Exocytosis and endocytosis are key mechanisms that maintain homeostasis in eukaryotic cells. These processes are often altered in brain cells during neurodegenerative diseases and are therefore a subject of study. The protein amisyn, also known as syntaxin-binding protein 6 (STXBP6), is expressed in astrocytes and is believed to act as an endogenous inhibitor of exocytosis. By doing so, it may limit the development of reactive astrocytes, neurodegeneration, and neuroinflammation, processes involving lysosomes. Lysosomal fusion is inhibited by norepinephrine, which is released from neurons of the locus coeruleus. Norepinephrine is thought to influence amisyn function through adrenergic receptors, a topic we explored in this thesis. Our research involved molecular cloning, electrophysiology, and optophysiology. In the molecular cloning section, we prepared recombinant constructs by excising the green fluorescent protein gene from the vectors pEGFP-N1_ANP-emd and PL339-pCMV_amisyn-PH-EGFP and replacing it with a red fluorescent marker. This yielded the vectors pEGFP-N1_ANP-mCherry and PL339-pCMV_amisyn-PH-mCherry. These vectors were subsequently used for (co)transfection of mouse astrocytes, where we investigated the localization of the amisyn protein and ANP protein, the latter serving as a marker for secretory vesicles. In the electrophysiology section, we analyzed the impact of amisyn on exocytosis and endocytosis by measuring vesicle fusion and fission through membrane capacitance recordings. In cells transfected with wild-type amisyn, which partially localizes to the membrane, we observed a decreased frequency of exocytotic secretory events. This was not observed in cells transfected with a mutant form of amisyn, where membrane localization was disrupted due to mutations in the amino acid sequence. In the optophysiological measurements, using confocal fluorescence microscopy, we demonstrated the localization of amisyn and ANP proteins within the cytoplasm of astrocytes.
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