Abstract
Purpose:
Rgcs1 is a QTL that influences ganglion cell death after optic nerve crush. Spink2, a gene of interest in this locus, is upregulated in mouse retina after crush. SPINK 2 is a serine protease inhibitor Kazal type 2. Previous studies suggest that these inhibitors function as suppressors of autophagic flux (AF). High levels of SPINK 2 may therefore alter the AF in RGCs, a process that has been shown to increase their susceptibility to optic nerve damage.
Methods:
Resistant (DBA/2J) and susceptible (Balb/c) mice were used to perform optic nerve crush. All crush studies compare the crushed retina to the control (non-crushed) retina. Changes in transcript abundance of autophagy markers Beclin 1, Atg5, Atg7 and Pik3c3 were examined 7 days post-crush using qPCR. Activated caspase 3/7 was measured using a cell death assay with D407 cells in the presence or absence of SPINK 2 and/or staurosporine. Spinning disc microscopy was used to image GFP-LC3 labeled autophagosomes in rapamycin-treated D407 RPE cells in the presence or absence of SPINK 2. Fusion of the autophagosome and lysosome was analyzed using tfLC3 (GFP-RFP-LC3) in the presence or absence of Rapamycin and/or SPINK 2. Quantification and kinetics of live imaging LC3 experiments were analyzed using Imaris software.
Results:
Transcript levels of autophagy markers were upregulated post-crush, and were significantly higher in resistant DBA/2J mice. D407 cells expressing Spink2 exhibited an increase in caspase activity in response to staurosporine, relative to control GFP transfected cells. Rapamycin caused a significant increase in LC3 containing vesicles within 30 minutes in cells with and without SPINK 2. D407 cells expressing Spink2 accumulated significantly more vesicles. These remained small even 4 hours after rapamycin treatment, suggesting impaired fusion between autophagosomes and lysosomes. Further experiments directly examining this fusion event are underway and results will be reported.
Conclusions:
SPINK 2 increases cell susceptibility to apoptotic stimuli, possibly by impairing the AF response. This provides a rationale for the difference in ganglion cell susceptibility associated with the Rgcs1 locus.
Keywords: 426 apoptosis/cell death •
531 ganglion cells •
539 genetics