Identification of naturally occurring fatty acids of the myelin sheath that resolve neuroinflammation.

Lipids constitute 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain, and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids' saturated fatty acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as therapeutics for MS.

Ketamine activates cell cycle signaling and apoptosis in the neonatal rat brain.

BACKGROUND: Prolonged exposure to ketamine results in accelerated neurodegeneration and neurocognitive deficits in the neonatal rats. Experimental models of neurodegeneration have implicated reentry of postmitotic neurons into the cell cycle, leading to cell death. The authors hypothesize that the ketamine-induced neuroapoptosis is partially due to aberrant cycle cell reentry. To explore this hypothesis, the authors characterized the effect of ketamine on the cell cycle signaling pathway in the developing rodent brain in vivo and in vitro. METHODS: Postnatal day 7 rat pups and primary neurons were used for the experiments. Each rat pup received five intraperitoneal doses of either saline or ketamine (5, 10, and 20 mg/kg/dose) at 90-min intervals over 6 h. Primary neurons were exposed to varying concentrations of ketamine to determine the dose and duration effects. The expression of cell cycle proteins (cyclin D1, cyclin-dependent kinase 4, and E2F1), Bcl2-interacting mediator of cell death (Bim), and activated caspase-3 was determined. The effect of cyclin D1 knockdown by small interfering RNA was also examined in primary neurons incubated in ketamine. RESULTS: Ketamine mediated a dose- and time-dependent increase in expression of cell cycle proteins and activated caspase-3. Cyclin D1, cyclin-dependent kinase 4, E2F1, Bim, and cleaved caspase-3 expression increased at 12 h and peaked at 24 h in vitro. Knockdown of cyclin D1 by small interfering RNA attenuated Bim and cleaved caspase-3 expression. CONCLUSION: These findings support a model in which ketamine induces aberrant cell cycle reentry, leading to apoptotic cell death in the developing rat brain.

PTEN/mTOR and axon regeneration.

How axon regeneration is controlled in both PNS and CNS remains elusive. Mechanistic studies of axon growth during development and axon regeneration after injury reveal the PTEN dependent molecular mechanism as a commonality. This pathway could impact the processes occurring in the neuronal soma, such as mTOR-regulated protein translation, and in the axons, such as cytoskeleton assembly. In this review, we will discuss the current understanding of the involvement of these processes in the regulation of axon growth and the potential implication in promoting axon regeneration after injury.

Epitope spreading to citrullinated antigens in mouse models of autoimmune arthritis and demyelination.

INTRODUCTION: Anti-citrullinated protein antibodies have a diagnostic role in rheumatoid arthritis (RA); however, little is known about their origins and contribution to pathogenesis. Citrullination is the post-translational conversion of arginine to citrulline by peptidyl arginine deiminase, and increased citrullination of proteins is observed in the joint tissue in RA and in brain tissue in multiple sclerosis (MS). METHODS: We applied synovial and myelin protein arrays to examine epitope spreading of B cell responses to citrullinated epitopes in both the collagen-induced arthritis (CIA) model for RA and the experimental autoimmune encephalomyelitis (EAE) model for MS. Synovial and myelin protein arrays contain a spectrum of proteins and peptides, including native and citrullinated forms, representing candidate autoantigens in RA and MS, respectively. We applied these arrays to characterise the specificity of autoantibodies in serial serum samples derived from mice with acute and chronic stages of CIA and EAE. RESULTS: In samples from pre-disease CIA and acute-disease EAE, we observed autoantibody targeting of the immunising antigen and responses to a limited set of citrullinated epitopes. Over the course of diseases, the autoantibody responses expanded to target multiple citrullinated epitopes in both CIA and EAE. Using immunoblotting and mass spectrometry analysis, we identified citrullination of multiple polypeptides in CIA joint and EAE brain tissue that have not previously been described as citrullinated. CONCLUSIONS: Our results suggest that anti-citrulline antibody responses develop in the early stages of CIA and EAE, and that autoimmune inflammation results in citrullination of joint proteins in CIA and brain proteins in EAE, thereby creating neoantigens that become additional targets in epitope spreading of autoimmune responses.

Fluid supported lipid bilayers containing monosialoganglioside GM1: a QCM-D and FRAP study.

In an effort to use model fluid membranes for immunological studies, we compared the formation of planar phospholipid bilayers supported on silicon dioxide surfaces with and without incorporation of glycolipids as the antigen for in situ antibody binding. Dynamic light scattering measurements did not differentiate the hydrodynamic volumes of extruded small unilamellar vesicles (E-SUVs) containing physiologically relevant concentrations (0.5-5 mol%) of monosialoganglioside GM1 (GM1) from exclusive egg yolk L-alpha-phosphatidylcholine (egg PC) E-SUVs. However, quantifiable differences in deposition mass and dissipative energy loss emerged in the transformation of 5 mol% GM1/95 mol% egg PC E-SUVs to planar supported lipid bilayers (PSLBs) by vesicle fusion on thermally evaporated SiO2, as monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. Compared to the 100 mol% egg PC bilayers on the same surface, E-SUVs containing 5 mol% GM1 reached a approximately 12% higher mass and a lower dissipative energy loss during bilayer transformation. PSLBs with 5 mol% GM1 are approximately 18% heavier than 100 mol% egg PC and approximately 11% smaller in projected area per lipid, indicating an increased rigidity and a tighter packing. Subsequent binding of polyclonal immunoglobulin G anti-GM1 to the PSLBs was performed in situ and showed specificity. The anti-GM1 to GM1 ratios at equilibrium were roughly proportional to the concentrations of anti-GM1 administered in the solution. Fluorescence recovery after photobleaching was utilized to verify the retained, albeit reduced lateral fluidity of the supported membranes. Five moles percentage of GM1 membranes (GM1 to PC ratio approximately 1:19) decorated with 1 mol% N-(Texas Red sulfonyl)-1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine (Texas Red DHPE) exhibited an approximately 16% lower diffusion coefficient of 1.32+/-0.06 microm2/s, compared to 1.58+/-0.04 microm2/s for egg PC membranes without GM1 (p<0.01). The changes in vesicle properties and membrane lateral fluidity are attributed to the interactions of GM1 with itself and GM1 with other membrane lipids. This system allows for molecules of interest such as GM1 to exist on a more biologically relevant surface than those used in conventional methods such as ELISA. Our analysis of rabbit serum antibodies binding to GM1 demonstrates this platform can be used to test for the presence of anti-lipid antibodies in serum.

Lipid microarrays identify key mediators of autoimmune brain inflammation.

Recent studies suggest that increased T-cell and autoantibody reactivity to lipids may be present in the autoimmune demyelinating disease multiple sclerosis. To perform large-scale multiplex analysis of antibody responses to lipids in multiple sclerosis, we developed microarrays composed of lipids present in the myelin sheath, including ganglioside, sulfatide, cerebroside, sphingomyelin and total brain lipid fractions. Lipid-array analysis showed lipid-specific antibodies against sulfatide, sphingomyelin and oxidized lipids in cerebrospinal fluid (CSF) derived from individuals with multiple sclerosis. Sulfatide-specific antibodies were also detected in SJL/J mice with acute experimental autoimmune encephalomyelitis (EAE). Immunization of mice with sulfatide plus myelin peptide resulted in a more severe disease course of EAE, and administration of sulfatide-specific antibody exacerbated EAE. Thus, autoimmune responses to sulfatide and other lipids are present in individuals with multiple sclerosis and in EAE, and may contribute to the pathogenesis of autoimmune demyelination.