Dataset Information

Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

ABSTRACT: To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC.Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.

SUBMITTER: Smit-McBride Z

PROVIDER: S-EPMC4323684 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

Smit-McBride Zeljka Z Oltjen Sharon L SL Radu Roxana A RA Estep Jason J Nguyen Anthony T AT Gong Qizhi Q Hjelmeland Leonard M LM

Molecular vision 20150205

<h4>Purpose</h4>To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.<h4>Methods</h4>Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and weste ...[more]

PMID: 25684976

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Project description:Purpose:The Grm6nob8 mouse carries a missense mutation in the Grm6 gene (p.Met66Leu), and exhibits a reduced b-wave of the electroretinogram (ERG), abnormal localization of metabotropic glutamate receptor 6 (mGluR6) to the depolarizing bipolar cell (DBC) soma, and a reduced level of mGluR6 at the DBC dendritic tips. Although the underlying mechanism remains unknown, one possible explanation is that DBCs cannot efficiently traffic the mutant mGluR6. In that scenario, reducing the total amount of mutant mGluR6 protein might normalize localization, and thus, improve the ERG phenotype as well. The second purpose of this study was to determine whether the abnormal cellular distribution of mutant mGluR6 in Grm6nob8 retinas might induce late onset DBC degeneration. Methods:We crossed Grm6nob8 animals with Grm6nob3 mice, which carry a null mutation in Grm6, to generate Grm6nob3/nob8 compound heterozygotes. We used western blotting to measure the total mGluR6 content, and immunohistochemistry to document mGluR6 localization within DBCs. In addition, we examined outer retinal function with ERG and retinal architecture in vivo with spectral domain optical coherence tomography (SD-OCT). Results:The retinal content of mGluR6 was reduced in the retinas of the Grm6nob3/nob8 compound heterozygotes compared to the Grm6nob8 homozygotes. The cellular distribution of mGluR6 in the Grm6nob3/nob8 compound heterozygotes matched that of the Grm6nob8 homozygotes, with extensive expression throughout the DBC cell body and limited expression at the DBC dendritic tips. The dark-adapted ERG b-waves of the Grm6nob3/nob8 mice were reduced in comparison to those of the Grm6nob8 homozygotes at postnatal day 21 and 28. The overall ERG waveforms obtained from 4- through 68-week old Grm6nob8 mice were in general agreement for dark- and light-adapted conditions. The maximum response and sensitivity of the dark-adapted ERG b-wave did not change statistically significantly with age. SD-OCT revealed the maintained laminar structure of the retina, including a clear inner nuclear layer (INL) at each age examined (from 11 to 57 weeks old), although the INL in the mice older than 39 weeks of age was somewhat thinner than that seen at 11 weeks. Conclusions:Mislocalization of mutant mGluR6 is not normalized by reducing the total mGluR6. Mislocalized mutant mGluR6 does not trigger substantial loss of DBCs.

Dataset Information

Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

Publications

Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

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