Ldlr-Related Protein (Lrp1) expression in the retina of knockout mice lacking a functional low-density lipoprotein receptor (Ldlr) gene




Olivia Rose Ballone

Introduction

The macula is the part of the human retina responsible for central vision. It has a very high concentration of photoreceptor cells which detect light and send signals to the brain via inner retinal cells. AMD is caused by a gradual loss of function in the retinal pigment epithelium (RPE), the support cells directly underlying photoreceptors in the eye. Photoreceptor death results from loss of RPE support. AMD patient eyes are characterized by accumulation of cholesterol in and beneath the RPE of the macula in greater amounts than in the periphery (4, 5). AMD patients have drusen formation beneath the RPE. Drusen are localized, basal RPE deposits containing a complex mix of lipids including cholesterol.
It has been shown that variants of genes encoding proteins involved in lipid transport and cholesterol pathways are risk factors for AMD development and/or progression (1, 3). Low density lipoprotein receptor (Ldlr) and Ldlr-Related Protein (Lrp1) are cholesterol particle uptake receptors which can both clear LDL from extracellular space. In liver cells, Ldlr and Lrp1 are co-expressed and in KO mice they have been shown to compensate for one another (6).
The impact of the knockout of Ldlr in the mice retina has not yet been characterized. We found that RPE cells appear normal in these mice. Since we do not see gross abnormalities in the RPE of Ldlr KO mice, we hypothesize that Lrp1 may compensate for the absence Ldlr in the RPE, as is seen in liver cells.

Materials and Methods
Ldlr-KO mice were developed by the Jackson Laboratory to have a Neomycin resistance (Neo) cassette inserted into exon 4 of the Ldlr gene. This insertional modification results in the production of a non-functional, truncated protein (2). DNA was isolated from homogenized tails of both wild type and Ldlr KO mice. DNA isolated from the WT and KO mice was amplified by PCR using a forward primer in intron 3 and reverse primer in intron 4 of the Ldlr gene. PCR product was visualized on a 1% agarose gel, purified, and sent out for sequencing by Genewiz. WT and KO mice were sacrificed by CO2 asphyxiation and their eyes were enucleated. Eye cups were collected from dissection of the whole eyes and homogenized in lysis buffer. RNA was purified from homogenized eye cups. RT-PCR was performed on RNA isolated from eye cups from WT and KO mice using primers spanning exons 3-5 and 2-3 of the Ldlr gene. A third primer set was designed to detect the presence of the Neo-cassette in the Ldlr transcript. Another primer set spanning exon 23-25 in the Lrp1 gene was designed. RT-PCR products were visualized on a 1% agarose gel, purified, and sent out for Sanger sequencing.

Results
PCR amplification of tail DNA isolated from WT and KO mice was performed using a forward primer in intron 3 and reverse primer in intron 4 of the Ldlr gene. The results show that the PCR product generated from WT mice is the expected size, whereas the KO PCR product is significantly larger (Fig. 1). The difference in the sizes of the PCR products generated likely reflects the presence of the Neo-cassette insert in exon 4 in the KO mice. Sanger sequencing of the band created by the KO mice confirms the presence of the primed region of intron 3, the first 202 bp of exon 4, a 5’ HPRT insertion vector, the Neo-cassette sequence and a 3’ insertion vector, followed by the remaining sequence of exon 4, and the primed region of intron 4. The PCR product produced from DNA isolated from WT mice represents the sequence from intron 3 through intron 4. The faint signal at 541 bp observed in the PCR reaction performed on DNA isolated from the KO animals was determined to be intron 3 through intron 4 (the WT product), and is likely the result of a pipetting error when loading the gel.
RT-PCR was performed using primers spanning exons 3 to 5, 2 to 3, 3 to Neo-cassette (in exon 4) of the Ldlr gene, and 23 to 25 in the Lrp1 gene and the products generated is presented in in Figure 2 (2A, 2B, 2C, and 2D respectively).The products generated from the RNA isolated from the WT and KO mice using primers spanning exons 3 through 5 was 603 bp and 162 bp respectively (Fig. 2A). The product generated from the WT mice was the predicted size, and was confirmed by Sanger sequencing (Fig. 3, shown in blue). The product generated from the KO mice represents the presence of a transcript in which exon 4 was skipped (Fig. 3, shown in red). This was confirmed by Sanger sequencing. The RT-PCR products generated from the RNA isolated from the WT and KO mice using primers in exon 2 and 3 of the Ldlr gene generated products of the same size as expected (Fig. 2B). This predicted product was confirmed by Sanger sequencing. RT-PCR analysis with primer in exon 3 and primer in the Neo-cassette confirmed the presence of said transcript in the KO mice but not in the WT mice. RT-PCR performed on the RNA isolated from the WT and KO mice using primers specific for the Lrp1 transcript did not exhibit any significant quantitative differences (Fig. 2D). Sanger sequencing confirmed that these bands are a product of the Lrp1 transcript. RT-PCR performed on the RNA isolated from the WT and KO mice using primers specific for the Lrp1 transcript did not exhibit any significant quantitative differences (Fig. 2D). Sanger sequencing confirmed that these bands are a product of the Lrp1 transcript.

Discussion
The results of this study indicate that there is not a noticeable upregulation of the Lrp1 transcript in the KO eye cups compared to the WT eye cups. Although this finding does not support the hypothesis that Lrp1 will be upregulated in Ldlr-KO mice RPE, the performance of quantitative RT-PCR reactions may enable the detection of a more subtle change in the level of Lrp1 transcript. Further research into the Abca1 transcript, which codes for a cholesterol efflux regulatory protein, could prove useful in elucidating the lipid-cholesterol compensatory pathway in the RPE of Ldlr-KO mice. The Abca1 transcript has been shown to be upregulated in Ldlr-KO mice liver.
Understanding the cholesterol pathway is important as misregulation may play a role in AMD.

References
Curcio, Christine A. "Soft drusen in age-related macular degeneration: biology and targeting via the oil spill strategies." Investigative Ophthalmology & Visual Science 59.4 (2018): AMD160-AMD181.
The Jackson Laboratory. “MOUSE STRAIN DATASHEET - 006952: B6.129-Akt2tm1.1Mbb Ldlrtm1Her/J”.”
Noske, Uta Maria, et al. "Lipid metabolism in retinal pigment epithelium (RPE): a possible role of LDL receptors." Der Ophthalmologe 95.12 (1998): 814-819.
Pennington, Katie L., and Margaret M. DeAngelis. "Epidemiology of age-related macular degeneration (AMD): associations with cardiovascular disease phenotypes and lipid factors." Eye and vision 3.1 (2016): 34.
Rudolf, M., et al. "Accumulation of lipid particles in Bruch's membrane of LDL receptor knockout mice as a model of age-related macular degeneration." Der Ophthalmologe: Zeitschrift der Deutschen Ophthalmologischen Gesellschaft 101.7 (2004): 715-719.
van de Sluis, Bart, Melinde Wijers, and Joachim Herz. "News on the molecular regulation and function of hepatic LDLR and LRP1." Current opinion in lipidology 28.3 (2017): 241.




Figures


Figure 1- PCR Amplification of DNA isolated from WT and KO mice tails using primers spanning intron 3 to intron 4.


Figure 2- Gel imaging of RT-PCR reaction products. (A) RT-PCR products using primers spanning exons 3 to 5 in LDLR gene. (B) RT-PCR products using primers spanning exons 2 to 3 in LDLR gene. (C) RT-PCR products using primers spanning exon 3 to Neo-cassette in exon 4 in the LDLR gene. (D) RT-PCR products using primers spanning exon 23 to 25 in the LRP1 gene.


Figure 3- Exon-Intron graph of exon 2 through 5 of the LDLR gene. Red star indicates the Neo-cassette insert in exon 4 of KO mice. Red splice diagram represents KO RT-PCR product when primed with forward primer in exon 3 and reverse primer in exon 5 (represented by arrows). Blue splice diagram represents WT RT-PCR product when primed with these primers.


Abstract
Age-related macular degeneration (AMD) is the leading cause of permanent vision loss in the elderly population of developed countries. AMD patients exhibit a buildup of cholesterol in and beneath the macula of the eye (1). Understanding the formation of these lipid rich deposits could help in the development of prevention and treatment strategies for AMD.
This project aims to study Ldlr-Related Protein (Lrp1) expression in the retina of low-density lipoprotein receptor (Ldlr) knockout mice. Both Ldlr and Lrp1 genes code for membrane receptor proteins involved in cholesterol transport. It is published that Lrp1 transcription is upregulated in the liver of Ldlr-KO mice (6). However, this lipid-cholesterol pathway has not yet been characterized in the retina of Ldlr-knockout mice.
The genotypes of the wild type (WT) and Ldlr-knockout (KO) mice were confirmed by PCR amplification, and Sanger sequencing. RT-PCR reactions were performed with three different Ldlr primer sets and one Lrp1 primer set on RNA purified from WT and KO mice eye cups. These products were visualized on gels and sent out for sequencing to confirm their identity. No change in expression of Lrp1 was detectable in these RT-PCR products.

Full Paper

Acknowledgments

I would like to thank Dr. Finnemann for providing me with the mice used for this project and assisting me with my project design. I would like to thank Anthony Evans and Devin Rocks for their patience and continuous support. Finally, I would like to thank Dr. Rubin for his guidance and helpful feedback throughout this project.


This document was last modified 05/15/2019.
This site is powered by the versatile Zope platform.
This is a project of the Biology Department of Fordham University
Biotechniques.org Home