Cell Type-Selective Loss of Peroxisomal β-Oxidation Impairs Bipolar Cell but Not Photoreceptor Survival in the Retina

Abstract

Retinal degeneration is a common feature in peroxisomal disorders leading to blindness. Peroxisomes are present in the different cell types of the retina; however, their precise contribution to retinal integrity is still unclear. We previously showed that mice lacking the central peroxisomal β-oxidation enzyme, multifunctional protein 2 (MFP2), develop an early onset retinal decay including photoreceptor cell death. To decipher the function of peroxisomal β-oxidation in photoreceptors, we generated cell type selective Mfp2 knockout mice, using the Crx promotor targeting photoreceptors and bipolar cells. Surprisingly, Crx-Mfp2−/− mice maintained photoreceptor length and number until the age of 1 year. A negative electroretinogram was indicative of preserved photoreceptor phototransduction, but impaired downstream bipolar cell signaling from the age of 6 months. The photoreceptor ribbon synapse was affected, containing free-floating ribbons and vesicles with altered size and density. The bipolar cell interneurons sprouted into the ONL and died. Whereas docosahexaenoic acid levels were normal in the neural retina, levels of lipids containing very long chain polyunsaturated fatty acids were highly increased. Crx-Pex5−/− mice, in which all peroxisomal functions are inactivated in photoreceptors and bipolar cells, developed the same phenotype as Crx-Mfp2−/− mice. In conclusion, the early photoreceptor death in global Mfp2−/− mice is not driven cell autonomously. However, peroxisomal β-oxidation is essential for the integrity of photoreceptor ribbon synapses and of bipolar cells.

Cone Photoreceptor Loss in Light-Damaged Albino Rats

“…Despite the continued ONL thinning, there was significant recovery of the a- and b-waves, indicating that there may be some plasticity in the signaling of the retina. This type of plasticity or recovery has been noted before in rodent models of degeneration [ 14 , 27 ], and bipolar cell sprouting is a potential contributing factor [ 28 , 29 ]. An electron microscopic examination of the outer plexiform layer could confirm synaptic remodeling.…”

ERG and Behavioral CFF in Light-Damaged Albino Rats

“…At the end of our study, ONL thickness showed a persistent 22% loss, but the b-wave had recovered. Other investigators have noted b-wave recovery after photoreceptor loss [17,23,24] and retinal remodeling is known to occur following photoreceptor loss [25][26][27], which may explain the recovery of b-wave amplitudes. Considering the similar loss of the b-wave amplitude in the RCS (PN23) and light-damaged albino rats (R6), we expected to see greater reductions in ERG CFF in the light-damaged rats.…”

Unexpected failure of rod bipolar cell targeting using L7Cre-2 mice

“…However, the exact mechanistic association between MFP2 deficiency and retinopathy remained unclear (Das et al, 2021b). To decipher which cell-type was responsible for this phenotype, mice lacking MFP2 specifically in both photoreceptors and bipolar cells (Crx-Mfp2 − /− ) were studied (Swinkels et al, 2022). These mice presented with impaired photoreceptor synaptic integrity and reduced rod bipolar cell (RBC) survival and functioning.…”

“…Both mouse strains were of C57BL6 background. Genotyping was performed as previously described (Swinkels et al, 2022). To investigate the Cre-recombination pattern in L7-Mfp2 − /− mice, they were crossed with mT/mG reporter mice (Muzumdar et al, 2007), kindly supplied by the laboratory of Angiogenesis and Vascular Metabolism (KU Leuven-VIB).…”

“…In situ hybridization was executed on paraffin eye sections (7 μm) using the RNAScope® 2.5 HD Chromogenic Detection Kit (Advanced Cell Diagnostics), as previously described (Das et al, 2021b;Swinkels et al, 2022). Afterwards, sections were overnight incubated with primary antibodies to protein kinase Cα (PKCα) (1/100, Santa Cruz SC-17769), followed by probing with Alexa Fluor 488 goat anti-mouse IgG (1/200, Invitrogen) and counterstaining with Hoechst 33342 (1/500, Sigma).…”