Knock, Knock, Who's There? Not SCN1A in Brain Organoids

Abstract

Unraveling the Molecular Landscape of SCN1A Gene Knockout in Cerebral Organoids: A Multiomics Approach Utilizing Proteomics, Lipidomics, and Transcriptomics

Koh, B., Kim, Y.E., Park, S.B., Kim, S.S., Lee, J., Jo, J.H., Lee, K., Bae, D.H., Kim, T.Y., Cho, S.H. and Bae, M.A., 2024. ACS omega, 9(38): 39804-39816.

This study investigates the impact of sodium channel protein type 1 subunit alpha (SCN1A) gene knockout (SCN1A KO) on brain development and function using cerebral organoids coupled with a multiomics approach. From comprehensive omics analyses, we found that SCN1A KO organoids exhibit decreased growth, dysregulated neurotransmitter levels, and altered lipidomic, proteomic, and transcriptomic profiles compared to controls under matrix-free differentiation conditions. Neurochemical analysis reveals reduced levels of key neurotransmitters, and lipidomic analysis highlights changes in ether phospholipids and sphingomyelin. Furthermore, quantitative profiling of the SCN1A KO organoid proteome shows perturbations in cholesterol metabolism and sodium ion transportation, potentially affecting synaptic transmission. These findings suggest dysregulation of cholesterol metabolism and sodium ion transport, with implications for synaptic transmission. Overall, these insights shed light on the molecular mechanisms underlying SCN1A-associated disorders, such as Dravet syndrome, and offer potential avenues for therapeutic intervention.

Commentary

SCN1A encodes the voltage-gated sodium channel α subunit Na_v1.1, which is highly expressed in the central nervous system. ¹ Heterozygous loss-of-function mutations in SCN1A are associated with several forms of early-life epilepsy, including generalized epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (DS). Multiple models have been generated and are used for studying SCN1A-derived epilepsy, including rodent models and induced pluripotent stem cells (iPSCs). Heterozygous Scn1a knockout (KO) mice, a model of DS, exhibit increased susceptibility to hyperthermia-induced seizures, spontaneous seizures, premature mortality, and numerous behavioral comorbidities.^2,3 Moreover, studies using mouse models have revealed altered excitability ² and established the important contribution of various interneurons to impaired neuronal excitability in DS.^4,5

One advantage of iPSCs from human patients is that they can be differentiated into specific cell types to study human brain development. Previous studies using DS-patient iPSC-derived GABAergic neurons demonstrated impaired sodium current activation and reduced action potential generation,^6,7 which is consistent with the findings from mouse models. Furthermore, recent studies using DS-patient iPSC-derived neurons provide insight into the molecular changes that may occur in the brain in DS, including alterations in the chromatin ⁷ and transcriptomic ⁸ landscape. While iPSCs offer the opportunity to study human neurons, there are limitations to their use to investigate the structure and complexity of the human brain’s neural circuitry. However, recent advances have led to the generation of the three-dimensional (3D) human brain organoid, which recapitulates some structural and functional connectivity of the human brain. Human brain organoids can be used to study early human brain development in both healthy and disease conditions.

In the current study, Koh et al., generated a homozygous SCN1A KO cerebral organoid model to study human brain development in the absence of SCN1A. ⁹ While this model may not directly reflect the molecular changes that occur in DS, which is associated with heterozygous loss-of-function SCN1A mutations, it does provide insight into SCN1A-mediated alterations that might occur during early human brain development. First, compared to control organoids, Koh et al., observed reduced growth in SCN1A KO organoids at Day 120. ⁹ Neurotransmitter levels (cholinergic, serotonergic, dopaminergic, and GABAergic) were quantified at different stages of organoid development (D25, D70, D120). Notably, while levels of various neurotransmitters increased over time in the control organoids, only levels of GABA were found to be significantly increased in the SCN1A KO organoids. Furthermore, GABA levels were significantly higher in the SCN1A KO organoids compared to controls at each developmental stage. In addition, compared to control organoids, upregulation of ether phospholipids and neurosteroids (11-desoxycortisol and 21-desoxycortisol) levels and downregulation of progesterone and pregnenolone were observed in Day 120 SCN1A KO organoids. Transcriptomic analyses identified differentially expressed genes involved in signaling receptor activity, signal receptor binding, and molecular transducer activity in Day 120 SCN1A KO organoids. In future studies, it would be valuable to perform single-cell transcriptomic analyses to establish cell type contributions to changes in gene expression in SCN1A KO organoids.

Finally, proteomic analyses were performed on Day 0 and Day 120 organoids. In Day 0 organoids, only 14 proteins were differentially expressed in the SCN1A KO organoids, and gene ontology (GO) analysis suggested that these proteins are involved in cholesterol synthesis and fatty acid biosynthetic processes. A greater number of differentially expressed proteins (192 upregulated, 94 downregulated) were identified in Day 120 SCN1A KO organoids. Hierarchical clustering analyses identified 4 clusters based on expression patterns. For example, Cluster 1 (62 proteins) comprised proteins that were significantly increased only in the control organoids from Day 0 to Day 120. Interestingly, the proteins in Cluster 1 are important for cholesterol homeostasis, lipid catabolic processes, and extracellular matrix formation, which functionally overlap with the GO analysis of differentially expressed proteins in Day 0 SCN1A KO organoids. This observation suggests sustained alterations in cholesterol processes in SCN1A KO organoids during development, which might explain, in part, the dysregulation of neurosteroid levels, as cholesterol is a precursor for steroid hormones. Interestingly, soticlestat, a cholesterol 24-hydroxylase inhibitor, has been shown to reduce spontaneous seizures and premature mortality in a mouse model of DS. ¹⁰ In silico analyses revealed that the proteins identified in Clusters 3 and 4 were associated with synaptic transmission, synapse organization, and sodium channels. ⁹ Intriguingly, the sodium channel gene SCN2A was significantly increased in expression in Day 120 SCN1A KO organoids. While sodium channel expression has not been examined in homozygous Scn1a KO mice, mRNA expression of the other brain-expressed sodium channel genes in heterozygous Scn1a KO mice is comparable to wild-type littermates. ¹¹

The current study by Koh et al., generated a homozygous SCN1A KO cerebral organoid model and utilized a multiomics approach to better understand the role of SCN1A in human brain development. ⁹ While this approach provided valuable insight into the molecular landscape of a homozygous SCN1A KO cerebral organoid, caution should be taken when extrapolating these findings to DS, which is associated with heterozygous loss-of-function mutations in SCN1A. It also remains to be determined whether this model exhibits electrophysiological alterations. A recent study generated ventral forebrain organoids from iPSCs from 3 DS patients to demonstrate feasibility of generating the model, ¹² but to date, there has been no functional characterization of SCN1A KO organoids. In future studies, it would be beneficial to establish whether there is increased neuronal excitability in SCN1A KO cerebral organoids and whether this alteration arises from GABAergic interneuron dysfunction as previously observed in both iPSC-derived neurons and mouse models of DS. In future studies, it would also be important to extend the molecular and functional characterization to heterozygous SCN1A KO organoids. While the functional properties of organoids lacking SCN1A have not yet been established, Koh et al., generated rich multiomics datasets from a homozygous SCN1A KO cerebral organoid that will be useful for studying human brain development in the absence of SCN1A. In future studies, it would also be valuable to extend characterization of SCN1A KO organoids to later developmental stages. However, it is interesting to note that the expression of some proteins was already altered in SCN1A KO cerebral organoids at an early development timepoint (Day 0), ⁹ which might suggest that these proteins contribute to the early phenotypes observed in SCN1A KO organoids. Finally, organoids can also be fused to generate assembloids or spheroids to further study the contribution of different cell types and brain structures, and thus, provide a more complex model to study human brain development.