Spatio-temporal analysis of Ca2+-dependent enzymatic activity in models of retinal degeneration

Abstract

Calcium (Ca2+) levels in neurons are strictly regulated and Ca2+ dysregulation has previously been linked to neuronal cell death. In the mammalian retina, Ca2+ dysregulation may be connected to both cone and rod photoreceptor cell death (e.g. Arango-Gonzalez et al., 2014, Kulkarni et al., 2016). The interpretation of this research has, however, been hampered by spatial inhomogeneity of retinal degenerative events, depending on the model used. Here we examined the spatio-temporal activation patterns of the Ca2+-dependent cysteine protease calpain in comparison with cell death patterns across three different mutant mouse lines, and wildtype (wt) mice. We determined the spatial activation of calpain during disease progression in the primary cone degeneration model cpfl1 and in the primary rod degeneration models rd1 and rd10, relative to wt controls and showed an increase in calpain activity in the rd1 and rd10 models in a spatio-temporal pattern. We used the TUNEL assay to stain for dying cells and found the spatiotemporal cell death pattern corresponding to that of calpain activity. Similarly, we observed up-regulation of the calpain-2 isoform, whereas no up-regulation of calpain-1 was observed. In addition, we found an upregulation of apoptosis-inducing-factor (AIF) in the disease models compared to wt, while increases in caspase-3 activation were mainly found in cones late in the rd1 degeneration. Together these results indicate that calpain activity plays a role in the non-apoptotic photoreceptor cell death seen during retinal degeneration. 20 To further link the effects of Ca2+ dynamics to photoreceptor degeneration, we used a transgenic mouse line that expresses the HR2.1:TN-XL (TN-XL) Ca2+ biosensor in cones. We used two-photon microscopy to study calcium levels and light-evoked responses in cone axon terminals in acute retinal slices (Kulkarni et al., 2015) before and after the application of the Ca2+ channel blockers L-cis (blocker of outer segment CNG channels) and D-cis (blocker of synaptic voltage gated calcium channels) diltiazem. Our data suggest that both diltiazem enantiomers lower baseline Ca2+ levels in cone terminals, while light-induced responses were altered only by the L-cis enantiomer. To link Ca2+ dysregulation to calpain activation, we developed two-photon live imaging of calpain activity. With this approach, we disentangled part of the interaction between Ca2+-mediated processes, calpain regulation, and photoreceptor cell death. Our data suggests a causal link between Ca2+ dysregulation and cell death, high-lighting a therapeutic target for future compound development.