Lysosomal storage, mitochondrial pathology, and autophagy in knockout of tripeptidyl peptidase 1 in human neuroblastoma cells in vitro.

Deficiency of tripeptidyl-peptidase 1 (TPP1; EC 3.4.14.9), a lysosomal enzyme encoded by the CLN2 gene, is associated with the lysosomal storage disorder - classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease). The classic form of CLN2 disease leads to the accumulation of autofluorescent lysosomal storage and a massive loss of neurons with gliosis in the brain. The major component of the storage is subunit c of mitochondrial ATP synthase, a highly hydrophobic, 75-aminoacid polypeptide. We developed an in vitro model of CLN2 disease by knocking out CLN2 in the human neuroblastoma cells SH-SY5Y by using CRISPR/cas9 technology. We focused on defining the pattern of deposition of subunit c, factors contributing to subunit c accumulation, and subcellular morphometry to identify differences between the model cells knockout (KO) and controls. Implementation of acetone for cell fixation allowed us to: i. identify higher levels of subunit c in the mitochondria of KO cells than controls; ii. characterize in detail subunit c inclusions, also present in controls; iii. identify other mitochondrial proteins colocalizing with subunit c in inclusions; and iv. detect mitochondrial pathology in degenerating cells often accompanied by deposition of subunit c. Differentiation of cells with retinoic acid and brain-derived neurotrophic factor led to a substantial increase in the levels of subunit c and to significant differences in the levels of autophagy-related proteins between KO and control cells. Inhibition of induced autophagy by bafilomycin A1 (Baf.A1) decreased subunit c levels in controls but not in KO cells, whereas the levels of subunit c were unaffected by Baf.A1 treatment upon basal autophagy. Finally, subcellular morphometry showed differences in the number and size of vesicular structures immunostained for autophagy-related proteins between KO cells and controls upon both induced and basal autophagy, further supporting the association of TPP1 deficiency with autophagy.