RGS14 protects against seizure-induced mitochondrial oxidative stress and pathology in the hippocampus Open Access
Harbin, Nicholas (Summer 2023)
Published
Abstract
RGS14 is a multifunctional scaffolding protein that is highly enriched within pyramidal cells (PCs) of hippocampal area CA2. In these neurons, RGS14 suppresses glutamate-induced calcium influx and related G protein and ERK signaling in dendritic spines to restrain postsynaptic signaling and plasticity. Previous findings show that, CA2 PCs are resistant to degeneration (unlike CA1 and CA3 PCs), modulate hippocampal excitability, generate epileptiform activity, and promote hippocampal pathology in animal models and patients with temporal lobe epilepsy (TLE). While RGS14 is protective against peripheral injury, similar roles for RGS14 during pathological injury in hippocampus remain unexplored. Because RGS14 suppresses CA2 excitability and signaling, we hypothesized that RGS14 would moderate seizure behavior and hippocampal pathology following seizure activity, possibly affording protection to CA2 PCs. Using kainic acid (KA) to induce status epilepticus (KA-SE) in mice, we show that the loss of RGS14 (RGS14 KO) accelerated onset of limbic motor seizures and mortality compared to wild type (WT) mice, and that KA-SE upregulated RGS14 protein expression in CA2 and CA1 PCs of WT. Our proteomics data show that the loss of RGS14 impacted the expression of a number of proteins at baseline and after KA-SE, many of which associated unexpectedly with mitochondrial function and oxidative stress. RGS14 was shown to localize to the mitochondria in CA2 PCs of mice and reduce mitochondrial respiration in vitro. As a readout of oxidative stress, we found that RGS14 KO dramatically increased 3-nitrotyrosine levels in CA2 PCs, which was greatly exacerbated following KA-SE and correlated with a lack of superoxide dismutase 2 (SOD2) induction. Assessing for hallmarks of seizure pathology in RGS14 KO, we unexpectedly found no differences in neuronal injury in CA2 PCs. However, we observed a striking and unexpected lack of gliosis in CA1 and CA2 compared to WT. Together, our data demonstrate a newly appreciated role for RGS14 in protecting against intense seizure activity and pathology in hippocampus. Our findings are consistent with a model where RGS14 limits seizure onset and mortality and, after seizure, is upregulated to support mitochondrial function, prevent oxidative stress in CA2 PCs, and promote glial activation in hippocampus.
Table of Contents
Chapter 1: Introduction
1.1. Overview of dissertation 1
1.2. Synaptic transmission and neuronal signaling 2
1.3. G-protein signaling in neurons 7
1.4. Regulation of G-protein signaling by RGS 14
1.5. RGS14 expression and regulation of cell signaling 18
1.6. Roles of RGS14 in neuronal function and behavior 23
1.7. Excitability, epilepsy, and temporal lobe epilepsy (TLE) 29
1.8. Animal models of TLE and hippocampal pathology 32
1.9. Area CA2 function, emerging role in TLE, and implications for RGS14 38
1.10. Research focus of the dissertation project 43
Chapter 2: Materials and Methods 46
Chapter 3: Results and Findings of Dissertation
3.1. Loss of RGS14 increases susceptibility to KA-induced seizures by expediting entry into SE and mortality 59
3.2. RGS14 protein expression is upregulated following status epilepticus 62
3.3. Metabolic and mitochondrial proteins are differentially expressed between WT and RGS14 KO mice 1 day after KA-SE 67
3.4. WGCNA Analysis reveals significant alterations of protein modules related to cellular/lipid metabolism and astrocytes in RGS14 KO hippocampi 76
3.5. RGS14 localizes to mitochondria in CA2 PCs and reduces mitochondrial respiration in vitro 82
3.6. RGS14 is necessary for SOD2 induction in area CA2 and prevents 3-nitrotyrosine accumulation 85
3.7. Loss of RGS14 does not alter early hippocampal neuronal injury in areas CA2 or CA1 after KA-SE 90
3.8. RGS14 is required for glial response to KA-SE 93
Chapter 4: Discussion of Dissertation Findings and Future Directions
4.1. Summary of findings 103
4.2. Neuronal mechanisms by which loss of RGS14 primes the hippocampus and the brain to seizure sensitivity caused by KA 104
4.3. Factors that may influence susceptibility to seizures and pathology based on hippocampal proteomics 111
4.4. Mechanisms by which RGS14 regulates mitochondrial function and oxidative stress 119
4.5. Mechanisms that RGS14 may regulate to promote glial response 126
4.6. Possible protective roles of RGS14 in CA2 hippocampus and hippocampal TLE pathogenesis 130
4.7. Future directions 134
4.7.1. In vitro measurement of epileptiform activity in acute hippocampal slices 135
4.7.2. In vivo EEG measurements of epileptiform activity and physiological monitoring of respiratory and cardiac function 136
4.7.3. Evaluation of RGS14’s effects on neuronal mitochondria 138
4.7.4. Characterizing RGS14’s role in glial response 139
4.7.5. Monitoring the development of TLE and associated pathology after SE 141
4.8. Conclusions and working model of CA2 regulation by RGS14 142
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