Defective autophagy in a fibroin secretion-deficient silkworm mutant

The silkworm <i>Bombyx mori</i> is an economically important insect for silk production. Its silk glands are responsible for the synthesis and secretion of silk proteins. The naked pupa (<i>Nd</i>), a fibroin heavy chain mutant strain of silkworm, was found to exhibit severe atrophy, degeneration of the posterior silk gland (PSG), and abnormal secretion of fibroin proteins, thereby producing little or no silk. Here, we found that the autophagic marker Atg8-PE was upregulated through the target of rapamycin complex 1 signaling pathway in <i>Nd</i>. However, as autophagy substrates, SQSTM1/p62 and ubiquitinated protein levels increased in <i>Nd</i>. Furthermore, treatment with BafA1 showed no effect on the protein levels of SQSTM1/p62, indicating impaired autophagic flux in <i>Nd</i>. Abnormal acidification of lysosomes was further detected, which resulted in a decreased proportion of matured CtsL1 (cathepsin L1). Thus, the substrate in autolysosomes cannot be degraded within a rapid time frame, resulting in the accumulation of protein aggregates, which cause atrophy and degeneration of the PSG. We also found that acidic nanoparticles rescued lysosomal acidification and relieved the degenerative changes of <i>Nd</i>-PSG. The findings of this study suggest that the <i>Nd</i> mutant silkworm can be used as an animal model for studying protein aggregation diseases.<b>Abbreviations</b>: AD: Alzheimer disease; aNP: acidic nanoparticle; APP: amyloid beta precursor protein; Atg8: autophagy related 8; BACE1: beta-secretase 1; BafA1: bafilomycin A₁; CtsL1: cathepsin L1; CRY: crystallin; ER: endoplasmic reticulum; FibH: fibroin heavy chain; FibL: fibroin light chain; FUS: FUS RNA binding protein; HD: Huntington disease; HRP: horseradish peroxidase; <i>Nd</i>: naked pupa; OSBPL2: oxysterol binding protein like 2; PD: Parkinson disease; PE: phosphatidylethanolamine; p-EIF4EBP: phosphorylated eukaryotic initiation factor 4E binding protein; PROM1: prominin 1; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B; PSEN: presenilin; PSG: posterior silk gland; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: standard error of the mean; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; TARDBP: TAR DNA binding protein; TORC1: target of rapamycin complex 1; UBQLN2: ubiquilin 2; V-ATPase: vacuolar-type ATPase.