Generation of neurons from LAMP2-deficient iPSCs for modeling lysosomal dysfunction induced neuropathogenesis

Abstract

Lysosomes are organelles responsible for the degradation of obsolete cellular constituents. The LAMP2 protein, which associated with the lysosomal membrane, helps maintain lysosomal membrane integrity and mediate autophagy. Clinically, primary LAMP2 deficiency is associated with the Danon disease, in which affected individual may manifest severe cardiomyopathy and certain degree of mental retardation. Owing to the difficulties in obtaining and culturing human neurons, the pathological role of LAMP2 deficiency in the neural injury remains unclear.

The aim of this study was to use the cortical neurons derived from LAMP2-deficient iPSCs to evaluate the effects of lysosomal instability on neuronal dysfunction. In brief, iPSCs were generated from a patient with LAMP2 gene mutation. The resultant iPSCs have been differentiated into cortical neurons. Western blot and immunofluorescent analysis confirmed that the patient-specific iPSCs-derived neurons were lack of LAMP2 production. Interestingly, this LAMP2-deficient neurons showed the leakage of the lysosomal enzyme-cathepsin L. The results from MTT assay, TUNEL assay, activated caspase-3/7 detection and Western blot showed that in the presence of oxidative stress, the cell viability of LAMP2-deficient neurons were largely decreased with further leakage of lysosomal cathepsin L and increased active caspase-3/7 activity. Surprisingly, there was no obvious accumulation of LC3 in the LAMP2-deficient neurons, indicating the oxidative stress induced cell death was possibly not due to autophagy dysfunction. Furthermore, iPSC-derived neurons were treated with cathepsin L inhibitor. The MTT and TUNEL assay demonstrated the reduction in cell death, apoptotic rate and the level of activated caspase-3/7 of LAMP2-deficient neurons. These suggest that the leakage of lysosomal cathepsin L caused by LAMP2 deficiency is the upstream effector in apoptosis pathway in the LAMP2-deficient iPSC-derived cortical neurons under mild oxidative stress.