Fig 2: Loss of RGS14 exacerbates hippocampal neuronal injury after KA-SE.(A-B) Representative FluroJade B (FJB) staining in WT (A) and RGS14 KO (B) dorsal hippocampi 1 day following KA-SE (A). Dashed box represents the injured region of area CA3. Scale bar, 50 µm. (C-D) Representative images of area CA3 illustrates increased number of degenerating pyramidal cells in RGS14 KO mice (D) compared to WT mice (C). Scale bar, 20 µm. (E) Mean number of FJB+ neurons in area CA3 of the dorsal hippocampus after KA-SE (n = 4 per genotype) (WT, 2.00 ± 1.41; RGS14 KO, 20.00 ± 5.73). Statistical Analysis: (E) Unpaired t-test was used to compare mean number of FJB+ neurons (WT vs RGS14 KO, *p < 0.05). Error bars represent the SEM. (1 column)

Fig 3: RGS14 is necessary for SOD2 induction in area CA2 and prevents 3-nitrotyrosine accumulation.(A) Representative IHC images of superoxide dismutase 2 (SOD2) expression in WT and RGS14 KO CA2 one day after saline or KA-SE. Note the induction of SOD2 expression after KA-SE in WT but not RGS14 KO CA2. SP and SR represent regions of analysis for cell body (SP) and dendritic (SR) layers of CA2. Scale bar, 20 µm. (B) Mean SOD2 immunofluorescence in the cell bodies (SP) of CA2 expressed relative to saline-treated WT mean (SAL: WT, n=3, 1.00 ± 0.76; RGS14 KO, n=4, 10.09 ± 4.93; 1d KA: WT, n=4, 71.55 ± 19.06; RGS14 KO, n=4, 9.28 ± 5.76). (C) Mean SOD2 immunofluorescence in the proximal dendrites (SR) of CA2 expressed relative to saline-treated WT mean (SAL: WT, 1.00 ± 0.26; RGS14 KO, 0.70 ± 0.21; 1d KA: WT, 4.65 ± 2.00; RGS14 KO, 0.84 ± 0.29). (D) Representative IHC images of 3-nitrotryosine (3-NT) staining in WT and RGS14 KO CA2 one day after saline or KA-SE. 3-NT is detected in striking abundance in RGS14 KO mice compared to WT mice, an effect that is exacerbated following KA-SE. Scale bar, 20 µm. (E) Mean immunofluorescence of 3-NT staining in area CA2 expressed relative to saline-treated WT mean (SAL: WT, n=4, 1.00 ± 0.32; RGS14 KO, n=3, 532.56 ± 83.88; 1d KA: WT, n=4, 7.33 ± 3.86; RGS14 KO, n=3, 5213.93 ± 1908.17). Statistical analysis: (B, C, E) Two-way ANOVA with Tukey post-hoc comparisons used to compare group means (B, two-way ANOVA, F = 5.68 for genotype, *p < 0.05; F = 9.77 for treatment, *p < 0.05; F = 10.23 for interaction, **p < 0.01; Tukey’s, WT SAL vs WT KA, **p < 0.01; WT KA vs KO KA, **p < 0.01) (E, two-way ANOVA, F = 12.89 for genotype, **p < 0.01; F = 8.61 for treatment, *p < 0.05; F = 8.56 for interaction, *p < 0.05; Tukey’s, KO SAL vs WT KA, *p < 0.05; WT KA vs KO KA, **p < 0.01). Error bars represent the SEM. SP, stratum pyramidale; SR, stratum radiatum. 3-NT, 3-nitrotyrosine. (1.5 column)

Fig 4: Proposed working model for RGS14 regulation of CA2 pyramidal neurons following seizure.Diagram of a CA2 dendritic spine illustrating known and speculative roles for RGS14 in intracellular regulation. RGS14 at the plasma membrane and in the cytosol can negatively regulate both GPCR- and MAPK-dependent signaling, while buffering calcium transients from NMDA receptors and voltage-gated calcium channels (VGCC) through an unknown mechanism. In addition to its localization at the membrane and in cytosolic spaces, RGS14 localizes to mitochondria in CA2 pyramidal cells. By its effect on signaling and calcium buffering at the membrane/cytosol, its localization to the mitochondria, or through an unknown mechanism, RGS14 is capable of influencing metabolic/mitochondrial protein expression, including SOD2. Although speculative, RGS14 may buffer mitochondrial calcium in a similar fashion to buffering membrane calcium transients of dendritic spines. Regulatory capacity by RGS14 culminates to provide balance of reactive metabolites (O2−) and mitochondrial function. Additionally, KA seizure activity upregulates RGS14, which may be necessary for promoting microglial activation possibly by the synthesis and release of activating factors. When RGS14 is absent, this leads to overwhelming abundance of 3-nitrotyrosine (3-NT), an indicator of oxidative stress, which may be enhanced after seizure due to the loss of SOD2 upregulation and 3-NT inactivation of SOD2. Exacerbated oxidative stress in RGS14 KO likely causes cellular dysfunction and alters the excitatory properties of CA2 pyramidal cells, leading to the enhanced hippocampal excitability and accelerated onset of SE and mortality. Abbreviations: GPCR, G-protein coupled receptor; RTK, receptor tyrosine kinase; MAPK, mitogen-activated protein kinase; NMDA, N-methyl-d-aspartate; KA, kainic acid; Glu, glutamate; VGCC, voltage-gated calcium channel; Ca2+, calcium; SOD2, superoxide dismutase 2; O2−, superoxide. Created with BioRender.com. (1.5 column).

Fig 5: Differential expression analysis of the hippocampal proteome reveals altered mitochondrial and metabolic protein expression in RGS14 KO versus WT mice.(A) Volcano plot showing differentially expressed proteins (DEPs) one day after SAL (yellow) or KA-SE (red) treatment in RGS14 KO hippocampus relative to WT plotted by their −log10(p-value) and log2 fold change (KO/WT). Proteins were considered significantly downregulated if the adjusted p-value <0.05, log2 fold change (KO/WT) < 0 and significantly upregulated if the adjusted p-value <0.05, log2 fold change (KO/WT) > 0 (n = 5 per genotype/condition). Labeled dots represent DEPs involved with cellular metabolism and localize to the mitochondria. (B) Diagram illustrating between-genotype, within-treatment comparisons used to determine DEPs. (C) Venn diagram showing the number of upregulated or downregulated proteins in RGS14 KO relative to WT after SAL or KA-SE and DEPs common to both treatment groups. (D-E) Gene ontology analysis demonstrates similar biological function and processes of DEPs that are either downregulated (DOWN) or upregulated (UP) in RGS14 KO hippocampi one day after SAL (D) or KA-SE (E). Red arrows indicate ontologies of interest that may influence seizure susceptibility (D) or mitochondrial metabolism and oxidative stress (E). (2 column). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)