Periodic Reporting for period 1 - ExoDevo (Extracellular vesicles-mediated cross-talk during human brain development and disease)
Reporting period: 2023-01-01 to 2025-06-30
Cellular cross-talk is an essential process influenced by numerous factors including secreted vesicles that transfer nucleic acids, lipids, and proteins between cells. Extracellular vesicles (EVs) have been the center of many studies focusing on neuron-to-neuron communication while the role of EVs in progenitor-to-neuron and -astrocyte communication occurring during brain development has not been systematically investigated. Extracellular signals regulating the development of the brain are key players altered in many neurodevelopmental disorders (NDDs). Strikingly, we have found that more than 60% of the genes associated with NDDs encode proteins that are loaded into EVs.
With ExoDevo, inspired by new cell-non-autonomous mechanisms that we have identified as the cause of NDDs, I will investigate the role of EVs during brain development. I will focus on the physiological function of EVs that mediate the signals for cell-to-cell cross-talk and combine transcriptomic, proteomic, imaging, and functional analysis of EVs derived from human cerebral organoids. This will open new avenues to tackle fundamental questions, such as how different cells communicate and feedback at different times and distances in the highly dynamic process of brain development. Ultimately, this will be investigated in human models of NDDs and will allow me to identify pathologically altered cellular cross-talk mediated by EVs. This knowledge of the cellular processes governing EVs’ biology will provide the basis to better understand novel mechanisms underlying brain development and neurodevelopmental human pathologies and explore new deliverable compounds for therapy. My expertise in human brain development and diseases together with the possibility of combining multiple technologies will be indispensable to achieve these essential goals. Meanwhile, exploring these novel aspects of brain development will bring me beyond my current research focus and broaden my perspectives on NDDs.
With ExoDevo, inspired by new cell-non-autonomous mechanisms that we have identified as the cause of NDDs, I will investigate the role of EVs during brain development. I will focus on the physiological function of EVs that mediate the signals for cell-to-cell cross-talk and combine transcriptomic, proteomic, imaging, and functional analysis of EVs derived from human cerebral organoids. This will open new avenues to tackle fundamental questions, such as how different cells communicate and feedback at different times and distances in the highly dynamic process of brain development. Ultimately, this will be investigated in human models of NDDs and will allow me to identify pathologically altered cellular cross-talk mediated by EVs. This knowledge of the cellular processes governing EVs’ biology will provide the basis to better understand novel mechanisms underlying brain development and neurodevelopmental human pathologies and explore new deliverable compounds for therapy. My expertise in human brain development and diseases together with the possibility of combining multiple technologies will be indispensable to achieve these essential goals. Meanwhile, exploring these novel aspects of brain development will bring me beyond my current research focus and broaden my perspectives on NDDs.
We investigated the critical role of extracellular vesicles (EVs) as a communication system that coordinates the molecular and cellular events underlying human brain development. Extracellular vesicles are small, membrane-bound particles secreted by cells and have emerged as a crucial mode of intercellular signaling, capable of transporting a wide range of cargo across different cell types and spatial locations. Our study explores how EV-mediated signaling modulates brain development by delivering molecular cues.
To investigate the role of EVs in human neural development, we employed both 2D cell cultures and 3D cerebral organoids (COs) and analyzed EV release, composition, uptake, and function across different cell types (and developmental stages. Key findings include:
• EV composition and release are highly dynamic, varying with cell type, developmental stage, and environment.
• EV uptake is cell-type specific, with cells preferentially taking up EVs from developmentally related cells and the cellular localization of EVs varies by cell type, suggesting different signaling mechanisms.
• EVs, particularly those derived from distinct brain regions (dorsal vs ventral), carry transcription factors (TFs) that can induce functional changes in recipient cells, influencing transcriptional programs.
First, we show that EV marker expression varies by cell type: some markers were ubiquitously expressed (CD63, CD81, and PDCD6IP), while others were more restricted. For instance, CD82 and CD9 were less abundant. This suggests cell-type-specific patterns in EV biogenesis and cargo loading.
We then monitored the dynamic of EV composition during development: EV composition is dynamic and changes throughout neural development, as evidenced in 3D COs. E
Then we investigated the Cell-Type Specific EV Uptake: EV uptake is not random; it is preferentially targeted towards specific cell types. NPCs preferentially uptake EVs from young and mature neurons (nEVs), while mature neurons preferentially take up EVs from astrocytes (aEVs). Astrocytes internalize both EVs from progenitors (pEVs) and neurons (nEVs), with a preference for mature nEVs. This suggests a selective communication mechanism, where cells preferentially uptake EVs from their developmentally related partners.
We found that EVs carry transcription factors (TFs) and Affect Gene Expression. dEVs have a larger number of TFs than vEVs, many of which are critical during neurogenesis. TFs in EVs do not strictly correspond with their expression levels in the donor cells, suggesting regulated secretion. When NPCs are treated with dEVs and vEVs, their transcriptome is altered, with particular dysregulation of patterning pathways (WNT and TGF-β). This indicates that EVs can actively influence gene expression in recipient cells through the delivery of TFs. The study identified specific TFs like YAP1, CUX1, and NR2F2, delivered by EVs, which then cause changes in target gene expression in recipient cells. Live-cell imaging shows that EVs are internalized by NPCs and are uniquely localized in the nucleus, particularly after cell division. This nuclear localization suggests a specific mechanism for altering transcriptional activity in NPCs.
Finally, we focused on the mechanisms of EV uptake. We addressed various ways EVs can interact with receiving cells such as membrane fusion, receptor binding, and internalization through endocytosis. EVs from donor cells were not selectively loaded with classic transmembrane proteins, however, recipient cells showed a clear preference for EVs from developmentally related cells. Cellular localization of EVs in the recipient cell varies and suggests different mechanisms of uptake depending on the target cell type. The response of cells to EVs depends on the dose of EVs used, and further investigation into dose manipulation may reveal further details of the uptake patterns.
To investigate the role of EVs in human neural development, we employed both 2D cell cultures and 3D cerebral organoids (COs) and analyzed EV release, composition, uptake, and function across different cell types (and developmental stages. Key findings include:
• EV composition and release are highly dynamic, varying with cell type, developmental stage, and environment.
• EV uptake is cell-type specific, with cells preferentially taking up EVs from developmentally related cells and the cellular localization of EVs varies by cell type, suggesting different signaling mechanisms.
• EVs, particularly those derived from distinct brain regions (dorsal vs ventral), carry transcription factors (TFs) that can induce functional changes in recipient cells, influencing transcriptional programs.
First, we show that EV marker expression varies by cell type: some markers were ubiquitously expressed (CD63, CD81, and PDCD6IP), while others were more restricted. For instance, CD82 and CD9 were less abundant. This suggests cell-type-specific patterns in EV biogenesis and cargo loading.
We then monitored the dynamic of EV composition during development: EV composition is dynamic and changes throughout neural development, as evidenced in 3D COs. E
Then we investigated the Cell-Type Specific EV Uptake: EV uptake is not random; it is preferentially targeted towards specific cell types. NPCs preferentially uptake EVs from young and mature neurons (nEVs), while mature neurons preferentially take up EVs from astrocytes (aEVs). Astrocytes internalize both EVs from progenitors (pEVs) and neurons (nEVs), with a preference for mature nEVs. This suggests a selective communication mechanism, where cells preferentially uptake EVs from their developmentally related partners.
We found that EVs carry transcription factors (TFs) and Affect Gene Expression. dEVs have a larger number of TFs than vEVs, many of which are critical during neurogenesis. TFs in EVs do not strictly correspond with their expression levels in the donor cells, suggesting regulated secretion. When NPCs are treated with dEVs and vEVs, their transcriptome is altered, with particular dysregulation of patterning pathways (WNT and TGF-β). This indicates that EVs can actively influence gene expression in recipient cells through the delivery of TFs. The study identified specific TFs like YAP1, CUX1, and NR2F2, delivered by EVs, which then cause changes in target gene expression in recipient cells. Live-cell imaging shows that EVs are internalized by NPCs and are uniquely localized in the nucleus, particularly after cell division. This nuclear localization suggests a specific mechanism for altering transcriptional activity in NPCs.
Finally, we focused on the mechanisms of EV uptake. We addressed various ways EVs can interact with receiving cells such as membrane fusion, receptor binding, and internalization through endocytosis. EVs from donor cells were not selectively loaded with classic transmembrane proteins, however, recipient cells showed a clear preference for EVs from developmentally related cells. Cellular localization of EVs in the recipient cell varies and suggests different mechanisms of uptake depending on the target cell type. The response of cells to EVs depends on the dose of EVs used, and further investigation into dose manipulation may reveal further details of the uptake patterns.
This study represents a breakthrough because it reveals a highly organized and previously unappreciated system for cellular communication in the developing human brain. Despite their known roles in other systems, EVs have remained underexplored in the context of brain development. This study is ground-breaking because it identifies specific roles of EVs in the development of the human brain and uncovers their potential involvement in fundamental developmental processes, such as cortical layering, synaptic pruning, and glial cell communication. It advances the field by addressing a critical gap in our understanding of how cellular coordination in the brain is achieved. Traditionally, brain development has been studied through the lens of genetic programming, soluble signaling molecules, and cell-cell interactions via direct contact. However, our study highlights EVs as a previously underestimated mode of communication. Our work likely represents a breakthrough for several reasons, as it tackles one of the most complex and fundamental questions in neuroscience: how does the human brain develop and organize itself so precisely? EVs as mediators of this process introduce a novel and underexplored layer of intercellular communication, offering insights that could transform our understanding of brain development and disease.