Abstract
SPG76 is a complicated form of hereditary spastic paraplegia (HSP) associated with mutations in the CAPN1 gene. The encoded protein, calpain 1, is a calcium-activated cysteine protease that catalyzes the proteolytic cleavage of a variety of cellular proteins and is involved in a wide range of biological processes. Calpain 1 and calpain 2 isoforms are highly expressed in various tissues and have opposite effects on survival: calpain 1 induces the activation of Akt and ERK pro-survival pathways, regulates autophagy and is neuroprotective, while calpain 2 induces neurodegeneration. We characterized fibroblast cells derived from two SPG76 patients carrying a homozygous mutation (p.Tyr320Leufs73*) in the CAPN1 gene that leads to the absence of the protein. Loss of calpain 1 in SPG76 patient's derived cells increased calpain 2 activation and induced autophagosome formation and accumulation, inhibited Akt and ERK1/2 pro-survival pathways, reducing GSK3β inhibition, and increased cell susceptibility to ER stress. In ER stress conditions, SPG76 cells presented unfolded protein response (UPR) activation, increased apoptosis and cell death. We analysed the potential of targeting calpain 2 and the Akt pro-survival pathway to rescue the SPG76 deranged pathways in patient's derived cells. We found that the calpain inhibitors olesoxime and MDL28170, naringenin and the GSK3β inhibitor tideglusib were the most effective in increasing Akt activation and GSK3β inhibition and in rescuing apoptosis and cell death in SPG76 cells. Among these, olesoxime and MDL28170 reduced calpain activity, rescued apoptosis and locomotor deficits in vivo in a CalpB KO Drosophila model that replicates the SPG76 phenotype.
