In the current research, 6 patients with anti-NMDAR encephalitis NORSE (R1-R6), 5 with C-NORSE (E1-E5), and 5 controls (C1-C5) were enrolled. The grouping and clinical features of the patients are presented in Table 1.
The severity, outcomes and relapses of patients with anti-NMDAR encephalitis are correlated with the anti-NDMAR antibody level of CSF [16, 17]. To elucidate different stages of anti-NMDAR encephalitis NORSE, patients without history of immunotherapy (R4, R5) and refractory to IVIG (R6, without clinical improvement or antibody reduction), and those with history of immunotherapy and antibody reduction (R2, R3) or negative conversion (R1) were included (Fig. S1). All the anti-NMDAR encephalitis NORSE patients received AEDs. Among all the C-NORSE patients, no definite pathogenic factor was confirmed. Patients with definite vascular diseases (e.g., cerebral infarction, hemorrhage, cerebral venous sinus thrombosis, cerebral arteriovenous fistula, and vascular malformation) were selected as controls.
The cranial images and electroencephalogram (EEG) findings of patients are illustrated in Figs. S2-S3. For patients with anti-NMDAR encephalitis NORSE, EEG revealed diffused delta waves with increased amplitude. CT/MRI of brain was unremarkable or with leukoaraiosis. The MRI findings in the C-NROSE were not specific with scattered abnormal signals in the cortex or subcortex. Besides, a number of patients were found with complete normal MRI findings.
Proteomic up-regulation index in CSF was correlated with the severity of patients with anti-NMDAR encephalitis NORSE and C-NORSE
To determine the underlying mechanisms of anti-NMDAR encephalitis NORSE and C-NORSE and whether C-NORSE is also an immune-mediated inflammatory disease, FAIMS-based quantitative proteomic analysis of CSF samples was conducted (Fig. 1A). 12239 unique peptides, 1797 proteins, and 1675 quantifiable proteins were identified (Fig. 1B, Additional file 1). The peptides were distributed between 7-20 amino acids, which were coincident with trypsin digestion. In nearly 30% of proteins, sequence coverage was above 20%. Proteins with fold-change over 1.5 and P-value less than 0.05 were considered as significantly changed. 63 and 38 proteins were up- and down-regulated in CSF samples in patients with anti-NMDAR encephalitis NORSE, respectively (Fig.1B, Additional file 2); 42 and 14 proteins were up- and down-regulated in CSF samples in patients with C-NORSE, respectively (Fig. 1B, Additional file 3).
Putting proteomes and clinical characteristics together, we found that up-regulated proteins were positively associated with the length of stay in ICU and mRS score at discharge. These two factors reflected severity of disease and neurological function of the patients. Compared to patients of R1, R2, and R3, proteins were more significantly up-regulated in R4, R5, and R6, accompanying with longer duration in ICU and higher mRS score at discharge. The same pattern was also observed between E1 and E2 versus E3, E4 and E5 (Fig. 2A). In addition, the mean fold-change of all up- or down-regulated proteins was calculated as the proteomic score. Since some patients’ ICU stay period were not quite accurate because of pass away or discharge ahead, time needed to awake for patients in a coma was used as another indicator of disease severity. Interestingly, the proteomic score of up-regulated proteins showed approximately the same trend with the time from sample collection to awoke (Fig. 2B-C). To narrow-down the highly correlated proteins, Spearman’s or Pearson’s correlation analysis was undertaken for each changed proteins. 7 proteins in anti-NMDAR encephalitis NORSE group, IGLV3-27, IGKV1-39, LI6R, SECTM1, ICAM1, GRN, and CD82, were found. These proteins’ mean fold change or named as refined proteomic score was highly logarithmic correlated with time needed to awake (R2=0.9739, Fig. S4). In C-NORSE group, 9 proteins were found, including HIST2H2AC, PPP3CC, PDXP, SLC9A6, HDHD1, CPLX2, SH3BGRL, PSMA3, and CAPZB (Fig. S5). There were no significant differences in the profile of down-regulated proteins and the down-proteomic scores among the patients within anti-NMDAR encephalitis NORSE group, and within C-NORSE group as well (Fig. 2A, 2B).
The distribution of changed proteins in CSF samples was quite different between two groups. Venny analysis showed barely overlap of up- or down-regulated proteins (Fig. 2D). Subcellular localization of the changed proteins revealed that in anti-NMDAR encephalitis NORSE group, about 57% were extracellular proteins, while in C-NORSE group, 29%, 25%, and 18% were cytoplasmic, extracellular, and nuclear proteins, respectively (Fig. S6). Principal component analysis confirmed this discrepancy. There was no obvious clustering among all the samples (Fig. S7), while samples with high up-proteomic score were clustered well into different groups according to the type of disease (Fig. S8). These data imply that anti-NMDAR encephalitis NORSE and C-NORSE could underlie different mechanisms, leading to SE.
Proteins involved in humoral immune response, epigenetic regulation, and wound healing changed in CSF samples of anti-NMDAR encephalitis NORSE
GO enrichment analysis of changed proteins in anti-NMDAR encephalitis group showed that up-regulated proteins were highly enriched in humoral immune response and epigenetic regulation of gene expression, including immunoglobulins, HLA-A, HLA-B, F5, APOE, PCSK9, CD74, GRN, HIST1H4A, HIST2H2AC, H3F3A, ENPP2, GANAB, GALNT1, PLD4, PLAUR, ERAP2, PRDX4, PLOD1, etc. (Fig. 3A). Down-regulated proteins were involved in negative regulation of coagulation and wound healing, such as CFH, MGP, FGA, S100A8, FGB, MAPK1, AHSG, EPB42, SERPINE1, CTSG, HRG, SPTAN1, FGG, KNG1, LTF, CKB, APOA2, ZC4H2,GP1BA, PLCB3, FBN1, CTGF, DDAH1, B3GAT1, DNER, PLS3, GLUL, etc. (Fig. 3A). The majority of these proteins were estimated in a network (Fig. 3B). We further analyzed top three samples with high up-proteomic score, R4, R5 and R6, and more changed proteins were observed (Fig. S9, S10). However, the pathways mentioned above were highly enriched as well (Fig. S11).
As expected, high level of immunoglobulin appeared in CSF samples of patients with anti-NMDAR encephalitis NORSE (Fig. 1B, 2A, S1). To determine the immuno-reactivity and antibody profile of CSF, immunome protein microarray assay, which consists of more than 1600 proteins, was performed. R3, R4, R5, and R6 showed a high reaction, and 48 targeted biomarkers were identified (Fig. 3C, Additional file 4-6), demonstrating a comprehensive immune response in anti-NMDAR encephalitis NORSE patients. To indicate the inflammatory response level, cytokine protein microarray was also carried out. It was noted that 10 cytokines, including interferon-γ (IFN-γ), interleukin (IL)-1α, IL-1β, IL-10, IL-13, IL-4, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) were analyzed. Unexpectedly, none of them were significantly up-regulated either in anti-NMDAR encephalitis NORSE group or C-NORSE group (Fig. 3D, Additional file 7). Besides, IL-6, an important proinflammatory cytokine, was down-regulated, though its receptor IL-6R was increased in R3, R4, R5, E4, and E6.
CSF proteomes of C-NORSE indicated a neurodegeneration process
GO enrichment analysis of up-regulated proteins of C-NORSE group showed that pathways of regulation of innate immune response and lymphocyte mediated immunity were enriched, such as SCL9A6, HLA-B, UBQLN1, SYN1, PAK1, FCER2, LEP, PLAUR, STAT1, PSMA3, and complements C4A and C4B (Fig. 4A, upper panel). The reduced proteins were enriched in Fc receptor signaling pathway and immune response mediated by immunoglobulin (Fig. 4A, lower panel). This was consistent with the low reactivity in immunome protein microarray analysis (Fig. 3C, Fig. S12).
In addition to immune response, axon extension, dendrite, and synaptic-involved biological processes were highly enriched (Fig. 4A). 41 of 42 up-regulated proteins were related to synapse, especially presynapse (Fig. 4B). Moreover, 36 of these proteins were related to ubiquitination (Fig. 4C); 29 formed several networks or interactions (Fig. 4D). Furthermore, cell apoptosis, phagocytosis, peptidase, and proteasome pathways were highly enriched (Fig. 4A). STAT cascade, which is important for regulation of cell apoptosis, appeared. In samples with top three up-proteomic scores, E3 , E4, and E5 (Fig. S13), cell apoptosis-associated biological processes could be observed (Fig. S14). The above-mentioned findings imply that C-NORSE might undergo a neurodegeneration process. To verify this hypothesis, a mouse model of SE was established. Surprisingly, two synapse proteins detected, PAK1 and SYN1, were increased consistently with FAIMS (Fig. S15) and an obvious ubiquitination and cell apoptosis could be observed in the stratum pyramidale of CA1 to CA3 regions of hippocampus (Fig. 5).