Extracellular vesicles released by microglia and macrophages carry endocannabinoids which foster oligodendrocyte differentiation.

Introduction: Microglia and macrophages can influence the evolution of myelin lesions through the production of extracellular vesicles (EVs). While microglial EVs promote in vitro differentiation of oligodendrocyte precursor cells (OPCs), whether EVs derived from macrophages aid or limit OPC maturation is unknown. Methods: Immunofluorescence analysis for the myelin protein MBP was employed to evaluate the impact of EVs from primary rat macrophages on cultured OPC differentiation. Raman spectroscopy and liquid chromatography-mass spectrometry was used to define the promyelinating lipid components of myelin EVs obtained in vitro and isolated from human plasma. Results and discussion: Here we show that macrophage-derived EVs do not promote OPC differentiation, and those released from macrophages polarized towards an inflammatory state inhibit OPC maturation. However, their lipid cargo promotes OPC maturation in a similar manner to microglial EVs. We identify the promyelinating endocannabinoids anandamide and 2-arachidonoylglycerol in EVs released by both macrophages and microglia in vitro and circulating in human plasma. Analysis of OPC differentiation in the presence of the endocannabinoid receptor antagonists SR141716A and AM630 reveals a key role of vesicular endocannabinoids in OPC maturation. From this study, EV-associated endocannabinoids emerge as important mediators in microglia/macrophage-oligodendrocyte crosstalk, which may be exploited to enhance myelin repair.

Predicting disease severity in multiple sclerosis using multimodal data and machine learning.

BACKGROUND: Multiple sclerosis patients would benefit from machine learning algorithms that integrates clinical, imaging and multimodal biomarkers to define the risk of disease activity. METHODS: We have analysed a prospective multi-centric cohort of 322 MS patients and 98 healthy controls from four MS centres, collecting disability scales at baseline and 2 years later. Imaging data included brain MRI and optical coherence tomography, and omics included genotyping, cytomics and phosphoproteomic data from peripheral blood mononuclear cells. Predictors of clinical outcomes were searched using Random Forest algorithms. Assessment of the algorithm performance was conducted in an independent prospective cohort of 271 MS patients from a single centre. RESULTS: We found algorithms for predicting confirmed disability accumulation for the different scales, no evidence of disease activity (NEDA), onset of immunotherapy and the escalation from low- to high-efficacy therapy with intermediate to high-accuracy. This accuracy was achieved for most of the predictors using clinical data alone or in combination with imaging data. Still, in some cases, the addition of omics data slightly increased algorithm performance. Accuracies were comparable in both cohorts. CONCLUSION: Combining clinical, imaging and omics data with machine learning helps identify MS patients at risk of disability worsening.

A large cohort of myotonia congenita probands: novel mutations and a high-frequency mutation region in exons 4 and 5 of the CLCN1 gene.

Myotonia congenita is a genetic disease characterized by impaired muscle relaxation after forceful contraction (myotonia) and caused by mutations in the chloride channel voltage-sensitive 1 (CLCN1) gene, encoding the voltage-gated chloride channel of skeletal muscle (ClC-1). In a large cohort of clinically diagnosed unrelated probands, we identified 75 different CLCN1 mutations in 106 individuals, among which 29 were novel mutations and 46 had already been reported. Despite the newly described mutations being scattered throughout the gene, in our patients, mutations were mostly found in exons 4 and 5. Most of the novel mutations located in the region comprising the intramembrane helices are involved in the ion-conducting pathway and predicted to affect channel function. We report for the first time that two mutations, inherited on the same allele as a heterozygous trait, abrogate disease expression, although when inherited singularly they were pathogenic. Such a mode of inheritance might explain the incomplete penetrance reported for autosomal dominant mutations in particular families.

Immune cell subpopulations and serum neurofilament light chain are associated with increased risk of disease worsening in multiple sclerosis.

Changes is lymphocyte subpopulations in peripheral blood have been proposed as biomarkers for evaluation of disease activity in multiple sclerosis (MS). Serum neurofilament light chain (sNfL) is a biomarker reflecting neuro-axonal injury in MS that could be used to monitor disease activity, response to drugs and to prognosticate disease course. Here we show a moderate correlation between sNfL and lymphocyte cell subpopulations, and our data furthermore suggest that sNfL and specific immune cell subpopulations together could predict future disease worsening in MS.

Intranasal delivery of mesenchymal stem cell secretome repairs the brain of Alzheimer's mice.

The multiplicity of systems affected in Alzheimer's disease (AD) brains calls for multi-target therapies. Although mesenchymal stem cells (MSC) are promising candidates, their clinical application is limited because of risks related to their direct implantation in the host. This could be overcome by exploiting their paracrine action. We herein demonstrate that in vivo systemic administration of secretome collected from MSC exposed in vitro to AD mouse brain homogenates (MSC-CS), fully replicates the cell-mediated neuroreparative effects in APP/PS1 AD mice. We found a complete but transient memory recovery by 7 days, which vanished by 14 days, after a single MSC-CS intravenous administration in 12-month or 22-24-month-old mice. Treatment significantly reduced plaque load, microglia activation, and expression of cytokines in astrocytes in younger, but not aged, mice at 7 days. To optimize efficacy, we established a sustained treatment protocol in aged mice through intranasal route. Once-weekly intranasal administration of MSC-CS induced persistent memory recovery, with dramatic reduction of plaques surrounded by a lower density of ß-amyloid oligomers. Gliosis and the phagocytic marker CD68 were decreased. We found a higher neuronal density in cortex and hippocampus, associated with a reduction in hippocampal shrinkage and a longer lifespan indicating healthier conditions of MSC-CS-treated compared to vehicle-treated APP/PS1 mice. Our data prove that MSC-CS displays a great multi-level therapeutic potential, and lay the foundation for identifying the therapeutic secretome bioreactors leading to the development of an efficacious multi-reparative cocktail drug, towards abrogating the need for MSC implantation and risks related to their direct use.

CCL21 overexpressed on lymphatic vessels drives thymic hyperplasia in myasthenia.

OBJECTIVE: Myasthenia gravis (MG), a neuromuscular disease mediated by anti-acetylcholine receptor (AChR) autoantibodies, is associated with thymic hyperplasia characterized by ectopic germinal centers that contain pathogenic antibody-producing B cells. Our thymic transcriptome study demonstrated increased expression of CCL21, a recruiter of immune cells. Accordingly, we are investigating its implication in MG pathogenesis. METHODS: The expression of CCL21 and its CCR7 receptor was analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting, respectively. Chemotaxis of T and B cells to CCL21 was measured by transwell assay. The nature of the thymic cells overexpressing CCL21 was investigated by immunochemistry and laser-capture microdissection combined with real-time PCR. RESULTS: We demonstrate that CCL21 is overexpressed specifically in hyperplastic MG thymuses, whereas there is no variation in CCR7 levels on blood cells. We show that although CCL21 attracts both human T and B cells, it acts more strongly on naive B cells. CCL21 overexpression is normalized in corticoid-treated MG patients, suggesting that targeting this chemokine could represent a new selective treatment, decreasing the abnormal peripheral lymphocyte recruitment. Moreover, we locate protein and messenger RNA overexpression of CCL21 to specific endothelial vessels. Investigation of the nature of these vessels demonstrated different angiogenic processes in MG thymuses: high endothelial venule angiogenesis and lymphangiogenesis. Unexpectedly, CCL21 overexpression originates from afferent lymphatic endothelial vessels. INTERPRETATION: We postulate that thymic overexpression of CCL21 on specialized lymphatic vessels results in abnormal peripheral lymphocyte recruitment, bringing naive B cells in contact with the inflammatory environment characteristic of MG thymuses, where they can be sensitized against AChR.

Regulatory and pathogenic mechanisms in human autoimmune myasthenia gravis.

The thymus is frequently hyperplastic in young female myasthenia gravis (MG) patients presenting with anti-acetylcholine receptor (AChR) antibodies. This thymic pathology is characterized by the presence of ectopic germinal centers (GCs) containing B cells involved at least partially in the production of pathogenic anti-AChR antibodies. Our recent studies have furthered our understanding of the mechanisms leading to GC formation in the hyperplastic thymus. First, we showed that CXCL13 and CCL21, chemokines involved in GC formation, are overexpressed in MG thymus. Second, we demonstrated an increase in pro-inflammatory activity in the thymus from MG patients and its partial normalization by glucocorticoids, as evidenced by gene expression profile. Third, we found that pro-inflammatory cytokines are able to upregulate the expression of AChR subunits in thymic epithelial and myoid cells. Fourth, we showed that the function of T regulatory (Treg) cells, whose role is to downregulate the immune response, is severely impaired in the thymus of MG patients; such a defect could explain the chronic immune activation observed consistently in MG thymic hyperplasia. Altogether, these new data suggest that CXCL13 and CCL21, which are produced in excess in MG thymus, attract peripheral B cells and activated T cells, which are maintained chronically activated in the inflammatory thymic environment because of the defect in suppressive activity of Treg cells. Presence of AChR in the thymus and upregulation of its expression by the pro-inflammatory environment contribute to the triggering and maintenance of the anti-AChR autoimmune response.

Characterization of mouse spinal cord vascular network by means of synchrotron radiation X-ray phase contrast tomography.

High resolution Synchrotron-based X-ray Phase Contrast Tomography (XPCT) allows the simultaneous detection of three dimensional neuronal and vascular networks without using contrast agents or invasive casting preparation. We show and discuss the different features observed in reconstructed XPCT volumes of the ex vivo mouse spinal cord in the lumbo-sacral region, including motor neurons and blood vessels. We report the application of an intensity-based segmentation method to detect and quantitatively characterize the modification in the vascular networks in terms of reduction in experimental visibility. In particular, we apply our approach to the case of the experimental autoimmune encephalomyelitis (EAE), i.e. human multiple sclerosis animal model.

The immunomodulatory function of mesenchymal stem cells: mode of action and pathways.

Mesenchymal stem cells (MSCs) are being increasingly investigated as a therapeutic alternative, not only for their possible regenerative potential but also for their immunomodulatory action, which is being exploited for controlling diseases associated with inflammation. Understanding their direct and indirect target cells, as well as their mode of action and relevant pathways, is a prerequisite for the appropriate and optimal use of MSCs in therapy. Here, we review recent findings on the effects of MSCs on adaptive and innate immune cells. We also consider the impact of the environment on MSC profile, both anti- and proinflammatory, and the mechanisms and molecular pathways through which their effects are mediated, both at the MSC and target cell levels.

Mesenchymal stem cells for the treatment of neurological diseases: Immunoregulation beyond neuroprotection.

An inflammatory response is often observed in neurological diseases, being characterized sometimes by activation of adaptive cells (T and B lymphocytes) and, almost inexorably, of cells of the innate immunity (microglial cells, macrophages). Mesenchymal stromal/stem cells represent a promising therapeutic approach for the treatment of intractable neurological diseases given the possibility that they affect neurodegeneration both directly and indirectly, through their potent immunomodulatory effect. Here we will review the evidence, mostly deriving from preclinical studies, that MSC, beyond their ability to foster neurorepair, can ameliorate neurodegenerative diseases through their effect on associated immune responses.

AMDA 2.13: A major update for automated cross-platform microarray data analysis.

Microarray platforms require analytical pipelines with modules for data pre-processing including data normalization, statistical analysis for identification of differentially expressed genes, cluster analysis, and functional annotation. We previously developed the Automated Microarray Data Analysis (AMDA, version 2.3.5) pipeline to process Affymetrix 3' IVT GeneChips. The availability of newer technologies that demand open-source tools for microarray data analysis has impelled us to develop an updated multi-platform version, AMDA 2.13. It includes additional quality control metrics, annotation-driven (annotation grade of Affymetrix NetAffx) and signal-driven (Inter-Quartile Range) gene filtering, and approaches to experimental design. To enhance understanding of biological data, differentially expressed genes have been mapped into KEGG pathways. Finally, a more stable and user-friendly interface was designed to integrate the requirements for different platforms. AMDA 2.13 allows the analysis of Affymetrix (cartridges and plates) and whole transcript probe design (Gene 1.0/1.1 ST and Exon 1.0 ST GeneChips), Illumina Bead Arrays, and one-channel Agilent 4×44 arrays. Relative to early versions, it supports various experimental designs and delivers more insightful biological understanding and up-to-date annotations.

Hind limb muscle atrophy precedes cerebral neuronal degeneration in G93A-SOD1 mouse model of amyotrophic lateral sclerosis: a longitudinal MRI study.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, neurodegenerative disorder caused by the degeneration of motor neurons in the CNS, which results in complete paralysis of skeletal muscles. Recent experimental studies have suggested that the disease could initiate in skeletal muscle, rather than in the motor neurons. To establish the timeframe of motor neuron degeneration in relation to muscle atrophy in motor neuron disease, we have used MRI to monitor changes throughout disease in brain and skeletal muscle of G93A-SOD1 mice, a purported model of ALS. Longitudinal MRI examination of the same animals indicated that muscle volume in the G93A-SOD1 mice was significantly reduced from as early as week 8 of life, 4 weeks prior to clinical onset. Progressive muscle atrophy from week 8 onwards was confirmed by histological analysis. In contrast, brain MRI indicated that neurodegeneration occurs later in G93A-SOD1 mice, with hyperintensity MRI signals detected only at weeks 10-18. Neurodegenerative changes were observed only in the motor nuclei areas of the brainstem; MRI changes indicative of neurodegeneration were not detected in the motor cortex where first motor neurons originate, even at the late disease stage. This longitudinal MRI study establishes unequivocally that, in the experimental murine model of ALS, muscle degeneration occurs before any evidence of neurodegeneration and clinical signs, supporting the postulate that motor neuron disease can initiate from muscle damage and result from retrograde dying-back of the motor neurons.

Thymic remodeling associated with hyperplasia in myasthenia gravis.

Acquired myasthenia gravis (MG), a neurological autoimmune disease, is caused by autoantibodies against components of the neuromuscular junction that lead to disabling muscle fatigability. The thymus is clearly involved in the pathogenesis of early-onset MG with anti-acetylcholine receptor antibodies, and thymic hyperplasia of lympho-proliferative origin is a hallmark of the disease. In this review, we describe the structural and cellular changes associated with thymic hyperplasia, its main characteristics being the development of ectopic germinal centers (GCs) associated with active neoangiogenic processes, such as development of high endothelial venules and lymphangiogenesis. What triggers such thymic abnormalities in MG is not yet clear. A thymic transcriptome analysis has demonstrated a strong inflammatory signature in MG that could orchestrate the development of thymic hyperplasia. In this context, thymic epithelial cells (TECs) seem to play a central role, either by contributing or responding to the inflammatory environment and up-regulating the autoimmune response. In particular, MG TECs clearly overexpress various cytokines, among which chemokines play a crucial role in the recruitment of peripheral lymphocytes to the thymus via the newly expanded vessel network, thereby leading to the development of ectopic GCs. Clearly, a better understanding of major events that lead to thymic hyperplasia will help optimize strategies toward more specific therapy for MG.

Autoimmune spread to myelin is associated with experimental autoimmune encephalomyelitis induced by a neuronal protein, beta-synuclein.

Accumulating evidence suggests that autoimmunity against neuronal proteins is important for MS pathogenesis. We have characterized T- and B-cell responses associated with experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats with recombinant beta-Synuclein (betaSync), a neuronal component. The encephalitogenic betaSync-specific T cells recognize a single immunodominant region with an epitope delineated at amino acids 97-105; B-cell specificity is more widespread, albeit directed mostly to the C-terminus of betaSync. Most interestingly, betaSync-induced autoimmune T- and B-cell responses spread not only to other neuronal antigens but also to myelin encephalitogens, raising the possibility that anti-neuronal immune attacks could also result in demyelination.

The myelin-associated oligodendrocytic basic protein region MOBP15-36 encompasses the immunodominant major encephalitogenic epitope(s) for SJL/J mice and predicted epitope(s) for multiple sclerosis-associated HLA-DRB1*1501.

Autoimmune response to the myelin-associated oligodendrocytic basic protein (MOBP), a CNS-specific myelin constituent, was recently suggested to play a role in the pathogenesis of multiple sclerosis (MS). The pathogenic autoimmune response to MOBP and the associated pathology in the CNS have not yet been fully investigated. In this study, we have characterized the clinical manifestations, pathology, T cell epitope-specificity, and TCRs associated with experimental autoimmune encephalomyelitis (EAE) induced in SJL/J mice with recombinant mouse MOBP (long isoform, 170 aa). Analysis of encephalitogenic MOBP-reactive T cells for reactivity to overlapping MOBP peptides defined MOBP15-36 as their major immunodominant epitope. Accordingly, MOBP15-36 was demonstrated to be the major encephalitogenic MOBP epitope for SJL/J mice, inducing severe/chronic clinical EAE associated with intense perivascular and parenchymal infiltrations, widespread demyelination, axonal loss, and remarkable optic neuritis. Molecular modeling of the interaction of I-A(s) with MOBP15-36, together with analysis of the MOBP15-36-specific T cell response to truncated peptides, suggests MOBP20-28 as the core sequence for I-A(s)-restricted recognition of the encephalitogenic region MOBP15-36. Although highly focused in their epitope specificity, the encephalitogenic MOBP-reactive T cells displayed a widespread usage of TCR Vbeta genes. These results would therefore favor epitope-directed, rather than TCR-targeted, approaches to therapy of MOBP-associated pathogenic autoimmunity. Localization by molecular modeling of a potential HLA-DRB1*1501-associated MOBP epitope within the encephalitogenic MOBP15-36 sequence suggests the potential relevance of T cell reactivity against MOBP15-36 to MS. The reactivity to MOBP15-36 detected in MS shown here and in another study further emphasizes the potential significance of this epitope for MS.