Emerging single cell sequencing approaches pave new paths for the epilepsy treatment 21.11.20224’ Neurosciences Review Emerging single cell sequencing approaches pave new paths for the epilepsy treatmentUnderstanding how our brains work in health and disease is enormously challenging due to great variety and number of neuronal cells and the functional connections that they form with each other. As summarized by Michio Kaku, “The brain weighs only three pounds, yet it is the most complex object in the solar system” (1).To elucidate how our brains function is especially important for the development of treatments for neurological disorders and diseases that pose a great burden to patients and often have limited treatment options. Epilepsy is one of the chronic diseases of the brain, characterized by spontaneous seizure episodes of varying frequency, affecting around 50 million people globally (2). Despite the high prevalence, there is a lack of knowledge about the epilepsy pathophysiology on the cellular and molecular level which might be crucial for the development of new treatments.We bring you here a selection of noteworthy studies that applied transcriptomic analysis at single cell resolution, proving that single cell sequencing can propel the research on epilepsy forward.Neuronal subtypes and genes underlying epileptogenesisPfisterer et al. (3) have used single nucleus transcriptomic analysis to characterize neuronal transcriptomes derived from temporal cortex of multiple temporal lobe epilepsy and non-epileptic subjects. The study has identified several subtypes of neurons with differential effect of epilepsy on neuronal transcriptome in thousands of genes in specific neuronal subtypes (in total, ~6,900 and 13,700 differentially expressed genes for GABAergic and principal neurons, respectively). Importantly, the majority of the identified dysregulated genes are novel epilepsy-associated genes, expanding our knowledge on the mechanisms of epileptogenesis. These finding owe to large part to single cell transcriptomics approach, that allowed for overcoming of the limitations of the bulk transcriptomics studies.Figure 1. Preparation of single-nucleus samples for transcriptomic analysis of the epileptic and nonepileptic temporal cortex (Image modified from Pfisterer et al. (3), http://creativecommons.org/licenses/by/4.0/).De-differentiated populations of glia in epilepsyThe approach of Pai et al. (4) has focused on elucidating the role of glia in the pathophysiology of epilepsy. Two distinct glial cell types, astrocytes and oligodendroglial-lineage populations were isolated using fluorescence activated nuclei sorting with positive selection nuclear markers from surgical epilepsy samples. The single nucleus RNA-seq study has uncovered two abnormal populations of epilepsy glia: a rare population of oligodendroglial progenitors with enriched inflammatory and microglial signature and a prominent population of hybrid glia expressing both oligodendroglial progenitor and reactive astrocyte -associated genes. The newly described hybrid glial populations have shown expression of dual markers confirming the de-differentiated phenotype also in immunofluorescence assays. As the current epilepsy treatments focus on decreasing the neuronal excitability have limited clinical efficiency, understanding the role of glial subtypes in epilepsy might lead to new therapies in the future.Figure 2. Single-cell transcriptomics of human temporal lobe epilepsy revealing mixed lineage glial subpopulations (Image modified from Pai et al. (4), http://creativecommons.org/licenses/by/4.0/).Pro-inflammatory signaling in epilepsyThe remarkable study by Kumar et al. (5) has focused on epileptogenic triggers, the processes that generate and perpetuate epilepsy. Brain tissues obtained during resective epileptic surgeries were analyzed using single-cell CITE-seq approach that enables simultaneous profiling of transcripts and cell-surface proteins. The obtained data showed that microglia, the innate immune cells in the central nervous system, exhibit a pro-inflammatory phenotype and that direct physical interactions between microglia and infiltrated T cells occur in the drug-refractory epilepsy brains. As the treatment of the drug-refractory epilepsy currently relies only on resective epilepsy surgery, the insights into the immune microenvironment in epileptic tissue may enable the development of non-invasive therapies.Figure 3. Microglia and infiltrating immune cells in brain tissue from epileptic patients (Image modified from Kumar et al. (5), http://creativecommons.org/licenses/by/4.0/).Are you thinking about how to apply single cell sequencing to your research project? Get in touch with our experts at info@singleronbio.com or through our contact numbers.ReferencesMichio Kaku.The Future of the Mind: The Scientific Quest to Understand, Enhance, and Empower the Mind. Doubleday, 2015.World Health Organization, accessed 18 November 2022, https://www.who.int/news-room/fact-sheets/detail/epilepsyPfisterer, U., Petukhov, V., Demharter, S. et al. Identification of epilepsy-associated neuronal subtypes and gene expression underlying epileptogenesis. Nat Commun 11, 5038 (2020). https://doi.org/10.1038/s41467-020-18752-7Pai, B., Tome-Garcia, J., Cheng, W.S. et al. High-resolution transcriptomics informs glial pathology in human temporal lobe epilepsy. acta neuropathol commun 10, 149 (2022). https://doi.org/10.1186/s40478-022-01453-1Kumar, P., Lim, A., Hazirah, S.N. et al. Single-cell transcriptomics and surface epitope detection in human brain epileptic lesions identifies pro-inflammatory signaling. Nat Neurosci 25, 956–966 (2022). https://doi.org/10.1038/s41593-022-01095-5 A post by Singleron teamCheck out our latest blog posts Learn more 23.12.12 Annual Research Roundup: 2023's Most Impactful Publications! 2023 was a busy and successful year for our scientific community. As 2023 comes to an end, it is time to look back at some of theimpactful publications from this year. Read more 23.11.22 Decoding the Biological Meaning of Your Data: The Power of Accurate Automated Cell Type Annotation Automated single cell RNA sequencing annotation streamlines analysis, saving time and improving reproducibility. 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