Abstract
Purpose :
CNTF has been shown to enhance photoreceptor survival in various retinal degeneration models. We have shown previously that in a mouse model of retinitis pigmentosa with the rds/peripherin 2 (P216L) mutation, constitutive expression of secreted CNTF from the RPE increases photoreceptor viability without improvement of ERG. Here, we examine synaptic connections in the outer plexiform layer of the rds (P216L) mutant retina and evaluate impacts of CNTF treatment on photoreceptor to interneuron connections.
Methods :
A lentiviral vector (LV-hCNTF) expressing the same recombinant CNTF used in clinical trials was produced and delivered subretinally to rds mice at postnatal day 25 (P25). Infected eyes were harvested at P31 or P56 and examined for photoreceptor pre- and post-synaptic markers along with retinal cell type markers using confocal microscopy. RNA sequencing analyses were performed for whole retinal samples after 24-hour CNTF exposure at P26 and 10-day CNTF treatments at P35.
Results :
Compared to the wild type, rds mutant shows significantly diminished ribbon synaptic markers Ribeye and Bassoon in photoreceptor cells. Presynaptic terminal marker PSD95 and postsynaptic marker mGluR6 were also decreased in the mutant. Furthermore, mGluR6 was mislocalized in bipolar cell soma. LV-hCNTF infection resulted in restored density of Ribeye and Bassoon, resembling that of the wild-type. CNTF treatment for 6 days increased the levels of PSD95 and mGluR6 proteins, but did not completely alleviate mGluR6 mislocalization in bipolar neurons. However, 30-day CNTF treatment appears to restore mislocalized mGluR6 to the synaptic zone. RNAseq analysis reveals altered expression of numerous synaptic related genes in the mutant by P26, and a 24-hour CNTF treatment restored the transcript levels of Ribeye, Bassoon, and mGluR6 towards the WT levels. Interestingly, after 10-day CNTF treatment, expression of several genes critical in photoreceptor to bipolar functional connectivity remain abnormal.
Conclusions :
Photoreceptor degeneration elicits drastic changes in synaptic connections, including loss of presynaptic ribbons and disassembling of postsynaptic complexes. Treatment with CNTF can improve synaptic structures at the morphological level and partially correct abnormal gene expression relevant to OPL synapses. However, full impacts of CNTF on functional photoreceptor to bipolar connectivity remain to be evaluated.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.