Autolysosome biogenesis and developmental senescence are regulated by both Spns1 and v-ATPase

Abstract

Spns1 (Spinster homolog 1 [Drosophila]) in vertebrates, as well as Spin (Spinster) in Drosophila, is a hypothetical lysosomal H C -carbohydrate transporter, which functions at a late stage of macroautophagy (hereafter autophagy). The Spin/Spns1 defect induces aberrant autolysosome formation that leads to developmental senescence in the embryonic stage and premature aging symptoms in adulthood. However, the molecular mechanism by which loss of Spin/Spns1 leads to the specific pathogenesis remains to be elucidated. Using chemical, genetic and CRISPR/Cas9-mediated genome-editing approaches in zebrafish, we investigated and determined a mechanism that suppresses embryonic senescence as well as autolysosomal impairment mediated by Spns1 deficiency. Unexpectedly, we found that a concurrent disruption of the vacuolar-type H C -ATPase (v-ATPase) subunit gene, atp6v0ca (ATPase, H C transporting, lysosomal, V0 subunit ca) led to suppression of the senescence induced by the Spns1 defect, whereas the sole loss of Atp6v0ca led to senescent embryos similar to the single spns1 mutation. Moreover, we discovered that the combined stable defect seen in the presence of both the spns1 and atp6v0ca mutant genes still subsequently induced premature autophagosome-lysosome fusion marked by insufficient acidity, while extending developmental life span, compared with the solely mutated spns1 defect. Our data suggest that Spns1 and the v-ATPase orchestrate proper autolysosomal biogenesis with optimal acidification that is critically linked to developmental senescence and survival.

L-leucine and SPNS1 coordinately ameliorate dysfunction of autophagy in mouse and human Niemann-Pick type C disease

“…Studies on zebrafish mutants with the cardia bifida (two hearts) phenotype led to identification of the zebrafish homolog of Spns2 , which was shown to encode a sphigosine-1-phosphate (S1P) transporter with a role in S1P secretion 30 , 31 . Another study in zebrafish revealed that developmental senescence due to Spns1 deficiency can be suppressed by a concurrent disruption of the vacuolar-type H + -ATPase (v-ATPase) subunit gene, atp6v0ca 32 , implying that loss of Spns1 resulted in an elevated v-ATPase activity, which may have partly contributed to the Spns1 -loss phenotype. However, we also obtained a result in conflict with this report: SPNS1 overexpression increased, rather than decreased, the amount of ATP6V0A4, though the effect was statistically non-significant (Figs 2N , S3 and S15 ).…”

Studying Autophagy in Zebrafish

“…So far only one study has used this system to create mutant lines. CRISPR/Cas9-based mutagenesis in spns1 and atp6v0ca genes induced premature autophagosome-lysosome fusion marked by insufficient acidity leading to developmental senescence and death [ 45 ]. spns1 is thought to function as a lysosomal H + -carbohydrate symporter, which functions at a late and terminal stage of autophagy [ 46 , 47 ] .…”

“…Moreover, knockdown of optineurin, an ubiquitin-binding autophagy-receptor protein, was shown to cause motor axonopathy due to defective autophagic clearance of accumulated SOD1-G93A aggregates [ 64 ], defective vesicle trafficking in the axons [ 65 ], and increased susceptibility to Salmonella enterica infection [ 66 ]. Morpholino-mediated depletion of Spns1, a lysosomal transporter, was found to upregulate embryonic cellular senescence [ 46 ] and this was counteracted by the depletion of the lysosomal v-ATPase, which together suppresses developmental senescence and increases life-span [ 45 ]. Transient depletion of p62/sqstm1, another ubiquitin-binding autophagy receptor protein, in zebrafish embryos was shown to increase susceptibility to Shigella flexneri and Mycobacterium marinum in the host, indicating the role of autophagy against bacterial infection [ 67 , 68 ].…”

CRISPR system for genome engineering: the application for autophagy study

“…Currently, most works for genome editing of the Atgs were focued on the gene knockout as well as knockin ( 67 ). The effects of several Atg genes knockout have been well studied from the formation of autophagosomes to autolysosomal biogenesis ( 68 , 69 ). Using CRISPR/Cas9 to delete of the canonical autophagy-essential genes ATG5 , ATG7 , ATG16L and ULK1 have also been reported in in vitro or in vivo models ( 70 – 74 ).…”