Abstract
Purpose :
In zebrafish, a BH3-only SNARE protein, BNip1, induces photoreceptor (PhR) apoptosis in response to vesicular fusion defects (Nishiwaki et al. (2013) Dev Cell25, 374-387). The aim of our research is to elucidate physiological roles of BNip1 in PhR apoptosis.
Methods :
First, we examined which developmental stage β-SNAP is required to prevent BNip1-dependent PhR apoptosis. In zebrafish b-SNAP mutants, PhRs undergo apoptosis in the period of 60–84 hours post-fertilization (hpf). We induced β-SNAP expression in the mutants under the control of heat-shock promoter in different time windows, and examined which time window β-SNAP rescues PhR apoptosis. Second, we evaluated levels of vesicular transport in PhRs by calculating a rate of outer segment (OS) growth during development, and examined its correlation with PhR apoptosis in the mutants. Third, to decrease protein transport to the OS, we inhibited vesicular transport regulators through the connecting cilium such as IFT88, and also applied a mTOR inhibitor, Rapamycin, and then examined PhR survival in β-SNAP mutants.
Results :
Overexpression of β-SNAP after 48 hpf rescued PhR apoptosis in β-SNAP mutants, whereas overexpression after 72 hpf did not recover PhR survival, suggesting that β-SNAP activity from 48 to 72 hpf is enough to prevent BNip1-dependent PhR apoptosis. Next, we introduced β-SNAP expression in the mutants only from 1 to 5 days post-fertilization (dpf). In this case, PhRs continue to survive at least until 21 dpf, suggesting that β-SNAP activity in 2-5 dpf is enough to prevent BNip1-dependent PhR apoptosis. Consistently, the OS rapidly grew in 2-5 dpf indicating that protein transport levels correlates with BNip1-dependent PhR apoptosis. Indeed, the decline of protein transport or synthesis rescued PhR apoptosis in the mutants.
Conclusions :
Our findings indicate that BNip1-dependent PhR apoptosis in zebrafish β-SNAP mutants links to intracellular vesicular transport. Thus, BNip1 functions as a safe guard that inhibits excessive activation of vesicular transport in PhRs. BNip1 may cooperate with ER stress response to determine an appropriate range of vesicular transport levels.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.