Exosome RNA Exosome RNA Research & Industry News
Extracellular vesicles, or EVs, are tiny membrane-bound particles released by cells. They carry proteins, RNA, lipids, and other molecules between cells, allowing them to communicate with one another. In the developing brain, this communication can help guide immature cells toward specific cell types and influence how neural circuits are formed.
Researchers at LMU Munich have now found that disrupted extracellular vesicle signaling may contribute to progressive myoclonus epilepsy type 1, or EPM1, a rare neurological disorder caused by mutations in the CSTB gene.
Modeling EPM1 with cerebral organoids
EPM1 is caused by mutations that reduce the function of cystatin B, a protein encoded by the CSTB gene. People with the condition develop symptoms including muscle jerks, seizures, and problems with movement and coordination.
To investigate how loss of CSTB affects the developing brain, the researchers created cerebral organoids from cells obtained from patients with EPM1. Cerebral organoids are three-dimensional collections of neural cells grown in the laboratory that reproduce some features of early brain development.
The EPM1 organoids showed increased electrical activity and an abnormal balance between excitatory and inhibitory signals.
Maintaining the correct balance between these signals is essential for normal brain function. Excitatory neurons increase the likelihood that other neurons will become active, while inhibitory neurons help restrain neural activity. Disruption of this balance can contribute to excessive electrical activity associated with epilepsy.
RNA sequencing reveals altered neural cell development
The researchers used single-cell RNA sequencing to examine gene expression in individual cells from ventral regions of the cerebral organoids.
During normal brain development, progenitor cells in different regions receive molecular signals that guide them toward particular neuronal identities. Ventral progenitors, for example, contribute to the formation of several types of inhibitory neurons.
The RNA sequencing results revealed that cells in EPM1 organoids were not developing according to their expected ventral identity. Instead, some progenitor cells shifted toward gene expression patterns associated with dorsal neurons.
This abnormal cell fate specification suggested that an important signaling mechanism responsible for establishing regional identity in the developing brain had been disrupted.
Defective EV-mediated transport of SHH alters neural fate specification in EPM1 epilepsy
All data represent pooled results from two control iPSC lines (CTRL.1 and CTRL.2) and two patient iPSC lines (EPM1.1 and EPM1.2). (A) Volcano plot showing proteins significantly enriched (green) in CSTB co-IP compared to control (IgG). (B) GO enrichment analysis of significantly enriched proteins in CSTB co-IP compared to control (IgG). (C) Reverse transcription qPCR (RT-qPCR) analysis of SHH pathway target genes (GLI1, NKX2-1, PTCH1, PTCH2, OLIG2, and CCND1) in CTRL and EPM1 vNPCs. (D) Quantification of intracellular and extracellular SHH protein levels in CTRL versus EPM1 vNPCs. Scheme created in BioRender (https://BioRender.com/mj7rl71). (E) Quantification of intracellular and extracellular SHH protein levels in EPM1 versus CSTB-overexpressing EPM1 vNPCs. Scheme created in BioRender (https://BioRender.com/mj7rl71). (F) Clustered heatmap showing the expression levels of the differentially regulated genes in NPCs following the acute treatment of vEVs, vEPM1-EVs, or no treatment. GO terms enriched in each cluster are listed on the right. Scheme created in BioRender (https://BioRender.com/tpd7euk). (G) RT-qPCR analysis of SHH pathway target genes (GLI1, NKX2-1, PTCH1, PTCH2, OLIG2, and CCND1) in CTRL vNPCs following treatment (1 week) with CTRL-EVs versus EPM1-EVs. Scheme created in BioRender (https://BioRender.com/mj7rl71). (H) Volcano plot showing pseudobulk analysis for the progenitor cluster of scRNA-seq data from vCOs. Genes enriched in vCTRL are highlighted in green, and genes enriched in vEPM1 are highlighted in purple. (I) Volcano plot showing pseudobulk analysis for the Progenitor cluster of scRNA-seq data from mosaic vCOs. Genes enriched in mvCTRL are highlighted in green, and genes enriched in mvEPM1 are highlighted in purple. (J) Representative images of MEIS2 and KI67 immunostaining in vEPM1 and SHH-treated vEPM1 COs and quantification of the percentage of positive ventricles for each condition. DAPI staining is presented as a nuclear counterstain. Scale bars, 100 μm. n = 9 to 11. Statistical significance was determined using an exact binomial test. ****P < 0.0001. (K) Representative images of NKX2-1 immunostaining in vEPM1 and SHH-treated vEPM1 COs and quantification of the percentage of positive ventricles for each condition. DAPI staining is presented as a nuclear counterstain. Scale bas, 100 μm. n = 9 to 11. Statistical significance was determined using an exact binomial test. ****P < 0.0001. (L) Representative images of PAX6 immunostaining in vEPM1 and SHH-treated vEPM1 COs and quantification of the percentage of positive ventricles for each condition. DAPI staining is presented as a nuclear counterstain. Scale bars, 100 μm. n = 9 to 11. Statistical significance was determined using an exact binomial test. ****P < 0.0001.
Extracellular vesicles influence the neural signaling environment
The researchers traced this developmental problem to changes in the extracellular environment surrounding the cells.
Extracellular vesicles normally carry signaling molecules between cells, helping coordinate development across tissues. In EPM1 organoids, EV production and cargo were altered, reducing the effectiveness of this communication system.
One particularly important molecule affected was Sonic Hedgehog, or SHH.
Despite its unusual name, SHH is a major developmental signaling protein. During brain development, it helps establish ventral brain identity and directs neural progenitor cells toward specific cell types.
The researchers found that CSTB physically interacts with SHH. When CSTB was deficient, the amount of SHH contained in and released from cells was reduced.
This suggests that CSTB has a previously unrecognized role in maintaining the signaling environment needed for normal brain patterning.
Connecting CSTB, SHH, and extracellular vesicles
The results support a model in which CSTB helps maintain normal SHH signaling through extracellular vesicles.
When CSTB function is impaired, EV dynamics and protein cargo change. Less SHH is available within the extracellular environment, weakening signals that normally maintain ventral neural identity.
As a result, some neural progenitor cells develop toward inappropriate cell fates. This altered development may contribute to the imbalance between excitatory and inhibitory neurons observed in EPM1.
The researchers describe this relationship as a CSTB-SHH-EV signaling axis.
A potential new direction for EPM1 research
These findings expand the potential role of extracellular vesicles in neurological disease. Rather than functioning only as carriers of cellular material, EVs may actively shape the developmental environment that determines what types of neurons are produced.
The results also suggest that some neurological disorders caused by genetic mutations may involve changes in communication between cells, not only defects within individual cells.
If researchers can better understand how CSTB controls SHH packaging, release, and transport through extracellular vesicles, it may eventually be possible to identify ways to restore normal developmental signaling.
The CSTB-SHH-EV pathway could therefore provide a new target for investigating therapies aimed at correcting the excitatory and inhibitory imbalance associated with EPM1 and improving understanding of how extracellular vesicle signaling contributes to brain development and epilepsy.
