Using fibre photometry with a fluorescence resonance energy transfer-based biosensor to test efficacy of calpain inhibitors

Calcium-activated proteases, known as calpains, cleave proteins into smaller protein fragments, altering protein structure and function. Calpain overactivity is a pathological hallmark of many human diseases, including neurodegenerative diseases, which are characterized by excessive proteolytic cleavage and formation of protein aggregates. Current methods for determining calpain activity, such as examination of the presence or absence of calpain substrates in post-mortem tissue, are low-throughput, costly and retrospective, hampering the search for potentially therapeutically efficacious calpain inhibitor compounds. Here, we describe a novel methodology that allows real-time examination of calpain activity within the brain of the awake mouse. We used an adeno-associated viral (AAV) vector to express a fluorescence resonance energy transfer (FRET)-based calpain sensor in cerebellar neurons of male mice and measured real-time changes in FRET signal as a proxy for calpain activity via an implanted fibre optic cannula. This methodology was used to compare within-animal responses to different doses and administration routes of several calpain inhibitor compounds, including calpeptin, BLD-2736 and SNJ-1945. Using our AAV-calpain sensor and fibre photometry approach, we were able to obtain high-quality FRET recordings that were reproducible both between experimental animals and within experimental animals, allowing direct comparison of different calpain inhibitor compounds, enabling identification of the most efficacious treatment strategy that could be progressed further with preclinical treatment studies. As a positive control, we also tested changes in FRET signal in response to ionomycin, a known calcium ionophore. To our knowledge, this is the first report of real-time measurement of neuronal calpain activity in vivo. Future experiments could exploit this novel methodology to obtain calpain activity measurements alongside other pharmacological and behavioural/physiological measurements to increase the translational validity of preclinical models for drug development in diseases characterized by calpain overactivity.