Manis, A. | bioRxiv Science

bioRxiv2025

Molecular deconstruction of the pre-B&oumltzinger Complex/Nucleus Ambiguus (preB&oumltC/NA) region: cellular constituencies and transcriptional responses to repeated seizures in the rat hindbrain

Epilepsy affects millions worldwide, but a significant portion suffers from uncontrollable epilepsy. Repeated seizures have many consequences, including a high risk of post-ictal cardiorespiratory failure and Sudden Unexpected Death in Epilepsy (SUDEP). Major risk factors for SUDEP include biological sex in addition to the occurrence of generalized tonic-clonic seizures (GTCSs). How repeated seizures lead to cardiorespiratory dysfunction remains unknown. A key factor in many neurological diseases is neuroinflammation, predominantly mediated by microglia and astrocytes that become dysfunctional. Mechanistically, questions remain how they affect neuronal function in epilepsy and contribute to cardiorespiratory dysfunction and increased SUDEP risk. Previously, we have shown that repeated seizures in our novel rat model with genetic mutations in kcnj16, an inwardly rectifying K+ channel, in the Dahl salt sensitive rat (SSkcnj16-/-) led to increased neuroinflammation in key ventilatory regions at 3 and 5 days of seizures. Specifically, there was increased recruitment of various inflammatory mediators, increased recruitment of activated microglia, with improvement in post-ictal respiratory dysfunction and mortality with usage of anti-inflammatory agents. Here we tested the hypothesis that repeated seizures lead to differential neuroinflammatory activation after repeated seizures in CNS regions of ventilatory control. Male SSkcnj16-/- rats were subjected to 0 (Naive), 3, 7 or 10 days of seizure, and subsequently, the pre-Botzinger Complex/Nucleus Ambiguus (preBotC/NA) was isolated and sent for nuclei isolation and sequencing. Seurat was utilized to filter and process the data, integrate across conditions and allow for differential gene expression (DEG) analysis. Afterwards, pathways enrichment analysis was performed allowing for determination of unique pathways recruited across cell types for each seizure condition. Overall, we were able to identify 18 unique cell types based on transcriptomic signatures, with 8 different neuronal populations, grouped based on Type 1, Type 2 or a mixed Type 1 & Type 2 genetic expression, indicating rhythm generation or pattern generation, respectively. We found that majority of the neuronal clusters were Type 1 or mixed type, indicating predominantly rhythmogenic neuronal populations. Importantly, these critical neuronal populations showed significant upregulation in various metabolic and neurological disease pathways at the 3 and 7 Day timepoints. Furthermore, we identified various glial cells, including microglia and astrocytes and saw increased recruitment in various Inflammatory pathways, Metabolic pathways and Chemokine related pathways after 3 and 7Days of seizures, confirming our previous results. Consequently, our results show for the first time, transcriptomic characterization of crucial rhythmogenic neuronal populations after repeated seizures and the changes that may underlie their dysfunction in SUDEP, mediated in part through the network change in upregulated inflammatory pathways in surrounding glial cells.

neuroscience↗

bioRxiv2024

Coordinated Regulation of Renal Glucose Reabsorption and Gluconeogenesis by mTORC2 and Potassium

BackgroundThe kidney proximal tubule is uniquely responsible for reabsorption of filtered glucose and gluconeogenesis (GNG). Insulin stimulates glucose transport and suppresses GNG in the proximal tubule, however, the signaling mechanisms and coordinated regulation of these processes remain poorly understood. The kinase complex mTORC2 is critical for regulation of growth, metabolism, solute transport, and electrolyte homeostasis in response to a wide array of inputs. Here we examined its role in the regulation of renal glucose reabsorption and GNG. MethodsRictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. These animals were subjected to fasting, refeeding, and variation in dietary K+. Metabolic parameters including glucose homeostasis and renal function were assessed in balance cages. Kidneys and livers were also harvested for molecular analysis of gluconeogenic enzymes, mTORC2-regulated targets, and plasma membrane glucose transporters. ResultsOn a normal chow diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane showed lower SGLT2 and SGLT1 in the fed state, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes in the fasted and fed states. These effects were correlated with reduced downstream phosphorylation of Akt and the transcription factor FOXO4, identifying a novel role of FOXO4 in the kidney. Interestingly, high dietary K+ prevented glycosuria and excessive GNG in TRKO mice, despite persistent reduction in mTORC2 substrate phosphorylation. ConclusionRenal tubule mTORC2 is critical for coordinated regulation of sodium-glucose cotransport by SGLT2 and SGLT1 as well as renal GNG. Dietary K+ promotes glucose reabsorption and suppresses GNG independently of insulin signaling and mTORC2, potentially providing an alternative signaling mechanism in states of insulin resistance. SIGNIFICANCE STATEMENTThe kidney contributes to regulation of blood glucose through reabsorption of filtered glucose and gluconeogenesis. This study shows that mTORC2 and dietary potassium coordinate the regulation of sodium-glucose cotransport and glucose production in the kidney via independent mechanisms. New insights into the regulation of these processes in the kidney offer promising implications for diabetes mellitus management and treatment.

physiology↗