Hippocampal Neurons - The Destructive Force Behind Epilepsy and Mental Disorders

Background Introduction

Tuberous Sclerosis Complex (TSC) is a complex multi-system genetic disorder that manifests with lesions in the brain, skin, heart, kidneys, and other organs as the patient ages. Clinically, TSC presents with symptoms such as epilepsy and developmental delay. TSC is caused by loss-of-function mutations in the TSC1 or TSC2 genes, which encode the proteins Hamartin and Tuberin, respectively. These mutations lead to dysfunction of the TSC1-TSC2 complex, resulting in the activation of the mammalian target of rapamycin (mTOR) pathway. Overactivation of this pathway has been shown to be associated with various genetic and acquired epilepsies.

GABAergic interneurons play a crucial role in maintaining neural circuit balance, regulating excitation-inhibition, and modulating cognitive functions. In TSC, dysfunction of GABAergic neurons is considered a significant cause of network activity disorders and related neurological symptoms. This dysfunction may be attributable to specific cell types. This study focuses on identifying specific subpopulations of interneurons in TSC, particularly those derived from the medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE).

Authors and Sources

This paper, titled “Impaired GABAergic Regulation and Developmental Immaturity in Interneurons Derived from the Medial Ganglionic Eminence in the Tuberous Sclerosis Complex,” was authored by Mirte Scheper, Frederik N. F. Sørensen, Gabriele Ruffolo, among others. They hail from the Amsterdam University Medical Center, the University of Copenhagen Faculty of Health and Medical Sciences, Sapienza University, the UCL Queen Square Institute of Neurology, the University Medical Center Utrecht, and the University of Queensland. The paper was published in the 2024 issue of Acta Neuropathologica, volume 147, page 80.

Research Process and Methods

To gain deeper insights into GABAergic neuron dysfunction in TSC, this study employed single-nuclei RNA sequencing (snRNA-seq) to analyze brain samples from TSC patients and control groups.

Sample Collection and Processing

The study used surgical and postmortem brain tissues obtained from the Amsterdam University Medical Center, University Medical Center Utrecht, and Queensland Children’s Hospital. Control group samples were obtained postmortem within nine hours from age-matched individuals with no history of epilepsy or other neurological disorders. The prefrontal cortex or middle frontal gyrus regions were selected for analysis from all control group samples.

Single-nuclei RNA Sequencing

Nuclei were extracted from frozen tissues and sorted using fluorescence-activated cell sorting (FACS). RNA sequencing libraries were prepared using the Chromium Single Cell 3’ Reagent Kits v3.1 from 10× Genomics and sequenced on the Illumina NovaSeq 6000 platform. Data preprocessing and analysis were performed, with the expression matrix containing unique molecular identifiers (UMIs) imported into Seurat software for further processing.

Immunohistochemistry and Electrophysiology

To further validate the findings, immunohistochemistry was used to label sst+ interneurons in the brains of TSC patients. Additionally, functional assessments were conducted using African clawed frog oocytes to test the impact of GABAA receptor subunits on GABA affinity.

Main Research Findings

Dysfunction of GABAergic Interneurons

Single-nuclei RNA sequencing revealed that sst+ interneurons in TSC patients exhibited immature characteristics, including abnormal NKCC1/KCC2 ratios. This imbalance in chloride ion homeostasis indicated reduced inhibitory properties of GABAergic signaling. Moreover, downregulation of the GABAA receptor α1 subunit and upregulation of the α2 subunit in sst+ interneurons highlighted functional and developmental dysregulation.

Data Analysis and Subgroup Identification

Using UMAP (Uniform Manifold Approximation and Projection) analysis, interneurons were clustered into multiple groups and further classified by specific marker expression. The study showed that MGE- and CGE-derived interneurons in TSC exhibited distinct gene expression patterns and maturity levels.

Immature Functional Characteristics

Electrophysiological experiments demonstrated that GABAA receptor function tests on membranes from TSC brain tissue showed reduced GABA affinity across various subunit compositions. This further supports the immature characteristics of sst+ interneurons in TSC, with their functional abnormalities potentially contributing to epilepsy and other neurological impairments in TSC patients.

Localization and Migration Issues in Brain Tissue

The study also found that sst+ interneurons in TSC patients were primarily located in the deeper layers of the cortex (L5/6), whereas in the control group, these neurons were evenly distributed across the superficial and deep layers. This suggests possible migration issues of these interneurons in TSC, related to cortical layering abnormalities.

Conclusions and Significance

This study revealed the dysregulation of GABAergic interneurons in TSC, particularly the immature characteristics of sst+ interneurons derived from the MGE. Through comprehensive molecular and functional analyses, the study demonstrated that abnormalities in these neurons might be critical to the disrupted neural network activity, leading to epilepsy and various neuropsychiatric symptoms in TSC patients.

Research Highlights

The highlights of this study include the detailed analysis of GABAergic interneuron dysregulation in TSC using single-nuclei RNA sequencing, particularly the immaturity of sst+ interneurons and their functional and localization abnormalities. Additionally, the study validated these key findings through multiple experimental approaches, providing important references for future therapeutic strategies targeting TSC.

This study not only expands the understanding of neural network dysregulation mechanisms in TSC but also offers new perspectives for the development of precision medicine. Future research should delve deeper into the electrophysiological and molecular levels of sst+ interneurons to uncover their specific functional roles and interaction mechanisms, providing new therapeutic targets for TSC and related neuropsychiatric disorders.