GPRC5B is a membrane glycoprotein robustly expressed in mouse cerebellar Purkinje cells (PCs). Its function is unknown. In Gprc5b-/- mice that lack GPRC5B, PCs develop distal axonal swellings in deep cerebellar nuclei (DCN). Numerous misshapen mitochondria, which generated excessive amounts of reactive oxygen species (ROS), accumulated in these distal axonal swellings. In primary cell cultures of Gprc5b-/- PCs, pharmacological reduction of ROS prevented the appearance of such swellings. To examine the physiological role of GPRC5B in PCs, we analyzed cerebellar synaptic transmission and cerebellum-dependent motor learning in Gprc5b-/- mice. Patch-clamp recordings in cerebellum slices in vitro revealed that the induction of long-term depression (LTD) at parallel fiber-PC synapses was normal in adult Gprc5b-/- mice, whereas the induction of long-term potentiation (LTP) at mossy fiber-DCN neuron synapses was attenuated in juvenile Gprc5b-/- mice. In Gprc5b-/- mice, long-term motor learning was impaired in both the rotarod test and the horizontal optokinetic response eye movement (HOKR) test. These observations suggest that GPRC5B plays not only an important role in the development of distal axons of PCs and formation of synapses with DCN neurons, but also in the synaptic plasticity that underlies long-term motor learning.

Opalin, a central nervous system-specific myelin protein phylogenetically unique to mammals, has been suggested to play a role in mammalian-specific myelin. To elucidate the role of Opalin in mammalian myelin, we disrupted the Opalin gene in mice and analyzed the impacts on myelination and behavior. Opalin-knockout (Opalin-/-) mice were born at a Mendelian ratio and had a normal body shape and weight. Interestingly, Opalin-/- mice had no obvious abnormalities in major myelin protein compositions, expression of oligodendrocyte lineage markers, or domain organization of myelinated axons compared with WT mice (Opalin+/+) mice. Electron microscopic observation of the optic nerves did not reveal obvious differences between Opalin+/+ and Opalin-/- mice in terms of fine structures of paranodal loops, transverse bands, and multi-lamellae of myelinated axons. Moreover, sensory reflex, circadian rhythm, and locomotor activity in the home cage, as well as depression-like behavior, in the Opalin-/- mice were indistinguishable from the Opalin+/+ mice. Nevertheless, a subtle but significant impact on exploratory activity became apparent in Opalin-/- mice exposed to a novel environment. These results suggest that Opalin is not critical for central nervous system myelination or basic sensory and motor activities under conventional breeding conditions, although it might be required for fine-tuning of exploratory behavior.

Atherosclerosis is a major cause of cerebral and cardiovascular diseases. Intravascular plaques, a well-known pathological finding of atherosclerosis, have a necrotic core composed of macrophages and dead cells. Intraplaque macrophages, which are classified into various subtypes, play key roles in maintenance of normal cellular microenvironment. Excessive uptake of oxidized low-density lipoprotein causes conversion of macrophages to foam cells, and consequent progression/exacerbation of atherosclerosis. G-protein-coupled receptor 55 (GPR55) signaling has been reported to associate with atherosclerosis progression. We demonstrated recently that lysophosphatidylglucoside (lysoPtdGlc) is a specific ligand of GPR55, although in general physiological ligands of GPR55 are poorly understood. Phosphatidylglucoside is expressed on human monocytes and can be converted to lysoPtdGlc. In the present study, we examined possible involvement of lysoPtdGlc/GPR55 signaling in foam cell formation. In monocyte-derived M2c macrophages, lysoPtdGlc/GPR55 signaling inhibited translocation of ATP binding cassette subfamily A member 1 to plasma membrane, and cholesterol efflux. Such inhibitory effect was reversed by GPR55 antagonist ML193. LysoPtdGlc/GPR55 signaling in M2c macrophages was involved in excessive lipid accumulation, thereby promoting foam cell formation. Our findings suggest that lysoPtdGlc/GPR55 signaling is a potential therapeutic target for inhibition of atherosclerosis progression.

Microglia/macrophages and lymphocytes (T-cells) accumulate around motor and primary sensory neurons that are regenerating axons but there is little or no microglial activation or T-cell accumulation around axotomised intrinsic CNS neurons, which do not normally regenerate axons. We aimed to establish whether there was an inflammatory response around the perikarya of CNS neurons that were induced to regenerate axons through a peripheral nerve graft.

Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated inflammation, which attacks the myelin sheath. MS pursues a relapsing and remitting course with varying intervals between symptoms. The main clinical pathological features include inflammation, myelin sheath destruction and plaque formation in the central nervous system (CNS). We previously reported that cystatin F (CysF) expression is induced in demyelinating lesions that are accompanied by active remyelination (referred to as shadow plaques) but is down-regulated in chronic demyelinated lesions (plaques) in the spinal cord of MS patients and in several murine models of demyelinating disease. CysF is a cathepsin protease inhibitor whose major target is cathepsin C (CatC), which is co-expressed in demyelinating regions in Plp4e/- mice, a model of chronic demyelination. Here, we report the time course of CatC and CysF expression and describe the symptoms in a mouse experimental autoimmune encephalomyelitis (EAE) model using CatC knockdown (KD) and CatC over-expression (OE) mice. In myelin oligodendrocyte glycoprotein (MOG)-EAE, CatC positive cells were found to infiltrate the CNS at an early stage prior to any clinical signs, in comparison to WT mice. CysF expression was not observed at this early stage, but appeared later within shadow plaques. CatC expression was found in chronic demyelinated lesions but was not associated with CysF expression, and CatCKD EAE mouse showed delayed demyelination. Whereas, CatCOE in microglia significantly increased severity of demyelination in the MOG-EAE model. Thus, these results demonstrate that CatC plays a major role in MOG-EAE.

Previous work has shown that motor skill learning stimulates and requires generation of myelinating oligodendrocytes (OLs) from their precursor cells (OLPs) in the brains of adult mice. In the present study we ask whether OL production is also required for non-motor learning and cognition, using T-maze and radial-arm-maze tasks that tax spatial working memory. We find that maze training stimulates OLP proliferation and OL production in the medial prefrontal cortex (mPFC), anterior corpus callosum (genu), dorsal thalamus and hippocampal formation of adult male mice; myelin sheath formation is also stimulated in the genu. Genetic blockade of OL differentiation and neo-myelination in Myrf conditional-knockout mice strongly impairs training-induced improvements in maze performance. We find a strong positive correlation between the performance of individual wild type mice and the scale of OLP proliferation and OL generation during training, but not with the number or intensity of c-Fos+ neurons in their mPFC, underscoring the important role played by OL lineage cells in cognitive processing.

Secretory lysosomes are required for the specialised functions of various types of differentiated cells. In osteoclasts, the lysosomal proton pump V-ATPase (vacuolar-type ATPase) is targeted to the plasma membrane via secretory lysosomes and subsequently acidifies the extracellular compartment, providing optimal conditions for bone resorption. However, little is known about the mechanism underlying this trafficking of secretory lysosomes. Here, we demonstrate that the lysosome-specific a3 isoform of the V-ATPase a subunit plays an indispensable role in secretory lysosome trafficking, together with Rab7, a small GTPase involved in organelle trafficking. In osteoclasts lacking a3, lysosomes were not transported to the cell periphery, and Rab7 was not localised to lysosomes but diffused throughout the cytoplasm. Expression of dominant-negative (GDP-bound form) Rab7 inhibited lysosome trafficking in wild-type cells. Furthermore, a3 directly interacted with the GDP-bound forms of Rab7 and Rab27A. These findings reveal a novel role for the proton pump V-ATPase in secretory lysosome trafficking and an unexpected mechanistic link with Rab GTPases.

Cerebral malaria is a complication of Plasmodium falciparum infection characterized by sudden coma, death, or neurodisability. Studies using a mouse model of experimental cerebral malaria (ECM) have indicated that blood-brain barrier disruption and CD8 T cell recruitment contribute to disease, but the spatiotemporal mechanisms are poorly understood. We show by ultra-high-field MRI and multiphoton microscopy that the olfactory bulb is physically and functionally damaged (loss of smell) by Plasmodium parasites during ECM. The trabecular small capillaries comprising the olfactory bulb show parasite accumulation and cell occlusion followed by microbleeding, events associated with high fever and cytokine storm. Specifically, the olfactory upregulates chemokine CCL21, and loss or functional blockade of its receptors CCR7 and CXCR3 results in decreased CD8 T cell activation and recruitment, respectively, as well as prolonged survival. Thus, early detection of olfaction loss and blockade of pathological cell recruitment may offer potential therapeutic strategies for ECM.

Myelination speeds conduction of the nerve impulse, enhancing cognitive power. Changes of white matter structure contribute to learning, and are often assumed to reflect an altered number of myelin wraps. We now show that, in rat optic nerve and cerebral cortical axons, the node of Ranvier length varies over a 4.4-fold and 8.7-fold range respectively and that variation of the node length is much less along axons than between axons. Modelling predicts that these node length differences will alter conduction speed by ~20%, similar to the changes produced by altering the number of myelin wraps or the internode length. For a given change of conduction speed, the membrane area change needed at the node is >270-fold less than that needed in the myelin sheath. Thus, axon-specific adjustment of node of Ranvier length is potentially an energy-efficient and rapid mechanism for tuning the arrival time of information in the CNS.

Oligodendrocyte precursors (OPs) continue to proliferate and generate myelinating oligodendrocytes (OLs) well into adulthood. It is not known whether adult-born OLs ensheath previously unmyelinated axons or remodel existing myelin. We quantified OP division and OL production in different regions of the adult mouse CNS including the 4-month-old optic nerve, in which practically all axons are already myelinated. Even there, all OPs were dividing and generating new OLs and myelin at a rate higher than can be explained by first-time myelination of naked axons. We conclude that adult-born OLs in the optic nerve are engaged in myelin remodeling, either replacing OLs that die in service or intercalating among existing myelin sheaths. The latter would predict that average internode length should decrease with age. Consistent with that, we found that adult-born OLs elaborated much shorter but many more internodes than OLs generated during early postnatal life.

Glycosphingolipids and cholesterol are principal components of plasmamembrane microdomains, i.e. lipid rafts. Recent studies revealed the possible presence of a variety of microdomains that distinctly differ in terms of their molecular composition and functions. To understand their precise structures and functions, we produced monoclonal antibodies (MAbs) by immunizing mice to the microdomains prepared from a fraction of detergent-insoluble membrane (DIM) of HL60 cells. Biochemical characterization of the antigen epitopes led to classification of the MAbs into two groups. One group consists of MAbs that react with lipids such as phosphatidylglucoside, lysophosphatidylinositol, and gangliosides (GM1a and GD1b), and the other consists of MAbs that react with proteins such as annexin I, aminopeptidase N and acrogranin. Immunofluorescence staining of HL60 cells with the MAbs, except for the MAbs that recognize lysophosphatidylinositol or annexin I, resulted in patchy-like images of the cell membranes. Interestingly, MAbs belonging to the former group had the potential to induce cell proliferation/differentiation in vitro. Our MAbs against the DIM fraction of HL60 cells can be valuable tools for the study of membrane microdomains.

Neutrophils undergo spontaneous apoptosis within 24-48 h after leaving bone marrow. Apoptotic neutrophils are subsequently phagocytosed and cleared by macrophages, thereby maintaining neutrophil homeostasis. Previous studies have demonstrated involvement of lysophosphatidylglucoside (lysoPtdGlc), a degradation product of PtdGlc, in modality-specific repulsive guidance of spinal sensory axons, via its specific receptor GPR55. In the present study, using human monocytic cell line THP-1 as a model, we demonstrated that lysoPtdGlc induces monocyte/macrophage migration with typical bell-haped curve and a peak at concentration 10-9 M. Lysophosphatidylinositol (lysoPtdIns), a known GPR55 ligand, induced migration at higher concentration (10-7 M). LysoPtdGlc-treated cells had a polarized shape, whereas lysoPtdIns-treated cells had a spherical shape. In EZ-TAXIScan (chemotaxis) assay, lysoPtdGlc induced chemotactic migration activity of THP-1 cells, while lysoPtdIns induced random migration activity. GPR55 antagonist ML193 inhibited lysoPtdGlc-induced THP-1 cell migration, whereas lysoPtdIns-induced migration was inhibited by CB2-receptor inverse agonist. SiRNA experiments showed that GPR55 mediated lysoPtdGlc-induced migration, while lysoPtdIns-induced migration was mediated by CB2 receptor. Our findings, taken together, suggest that lysoPtdGlc functions as a chemotactic molecule for human monocytes/macrophages via GPR55 receptor, while lysoPtdIns induces random migration activity via CB2 receptor.

Swine hemagglutinating encephalomyelitis virus (HEV) has been shown to have a capability to propagate via neural circuits to the central nervous system after peripheral inoculation, resulting in acute deadly encephalomyelitis in natural host piglets as well as in experimental younger rodents. This study has systematically examined the assembly and dissemination of HEV 67N in the primary motor cortex of infected rats and provides additional evidence indicating that membranous-coating-mediated endo-/exocytosis can be used by HEV for its transsynaptic transfer. In addition, our results suggested that this transsynaptic pathway could adapted for larger granular materials, such as viruses. These findings should help in understanding the mechanisms underlying coronavirus infections as well as the intercellular exchanges occurring at the synaptic junctions.

Myelin, made by oligodendrocytes, is essential for rapid information transfer in the central nervous system. Oligodendrocyte precursors (OPs) receive glutamatergic synaptic input from axons but how this affects their development is unclear. Murine OPs in white matter express AMPA receptor (AMPAR) subunits GluA2, GluA3 and GluA4. We generated mice in which OPs lack both GluA2 and GluA3, or all three subunits GluA2/3/4, which respectively reduced or abolished AMPAR-mediated input to OPs. In both double- and triple-knockouts OP proliferation and number were unchanged but ~25% fewer oligodendrocytes survived in the subcortical white matter during development. In triple knockouts, this shortfall persisted into adulthood. The oligodendrocyte deficit resulted in ~20% fewer myelin sheaths but the average length, number and thickness of myelin internodes made by individual oligodendrocytes appeared normal. Thus, AMPAR-mediated signalling from active axons stimulates myelin production in developing white matter by enhancing oligodendrocyte survival, without influencing myelin synthesis per se.