Calcium acts as a double-edged sword, playing critical roles in both neuronal degeneration (Goodman et al., 2002; Bianchi et al., 2004; O'Hagan et al., 2005; Xu et al., 2001; Calixto et al., 2012) and regeneration (Hammarlund et al., 2009; Tang and Chisholm, 2016; Caneo et al., 2019). We previously showed in C. elegans that diapause prevents degeneration and even promotes regeneration of Touch Receptor Neurons (TRNs) expressing mutated
mec-4d channels (Caneo et al., 2019). In this work, we aim to understand how intracellular calcium is managed during neuroprotection. To monitor calcium, we performed 2-photon imaging of TRNs expressing GCaMP6m and tagRFP in wild type and
mec-4d worms at L2 stage as well as dauers. Consistent with a contribution of calcium in
mec-4d -induced neurodegeneration, we found an increase in intracellular calcium in L2
mec-4d worms compared to WT controls. To our surprise, basal calcium was also increased in diapausing
mec-4d compared to L2
mec-4d animals. This suggests that non-physiological calcium concentrations are maintained in the soma while TRNs regenerate. Enhanced calcium buffering by mitochondria may underlie the regenerative capacity in high calcium. We next investigated mitochondrial alterations in
mec-4d worms using a GFP fused with an MLS in the TRN (Fatouros et al., 2012). In
mec-4d L2 animals, there is a 23% decrease in mitochondrial length along with a 22% reduction in number compared to WT L2; in contrast, WT dauers exhibit 15% longer but 16% fewer mitochondria compared to WT L2 animals. Notably,
mec-4d dauers show an increase of 31% and 36% in mitochondrial length and number, respectively, compared to
mec-4d L2 animals. Taken together, these findings suggest that
mec-4d mutation disrupts mitochondrial function, and entering diapause compensates for mitochondrial dysfunction. We next wondered whether calcium might be acting in axonal protection. Using RNAi, we silenced the calcium transporters
sca-1, from the endoplasmic reticulum, and
mcu-1, from the mitochondria, in the TRNs and assessed axon morphology at 72 hours after hatching. Reducing the expression of
sca-1 and
mcu-1 resulted in a reduction of WT-like axons (p-value=0.0007), suggesting that calcium transport from the cytoplasm into these organelles is key for neuronal protection. Thus, our results provide new insights into the calcium-dependent mechanisms that tilt the balance between neuronal death and repair.