Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

syndrome are predicted to disrupt calcium binding, so calcium binding by fibrillin seems to play an important role. It can be crucial for protein-protein interaction, stabilization of the molecule, protection against proteolysis and maturation of a proform of fibrillin. Single mutation in EGF-like, calcium binding domain of EFEMP1 might disrupt potential protein-protein interactions and results in misfolding and accumulation of EFEMP1. Protein-protein interactions through EGF-like domains are well known, for example Notch-Serrate proteins interact that way during development of the nervous system in Drosophila melanogaster (Rebay et al., 1991). This type of interactions is also known from the vertebrates (Notch/Delta signalling pathway). There is a possibility that EFEMP1 accumulates because of inability to bind its normal secretion partner. It has been recently discovered, that EFEMP1 binds tissue inhibitor of metalloproteinases-3 (TIMP-3), a matrix-bound inhibitor of matrix metalloproteinases (Klenotic et al., 2004). It is known, that mutations in the Timp-3 gene cause Sorsby fundus dystrophy (SFD), other hereditary macular degenerative disease ( Langton et al., 2000).

EFEMP1 has been recently named fibulin 3 (Giltay et al., 1999) and qualified to the family of fibulins, extracellular matrix proteins (Argraves et al., 2003; Timpl et al., 2003). Six know members, including FBLN3 (EFEMP1), has been characterized from mammalian species. All of them share an elongated structure and many calcium-binding sites, owing to the presence of tandem arrays of epidermal growth factor-like domains. The structure of most of the fibulins can be modified by alternative messanger RNA splicing. They are hypothesized to function as intramolecular bridges that stabilize the organization of extracellular matrix structures (Argraves et al., 2003). The involvement of mutant fibulins in the development of macular degeneration has been confirmed lately by the identification of mutations in FBLN 5 and 6 in subsets of patients with AMD (Schultz et al., 2003; Stone et al., 2004) .

EFEMP1 is known to be a secreted protein, but the retinal cells that express its gene have not yet been identified. Our goal was to localize the EFEMP1 transcripts in different types of human retinal cells. We also wanted to analyze the compartmentalization of the wild type and mutated EFEMP1 proteins.

2EFEMP1 Displays an Unexpected Expression Pattern in Diseased Human Retina

We have cloned EFEMP1 genes (mutated and wild type) into pBluescript vector and performed in situ hybridization on human retinas. This technique allows specific nucleic acid sequences to be detected in morphologically preserved cells or tissue sections. In combination with immunohistochemistry it can relate microscopic topological information to gene activity at the DNA, mRNA, and protein level. In order to obtain probes for in situ hybridization we have performed in vitro transcription with T3 or T7 polymerase to produce sense or antisense probes. Sense probes served as a negative control as they cannot hybridize with EFEMP1 mRNA. In vitro transcription was conducted in presence of radioactive compound — 35S. Radioactive

riboprobes were synthesized and hydrolyzed to finally produce fragments of around 150 base pairs, which were then used for hybridization. The length of a probe is usually a compromise between the strength of the signal (long probe) and the penetration into the tissue (short probe).

To determine expression patterns of the EFEMP1 gene, in situ hybridization studies were carried out on pre-treated paraffin sections of normal and Malattia Leventinese (ML) human retinas. We have used melanoma affected eyes as a control for all experiments. In normal eye EFEMP1 transcripts were localized mainly in the retinal ganglion cells and the photoreceptors. In the diseased human eyes, transcripts were principally found in the RPE accompanied with drastic morphological changes of this tissue. This upregulation is not specific for the ML eye except in the ciliary body. In situ hybridization studies revealed specific localization of EFEMP1 transcripts in ML and control human retinas. Despite being a widely expressed gene, EFEMP1 pattern shows tissue specificity and differs between normal and diseased eyes. To compare the level of EFEMP1 gene expression between different regions of RPE in ML eye, laser capture microdissections (LCM) were combined with RNA isolation and real-time PCR. Significant differences were observed between different regions of RPE in ML eye.

Fusion proteins containing wild type and mutated (R345W) EFEMP1 (the shortest and the longest splice variants) with C-terminal GFP or flag peptide under the control of CMV promoter were constructed in order to determine cellular localization of the protein. Plasmids were transfected into VERO (African green monkey kidney) and ARPE-19 (human RPE) cells. While long variants of the protein showed an uniform, cytoplasmic localization, short variants were localized within the vacuoles and the cells expressing the transgenes died few hours after transfection. Western-blot analysis showed that the long variant of the protein is stable and gives a strong signal whereas the short variant is degraded. These observation led us to conclusions, that the short variant of EFEMP1 (43 kDa) probably doesn’t exist in substantial amounts at the protein level. We have been unable to detect any striking difference in cell compartmentalization between the wild type and the mutated EFEMP1 during these studies.

Both abnormal expression of the EFEMP1 gene and mutation and accumulation of EFEMP1 protein (inside or outside the cells) might contribute to the Malattia Leventinese pathology.

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Role of ELOVL4 in Fatty Acid Metabolism

Vidyullatha Vasireddy, Majchrzak Sharon, Norman Salem, Jr,

and Radha Ayyagari

1 Introduction

Stargardt like macular dystrophy (STGD3) is an inherited form of early onset autosomal dominant macular degeneration. Pathophysiology of STGD3 is associated with the loss of central vision and RPE atrophy. Accumulation of lipofuscin was observed with the progression of the disease (Stone et al., 1994; Griesinger et al., 2000). Mutations in a novel gene, elongation of very long chain fatty acid-4 (ELOVL4) were found to be associated with STGD3 (Zhang et al., 2001).

ELOVL4 is homologous to the ELO family of proteins that play a role in fatty acid metabolism in yeast (Tvrdik et al., 2000). Human ELOVL4 protein has 314 amino acids with a putative dilysine (KXKXX) endoplasmic reticulum retention signal at the C-terminus. So far a 5 bp-deletion mutation and two additional mutations, all resulting in truncation of the protein have been identified in the ELOVL4 gene (Bernstein et al., 2001; Maugeri et al., 2004). The 5 bp deletion mutation in the exon 6 of ELOVL4 causes a frame shift, leading to the loss of 51 amino acids and a premature truncation of the protein (Zhang et al., 2001). Co-expression of mutant and wild type ELOVL4 in COS-7 cells revealed that the mutant protein interacts with the wild type, forming higher molecular weight aggregates (Grayson and Molday, 2005; Vasireddy et al., 2005).

To better understand the mechanism underlying the pathology of STGD3, we generated and characterized two mouse models: a knock-in mouse model carrying the Elovl4 5-bp deletion (Elovl4+/del ) and an Elovl4 knock-out mouse model. Elovl4+/del were viable and developed progressive photoreceptor degeneration, while the heterozygous Elovl4 knock-out mice had normal retinal function (Raz-Prag et al., 2006; Vasireddy et al., 2006). The homozygous knock-out mice and the 5-bp deletion knock-in mice carrying the deletion in the homozygous state (Elovl4del/del ) died within a few hours after birth due to an defect in the

R. Ayyagari

Ophthalmology and Visual Sciences, W. K. Kellogg Eye Center, University of Michigan, Ann Arbor, Tel: 313-647-6345, Fax: 313-936-7231

e-mail: ayyagari@umich.edu

epidermal barrier (Vasireddy et al., 2007). Through studies on these mice,we have demonstrated that ELOVL4 is essential for the synthesis of epidermal very long-chain FA (VLFA), and is required for generating omega-O-acylceramides, which are essential for the formation of epidermal permeability barrier (Vasireddy et al., 2007). Subsequently, these observations were confirmed by studies reported by other investigators (Cameron et al., 2007; McMahon et al., 2007).

In the mouse, expression of Elovl4 has been observed in retina, brain, whole skin, and testis (Mandal et al., 2004). Similar to retina and skin, brain tissue is also known to have a unique unsaturated fatty acid composition. In the skin, lack of functional ELOVL4 resulted in depletion of fatty acids with chain length longer than C28, however, the function of ELOVL4 in brain and ocular tissue is not known. To study the effect of an absence of functional ELOVL4 on brain and eye, we analyzed the fatty acid composition of these tissues from homozygous Elovl4 5-bp deletion knock-in mice and compared with the litter mate controls.

2 Materials and Methods

2.1 Animal Maintenance and Tissue Collection

Design of the construct and generation of Elovl4+/del has been described elsewhere (Vasireddy et al., 2006). Experiments were conducted in accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research and protocols used were approved by the Animal Care and Use Committee of the University of Michigan. All the animals were maintained in a 12-hour dark and light cycle. For the present studies, P0 pups were dissected on day 0. Total eye balls and brain were collected, weighed and stored at -80◦C until fatty acid analysis was performed. Tail samples were collected for genotyping of the animals.

2.2 Extraction and Analysis of Fatty Acid Profiles

To determine the effect of Elovl4 5-bp deletion mutation on fatty acid composition in the eye and brain, the levels of saturated and unsaturated fatty acids in these tissues from P0 pups of Elovl4 del/del mice (N=10) were determined and compared with wild type littermate controls (N=10). The tissues were weighed and lipid extraction was performed according to the Bligh and Dyer method (Bligh and Dyer, 1959). Butylated hydroxytoluene (BHT) was added to each sample, along with the internal standard 22:3n-3 methyl ester. Retinas were subsequently transmethylated using the BF3-methanol method of Morrison and Smith (Morrison and Smith, 1964) as modified by Salem et al (Salem et al., 1996), with the co-solvent hexane (Salem et al., 1996). The methyl ester samples were analyzed by gas chromatography as previously described (Salem et al., 1996).

2.3 Statistical Analysis

Data of eye ball fatty acids are presented as the mean ± standard deviation (SD) and the Fatty acid profiles of the brain and diet are presented as mean± Standard Error (SE). Comparisons of means between two experimental groups were performed using the two tailed, independent Student’s t test. To compare the mean among more than two different groups, Analysis of variance (ANOVA) was used.

3 Results

The Elovl4 5-bp deletion knock in mice with homozygous and heterozygous genotypes were born in an expected Mandalian ratio. Pups carrying the different genotypes were indistinguishable at birth. Total eye balls and brains were collected from new born pups for fatty acid analysis.

Comparison of the fatty acid profile of the eye balls of P0 pups from mice carrying the 5-bp deletion in the homozygous state and litter mate heterozygous pups and wild type controls showed a significant alteration in the levels of several n–3 fatty acids including 20:5 n–3 (P = 0.006), 22:5n–3 (P<0.01), 24:5 n–3 (P = 0.003) and 24:6 n–3 (P = 0.046) (Table 1). The levels of 20:5 n–3, 22:5 n–3, 24:5 n–3 and 24:6 n–3 FA were found to be decreased in Elovl4 del/del pups when compared with the Wt controls as well as Elovl4 +/del pups. Where as the levels of these FA were found to be increased in Elovl4 +/del animals when compared with the Wt animals. However, there was no significant alteration in the levels of 22:6n–3 in the eye balls of these pups. Although the exact role of ELOVL4 in the fatty acid metabolism in these tissues is not known, the altered fatty acid levels indicate a possible role of ELOVL4 in long chain fatty acid metabolism in ocular tissue.

The fatty acid profile of brain tissue did not show significant alteration in the levels of the saturated, monounsaturated or polyunsaturated fatty acids in Elovl4del/del animals when compared with the Wt controls (Table 2).

4 Discussion

The present study is designed to investigate the effect of the lack of a functional ELOVL4 on the fatty acid composition of the brain, and eye balls, using mice carrying the 5-bp deletion in the homozygous state. In mammals, fatty acids consisting up to 16 carbon atoms (C16) are synthesized by fatty acid synthase complex. Significant amounts of these synthesized fatty acids and the dietary fatty acids are further elongated to long chain fatty acids and to very long chain fatty acids (VLFA). Formation of VLFA takes place in the ER by membrane bound elongases. Six Elovls (ELOVL1-6), which are proposed play a role in the condensation step of the fatty acid chain elongation have been identified. Elovl1 was observed to be involved in the synthesis of saturated C26 fatty acids

Table 1 Fatty acid profiles of the eye balls of Elovl4 del/del and Elovl4 +/del pups in comparison to Wt controls

and sphingolipid formation (Salem et al., 1996; Tvrdik et al., 2000). Elovl2 was hypothesized to have a role in PUFA synthesis. Both mouse and human Elovl2 were shown to elongate arachidonic acid (20:4n–6), eicosapentaenoic acid (20:5n–3), docosatetraenoic acid (22:4n–6) and docosapentaenoic acid (22:5n–3). Ablation of Elovl3 also showed an impaired epidermal permeability barrier and synthesis of VLCFA in the skin (Westerberg et al., 2004) Elovl5, is involved in the elongation of various polyunsaturated long-chain fatty acids of C18–C20 (Leonard et al., 2002). Elovl6 has been reported to be involved in the elongation of saturated fatty acids with 12–16 carbons to C18 and may not participate in the

Table 2 Brain Fatty acid profiles of Elovl4 del/del pups in comparison to the Wt littermate controls. Data are expressed as the mean ± sem ug/mg tissue wet weight

elongation of fatty acids with chain lengths longer than C18 (Moon et al., 2001). These observations indicate that the ELOVL family of proteins participates in fatty acid elongation in mammals similar to the Elo group of enzymes in yeast.

Although Elovl4 was initially discovered as a gene associated with Stargardt-like macular degeneration (Zhang et al., 2001), significant amount of Elovl4 expression was observed in non-ocular tissues, like brain and skin. (Mandal et al., 2004). The high expression of ELOVL4 in the retina leads to the speculation that it might be involved in the elongation steps required for the synthesis of docosahexaenoic acid (22:6n–3) the major long-chain fatty acid present in the retina (Zhang et al., 2001).

Depletion of very long-chain fatty acids with chain length longer than C26, OH-ceramides and glycosyl-ceramides was observed in the skin of mice lacking

288 V. Vasireddy et al.

functional ELOVL4 suggesting a role for ELOVL4 in the synthesis of FAs with chain length longer than C26 (Vasireddy et al., 2007). The retinal FA profile of mice carrying the 5-bp deletion Elvol4 mutation in the heterozygous state showed a significant decrease in the levels of 20:5n–3 (P <0.028), 22:5 n–3 (P <0.04), 24:6 n3 (P <0.005). Consistent with these results, a significant decrease in the 20:5 n–3(P <0.02), 22:5 n–3 (P <0.01), 24:5 n–3 (P <0.01), 24:6 n–3 (P <0.01) fatty acid levels was observed in the eye balls of Elovl4 pups compared to the age matched littermate wild type controls (Table 1). This data clearly indicates a role for ELOVL4 in long-chain fatty acid metabolism in the ocular tissue. The analysis of fatty acid profiles in the retina and eye balls of heterozygous and homozygous 5-bp deletion knock-in mice respectively did not include very long chain fatty acids and ceramides. Therefore, the effect of the absence of functional ELOVL4 on these classes of fatty acids in the ocular tissue is not known.

The analysis of the fatty acid profiles of the brain of Elovl4 pups did not show significant alteration in the levels of saturated, mono-unsaturated and polyunsaturated fatty acids. In the brain tissue, in addition to ELOVL4 gene, Elovl1, Elovl3, Elovl4, Elovl5 are expressed and the level of expression of Elovl4 was found to be less than the expression of Elovl1 and Elovl6. The unaltered long chain fatty acid profiles of brain tissue from Elovl4 pups also indicate that the ELOVL4 may not be the major fatty acid elongase in the brain. The brain fatty acid profiles of mice carrying the Elovl4 Y270X mutation in the homozygous state were also reported to be not significantly different from the control mice (Cameron et al., 2007). These results are consistent with our observation on the brain lipid profile of 5-bp deletion knock in mice.

Though the general function of the Elovl genes is partially understood, very little is known about the role of fatty acids of specific chain length in cellular and developmental processes. Although the altered key epidermal FA profiles of Elovl4del/del mice and Elovl4 deficient mice are now identified, we are still at an early stage in understanding the biological function of ELOVL4 and its specific role in the regulation of fatty acid chain elongation. The analysis of the epidermal fatty acids of Elovl4del/del showed an alteration in the VLFA and ceramide metabolism. We do not know whether similar alterations will be observed in the eyes and brain of Elovl4 pups. The present study was not designed to evaluate the levels of VLFAs or ceramides in the retina and brain. Hence, additional studies are needed to establish the role of Elovl4 in fatty acid metabolism of the brain and eye balls of these pups. Further detailed analysis of the VLFA profiles in these pups may aid in the understanding of the effect of ELOVL4 mutation on VLFA metabolism.

Acknowledgments The authors would like to thank Austra Liepa (University of Michigan) for generation and maintenance of the animals. This work was supported by grants to RA from NIH (R01EY13198) and the Foundation Fighting Blindness; a research grant to RA, core grant to the University of Michigan, Department of Ophthalmology (P30EY007003) and Vision Research (P30EY07060). The project was supported in part by the Intramural Research Program of the National Institutes of Health, NIAAA.

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