Proteomic differences in hippocampus and cortex of sudden unexplained death in childhood

Abstract

Sudden unexplained death in childhood (SUDC) is death of a child over 1 year of age that is unexplained after review of clinical history, circumstances of death, and complete autopsy with ancillary testing. Multiple etiologies may cause SUDC. SUDC and sudden unexpected death in epilepsy (SUDEP) share clinical and pathological features, suggesting some similarities in mechanism of death and possible abnormalities in hippocampus and cortex. To identify molecular signaling pathways, we performed label-free quantitative mass spectrometry on microdissected frontal cortex, hippocampal dentate gyrus (DG), and cornu ammonis (CA1-3) in SUDC (n = 19) and pediatric control cases (n = 19) with an explained cause of death. At a 5% false discovery rate (FDR), we found differential expression of 660 proteins in frontal cortex, 170 in DG, and 57 in CA1-3. Pathway analysis of altered proteins identified top signaling pathways associated with activated oxidative phosphorylation (p = 6.3 × 10^-15, z = 4.08) and inhibited EIF2 signaling (p = 2.0 × 10^-21, z = - 2.56) in frontal cortex, and activated acute phase response in DG (p = 8.5 × 10^-6, z = 2.65) and CA1-3 (p = 4.7 × 10^-6, z = 2.00). Weighted gene correlation network analysis (WGCNA) of clinical history indicated that SUDC-positive post-mortem virology (n = 4/17) had the most significant module in each brain region, with the top most significant associated with decreased mRNA metabolic processes (p = 2.8 × 10^-5) in frontal cortex. Additional modules were associated with clinical history, including fever within 24 h of death (top: increased mitochondrial fission in DG, p = 1.8 × 10^-3) and febrile seizure history (top: decreased small molecule metabolic processes in frontal cortex, p = 8.8 × 10^-5) in all brain regions, neuropathological hippocampal findings in the DG (top: decreased focal adhesion, p = 1.9 × 10^-3). Overall, cortical and hippocampal protein changes were present in SUDC cases and some correlated with clinical features. Our studies support that proteomic studies of SUDC cohorts can advance our understanding of the pathogenesis of these tragedies and may inform the development of preventive strategies.

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

Fig. 1

Detected proteins and PCA from proteomics analyses in frontal cortex, dentate gyrus, and hippocampal CA1-3. a Number of proteins detected in each brain region by label-free quantitative MS is indicated, with an overlap of 2498 proteins detected in all brain regions. b The PCA in the frontal cortex shows separation of control (n = 15) and SUDC (n = 19) cases. In the hippocampus, the variation is indicated for c the dentate gyrus (control n = 16, SUDC n = 18) and d the hippocampal CA1-3 (control n = 17, SUDC n = 18). An analysis of group separation in PCA1 for each brain region is depicted by a box plot with bars indicating minimum and maximum values, evaluated by an unpaired two-tailed t test. PCA1 principal component analysis 1, PCA2 principal component analysis 2

Fig. 2

Differential proteomic expression analyses in frontal cortex, dentate gyrus, and hippocampal CA1-3. a Volcano plots indicate significantly different proteins in SUDC when compared to control cases after a student’s two-tailed t test with permutation correction at a 5% FDR in a frontal cortex, b dentate gyrus, and c hippocampal CA1-3. Cell type-specific protein annotation is indicated, with the “General—Neuron” annotation including both excitatory and inhibitory neuron annotations. IPA pathway enrichment analysis of the significantly altered proteins identified the indicated pathways that are activated (red) or inhibited (blue) with a significant z score ( ≥|2|) and p value of overlap (p < -log₁₀(0.05)) for d frontal cortex, e dentate gyrus, and f hippocampal CA1-3

Fig. 3

Inter-regional correlation analyses. a Among significant proteins in each brain region analyzed, there were 6 proteins significantly altered in all 3 brain regions. There were more similarities when comparing 2 brain regions, with the most similar proteins between the frontal cortex and dentate gyrus (39 proteins) and highest percentage of shared proteins between the frontal cortex and hippocampal CA1-3 (46% hippocampal CA1-3 proteins). b Of the significant proteins detected in all regions (n = 683 significant and detected/811 total significant), a correlation analysis of frontal cortex and dentate gyrus indicated a significant positive correlation (p < 0.0001, R² = 0.31) with 495 proteins (72.5% proteins) changing in the same direction (up/down, purple) and 188 proteins changing in the opposite direction (i.e. up in frontal cortex and down in dentate gyrus; yellow). c Of the significant proteins detected in all regions (n = 683), a correlation analysis of frontal cortex and hippocampal CA1-3 indicated a significant positive correlation (p < 0.0001, R² = 0.63) with 606 proteins (88.7%) changing in the same direction (up/down, purple) and 77 proteins changing in the opposite direction (yellow). d Of the significant proteins detected in all regions (n = 683), a correlation analysis of dentate gyrus and hippocampal CA1-3 indicated a significant positive correlation (p < 0.0001, R² = 0.34) with 474 proteins (69.4%) changing in the same direction (up/down, purple) and 209 proteins changing in the opposite direction (yellow)

Fig. 4

WGCNA of clinical variables in frontal cortex, dentate gyrus, and hippocampal CA1-3. A correlation analysis of clinical variables to proteomics indicated significant modules and associated GO annotations in the a frontal cortex b dentate gyrus, and c hippocampal CA1-3. 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 annotations are noted on the right (FDR < 5% with at least 5 proteins) and detailed in Supplementary Table 13–15, online resource. Several modules did not have a significant GO annotation and are noted as “n.s.” FS febrile seizure, NP HP findings neuropathology hippocampal findings