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Additional file 4: Supplementary Figure 3. Range of CSF markers in individuals divided by CSF Aβ42/Aβ40 ratio or CDR score. Significant differences are indicated with stars, *** p

Additional file 8: Supplementary Figure 7. Boxplots of proteins displaying significant differences between individuals divided by NfL concentration and CSF Aβ42/Aβ40. Upon stratification of individuals based on both CSF Aβ42/Aβ40 ratio and NfL concentration two trends in protein profiles could be identified; higher protein levels in Nf + individuals, independently of Aβ42/Aβ40 ratio and higher protein levels in the Nf + A+ group.

Additional file 5: Supplementary Figure 4. Boxplots of proteins displaying significant differences between A+ individuals and A- individuals.

Additional file 10: Supplementary Figure 9. Heatmap of tissue expression for all studied proteins across 37 human tissues. When comparing the expression profiles of the 104 analysed proteins across 37 different human tissues, the responding genes could be divided into four clusters. The first cluster showed a general expression in all tissue types (Cluster 1) and the second cluster displayed elevated expression in the liver compared to other tissues (Cluster 2). A third cluster had higher expression in the brain (Cluster 3) and the last was a mixed group with high expression in brain or other tissue types (Cluster 4).

Additional file 7: Supplementary Figure 6. Sex differences for RIMS3 and VCAM1. Significant differences between female and male protein levels could be identified using the Wilcoxon rank sum test. However, sex showed no significant interaction with the CSF markers.

Additional file 9: Supplementary Figure 8. Correlations to NRGN concentration and comparison of NRGN levels between A+ and A- individuals. (A) Visualization of Pearson R for correlations between NRGN concentration and the 104 measured proteins. Thirty-seven proteins displayed a Pearson R > 0.5 and are annotated by their HGNC ID. (B) Higher concentration of NRGN was observed in A+ individuals compared to the A- individuals.

Additional file 3: Supplementary Figure 2. Correlation scatterplots between suggested markers for AD, neurodegeneration or synaptic dysfunction. All correlations with a Spearman rho value> 0.6 are displayed in red.

Additional file 6: Supplementary Figure 5. T-tau association with GAP43 levels for individuals divided by CDR score. (A) Scatterplot of GAP43 levels and t-tau concentration. Both individuals with CDR ≥ 0.5 and CDR = 0 display significant associations of GAP43 levels with t-tau concentration (CDR ≥ 0.5: Spearman rho = 0.72; p = 3E-08; CDR = 0: Spearman rho = 0.81; p = 4E-56). (B) Linear regression revealed no significant difference between slopes of CDR ≥ 0.5 and CDR = 0 individuals for the association of GAP43 with t-tau concentration.

Additional file 2: Supplementary Figure 1. Dichotomization of individuals into groups based on NfL concentration and APOE ε4 carrier status.

Additional file 5: Supplementary Figure 4. Antibodies used for development of NF-M sandwich assay aligned to the neurofilament amino acid sequences. The neurofilaments are shown in green and the size of the domains (head, rod and tail) are displayed at the top. The highly conserved rod domain is highlighted in grey. Position and length of the amino acid sequences used to generate the antibodies are shown in blue and the exact amino acid positions are given in brackets, if known. Antibody 13–1300 targets all three neurofilaments. The smallest epitope identified for HPA022845 corresponds to amino acids 746–749 [50].

Additional file 4: Supplementary Figure 3. Correlation between NF-M assays. (A) Correlation between HPA022845 and HPA023138 single-binder data. (B) Correlation between HPA022845 sandwich assays. HPA022845 was used as capture antibody together with two different detection antibodies, 20,664–1-AP and 13–0700. (C) Correlation between one HPA22845 sandwich assay and HPA023138 sandwich assay. HPA022845 was used as capture antibody together with 20,664–1-AP as detection antibody, and HPA023138 was used as capture antibody together with 34–1000 as detection antibody.

Additional file 1: Supplementary Table 1. List of 70 proteins included in statistical analysis. Sorted by p-value (lowest to highest). Supplementary Table 2.P-values of age effect from generalized linear models. Sorted by order of Table 2. Supplementary Table 3. Table of unadjusted p-values for group separations in the second cohort. Sorted by order of Table 2. Supplementary Table 4. Antibodies used for development of NF-M sandwich assay.

Additional file 2: Supplementary Figure 1. CSF levels of proteins in Table 2. Statistically significant differences (p-values) are found in Table 2. NC – Non-carriers, PMC – Presymptomatic mutation carriers, PPA – Primary progressive aphasia, bvFTD – Behavioural variant FTD.

Additional file 3: Supplementary Figure 2. Validation of NF-M antibody binding. (A) Detection of NF-M with HPA022845 as capture antibody and 20,664–1-AP as detection antibody. No cross-reactivity with antibodies targeting NF-L or NF-H was observed. (B) Detection of NF-M with HPA022845 as capture antibody and 13–0700 as detection antibody. No cross-reactivity with antibodies targeting NF-L or NF-H was observed. (C) Detection of NF-M with HPA023138 as capture antibody and 34–1000 as detection antibody. No cross-reactivity with antibodies targeting NF-L or NF-H was observed. (D-F) Comparison between sandwich assay data and single-binder data. Rho(D) = 0.85, p(D) = 1E− 7, rho(E) = 0.86, p(E) = 1E− 7, rho(F) = 0.82, p(F) = 1E− 6.

Figure S1. Total protein concentration. Figure S2. Levels on FN1 and CCL2 in BAL. Figure S3. Correlation of paired antibodies. Figure S4. FN1 and CCL2 correlation. Figure S5. Correlations of protein levels in BAL fluid and serum. Figure S6. Correlations of protein levels in unprocessed and concentrated BAL. (DOCX 685 kb)

Narcolepsy is a chronic sleep disorder with a yet unknown cause, but the specific loss of hypocretin-producing neurons together with a strong human leukocyte antigen (HLA) association has led to the hypothesis that autoimmune mechanisms might be involved. Here, we describe an extensive effort to profile autoimmunity repertoires in serum with the aim to find disease-related autoantigens. Initially, 57 serum samples from vaccine-associated and sporadic narcolepsy patients and controls were screened for IgG reactivity towards 10 846 fragments of human proteins using planar microarrays. The discovered differential reactivities were verified on suspension bead arrays in the same sample collection followed by further investigation of 14 antigens in 176 independent samples, including 57 narcolepsy patients. Among these 14 antigens, methyltransferase-like 22 (METTL22) and 5'-nucleotidase cytosolic IA (NT5C1A) were recognized at a higher frequency in narcolepsy patients of both sample sets. Upon sequence analysis of the 14 proteins, polymerase family, member 3 (PARP3), acyl-CoA-binding domain containing 7 (ARID4B), glutaminase 2 (GLS2) and cyclin-dependent kinase-like 1 (CDKL1) were found to contain amino acid sequences with homology to proteins found in the H1N1 vaccine. These findings could become useful elements of further clinical assays that aim towards a better phenotypic understanding of narcolepsy and its triggers.

Narcolepsy is a chronic sleep disorder with a yet unknown cause, but the specific loss of hypocretin-producing neurons together with a strong human leukocyte antigen (HLA) association has led to the hypothesis that autoimmune mechanisms might be involved. Here, we describe an extensive effort to profile autoimmunity repertoires in serum with the aim to find disease-related autoantigens. Initially, 57 serum samples from vaccine-associated and sporadic narcolepsy patients and controls were screened for IgG reactivity towards 10 846 fragments of human proteins using planar microarrays. The discovered differential reactivities were verified on suspension bead arrays in the same sample collection followed by further investigation of 14 antigens in 176 independent samples, including 57 narcolepsy patients. Among these 14 antigens, methyltransferase-like 22 (METTL22) and 5'-nucleotidase cytosolic IA (NT5C1A) were recognized at a higher frequency in narcolepsy patients of both sample sets. Upon sequence analysis of the 14 proteins, polymerase family, member 3 (PARP3), acyl-CoA-binding domain containing 7 (ARID4B), glutaminase 2 (GLS2) and cyclin-dependent kinase-like 1 (CDKL1) were found to contain amino acid sequences with homology to proteins found in the H1N1 vaccine. These findings could become useful elements of further clinical assays that aim towards a better phenotypic understanding of narcolepsy and its triggers.