Raphe and ventrolateral medulla proteomics in sudden unexplained death in childhood with febrile seizure history

Abstract

Sudden unexplained death in childhood (SUDC) is death of a child ≥ 12 months old that is unexplained after autopsy and detailed analyses. Among SUDC cases, ~ 30% have febrile seizure (FS) history, versus 2-5% in the general population. SUDC cases share features with sudden unexpected death in epilepsy (SUDEP) and sudden infant death syndrome (SIDS), in which brainstem autonomic dysfunction is implicated. To understand whether brainstem protein changes are associated with FS history in SUDC, we performed label-free quantitative mass spectrometry on microdissected midbrain dorsal raphe, medullary raphe, and the ventrolateral medulla (n = 8 SUDC-noFS, n = 11 SUDC-FS). Differential expression analysis between SUDC-FS and SUDC-noFS at p < 0.05 identified 178 altered proteins in dorsal raphe, 344 in medullary raphe, and 100 in the ventrolateral medulla. These proteins were most significantly associated with increased eukaryotic translation initiation (p = 3.09 × 10^-7, z = 1.00), eukaryotic translation elongation (p = 6.31 × 10^-49, z = 6.01), and coagulation system (p = 1.32 × 10^-5, z = 1.00). The medullary raphe had the strongest enrichment for altered signaling pathways, including with comparisons to three other brain regions previously analyzed (frontal cortex, hippocampal dentate gyrus, cornu ammonus). Immunofluorescent tissue analysis of serotonin receptors identified 2.1-fold increased 5HT2A in the medullary raphe of SUDC-FS (p = 0.025). Weighted gene correlation network analysis (WGCNA) of case history indicated that longer FS history duration significantly correlated with protein levels in the medullary raphe and ventrolateral medulla; the most significant gene ontology biological processes were decreased cellular respiration (p = 9.8 × 10^-5, corr = - 0.80) in medullary raphe and decreased synaptic vesicle cycle (p = 1.60 × 10^-7, corr = - 0.90) in the ventrolateral medulla. Overall, FS in SUDC was associated with more protein differences in the medullary raphe and was related with increased translation-related signaling pathways. Future studies should assess whether these changes result from FS or may in some way predispose to FS or SUDC.

Keywords: Febrile seizures; Laser capture microdissection; Proteomics; SUDC.

Fig. 1

Dissected brainstem regions and differential expression analyses. a–c) overview schematic of regions microdissected in the midbrain dorsal raphe, medullary raphe, and ventrolateral medulla. After proteomic analysis, principal component analysis (PCA) shows distribution of SUDC cases with febrile seizure history (SUDC-FS; orange) and SUDC cases without febrile seizure history (SUDC-noFS; yellow) in the midbrain dorsal raphe d), medullary raphe e), and ventrolateral medulla f). There was no segregation of cases by FS history in any of the brain regions analyzed, in PCA1: midbrain dorsal raphe (p = 0.93, unpaired t-test), medullary raphe (p = 0.12), ventrolateral medulla (p= 0.24) or PCA2: midbrain dorsal raphe (p = 0.055), medullary raphe (p = 0.33), ventrolateral medulla (p = 0.72). g) Differential expression analysis identified 178 altered proteins (68 increased, 110 decreased) in the midbrain dorsal raphe at p < 0.05. h In the medullary raphe, 344 proteins were altered (217 increased, 127 decreased). i In the ventrolateral medulla, 100 proteins were altered (53 increased, 47 decreased). The top 5 significantly increased and decreased proteins are annotated by gene name, as well as protein of interest SLC2A13. Dotted lines correspond to p < 0.05 and fold change at 1.5. Cell type annotation for each protein is indicated by color. Additional protein information is available in Supplementary Tables 2, 3, 4

Fig. 2

Signaling pathways associated with differentially expressed proteins in the brainstem. a in the midbrain dorsal raphe, 90 signaling pathways (p value of overlap < 0.05) were associated with the altered proteins, b in the medullary raphe, 201 signaling pathways were associated with the altered proteins, c in the ventrolateral medulla, 82 signaling pathways were associated with altered proteins. The top 20 significant signaling pathways are depicted. Color indicates z-score, with red increased and blue decreased. The ratio of proteins altered in a pathway are indicated by circle size

Fig. 3

Signaling pathways altered in multiple brain regions. Among the six brain regions (DR = dorsal raphe, MR = medullary raphe, VLM = ventrolateral medulla, FC = frontal cortex, DG = dentate gyrus, HP = hippocampal CA1-3) evaluated in SUDC, there were 62 signaling pathways that were significantly altered in at least 2 brain regions (p value) and impacted by fold-change in at least one brain region (z-score). Pathways are sorted by most significant in the medullary raphe. Z-score is indicated by color, increased (red), decreased (blue). P value of overlap is indicated for the signaling pathways at p < 0.05

Fig. 4

Histological localization and quantification of top protein candidates SLC2A13 and SEZ6L2 in the brainstem. a By LC–MS/MS in the midbrain dorsal raphe, SLC2A13 was among the top significant proteins and had the largest fold change with a 24.9-fold decrease in SUDC-FS when compared to SUDC-noFS (p = 3.97 × 10^–3), b IF for SLC2A13 in the midbrain dorsal raphe showed a similar trend with a 1.5-fold decrease in SUDC-FS (p = 0.23) from semiquantitative analysis, c IF for TPH2 on the same slides for SLC2A13 in the midbrain dorsal raphe showed no difference between SUDC-FS and SUDC-noFS, similar to proteomics, d colocalization analysis of SLC2A13 and TPH2 in the midbrain dorsal raphe showed a moderate correlation by Mander coefficient of the green channel (TPH2), which was different between SUDC-FS and SUDC-noFS (p = 0.026). There was a higher correlation in the red channel (SLC2A13) for both SUDC-FS and SUDC-noFS. e By LC–MS/MS in the medullary raphe, SEZ6L2 was among the top significant proteins and had the largest fold change with a 3.9-fold increase in SUDC-FS (p = 4.75 × 10^–5), f IF for SEZ6L2 in the medullary raphe showed a similar trend with a 1.3-fold increase (p = 0.49) from semiquantitative analysis, g IF for TPH2 on the same slides for SEZ6L2 in the medullary raphe showed no difference between SUDC-FS and SUDC-noFS, similar to proteomics, h colocalization analysis of SEZ6L2 and TPH2 in the medullary raphe showed a low correlation in the red channel and moderate correlation in the green channel, which was not different between SUDC-FS and SUDC-noFS, i representative images from IF in the midbrain dorsal raphe are shown for SLC2A13 (red) and TPH2 (green) in SUDC-FS and SUDC-noFS. TPH2 indicates the region with serotonergic neurons that was microdissected for proteomic analysis. SLC2A13 was present in TPH2( +) cells, as well as in other neighboring TPH2(−) cells, j representative images from IF in the medullary raphe are shown for SEZ6L2 (red) and TPH2 (green) in SUDC-FS and SUDC-noFS. TPH2 indicates the region with serotonergic neurons that was microdissected for proteomic analysis. SEZ6L2 was present in TPH2( +) cells, as well as in other neighboring TPH2(−) cells. Scale bar 100 um. Error bars indicate SEM

Fig. 5

Serotonin 5HT1A and 5HT2A receptor histological localization and quantification in the midbrain dorsal raphe. From IHC semiquantitative analysis, there was no difference in a serotonin receptor 5HT1A (p = 0.77) when comparing SUDC-FS and SUDC-noFS, b TPH2 from IF on the same tissue sections with 5HT1A was evaluated in colocalization analyses, c colocalization analysis of 5HT1A and TPH2 showed a moderate correlation by Mander coefficient of the red and green channels, with no difference between SUDC-FS and SUDC-noFS, d there was no difference in serotonin receptor 5HT2A (p = 0.29) when comparing SUDC-FS and SUDC-noFS, e TPH2 from IF on the same tissue sections with 5HT2A was evaluated in colocalization analyses, f colocalization analysis of 5HT2A and TPH2 showed lower correlations in the red and green channels, with no difference between SUDC-FS and SUDC-noFS. Representative images from IF are shown for 5HT1A (red) g, 5HT2A (red) h, and TPH2 (green) in SUDC-FS and SUDC-noFS. TPH2 indicates the region with serotonergic neurons that was microdissected for proteomic analysis. Scale bar 100 um. Error bars indicate SEM

Fig. 6

Serotonin 5HT1A and 5HT2A receptor histological localization and quantification in the medullary raphe. From IF semiquantitative analysis, there was no difference in a serotonin receptor 5HT1A (p = 0.83) when comparing SUDC-FS and SUDC-noFS, b TPH2 from IF on the same tissue sections with 5HT1A was evaluated in colocalization analyses, c colocalization analysis of 5HT1A and TPH2 showed a low correlation by Mander coefficient in the red channel and a moderate correlation in the green channel, with no difference between SUDC-FS and SUDC-noFS d serotonin receptor 5HT2A was 2.1-fold increased (p = 0.025) when comparing SUDC-FS and SUDC-noFS, e TPH2 from IF on the same tissue sections with 5HT2A was evaluated in colocalization analyses, f colocalization analysis of 5HT2A and TPH2 showed no correlation of the red and green channels. Representative images from IF are shown for 5HT1A (red) g, 5HT2A (red) h, and TPH2 (green) in SUDC-FS and SUDC-noFS. TPH2 indicates the region with serotonergic neurons that was microdissected for proteomic analysis. Scale bar 100 um. Error bars indicate SEM

Fig. 7

Serotonin 5HT1A and 5HT2A receptor histological localization and quantification in the ventrolateral medulla. From IF semiquantitative analysis, there was no difference in a serotonin receptor 5HT1A (p = 0.53) when comparing SUDC-FS and SUDC-noFS, b TPH2 from IF on the same tissue sections with 5HT1A was evaluated in colocalization analyses, c colocalization analysis of 5HT1A and TPH2 showed a low correlation by Mander coefficient in the red channel and a moderate correlation in the green channel, with no difference between SUDC-FS and SUDC-noFS, d There was no difference in serotonin receptor 5HT2A (p = 0.29) when comparing SUDC-FS and SUDC-noFS, e TPH2 from IF on the same tissue sections with 5HT2A was evaluated in colocalization analyses, f colocalization analysis of 5HT2A and TPH2 showed no correlation of the red and green channels. Representative images from IHC are shown for 5HT1A (red) g, 5HT2A (red) h, and TPH2 (green) in SUDC-FS and SUDC-noFS. Scale bar 100 um. Error bars indicate SEM

Fig. 8

WGCNA of case history in midbrain dorsal raphe, medullary raphe, and the ventrolateral medulla. A correlation analysis of case history variables to proteomics indicated significant modules and associated GO BP annotations in the a midbrain dorsal raphe, b medullary raphe, and c ventrolateral medulla. Modules are clustered by eigenprotein adjacency (relatedness to other modules) on the left. Name of module is indicated by “M-color” and corresponding color block. P values are indicated for those modules with p < 0.05 correlation. Positive correlation is indicated in red and negative correlation in blue. Top module GO BP annotations are noted on the right (FDR < 5% with at least 5 proteins) and detailed in Supplementary Tables 14, 15, 16. Several modules did not have a significant GO BP annotation and are noted as “n.s.” = not significant. FS = febrile seizure. FS duration = FS history duration