Date

Thursday 23 Apr 2026

Time

15:00 - 15:30

Location

BE017

Supervisor

Sander van Kasteren

2nd reviewer

Mario van der Stelt

Jury

Mark Overhand

The brain is a complex organ whose molecular mechanisms remain poorly understood. The underlying causes of brain disorders, including psychiatric and neurodegenerative diseases, are still largely unknown. Two classes of biomolecules in the brain that have proven difficult to study are fatty acids (FAs) and endocannabinoids (ECs). To address this challenge, clickable cyclopropene probes (cp-probes) of FAs and ECs have been developed, capable of undergoing bioorthogonal reactions with fluorophore-conjugated tetrazines. To study these probes in a brain model, this thesis aimed and succeeded in the development of an imaging protocol studying cpFAs and cpECs in human induced pluripotent stem cells derived adherent cortical brain organoids (ACOs). Within this assay, probe uptake, bioorthogonal click labeling with a BODIPY–tetrazine, and co-staining of astrocytes and neurons were successfully implemented. The primary objective of the imaging protocol was to investigate the partitioning of the cpFAs and cpECs among different regions and cell types in ACOs. The results demonstrated that cpAEA and cp2AG colocalize more with neurons than with astrocytes, indicating their importance in neurotransmitter regulation. Further experiments revealed that MAGL inhibition reduced cp2AG fluorescence, confirming active hydrolysis in ACOs, whereas FAAH inhibition had minimal impact on cpAEA, likely due to limited enzyme availability in ACOs. In the last experiment, cpFAs displayed cell type partitioning, with cpPOA, cpSA, and cpLA favoring astrocytes, and others favoring neurons. These findings could provide insight into the functional roles of certain cpFAs, while the established properties of specific cpFAs may also help to discover the physiological roles of astrocytes and neurons. Together, these findings indicate that the optimized imaging protocol can visualize the cellular dynamics of endocannabinoids and fatty acids, providing a valuable platform for investigating neuronal and glial pathways implicated in psychiatric disorders.