prevalence was reported as 1:3000-4000 worldwide, making it the main reason for blindness in the working population in industrial countries. The mutations of over 70 genes have been related to this genetic disorder, and there is generally no effective treatment, except for gene therapy for the RPE65 mutations. Hence, new molecular targets are required for novel treatment development. The signaling molecule cGMP and its dependent protein kinase G (cGMP-PKG) have been regarded as one of the
prime effectors to drive the disease. However, the insights into the downstream signaling of the system, are still unclear. This thesis aimed to explore the cGMP-PKG-dependent transcriptome and proteome.
The Paper I showed the cGMP-PKG-dependent transcriptome in this study. Applying RNA sequencing to study the retinal explants from the diseased rd1 models and WT with cGMP-PKG manipulation, I identified the cGMP-PKG-dependent genes and proposed that this system may negatively regulate oxidative phosphorylation and mitochondrial pathways, which may affect retinal degeneration.
The paper II investigated the cGMP-PKG phosphoproteome. The phosphorylated peptide enrichment and mass-spectrometry were applied to explore the cGMP-PKG-dependent phosphoproteome within rd1 retinal explants with PKG inhibition or not. I identified a list of cGMP-PKG-dominated phosphorylations and picked up RAF1 proto-oncogene, serine/threonine kinase (RAF1) for further validation. This suggested that RAF1 may be involved in retinal degeneration, although in an as yet unclear mechanism.
The Paper III investigated cGMP-PKG-dependent kinase activity profiling and the phosphoproteome with a microarray-based technique and mass-spectrometry, respectively. The rd10 model, with a different mutation in the gene for PDE6 was used. This yielded the lists of cGMP-PKG-dependent kinase and phosphorylations, which were partially compatible with Paper II. Also, this showed that Ca2+/calmodulindependent protein kinase II and IV (CaMK2, CaMK4) may play a role during retinal degeneration.
Paper IV focused on cyclin-dependent kinase 1 (CDK1), which was identified from Paper II, namely, and investigated if it has effects on retinal degeneration. The data showed that CDK1 participates in the late stage of retinal degeneration, and also provided a link between this enzyme and the cGMP-PKG system.
The Paper V validated another target, pyruvate kinase isozyme M2 (PKM2) identified in the previous transcriptome study. The PKM2 within retinas was activated from two disease models, namely rd2 and rd10 in a pharmacological manner during explant culture. I observed that PKM2 activation in rd10 alleviated the photoreceptor degeneration while no difference was noticed in rd2 under treatment.
All in all, this thesis provides novel insights about cGMP-PKG-dependent targets, which may have a role during photoreceptor degeneration and cast light on the therapeutic development of this retinal disease.
- Department of Clinical Sciences, Lund
- Ekström, Per, Supervisor
- Perez, Maria Thereza, Assistant supervisor
|Award date||2022 Dec 7|
|Place of Publication||Lund|
|Publication status||Published - 2022|
Place: Ögonkliniken A, Kioskgatan 1, Skånes Universitetssjukhus i Lund. Join by Zoom: https://lu-se.zoom.us/j/63641299114
Name: Grimm, Christian
Affiliation: Lab for Retinal Cell Biology Department of Ophthalmology, University of Zurich
- Cell and Molecular Biology
- retinal degeneration
- cell death
- organotypic explant culture
- RNA sequencing
- mass spectrometry