Abstract
Purpose :
Stargardt disease (STGD1) is an autosomal recessive macular dystrophy caused by mutations in ABCA4, an ATP-binding cassette (ABC) protein that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from the lumen to cytoplasmic leaflet of photoreceptor outer segment disc membranes. The N965S missense mutation in nucleotide binding domain 1 (NBD1) of ABCA4 is the most common STGD1 variant found in the Danish population and is frequently found in STGD1 patients of Chinese decent. The purpose of this study is to determine the molecular basis for STGD1 associated with the N965S mutation by investigating the effect of this mutation on the expression, cellular localization and functional activity of the ABCA4, and photoreceptor cell survival in a mouse harboring this disease mutation.
Methods :
An ABCA4-N965S knockin mouse was generated by homologous recombination. The localization of ABCA4 was determined by confocal scanning microscopy. The level of expression was determined by western blotting after solubilization in CHAPS detergent. The functional activity of affinity purified ABCA4 was determined by a luminescence ATPase assay. A2E levels were measured by HPLC.
Results :
In mice homozygous for the N965S mutation, ABCA4 was found both in photoreceptor outer segments (OS) and the inner segments (IS), with the latter co-localizing with ER markers. Heterozygous mice showed a similar distribution but with a higher level of ABCA4 in the OS. N965S ABCA4 expressed at 50% of WT ABCA4 levels and exhibited reduced basal ATPase activity and no stimulation of its ATPase activity by N-Ret-PE. A2E levels obtained from RPE cells increased with age with the N965S homozygous mice showing a significantly larger increase in A2E levels compared to heterozygous and WT mice. Photoreceptors stress and degeneration was not evident in young mice. The effect of the N965S mutations on older mice is currently under investigation and will be presented.
Conclusions :
Loss in function and partial protein misfolding are mechanisms underlying STGD1 disease associated with the N965S mutation in ABCA4. The N965S ABCA4 mutant mouse is a valuable model to develop and test drug and gene based treatments for STGD1.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.