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Inflammation in multiple sclerosis induces a specific reactive astrocyte state driving non‐cell‐autonomous neuronal damage

2022; Springer Science+Business Media; Volume: 12; Issue: 5 Linguagem: Inglês

10.1002/ctm2.837

2001-1326

Clara Matute‐Blanch, Verónica Brito, Luciana Midaglia, Luisa María Villar, Gerardo Garcia-Díaz Barriga, Alerie Guzman de la Fuente, Eva Borràs, Sara Fernández‐García, Laura Calvo‐Barreiro, Andrés Miguez, Lucienne Costa‐Frossard, Rucsanda Pinteac, Eduard Sabidó, Jordi Alberch, Denise Fitzgerald, Xavier Montalbán, Manuel Comabella,

Alzheimer's disease research and treatments

Dear Editor An in-depth understanding of the neurodegenerative component of multiple sclerosis (MS) is crucial for the design of therapeutic approaches that may stop disease progression. Astrocytes have emerged as key contributors to the pathogenesis of MS.1 However, the mechanisms underlying the regulation of maladaptive astrocytic responses remain unknown. In this report, we show that a high inflammatory activity in MS patients at disease onset induces a specific reactive astrocyte state that triggers synaptopathy and contributes to neuronal damage in vitro and ex vivo suggesting potential mechanisms that may ultimately lead to neurodegeneration. To investigate whether astrocytes are essential contributors to neuronal damage in MS, we cultured purified astrocytes with cerebrospinal fluid (CSF) samples from MS patients with high inflammatory activity at disease onset (MS-High, Table S1). Then, we examined the effect of astrocytic secretomes on neurons (Figure 1A). Astrocytes became reactive upon high inflammatory CSF exposure (Figure 1B) and induced morphological alterations typically observed in neurodegenerative disorders, such as a less complex dendritic tree due to decreased arborisation (Figure 1C, D). Moreover, these abnormalities were accompanied with synaptic plasticity impairment (Figure 1E, F). Considering that a high lesion load at disease onset has been associated with an increased risk of neurological disability development,2 we assessed whether the non-cell-autonomous effect on neuronal plasticity could be influenced by the degree of inflammatory activity of MS patients (Figure 2A and Table S1). Interestingly, we observed a direct correlation between the degree of inflammatory exposure and the extent of both astrocyte-mediated synaptopathy (Figure 2B, C) and dendrite arborisation impairment (Figure 2D, E). We next characterised the secretomes from astrocytes exposed to high inflammatory MS microenvironment and found an altered pro-inflammatory profile comprised of 23 upregulated factors (Figure 3A). Functional enrichment and interactome analysis revealed a set of pro-inflammatory pathways enriched following the MS-High CSF exposure (Figure S1). Moreover, nuclear factor NF-kappa-B p105 subunit (Nfkb1) and cellular tumour antigen p53 (Trp53) were identified as the transcription factors regulating the MS-High-associated astrocyte secretome (Figure 3B), both involved in NF-ĸB signalling. SerpinE1, also known as plasminogen activator inhibitor 1 (PAI-1), which has been shown to exacerbate axonal damage and demyelination in MS animal models3 and be regulated by NF-ĸB in neuroinflammation,4 was significantly increased in the MS-High secretomes (Figure 3A and Table S2). Considering its potential role as a mediator of neurodegeneration, we validated by ELISA SerpinE1 increased levels in secretomes from astrocytes exposed to MS-High condition (Figure 3C). By using omics technologies, we studied whether secretomes that alter neuronal plasticity are associated with a specific reactive astrocyte state in MS patients with high inflammatory activity. Astrocytes stimulated with MS-High CSF exhibited a specific reactive gene (Figure 3D) and protein (Figure 3E) expression profile. We identified a MS-High-associated reactive gene signature comprised of 7 differentially expressed genes (Figure 3F) that were validated by qPCR (Figure 3G). This reactive gene expression fingerprint was mostly comprised of downregulated immediate early response genes (Nr4a1, Klf6, Egr2 and Fosb). Interestingly, Nr4a1 and Klf6 have been reported to promote anti-inflammatory responses by specifically repressing NF-ĸB activity.5, 6 To further decipher the MS-High-specific reactive astrocyte state, we performed a functional enrichment analysis integrating all datasets obtained from CSF exposed astrocytes: secretomes and gene/protein expression. Overall, this revealed a prominent inflammatory signature in MS-High astrocytes (Figure S2). Furthermore, Nfkb1, Trp53 and transcription factor p65 (Rela), were identified as the transcription factors regulating the MS-High astrocyte-specific fingerprint (Figure 3H). The inhibition of NF-кB activation in astrocytes ameliorated immune infiltrate, axonal damage and demyelination, by preventing the establishment of the astrocyte-mediated pro-inflammatory microenvironment that leads to disease progression in EAE.7, 8 Moreover, a common MS risk variant (rs7665090) has been found associated to increased NF-кB signalling in astrocytes, driving increased lymphocyte infiltrate and lesion size.9 These findings provide evidence that a high inflammatory microenvironment in MS patients may mediate disease progression by enhancing NF-кB signalling in astrocytes, which modifies their secretome content resulting in both immune-mediated neurodegeneration and potential direct neurotoxic effects. Next, we investigated whether this reactive astrocyte state is associated with a specific CSF proteome in MS patients with high inflammatory activity. LC/MS analysis showed that CSF from MS-High patients have a specific proteome profile (Figure 4A, B). To elucidate the mechanisms underlying astrocyte reactivity, we performed an integrative omics data analysis at CSF, reactive astrocytes, and secretomes levels. Mitogen-activated protein kinase (ERK)-1/2 cascade, NF-ĸB-inducing kinase (NIK)/NF-ĸB signalling, Nfkb1 and Trp53 were found as the most significantly enriched pathways and transcription factors by the exposure to a highly inflammatory MS microenvironment (Figure 4C and Table S3). These data reinforced the role of an enhanced NF-ĸB signalling in the MS-High reactive astrocytes. Remarkably, we identified the prognostic biomarker chitinase 3-like 1 (CHI3L1)10 upregulated in the MS-High CSF (Figure 4B) and represented in the aforementioned pathways (Table S3). To investigate whether CHI3L1 could be a mediator of the MS-High reactive astrocyte state, we stimulated astrocytes with CHI3L1 at concentrations above the cut-off value that demonstrated prognostic implications in MS patients10 (Figure 4D). CHI3L1 stimulation (600 ng/ml) downregulated Nr4a1 and Klf6 expression, both involved in the inhibition of NF-ĸB signalling5, 6 (Figure 4E and Figure S3). Protein interactome computation revealed an interaction between the NF-ĸB transcription module (Nr4a1 and Klf6) potentially controlled by CHI3L1 (Figure 4F). Noteworthy, we also found interactions between Mapk3 (ERK1), Mapk1 (ERK2) and Nr4a1 in the NF-ĸB transcription module, which might be regulated by CHI3L1 and SerpinE1 (Figure 4G). Finally, to address whether CHI3L1 could be a potential driver of astrocyte-mediated neuronal damage we used P7 murine myelinating organotypic brain slice cultures that generate compact myelin ex vivo and mimic in vivo microenvironment. After 48 h, CHI3L1 (600 ng/ml) induced axonal damage reducing total neurofilament area (Figure 4H). Our findings provide evidence that the degree of inflammatory activity in MS patients at disease onset has the potential to induce a specific reactive state in astrocytes that trigger neuronal damage (Figure S4). This reactive state, mainly associated with the NF-ĸB signalling, could be exploited as a prognostic biomarker that reflects a potential detrimental effect of MS astrocytes on neuronal plasticity. The authors thank the Advanced Optic Microscopy Unit (Campus Casanova) of the Centre Científic i Tecnològic (CCIT, Universitat de Barcelona) for their advice with microscopy techniques. The authors report no competing interests. G.G.D.B. is now an employee of Evotec. The study was funded by the 'Red Española de Esclerosis Múltiple (REEM)' sponsored by the 'Fondo de Investigación Sanitaria' (FIS; project reference: PI15/01111), Ministry of Science and Innovation, Spain; the 'Ajuts per donar Suport als Grups de Recerca de Catalunya', sponsored by the 'Agència de Gestió d'Ajuts Universitaris i de Recerca' (AGAUR), Generalitat de Catalunya, Spain; and Wellcome Trust (110138/Z/15/Z, to D.C.F.), United Kingdom. Table S1. Demographic data of the MS patients and controls included in the study Figure S1. Astrocytes exposed to CSF from MS patients with high inflammatory activity exhibit an altered pro-inflammatory secretome Table S2. GO and KEGG functional analysis for the secretome content of astrocytes exposed to the MS-High compared to the MS-Low condition Figure S2. Astrocytes exposed to MS-High-derived CSF exhibit a pro-inflammatory signature mainly associated with NF-kB signalling pathway Table S3. Bioinformatic analysis integrating the MS-High-exposed astrocyte-specific fingerprint compared to the MS-Low exposure Figure S3. Expression levels of genes associated with the specific astrocyte-derived gene expression signature following CHI3L1 stimulation Figure S4. Schematic flowchart summarising the main results of the study Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.