Examination of VLC-PUFA-deficient photoreceptor terminals.

PURPOSE: Juvenile-onset autosomal dominant Stargardt-like macular dystrophy (STGD3) is caused by mutations in ELOVL4 (elongation of very long fatty acids-4), an elongase necessary for the biosynthesis of very long chain fatty acids (VLC-FAs ≥ C26). Photoreceptors are enriched with VLC polyunsaturated fatty acids (VLC-PUFAs), which are necessary for long-term survival of rod photoreceptors. The purpose of these studies was to determine the effect of deletion of VLC-PUFAs on rod synaptic function in retinas of mice conditionally depleted (KO) of Elovl4. METHODS: Retina function was assessed in wild-type (WT) and KO by electroretinography. Outer plexiform structure was evaluated by immunofluorescence and transmission electron microscopy. Single-cell recordings measured rod ion channel operation and rod bipolar glutamate signaling. Sucrose gradient centrifugation was used to isolate synaptosomes from bovine retina. Proteins and lipids were analyzed by Western blotting and tandem mass spectroscopy, respectively. RESULTS: Inner retinal responses (b-wave, oscillatory potentials, and scotopic threshold responses) of the ERG were decreased in the KO mice compared to controls. However the rod ion channel operation and bipolar glutamate responses were comparable between groups. Biochemical analysis revealed that conventional and ribbon synapses have VLC-PUFAs. Ultrastructural analysis showed that the outer plexiform layer was disorganized and the diameter of vesicles in rod terminals was smaller in the KO mice. CONCLUSIONS: Very long chain PUFAs affect rod function by contributing to synaptic vesicle size, which may alter the dynamics of synaptic transmission, ultimately resulting in a loss of neuronal connectivity and death of rod photoreceptors.

Effect of reduced retinal VLC-PUFA on rod and cone photoreceptors.

PURPOSE: Autosomal dominant Stargardt-like macular dystrophy (STGD3) is a juvenile-onset disease that is caused by mutations in Elovl4 (elongation of very long fatty acids-4). The Elovl4 catalyzes the first step in the conversion of C24 and longer fatty acids (FAs) to very long-chain FAs (VLC-FAs, ≥C26). Photoreceptors are particularly rich in VLC polyunsaturated FAs (VLC-PUFA). To explore the role of VLC-PUFAs in photoreceptors, we conditionally deleted Elovl4 in the mouse retina. METHODS: Proteins were analyzed by Western blotting and lipids by gas chromatography (GC)-mass spectrometry, GC-flame ionization detection, and tandem mass spectrometry. Retina function was assessed by electroretinography (ERG), and structure was evaluated by bright field, immunofluorescence, and transmission electron microscopy. RESULTS: Conditional deletion (KO) of retinal Elovl4 reduced RNA and protein levels by 91% and 96%, respectively. Total retina VLC-PUFAs were reduced by 88% compared to the wild type (WT) levels. Retinal VLC-PUFAs incorporated in phosphatidylcholine were less abundant at 12 months compared to 8-week-old levels. Amplitudes of the ERG a-wave were reduced by 22%, consistent with photoreceptor degeneration (11% loss of photoreceptors). Analysis of the rod a-wave responses gave no evidence of a role for VLC-PUFA in visual transduction. However, there were significant reductions in rod b-wave amplitudes (>30%) that could not be explained by loss of rod photoreceptors. There was no effect of VLC-PUFA reduction on cone ERG responses, and cone density was not different between the WT and KO mice at 12 months of age. CONCLUSIONS: The VLC-PUFAs are important for rod, but not cone, function and for rod photoreceptor longevity.