-bound arginine to citrulline in most tissues of the body and are garnering increased interest for their physiological and pathological roles. Although it has been shown that oligodendrocytes of the CNS express the PAD isoenzyme type 2, nothing is presently known about PAD expression in Schwann cells, the myelinating cells of the PNS. To evaluate PAD expression in the PNS, cultivated rat and human Schwann cells and slices of fetal, juvenile, and normal and regenerated adult sciatic nerves were examined with RT-PCR, Western blot, and immunohistochemical analysis. Samples from cerebellar cultures and skin served as positive controls. One of the principle findings was that cultivated Schwann cells expressed significant levels of mRNA and protein for the PAD isoenzymes 2 and 3. PAD1 and PAD4, however, were not expressed in any types of Schwann cells. Using double immunofluorescence, the majority of PAD2 staining was localized in immature cell stages. Moreover, increased amounts of PAD2, PAD3, and peptidyl-citrulline were also found in human fetal and rat juvenile and regenerated sciatic nerves as compared to similar normal adult specimens. Neuronal and inducible nitric oxide synthases, enzymes that convert free arginine to citrulline, were also expressed in Schwann cells; however, their massive induction by LPS/K(+), was not reflected in an enhanced peptidyl-citrulline immunosignal. These data suggest that, similar to the CNS, citrullination of proteins may also exert a specific role in thecourse of PNS development and repair.     

Gas6 increases myelination by oligodendrocytes and its deficiency delays recovery following cuprizone-induced demyelination

Multiple sclerosis (MS) is a complex demyelinating disease of the central nervous system. Current research has shown that at least in some cases, the primary insult in MS could be directed at the oligodendrocyte, and that the earliest immune responses are primarily via innate immune cells. We have identified a family of receptor protein tyrosine kinases, known as the TAM receptors (Tyro3, Axl and Mertk), as potentially important in regulating both the oligodendrocyte and immune responses. We have previously shown that Gas6, a ligand for the TAM receptors, can affect the severity of demyelination in mice, with a loss of signalling via Gas6 leading to decreased oligodendrocyte survival and increased microglial activation during cuprizone-induced demyelination. We hypothesised TAM receptor signalling would also influence the extent of recovery in mice following demyelination. A significant effect of the absence of Gas6 was detected upon remyelination, with a lower level of myelination after 4 weeks of recovery in comparison with wild-type mice. The delay in remyelination was accompanied by a reduction in oligodendrocyte numbers. To understand the molecular mechanisms that drive the observed effects, we also examined the effect of exogenous Gas6 in in vitro myelination assays. We found that Gas6 significantly increased myelination in a dose-dependent manner, suggesting that TAM receptor signalling could be directly involved in myelination by oligodendrocytes. The reduced rate of remyelination in the absence of Gas6 could thus result from a lack of Gas6 at a critical time during myelin production after injury. These findings establish Gas6 as an important regulator of both CNS demyelination and remyelination.     

Peptidylarginine deiminase expression and activity in PAD2 knock-out and PAD4-low mice

Citrullination, the conversion of peptidylarginine to peptidylcitrulline is catalyzed by peptidylarginine deiminases (PAD). The expression of PAD isoforms displays great variation among different tissues as demonstrated by PAD mRNA analyses. Here we have analyzed the differential expression of PAD2, PAD4 and PAD6 in mouse tissues at the protein level and by enzymatic activity assays using PAD2 and PAD4 knock-out strains. As expected, no PAD2 expression was detected in the PAD2−/− mice. In contrast, the PAD4 protein was observed in several tissues of the PAD4 knock-out mice, albeit at reduced levels in most tissues, and are therefore referred to as PAD4-low mice. In material from PAD2−/− mice, except for leukocyte lysates, hardly any PAD activity was found and no citrullinated proteins were detected after incubation in the presence of calcium. PAD activity in the PAD4-low mice was similar to that in wild-type mice. In both PAD knock-out strains the expression of PAD6 appeared to be up-regulated in all tissues analyzed, with the exception of spleen and testis. Our data demonstrate that the PAD2 protein is expressed in brain, spinal cord, spleen, skeletal muscle and leukocytes, but not detectably in liver, lung, kidney and testis. PAD4 was detected in each of these tissues, although the expression levels varied. In all tissues where PAD2 was detected, except for blood cells, this PAD isoform appeared to be responsible for virtually all peptidylarginine deiminase activity.     

Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity

Tumor suppressor genes are frequently silenced in cancer cells by enzymes catalyzing epigenetic histone modifications. The peptidylarginine deiminase family member PAD4 (also called PADI4) is markedly overexpressed in a majority of human cancers, suggesting that PAD4 is a putative target for cancer treatment. Here, we have generated novel PAD inhibitors with low micromolar IC(50) in PAD activity and cancer cell growth inhibition. The lead compound YW3-56 alters the expression of genes controlling the cell cycle and cell death, including SESN2 that encodes an upstream inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Guided by the gene expression profile analyses with YW3-56, we found that PAD4 functions as a corepressor of p53 to regulate SESN2 expression by histone citrullination in cancer cells. Consistent with the mTORC1 inhibition by SESN2, the phosphorylation of its substrates including p70S6 kinase (p70S6K) and 4E-BP1 was decreased. Furthermore, macroautophagy is perturbed after YW3-56 treatment in cancer cells. In a mouse xenograft model, YW3-56 demonstrates cancer growth inhibition activity with little if any detectable adverse effect to vital organs, whereas a combination of PAD4 and histone deacetylase inhibitors further decreases tumor growth. Taken together, our work found that PAD4 regulates the mTORC1 signaling pathway and that PAD inhibitors are potential anticancer reagents that activate tumor suppressor gene expression alone or in combination with histone deacetylase inhibitors.     

Melatonin Promotes Oligodendroglial Maturation of Injured White Matter in Neonatal Rats

Objective

To investigate the effects of melatonin treatment in a rat model of white matter damage (WMD) in the developing brain. Additionally, we aim to delineate the cellular mechanisms of melatonin effect on the oligodendroglial cell lineage.

Methods

A unilateral ligation of the uterine artery in pregnant rat at the embryonic day 17 induces fetal hypoxia and subsequent growth restriction (GR) in neonatal pups. GR and control pups received a daily intra-peritoneal injection of melatonin from birth to post-natal day (P) 3.

Results

Melatonin administration was associated with a dramatic decrease in microglial activation and astroglial reaction compared to untreated GR pups. At P14, melatonin prevented white matter myelination defects with an increased number of mature oligodendrocytes (APC-immunoreactive) in treated GR pups. Conversely, melatonin was not found to be associated with an increased density of total oligodendrocytes (Olig2-immunoreactive), suggesting that melatonin is able to promote oligodendrocyte maturation but not proliferation. These effects appear to be melatonin-receptor dependent and were reproduced in vitro.

Interpretation

These data suggest that melatonin has a strong protective effect on developing damaged white matter through decreased microglial activation and oligodendroglial maturation leading to a normalization of the myelination process. Consequently, melatonin should be a considered as an effective neuroprotective candidate not only in perinatal brain damage but also in inflammatory and demyelinating diseases observed in adults.     

Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury

Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages.