CSF and serum NfL levels were highly correlated, and serum concentrations were increased in RRMS. Serum NfL levels correlated with MRI markers of WM disease severity. Our findings further support longitudinal studies of serum NfL as a potential biomarker of on-going disease progression and as a potential surrogate to quantify effects of neuroprotective drugs in clinical trials.

SummaryRecent studies highlighted the importance of astrocytes in neuroinflammatory diseases, interacting closely with other CNS cells but also with the immune system. However, due to the difficulty in obtaining human astrocytes, their role in these pathologies is still poorly characterized. Here, we develop a serum-free protocol to differentiate human induced pluripotent stem cells (hiPSCs) into astrocytes. Gene expression and functional assays show that our protocol consistently yields a highly enriched population of resting mature astrocytes across the 13 hiPSC lines differentiated. Using this model, we first highlight the importance of serum-free media for astrocyte culture to generate resting astrocytes. Second, we assess the astrocytic response to IL-1β, TNF-α, and IL-6, all cytokines important in neuroinflammation, such as multiple sclerosis. Our study reveals very specific profiles of reactive astrocytes depending on the triggering stimulus. This model provides ideal conditions for in-depth and unbiased characterization of astrocyte reactivity in neuroinflammatory conditions.

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Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases.

BackgroundIncreasing evidences link T helper 17 (Th17) cells with multiple sclerosis (MS). In this context, interleukin-22 (IL-22), a Th17-linked cytokine, has been implicated in blood brain barrier breakdown and lymphocyte infiltration. Furthermore, polymorphism between MS patients and controls has been recently described in the gene coding for IL-22 binding protein (IL-22BP). Here, we aimed to better characterize IL-22 in the context of MS.MethodsIL-22 and IL-22BP expressions were assessed by ELISA and qPCR in the following compartments of MS patients and control subjects: (1) the serum, (2) the cerebrospinal fluid, and (3) immune cells of peripheral blood. Identification of the IL-22 receptor subunit, IL-22R1, was performed by immunohistochemistry and immunofluorescence in human brain tissues and human primary astrocytes. The role of IL-22 on human primary astrocytes was evaluated using 7-AAD and annexin V, markers of cell viability and apoptosis, respectively.ResultsIn a cohort of 141 MS patients and healthy control (HC) subjects, we found that serum levels of IL-22 were significantly higher in relapsing MS patients than in HC but also remitting and progressive MS patients. Monocytes and monocyte-derived dendritic cells contained an enhanced expression of mRNA coding for IL-22BP as compared to HC.Using immunohistochemistry and confocal microscopy, we found that IL-22 and its receptor were detected on astrocytes of brain tissues from both control subjects and MS patients, although in the latter, the expression was higher around blood vessels and in MS plaques.Cytometry-based functional assays revealed that addition of IL-22 improved the survival of human primary astrocytes. Furthermore, tumor necrosis factor α-treated astrocytes had a better long-term survival capacity upon IL-22 co-treatment. This protective effect of IL-22 seemed to be conferred, at least partially, by a decreased apoptosis.ConclusionsWe show that (1) there is a dysregulation in the expression of IL-22 and its antagonist, IL-22BP, in MS patients, (2) IL-22 targets specifically astrocytes in the human brain, and (3) this cytokine confers an increased survival of the latter cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0335-3) contains supplementary material, which is available to authorized users.

ObjectiveTo assess whether chronic white matter inflammation in multiple sclerosis (MS) patients - as detected in-vivo by paramagnetic rim MRI lesions (PRL) - is associated with higher serum neurofilament light chain (sNfL) levels, a marker of neuro-axonal damage.MethodsIn 118 MS patients with no gadolinium-enhancing lesions or recent relapses, we analyzed 3D-submillimeter phase MRI and sNfL levels. Histopathological evaluation was performed in 25 MS lesions from 20 additional autopsy MS patients.ResultsIn univariable analyses, participants with ≥2 PRL (“PRL ≥2”, n=43) compared to those with ≤1 PRL (“PRL 0–1,” n=75) had higher age-adjusted sNfL percentiles (median, 91 and 68; p<0.001) and higher MS disease severity scale (MSSS median, 4.3 and 2.4; p=0.003). In multivariable analyses, sNfL percentile levels were higher in PRL ≥2 cases (βadd: 16.3; 95% CI: 4.6–28.0; p<0.01), whereas disease-modifying treatment (DMT), EDSS, and T2 lesion load did not affect sNfL. In a similar model, sNfL percentile levels were highest in cases with ≥4 PRL (n=30; βadd: 30.4; 95% CI, 15.6–45.2; p<0.01). Subsequent multivariable analysis revealed that PRL ≥2 cases had also higher MSSS (βadd: 1.1; 95% CI, 0.3–1.9; p<0.01), whereas MSSS was not affected by DMT or T2 lesion load. On histopathology, both chronic active and smoldering lesions exhibited more severe acute axonal damage at the lesion edge than in the lesion center (edge vs center: p=0.004 and p=0.0002, respectively).InterpretationChronic white matter inflammation was associated with increased levels of sNfL and disease severity in non-acute MS patients, suggesting that PRL contribute to clinically relevant, inflammation-driven neurodegeneration.

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