Uncoupling of Photoreceptor Peripherin/rds Fusogenic Activity from Biosynthesis, Subunit Assembly, and Targeting A POTENTIAL MECHANISM FOR PATHOGENIC EFFECTS*

Inherited defects in the RDS gene cause a multiplicity of progressive retinal diseases in humans. The gene product, peripherin/rds (P/rds), is a member of the tetraspanin protein family required for normal vertebrate photoreceptor outer segment (OS) architecture. Although its molecular function remains uncertain, P/rds has been suggested to catalyze membrane fusion events required for the OS renewal process. This study investigates the importance of two charged residues within a predicted C-terminal helical region for protein biosynthesis, localization, and interaction with model membranes. Targeted mutagenesis was utilized to neutralize charges at Glu321 and Lys324 individually and in combination to generate three mutant variants. Studies were conducted on variants expressed as 1) full-length P/rds in COS-1 cells, 2) glutathione S-transferase fusion proteins in Escherichia coli, and 3) membrane-associated green fluorescent protein fusion proteins in transgenic Xenopus laevis. None of the mutations affected biosynthesis of full-length P/rds in COS-1 cells as assessed by Western blotting, sedimentation velocity, and immunofluorescence microscopy. Although all mutations reside within a recently identified localization signal, none altered the ability of this region to direct OS targeting in transgenic X. laevis retinas. In contrast, individual or simultaneous neutralization of the charged amino acids Glu321 and Lys324 abolished the ability of the C-terminal domain to promote model membrane fusion as assayed by lipid mixing. These results demonstrate that, although overlapping, C-terminal determinants responsible for OS targeting and fusogenicity are separable and that fusogenic activity has been uncoupled from other protein properties. The observation that subunit assembly and OS targeting can both proceed normally in the absence of fusogenic activity suggests that properly assembled and targeted yet functionally altered proteins could potentially generate pathogenic effects within the vertebrate photoreceptor

Uncoupling of photoreceptor peripherin/rds fusogenic activity f0rom Biosynthesis, Subunit Assembly, and Targeting. A Potential Mechanism for Pathogenic Effects

Inherited defects in the RDS gene cause a multiplicity of progressive retinal diseases in humans. The gene product, peripherin/rds (P/rds), is a member of the tetraspanin protein family required for normal vertebrate photoreceptor outer segment (OS) architecture. Although its molecular function remains uncertain, P/rds has been suggested to catalyze membrane fusion events required for the OS renewal process. This study investigates the importance of two charged residues within a predicted C-terminal helical region for protein biosynthesis, localization, and interaction with model membranes. Targeted mutagenesis was utilized to neutralize charges at Glu321 and Lys 324 individually and in combination to generate three mutant variants. Studies were conducted on variants expressed as 1) full-length P/rds in COS-1 cells, 2) glutathione S-transferase fusion proteins in Escherichia coli, and 3) membrane-associated green fluorescent protein fusion proteins in transgenic Xenopus laevis. None of the mutations affected biosynthesis of full-length P/rds in COS-1 cells as assessed by Western blotting, sedimentation velocity, and immunofluorescence microscopy. Although all mutations reside within a recently identified localization signal, none altered the ability of this region to direct OS targeting in transgenic X. laevis retinas. In contrast, individual or simultaneous neutralization of the charged amino acids Glu 321 and Lys324 abolished the ability of the C-terminal domain to promote model membrane fusion as assayed by lipid mixing. These results demonstrate that, although overlapping, C-terminal determinants responsible for OS targeting and fusogenicity are separable and that fusogenic activity has been uncoupled from other protein properties. The observation that subunit assembly and OS targeting can both proceed normally in the absence of fusogenic activity suggests that properly assembled and targeted yet functionally altered proteins could potentially generate pathogenic effects within the vertebrate photoreceptor

Protective Gene Expression Changes Elicited by an Inherited Defect in Photoreceptor Structure

Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE), and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds) mouse model. Global gene expression profiling was performed on flow-sorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold) differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P) 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model

Strategy and sample generation for gene expression profiling of structurally abnormal rod photoreceptors.

<p>(A) Photoreceptors in the <i>rds</i> mouse fail to elaborate OSs. OS disk membrane biosynthesis begins at ∼P10, OSs are well established by P14, and reach full length by ∼P21. (B) <i>Left panels</i>: immunofluorescence analyses of retinal cryosections; DAPI-stained nuclei appear blue. eGFP labeled photoreceptors (green) were observed in WT, Nrl-eGFP and <i>rds</i>:Nrl-eGFP retinas; however, OSs labeled with anti-P/rds antibody PabMPCT (red), were only detected in WT and Nrl-eGFP retinas. <i>Right panels</i>: fluorescence-activated cell sorting was utilized to enrich rod photoreceptors from trypsin-dissociated mouse retinas. Typical histograms, generated using a BD Biosciences FACSVantage SE cell sorter, illustrate GFP-containing rod photoreceptors detected in dissociated (P9) retinas from Nrl-eGFP and <i>rds</i>:Nrl-eGFP, but not WT mice. Collected rod cell fractions, typically ∼500,000 cells per retina, are indicated (brackets).</p

Activated microglia are present in mature <i>rds</i> retina.

<p>(A) Immunofluorescent micrographs of age-matched <i>rds</i> and WT murine ocular cryosections are shown; DAPI-stained nuclei appear blue. Labeling of microglia by anti-Iba1 and anti-CD11b antibodies (red, <i>left and middle panels</i>) shows distributions of resting filamentous (arrowheads) and activated ameboid (arrows) microglia. GFAP labeling (red, <i>right panels</i>) shows distributions of activated Müller glial cells and reflects retinal stress. (B) Quantitative analysis of activated microglial migration in <i>rds</i> (filled bars) and WT (unfilled bars) retinas. Iba1 labeled cells were tabulated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031371#s4" target="_blank">Methods</a> (averages are reported s.e.m.; <i>n = 3</i>). The results show migration of activated microglia into nuclear layers of the P21 <i>rds</i> retina.</p

Gene expression analysis of GeneChip hybridizations using IPA: List of cellular functional pathways which show major changes in genetic regulation, as a result of the <i>rds</i> defect.

<p>This list was generated with input of all genes which are differentially regulated at one or more time points.</p

Transient upregulation may reflect homeostatic mechanisms.

<p>Differential regulation of numerous genes was observed at P14; however, expression returned to baseline levels by P21. This temporal pattern included genes mainly representing cellular organization and lipid metabolism functional pathways.</p

Quantitative PCR analyses for validation of gene expression changes in the <i>rds</i> model.

<p>For each gene, the maximal fold change value (<i>rds</i>:Nrl-eGFP vs. Nrl-eGFP) between P6 and P21 was plotted. Error bars indicate the standard error of the mean (s.e.m.; <i>n = 3</i>). Genes were classified into functional pathways, using Ingenuity Pathways Analysis software. Expression of most genes changed less than two-fold, and dysregulation in a variety of functional pathways was seen.</p

Neurotrophic factors are upregulated in the <i>rds</i> retina.

<p>Maximal Fold change values from qPCR study show strong upregulation of <i>CNTF</i> and <i>GDNF</i>, which are known to have a neuroprotective role for photoreceptors. Error bars indicate standard error of the mean (<i>n = 3</i>).</p