Multi-proteomic Analysis Revealed Distinct Protein Profiles in Cerebrospinal Fluid of Patients Between Anti-NMDAR Encephalitis NORSE and Cryptogenic NORSE

Abstract

Background: New-onset refractory status epilepticus (NORSE) caused by anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis and cryptogenic etiologies are various in clinical features. The underlying mechanisms of the diseases have still remained elusive.Methods: 6 patients with anti-NMDAR encephalitis NORSE, 5 with cryptogenic NORSE (C-NORSE), and 5 controls were enrolled. Clinical data, including clinical features, cerebrospinal uid (CSF) samples, and cranial images were collected. CSF samples were tested for FAIMS based quantitative proteomic analysis, immunome protein microarray, and in ammatory cytokine array. Bioinformatics and immunostaining were used for interpretation and veri cation.Results: The clinical features of anti-NMDAR encephalitis NORSE and C-NORSE patients were similar to those reported previously. Proteomic analysis revealed that 101 proteins (63 up-regulated and 38 downregulated) and 56 proteins (42 up-regulated and 14 down-regulated) changed in CSF samples of anti-NMDAR encephalitis NORSE and C-NORSE patients, respectively. The mean fold-change of the upregulated proteins, namely up-proteomic score in this study, was positively associated with the severity of disease, including ICU stay, mRS score at discharge, and time taken for patients awaking from a coma. The distribution of changed proteins in CSF between the two diseases were quite different. Pathways of humoral immune response, wound healing, and epigenetic regulation of transcription were up-regulated in anti-NMDAR encephalitis NORSE. Pathways of innate and lymphocyte mediated immune response, synaptic functions, ubiquitination, and cell apoptosis were up-regulated in C-NORSE group, suggesting that C-NORSE could undergo neurodegeneration. This was consistent with a mouse model of SE, which showed high ubiquitination and cell apoptosis in CA1 to CA3 regions of hippocampus. Immunome microarray analysis demonstrated high autoantibody targeting 48 proteins in CSF samples of anti-NMDAR encephalitis NORSE. However, in C-NORSE, the reaction was kept at a very low level.In ammatory cytokine array, unexpectedly, showed no remarkable changes except for decreased level of interleukin-6 (IL-6) in both anti-NMDAR encephalitis NORSE and C-NORSE.Conclusions: The present study suggests that the up-proteomic score of CSF could be a promising indicator for assessment of the severity of anti-NMDAR encephalitis NORSE and C-NORSE. The distinct CSF proteomes imply different pathogenic mechanisms of the two diseases, and immunotherapy strategies as well.

Cytokines inNew‐OnsetRefractory Status Epilepticus Predict Outcomes

“…That might also explain the correlation previously reported between the CSF CXCL8 levels and the up-proteomic score that has been presented as a promising indicator for the assessment of the severity of NORSE. 15 Those data and our results suggest the possible benefit of anti-CXCL8 therapy to reduce seizure severity and prevent the development of post-NORSE epilepsy and worse outcomes. Although anti-CXCL8 therapy has not been reported for the treatment of patients with SE, in a mouse model of SE refractory to benzodiazepines, the administration of reparixin, a molecule able to specifically block the activation of CXCR1/CXCR2 by CXCL8, showed a faster SE decay, a reduced incidence of acute symptomatic seizures after SE, and a delayed time to spontaneous seizure onset compared with control animals.…”

“…17,41,42 The increase of CXCL8 levels in the serum of patients with cNORSE found in our study mirrors the recent findings of a multi-protein analysis study that suggested the involvement of the innate and lymphocyte-mediated immune response in cNORSE pathogenesis. 15 Studies have shown that CXCL8 receptors are expressed by neurons and the acute application of CXCL8 to neurons enhanced neuronal excitability by inducing an intracellular calcium elevation and increasing sodium currents. 43,44 Chronic treatment with MIP-1α, a chemokine involved in macrophage and neutrophil recruitment, was also found able to modulate intracellular calcium dynamics and increase hippocampus NMDA receptor levels.…”

“…These cases were used to compare CSF and serum samples within the same patient. All patients were adults except four children or teenagers (9,12,15, and 17 years old) in the NORSE group. 39% (24/61) of patients with NORSE presented with a preceding febrile illness before SE onset and therefore qualified as FIRES.…”

“…8,[10][11][12][13][14] A recent quantitative proteomic analysis of CSF samples suggested the involvement of the innate and lymphocyte-mediated immune response in patients with NORSE for whom no etiology was identified despite an extensive workup. 15 There is less evidence for disturbance in serum cytokines in patients with NORSE. 8,11 No study has evaluated the direct association between cytokine/chemokine levels and the outcome of patients with NORSE.…”

“…10,16 The CSF IL-6 and CXCL8 levels were found positively associated with an up-proteomic score that has been suggested as a promising indicator for assessment of the severity of NORSE in a cohort of 11 patients. 15 Pro-inflammatory cytokines may enhance seizure recurrence and subsequent sequelae. 8,[17][18][19] Elevated pro-inflammatory cytokines in young children with febrile SE were correlated with T2 magnetic resonance imaging (MRI) hippocampal hyperintensity and with the development of hippocampal sclerosis.…”