Purpose:
Phr1 is a novel regulator of topographic mapping of retinal axons to their central targets in the brain. When Phr1 is conditionally knocked out in mouse retinal ganglion cells (RGC), axons are mistargeted in the lateral geniculate nucleus and the superior colliculus. Loss of Phr1, a presumed E3 ubiquitin ligase, may result in increased levels of proteins which it normally degrades. In order to 1) determine if Phr1 is localized to axons and 2) detect potential Phr1 effectors, proteomics analysis by mass spectrometry was performed on optic nerves from control mice and Phr1-mutant mice.
Methods:
Optic nerves from mice were obtained by dissection, and proteins extracted and digested. Mass spectrometry was performed and statistically significant differences in peptide and protein content between mutant and control samples were identified. Immunoblotting of optic nerve samples was used to confirm differences in protein expression.
Results:
Gel electrophoresis identified a specific band which was targeted for proteomic analysis, which demonstrated the presence of Phr1 in control optic nerve (Scaffold Protein Identification Probability = 100%). Analysis of complex mixtures from mutant and control optic nerves found levels of a single protein, heterogeneous nuclear ribonucleoprotein M (hnRNP-M), to be statistically different between samples, with a 2.2-fold enrichment in the mutant (p=4.16E-5). This differential expression was confirmed by immunoblotting.
Conclusions:
Phr1 is a low abundance protein with presumed ubiquitin ligase function, unclear localization, and potential interactions with hundreds of proteins. The use of proteomics to study Phr1 is a novel application of emerging technology where conventional protein methods are inadequate. Enrichment of hnRNP-M in Phr1-mutant optic nerve suggests that Phr1 may regulate RGC axonal targeting by altering levels of hnRNP-M, a protein involved in pre-mRNA splicing. A greater understanding of Phr1 and hnRNP-M may benefit our understanding of visual development and axonal targeting in the nervous system.
Keywords: proteomics • ganglion cells • thalamus/lateral geniculate nucleus