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PROTEOME OF BOVINE MITOCHONDRIA AND ROD OUTER SEGMENT DISKS: COMMONALITIES AND DIFFERENCES.

ABSTRACT: The retinal rod outer segment (OS) isa stack of diskssurrounded by the plasma membrane,housing proteins related to phototransduction as well asmitochondrial proteins involved in oxidative phosphorylation (OxPhos). This prompted us to compare the proteome of bovine OS disks and mitochondria, to assess the significant top gene signatures of each sample.The two proteomes, obtained byLTQ-OrbitrapVelos mass spectrometry, were compared by statistical analyses. 4139 proteins were identified, 2045 of which overlapping in the two sets. Non-hierarchical Spearman’s correlogram revealed that the groups were clearly discriminated. Partial Least Square Discriminant plus Support Vector Machine analysis identified the major discriminative proteins, implied in photo-transduction and lipid metabolism, respectively. Gene ontology analysis identified top gene signatures of disk proteome, enriched in vesiculation, glycolysis, and OxPhos proteins. The tricarboxylic acid (TCA) cycle and the electron transport proteinswere similarly enriched in the two samples: Notably thelatterwas up regulated in disks.Data suggest that the OxPhos proteins may derive from mitochondria becoming a true OS component, and acquiring different functionality once in ectopic location. Understanding theyet unanswered issue of chemical energy supply in the rod OS, can positively influence our vision of retinal physiology and pathology.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Retinal Rod, Liver

SUBMITTER: Chiara Lavarello

LAB HEAD: Andrea Petretto

PROVIDER: PXD008087 | Pride | 2022-03-01

REPOSITORIES: Pride

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Project description:Photoreceptor disorders are collectively known as retinal degeneration (RD), and include retinitis pigmentosa (RP), cone-rod dystrophy and age related macular degeneration (AMD). These disorders are largely genetic in origin; individual mutations in any one of >200 genes cause RD, making mutation specific therapies prohibitively expensive. A better treatment plan, particularly for late stage disease, may involve stem cell transplants into the photoreceptor or ganglion cell layers of the retina. Stem cells from young mouse retinas can be transplanted, and can form photoreceptors in adult retinas. These cells can be grown in tissue culture, but can no longer form photoreceptors. We have used microarrays to investigate differences in gene expression between cultured retinal progenitor cells (RPCs) that have lost photoreceptor potential, postnatal day 1 (pn1) retinas and the postnatal day 5 (pn5) retinas that contain transplantable photoreceptors. We have also compared FACS sorted Rho-eGFP expressing rod photoreceptors from pn5 retinas with Rho-eGFP negative cells from the same retinas. We have identified over 300 genes upregulated in rod photoreceptor development in multiple comparisons, 37 of which have been previously identified as causative of retinal disease when mutated. It is anticipated that this research should bring us closer to growing photoreceptors in culture and therefore better treatments for RD. This dataset is also a resource for those seeking to identify novel retinopathy genes in RD patients. We extracted whole retinas from postnatal day 1 (Pn1) and postnatal day 5 (Pn5) mice, and compared them with cultured RPCs derived from pn5 retinas, using Affymetrix mouse 430A_2 arrays. We also extracted cells from Rho-eGFP Pn5 retinas and FACS sorted them. GFP+ve cells represent immature rod photoreceptors, as they express the Rho-eGFP fusion protein, which is only expressed in rods. GFP-ve cells represent all other retinal neurons. These samples were amplified and compared using Affymetrix mouse 430A_2arrays, by Source Biosciences GMBH, Berlin, Germany. Results from immature rods were then compared with those from other retinal neurons, while results from whole Pn5 retinas were compared with Pn1 retinas (which don't yet express rod specific genes), and RPCs, which are glial precursors. RPCs were also compared with Pn1 retinas. Genes which showed changed expression profiles in at least 3/4 of comparisons were prioritised for further investigation.

Project description:<h4>Summary</h4>Primary cilia are key sensory organelles whose dysfunction leads to ciliopathy disorders such as Bardet-Biedl syndrome (BBS). Retinal degeneration is common in ciliopathies, since the outer segments (OSs) of photoreceptors are highly specialized primary cilia. BBS1, encoded by the most commonly mutated BBS-associated gene, is part of the BBSome protein complex. Using a new bbs1 zebrafish mutant, we show that retinal development and photoreceptor differentiation are unaffected by Bbs1-loss, supported by an initially unaffected transcriptome. Quantitative proteomics and lipidomics on isolated OSs show that Bbs1 is required for BBSome-entry into OSs and that Bbs1-loss leads to accumulation of membrane-associated proteins in OSs, with enrichment in proteins involved in lipid homeostasis. Disruption of the tightly regulated OS lipid composition with increased OS cholesterol content are paralleled by early functional visual deficits, which precede progressive OS morphological anomalies. Our findings identify a new role for Bbs1/BBSome in OS lipid homeostasis and suggest a new pathomechanism underlying retinal degeneration in BBS. Data availability:The proteomic data generated in this study have been deposited in PRIDE repository under accession code <a href='https://www.ebi.ac.uk/pride/archive/projects/PXD026646' rel='noopener noreferrer' target='_blank'>PXD026646</a> for the OS proteome and <a href='https://www.ebi.ac.uk/pride/archive/projects/PXD030522' rel='noopener noreferrer' target='_blank'>PXD030522</a> for the whole eye lysate proteome [<a href='https://www.ebi.ac.uk/pride' rel='noopener noreferrer' target='_blank'>https://www.ebi.ac.uk/pride</a>].The RNAseq data generated in this study have been deposited in the SRA repository under the BioProject accession number PRJNA789116 [<a href='https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA789116' rel='noopener noreferrer' target='_blank'>https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA789116</a>].

			Action	DRS
	DiskFr1_01.raw	Raw
	DiskFr1_02.raw	Raw
	DiskFr1_03.raw	Raw
	DiskFr2_01.raw	Raw
	DiskFr2_02.raw	Raw

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PROTEOME OF BOVINE MITOCHONDRIA AND ROD OUTER SEGMENT DISKS: COMMONALITIES AND DIFFERENCES.

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