Human tissue
Post-mortem human tissue samples
Eyeballs from individuals diagnosed with MS were obtained from the Netherlands Brain Bank, and those from control donors without recognizable neuropathological changes were obtained from Johns Hopkins University. Detailed information about donors used for snRNA-seq, immunohistochemistry, qPCR and smFiSH are provided in Supplementary Table 1. The macula region of these donors was used for snRNA-seq while smFISH was performed on tissue adjacent to the macula. qPCR and immunohistochemistry were conducted with the peripheral retina. Paraffin-embedded cortex tissue for histopathology was obtained from the UK Multiple Sclerosis Tissue Bank at Imperial College London. Samples were classified as chronic active lesions or normal appearing grey matter according to the histopathological assessment provided by the UK Multiple Sclerosis Tissue Bank’s histopathology reports. Although no statistical methods were used to predetermine sample size, our sample size is comparable to those reported in previous studies12,13,60.
Nucleus isolation and library preparation
Eyeballs were enucleated from deceased healthy controls and people with MS. Only tissue from patients with a post-mortem interval ≤24 h was used. To isolate the macula region, fresh-frozen eyeballs were positioned in a CM3050 S cryostat (Leica Microsystems) at –20 °C, with the lens facing downward and the optic disc oriented toward the examiner. The macula was identified as a distinct yellow spot on the retina, and the tissue was stored at −80 °C until use. All of the subsequent steps were performed on ice according to a previously published protocol with minor adaptations. In brief, on each experimental day, the frozen tissue was transferred to ice-cold NP40 lysis buffer (0.1% NP-40 (Thermo Fisher Scientific, 85124), 10 mM Tris pH 8.0, 1 mM CaCl2, 8 mM MgCl2, 15 mM NaCl, 0.02 U µl–1 DNase I (Merck Millipore, D4527)). The retina was transferred to a Dounce homogenizer in 1 ml lysis buffer supplemented with 0.2 U µl−1 Ribolock RNase Inhibitor (Thermo Fisher Scientific, EO0382) and homogenized 20 times with both loose and tight pestles. The homogenate was passed through a 100 µm cell strainer and centrifuged at 500g for 5 min. All buffers, except for the washing buffer, were supplemented with 0.16 U µl−1 Ribolock RNase inhibitor. Pelleted nuclei were resuspended in staining buffer (Tris base buffer: 10 mM Tris pH 8.0, 1 mM CaCl2, 8 mM MgCl2, 15 mM NaCl, 1 U ml−1 DNase I) containing 0.02% Tween-20 and 2% BSA, with primary antibodies against NeuN (1:250, Merck Millipore, ABN91) and RBPMS (1:250, Abcam, ab194213) for 15 min at 4 °C. Nuclei were washed, centrifuged at 500g for 5 min and stained with secondary antibodies (anti-chicken 647, 1:500; Jackson ImmunoResearch, 703-605-155; and anti-rabbit PE, 1:200, BioLegend, Poly4064) for 15 min at 4 °C. After another wash step, nuclei were filtered through a 70 µm strainer, resuspended in sorting buffer (Tris base buffer with 2% BSA), and Hoechst (1:2,000) was added to visualize nuclei. NeuN+RBPMS+ nuclei were sorted using the BD FACS Aria III device running BD FACSDiva v.9.0.1 into Ames medium (Sigma-Aldrich, A1420) with 1.5% BSA. Sorted NeuN+RBPMS+ nuclei were pelleted at 500g for 5 min at 4 °C, resuspended in approximately 20 µl of 1% BSA in PBS, visually inspected, counted in a Neubauer chamber and adjusted to a concentration of around 1,000 nuclei per µl. Nuclei were then loaded onto the 10x Chromium Single Cell Chip G (10x Genomics) with a targeted recovery of about 12,000 nuclei per channel. Libraries were generated according to the manufacturer’s protocol using the Chromium Single Cell 3′ Reagent Kit version 3.1 (dual index), measured using the Agilent Bioanalyzer (TapeStation 4150) and sequenced on the Illumina NovaSeq 6000 platform (paired-end), aiming for a sequencing depth of around 30,000 reads per nucleus.
Data preprocessing and quality control
Count matrices for each sample were generated by aligning each library to the human reference mRNA transcriptome GRCh38-2020-A using Cell Ranger (v.7.0.1), including both exonic and intronic reads61. The Cell Ranger output was processed using CellBender v.0.3.0 with the default settings (epochs = 150, fpr = 0.01, learning rate = 10−4) to remove ambient RNA and other background noise62. Downstream analysis was performed with Seurat (v.5)63 in R Studio (R v.4.4.1). For each RNA count matrix, the following steps were carried out: cell counts were normalized to a total library size of 10,000 and log transformed (Seurat, NormalizeData). The top 2,000 variable features were identified (Seurat, FindVariableFeatures), followed by data scaling (Seurat, ScaleData) and dimensionality reduction (Seurat, RunPCA, npcs = 50). RPCA integration was performed to integrate the principal components (Seurat, IntegrateLayers) and used as input for k nearest-neighbour graph construction (Seurat, FindNeighbors, dims = 50) and Leiden clustering with a resolution of 2.5 (Seurat, FindClusters), initially deliberately overclustering the dataset. An initial dataset of 351,737 nuclei was subjected to quality control. Potential doublets were identified using the scDblFinder64 package. Given the defined cluster structure in our dataset, we used a cluster-based approach for doublet identification, estimating the standard 10x doublet rate of 0.8% per 1,000 nuclei. To remove non-RGC nuclei, we filtered the dataset for clusters with high expression of RBPMS, the main RGC marker gene. After subsampling, nuclei with abnormally high (greater than mean + 3 s.d.) gene counts, fewer than 2,300 gene counts, a doublet score of >0.5, as well as mitochondrial counts of >5% were removed.
Clustering and cell type annotation
After removing low-quality and non-RGC nuclei from the dataset, we repeated the normalization and clustering pipeline at a resolution of 0.5 (RunPCA, npcs = 30; FindNeighbors, dims = 30). For each cluster, we calculated differentially expressed marker genes compared with every other cluster (Seurat, FindMarkers). Clusters were merged if ≤5 differentially expressed genes were found between them, with an average log2-transformed fold change of >2 or <–2 and a P value < 0.05. Moreover, if a cluster contained fewer than 200 nuclei in the control condition or was absent in two or more samples, it was fused with its nearest neighbour based on a Euclidean distance matrix constructed in gene expression space (Seurat, BuildClusterTree; Supplementary Fig. 1e). The remaining 27 clusters were annotated manually based on their respective marker gene expression, using three published healthy human RGC datasets as reference8,9,10. To avoid disease-related transcriptional changes influencing cell type annotation, marker genes were identified exclusively from healthy control samples using Seurat’s FindAllMarkers function. Of the top 30 markers of each cluster, 2–3 are shown in Extended Data Fig. 1e. For each reference dataset, the top 50 marker genes per cell type were selected and used for gene set enrichment analysis (GSEA) through the ClusterProfiler65 package, applying the ranked cluster markers from the healthy control samples of our dataset. For the enrichment analysis in Extended Data Fig. 1c and the cell type marker identification in Extended Data Fig. 1e, the three midget OFF RGC subtypes (MG-OFF1, MG-OFF2, MG-OFF3) and the four midget ON RGC subtypes (MG-ON1, MG-ON2, MG-ON3, MG-ON4) were combined into single midget OFF and midget ON types. This consolidation streamlined marker gene identification and cell type annotation. The merged midget ON and midget OFF RGC clusters were also used for the UMAP representation in Fig. 1c.
Differential gene expression analysis
To analyse the transcriptional response of all RGCs during MS, we performed sample- and condition-wise pseudobulk aggregation of unnormalized counts (Seurat, aggregateExpression). The DESeq2 package66 (v.1.44) was used for normalization and differential gene expression analysis, considering genes with false discovery rate adjusted P value < 0.05 as differentially expressed. Enrichment analysis of biological themes was performed with the ClusterProfiler65 (v4.12.6) package.
Analysis of resilient and susceptible RGC subtypes
To assess differences in RGC vulnerability among cell types in MS, we calculated the relative frequency of each cell type in both control and MS RGC populations. Outliers were identified and excluded using Grubbs’ test. Significant differences in the relative frequencies between control and MS samples were assessed using Mann–Whitney U-tests. Clusters with significantly lower relative frequencies in MS compared with controls (P < 0.05, log2-transformed fold change < 0) were defined as susceptible. Clusters with a significantly higher relative frequency in MS compared with controls (P < 0.05, log2-transformed fold change > 0) were defined as resilient. Clusters falling into either term but not reaching significance (P > 0.05) were classified as intermediate susceptible (log2-transformed fold change < 0) or intermediate-resilient (log2-transformed fold change > 0). We performed sample- and condition-wise pseudobulk aggregation of significantly de-enriched (MG-ON4, MG-OFF2, ipRGC) and significantly enriched clusters (PG-ON, RGC26, RGC13) to compare transcriptional profiles between susceptible and resilient clusters. DESeq2 analysis was conducted to determine differentially expressed genes between resilient MS, resilient control, susceptible MS and susceptible control pseudobulks. Gene regulation modules were defined as follows: module A, genes upregulated in resilient RGCs compared to susceptible RGCs; module B, genes upregulated in susceptible RGCs compared with resilient RGCs; module C, genes upregulated in resilient RGCs during MS; module D, genes downregulated in resilient RGCs during MS; module E, genes upregulated in susceptible RGCs during MS; module F, genes downregulated in susceptible RGCs during MS. Gene Ontology analysis with the genes in each module was performed using the clusterProfiler65 package. To calculate resilience and vulnerability scores for the susceptible, intermediate susceptible, intermediate resilient and resilient pseudobulks of each donor and condition, we applied the AUCell algorithm67, using the top 200 module-defining genes from modules A–E as input, after pseudobulk aggregation and normalization of gene expression. To calculate intrinsic resilience signatures for each pseudobulked RGC subtype and donor, we used the top 200 genes of module A (intrinsic resilience signature) and module B (intrinsic susceptibility signature) as the input for AUCell analysis, after aggregation and normalization of gene expression by donor and cell type.
Linear regression analysis of intrinsic susceptibility and resilience genes
To identify genes of which the expression correlated with resilience, we extended the analysis to include all RGC types rather than focusing solely on the most resilient or susceptible clusters. Clusters were ranked from most susceptible to most resilient based on the signed negative logarithmic P value of their relative frequency in MS compared with controls. We focused on the ten samples from control individuals. To minimize biases arising from differences in cluster size across RGC types, we standardized cluster sizes by bootstrapping the dataset, randomly selecting 100 nuclei from each cluster within each sample. Clusters containing fewer than 10 nuclei were excluded. For clusters with fewer than 100 nuclei, sampling was performed with replacement; for larger clusters, it was performed without replacement. This process was repeated five times, and pseudobulk count vectors were summed across iterations. The DESeq2 package (v.1.44) was used to normalize gene counts of the size-adjusted pseudobulks, accounting for differences in sequencing depth and library size across samples. Variance stabilization was performed using the rlog function within DEseq2. For each gene, a linear regression was performed using the lm function in R (formula: normalized expression ~ resilience), modelling the normalized gene expression in a given cluster and sample as a function of the cluster resilience score, as defined previously by the signed negative logarithmic P value of a cluster’s relative frequency in MS compared with controls. P values for significance were adjusted for multiple comparisons using the FDR method.
Single-molecule fluorescence multiplex in situ RNA hybridization
smFISH was performed on representative post-mortem, unfixed MS and control cryosections (16 µm thickness) using the RNAscope Multiplex Fluorescent Detection Kit v2 (ACD Biotechne, 323100) according to the manufacturer’s protocol. The following human RNAscope assay probes were used (ACD Biotechne): POU4F1 (438441), TBR1 (425571-C2), FOXP2 (407261-C2), GPR149 (1040901-C3) and CFH (428731-C3). Probes were labelled with TSA Vivid Fluorophores (fluorescein, cyanine 3, cyanine 5, Akoya Biosciences). Each run included quality-control slides stained with the human 3-plex RNAscope positive-control probe (320861) and 3-plex negative-control probe (320871) from ACD Biotechne.
Image acquisition and quantification of fluorescence multiplex in situ RNA hybridization
Multiplex fluorescence images were acquired using the Leica DM6 B microscope equipped with a Leica K5 camera and the THUNDER imaging system for preprocessing. Images were captured at ×20 magnification as z stacks to ensure thorough signal capture within tissue sections. Acquisition was performed with LAS-X software, and images were exported as LIF files for subsequent analysis with QuPath68 (v.0.4.3). In QuPath, analysis was performed using the subcellular spot detection tool. The RGC layer was first selected, followed by cell detection based on the DAPI channel. Subcellular spot detection was then conducted for POU4F1, FOXP2, TBR1, CFH and GPR149. Double-positive cells (such as POU4F1-positive cells co-expressing specific subpopulation markers) were identified using an initial single-measurement classifier, followed by a composite classifier.
RT–qPCR
We reverse-transcribed RNA to complementary DNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1632) according to the manufacturer’s instructions. Samples were measured on the QuantStudio Flex Real-Time PCR System using TaqMan Gene Expression Assays (Thermo Fisher Scientific) for Cfh (mouse, Mm01299248_m1), RBPMS (human, Hs01060992_m1) and RCVRN (human, Hs00610056_m1). All analyses were performed in triplicate. We calculated gene expression as \({2}^{-\varDelta {C}_{{\rm{t}}}}\) relative to Tbp (mouse, Mm01277042_m1) or GAPDH (human, Hs99999905_m1) as the endogenous control.
Human histopathology of post-mortem tissues
Tissue paraffin sections were cut at 3 μm and stained with haematoxylin and eosin (H&E) according to standard procedures. For immunohistochemical staining, brain tissue sections were processed as follows. After dewaxing and inactivation of endogenous peroxidases (3% hydrogen peroxide), antibody-specific antigen retrieval was performed using the Ventana BenchMark XT autostainer. The sections were blocked with rabbit serum and then incubated with the primary antibody against human CFH (1:40; R&D, AF4779). For detection of specific binding, the anti-goat Histofine Simple Stain MAX PO immune-enzyme polymer (Nichirei, 414161F) was used as the secondary antibody. For DAB staining, the UltraView Universal DAB Detection Kit (Roche, 760-500) was used. Counterstaining and bluing were performed using haematoxylin (Ventana Roche, 760-2021) and Bluing Reagent (Ventana Roche, 760-2037) for 4 min. Subsequently, stained sections were mounted in mounting medium. For quantification, DAB-positive neurons were counted manually per area of cortical tissue within ImageJ (Fiji, v.2.14.0). Neurons were identified on the basis of their characteristic morphology in H&E-stained sections. Information about donors used for histopathology is provided in Supplementary Table 2.
Animal models
Mice
All mice, including C57BL/6J WT (The Jackson Laboratory), SJL/JRj WT (Janvier Labs), C3 knockout (B6.129S4-C3tm1Crr/J, The Jackson Laboratory) and Cfhflox/flox mice were maintained under specific pathogen-free conditions in the central animal facility of the University Medical Center Hamburg-Eppendorf (UKE). Adult mice aged 8–20 weeks were used for experiments. For EAE experiments involving C57BL/6J WT, SJL/JRj WT and C3-KO mice, only female animals were included. To assess potential sex dependent effects, an additional cohort of male C57BL/6J mice was used to compare neuronal mHDM-FH expression with a GFP control. For experiments with Cfhflox/flox mice both sexes were used. The mice were kept under a 12 h light–12 h dark cycle at 22 ± 2 °C and 40–60% relative humidity with ad libitum access to standard chow (Altromin, 1328P) and water. EAE mice additionally received DietGel Recovery (Ssniff, H007-72065). For EAE experiments, if both sexes were included, equal numbers of male and female mice were allocated to each experimental group. Furthermore, animals from each litter were evenly distributed across groups to minimize potential litter effects. Within these constraints, mice were randomly assigned to receive either effector-AAVs or control AAVs. Sample sizes were selected based on the principles of the 3Rs (replacement, reduction and refinement), the use of genetically homogeneous inbred mouse strains and sample sizes commonly used in previous studies employing the EAE model that detected comparable biological effects25,37,38. Clinical EAE scoring was conducted in a blinded manner, with researchers unaware of both the genotype and the injected AAV to prevent observer bias.
Generation of Cfh neuronal knockout mice
Cfhtm1a(EUCOMM)Wtsi sperm (The Jackson Laboratory) were revitalized on C57BL/6 wild-type female mice (The Jackson Laboratory), resulting in litters positive for the Cfhtm1a(EUCOMM)Wtsi allele. To obtain the conditional (tm1c) allele (Cfh-flox), these animals were bred with ACTB:FLPe B6J mice expressing the Flp recombinase (The Jackson laboratory, 005703)69. To generate neuronal Cfh-KO mice, we performed retrobulbar intravenous injections of an AAV (serotype PhP.eB) carrying cre recombinase under the control of the hSYN1 promoter in Cfhflox/flox mice.
EAE model
For experiments involving AAV-mediated expression, mice received retrobulbar injections of 100 μl of the respective AAV (serotype PhP.eB) at a titre of 3 × 1011 viral genomes in PBS 3 weeks before EAE induction. Mice were monitored daily and weighed starting 1 week after the injection. Mice injected with effector constructs were compared to littermates receiving GFP-only control AAVs. To induce EAE in C57BL/6 mice, they were immunized subcutaneously with 200 μg MOG35–55 peptide (Schafer-N) emulsified in Complete Freund’s adjuvant (CFA) (Difco, DF0639-60-6) containing 2 mg ml−1 Mycobacterium tuberculosis (Difco, DF3114-33-8). Moreover, 300 ng of pertussis toxin (Calbiochem, CAS70323-44-3, 516562) was administered intraperitoneally on the day of immunization and again 48 h later. For experiments in SJL mice, EAE was induced by subcutaneous immunization with 100 µl of PLP139–151 peptide (1.5 mg ml−1) emulsified 1:1 with 100 µl of CFA (Difco, DF0639-60-6) containing M. tuberculosis H37RA (1 mg ml−1, 100 µg per mouse). Clinical signs were assessed daily using the following scoring system: 0, no clinical deficits; 1, tail weakness; 2, hind limb paresis; 3, partial hind limb paralysis; 3.5, full hind limb paralysis; 4, full hind limb paralysis with forelimb paresis; and 5, premorbid state or death. Animals reaching a clinical score of ≥4 were euthanized in accordance with the regulations of the German Animal Welfare Act. All EAE experiments were conducted with investigators blinded to the genotype and treatment conditions.
Visual assessment in mice
To evaluate visual acuity and contrast sensitivity in EAE mice, automated optomotor testing was performed using the OptoDrum system (Striatech)70,71. Mice were placed individually onto an elevated central platform surrounded by computer monitors and monitored from above by a camera. The optomotor reflex, a head-tracking movement elicited by rotating visual stimuli, was induced by a moving vertical black-and-white grating displayed on the surrounding screens. Visual acuity was determined by progressively increasing the spatial frequency (that is, the number of stripes per degree of visual angle) until the optomotor reflex could no longer be elicited. Contrast sensitivity was assessed by gradually reducing the contrast of the grating until the reflex was no longer observed. For each stimulus condition (spatial frequency or contrast level), two successful responses were required to advance to the next step, whereas three consecutive failures were defined as an unsuccessful trial.
Analysis of published EAE sequencing datasets
Raw count matrices and metadata from publicly available RNA-seq datasets were retrieved from the Gene Expression Omnibus (GEO) repository. The GSE118948 dataset includes single-cell sequencing data of CD45+ cells isolated from the spinal cords of EAE mice 15 days after immunization. Analysis of the single-cell data was performed using Seurat, with cell type annotations from the original publication22. Differential expression analysis was conducted with the FindMarkers function within Seurat using a Wilcoxon rank sum test to compare gene expression in healthy mice and those with acute EAE across T cells, neutrophils, dendritic cells, macrophages, monocytes, CNS-associated monocytes and microglia. Bulk RNA-seq data from GEO GSE194071 (ref. 23) were used to examine spinal cord microglia in acute and chronic recovery EAE mice, while GSE100329 (ref. 24) provided data for analysing spinal cord astrocytes in acute and chronic progressive EAE mice. Moreover, GSE104899 (ref. 25) and GSE279707 (ref. 26) were used to study spinal cord motor neurons in acute EAE mice. All bulk sequencing data were analysed using DESeq2 (v.1.44), and signed negative logarithmic FDR-adjusted P values were calculated for visualization.
Immunohistochemistry of spinal cord tissue
Mouse spinal cord tissue was obtained and processed as described previously72. Images were acquired using a Zeiss LSM 900 Airyscan 2 laser-scanning confocal microscope equipped with ZEN blue software v.3.9. Antibodies and their dilutions used for immunohistochemistry are specified in Supplementary Table 3.
Immunohistochemistry analysis of mouse retinal whole mounts
After transcardial perfusion of mice with ice-cold 4% PFA, the orientation of the eye was marked in situ, and eyes were enucleated and post-fixed in 4% PFA for 1 h. The eyeballs were then transferred to ice-cold PBS. Whole-mount dissection of retinas was performed under a dissection microscope. For immunohistochemistry, retinas were washed in PBS 0.5% Triton X-100 and permeabilized by freezing them at −70 °C for 15 min. After thawing, the retinas were rinsed with PBS 0.5% Triton X-100 and incubated overnight with primary antibodies at 4 °C in blocking buffer (PBS with 2% Triton X-100 and 2% normal donkey serum). The retinas were subsequently washed in PBS 0.5% Triton X-100 and incubated with secondary antibodies in PBS with 2% Triton X-100 for 2 h at room temperature. The retinas were washed with PBS and mounted vitreal side up. All antibodies used are listed in Supplementary Table 3. Retinal whole mounts were imaged using the Zeiss Axiocam 705 mono microscope. Semi-automated quantification of GFP-positive RGCs was performed within Fiji (ImageJ, v.2.14.0). Background subtraction was applied to correct for uneven illumination and contrast enhancement was performed to optimize illumination. Thresholding used the default autothreshold method and the image was then converted into a binary mask. To reduce noise, the Despeckle function was applied. Segmentation was refined with the Watershed algorithm to separate cells. Particle analysis was conducted on segmented objects with inclusion of particles with a size of 30–300 pixels and circularity range of 0.4–1.0.
Luxol fast blue staining
Myelination in EAE mice was assessed by Luxol Fast Blue staining in transverse sections of the dorsal columns in the cervical, thoracic and lumbar spinal cord as previously described73. The slides were imaged on the NanoZoomer 2.0-RS digital slide scanner with NDP.view2 software (Hamamatsu). A customized counting mask within Fiji (ImageJ, v.2.14.0) was created to quantify the area of Luxol Fast Blue-positive axons in the white matter of the spinal cord. The Luxol Fast Blue-positive area was normalized to the total analysed area within the dorsal column.
Immunoblot
Spinal cords of healthy and EAE mice were homogenized in 2 ml radioimmunoprecipitation buffer (50 mM Tris, 150 mM NaCl, 0.5 mM EDTA, 10% SDS, 1% NP-40, 10% sodium deoxycholate, with protease and phosphate inhibitor cocktails (cOmplete, Sigma-Aldrich, 11836170001)) using a tissue grinder and incubated at 4 °C for 30 min on a rotating wheel. The lysates were centrifuged for 5 min to remove the cell debris. Protein concentrations were determined by BCA assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, 23228 and 23224) according to the manufacturer’s protocol, and 25 µg of protein were loaded onto SDS–PAGE (NuPAGE, Thermo Fisher Scientific, NW04125BOX) followed by wet transfer to polyvinylidene fluoride membranes. Blocking was performed using 5% BSA for 1 h at room temperature. Membranes were incubated with primary antibodies overnight at 4 °C. Horseradish-peroxidase-coupled secondary antibodies were applied for 1 h at room temperature, and chemiluminescence was visualized using WesternSure PREMIUM Chemiluminescent Substrate (LI-COR, 926-95000) according to the manufacturer’s protocol. A list of all of the antibodies used is provided in Supplementary Table 3. All uncropped western blot membranes are provided in Supplementary Fig. 18.
Immune cell isolation for flow cytometry
Spinal cord tissue was collected after transcardial PBS perfusion and dissociated into single-cell suspensions using 1 mg ml−1 collagenase A (Roche, 11088793001) and 0.1 mg ml−1 DNase I from bovine pancreas (Merck Millipore, 260913) with the gentleMACS Octo Dissociator (Miltenyi Biotec, program: Multi_F). The dissociated tissue was passed through a 70 µm cell strainer, and immune and glial cells were enriched using a discontinuous density gradient. After centrifugation at 2,500 rpm, 4 °C for 30 min, cells were collected from the interphase between the 30% Percoll and 70% Percoll layers. Non-specific Fc-receptor-mediated antibody binding was blocked by pre-incubation with TruStain FcX anti-mouse CD16/32 antibody (BioLegend, 101320) for 10 min at 4 °C, followed by surface antibody staining in Brilliant Stain Buffer (BD Biosciences, 563794) for 30 min at 4 °C. Dead cells were excluded from analysis using Zombie Green Fixable Viability Stain (BioLegend, 423112). For intracellular staining of CD206 and CD68, cell suspensions were fixed for 20 min at room temperature using fixation buffer (BioLegend, 420801), followed by 20 min incubation with anti-CD68 antibody in intracellular staining permeabilization wash buffer (BioLegend, 421002). Absolute cell numbers for CD45high leukocytes and CD45med microglia were determined using Precision Count Beads (BioLegend, 424902). We obtained data using the BD Symphony A3 flow cytometer (BD Biosciences) and analysed data using FlowJo v.10.9 (BD Biosciences).
Viral vectors
Vector construction
All primers and oligonucleotides used in this study are listed in Supplementary Table 4. For expression of synthetic minimal CFH-derived constructs, clonal genes were synthesized by Twist Bioscience (Supplementary Table 4 (gene synthesis tab)). In mHDM-FH, the N-terminal SCR1–5 are linked to C-terminal SCR18–20 through an SGSG linker. Amino acids 24–152 of mouse CFHR-1 were used as a dimerization unit to increase protein half-life. The amino acid sequence of mHDM-FH was derived from a previous study31. CR2-FH is a fusion protein consisting of a complement receptor 2 fragment linked to the N-terminal SCR domains 1–5 of CFH. The amino acid sequence of CR2-FH contains the signal peptide of mouse IgG κ light chain for optimal secretion, amino acid residues 1–257 of mature mouse CR2, a (G4S)2 linker, followed by the 5 N-terminal SCR domains of mouse factor H33. Both sequences were codon-optimized for expression in mouse cells. A Pfl23II restriction-enzyme-recognition site was attached to the 5′ end, and BamHI and SalI sites were added to the 3′ end. Clonal genes were digested with Pfl23II and SalI for ligation into a customized pAAV backbone with a hSYN1 promoter derived from pAAV-hSYN1-mTurquoise2 (gift from V. Gradinaru, Addgene 99125), an eGFP:KASH cassette (AAV:ITR-U6-sgRNA(backbone)-hSyn-cre-2A-EGFP-KASH-WPRE-shortPA-ITR, gift from Feng Zhang, Addgene 60231) and a P2A-T2A cleavage peptide. A control plasmid containing a stop codon was cloned downstream of the P2A-T2A cleavage peptide. For lentiviral expression in primary neuronal cultures, clonal genes were digested with Pfl23II and BamHI for ligation into a custom lentiviral backbone with an hSYN1 promoter, a mScarlet cassette and a P2A cleavage peptide. For lentiviral expression of full-length mouse CFH, two separate PCR fragments were generated and linked with a triple-ligation strategy. The full-length Cfh sequence was amplified from mouse kidney cDNA with primers containing Pfl23II and XbaI, or XbaI and SgsI restriction sites, respectively. The inserts were ligated into the same lentiviral backbone described above. These constructs were subsequently used as templates to generate different mHDM-FH and CFH variants using the primers in Supplementary Table 4. To create constructs lacking the signal peptide, forward primers excluding the first 54 base pairs (P15–P18) were used. To exclude SCR20, we used reverse primers complementary to SCR19 (P10–P12) and inserted the construct upstream of the dimerization domain. The 2×KDEL sequences for retention in the secretory pathway were included in the reverse primers (P19–P20). For expressing the SCR20 alone (P13–P14), SCR20 was added downstream of an Lck-transmembrane domain (P8–P9) for intracellular retention at the cell membrane. The Lck domain was amplified from PZac2.1 hSYN1-Lck-13×Linker-BioID2-BioID2-HA (gift from B. Khakh, Addgene 176854). The lentiviral IFNγ overexpression construct was generated as previously described37. We used an AAV carrying cre recombinase under the hSYN1 promoter to generate conditional knockouts in neuronal cultures (pAAV-hSYN1-cre-P2A-dTomato, gift from R. Larsen (Addgene 107738). To generate conditional knockouts in mice, the plasmid pENN.AAV.hSYN.HI.eGFP-cre.WPRE.SV40, a gift from J. M. Wilson (Addgene 105540) was used. For expression of TurboID constructs, the sequence for the biotin ligase was amplified from pAAV-TBG-Cyto-TurboID (gift from J. Long, Addgene 149414) using primers for a C-terminal HA protein tag and inserted into a lentiviral backbone with a hSYN1 promoter using NheI and BamHI (Turbo-Cyto; P21-P22) or Nhel and EcoRI (Turbo-ER; P23-P24). For ER retention of the TurboID-ER construct, we included an N-terminal signal peptide and a C-terminal KDEL sequence, separated by a GGGGS linker. For in vivo delivery, the TurboID-ER cassette was transferred into a pAAV backbone with a hSYN1 promoter using NheI and SgsI (P25–P26). The following constructs were generated and used for in vitro or in vivo experiments in this study: Lenti-hS-mmCfh-P2a-mScarlet, Lenti-hS-mmCfhΔ20-P2a-mScarlet, Lenti-hS-mScarlet-P2a-mmCfh-2×KDEL, Lenti-hS-mmCfhΔSP-P2a-mScarlet, Lenti-hS-mmCfhΔ20ΔSP-P2a-mScarlet, Lenti-hS-mScarlet-P2a-CR2-FH, Lenti-hS-mHDM-FH-P2a-mScarlet, Lenti-hS-mHDM-FHΔ20-P2a-mScarlet, Lenti-hS-mScarlet-P2a-mHDM-FH-2xKDEL, Lenti-hS-mScarlet-P2a-mHDM-FHΔ20-2×KDEL, Lenti-hS-mHDMΔSP-FH-P2a-mScarlet, Lenti-hS-mHDM-FHΔ20ΔSP-P2a-mScarlet, Lenti-hS-Lck-scr20-P2a-mScarlet, Lenti-CMV-mmIfng-P2a-mScarlet, Lenti-hS-mScarlet, Lenti-CMV-mScarlet, Lenti-hS-TurboID-ER, Lenti-hS-TurboID-Cyto, pAAV-hS-eGFP(-KASH)-P2a(-T2a)-mHDM-FH, pAAV-hS-eGFP-P2a-mHDM-FHΔ20, pAAV-hS-eGFP-P2a-mHDM-FHΔSP, pAAV-hS-eGFP-P2a-Lck-scr20, pAAV-hS-eGFP(-KASH), pAAV-hS-eGFP-KASH-P2a-T2a-CR2-FH, pAAV-hS-TurboID-ER. All final products were confirmed by Sanger sequencing.
Lentiviral production
To produce VSV-G-pseudotyped lentiviruses, HEK293T cells were seeded out at 6 × 104 per cm2 1 day before transfection in DMEM containing glutamine and high glucose (Thermo Fisher Scientific), together with helper plasmids pMDLg/pRRE, pRSV-Rev and pMD2.G. pMDLg/pRRE (gift from D. Trono, Addgene 12251, 12253, 12259). For a 10 cm cell culture plate, we used 15 µg transfer plasmid, 10 µg pMDLg/pRRE, 5 µg pRSV-Rev and 2 µg pMD2.G. Plasmids were mixed in 1× HEPES-buffered saline (HBS) and 125 mM CaCl2 solution and applied to the HEK293T cells in the presence of 25 µM chloroquine diphosphate. After 12 h, the medium was changed and the supernatant was collected at 36 h after transfection, filtered through a 0.45 µm PES filter and immediately snap-frozen and stored at −80 °C. For CFH expression lentivirus production, lentiviral particles were concentrated using the Lenti-X Concentrator (Takara Bio, 631478) according to the manufacturer’s instructions. Primary neurons were transduced at 5–8 days in vitro (d.i.v.) with 70–80% efficiency, visually confirmed by fluorescent protein expression.
AAV production
For vector production, we selected the PhP.eB serotype, which has shown high efficiency in transducing the CNS74. AAV particles were produced according to the standard procedures of the UKE vector facility38,75.
Cell culture
Primary neuronal cultures
For primary cortical cultures, pregnant C57BL/6J or Cfhflox/flox mice were euthanized, and cortices were isolated and dissociated. Cells were plated at a density of 6 × 104 cells per cm2 on poly-d-lysine-coated wells (5 µM, Sigma-Aldrich) and maintained in PNGM medium (Lonza, CC4461) at 37 °C, 5% CO2 and 98% relative humidity. Experiments were performed using cultures between 14 and 23 d.i.v. To generate Cfh-cKO neurons, neuronal cultures from Cfhflox/flox mice were transduced at 4 d.i.v. with an AAV PhP.eB carrying cre recombinase (Addgene 107738) under the control of an hSYN1 promoter at a multiplicity of transfection (MOI) of 30,000. The transduction efficiency, estimated visually based on eGFP expression, was 70–80%. Chronic IFNγ exposure was achieved by delivering a lentivirus containing an Ifng expression construct under the control of a CMV promoter at 7 d.i.v.37. Either mScarlet or GFP-only controls were used for all AAV and lentiviral expression experiments.
RNA-seq analysis of neuronal Cfh-cKO cultures
RNA from neuronal Cfh-cKO and control cultures with or without glutamate stimulation were isolated using the RNeasy Mini Kit (Qiagen). RNA-seq libraries were prepared using the TruSeq stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions. After pooling, libraries were sequenced on the NovaSeq 6000 sequencer (Illumina) generating 150 bp paired-end reads. Reads were aligned to the mouse reference genome (mm10, 2020A) using STAR v.2.5.2b with the default parameters. Overlaps with annotated gene loci were counted using featureCounts v.1.5.1. Differential expression analysis was performed using DESeq2 (v.1.44), with genes showing a FDR-adjusted P < 0.05 considered differentially expressed. Gene lists were annotated using biomaRt (v.2.60.1). We then performed an AUCell (v.1.26) analysis to directly compare a transcriptional excitotoxicity signature between WT control, Cfh-cKO control, and glutamate-stimulated WT and Cfh-cKO neuronal cultures. The transcriptional excitotoxicity signature was defined as all differentially upregulated genes in our glutamate-stimulated WT neuronal cultures in comparison to the WT control condition. Moreover, we performed GSEA with the top 200 glutamate-induced genes in the Cfh-cKO glutamate versus WT glutamate condition.
Pfa1 and HEK293T cell lines
Immortalized mouse fibroblasts (Pfa1 cells) were received from the originating laboratory (Conrad laboratory, Helmholtz Institute)41,76 and were not further authenticated. HEK293T cells were purchased from an authorized vendor (ACC 635, DMSZ). Pfa1 cells and HEK293T cells were maintained in DMEM high glucose (4.5 g l−1 glucose, 21969-035, Gibco) supplemented with 10% FBS, 2 mM l-glutamine and 1% penicillin–streptomycin at 37 °C with 5% CO2. Cells were passaged after reaching approximately 80% confluency and were routinely tested for mycoplasma contamination. For experiments, they were seeded at 30,000 cells per cm2 1 day before stimulation. The respective compounds and stimulation times are specified in the corresponding figure legends. All cell lines were regularly checked for mycoplasma contamination using the VenorGeM Advance kit (Minerva biolabs, 11-7024) according to the manufacturer’s instructions. Cell lines were free of mycoplasma contamination.
Cell viability assay
The RealTime-Glo MT Cell Viability Substrate and NanoLuc Enzyme (Promega, G9711) were combined, added to neuronal cultures and incubated for 5 h to allow equilibration of the luminescence signal before treatment application. Toxicity was assessed after exposure to 50 μM glutamate, 100 nM MDA (Sigma-Aldrich, 63287) or 6 µM RSL3 (Sigma-Aldrich, SML2234). Luminescence was recorded immediately before the stressors were applied and 15 h later using the Spark 10 M multimode microplate reader (Tecan) at 37 °C and 5% CO2. At least four technical replicates were included for each condition; each well’s datapoints were normalized to its final luminescence value before stressor application. Subsequently, the data were normalized to the mean of the control wells at each timepoint to account for well-to-well variability in cell seeding and to the maximal neuronal death (2 mM glutamate) condition. Statistical comparisons were performed using end-point measurements.
For the medium change, CFH antibody and IFNγ experiments, we measured cell viability by quantifying condensed nuclear signal. Neuronal cultures were exposed to 50 µM glutamate, 100 nM MDA or 6 µM RSL3 for 10 h. Where indicated, additional treatments were applied 30 min before the stressors. Hoechst 33342, a cell-permeant nuclear counterstain, was added to the culture wells at a final concentration of 20 µM and incubated for 1 h. After incubation, cells were washed twice with preconditioned medium before imaging. Image acquisition was performed using the Zeiss LSM 900 Airyscan 2 confocal microscope with a ×40 magnification objective. Using Fiji, we generated a mask to capture all nuclei and measured the Hoechst 33342 fluorescence intensity. We then defined condensed nuclei using a threshold corresponding to the mean signal of the control conditions plus 2.5 s.d. This was used to estimate the number of dying and surviving neurons after the respective stimuli were applied.
ROS imaging
ROS levels in response to glutamate, MDA and RSL3-induced stress were measured using CellROX Green Reagent (Thermo Fisher Scientific, C10444). Neuronal cultures were stimulated with 50 μM glutamate for 2 h, 100 nM MDA for 10 h or 6 µM RSL3 for 10 h. CellROX Green was added to each well at a final concentration of 5 μM after 30 min of stressor exposure, alongside Hoechst 33342, as a nuclear counterstain for visualization. After the incubation, cells were washed twice with preconditioned medium to remove excess reagents and imaged using the Zeiss LSM 900 Airyscan 2 confocal microscope at ×20 magnification. Nuclear fluorescence of the CellROX signal was quantified using Fiji (ImageJ, v.2.14.0) to assess ROS levels.
BODIPY C11 imaging
To assess lipid peroxidation, the BODIPY C11 probe (Image-iT, Thermo Fisher Scientific, D3861) was used. Neuronal cultures were exposed to 50 µM glutamate, 100 nM MDA or 6 µM RSL3 for 10 h. Where indicated, additional treatments were applied 30 min before the stressors. BODIPY C11 was added to the culture wells at a final concentration of 20 µM and incubated for 1 h. Simultaneously, Hoechst 33342, a cell-permeant nuclear counterstain, was included. After incubation, cells were washed twice with pre-conditioned medium before imaging. Image acquisition was performed using the Zeiss LSM 900 Airyscan 2 confocal microscope with a ×40 magnification objective. Lipid peroxidation was quantified using Fiji (ImageJ, v.2.14.0) software. In lentiviral expression experiments, the ratio of oxidized to non-oxidized lipids was calculated due to the negligible or undetectable mScarlet signal from lentiviral transduction under the microscopy settings used. Owing to the stronger tdTomato signal emitted after AAV transduction of our pAAV-hSYN1-cre-P2A-dTomato constructs, only the MFI of the oxidized lipids was quantified in these experiments. For time-resolved and subcellularly resolved lipid peroxidation analyses, we used the following live cell imaging dyes: 1 µM MitoTracker Deep Red FM (Thermo Fisher Scientific), 1 µM ER-Tracker Blue-White DPX (Thermo Fisher Scientific), 500 nM SiR-lysosome kit (Cytoskeleton), 10 µg ml−1 Hoechst 33342 (Thermo Fisher Scientific) and 500 nM SiR700 actine kit (Cytoskeleton). Dyes were applied in the indicated concentrations and washed out together with the BODIPY C11 probe. All imaging experiments, except for the ER-Tracker, included Hoechst 33342 staining. The oxidized BODIPY C11 signal in the overlap with the respective cell organelle marker was quantified.
Quantification of complement component levels by ELISA
Peripheral blood was collected from the vena cava at the time of euthanasia. The samples were centrifuged at 1,000g for 10 min at 4 °C, and plasma was stored at −80 °C until use. Spinal cord tissue was collected after perfusion with ice-cold PBS and snap-frozen in liquid nitrogen. Tissues were lysed in protein extraction buffer (100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate) supplemented with phosphatase and protease inhibitor cocktails and PMSF (final concentration 1 mM) added immediately before use. ELISAs were performed according to the manufacturers’ instructions. CFH levels were determined using the Mouse Complement Factor H ELISA Kit (Hycultec, RD-CFH-Mu); plasma samples were diluted 1:100,000 in PBS, and cell culture supernatants were used undiluted. Total C3 concentrations were measured using the Mouse C3 ELISA Kit (Abcam, ab263884); plasma was diluted 1:100,000 in sample diluent NS, and tissue lysates were diluted 1:1,000 in 1× cell extraction buffer PTR. C3a levels were quantified using the Mouse C3a ELISA Kit (MyBioSource, MBS2701721); plasma was diluted 1:500 in PBS, and tissue lysates were diluted 1:20 in reagent diluent. C5a concentrations were measured using the Mouse C5a DuoSet ELISA Kit (R&D Systems, DY2150); plasma was diluted 1:100 in PBS, and tissue lysates were diluted 1:10 in reagent diluent. To assess complement activation, C3 consumption was calculated by dividing total C3a concentrations by total C3 concentrations for each individual sample (C3a/C3 ratio). Standard curves for all ELISAs were generated using the provided recombinant standards and fitted with a four-parameter logistic regression model.
Immunocytochemistry
Neuronal cultures were grown on 12-mm-diameter coverslips and stimulated with 50 μM glutamate, 100 nM MDA or 6 µM RSL3 for 6 h. After stimulation, cells were fixed in 4% PFA for 15 min and blocked in 10% normal donkey serum containing 0.1% Triton X-100. Immunolabelling was performed using the specified antibodies (details are provided in Supplementary Table 3). Images were captured using the Zeiss LSM900 Airyscan 2 laser-scanning confocal microscope. Co-localization was analysed using Fiji (ImageJ, v.2.14.0). The percentage of overlap with CFH (from total CFH within the cell) was reported.
PLA between CFH and MDA-modified proteins
To investigate the interaction between CFH and MDA-modified proteins, we performed a Duolink in situ proximity ligation assay (PLA) according to the manufacturer’s instructions (Merck). In brief, PLA technology uses pairs of primary antibodies conjugated to complementary oligonucleotides that, when bound within around 40 nm of each other, enable rolling-circle amplification and generate a discrete fluorescence signal after hybridization with labelled probes. For detection of CFH–MDA interactions, a Cfh-KO-validated goat anti-CFH antibody (Quidel, A313, 1:200) was combined with the PLA Goat MINUS probe (Merck, DUO92006), and a rabbit anti-MDA-modified protein antibody (Abcam, ab27642, 1:200) was paired with the PLA Rabbit PLUS probe (Merck, DUO92002). Signal amplification was achieved using the Duolink in situ detection reagent, far-red channel (Merck, DUO92013). To identify neurons, actin co-staining was performed in parallel. Images were acquired using the Zeiss LSM900 Airyscan 2 laser-scanning confocal microscope. Puncta per neuron were counted and quantified.
Proximity labelling with TurboID
Proximity-dependent biotinylation was performed using the biotin ligase TurboID targeted either to the ER lumen or to the cytoplasm. Cytosolic TurboID (TurboID-Cyto) expresses an HA-tagged TurboID biotin ligase under the hSYN1 promoter for cytosolic localization. ER-TurboID contains an HA-tagged TurboID (TurboID-ER) with an N-terminal signal peptide (SP) and a C-terminal KDEL ER-retention motif under the hSYN1 promoter. Cells were transduced with lentiviruses expressing either cytoplasmatic or ER-targeted TurboID at d.i.v. 7. Proximity labelling was induced by supplementing the cell culture medium with 50 µM biotin (IBA Lifescience, 6-6325-001) for 30 min at 37 °C. Cells were then rapidly cooled on ice, washed with ice-cold PBS and lysed in denaturing lysis buffer (8 M urea, 10 mM Tris buffer, 100 mM NaH2PO4). The lysates were incubated with streptavidin-coated magnetic beads (Thermo Fisher Scientific, 88817) overnight, washed twice with RIPA buffer, once with 1 M KCl, once with 0.1 M Na2CO3, once with 2 M urea in 10 mM Tris-HCl (pH 8.0) and twice again with RIPA buffer. Bound proteins were eluted by boiling in SDS sample buffer at 95 °C for 5 min for immunoblot analysis. For in vivo proximity labelling, mice received retrobulbar injections of 100 μl of an AAV carrying ER-retained TurboID (serotype PhP.eB) at a titre of 3 × 1011 viral genomes in PBS 3 weeks before EAE induction. For biotin pulsing, chow was soaked with 2.5 mg biotin (IBA Lifescience, 6-6325-001) per mouse and day dissolved in water starting 3 days before termination. At the day of perfusion, mice received an i.p. injection of 2.5 mg biotin dissolved in 100 µl PBS in addition. Spinal cord tissue was collected after transcardial perfusion with 10 ml ice-cold PBS. Tissues were homogenized in lysis buffer using a tissue disruptor (twice for 1 min at 4 °C with intermittent cooling on ice) and lysates were cleared by centrifugation (20,000g, 30 min, 4 °C). Supernatants were incubated overnight at 4 °C with equilibrated streptavidin magnetic beads (100 µl per sample) under rotation. Beads were subsequently washed twice with RIPA buffer, once with 1 M KCl, once with 0.1 M Na2CO3, once with 2 M urea in 10 mM Tris-HCl (pH 8.0), and twice again with RIPA buffer. Bound proteins were eluted in 2× reducing sample buffer by heating at 95 °C for 5 min before downstream analysis.
LC–MS/MS based proteomics
Samples were boiled at 95 °C for 5 min and sonicated with a probe sonicator. The digestion was performed in a 96-well LoBind plate (Eppendorf) semi-automated on the Andrew+ Pipetting Robot (Waters). Disulfide bonds were reduced in 10 mM dithiothreitol for 30 min at 56 °C while shaking at 800 rpm and alkylated in presence of 20 mM iodoacetamide for 30 min at 37 °C. Carboxylate-modified magnetic E3 and E7 speed beads (Cytvia Sera-Mag) at a 1:1 ratio in liquid chromatography–mass spectrometry (LC–MS)-grade water were added in a 10:1 (beads/protein) ratio adapted from the SP3-protocol workflow77. Protein binding was performed at 50% acetonitrile during shaking at 600 rpm for 18 min. Magnetic beads were washed twice with 80% ethanol and 100% acetonitrile. Digestion with trypsin in 100 mM AmBiCa was performed (sequencing grade, Promega) at a 1:100 (enzyme:protein) ratio at 37 °C overnight while shaking at 500 rpm. Trifluoroacetic acid was added to a final concentration of 1% and shaken at 500 rpm for 5 min. The supernatant containing tryptic peptides was transferred into a new 96-well LoBind plate, ready for subsequent LC–MS/MS analysis. Chromatography separation of tryptic peptides was achieved with a two-buffer system (buffer A: 0.1% formic acid in H2O, buffer B: 0.1% formic acid in 80% acetonitrile) on a UHPLC (Vanquish neo UHPLC system, Thermo Fisher Scientific) at a flow rate of 0.4 µl min−1. Attached to the UHPLC was a peptide trap (300 µm × 5 mm, C18, PepMap Neo Trap Cartridge, Thermo Fisher Scientific) for online desalting and purification, followed by a 25 cm C18 reversed-phase column (120 Å, 1.7 µm, 75 µm × 250 mm, Aurora Ultimate, IonOptics). Peptides were separated using a 70 min method with linearly increasing acetonitrile concentration from 3% to 34% buffer B over 60 min. MS/MS measurements were performed on a quadrupole-orbitrap hybrid mass spectrometer (Exploris 480, Thermo Fisher Scientific). Eluting peptides were ionized using a nano-electrospray ionization source (nano-ESI) with a spray voltage of 1,700 and analysed in data-independent acquisition (DIA) mode. For each MS1 scan, the ion-accumulation time was set to automatic, with an AGC target of 300%. The scan was set to m/z 400–800 with a resolution of 120,000 at m/z 200. Within a precursor mass range of m/z 400–800, fragmentation in DIA mode with m/z 12 isolation windows and m/z 1 window overlaps was performed (total of 33 scan events). Fragmentation was performed at a normalized collision energy of 30% using higher-energy collisional dissociation. The Orbitrap resolution was set to 60,000. LC–MS/MS data were searched using the CHIMERYS algorithm integrated into the Proteome Discoverer software (v.3.1.0.638, Thermo Fisher Scientific) with the Inferys 3.0.0 as the prediction model against a reviewed Mus musculus Swissprot database. Carbamidomethylation was set as a fixed modification for cysteine residues. The oxidation of methionine was allowed as variable modification. A maximum number of one missing tryptic cleavage was set. Peptides between 7 and 30 amino acids were considered. A strict cut-off (FDR < 0.01) was set for peptide and protein identification.
Ethics
All experimental procedures involving animals complied with institutional guidelines and adhered to the German Animal Welfare Act. Ethical approval for animal experiments was granted by the State Authority of Hamburg, Germany (approval numbers 41/22, 53/24, 40/25). For human eye tissue obtained from the Johns Hopkins University, all procedures were approved within the IRB00374036 “Characterizing mechanisms involved in pathological processes of retina neurodegeneration and optic neuritis present in MS in post-mortem samples”. For the human tissue analyses, as the samples could no longer be linked to identifiable individuals, the work did not qualify as a “research project on humans” under § 9 para. 2 of the Hamburg Chamber of Commerce Act for the Health Professions. Consequently, consultation required under § 15 para. 1 of the Professional Code of Conduct for Physicians in Hamburg was not necessary. For cortex tissue obtained from the UK Multiple Sclerosis Tissue Bank at Imperial College London, all procedures used by the Tissue Bank in the procurement, storage and distribution of tissue have been approved by the relevant Multicentre Research Ethics Committee (08/MRE09/31+5).
Statistical analysis
All statistical analysis was performed in R (v.4.4.1). The statistical analyses used for snRNA-seq data are described in the respective sections of the Article. A detailed description of statistical methods for experimental data is provided in the figure legends. Unless stated otherwise, data are presented as mean values. Differences between two experimental groups were determined using unpaired two-tailed Student’s t-tests or Mann–Whitney U-tests. The reported n represents biologically independent samples and not technical replicates. Statistical analysis of the clinical scores in EAE experiments was performed using the Mann–Whitney U-test on the areas under the curve for each animal.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
