Validation of novel tools to monitor glycogen in living astrocytes

Abstract

Glycogen is the obligatory fuel of the brain, yet there is a lack of established assays to trace the highly dynamic nature of glycogen granules in live astrocytes in real time. Current assays are limited as they measure glycogen in fixed or homogenised tissue, and these procedures may deplete glycogen stores. In this study, we aimed to validate two novel glycogen tracing assays, 2-NBDG, a D-glucose fluorescent analogue, and hLaf-CyPet, a fluorescent laforin construct. We loaded dissociated cultured astrocytes with 500 μM 2-NBDG or expressed hLaf-CyPet by transfection or adenoviral vector transduction. We conducted live confocal imaging to trace changes in granule fluorescence over time while inducing glycogenolysis. 2-NBDG was prone to bleaching, and bleached 2-NBDG emissions were consistent with autofluorescence. For hLaf-CyPet, the rationale was that, with granule degradation, fluorescence redistributes in the cytosol. We observed substantial variation in responses to induced glycogenolysis, with only few putative granules that conformed to the expected cytosolic redistribution (2 out of 15
transfected cells). When transduced, we found granule and cytosolic fluorescence increased over time. We compared hLaf-CyPet labelling with an ESG anti-glycogen antibody in fixed dissociated astrocytes and organotypic slices, and found that in fixed dissociated astrocytes, hLaf-CyPet granules did not behave as expected for glycogen and fixed organotypic slices lacked hLaf-CyPet granules. In hLaf-CyPet expressing and ESG stained dissociated astrocytes, the fluorophores were mostly mutually exclusive but some specific granules colocalised suggesting hLaf-CyPet might label glycogen. We suggest that hLaf-CyPet either labels glycogen but renders the granule insoluble by restricting access to glycogen associated proteins or that overexpression of the construct overwhelms the ubiquitin proteasome system resulting in aggresomes. Immunolabelling confirmed that dissociated astrocytes have smaller and fewer granules than organotypic slices, which we suggest to be a consequence of impaired glycogen metabolism as neuronal interactions are lost in dissociated astrocyte cultures. Further work is required to identify the limitations of the hLaf-CyPet probe and determine whether cultured astrocytes can be used as a model to trace glycogen dynamics.

Date of Award	1 Oct 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Anja G Teschemacher (Supervisor) & Sergey Kasparov (Supervisor)